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Method Development in Electrospray Ionisation
Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry Study of Plant Oils - Macadamia Oil as
a Model
By
Ahmad Mokhtari-Fard
A thesis submitted in fulfilment of the requirements
for the degree of Doctor of Philosophy
School of Chemistry
The University of New South Wales
Sydney, Australia
July 2008
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils II
Declaration
I hereby declare that this thesis is my own work and that, to the best of my
knowledge and belief, it contains no material previously published or written by
another nor material which to substantial extent has been accepted for an award of
any other degree or diploma of a university or other institute of higher learning
except where due acknowledgement is made in the text of this thesis.
Ahmad Mokhtari-Fard
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils III
Abstract
A novel analytical method is developed to examine the chemical composition of
plant oils by electrospray ionisation high-resolution Fourier transform ion cyclotron
resonance mass spectrometry in both positive- and negative-ion modes. To date, this
is the first reported application of this technique for the study of macadamia nut oil.
Samples of macadamia nut oil from the Macadamia Integrifolia- Proteaceae family
(smooth shell) are examined. The fatty acid profile of the oil is obtained by this mass
spectrometric examination of the transesterified and hydrolysed oil samples. The
Fourier transform ion-cyclotron resonance mass spectrometry results are compared to
those obtained from similar samples using gas chromatography-mass spectrometry
techniques. High performance liquid chromatography and Fourier transform ion
cyclotron resonance mass spectrometry are used to separate and assign the isomers
present in the methanol extract of the oils in separate experiments.
Significant results in this study include:
- The first observation and identity of a number of oxidised triacylglycerols in
macadamia oil samples.
- The first observation of oxidised and free fatty acids, measured directly in
hydrolysed oil and in the methanol extract of macadamia oil.
- High resolution Fourier transform ion cyclotron resonance mass spectrometry
in broadband mode which enables isobars to be observed.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils IV
- Esterified oil Fourier transform ion cyclotron resonance mass spectrometry
results are consistent with our gas chromatography-mass spectrometry results
and with the results of similar studies on macadamia oil in the literature.
- A number of fatty acids with odd number of carbon atoms are observed in the
oil.
- In electrospray ionisation Fourier transform ion cyclotron resonance mass
spectrometry of oils, the sample preparation is straightforward. The sample is
dissolved in methanol or acetonitrile and the solution is introduced to the
electrospray source directly. Introducing oil samples to the gas
chromatograph-mass spectrometer needs the oils to be esterified prior to the
analysis.
- In this work, state-of-the-art mass spectrometry demonstrates distinct
advantages in comparison to gas chromatography measurements such as
direct identification of free fatty acids in oil samples, whereas this is not
possible in gas chromatography-mass spectrometry due to the required
esterification step prior to the analysis.
- High performance liquid chromatography fraction collection is combined
with Fourier transform ion cyclotron resonance mass spectrometry in off-line
mode and found to improve the sensitivity, selectivity and signal to noise
levels due to the lower number of compounds in each high performance
liquid chromatography fraction compared to the methanol extract of
macadamia oil sample. Also isomers of monoacylglycerols have been
resolved using the high performance liquid chromatography technique.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils V
Acknowledgments
Many people have assisted me in this study and I wish to record my appreciation
here for their assistance.
I owe thanks to my supervisor Associate Professor Gary Willett for providing
guidance, encouragement and inspiration during the research project and for help in
writing my thesis. I have been always impressed by his down-to-earth personality
and earnest attitude towards scientific research.
My special thanks go to my co-supervisor Mr. Athol Turner who assisted me to
obtain and inspired me with the research described in this thesis. As a good friend, he
always supported me with many helpful discussions and suggestions. I believe what I
have learnt from him will benefit the rest of my life and my career. His young heart
is a gift that I wish will beat for many more years.
I gratefully appreciate the support and supervision provided by Associate Professor
Stephen Colbran, specially during the last year of my PhD study.
I appreciate the guidance and friendship provided by Professor Bryn Hibbert. I will
always be honored to recognize him as an outstanding colleague.
I would like to pay a special tribute to many people who helped me to complete this
thesis. Dr. Rui Zhang, who is a wonderful teacher and taught me how to use the
FTICR mass spectrometer, Dr. Derek Smith, an expert in theoretical chemistry who
helped me in calibration calculations, Dr. Joe Brophy, who assisted me to obtain the
GC-MS results and discussed them with me and Dr. Keith Fisher who provided me
with helpful ideas and procedures for operating the FTICR mass spectrometer.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils VI
Special thanks are also paid to FTICR-MS group members including Khaled Edbey,
John Giffard and Nick Proschogo. Over the years, I have received a great deal of
help from all of them. I also appreciate help from Mansour Ahmad teaching me some
skills in working with fatty acids on the FTICR mass spectrometer.
I also pay a special tribute to Mr. Han Sit Chan for his support in solving many
computer and network problems that I encountered during this research project.
Finally, my special thanks go to my wife (Fariba) and two children (Nazanin and
Reza) for supporting me throughout my PhD studies. They are very tolerant and
patient and have always encouraged me to move forward during long years of my
study. I am a lucky person to have such a supportive family.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils VII
Dedication Dedicated to:
My wife and my children, (Fariba, Nazanin and Reza)
my parents,
(my mother and the memory of my father)
and my teachers.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils VIII
Table of Contents
Declaration................................................................................................... II Abstract.......................................................................................................III Acknowledgments ........................................................................................ V Dedication ................................................................................................. VII Table of Contents.....................................................................................VIII List of Tables...............................................................................................XI List of Figures ..........................................................................................XIII List of Abbreviations ............................................................................... XVI 1. Introduction .............................................................................................. 1
1.1- Definitions ............................................................................................................... 5 1.1.1- Lipids............................................................................................................................ 5 1.1.2- Fatty Acids .................................................................................................................. 13 1.1.3- Triacylglycerols........................................................................................................... 21
1.1.3.1- Determination of the Positional Distribution of FAs in Fats and Oils ..................... 23 1.2- Macadamia, From Nut to Shelf............................................................................... 24
1.2.1- Introduction................................................................................................................. 24 1.2.2– History and Production of Macadamia Nut .................................................................. 25 1.2.3– Botanical Description.................................................................................................. 27 1.2.4- Soil, Climate and Nutrition .......................................................................................... 28 1.2.5- Harvesting................................................................................................................... 28 1.2.6- Oil Extraction .............................................................................................................. 30 1.2.7– Industrial Macadamia Nut Oil Refining Processes........................................................ 32
1.3- Chemical Reactions Used in Sample Preparation for FTICR-MS Analysis.............. 34 1.3.1– Methanol Extraction of Macadamia Nut Oil to Remove the Triacylglycerols................ 34 1.3.2- Transesterification of Macadamia Nut Oil .................................................................... 35 1.3.3- Alkaline Hydrolysis of Macadamia Nut Oil.................................................................. 36
1.4- Mass Spectrometry of Lipids.................................................................................. 38 1.4.1-Gas Chromatography-Mass Spectrometry ..................................................................... 39 1.4.2- Electrospray Ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry............................................................................................................................................. 42
1.4.2.1- Electrospray Ionisation Source ............................................................................. 43 1.4.2.2- The ICR Cell........................................................................................................ 47
1.4.2.2.1- Ion Trapping................................................................................................. 48 1.4.2.2.2- Ion Cyclotron Motion.................................................................................... 50 1.4.2.2.3- Ion Cyclotron Excitation and Detection ......................................................... 51
1.4.2.3- Fourier Transform ................................................................................................ 55 1.4.2.4- Mass Calibration .................................................................................................. 56 1.4.2.5- Tandem Mass Spectrometry ................................................................................. 57
1.5- High Performance Liquid Chromatography ............................................................ 59 1.6- Kendrick Masses and Mass Defects in the Identification of Homologous Series...... 62 1.7- Normal Probability (Rankit) Plot ............................................................................ 63 1.8- Summary of the Method Development ................................................................... 63
2. Experimental........................................................................................... 68 2.1- Materials................................................................................................................ 69 2.2- Chemical Procedures.............................................................................................. 69
2.2.1- Methanol Extraction of Macadamia Oil........................................................................ 69 2.2.2- Hydrolysis of Macadamia Oil ...................................................................................... 70 2.2.3- Transesterification of Macadamia Oil........................................................................... 70 2.2.4- Sample Preparation for Mass Spectrometry .................................................................. 71
2.3- Instrumentation ...................................................................................................... 71
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils IX
2.3.1- Electrospray-ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometer............................................................................................................................................. 71
2.3.1.1- The Vacuum System ............................................................................................ 74 2.3.1.2- Electrospray Ionisation Source ............................................................................. 75 2.3.1.3- The “Infinity®” ICR cell ....................................................................................... 80 2.3.1.4- Ion Trapping ........................................................................................................ 82 2.3.1.5- Typical Source, Ion-transfer and ICR Cell Parameters Used in Positive- and Negative-ion Modes on the BioApex II 70e FTICR Mass Spectrometer ............................. 83 2.3.1.6- Superconducting Magnet ...................................................................................... 84 2.3.1.7- Collision-Induced Dissociation ............................................................................. 84 2.3.1.8- Pulse Sequence in FTICR-MS Experiments .......................................................... 85 2.3.1.9- Mass Calibration .................................................................................................. 87 2.3.1.10- The BioApex II FTICR Mass Spectrometer Control Software ............................. 88
2.3.2- High Performance Liquid Chromatography.................................................................. 88 2.3.2.1- Gradient Elution................................................................................................... 89
2.3.3- Gas Chromatography- Mass Spectrometry.................................................................... 91 3. Validation of the ESI FTICR-MS Method Developed for the Analysis of Plant Oils..................................................................................................... 92
3.1- Relation between Peak Intensities and Concentration of the Ions ............................ 93 3.2- Reproducibility of the ESI Source and the FTICR Mass Spectrometer .................... 95 3.3- Detection Limits of FTICR-MS in FA Measurement ............................................ 100 3.4- Effect of the Hexapole Ion Trap Delay on the Peak Intensities.............................. 100 3.5- Aging Stability of the Oil Samples ....................................................................... 101 3.6- Fragmentation during Ion Transfer ....................................................................... 102 3.7- Discussion of the Validity of the Mass Spectral Peak Assignments ....................... 105 3.8- Normal Probability (Rankit) Test of the Peak Intensities and Measured Masses .... 106
4. Positive-ion ESI FTICR-MS of Processed Macadamia Oil................. 108 4.1- Introduction ......................................................................................................... 109 4.2- Positive-ion ESI FTICR-MS of Processed Macadamia Oil.................................... 109
4.2.1- Free Fatty Acids and Monoacylglycerols Region, m/z 150-400................................... 112 4.2.2- Diacylglycerol Region, m/z 500-750 .......................................................................... 114 4.2.3- Triacylglycerol Region, m/z 800-1000 ....................................................................... 115
4.3- Positive-ion ESI FTICR-MS of Methanol Extract of Processed Macadamia Oil .... 117 4.4- Positive-ion ESI FTICR-MS of Hydrolysed Processed Macadamia Oil................. 121 4.5- Positive-ion ESI FTICR-MS of Esterified Processed Macadamia Oil.................... 123 4.6- Positive-ion ESI FTICR-MS of Esterified Methanol Extract of Processed Macadamia Oil .............................................................................................................................. 128 4.7- A Comparison of the Fatty Acids Observed in the Positive-ion ESI FTICR Mass Spectra of the Neat, Methanol Extract, Hydrolysed, Esterified and Esterified Methanol Extract of Processed Macadamia oil ............................................................................ 130
5. Negative-ion ESI FTICR-MS of Processed Macadamia Oil ............... 139 5.1- Introduction ......................................................................................................... 140 5.2- Negative-ion ESI FTICR Mass Spectra of Neat Processed Macadamia Oil ........... 141
5.2.1- Introduction............................................................................................................... 141 5.2.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400 .................................... 142 5.2.3- Diacylglycerol Region, m/z 500-750 .......................................................................... 145
5.2.3.1- Free Fatty Acid Dimers ...................................................................................... 147 5.2.3.2- Diacylglycerols .................................................................................................. 150
5.2.4- Triacylglycerol Region, m/z 800-1000 ....................................................................... 151 5.3- Negative-ion ESI FTICR Mass Spectra of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 154
5.3.1- Introduction............................................................................................................... 154 5.3.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400 .................................... 157
5.3.2.1- Kendrick Mass Defect (KMD) Values ................................................................ 161 5.3.3- Diacylglycerol Region, m/z 500-750 .......................................................................... 162 5.3.4- Triacylglycerol Region, m/z 800-1000 ....................................................................... 168
5.3.4.1- KMD Values of the Assignments in the TAG Region.......................................... 171
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils X
5.3.5- Stability of the Methanol Extract of Processed Macadamia Oil ................................... 172 5.4- Negative-ion ESI FTICR-MS of the Hydrolysed Processed Macadamia Oil.......... 175
5.4.1- Introduction............................................................................................................... 175 5.4.2- FTICR Mass Spectrum of Hydrolysed Processed Macadamia Oil ............................... 176 5.4.3- Comparison of FTICR Mass Spectra of Macadamia Oils ............................................ 180
5.5- A Summary of the Negative-ion ESI FTICR-MS of the Neat, Methanol Extract and Hydrolysed Processed Macadamia Oil......................................................................... 183
6. Gas Chromatography-Mass Spectrometry of Processed Macadamia Oil................................................................................................................... 189
6.1- Introduction ......................................................................................................... 190 6.2- GC-MS Analysis of Esterified Processed Macadamia Oil ..................................... 192 6.3- GC-MS Analysis of the Esterified Methanol Extract of Macadamia Oil ................ 193 6.4- A Summary of the Positive-ion and Negative-ion ESI FTICR-MS and GC-MS of the Hydrolysed and Esterified Neat Processed Macadamia Oil .......................................... 195 6.5- Conclusions ......................................................................................................... 200
7. Off-line ESI FTICR-MS of HPLC Fractions of the Methanol Extract of Processed Macadamia Oil ........................................................................ 202
7.1- Introduction ......................................................................................................... 203 7.2- HPLC of the Methanol Extract of Processed Macadamia Oil ................................ 205 7.3- ESI FTICR-MS of the HPLC Fractions 19 to 31 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 207 7.4- ESI FTICR-MS of the HPLC Fractions 32 to 50 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 212
7.4.1- Fractions 32 to 35 ...................................................................................................... 213 7.4.2- Fractions 36 to 38 ...................................................................................................... 213 7.4.3- Fractions 38 to 40 ...................................................................................................... 215 7.4.4- Fractions 44 to 48 ...................................................................................................... 217
7.5- ESI FTICR-MS of the HPLC Fractions 50 to 60 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 220 7.6- ESI FTICR-MS of the HPLC Fractions 60 to 63 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 222 7.7- ESI FTICR-MS of the HPLC Fractions 77 to 83 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 224 7.8- Positive- and Negative-ion FTICR Mass Spectra of the HPLC Blank Fractions of the Methanol Extract of Processed Macadamia Oil............................................................ 226 7.9- General Discussion: ............................................................................................. 228
8. Conclusions and Future Work ............................................................. 235 8.1- General Conclusion for this Study ........................................................................ 236 8.2- Future Work......................................................................................................... 239
9. References ............................................................................................. 244
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XI
List of Tables Table 1.1- Four nomenclature methods of FAs............................................................................... 15 Table 1.2- Some saturated and unsaturated FAs............................................................................ 17 Table 1.3- Composition of 100 g of macadamia nuts and the FA constituent of macadamia nut oil.
............................................................................................................................................. 25 Table 1.4- World production and consumption of macadamia nut-in-shell (NIS) and kernel in year
2000...................................................................................................................................... 26 Table 1.5- Different trapping methods usually used in FTICR mass spectrometry. ......................... 48 Table 2.1- Typical parameters used in positive- and negative-ion FTICR mass spectrometry
experiments.......................................................................................................................... 83 Table 2.2- Solvent programming for HPLC in the gradient elution of macadamia oil methanol
extract. ................................................................................................................................. 90 Table 3.1- Assignment of mass spectral peaks in the FTICR mass spectrum of a solution of fatty
acids mixture solution shown in Figure 3.1. .......................................................................... 96 Table 4.1. Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass
spectrum of neat macadamia oil shown in Fig. 4.1.............................................................. 111 Table 4.2. Assignment of the mass spectral peaks (>2%) in positive-ion ESI-FTICR mass spectrum of
the methanol extract of macadamia oil shown in Figure 4.5............................................... 119 Table 4.3. Assignment of the major mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass
spectrum of hydrolysed macadamia nut oil shown in Figure 4.6......................................... 122 Table 4.4- Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass
spectrum of esterified macadamia nut oil shown in Figure 4.7. .......................................... 124 Table 4.5. Assignment of the mass spectral peaks (>1%) in the positive-ion ESI-FTICR mass
spectrum of the esterified methanol extract of processed macadamia oil shown in Fig. 4.10............................................................................................................................................ 129
Table 4.6- A comparison of the fatty acid components observed in the positive-ion ESI-FT-ICR mass spectrometry experiments.................................................................................................. 134
Table 5.1- Assignment of the mass spectral peaks (>2%) in the expanded fatty acid region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.2. ....................................................................................................................... 144
Table 5.2- Assignment of the mass spectral peaks (>0.2% of the base peak in Figure 5.1) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of processed macadamia oil shown in Figure 5.3. ................................................................... 146
Table 5.3- Assignment of the mass spectral peaks (>2% of the base peak in Figure 5.1) in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.5. ............................................................. 152
Table 5.4- Assignment of selected mass spectral peaks (>2%) in the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Fig. 5.6. ....... 157
Table 5.5- Assignment of the mass spectral peaks (>2%) in the expanded FA region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.7............................................................................... 158
Table 5.6- Calculated Kendrick mass defects for homologous series in the negative-ion ESI FTICR mass spectrum of methanol extract of macadamia nut oil.................................................. 161
Table 5.7- Assignment of the mass spectral peaks (>2%) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.8............................................................................... 164
Table 5.8- Assignment of the mass spectral peaks (>2%) and KMD values in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.9.......................................................... 169
Table 5.9- Assignment of the mass spectral peaks (>2% of the base peak) in the negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil shown in Fig. 5.11. ............. 177
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XII
Table 5.10- Relative intensities of the assigned mass spectral peaks (>2% of the base peak) in the FTICR mass spectra of hydrolysed neat macadamia oil in both positive and negative-ion modes and the esterified oil in positive-ion mode. ............................................................. 188
Table 6.1- Assignment of the peaks in the GC-MS TIC of esterified processed macadamia oil...... 192 Table 6.2- Assignment of the peaks in the GC-MS TIC of esterified methanol extract of processed
macadamia oil. ................................................................................................................... 194 Table 6.3- A comparison of the FA ions observed in the positive-ion (Tables 4.3 & 4.4) and
negative-ion (Table 5.9) FTICR-MS as well as the GC-MS analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia nut oil............................................................... 198
Table 7.1. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.3. ........................................................... 210
Table 7.2. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.4. ........................................................... 211
Table 7.3. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 36 to 38 in Figure 7.7. ........................................................... 214
Table 7.4. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 38 to 40 in Figure 7.8. ........................................................... 216
Table 7.5. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectrum of the HPLC fraction 40 in Figure 7.9. .................................................................. 216
Table 7.6. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 44 to 48 in Figure 7.10. ......................................................... 218
Table 7.7. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 44 to 48 in Figure 7.11. ......................................................... 219
Table 7.8. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 50 to 60 in Figure 7.13. ......................................................... 222
Table 7.9. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 60 to 63 in Figure 7.14. ......................................................... 223
Table 7.10. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 77 to 83 in Figure 7.16. ......................................................... 225
Table 7.11. Assignment of the major mass spectral peaks in the ESI-FTICR mass spectra of the HPLC fractions of the methanol extract of macadamia nut oil. .................................................... 230
Table 7.12. A comparison of the retention times of the HPLC analysis of the methanol extract of macadamia nut oil with standard FA solutions analysed on same column in a previous study............................................................................................................................................ 234
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XIII
List of Figures Figure 1.1- A general classification of lipids...................................................................................... 6 Figure 1.2- Chemical structure of (a) glycerol and (b) a generalized structure of a triacylglycerol. ... 9 Figure 1.3- A naturally occurring TAG in cocoa butter. ..................................................................... 9 Figure 1.4- Structures of some common lipids. .............................................................................. 13 Figure 1.5- Hydrolysis of trioleyl glycerol to yield three oleic acid molecules and the glycerol
molecule............................................................................................................................... 19 Figure 1.6- General TAG molecule.................................................................................................. 21 Figure 1.7- Macadamia nuts on the tree ........................................................................................ 28 Figure 1.8- Cold press equipment used in the industrial production of seed and nut oil. Top: a batch
press, bottom: a hole-cylinder type oil expeller. .................................................................. 31 Figure 1.9- Saponifiable lipids in the hydrolysis reaction................................................................ 37 Figure 1.10- Non saponifiable lipids in the hydrolysis reaction of oils. ........................................... 38 Figure 1.11- Formation of charged drops at the tip of electrospray needle in high intensity electric
field. ..................................................................................................................................... 43 Figure 1.12- The formation of Rayleigh jets at the fissility greater than 1. Fine droplets dispatched
from the main drop are visible in shots c and d. ................................................................... 44 Figure 1.13- Schematic diagram of the electrospray needle, capillary and skimmer (not to scale). 46 Figure 1.14- Schematic diagram of the infinity ICR cell used in Bruker BioApex II. ......................... 47 Figure 1.15- Ions orbit in a plane perpendicular to the direction of a uniform magnetic field, B.
Positive (a) and negative (b) ions rotate in opposite directions in ion cyclotron motion....... 50 Figure 1.16- Incoherent and undetectable ion cyclotron orbital motion (a) is converted to a
coherent and detectable motion by applying an electric field and (b) is detected in the ICR cell at the frequency of the ions of particular m/z value....................................................... 52
Figure 2.1- The passively shielded Bruker BioApex II 70e Fourier transform ICR mass spectrometer used in this thesis. ................................................................................................................ 72
Figure 2.2- Schematic diagram of the Bruker BioApex II 70e ESI Fourier transfer ICR mass spectrometer used in this research....................................................................................... 73
Figure 2.3- The differential pumping on the Bruker BioApex II ESI FTICR mass spectrometer vacuum system. ................................................................................................................................. 74
Figure 2.4- Schematic diagram of the off-axis Analytica ESI source used in this research project... 76 Figure 2.5- The off-axis and on-axis spray needles inside the Analytica ESI cage............................ 77 Figure 2.6- The capillary tip and the end plate. . ............................................................................ 77 Figure 2.7- Schematic diagram of quartz capillary, skimmer and hexapole location in the ion optics
system . ................................................................................................................................ 79 Figure 2.8- Photograph showing the hexapole ion-trap in Analytica ESI source in the BioApex II
FTICR mass spectrometer...................................................................................................... 80 Figure 2.9- The infinity ICR cell in BioApex II FTICR mass spectrometer. ........................................ 81 Figure 2.10- Schematic diagram of the infinity ICR cell used in BioApex II FTICR mass spectrometer.
PV1, EV1 etc are parameters’ name . .................................................................................... 82 Figure 2.11- Pulse sequence used in FTICR-MS analyses for a simple experiment. ......................... 85 Figure 3.1- Negative-ion ESI FTICR mass spectrum of the test solution. ......................................... 96 Figure 3.2- Relative intensities of palmitate anion peaks versus measured masses in 17 consecutive
FTICR mass spectra of the test solution (normalised versus oleate anion peak as 1)............. 98 Figure 3.3- Relative intensities of stearate anion peaks versus measured masses in 17 consecutive
FTICR analyses of the test solution (normalised versus oleate anion peak as 1).................... 98 Figure 3.4- Average deviations from exact masses vs. average measured masses in 17 consecutive
FTICR mass spectra of the test solution. Each point represents the average of 17 mass measurements of a particular FA in the test solution............................................................ 99
Figure 3.5- Positive-ion FTICR mass spectra of methanol solution of neat processed macadamia oil in (a) December 2002, (b) July 2003 and (c) September 2003.............................................. 102
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XIV
Figure 3.6- Negative-ion FTICR mass spectra of the FA test solution at three capillary-skimmer voltages, a) 20 V, b) 125 V and c) 300 V............................................................................... 104
Figure 3.7- Rankit plot of the measured masses of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode. .............................................. 107
Figure 3.8- Rankit plot of the peak intensities of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode......................................................... 107
Figure 4.1- Positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil. ............................. 110 Figure 4.2- Expanded FA region of positive-ion ESI-FTICR mass spectrum of neat macadamia oil. 112 Figure 4.3- Expanded DAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut
oil. ...................................................................................................................................... 114 Figure 4.4- Expanded TAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut
oil. The thin vertical line underneath each peak is produced by the peak-picking routine in Bruker software.................................................................................................................. 116
Figure 4.5- Positive-ion ESI-FTICR mass spectrum of the methanol extract of macadamia nut oil. 118 Figure 4.6- Positive-ion FTICR mass spectrum of hydrolysed macadamia nut oil.......................... 121 Figure 4.7- Positive-ion ESI-FTICR mass spectrum of esterified macadamia oil............................. 123 Figure 4.8- Expanded FA and FAME region of the positive-ion ESI-FTICR mass spectrum of esterified
macadamia nut oil shown in Figure 4.7............................................................................... 125 Figure 4.9- Positive-ion FTICR mass spectrum of esterified macadamia oil in m/z 279.12 to m/z
279.23 region. Peaks 0.03 Da apart are resolved................................................................. 127 Figure 4.10- Positive-ion FTICR mass spectrum of esterified methanol extract of macadamia nut oil.
........................................................................................................................................... 128 Figure 4.11- A comparison of the ESI FTICR mass spectra of the FA region of (a) neat macadamia oil,
(b) methanol extract of macadamia oil, (c) hydrolysed macadamia oil and (d) esterified macadamia oil. ................................................................................................................... 132
Figure 4.12- A graphical comparison of the unsubstituted FA anions observed in the positive-ion FTICR mass spectra of the neat (Table 4.1), the methanol extract (Table 4.2), hydrolysed (Table 4.3) and the esterified (Table 4.4) processed macadamia nut oil.............................. 133
Figure 5.1- Negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil. ........... 142 Figure 5.2- Fatty acid region of the negative-ion ESI FTICR mass spectrum of neat processed
macadamia nut oil in Figure 5.1, m/z 150-400. ................................................................... 143 Figure 5.3- DAG region of the negative-ion ESI FTICR mass spectrum of neat processed macadamia
nut oil in Figure 5.1, m/z 500-750........................................................................................ 146 Figure 5.4- A comparison of the experimental (a) and simulated (b) isotopic distribution of the
C34H65O4¯ fatty acid dimer anion. ........................................................................................ 148 Figure 5.5- (a) TAG region of the negative-ion ESI FTICR mass spectrum of the neat processed
macadamia nut oil in Figure 5.1, m/z 800-1000, (b) expanded peaks in the TAG region, m/z 870-882............................................................................................................................... 151
Figure 5.6- Negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil. ................................................................................................................................ 156
Figure 5.7- FA region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 150-400.................................................... 158
Figure 5.8- DAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 500-750.................................................... 163
Figure 5.9- TAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 800-1000, (a) prior to internal calibration and (b) after internal calibration was carried out. ..................................................................... 169
Figure 5.10- A comparison of the negative-ion ESI FTICR mass spectra of the methanol extract of macadamia oil on two dates: (a) 22/01/2003 and (b) 19/08/2004...................................... 173
Figure 5.11- Negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil. ..... 177 Figure 5.12- Comparison of the negative-ion ESI FTICR mass spectrum of (a) hydrolysed processed
macadamia oil, (b) hydrolysed cold pressed oil batch 12 and (c) hydrolysed cold pressed oil batch 13.............................................................................................................................. 181
Figure 6.1- GC-MS TIC of esterified processed macadamia oil. ..................................................... 192 Figure 6.2- GC-MS TIC of esterified methanol extract of macadamia oil....................................... 194 Figure 7.1- HPLC chromatogram of the methanol extract of processed macadamia oil. ............... 206
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XV
Figure 7.2- Expansion of HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times of 19 to 31 minutes. ....................................................... 207
Figure 7.3- Positive-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil. ............................................................................ 208
Figure 7.4- Negative-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil. ............................................................................ 209
Figure 7.5- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times 32 to 50 minutes...................................................... 212
Figure 7.6- Negative-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 34 and (b) fraction 35 of the methanol extract of processed macadamia nut oil. ................................ 213
Figure 7.7- Positive-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 36, (b) fraction 37 and (c) fraction 38 of the methanol extract of processed macadamia nut oil...................... 214
Figure 7.8- Positive-ion ESI FTICR mass spectra of the HPLC fractions, (a) fraction 38, (b) fraction 39 and (c) fraction 40 of the methanol extract of processed macadamia nut oil...................... 215
Figure 7.9- Negative-ion ESI FTICR mass spectrum of the HPLC fraction 40 of the methanol extract of processed macadamia oil................................................................................................ 217
Figure 7.10- Positive-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.................................................................................... 217
Figure 7.11- Negative-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.................................................................................... 219
Figure 7.12- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 50 to 60 minutes...................................................... 220
Figure 7.13- Positive-ion ESI FTICR mass spectra of the HPLC fractions 50 to 60 of the methanol extract of processed macadamia oil.................................................................................... 221
Figure 7.14- Positive-ion ESI FTICR mass spectra of the HPLC fractions 60 to 63 of the methanol extract of processed macadamia oil.................................................................................... 223
Figure 7.15- Expanded HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 77 to 83 minutes............................................................................... 224
Figure 7.16- Positive-ion ESI FTICR mass spectra of the HPLC fractions 77 to 83 of the methanol extract of processed macadamia oil.................................................................................... 225
Figure 7.17- Positive-ion FTICR mass spectrum of the HPLC blank fraction. ................................. 226 Figure 7.18- Negative-ion FTICR mass spectrum of the HPLC blank fraction. . .............................. 227
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XVI
List of Abbreviations
APCI Atmospheric pressure chemical ionisation ATP Adenosine triphosphate CI Chemical ionisation CID Collision induced dissociation DAG Diacylglycerol ECD Electron capture detector ELSD Evaporative light scattering detector EI Electron ionisation ESI Electrospray ionisation FA Fatty acid FAME Fatty acid methyl ester FFA Free fatty acid FTICR Fourier transfer ion cyclotron resonance GC Gas chromatography HPLC High performance liquid chromatography ICR Ion cyclotron resonance IUPAC International Union of Pure and Applied Chemistry KMD Kendrick mass defect LC Liquid chromatography LDL Low density lipoprotein MAG Monoacylglycerol MALDI Matrix assisted laser desorption ionisation MS Mass spectrometry PEG Poly ethylene glycol PUFA Polyunsaturated fatty acid S/N Signal-to-noise TAG Triacylglycerol TOF Time of flight TOC Total ion chromatogram UHV Ultra high vacuum
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 1
Chapter 1
1. Introduction
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 2
Plant oils (edible oils) play an important role in human nutrition due to their every
day consumption and important biologically active compounds present in them. The
chemical composition of the different types of oil is considerably diverse, but in
general they contain significant amounts of triacylglycerols (approximately 97%) and
a large number of important minor chemical compounds. Plant oils are usually
characterized by standard techniques such as gas chromatography-mass spectrometry
(GC-MS) for fatty acid methyl ester (FAME) and reversed phase high performance
liquid chromatography (HPLC) for triacylglycerol (TAG) region. These two
techniques only discriminate between different types of oils such as macadamia,
canola and sunflower but not between different batches of same oil from the same
extracted lipid source, because in the case of FAME analysis the ratio of the fatty
acids present in each oil sample remain virtually constant. The aim of this research is
to develop a novel method for the analysis of oils and fats using electrospray
ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry
(FTICR-MS) which enables the direct analysis of complex mixtures, hence enabling
different batches of the same oil (containing different chemical finger prints) to be
chemically characterized. No other analytical technique has the capability of
analyzing oil samples which contain hundreds of compounds and this is highly
relevant to the worldwide edible oil industry. Traditionally, gas chromatography-
mass spectrometry (GC-MS) has been the favoured method for the analysis of oils
and fats and the majority of studies on the analysis of oils and fats have been
conducted using this technique.[1-5]
ESI FTICR-MS demonstrates several advantages in comparison to GC-MS
technique.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 3
- The high resolution and high mass accuracy of the ESI FTICR-MS technique
allows the direct analysis of macadamia nut oil without prior
chromatographic separation. The GC-MS technique, requiring the oil to be
esterified prior to gas chromatography analysis, significantly alters the
chemical composition of the oil, and is also a time-consuming process.
- The high resolution in broadband mode (average 50,000 full width at half
maximum (FWHM)) is a feature of FTICR-MS which enables isobars in
complex mixtures such as oils to be easily resolved.
- The high mass accuracy of FTICR-MS assists one in assigning possible
chemical formulae to selected mass spectral peaks.[6,7] This facility restricts
the number of possible candidates to a small group depending on the
resolution and the mass accuracy of the technique at the designated molecular
mass. As well, the selection criteria applied by the analyst include acceptable
mass measurement error, nominated participating atoms (elemental
composition including carbon, hydrogen, oxygen, nitrogen, phosphorus and
sulphur) and isotope patterns.[8] However, to postulate more accurate
structural formulae we need to carry out further separation and tandem mass
spectrometric analyses.
- Sample preparation and introduction in ESI FTICR-MS includes dissolution
of the sample in a suitable solvent such as methanol and introduction of the
solution into the ESI source; a comparatively simple process.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 4
- The sample is introduced to the ESI source at room temperature and
atmospheric pressure. No high temperature or low pressure inlet system is
required.
- Collision-Induced Dissociation (CID) can be used to provide information
regarding the molecular structure of components of the oil by selectively
fragmenting molecular ions.[9] GC-MS also provides structural information
by fragmentation of the analyte molecules by electron ionization (EI),
however, the fragmentation is not selective. A further drawback to GC-MS is
that double bonds are prone to migrate on the hydrocarbon chain under EI
conditions. Finally, oil samples need to be transesterified so that the
acylglycerols are converted to fatty acid methyl esters to permit the GC-MS
analysis.
The ESI FTICR-MS technique does not resolve molecular isomers, as separation is
based on m/z only. This problem is partly overcome by applying tandem mass
spectrometry in the form of CID to selectively break up the molecular ions into
smaller fragments.[10]
The alternative is to use HPLC in conjunction with ESI FTICR-MS in off-line or on-
line mode. A combination of HPLC and GC-MS has been used in the analysis of
conjugated linoleic acid isomers by Roach et al.[11]
In the off-line mode, the various compounds in the oil are separated by an HP liquid
chromatograph that is connected to a fraction collector which collects the output at
certain time intervals. The collected fractions are then analysed by ESI FTICR-MS.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 5
In the on-line mode, on the other hand, the output of the HPLC column is connected
directly to the ESI source of the mass spectrometer.
It should be emphasized here that ESI FTICR-MS is not considered to be a
replacement for the older techniques such as GC-MS, but instead as complementary.
The information collected using a combination of chromatography and FTICR-MS
techniques provide unparalleled precise understanding of the chemical constituents
of oils, especially for the characterization of trace compounds, and could eventually
be used to establish a library of the chemical composition of such oils.
1.1- Definitions 1.1.1- Lipids
Lipids are among the most important groups of biological molecules because of their
key roles in nutrition, metabolism and the energy needs of living cells. As a group of
organic compounds of various chemical compositions, lipids are usually divided into
four groups:
- Fatty acids (saturated and unsaturated)
- Glycerols (glycerol-containing lipids)
- Nonglycerol lipids (sphingolipids, steroids, waxes, lipid soluble vitamins)
- Complex lipids (lipoproteins)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 6
The term “Lipid” has not found a widely accepted definition. General organic
chemistry and biochemistry textbooks usually use solubility-based definitions and
have defined lipids as a group of naturally occurring hydrophobic compounds that
are readily soluble in non-polar solvents such as hexane, chloroform, toluene and
ether, and have a very low solubility in water.[12,13]
Figure 1.1 shows the main groups and sub-groups of lipids.
Lipids
Fatty acids Glycerides
Saturated Unsaturated Neutral glycerides
Phospho-glycerides
Nonglyceride lipids
Complex lipids
LipoproteinsSphingolipidsSteroids Waxes
Sphingomyelins Glycolipids
Figure 1.1- A general classification of lipids.
Lipids include a wide range of compounds such as fatty acids (FAs) and their
derivatives, carotenoids, terpenes, steroids, waxes and nonglyceride lipids as
illustrated in Figure 1.1. The structures of the compounds in different classes of
lipids are not necessarily similar.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 7
A more specific definition of lipids than one based on solubility is necessary. Most
lipid chemists tend to use the term “lipid” for FAs and their naturally occurring
derivatives such as esters and amides. This definition can be expanded to include
compounds closely related to fatty acid derivatives through biosynthetic pathways or
by their biochemical or functional properties such as prostanoids, aliphatic alcohols,
aliphatic ethers and cholesterol.
Christie has defined the term lipid to include: “Fatty acids and their derivatives, and
substances related biosynthetically or functionally to these compounds”[14] and this is
the definition adopted for this term in this thesis.
The most common type of lipids in nature consist of FAs and substituted FAs linked
by an ester bond to glycerol or other alcohols such as cholesterol, or by amide bonds
to sphingoid bases, or to other amines. Lipids may have moieties other than FAs,
phosphoric acid, organic bases, carbohydrates and various other compounds that can
be hydrolysed and released using a variety of hydrolytic procedures.
Simple lipids are defined as the lipids that, on hydrolysis, yield two or fewer types of
primary products per mole, and complex lipids are those that on hydrolysis, release
more than two primary molecules per mole of lipid.
Examples of a complex lipid can be either a glycerophospholipid, which contains a
phosphoric acid moiety and a glycerol backbone, or a glycolipid, which contains a
carbohydrate moiety.
In terms of energy storage and release, lipids mainly contain hydrogen, oxygen and
carbon, and represent a highly reduced form of carbon in organic molecules; upon
controlled oxidation in living cells, lipids produce a larger quantity of energy
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 8
compared to related molecules such as carbohydrates. Thus, in the living cell
metabolism and energy storage, lipids are the preferred molecules by nature due to
their higher energy release upon oxidation.
The lipids found in living systems are either amphipathic (containing both polar and
nonpolar groups) or hydrophobic (possessing only nonpolar groups). The
hydrophobic nature of lipid molecules enables membranes to behave as barriers to
polar molecules and water.[12]
Fats and oils are mixtures of naturally occurring acylglycerols. At room temperature,
fats are usually solids and oils are liquids. As one of their multiple functions, they
serve in energy storage and production in living systems. Although carbohydrates
(such as glucose) serve as the main energy source in the animal cell metabolism, an
equal weight of fat produces twice as much energy compared to carbohydrates. As
the fats are hydrophobic, the organism that carries fat does not have to carry the
hydration water that accompanies carbohydrates. Thus it is more efficient for an
organism to store fats as an optimal source of energy because it needs less mass for
the same amount of energy compared to carbohydrates and proteins and releases
double the amount of energy in bio-oxidation.[15]
The backbone of any triacylglycerol (TAG) molecule is glycerol. To produce a TAG
molecule, the three hydroxyl functional groups in a glycerol molecule are esterified
by three acyl groups. Figure 1.2 shows glycerol and a generalised structure of a TAG
molecule.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 9
OH
HO
OH
OR
O
O R''
O
R'
O
O
(a) Glycerol (b) Triacylglycerol
Figure 1.2- Chemical structure of (a) glycerol and (b) a generalized structure of a triacylglycerol. R, R’ and R” could be identical or different FA substituents.
Figure 1.3 shows a naturally occurring TAG in cocoa butter, 2-oleyl-1,3-distearyl
glycerol, in which two terminal substituted acyl groups are stearyl, and the third one
is oleyl.
O
O OC17H35
C17H35
C7H14
C8H17C
CC
O
OO
2-oleyl-1,3-distearyl glycerol
Figure 1.3- A naturally occurring TAG in cocoa butter.
Simple lipids include TAGs, diacylglycerols (DAGs), monoacylglycerols (MAGs),
steroids including sterols and sterol esters (cholesterol and cholesterol esters in
animal tissues), waxes, tocopherols (substituted benzopyranols) and free fatty acids
(FFAs).
Glycerophospholipids include a variety of compounds that are different in the
substituted groups on the glycerol phosphate. They include phosphatidic acid (1,2-
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 10
diacylglycerol-3-phosphate, trace compound in tissues, precursor of most other
glycerolipids), phosphatidylglycerol (1,2-diacylglycerol-phosphoryl-1’-glycerol,
lung-surfactant and important in plant chloroplasts), diphosphatidylglycerol
(cardiolipin, found in heart muscle mitochondria), phosphatidylcholine (1,2-
diacylglycerol-3-phosphorylcholine or lecithin, most abundant lipid in the
membranes of animal tissues), lysophosphatidylcholine (a powerful surfactant and a
water-soluble lipid), phosphatidylethanolamine (cephalin, the second most abundant
phospholipid in animal and plant tissues and major lipid in micro-organisms),
phosphatidylserine (N-acylphosphatidylserine, a weakly acidic lipid),
phosphatidylinositol (regulating vital processes in the cell), phosphonolipids (a
phosphonic acid esterified to glycerol) and ether lipids.
Glycoglycerolipids are found in photosynthetic plants and have mono- or di-
carbohydrate molecules (usually galactose or glucose) substituted on the glycerol
backbone. The other two hydroxyl groups on the glycerol are substituted by FA
groups.
Sphingomyelins and glycosphingolipids include long-chain bases that are linked by
an amide bond to a FA or to phosphorus-containing moieties such as sphingosines,
phytosphingosines, ceramides, sphingomyelins, ceramide phosphorylethanolamines,
neutral glycosylceramides and gangliosides.[14]
Waxes are esters of long-chain (C14 to C36) saturated and unsaturated fatty acids
with long-chain (C16 to C30) alcohols that are insoluble in water but soluble in
nonpolar organic solvents such as hexane.[15]
Figure 1.4 shows the structure of some common lipids.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 11
OH
OH
C15H31
O
O
HO
C15H31
O
O
C15H31
O
O
(a) Glycerol palmitate (MAG) (b) Glycerol dipalmitate (DAG)
HO
H
H
H
(c) A wax molecule (oleyl stearate) (d) Cholesterol
O
R3
R2
HO
R1
(e) Tocopherol (R1, R2 and R3 are H and/or CH3 for α, β, γ or δ tocopherol)
O
O
PO
OOH
OH
C15H31
O
C15H31
O
(f) Phosphatidic acid (1,2-Dipalmitoylglycerol-3-phosphate)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 12
O
O
PO
O OH
O
C15H31
O
C15H31
O
OH
OH
(g) Phosphatidyl glycerol (1,2-Dipalmitoylglycerol-3-phosphoryl-1'-glycerol)
RCOO CH
CH2
H2C
RCOO
O P O CH2
CHOH
H2C O P O CH2
HC
H2C
OOCR
OOCR
O- H+
OO
O- H+
(h) Cardiolipin (diphosphatidylglycerol)
RCOO CH
H2C
H2C
OOCR
O P O CH2CH2N+(CH3)3
O
O-
(i) Phosphatidylcholine
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 13
RCOO CH
H2C
H2C
OOCR
O P O CH2CH
O
O-
NH3+
COO-
(j) Phosphatidylserine (N-Acetylphosphatidylserine)
RCOO CH
H2C
H2C
OOCR
O P O
O
O- H+
OH
OHOH
OH
OH
(k) Phosphatidylinositol
Figure 1.4- Structures of some common lipids. R groups denote any organic substituent.
1.1.2- Fatty Acids
Fatty acids, as they occur in nature, are compounds consisting of a hydrocarbon
chain and a carboxylic acid functional group (-COOH). The general chemical
formula of FAs is RCOOH, in which R denotes a hydrocarbon chain, (RCO known
as an acyl group), usually with an even number of carbon atoms (as it is synthesized
using acetyl groups in the living cells) containing typically 4 to 36 carbons (C4 to
C36). The hydrocarbon chain of fatty acids usually contains an even number of
carbon atoms (commonly 12-24), may be saturated or unsaturated, and can contain
various functional groups such as double bonds, hydroxyl groups, alkyl group
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 14
branches on their hydrocarbon chain and a few contain three carbon rings. FAs are
usually found in the fats.[15]
Industrially, FAs are produced by the hydrolysis of the ester linkages in a fat or oil
(both of which generally consist of more than 95% TAGs), with the removal of
glycerol.[16]
The term “fatty acid” is often used to refer to those carboxylic acids that occur
naturally in TAGs; however, many biochemists and chemists refer to all unbranched
carboxylic acids as FAs regardless of their origin and chain length. In this thesis the
term “fatty acid” refers to all carboxylic acids that can occur in natural fats and oils,
regardless of the carbon chain length or number or type of functional groups on the
carbon chain.
Saturated FAs do not contain any double bonds along the acyl chain. Examples of
some saturated FAs that are observed in fats and oils are:
• Butyric (butanoic acid): CH3(CH2)2COOH or C4:0
• Lauric (dodecanoic acid): CH3(CH2)10COOH or C12:0
• Myristic (tetradecanoic acid): CH3(CH2)12COOH or C14:0
• Palmitic (hexadecanoic acid): CH3(CH2)14COOH or C16:0
• Stearic (octadecanoic acid): CH3(CH2)16COOH or C18:0
• Arachidic (eicosanoic acid): CH3(CH2)18COOH C20:0
Unsaturated FAs contain one or more double bonds. In most of these fatty acids,
each double bond has 3n carbon atoms after it, for n an integer in the range of 1 up to
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 15
about 8, and in animal fats and plant oils it is usual that all double bonds are in a cis
configuration.
The cis and trans terminology is applicable only to double bonds with two different
substitutions, however, unsaturated FAs usually contain more than two different
substituents on each double bond. Thus the Z and E (replacing cis and trans
respectively) terminology describes the steric configuration of the fatty acid alkyl
chain more precisely, although the cis and trans prefixes are still commonly used by
lipid chemists.
Table 1.1 lists four nomenclature methods of FAs.
Table 1.1- Four nomenclature methods of FAs.
Nomenclature of Fatty Acids Names Abbreviations
Trivial Name IUPAC Name Carboxyl-reference
ω-reference
Palmitic acid Hexadecanoic 16:0 16:0 Stearic acid Octadecanoic 18:0 18:0 Oleic acid 9-Octadecenoic acid 18:1 ∆9 18:1 (ω-9)
Linoleic acid 9,12-Octadecadienoic acid 18:2 ∆9,12 18:2 (ω-6) α-Linolenic acid 9,12,15-Octadecatrienoic acid 18:3 ∆9,12,15 18:3 (ω-3)
Apart from the trivial and IUPAC naming systems, there are two nomenclature
systems to make clear where the double bonds are located in the FA molecules.
These two schemes are illustrated below:
Nomenclature 1:
- Specifies the chain length and number of double bonds, separated by a colon; for
example, the 18-carbon oleic acid with one double bond is abbreviated as C18:1. The
positions of double bonds are specified by superscript numbers following ∆ (delta).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 16
For example, C20:2 ∆9,12 indicates that the double bonds are located at the ninth and
twelfth carbon-carbon bonds counting from the carboxylic group as carbon 1.[15]
Nomenclature 2:
- Omega-3, omega-6 or omega-9 (ω-3, ω-6 or ω-9): The first double bond is the
third, the sixth or the ninth carbon-carbon bond respectively counting from the end of
the chain most distant from the carboxyl group (ω carbon atom).
Examples:
• α-Linolenic acid, C18:3,
CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH, is an ω-3 FA,
Δ9,12,15, while γ-linolenic acid, C18:3, is ω-6 FA, Δ6,9,12.
• Linoleic acid, C18:2, CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH, Δ9,12,
and arachidonic acid, C20:4 Δ5,8,11,14, are ω-6 FAs.
• Oleic acid, C18:1, CH3(CH2)7CH=CH(CH2)7COOH, Δ9, and erucic acid,
C22:1 Δ13, are ω-9 FAs.
(Note: α-Linolenic acid (n-3) is the trivial name for all-cis-octadeca-9,12,15-trienoic
acid and γ-Linolenic acid (n-6) is the trivial name for all-cis-octadeca-6,9,12-trienoic
acid. [17])
In this thesis the first nomenclature will be used when referring to labeling of FAs.
Table 1.2 briefly lists some saturated and some unsaturated FAs and their IUPAC
and common names.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 17
Table 1.2- Some saturated and unsaturated FAs.
R in RCOOH IUPAC name Common name
a) Saturated fatty acids
CH3- Ethanoic acid Acetic acid CH3CH2- Propanoic acid Propionic acid
CH3CH2CH2- Butanoic acid Butyric acid n-C4H9- Pentanoic acid Valeric acid n-C5H11- Hexanoic acid Caproic acid n-C6H13- Heptanoic acid Enanthic acid n-C7H15- Octanoic acid Caprylic acid n-C8H17- Nonanoic acid Pelargonic acid n-C9H19- Decanoic acid Capric acid n-C11H23- Dodecanoic acid Lauric acid n-C13H27- Tetradecanoic acid Myristic acid
n-C15H31- Hexadecanoic acid Palmitic acid
n-C17H35- Octadecanoic acid Stearic acid
n-C19H39- Eicosanoic acid
C20:0 Arachidic acid
b) Unsaturated fatty acids
CH3(CH2)7CH=CH(CH2)7- (Z)-9-Octadecenoic acid
C18:1 Oleic acid
CH3(CH2)4CH=CHCH2CH=CH(CH2)7- (9Z,12Z)-9,12-Octadecadienoic
acid C18:2 Linoleic acid
CH3(CH2CH=CH)3(CH2)7- (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid
C18:3 α-Linolenic acid
Hexadecanoic acid or palmitic acid (C16:0) is the most abundant saturated FA in
nature whereas cis-9-octadecenoic acid or oleic acid (C18:1) is the most abundant
monoenoic FA in nature.[12] The C18 polyunsaturated FAs linoleic or Z-9,Z-12-
octadecadienoic acid (C18:2) and α-linolenic acid or Z-9,Z-12,Z-15-octadecatrienoic
acid (C18:3) are essential FAs for animals. They are major components of plant
lipids including vegetable oils. These FAs are biosynthetic precursors of C20 and
C22 polyunsaturated FAs (containing 3-6 double bonds) in animal systems.
Arachidonic acid (C20:4) is derived from linoleic acid via sequential de-saturation
and chain elongation reactions[18] and is an important constituent of the membrane
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 18
phospholipids in mammalian tissues, and is a precursor in the synthesis of
prostaglandins and other eicosanoids.[12]
The human body readily makes saturated FAs and unsaturated FAs that have one
double bond (monounsaturated FAs), but does not have the appropriate enzymes to
synthesize polyunsaturated FAs. At least one function of the essential FAs is to serve
as a precursor for the synthesis of eicosanoids, such as prostaglandins, a class of
compounds with hormone-like effects in many physiological processes such as
immune system and blood pressure regulation.[12]
FAs occur in large amounts in biological systems, but only in small amounts in the
free state. They typically are esterified to glycerol (to form acylglycerols) or to
glycerol derivatives such as phosphoglycerols.[12] The majority of FAs in plants and
animals occur in the form of TAGs, either simple (same FAs attached to glycerol) or
mixed (different FAs attached to glycerol).[12]
In vertebrates, free FAs circulate in the blood bound noncovalently to a protein
carrier, serum albumin. However, FA derivatives are present in blood plasma mostly
as esters or amides.[15]
FA derivatives are an important source of energy for many tissues since they can
yield relatively large quantities of adenosine triphosphate (ATP). Typically, many
cell types can use either glucose or FA derivatives to produce energy. Heart and
skeletal muscles prefer FA derivatives as the source of energy. On the other hand, the
brain cannot use FA derivatives as a source of energy, relying instead on glucose, or
on ketones produced by the liver from FA metabolism during starvation.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 19
As fats are composed of more than 95% acylglycerols, hydrolysis of fats
(saponification reaction) produces free FA molecules and glycerol. For example,
trioleyl glycerol yields three oleic acid molecules and glycerol on hydrolysis. Figure
1.5 shows the hydrolysis reaction of this triacylglycerol molecule.
Figure 1.5- Hydrolysis of trioleyl glycerol to yield three oleic acid molecules and the glycerol molecule.
Most naturally-occurring FAs possess an even number of carbon atoms with
unbranched carbon chain. The carbon chains may be saturated or unsaturated with a
varying number of double bonds. Double bonds are almost always in cis (Z)
configuration.[12]
Trans FAs are unsaturated FA molecules containing trans double bonds, which make
the molecule less twisted compared to FAs with cis double bonds. These are
generally a byproduct of industrial processes involving catalytic hydrogenation
(hardening of the oil), or alternatively they are generated by bacterial hydrogenation
of unsaturated FAs in the rumen (first stomach in cud-chewing animals) of
ruminating animals. Industrial hydrogenation of edible oils is used to produce stable
and solid oil products at room temperature, to prolong the oil shelf life and to make
the oil transportation and storage easier. Often associated with this process is the
production of unsaturated trans FAs (~5-10%), of which elaidic acid (trans-C18:1
+ C7H14
C8H17
O
OH
O
OO
O
O
O
C17H35
C17H35
C17H35
Hydrolysis
Acid or base
OH
HO
OH
3
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 20
Δ9) is a major component. The bacterial process in the rumen yields predominantly
trans vaccenic acid (trans-C18:1 Δ11), and also conjugated linoleic acids such as Δ9-
cis,11-trans-18:2.[19] Furthermore, some regular kitchen food cooking and processing
techniques convert cis-unsaturated FAs naturally present in food to their trans form.
High dietary intake of trans FAs by humans is known to be combined with a high
probability of myocardial infarction, atherosclerosis, coronary heart disease and
prostate cancer occurrence.[20,21]
Dietary lipids are sources of essential FAs for the human body. FAs are important in
membrane biosynthesis, energy balance, eicosanoid production, acetyl-CoA
production via β-oxidation and production of acylglycerols such as TAG molecules
via esterification, which are a source of energy for the body. The FA substituents
present in the cell membrane phospholipids influence the functional properties of
membranes. Polyunsaturated FAs increase the membrane fluidity, while saturated
FAs decrease it.[22]
Common FAs found in plant tissues are even numbered straight-chain compounds of
C14 to C22 with a saturated alkyl chain or up to four double bonds with cis
configuration. These FAs are also found in animal tissues with a wider range of chain
lengths and up to six double bonds that are separated by methylene groups.
Branched-chain FAs are synthesized by many micro-organisms and occasionally can
be produced in animal tissues. FAs with other substituent groups are found in some
plants and micro-organisms. These substituents include acetylenic and conjugated
double bonds, cyclopropane, cyclopentane and furan rings, hydroxy-, epoxy-, and
keto- groups.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 21
1.1.3- Triacylglycerols
Triacylglycerols are composed of glycerol esterified with three FAs. Figure 1.6
illustrates a general molecular formula of TAG molecules.
C
C
C
H
H
H
H
OOCR"
RCOO
OOCR'
H
Figure 1.6- General TAG molecule
IUPAC rules are used to number the carbons of TAG molecules. The secondary
hydroxyl group is shown to the left of carbon 2. The carbon above it will be carbon 1
and the carbon below it will be carbon 3. The prefix “sn” is used to specify
“stereospecific numbering”. Thus, the name triacyl-sn-glycerol is technically more
correct than triacylglycerol. When the stereochemistry is not specified, the primary
hydroxyl groups are usually named α- and α’-positions and the secondary hydroxyl
group is named β-position.
In living cells, the TAG molecules are synthesized by sequential acylation of free
glycerol via enzyme catalysed reactions, or by the catabolism of glucose and the
enzyme-controlled stepwise esterification of the hydroxyl groups. The product of
each esterification step has defined stereochemistry, and the stereochemistry of the
esterification processes are highly controlled by enzymes.[15]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 22
The position and the type of the FA substituents on the glycerol backbone play a key
role in the digestion, absorption and metabolism of the lipid in the living cell. [23,24]
During the digestion of a TAG molecule in the body, FAs at the sn-1 and sn-3
positions are digested by TAG-hydrolysing enzymes (e.g., Lipases) in the intestine.
The result is two free fatty acid molecules and a MAG molecule with an attachment
at sn-2. Free palmitic acid and stearic acid form insoluble salts with calcium and
magnesium in the intestine and their absorption by the body is reduced. The sn-2
MAG does not form a salt and is readily absorbed.[24,25] MAGs with FA derivatives
on the sn-2 positions are re-esterified and released into blood as TAGs later, thus
they are conserved in this way. In human milk fat more than 60% of the palmitic acid
is esterified at sn-2 position, while in cow milk it is mainly at sn-1 and sn-3
positions.[26-28]
The “pancreatic lipase hydrolysis” was the first method used to study the location of
FAs on the position sn-2 of the TAG molecules from natural oils and fats.[29-33]
Complete positional distribution of FAs is now determined using complex
stereospecific hydrolysis procedures. Due to this historical priority of the analytical
procedure, there has been an inclination to presume that the FA esterified on the
secondary hydroxyl group should be more important than the two primary ones. The
secondary hydroxyl group is important in digestion of TAG molecules in the
intestine of animals. The sn-3 is important in the cellular control mechanism as it is
the last position to be acylated during TAG biosynthesis.[34]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 23
1.1.3.1- Determination of the Positional Distribution of FAs in Fats and Oils
The position of FA substituents on TAG molecules influences their metabolism of
the lipids in the living cell. As discussed above, it has been shown that the FA
substituents on the sn-2 position are absorbed better than on the other two positions.
A higher frequency of occurrence of saturated FAs such as palmitic acid on the sn-2
position in animals increases the atherogenic potential of the lipid without altering
the level of blood lipids.[35]
Brokerhoff devised the first stereospecific analysis procedure for TAGs in 1965.[36]
The basic method includes partial hydrolysis of the TAG sample to prepare the
phospholipid derivatives, then hydrolysis with phospholipase A of snake venom,
followed by a separation and transesterification step of the products to be examined
on GC. The composition of the position sn-2 is independently determined by
pancreatic lipolysis.
NMR has found an increasing application in the locating of the FAs on the TAG
molecules in edible oils during the last fifteen years.[37-40] Both 1H and 13C NMR
have been used as an analytical tool to characterize the structure of lipids and to
assess the quality of the lipid products.[38,41] The problem with the 1H NMR of TAG
molecules is the intensive signal overlap of the aliphatic protons, while 13C NMR
suffers complexity from the small chemical shift differences observed for some
carbon atoms, although 13C NMR spectra are much better resolved than the
corresponding 1H NMR Spectra.[42,43] The fact that 13C chemical shifts are
concentration-dependent makes the problem more complicated.[44] 2D NMR
techniques such as HH- and CH-COSY, HMBC and INADEQUATE have been
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 24
implicated to distinguish the position and configuration of double bonds in the acyl
chains.[45,46]
1.2- Macadamia, From Nut to Shelf
1.2.1- Introduction
Macadamia nuts are the only commercially developed indigenous Australian crop.
They were well known to the Australian aboriginal people as a reliable food
source.[47] Countries such as South Africa, United States (mainly in Hawaii and
California), Kenya, Israel, Brazil, Malawi and Guatemala have also ventured into
cultivation and commerce of macadamia products.
Although high in oil, macadamia nut has cholesterol-lowering properties and a
beneficial effect on the lipoprotein profile in humans.[48] A controlled randomized
crossover-designed study of thirty healthy men with normal blood cholesterol levels
showed a macadamia-nut-based diet lowered serum total cholesterol and LDL
cholesterol within 4 weeks.[49] Other researches also revealed the preventive effects
of macadamia oil on risk of coronary artery disease[50] and type-2 diabetes.[51] Table
1.3 shows the composition of 100 g of macadamia nuts and the FA constituent of
macadamia nut oil.[52]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 25
Table 1.3- Composition of 100 g of macadamia nuts and the FA constituent of macadamia nut oil.
Composition of 100 g of macadamia nuts (about 40 nuts)
Fatty acid composition of macadamia nut oil
Nutritional Values Minerals, mg Fatty Acid Percent
Calories kCal 727 K 373 Oleic 67.14
Protein/ g 9.23 P 171 Palmitoleic 19.11
Oil/ g 78.21 Mg 119 Palmitic 6.15
Carbohydrates/ g 7.9 Ca 36 Eicosenoic 1.74
Thiamine/ mg 0.22 Na 6.6 Stearic 1.64
Riboflavin/ mg 0.12 Fe 1.8 Arachidic 1.59
Niacin/ mg 1.16 Zn 1.44 Linoleate 1.34
Cholesterol None Mn 0.38 Myristic 0.75
Cu 0.33 Lauric 0.62
1.2.2– History and Production of Macadamia Nut
Leichardt discovered a non-fruiting variety of the Macadamia ternifolia in 1834.[47]
In 1857 von Mueller and his colleague Hill discovered a specimen of Macadamia
ternifolia species in the southern region of Queensland. The macadamia tree was
called the Kindal Kindal tree by the aborigines and they supplemented their diet with
the nuts from this tree.[47]
In 1882 Macadamia integrifolia was introduced to the Hawaiian Islands, where it
was developed industrially from 1922. Macadamia became fully established as a
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 26
commercial crop in Hawaii during 1940s, and then subsequently in California,
Australia and other tropical and sub-tropical countries.[53]
In 1970, the Hawaiian macadamia industry produced approximately 90% of the
world production of macadamia nuts.[54] The Australian macadamia industry
essentially began in 1888 in New South Wales but only began to flourish
commercially from 1970. Production of nut-in-shell (NIS) in 1971/1972 was 93
metric tons and this rose to 2000 metric tons in 1983 and 27500 metric tons NIS in
1997.
Annual production of Australian macadamia nut-in-shell has grown from 19000
metric tonnes in 1994 to 29700 metric tonnes in 2003. In 1997 the Australian
macadamia nut production surpassed USA that had been ranked as the major
producer since the 1940s. Table 1.4 lists the production and consumption of
macadamia nut-in-shell (NIS) in the world.[55]
Table 1.4- World production and consumption of macadamia nut-in-shell (NIS) and kernel in year 2000.
Macadamia NIS Production Macadamia NIS Consumption
Country/ Region NIS Tonnes
Kernel Tonnes Country/ Destination Kernel Tonnes
(estimate)
South Africa 12500 3400 North America 10880
Kenya 8800 1000 Japan 3300
Malawi 4000 1000 Europe 3050
Zimbabwe 900 120 Hong Kong / China 1650
Central America 17000 3100 Australia 1200
Hawaii 22000 5500 Exporter – Own Consumption 1950
Australia 30000 9100 Committed Carry Over 1200
Total 95200 23220
Total 23230
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 27
The macadamia industry in Hawaii is based on the smooth shell type Macadamia
integrifolia and its cultivars. The rough shell varieties of Macadamia ternifolia show
unpredictable behaviour, are slow to come into bearing in Hawaii and they produce
lower grade nuts.[56]
1.2.3– Botanical Description
The mature macadamia tree grows to a height of 12-15 m. The leaves are dark green
and shiny and branches bear long sweet smelling racemes of creamy white flowers.
In summer each spray of 40-50 flowers produces 4-15 ‘nutlets’ which, eventually
ripen into large clusters of flowers.[57]
Macadamia is an evergreen tree of the family Proteaceae. There are ten species of the
genus macadamia in this family. Only two of these ten species produce edible nuts:
Macadamia integrifolia Maiden and Betche, known as the smooth-shell type and
Macadamia tetraphylla L.A.S. Johnson, commonly referred to as the rough shell
type, and their hybrids.[53]
The macadamia kernel, laid in a thick hard shell and with a high content of glucose
and sucrose, is a very sweet nut.[58] The most common commercially grown
macadamia nuts are of the Macadamia integrifolia cultivars.[52] The kernels of the
latter eight species are not of commercial value as they are bitter and considered
inedible due to the presence of cyanogenic glycosides which release HCN on
hydrolysis.[59,60] Figure 1.7 illustrates macadamia nuts fully grown on the tree.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 28
Figure 1.7- Macadamia nuts on the tree (Courtesy of Australian Macadamia Society)
1.2.4- Soil, Climate and Nutrition
Macadamia trees need free draining soil rich in organic matter and low in
compactness.[61] The main macadamia growing areas in Australia are on the coastal
regions of northern New South Wales and southeast Queensland. Macadamia trees
grow best in a sub-tropical climate. The shallow root system of the macadamia tree is
sensitive to prevailing winds. It grows best in regions with good drainage levels.[52]
1.2.5- Harvesting
Macadamia nuts are usually harvested from the orchard floor by mechanical
sweepers following natural fruit abscission (fruit falling on the ground from the
trees). The flowering period of many cultivars of macadamia is short in Australian
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 29
orchards; however, the abscission can occur over several months period within a
single cultivar and also varies in timing between cultivars.[62,63]
It is essential that the nuts are picked up frequently during the abscission peak period,
and more frequently during wet weather, otherwise the quality of the nut and the oil
deteriorates. As the fallen nuts contain about 30 percent water in the husk and 25
percent in the nut, the damage by mould growth could readily take place. The husks
of the macadamia nuts are removed within 24 hours of harvesting, to facilitate
drying.
The drying is the most critical step in the nut-in-shell processing, as it can maximize
the quality and the shelf life of the oil by preventing undesirable physiological
activities which cause fermentation resulting in the spoilage of the kernel. During the
drying process that can take 2-3 weeks, the moisture content of the nuts is reduced
from about 25% at harvest time down to about 2%. An initial drying temperature of
about 38 °C is applied. If this temperature is exceeded, undesired browning of the
kernel may occur later in the cooking step. After 2-3 weeks, the temperature is raised
to about 50 °C. In the final days of drying this higher temperature does not affect the
quality of the kernels.
After dehydration, the nuts are cracked prior to cooking. The nuts are cracked
between stainless steel drums, and the kernels are separated from the shell by a
combination of sieving and air blasting. It is crucial not to damage the kernel during
the process of steel-cracking the shell. It has been found useful to centrifuge the nuts
before cracking to loosen the kernels inside the shell.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 30
The kernels are stable when kept at -20 °C, however, the quality of the extracted oil
declines with increasing the storage temperature and humidity. Storing kernels at -20
°C is not practical commercially; therefore the nuts need to be processed with a
minimum delay.
The kernels are sorted and graded. Grade 1 and 2 kernels are used in confectionary
and the poorer quality kernels are passed on to oil extraction processes.
In the more recent methods of kernel grading, air floating is used to grade the kernels
eliminating the need for further washing and drying prior to the oil extraction
processes.
1.2.6- Oil Extraction
The oil is extracted from macadamia nuts either by mechanical pressing or by solvent
extraction processes. In mechanical pressing, the kernels are compressed and the oil
is squeezed out of the crushed mass of nuts under a very high pressure. A preheating
step is necessary to break the oil-containing cell walls to release the oil content of the
cells. The oil is collected in an appropriate reservoir. The remaining organic material
(the cake) is usually used in animal feed or is extracted using organic solvents to
retrieve the remaining oil in large-scale systems.
A batch press, processing one batch of nuts at a time, range from small hand-driven
presses that an individual can use, to power-driven commercial presses capable of
processing many tons of nuts a day (Figure 1.8 top)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 31
Figure 1.8- Cold press equipment used in the industrial production of seed and nut oil. Top: a batch press, bottom: a hole-cylinder type oil expeller.[64]
Expellers or continuous screw presses, achieve the pressure needed to press the nuts
by means of a screw rotating inside a cylindrical tube. The kernels, that are
previously heated up to 80 °C, are loaded continuously into the expeller where they
are fed into a horizontal cylinder by means of a rotating screw (worm shaft). Figure
1.8 bottom shows a screw type expeller used in the cold-press extraction of
macadamia nut oil.
None of the expeller machines are able to remove all of the oil from the nuts, and the
remaining cake contains 3-8% oil. In small-scale production situations this is not
important as the resulting cake finds uses for animal feed. To extract the remaining
oil from the cake in large-scale oil production systems, it is necessary to use solvent
extraction.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 32
Solvent extraction is a high-technology process used to extract oil from crushed
kernel or from expeller cake. As the capital costs are high for solvent extraction
processes, solvent extraction is used only in large-scale oil extraction systems. The
process is either a batch or a continuous counter-current extraction of the crushed
kernel or the cake in contact with an organic solvent, usually hexane. In the next
step, the solvent is stripped off under vacuum in a recovery plant and is recycled to
the extraction system, and the extracted oil passes on for further processes such as
refining.[64]
1.2.7– Industrial Macadamia Nut Oil Refining Processes
Refining is the last step in the processing of macadamia nut oil and includes some or
all of the following treatments: filtering, neutralization, winterizing, bleaching,
deodorizing and degumming.
Many crude oils contain short-chain free FAs which can induce unpleasant odours
and flavours. The free FAs are neutralized by treating the oil with a solution of
sodium hydroxide at about 70 °C. The FAs form soap with sodium hydroxide and
dissolve in the aqueous phase of the mixture. In larger refineries, the formed soap is
collected and sold to soap manufacturers.
Winterizing involves applying low temperature to the oil for a certain length of time,
during which higher melting point acylglycerides are solidified and are removed by
filtration.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 33
As oils are extracted from different varieties of macadamia kernels, the colour of the
extracted oil is not always consistent. Some oils are somewhat darker in colour and
are bleached by the addition of a small amount of acid-treated activated clays or
activated carbon prior to filtration. Many commercial plants bleach the crude oil as
routine and then add a controlled amount of colour in order to produce a consistent
and standard final product.
Degumming consists of the processes during which the oil is treated with small
amounts of water and phosphoric acid at about 60 °C. Degumming removes
phospholipids, fibers, protein-like compounds, polysaccharides and mucilage
released as the plant cells rupture during oil extraction. The above treatment, with
heat, causes the gums to coagulate after which they may be removed by
centrifugation or settling.
Deodorizing is steam distillation of the oil under pressure and in absence of
atmospheric oxygen, usually under a nitrogen atmosphere. Deodorizing removes
aromatic acids, aldehydes, free FAs and molecules that are produced in the oil before
the refining processes were started. These compounds include peroxides and some
volatile free FAs that are usually generated as a result of the contact of the oil with
air at the distillation high temperatures and can introduce unpleasant odour in the oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 34
1.3- Chemical Reactions Used in Sample Preparation for FTICR-MS Analysis 1.3.1– Methanol Extraction of Macadamia Nut Oil to Remove the Triacylglycerols
In this study the methanol extraction of macadamia oil is carried out to extract the
minor constituents such as free FAs from the oil, while the less soluble TAG
molecules remain in the oil. The extracted solution contains minor quantities of the
more abundant TAG (or more soluble substituted TAG) molecules. The former
comprising more than 98% of the oil,[65] this results in an increase in the signal to
noise ratio of the minor components of the oil in FTICR-MS analysis.
The ideal extraction solvent:
1- Should not dissolve the TAG molecules, but dissolve the remaining
compounds in the oil.
2- Should be immiscible with the oil to produce a separate phase.
3- Should be volatile enough to readily evaporate from the solution at a
temperature of about 40 °C or lower to avoid the loss of volatile oil
components during solvent evaporation.
4- Should not contain any non-volatile impurities such as stabilizers.
5- Must be of the highest purity and absolutely free from FAs.
In practice, there is no organic solvent that acts ideally for this extraction process.
The TAG molecules dissolve to some extent in most common organic solvents such
as methanol, hexane and diethyl ether. To achieve a refined dissolution profile, a
variable temperature is applied.[66] TAG and DAG molecules are precipitated out by
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 35
reducing the temperature during the extraction process. Smaller molecules such as
FAs are solubilised into the methanol phase from the nut oil as the solubility of these
compounds in methanol remains reasonably high at the low extraction
temperature.[67]
1.3.2- Transesterification of Macadamia Nut Oil
The transesterification reaction is used in this thesis to esterify the acylglycerols in
macadamia nut oil to yield their FA methyl ester derivatives. This is used to generate
the FA profile of the acylglycerols in the oil. The products from this reaction are also
studied using GC-MS.
In the transesterification of the oil with methanol, the acylglycerol ester bonds cleave
to produce FA methyl esters and glycerol. The acyl groups of the acylglycerols are
released in the reaction as FA methyl esters (FAMEs). Free FAs are also esterified. A
specific example, Reaction 1, shows the transesterification reaction of glycerol
tripalmitate in methanol. The products are methyl palmitate and glycerol.
Glycerol tripalmitate
CH3OH
Methyl palmitate
+HO
OH
HO
Glycerol
C15H31
O
OO
O C15H31
O
O
KOH
C15H31
O
C15H31
O
3
Reaction 1- Transesterification of glycerol tripalmitate (a TAG) in methanol yields methyl palmitate and glycerol.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 36
The transesterification reaction of the macadamia oil effectively alters its chemical
composition by replacing the glycerol moiety in the mono, di and triacylglycerols
with methanol. As a result, most of the FTICR-MS peaks are observed in the lower
mass region of the spectrum of the esterified oil, resulting in a simpler spectrum of
fewer assigned compounds. On the other hand and most significantly, the profile of
the FFA, MAG, DAG and TAG molecules in the original oil are lost. Procedure 2.2.2
details the transesterification procedure used in this study.
1.3.3- Alkaline Hydrolysis of Macadamia Nut Oil
During the hydrolysis reaction of the macadamia oil in boiling methanolic KOH, the
ester bonds in the acylglycerols are broken and FA potassium salts (soaps) are
formed. This reaction is referred to as saponification due to its extensive application
in the soap industry. Reaction 3 shows as an example the hydrolysis (saponification)
reaction of glycerol palmitate in hot methanolic KOH solution. The procedure for the
hydrolysis of macadamia oil is explained in Procedure 2.2.3.
O
O
C15H31
O
O KOH C15H31
O
+K -O
HO
OH
HO
Glycerol tripalmitate
Glycerol
+MeOH
C15H31
O
C15H31
O
3
Potassium palmitate (soap molecule)
Reaction 2- Hydrolysis of glycerol tripalmitate in hot methanolic KOH solution produces potassium palmitate (soap) and glycerol.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 37
In general, saponifiable lipids all contain one or more ester, amide, or glycosidic
bonds. Within the major group of saponifiable lipids, there are several subgroups:
waxes, acylglycerols, phosphoglycerides such as phosphatidates, ether lipids such as
plasmalogens, sphingolipids such as ceramides, sphingomyelins and
glycosphingolipids including cerebrosides and gangliosides. Figure 1.9 illustrates
various types of saponifiable lipids (not necessarily found in macadamia oil).
Figure 1.9- Saponifiable lipids in the hydrolysis reaction.
In general, non saponifiable lipids do not react with hot methanolic KOH solution.
They are separated from the final product mixture by ether extraction. Figure 1.10
shows some of the compounds most likely found in the non saponifiable part of the
hydrolysis reaction of oils. However, the exact composition of the hydrolysis
reaction mixture depends on the lipid nature (animal or plant source).
Saponifiable Lipids
Waxes Glycerolipids
Acylglycerols Phosphoglycerides
Sphingomyelins
Cerebrosides
Plasmalogens
Glycosphingolipids
Gangliosides
Saponifiable Lipids
Waxes Sphingolipids Glycerolipids
Phosphoglycerides
Sphingomyelins
Plasmalogens
Glycosphingolipids
Gangliosides
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 38
Nonsaponifiable Lipids
Fat Soluble Vitamins
EicosanoidsCholesterol
Vitamin AVitamin DVitamin EVitamin K
Bile AcidsBile SaltsBile Esters
SteroidsHormones
ProstaglandinsLeukotrienesThromboxanes
Progestins, GlucocorticoidsMineralocorticoidsAndrogens, Estrogens etc.
α, β, γ and δ-Tocopherols
and tocotrienols
Nonsaponifiable Lipids
Fat Soluble Vitamins
EicosanoidsCholesterol
Vitamin AVitamin DVitamin EVitamin K
Bile AcidsBile SaltsBile Esters
SteroidsHormones
ProstaglandinsLeukotrienesThromboxanes
Progestins, GlucocorticoidsMineralocorticoidsAndrogens, Estrogens etc.
α, β, γ and δ-Tocopherols
and tocotrienols
Figure 1.10- Non saponifiable lipids in the hydrolysis reaction of oils.
1.4- Mass Spectrometry of Lipids
Numerous analytical techniques have been implemented in the identification of the
constituents of animal fats/oils and plant oils. Among these are wet chemistry
methods (derivatisation, enzymatic methods, hydrolysis, etc.), as well as instrumental
methods such as chromatography including high performance liquid chromatography
(HPLC),[68,69] capillary electrophoresis (CE),[70] thin layer chromatography (TLC),[71]
gas chromatography (GC)[69,72,73] and mass spectrometry techniques.[1]
Mass spectrometry (MS) is an established method in physics, chemistry,
biochemistry and medicine due to high sensitivity and selectivity. Since early 1990s
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 39
a considerable progress was achieved by the invention of soft ionization techniques
such as ESI and matrix assisted laser desorption-ionization (MALDI).[74,75]
A primitive application of MS in organic chemistry was the analysis of simple
organic compounds such as alkanes in late 1920s.[76,77] One of the first applications
of mass spectrometry to the analysis of lipids was on the investigation of the
mechanism of FA oxidation in the living cell (beta-oxidation) in 1944.[78]
Various studies on lipids from sector, ion-trap, single and triple quadrupole, and
time-of-flight instruments incorporating EI, fast atom bombardment (FAB),
atmospheric pressure chemical ionisation (APCI),[79] MALDI[75,80] and ESI ionisation
methods are reported.[79,81-84] Nano-electrospray ionisation combined with FTICR-
MS has recently found applications in lipid analysis.[85]
Various mass analysers are used in the analysis of lipids, using either static or
dynamic magnetic or electric fields, but all operate according to the same law. They
all measure dimensionless quantity of mass-to-charge ratio, m/z, that represents the
ratio of the mass number and the charge number of an ion in the mass analyser.[86]
Quadrupole,[87] ion-trap,[88] time of flight,[89] orbitrap[90] and Fourier transform ion
cyclotron resonance (FTICR)[91] are among the most implemented mass analysers.
Christie has listed 208 review articles published on the analysis of lipids during 2000
to 2007.[92]
1.4.1-Gas Chromatography-Mass Spectrometry
During the early years of mass spectrometry application in chemistry, the types of
samples that could be satisfactorily analyzed on MS were limited due to the
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 40
requirement of sample volatility for this gas-phase analytical technique. Gas
chromatography appeared to be an ideal solution to the problem by introducing gas
phase samples to the mass spectrometer and separating the constituents of a possible
composite sample. A major challenge was to develop methods to convert solid lipids
into gaseous derivatives capable of passing into GC and MS instruments.[1]
A non-polar to medium polarity stationary phase GC column separates TAGs
according to increasing acyl carbon number. A 70 eV energy EI is widely used as an
ionization source in GC-MS analysis. As highly unsaturated TAG molecules in some
oils tend to undergo degradation and polymerization reactions at high temperatures
applied in GC columns, GC-MS is not recommended in direct analysis of TAGs.[93]
A combination of GC and triple quadrupole mass spectrometer offers the possibility
of MS/MS (tandem MS) to provide more structural information of the analyzed lipid
molecules.[93,94]
The invention of soft ionization techniques such as ESI and MALDI made it possible
to produce gas-phase molecular ions for most lipids directly regardless of the
molecular weight, polarity and complexity of the molecules.[1] As a matter of fact,
before this invention, there was no direct method of MS analysis of intact lipids such
as oils and fats.
Among mass spectrometry methods, GC-MS has found the most application in the
analysis of lipids. However, a fundamental disadvantage of GC-MS is the
requirement of a volatile and thermally stable analyte. Derivatization can solve the
problems of analyte volatility in GC-MS analysis of simple lipids, however, it may
need multiple step reactions for complex biomolecules.[94]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 41
Another drawback of GC-MS technique is that double bonds on unsaturated FAs
tend to migrate during the fragmentation in the electron ionization source.[95]
Determining the location of double bonds in carbon chains of FAs has been a
challenging problem in mass spectrometry. At the internal energies necessary for
ionization in EI source, migration of the double bonds along unsaturated acyl chains
occurs.[95] To overcome this problem, the double bonds are fixed by attaching
protecting groups to the acyl chain.[96]
Vetter developed the pyrrolidide derivatives to locate double bonds on unsaturated
FAs.[97] Harvey proposed 3-picolinyl esters (an aromatic basic reagent) for
derivatization of unsaturated alkyl chains.[98] Other derivatization methods include
piperidyl and morpholinyl esters,[99] triazolopyridine,[100] nitrobenzyl esters,[101] and
oxazoline derivatives.[102]
The nitrobenzyl ester derivatization technique is reported to improve the signal
intensity in the identification of FA mixtures in negative-ion mode[101] and in the
identification of hydroxy FAs in both positive- and negative-ion modes.[72]
Christie has discussed derivatization of unsaturated lipids for analysis on GC-MS
extensively.[103] He has compared various methods of preparation of lipid derivatives
including methyl esters, pyrrolidides, picolinyl esters, isopropylidenes, TMS ethers
and deuteration. The 3-picolinyl esters appear to yield more easily interpretable mass
spectra than the pyrrolidides. The picolinyl esters contain an aromatic ring that is
easily detectable in UV detectors (used in HPLC instruments).[104] He recommends
the picolinyl esters as the best general purpose derivatives for locating the double
bonds on lipids by GC-MS.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 42
Applications of derivatization include, but are not limited to, encouraging
fragmentation along different pathways, improving the selectivity and sensitivity of
the ionization process, enhancing the abundance of the molecular ion, and reducing
the polarity of FAs to increase the volatility and to promote the stability of
molecules.[105]
The main disadvantage of all FA derivatisations is that the geometry about the
double bond cannot be determined.[95,96]
1.4.2- Electrospray Ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
ESI FTICR-MS is the main technique used in this study. GC-MS is also used as a
supporting technique. The present study has led to the first published application of
ESI FTICR-MS technique in the investigation of plant oils by Fard et al. in 2004.[73]
Wu and co-workers later published an article on the application of FTICR-MS in the
investigation of adulteration in vegetable oils in 2005.[106]
The advantages of the ESI FTICR-MS technique compared to other mass
spectrometry techniques are high resolution, high mass accuracy, high sensitivity and
simplicity of sample preparation and injection. In one report we have resolved more
than 180 different components in a positive-ion ESI FTICR mass spectrum of a
processed macadamia oil sample. Hughey and co-workers have resolved 11,127
compositionally distinct components in positive-ion and 6,118 compounds in
negative-ion ESI FTICR mass spectrum of a crude petroleum oil sample with an
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 43
average mass resolving power of 350,000. They claim that FTICR-MS offers a
hundred-fold higher peak capacity than the best single-stage GC or LC.[107]
1.4.2.1- Electrospray Ionisation Source
Lord Rayleigh carried out and published one of the first studies on charged droplets
in late 1800s.[108] In this research, he showed that the spherical shape of a charged
drop remains stable as long as the fissility does not exceed unity. The fissility is a
function of the radius of the drop, the surface tension of the liquid and the charge of
the drop. Figure 1-11 illustrates the formation of charged drops in a high electric
field.
Figure 1.11- Formation of charged drops at the tip of electrospray needle in high intensity electric field.
Once the fissility increases beyond unity, two fine jets tend to form at both ends of
the drop (Rayleigh jets) dispatching fine charged droplets out of the main drop.[108] In
this phenomenon, the drop loses about 0.3% of its mass and about 33% of the charge
Sample solution
Additional gas flow
Capillary needle
±4200 V
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 44
to reduce the fissility to less than unity. The generated fine droplets are of the size of
approximately 1.5 µm and the fissility of close to 1, so they are likely to undergo a
Rayleigh instability soon afterwards.[109]
Figure 1.12 illustrates the formation of Rayleigh jets at the increased fissility of a
charged drop. The main drop dispatches fine droplets out in shots c and d. In shot f
the drop obtains some stability until the fissility grows to greater than unity due to
further solvent evaporation.
Figure 1.12- The formation of Rayleigh jets at the fissility greater than 1. Fine droplets dispatched from the main drop are visible in shots c and d. The whole process takes about 200 µS. Scale bar=100 µM[109]
Although this speculation has been examined both theoretically[110,111] and
experimentally,[112] the mechanics of the break-up of the fine charged droplets and
the details of the Rayleigh jet fineness are still unclear.[109] The ion evaporation
model of Iribarne and Thomson[113,114] is also widely accepted. In this model
Columbic explosion of rapidly evaporating liquid droplets of high charge density
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 45
results in charged droplets with smaller sizes, in which this sequence continues until
field-assisted ion evaporation occurs.
In electrospray ionisation, the droplets of solution are produced by “pneumatic
nebulization” that is injecting the analyte solution through a needle at whose end a
high voltage is applied. This voltage is high enough to disperse the emerging solution
into a very fine spray of charged drops with the same polarity. The solvent
evaporates, shrinking the drop size and increasing the charge density at the drop
surface.
At the Rayleigh limit, the Columbic repulsion overcomes the drop surface tension
and the drop explodes. This explosion forms a series of smaller droplets with lower
charge densities on their surfaces. By further solvent evaporation, this process of
shrinking and exploding is repeated until individually charged naked analyte ions are
formed.
The charges are statistically distributed on the available charge sites on the analyte,
leading to possible formation of multiple-charge ions in proper conditions. Increasing
the rate of solvent evaporation increases the extent of formation of multiple-charged
ions. Decreasing the diameter of the needle and lowering the analyte flow rate will
create ions with higher m/z ratio, making this technique a gentle ionisation
method.[115]
Figure 1.13 shows a schematic diagram of an ESI source along with the skimmer.
The skimmer provides a differential pressure barrier between the source
compartment and the hexapole region of the ESI source despite a 90% ion loss in this
process.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 46
Figure 1.13- Schematic diagram of the electrospray needle, capillary and skimmer (not to scale).
It was during the early years of 20th century that the importance of electrospray was
first realised.[116] The application of electrospray ionisation to evaporate the charged
droplets to produce gas-phase ions of an analyte in a solution was first investigated
by Dole et al. in late 1960s and early 1970s.[117,118] Based on these results, Yamashita
and Fenn[119-121] and Aleksandrov et al.[122,123] successfully combined electrospray
ionization source with mass spectrometry in the mid 1980s.
Off-axis configuration of electrospray needle increases the efficiency of the
electrospray ionisation process. Larger droplets fall down due to the gravity while the
smaller charged droplets are more likely to be absorbed toward the capillary inlet. A
stream of hot drying nitrogen gas promotes solvent evaporation and blows larger
droplets away from the capillary inlet. Belov and co-workers have developed an
alternative aperture structure, the ion funnel, to improve the ion collection and
increase the signal to noise ratio.[124]
Electrospray needle
Skimmer
Capillary
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 47
1.4.2.2- The ICR Cell Figure 1.14 shows a schematic diagram of the Bruker infinity ICR cell used in
BioApex II FTICR mass spectrometer.
Figure 1.14- Schematic diagram of the infinity ICR cell used in Bruker BioApex II.
The ‘infinity’ cell is a modified ICR cell with a uniformed potential applied on the
two trapping plates at each end to solve the common problem in the ordinary ICR
cells, ‘z-ejection’, that occurs due to non linearity in the ion excitation.[125,126] An
example of this non-linear effect is coupling of axial and radial motions and may
cause undesired ion ejection along the z-axis of the ICR cell.[127] To eliminate such
undesired ion ejections, Caravatti and Allemann designed a pair of trapping plates
comprising eleven segments with different potentials applied to each segment to
match the potential gradient of the RF excitation field.[128] Such an ICR cell then
behaves like an infinitely long cell with respect to the electrical RF excitation field.
From the ions’ point of view inside the cell, the two trapping plates act as RF mirrors
imitating an infinitely long RF field. Caravatti and Allemann have shown that the
EV1
PV1 PV2
PV2 quench
EV2a
+ + + + + + +
Excite
Detect
EV2b
B
sidekick:
EV1
PV1 PV2
PV2 quench
EV2a
+ + + + + + +
+ + + + + + + + + + + + + +
EV2b
B
sidekick:
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 48
infinity cell eliminates the excitation-ejection efficiently at frequency of ωC+2ωt and
greatly improves the S/N ratio.[128]
1.4.2.2.1- Ion Trapping
Ion trapping in FTICR mass spectrometry can be carried out in numerous ways
including Sidekick™ ion-trapping, gated ion-trapping (static or dynamic) and
collision gas-assisted dynamic ion-trapping. Table 1.5 lists different trapping
methods commonly used in FTICR mass spectrometry.
Table 1.5- Different trapping methods usually used in FTICR mass spectrometry.
Trapping Methods Summary
Method Application
Sidekick Routine, high-speed applications such as auto-sampling, HPLC.
Gated (Static) Mainly used in combination with ECD and IRMPD experiments
Gated (Dynamic)
Ultra-high resolution requirements Good for ECD and IRMPD
Gas Assisted (Dynamic)
Ultra high sensitivity applications Used frequently with larger biopolymers.
The Sidekick™ ion trapping method is mainly used for fast routine mass analysis in
FTICR mass spectrometry. After the ion injection is implemented, a potential
difference DEV2 is applied on the two EV2 half-plates outside the first trapping plate
PV1. This potential difference applies a force on the ions entering the cell. This force
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 49
is perpendicular to the z-axis of the cell and pushes the ions to ion cyclotron orbits
larger in diameter. The advantage of Sidekick™ ion trapping method is that a wide
mass range of ions can be trapped in the cell. As most of the ions are pushed away
from the centre of the cell into larger cyclotron orbits, this method is not suitable for
the electron-capture dissociation (ECD) or infrared multiphoton dissociation
(IRMPD) experiments that require the ions to reside close to the z-axis in the cell.[10]
Gated Ion Trapping is based on the time-of-flight of the ions in the gated ICR cell on
a proper time scheduling. This ion-trapping method can be applied in either a static
or dynamic way. In static ion-trapping the trapping potential is set at a constant value
and the detection of the ions critically depends on the time of flight of the ions.
Dynamic ion-trapping applies a pulsed trapping potential on the trapping plates in the
ICR cell. The length of the applied pulse can be tuned manually to improve the
signal intensity. The gated trapping keeps the ions on the z-axis of the cell and is
convenient for ECD and IRMPD experiments. The drawbacks associated with this
method are limited mass measurement range for the ions and relatively low trapping
efficiency.
In the collision gas-assisted dynamic ion-trapping method a pulsed collision gas is
applied in the ICR cell to quench the ions and a pulsed potential on the trapping plate
is applied to trap the ions. This trapping method greatly reduces the ion energy range
that is exhibited by the time of flight of the ions. This method is applicable to a wide
mass range of ions. The pulsed quenching gas has to be pumped out of the ICR cell
prior to detection; therefore, this ion-trapping method is slow. The mass range is
limited by the time of flight effect.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 50
1.4.2.2.2- Ion Cyclotron Motion
An ion of mass, m, carrying charge, q, moving in a uniform magnetic field, B, orbits
about the magnetic field direction as shown in Figure 1.15.
Figure 1.15- Ions orbit in a plane perpendicular to the direction of a uniform magnetic field, B. Positive (a) and negative (b) ions rotate in opposite directions in this ion cyclotron motion.[129]
The "undisturbed" cyclotron (rotational) frequency, ωC, is expressed in SI units as:[91]
ωc = qBm
1-1
υc = Cω2π
= 71.535611×10 B
m/z 1-2
In which υ is the cyclotron frequency of the rotation in Hertz, B is the strength of the
magnetic field in Teslas; m is the mass of the charged particle in micrograms; z is the
charge of the ion in multiples of elementary charge.
a b
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 51
A noteworthy characteristic of this latter equation is that all ions of the same mass-
to-charge ratio, m/q, rotate at the same ICR frequency, independent of velocity. This
feature makes ICR especially appropriate to mass spectrometry, since the ion
frequency is reasonably insensitive to kinetic energy, so the translational energy
(focusing) is not crucial for accurate m/z determination.
1.4.2.2.3- Ion Cyclotron Excitation and Detection
As for nuclear magnetic precession,[130] ion cyclotron rotation is incoherent, and does
not produce an observable electrical signal, i.e., there is no net difference in the
charge induced on two parallel detector electrodes by the ion cyclotron rotation of
the ions. At the moment of formation in the ICR cell, an ion may start its cyclotron
rotation randomly at any point around either circle shown in Fig. 1.15. Thus, for a
packet of ions, any charge induced on one detector plate will, on average, be
cancelled out by an equal charge induced by an ion with opposite phase, so that the
net measured charge difference between the two plates is zero. Furthermore, the
cyclotron radius of the ions is too small to produce a measurable signal, even if all
ions possessed the same cyclotron phase.
Thus, prior to any detection phase in the ICR cell, there must be an excitation
produced by applying a spatially uniform electric field of amplitude E0 perpendicular
to the magnetic field direction, and rotating resonant to the cyclotron frequency of
ions of a particular m/z value. During this excitation period, the dimensions of the
initial ion packet remain unchanged[131] and the packet accelerates along a spiral
trajectory, as illustrated in Fig. 1.16 a.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 52
Figure 1.16- Incoherent and undetectable ion cyclotron orbital motion (a) is converted to a coherent and detectable motion by applying an electric field and (b) is detected in the ICR cell at the frequency of the ions of particular m/z value.[91]
The ion packet cyclotron radius subsequent to the resonant irradiation of duration
Texcite increases to (SI units)
r = 0 exciteE T2B
1-3
As described,[91] a coherently orbiting ion packet induces a potential difference on
the two detection plates and may be considered as a current source. The receiver
plates and the wirings have an inherent resistance and capacitance in parallel.[132,133]
The current amplitude is proportional to the number of spatially coherent orbiting
ions. At common ICR frequencies (>10 kHz) the S/N ratio is basically independent
of cyclotron frequency. However, at lower frequencies (<10 kHz), the signal is
expected to be a direct function of the frequency.[132,134] As a result, throughout most
of the broad frequency range of a standard FTICR-MS excitation, the relative current
induced on the detection plates is represented by the S/N ratio. In addition, the
a b
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 53
detection limit (i.e., the minimum number of detectable ions from an unchanged
signal in an individual 1 S acquisition period to produce a S/N ratio of 3:1) may be
calculated from [133]
N = d(p-p)
1
CVqA (r) 1-4
in which C is the capacitance of the detection circuit, Vd(p-p) is the peak-to-peak
amplitude of the detected voltage (for a particular calibrated spectrometer), and A1(r)
is a coefficient related to the configuration of the trap, approximately proportional to r
and could be determined graphically.[135] For example, for some typical operating
parameters, namely, a detection circuit capacitance of 50 pF, Vd(p-p) of 3×10-7 V, and
A1(r) = 0.5 (i.e., the ion is excited to approximately half of its maximum cyclotron
radius), an observed S/N ratio of 3:1 corresponds to a detection limit of ~187 ions.
For a molecule of mass ~300 being injected at a flow rate of 120 μl/h with 1 s
delay in the hexapole, 187 ions corresponds to a concentration of ~9×10-15 M in
the solution.
The ICR signal is proportional to the induced current,[132,133,136]
dΔQ/dt = -2q(dy/dt)/d 1-5
Note that ICR signal is independent of magnetic field strength. Further, the induced
current increases linearly with ion cyclotron radius, because the ion y-velocity
component, dy/dt, increases linearly with radius, so the ICR signal increases
linearly with the excited ion cyclotron radius.
The linearity of the ICR signal with ion cyclotron radius is important for several
reasons.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 54
(1) At any frequency, the ICR response is proportional to the excitation spectral
magnitude at that frequency, because the ICR signal varies linearly with the
excited ion cyclotron radius (that is a linear function of the product excitation
voltage amplitude × duration).
(2) A Fourier transform of the time-domain ICR response gives the same
"absorption" spectrum that is otherwise obtained by measuring power absorption
while sweeping slowly across the m/z range.[137]
(3) The "superposition" principle implies that the signals from any number of ions
of arbitrary m/z values simply add at the detector; as a result, ions of a wide m/z
range can be detected simultaneously. The two prior points combine to constitute
the "multi-channel" advantage of pulsed excitation followed by Fourier
transformation to yield a spectrum of N data points in 1/N the time it would
take to scan the spectrum one channel at a time.[137]
(4) The detected signal intensity increases linearly with ion charge, so that ICR is
more sensitive for multiply charged ions. For example, individual DNA ions of
108 Da (each with ~30,000 charges) have been detected by FTICR-MS.[138]
Although image current detection at room temperature is typically less sensitive than
ion counting techniques characteristic of ion beam instruments, FTICR-MS detection
is non-destructive.[137]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 55
1.4.2.3- Fourier Transform
The most unique advantage of FTICR as a mass analyser is that ion mass-to-charge
ratio is experimentally measured as a frequency. Because frequency can be measured
accurately (in low and medium frequencies), FTICR-MS offers inherently higher
resolution than other types of mass spectrometry.
Various other aspects of Fourier transform data reduction related to ICR peak shape
and position have been discussed in the literature and are not discussed in detail in
this thesis: Nyquist sampling and foldover,[139] fast Fourier transformation,[140] zero-
filling,[141] "windowing" or apodisation,[137] deconvolution,[142-144] oversampling,[145]
two-dimensional Hadamard[146] or Fourier[147] MS/MS.
Non-FT methods for obtaining a frequency-domain (and thus mass-domain)
spectrum from a digitised time-domain ICR signal include: the Hartley transform (a
way of performing a Fourier transform on real-only data),[148,149] the Bayesian
maximum entropy method (MEM),[150,151] linear prediction,[152,153] and filter
diagonalisation.[154] However, FT data reduction is still overwhelmingly preferred,
partly because non-FT methods typically need an order of magnitude more data
storage; their computation time for an N-point time-domain data set typically scales
as N2 or N3 (vs. NlogeN for the fast Fourier transform;[140]) and they typically
perform best when the number of spectral peaks is small and the peak shapes are
uniform and known.
Finally, it is worth noting that Fourier transforms also work backwards (from
frequency- to time-domain) as well as forward (from time- to frequency-domain).
Thus, it is possible to specify a desired magnitude vs. frequency excitation spectrum,
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 56
and apply an "inverse" Fourier transform to generate the corresponding time-domain
waveform to be applied to the excitation electrodes of an ICR trapped-ion cell. Such
“stored waveform inverse Fourier transform” (SWIFT) excitation[155] is now widely
used in both FTICR[156] and Paul (quadrupole)[157,158] ion traps, for mass-selective
excitation and ejection.
1.4.2.4- Mass Calibration
For precise mass measurement, two parameters need to be taken into account; mass
calibration and mass resolution. The mass resolution is determined by the
instrumental and experimental conditions such as the strength of the applied
magnetic field and the pressure in the ICR cell.
A mass calibration equation for FTICR-MS is defined as:[159]
m = obs
γf
+ 2obs
βf
1-5
Where γ = qB/2π and β = -2qGTVeff/4π2
In equation 1-5 fobs is the observed frequency, GT is the geometry factor for the ICR
cell, Veff is the effective cell trapping potential, q is the charge on the ion and B is the
magnetic flux density in the ICR cell.
To calibrate the cell parameters, a reference compound with a well-defined spectrum
is chosen and introduced into the FTICR mass spectrometer. Peaks over a wide mass
range are measured in high-resolution mode. The mass calibration function in the
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 57
FTICR-MS operating software then compares the measured frequencies (fobs) with
the expected frequencies (calculated) and adjusts the two calibration constants γ and
β until the difference between the respective frequencies are minimized. This is
usually referred to as external calibration.
The mass calibration can also be performed using internal references and is referred
to as internal calibration. Here a sample is mixed with reference compounds that
produce ions covering the mass range including those of the sample. By calibrating
the reference ion masses, the sample ions are also calibrated. The latter method is the
most accurate in FTICR-MS with reported accuracy of < 1 ppm.[7,160]
1.4.2.5- Tandem Mass Spectrometry
Tandem mass spectrometry is used to obtain more information regarding the
chemical structure of the ions by fragmenting them. In ESI FTICR-MS,
fragmentation could be induced before ions arrive at the ICR cell either by increasing
the capillary-skimmer potential difference in the ESI source or extending ion
trapping times in the hexapole ion trap. Alternatively, fragmentation could be
performed on the trapped ions in the ICR cell by introducing a collision gas into the
ICR cell through a molecular leak valve.
Fragmentation by increased capillary-skimmer potential and hexapole ion trapping
yields less controlled fragment cations compared to the accurately-controlled
fragmentation obtained by the collision gas method in the ICR cell. It can also lead to
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 58
subsequent dissociation of the primary fragmented ions by post fragmentation
acceleration.
In the collision gas CID method, ions are translationally excited by applying a pulsed
electric field in the ICR cell. Depending on the length of the exciting electric field,
two different approaches are available in CID experiments, either on-resonance
irradiation (ORI) or sustained off-resonance irradiation (SORI). In ORI experiments
a short duration (<500 μs) electric field pulse is applied to the ions in the ICR cell.
Subsequent inelastic collisions with a neutral gas molecule target result in
fragmentation. In SORI experiments a long off-resonance pulse is applied to the ions
in ICR cell (≥500 ms).[161] SORI-CID results in sequential activation of ions by
multiple collisions of low kinetic energy ions (<10 eV) with the neutral gas molecule
target.[162] As a consequence, only small increments of internal energy are transferred
to the ion throughout the duration of the electric field pulse.[129] The differences
between ORI and SORI CID involve the maximum translational energy obtained by
the ions, the excitation time and the number of collisions effecting dissociation, that
is the amplitude, time, and frequency of the pulses applied to excite the ions
translationally.[163]
Other applicable fragmenting methods in FTICR-MS are: surface-induced
dissociation (SID), electron-capture dissociation (ECD), infrared multiphoton
dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD).[161,164]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 59
1.5- High Performance Liquid Chromatography
HPLC methods have been widely used in the analysis of the natural products,[165,166]
lipids[167-169] and particularly plant oils.[170] HPLC combined with single or triple
quadrupole mass spectrometer has been described as the most desirable system for
the lipid ester analysis.[171] HPLC separation in both off-line and on-line mode is
described as a preliminary requirement to avoid the co-infusion of the molecules in
the ESI sources that serves as a cause of signal suppression that can result in failure
to detect the minor constituents of the sample.[168]
Numerous researchers have implemented HPLC separation combined with various
ionisation sources in a range of studies.
Lin and co-workers[172] have used two different gradient elution solvent systems with
UV detector and evaporative light scattering detector (ELSD). They state they have
successfully separated 49 free fatty acids and their methyl esters. On examining
Table 1 in this article, it is quite clear from the retention times of several compounds
that they would not be resolved well enough to be identified. For example, retention
times of 18.36 and 18.36 minutes are reported for free fatty acids linolenic acid and
myristic acid. It is clear that these compounds would not be separated in a mixture
and in order to identify the fatty acids it would be necessary to run individual
standards.
The factors affecting retention times such as chain length, functional groups
(hydroxyl, oxo, keto, aldehydes, double and triple carbon bonds etc.) and geometric
isomerism have been discussed quantitatively. By implementing two C18 reversed-
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 60
phase columns in series, they have reported separation of double bond positional
isomers. Batta et al. have studied the separation of underivatized arachidonic acid
isomers using two different solvent systems on a reversed-phase HPLC column with
UV detection at 214 nm.[173] Kempen has described a method to simultaneously
analyse arachidonate metabolites in cultured cancer cells using negative-ion
electrospray tandem mass spectrometry (LC/MS/MS).[174] Byrdwell has studied the
oxidation products of acylglycerols in canola oil using reversed-phase HPLC in
conjunction with a triple-quadrupole mass spectrometer in parallel with an ion-trap
mass spectrometer using ESI and APCI sources with ammonium formate added as
electrolyte.[84] They have reported the formation of diacylglycerol and acylium,
RCO+, fragments as well as ammonium adduct of intact molecular ions of TAG
oxidation products.
Bylund has investigated the metabolites of linoleic and arachidonic acids by human
cytochrome P450 enzymes using reversed-phase HPLC column combined with ion-
trap mass spectrometry.[175] They have reported identifying arachidonic acid
metabolites in complex mixtures formed by enzymes such as cytochrome P450.
Redden has separated and quantified the TAGs of borage oil and evening primrose
oil using reversed-phase HPLC with UV detection. Borage oil is reported to have 34
UV-detectable fractions and evening primrose to have 22 fractions.[176] Steenhorst
has employed normal-phase HPLC combined with mass spectrometry to separate the
non-volatile lipid oxidation products into classes according to the molecular
polarities.[177] The classes include epoxy-TAG, oxo-TAG, hydroperoxy-TAG,
hydroxy-TAG and 2.5 acylglycerols. A 2.5 acylglycerol is defined as a product of the
breakdown of TAG hydroperoxides yielding non-volatile acylglycerol species with
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 61
two intact fatty acid chains and one short chain ending in an aldehyde or hydroxy
group. The retention times of TAG oxidation products on the normal-phase HPLC
system and the signal intensity of the MS detector are reported stable enough to
enable quantitative analysis based on external calibration.
Holcapek has implemented reversed-phase HPLC combined with various detection
methods including mass spectrometry, UV detection at 205 nm and ELSD for the
determination of the compounds occurring during the production of biodiesel from
rapeseed oil.[178] Individual mono-, di-, tri-acylglycerols and methyl esters of oleic,
linoleic and linolenic acid and free fatty acids are reportedly separated using a
combined linear gradient with aqueous-organic and non-aqueous mobile phases.
Sajiki has described HPLC-MS as more useful and convenient compared to GC-MS
in the study of free-polyunsaturated fatty acids (f-PUFA) oxidative metabolites in
biological samples due to high specificity and high sensitivity and due to the fact that
it requires no complicated pre-treatments.[68] Jandera and co-workers have studied
TAGs and DAGs in 16 plant oils including hazelnut, pistachio, poppy-seed, almond,
palm, rapeseed, macadamia, soya bean, sunflower, linseed, evening primrose, corn,
Brazil-nut, amaranth, Dracocephalum moldavica, and Silybum arianum using
HPLC-MS equipped with APCI source and UV detection at 205 nm and two C18
columns connected in series.[79] They have reported observation of fatty acids with
odd numbers of carbon atoms such as margaric acid (C17:0) and heptadecenoic acid
(C17:1).
Lee and colleagues have interesterified (exchange of acyl groups among
triacylglycerols) macadamia oil by tributyrin and tricaprylin,[179] and have used
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 62
normal-phase and reversed-phase HPLC with ELSD to separate and identify the
newly-formed lipids.
Multidimensional HPLC is also used in the study of complex bioorganic samples
such as humic mixtures.[180-182] However, it would be difficult to use such HPLC
techniques in the analysis of the present samples because solvents used in the
reversed and normal phase HPLC are incompatible.
1.6- Kendrick Masses and Mass Defects in the Identification of Homologous Series
Calculating Kendrick mass defects (KMDs) for organic molecules enables us to
readily recognize homologous series (molecules which are different only in the
number of CH2 groups, and which posses a similar heteroatom composition and the
same number of double bonds, rings, etc.).
In 1963 Edward Kendrick proposed a mass scale based on CH2 = 14.00000 rather
than 14.01565 Da.[183] According to this model, the Kendrick mass of a molecule is
calculated as follows:
Kendrick mass = IUPAC mass × (14.00000/14.01565)
Calculating Kendrick masses enables us to calculate the Kendrick mass defects that
are calculated as follows:
Kendrick mass defect = Kendrick mass – Kendrick nominal mass
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 63
Kendrick nominal mass is obtained by rounding the Kendrick mass to the nearest
integer number. KMD values are used in the assignment of homologous series in the
negative-ion mass spectra in Chapter 6.
1.7- Normal Probability (Rankit) Plot
The normal probability (rankit) plot is a graphical technique for assessing whether a
data set is distributed normally.[184] A normal distribution of the data points suggests
that systematic error does not exist in the measurements.
To perform this test, data points are sorted in descending order and each point is
given a rank in the data set. Inverse of the standard normal cumulative distribution of
the probability of the occurrence of the data points (z values) are then sketched
versus data points. The points should reasonably fit onto a straight trend line
resulting in a close correlation coefficient (R) of the trend line to 1. Deviations from
this straight line (R far from 1) indicate deviations from normality. In large samples
from a normally distributed population, such a plot will approximate a straight
line.[185]
1.8- Summary of the Method Development
Plant oils are important as they are used in millions of tones per year in food
industries as edible oil and as a source for the production of biofuels. Section 2.1
describes in more detail the samples studied.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 64
In this thesis a novel method is developed to study plant oils (macadamia nut oil as a
model) by ESI FTICR mass spectrometry. This method would enable the effect of
processing steps and chemical reactions on oils to be directly studied. To develop
this method, a sample of processed macadamia nut oil is studied using FTICR-MS
technique. The industrial refining processes have removed most of the polar
compounds from the processed oil, resulting in a relatively simple oil in terms of the
variety of the chemical constituents. This oil is chosen as a model to develop the
method and to optimize the technique. The ESI FTICR mass spectrometric, GC and
HPLC results of the samples are included in the DVD attached to this thesis.
This technique is capable of providing fingerprint libraries of various unprocessed
oils from different cultivars, climates and sources by detecting the trace compounds
in the plant oils. Three-dimensional diagrams of oil constituent compounds of the
same cultivar and from the same source can be a representative of the variations
caused by environmental parameters such as temperature and air oxidation. These
libraries could be used to optimize the quality of the oil by modifying the parameters
affecting the oil quality during nut growth, harvesting, storage and oil extraction.
This method is not considered to be a replacement for other chromatography and
mass spectrometry methods such as GC-MS, but a complementary tool to analyse
oils which are often very complex mixtures of many hundreds of similar compounds
that do not readily lend themselves to GC.
Sample preparation in oil analysis is usually a tedious and time-consuming process in
other mass spectrometry techniques. In MALDI technique, the matrix needs to be
optimized, and introducing the sample to the instrument usually is a multi-step
procedure.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 65
In quadrupole GC-MS studies of oils, a preliminary esterification reaction is
necessary to increase the volatility of the TAGs, DAGs and MAGs by
transesterification of the acyl groups, and to reduce the polarity of free FA molecules
to be examined on the GC column. This reaction causes a dramatic change in the
composition of the TAG, DAG, MAG and free FA molecules present in the oil by
releasing all of the acyl groups from the glycerol backbones as their esters and also
esterification of all of the free FA molecules present in the oil. However, the GC-MS
provides crucial information regarding the total FA profile of oils.
One of the unique advantages of ESI FTICR-MS compared to GC-MS is its
capability to directly measure the free FA content of oil without any preliminary
chemical reactions; the GC-MS technique is only capable of measuring total FA
profile of oils. However, GC-MS technique is capable of resolving the isomers of
FAs whereas standard FTICR-MS is not. Combining HPLC with ESI FTICR-MS
allows both the free FA content of the oil to be measured and the FA isomers to be
resolved.
The second advantage of ESI FTICR-MS over GC-MS is that in ESI the parameters
are usually tuned to minimize the fragmentation, and most of the peaks are simply
representatives of the corresponding molecular ions, while in ordinary GC-MS
instruments with EI source, the fragmentations make the spectra more complicated,
and the double bonds tend to migrate on the unsaturated FA molecules, making the
interpretation of the mass spectra more complex.
The mass resolving power of the sector GC-MS techniques is usually less than 10,
and the mass accuracy is rarely better than 0.1 mass unit. The FTICR-MS technique
represents a resolving power of several orders of magnitude higher and a mass
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 66
accuracy in the range of a few parts per million. The sample preparation, on the other
hand, is as easy as dissolving the sample in a solvent (methanol or acetonitrile) and
injecting the solution to the instrument. A brief comparison of FTICR-MS technique
with the GC-MS reveals the following advantages.
- High mass accuracy (<5 ppm using a combination of external and internal
calibrations)
- High resolution (>50000 and up to ~1000000 or more depending on the
measured mass and experimental parameters).
- No need to perform derivatization reactions or complicated sample
preparations prior to mass analysis.
- Capability of direct detection and quantification of free FAs in the oil.
- Very high sensitivity, detection levels of 10-10 to 10-13 M.
As mentioned previously, FTICR-MS detects molecule ions according to their
masses and is not capable of resolving isomers such as cis and trans isomers. For
example, oleic acid (cis-9-octadecenoic acid) and elaidic acid (trans-9-octadecenoic
acid) cannot be resolved by FTICR mass spectrometry as they have identical
molecular masses. FTICR mass spectrometry is not able to resolve the substituents
on the glycerol in DAGs and TAGs. As an example, whether the sn-1 substituent
acyl on the glycerol is oleate and the sn-2 acyl is palmitate or vice versa is not
distinguished by this latter technique. These last points demonstrate the importance
of implementing chromatography as well as mass spectrometric analysis if isomeric
components in the oils are to be studied.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 67
The FTICR-MS instrument is costly and the complexity of the technique is higher
than the GC-MS technique and operation of the former needs more training and skill.
Standard regular chemistry methods such as titrations are also used to measure some
of the parameters of the oils such as free acid content.
A DVD is attached to this thesis contains the complete set of FTICR-MS, HPLC
fraction collection and GC-MS spectra and the spreadsheets of extracted data of
intensities, mass accuracies, reproducibility and rankit tests.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 68
Chapter 2
2. Experimental
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 69
2.1- Materials
One processed macadamia oil from Macadamia Integrifolia (smooth shell) from the
family Proteaceae was used as supplied by Bronson and Jacobs, Homebush Bay,
Sydney, Australia. Two unprocessed macadamia nut oil samples (Batch 12 and Batch
13) produced by a cold press method were used as received from same supplier.
Fatty acids and acylglycerol standards were of reagent grade (Fluka) and used as
supplied. Sodium iodide was of analytical grade (May & Baker Ltd. Dagenham,
England) Polyethylene glycol (PEG-300) was of analytical grade (Fluka). PEG-300
and sodium iodide both were used without further purification.
Methanol HPLC grade (Unichrom) was used for extraction, dissolution of the
samples, washing the instrument before and after FTICR-MS experiments and for
HPLC separations.
2.2- Chemical Procedures
2.2.1- Methanol Extraction of Macadamia Oil
Procedure 2.2.1- The oil sample (0.5 ml) was mixed with equal volume of methanol
(0.5 ml) at room temperature in a 1.5 ml Eppendorf tube. For the solid samples, the
mixture was warmed up in warm water to melt the oil. The mixture was vortex
shaken for one minute, and the sample was stored at 0 °C for 12 h to facilitate the
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 70
precipitation of triacylglycerols. The remaining solution was decanted and the
solvent evaporated in a stream of nitrogen gas at ~50 °C. The extract was weighed
and stored in a freezer at –20 °C under nitrogen gas to minimize possible reactions
with atmospheric oxygen and/or effects of ultraviolet light.
An extraction blank was prepared according to the same procedure.
2.2.2- Hydrolysis of Macadamia Oil
Procedure 2.2.2- The oil sample (100 mg) was dissolved in a solution of 0.1 M
potassium hydroxide in 95% (v/v) ethanol (2 ml) and the solution was refluxed for 1
hr or left overnight at room temperature. After cooling, water (5 ml) was added, and
the solution was extracted with diethyl ether (10 ml) to remove any non-saponifiable
material. Centrifugation was used if necessary to break any emulsion that sometimes
formed. The aqueous layer was acidified with dilute HCl and was extracted with
diethyl ether (3×5 ml). The combined ether layers containing the products was
washed with water (5 ml), dried over anhydrous sodium sulphate after which the
ether was removed. The resulting hydrolysis products were stored under nitrogen gas
at -20 °C to protect them from possible effects of atmospheric oxygen and light.
2.2.3- Transesterification of Macadamia Oil
Procedure 2.2.3- Acidic transesterification: The oil sample (100 mg) was dissolved
in toluene (1 ml) and methanol (2 ml) containing 1% (v/v) sulphuric acid was added.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 71
The mixture was left overnight at 50 °C, or alternatively refluxed for 2 hrs after
which, the mixture was cooled and diethyl ether was added (10 ml) followed by
water (5 ml). After thorough shaking, the ether layer was removed, washed with
dilute sodium bicarbonate solution (5 ml), then with water (5 ml) and then dried over
anhydrous sodium sulphate. After the ether was removed by evaporation, the methyl
esters were recovered by dissolving in n-hexane.
2.2.4- Sample Preparation for Mass Spectrometry
The solutions of the neat oil, the methanol extract of the oil (extract), hydrolysed oil,
hydrolysed extract, esterified oil and esterified extract were prepared for mass
analysis by dissolving 1 µl of each of the samples in 1 ml methanol. An aliquot 100
µl of this solution was diluted to 1 ml in methanol. The latter solution was analysed
by ESI FTICR mass spectrometry.
2.3- Instrumentation
2.3.1- Electrospray-ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
A Bruker BioApex II 70e FTICR mass spectrometer was used in this research. This
instrument is equipped with various external ionization sources including ESI, EI,
APCI, MALDI, nanospray and a supersonic expansion cluster ion source.
In this thesis, only the ESI source was used for the study of the macadamia nut oils.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 72
The BioApex II 70e FTICR mass spectrometer consists of several components
including a 7 T passively shielded superconducting magnet, the vacuum system, the
ion optics voltage supply, the electrospray ionization source and the PC compatible
data processing workstation.
Figure 2.1 shows a photograph of the Bruker BioApex II 70e FTICR mass
spectrometer and Figure 2.2 a schematic diagram of the ESI source, ion optics and
the ICR cell.
Figure 2.1- The passively shielded Bruker BioApex II 70e Fourier transform ICR mass spectrometer used in this thesis.
Magnet
Gas inlet
ESI source
Syringe pump
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 73
Figure 2.2- Schematic diagram of the Bruker BioApex II 70e ESI Fourier transfer ICR mass spectrometer used in this research.[186] Instrument control variables are used to control various voltages and pulses (eg XDFL controls the ion beam deflection in horizontal plane).
PL1 PL2 (DPL2) Pulsed
PL4 (DPL4) Pulsed
HVO
(XDFL)
Pulsed
(YDFL)
Gate Valve
FOCL1 FOCL2
PL9 Off axis Electrospray Needle
Quartz Capillary
Skimmer
Gold-plated hexapole
ESI Source Ion Transfer Optics
EV1
PV1 PV2
Excitation
Detection
EV2b
PV2 quench
EV2a (EV2,DEV2)
Infinity Cell
PL1 (DPL2) Pulsed
(DPL4) Pulsed
HVO
Pulsed
Gate Valve
FOCL1 FOCL2
PL9
ESI Source Ion Transfer Optics
EV1
PV1 PV2
Excitation
Detection
EV2b
PV2 quench
EV2a (EV2,DEV2)
EV1
PV1 PV2
Excitation
Detection
EV2b
PV2 quench
EV2a (EV2,DEV2)
PV1 PV2
Excitation
Detection
EV2b
PV2 quench
EV2a (EV2,DEV2)
Infinity Cell
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 74
2.3.1.1- The Vacuum System
The sample ions are formed at atmospheric pressure in the ESI source and are then
directed into the ICR cell which operates at ultra-high vacuum (UHV) i.e. ~10-10
mbar and requires substantial differential pumping for proper operation of the
instrument.
Figure 2.3 illustrates the vacuum system on the Bruker BioApex II 70e FTICR mass
spectrometer.
Figure 2.3- The differential pumping on the Bruker BioApex II ESI FTICR mass spectrometer vacuum system.[186]
The gas-phase ions formed at the tip of the electrospray needle travel through a
quartz capillary (coated with a thin film of platinum at both ends) into the capillary-
skimmer chamber. In this first chamber, the pressure is maintained at about 10-2-10-4
mbar by a turbo molecular pump (Edwards EXT250H) backed by a rotary pump. The
hexapole ion trap and an extraction plate are located next to the skimmer in the ion
Hexapole
10-2-10-4 mbar 10-5-10-7 mbar 10-9-10-11 mbar 10-7-10-9 mbar
Skimmer
Endplate
Passive iron shield
Preamplifier
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 75
source chamber. The pressure in this second chamber is maintained at 10-5-10-7 mbar
by a cryogenic pump (Edwards CoolStar 800).
The extracted ions are directed into the FTICR cell by a series of ion-steering and
ion-focusing lenses. In the ion-focusing region, the pressure is maintained at ~10-7-
10-9 mbar by a cryogenic pump (Edwards CoolStar 400).
Finally, ions enter the ICR cell that is housed in the UHV chamber. The pressure in
this chamber is maintained at 10-9~10-11 mbar by two cryogenic pumps (Edwards
CoolStar 800 and 400).
Two ion gauges (Granville Philips) are used to continually monitor the pressure in
the capillary-skimmer chamber and the ultra high vacuum chamber in real time.
2.3.1.2- Electrospray Ionisation Source
Figure 2.4 shows the schematic diagram of the Analytica off-axis ESI source used in
this research project.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 76
Figure 2.4- Schematic diagram of the off-axis Analytica ESI source used in this research project.
Figure 2.5 illustrates the off-axis and on-axis needles in the ESI cage and Figure 2.6
shows the inlet tip of the quartz capillary and the end plate. The protecting cap has
been removed to expose the capillary tip.
To the skimmer
Hot drying nitrogen gas flow
Nitrogen sheath gas flow
Larger droplets fall due to the gravity
Small charged droplets are drawn to the capillary tip
Quartz capillary
Off-axis spray needle (~45°)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 77
Figure 2.5- The off-axis and on-axis spray needles inside the Analytica ESI cage.
Figure 2.6- The capillary tip and the end plate. The protective cap is removed to expose the capillary tip.
On-axis needle
Off-axis needle
End plate
Capillary tip
Electrical connections
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 78
The solutions containing the oil components were injected into the ESI chamber
using a Cole-Palmer (Vernon Hills, Illinois) syringe pump at a constant flow rate of
90-150 μl/h (typically 120 μl/h). The off-axis spray needle inner diameter was 0.1
mm.
The needle was connected to the ground potential. For negative-ion detection a
potential of about +4 kV was applied to the end of the capillary that faces the needle.
The end plate and the mesh cage surrounding the spray needle were held at about 2.5
kV.
This needle was placed off-axis at a distance of about 3 cm from the end of the
quartz capillary. Originally an on-axis configuration was used for the needle spray,
but the off-axis needle was observed to increase the efficiency and reproducibility of
the ESI process. The larger droplets drop due to the gravity while the smaller charged
droplets were more likely to reach the capillary inlet. Also the capillary was kept
cleaner, as the bulk solution was basically directed away from the tip of the quartz
capillary inlet.
A stream of nitrogen gas at room temperature (known as sheath gas) was diverted out
of the needle sheath and pressure optimization was found to improve the ionization
formation of the spray for lipid ions.
A second countercurrent stream of hot drying nitrogen gas (270 °C) that flows out of
the space between the capillary tip and the end plate (see Figure 2.6) was applied to
promote the evaporation of the solvent and also had the added effect of directing
larger droplets away from the capillary inlet as shown in Figure 2.4.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 79
Figure 2.7 shows a schematic diagram of the quartz capillary, skimmer and hexapole
location in the ion optics system.
Figure 2.7- Schematic diagram of quartz capillary, skimmer and hexapole location in the ion optics system (not to scale).
A conical skimmer is located close to the exit end of the quartz capillary as shown in
Figure 2.7 and varied over 0-20 V relative to the ground. A voltage of – 400 to +400
V was applied to the ion exit end of the capillary versus the skimmer. A 2.0 s time
was used for the delay in the hexapole ion trap.
For negative-ion detection a voltage of +4 kV was applied to the end of the quartz
capillary that faces the spray needle (see Figure 2.4). For positive ion these potentials
are reversed. This creates an electric field gradient between the spray needle and the
capillary and initiates the ion-formation from the droplets that emerge from the spray
needle. The end plate and the mesh cylinder surrounding the spray needle were held
at about +2.5 kV versus the ground (see Figure 2.5). A voltage of –400 to +400 V
Capillary
Hexapole Ion optics
To pump To pump
Ion / gas flow
Skimmer
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 80
versus skimmer is applied to the exit end of the capillary to assist in collection and
focussing the generated ions from the spray needle.
In the Analytica ESI source a small hexapole (see Figure 2.8) is used to store ions
from the continuous needle spray. Once a sufficient number of ions have been
collected, typically over 1 to 3 s, they are pulsed toward the ICR cell by lowering a
voltage on a trapping electrode at the ICR cell end of the hexapole.
Figure 2.8- Photograph showing the hexapole ion-trap in Analytica ESI source in the BioApex II FTICR mass spectrometer.
2.3.1.3- The “Infinity®” ICR cell
The infinity® ICR cell implemented in Bruker BioApex II 70e FTICR mass
spectrometer is a special ICR cell developed by Bruker in 1991.[128] This cell is
Trapping plate
Hexapole trap rods
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 81
designed to apply a uniform potential on the trapping plates at each end of the cell to
solve the common “z-ejection” problem in simple ICR cells. Figure 2.9 shows a
photograph of the infinity ICR cell used in BioApex II FTICR mass spectrometer.
Figure 2.9- The infinity ICR cell in BioApex II FTICR mass spectrometer. The scale is in mm.
The trapping plates have segmented regions thereby allowing differential potentials
to be applied throughout the exciting pulse.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 82
Figure 2.10 shows a schematic diagram of the infinity ICR cell, where PV1 And PV2
are parameter names for the trap plates and EV2a and EV2b are for the side kick ion
trap elements. Typical values for these variables are listed in Table 2.1.
Figure 2.10- Schematic diagram of the infinity ICR cell used in BioApex II FTICR mass spectrometer. PV1, EV1 etc are parameters’ name for voltages see table 2.1.
All electro-plates are made of titanium and are gold-plated. The trapping plates (PV1
and PV2) are designed and arranged as described by Caravatti et al. in their
paper.[128] The additional plates outside the PV1 are EV1 and two EV2 half-plates
designed to trap ions via a Sidekick® frequency.
2.3.1.4- Ion Trapping
The ICR cell ion trapping in BioApex II FTICR mass spectrometer was carried out in
Sidekick™ ion-trapping mode in this thesis. The instrument is also capable of
EV1
PV1 PV2
PV2quench
EV2a
++++
+ ++
Excitation
Detection
EV2b
B
sidekick:
EV1
PV1 PV2
PV2quench
EV2a
++++
+ ++
++++++++
++ ++++
Excitation
Detection
EV2b
B
sidekick:
Magnetic field
From ESI source
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 83
applying the gated ion trapping (static or dynamic) and collision gas-assisted
dynamic ion trapping, but those were not used.
2.3.1.5- Typical Source, Ion-transfer and ICR Cell Parameters Used in Positive- and Negative-ion Modes on the BioApex II 70e FTICR Mass Spectrometer Table 2.1 lists typical parameters used in positive- and negative-ion FTICR mass
spectrometry experiments.
Table 2.1- Typical parameters used in positive- and negative-ion FTICR mass spectrometry experiments.
Parameter Positive ion Value (V)
Negative ion Value (V)
Capillary 35.0 -43.0 Skimmer 2.51 -5.00
Offset 0.27 -1.91
Source
Extract -4.51 -0.87 PL1 43.80 -10.20 PL2 36.5 -75.50
DPL2 22.30 14.30 DPL4 -2.60 -11.50
FOCL1 -9.90 -27.20 FOCL2 -29.60 55.90 XDFL 46.8 -26.2 YDFL 16.1 -26.0 EV1 -1.22 4.12 EV2 -1.158 1.770
Ion-transfer
DEV2 -16.00 9.45 PV1 -1.46 -1.22 PV2 2.01 -1.35 EV1 -1.22 4.12 EV2 -1.158 1.770
DEV2 -16.00 9.45
ICR Cell
PL3 0.5 0.9
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 84
2.3.1.6- Superconducting Magnet
The Magnex Magnet 7 T superconducting magnet used in this thesis uses passive
magnetic field shielding. The solenoid comprises a coil wound of NbTi
superconducting wire with single and/or multiple filaments in a copper protective
matrix. The operating temperature for this magnet is kept at 4.2 K by immersing it in
a cryostat of liquid helium (280 L). A liquid nitrogen cryostat surrounds the liquid
helium vessel (250L), and both of these vessels are insulated and separated by
vacuum cases.
The superconducting magnet has a bore diameter of 150 mm, in which the ICR cell
sits at the centre of the homogenous magnetic field. Cooling water circulates around
the ICR cell to keep the magnet cool during the vacuum bake out process of the ion
optics and the ICR cell.
2.3.1.7- Collision-Induced Dissociation
Argon gas was used as the collision agent during the CID experiments performed on
the FTICR mass spectrometer. It was introduced into the ICR cell through a
molecular leak valve at a constant flow rate.
The ion selection in the ICR cell is achieved by an RF excitation pulse containing a
‘mass envelope’ at the selected m/z that ejects all unwanted ions out of the ICR cell.
The selected m/z ions are then excited by an excitation pulse containing a single RF
frequency with an appropriate intensity. A delay is allowed for the collision between
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 85
the selected ion and the collision gas. This is followed by the excitation of the
product ions in the cell and the detection.
2.3.1.8- Pulse Sequence in FTICR-MS Experiments The computer program that operates the FTICR mass spectrometer has a sequence of
pulse-delay events. Each pulse-delay sequence executes a specific operation on the
FTICR mass spectrometer. The BioApex II FTICR mass spectrometer came with
several standard pulse sequence programs that can be easily modified to suit different
experimental requirements.
Figure 2.11 shows a standard pulse sequence for a simple FTICR-MS experiment.
Acquire
P1 P2 P4
D1 D2
Figure 2.11- Pulse sequence used in FTICR-MS analyses for a simple experiment. The basic elements in each pulse sequence program include ion generation, ion
trapping, ion excitation and ion detection. The event sequence shown in Figure 2-11
is as follows: prior to the ion generation, a quench pulse-delay P1/D1 is applied to one
of the trapping plates which effectively ejects all the ions from the ICR cell. An ion
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 86
generation pulse-delay P2/D2 is then followed to produce ions from the ion source. In
the external ESI ion source, the P2 pulse is the ion accumulation time in the hexapole
ion-trap.
After the ions are trapped in the ICR cell, a P3 pulse applies radio frequency (RF)
“chirp” (50-200 V peak-to-peak and 180o out of phase) to the two excitation plates
and excites ions whose natural cyclotron frequencies are resonant with the applied
RF field. Ions of the same mass that are generated randomly in time and space are
now brought into a phase-coherent motion, moving together in a packet. This phase-
coherent ion packet generates an ion-induced image current on the receiver plates,
and this image current is passed to a differential amplifier circuit, which facilitates
the detection and amplification of the signals.
If a single RF frequency is selected for the ion excitation, the result is defined as
mass detection, which is commonly referred to as “narrow band” mass spectrum.
Alternatively, the excitation and detection of ions in ICR cell can be carried out in a
wide mass range by sweeping the excitation frequency with a fast frequency chirp
over a selected mass range. Ions in this mass range are excited into a phase-coherent
motion in less than a millisecond. This chirp excitation is often referred as “broad
band” mass spectrum. The experiments in this thesis were carried out using the Chirp
Excitation method.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 87
2.3.1.9- Mass Calibration
In external mode calibration, polyethylene glycol, MW=300, (PEG300, a commercial
product used in the production of detergents) and sodium iodide were used in
positive-ion mode, and sodium iodide was also used in negative-ion mode. First
appropriate solutions of PEG300 and NaI (10-4 M to 10-5 M) in methanol were
introduced to the FTICR mass spectrometer, the instrument was calibrated by
identifying several peaks over a mass range of 100- 1100 Da using a “Masscal”
program and setting up appropriate calibration table. A mass accuracy of <10 ppm
was typically achieved using this external calibration method.
For high mass accuracy calibration internal mass calibration was carried out using
internal reference compounds. In this method, the observed peaks for the known
compounds in the samples were used to recalibrate the spectra and to calculate the
m/z of the unknown compounds. By using the internal calibration method, a mass
accuracy of <2 ppm was usually achieved.
It can be shown from the theory of FTICR-MS[91] that the number of collected data
points is the highest in the lower mass region of the spectrum, hence, the mass
accuracy is affected by the actual number of data points obtained over the measured
mass and the peak symmetry. Therefore, it is expected to be able to measure the
FTICR-MS peaks with a higher precision and accuracy for fatty acids compared to
TAGs.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 88
2.3.1.10- The BioApex II FTICR Mass Spectrometer Control Software
The operation of the BioApex II FTICR mass spectrometer and the mass
spectrometry data acquisition were conducted by XMASS software package supplied
by Bruker Daltonics Company (Version 6.0.1). This program provides control of the
voltages on different components of the FTICR mass spectrometer, including the ion
source, ion optics and ICR cell. It also possesses many options for data processing.
Details of the functions and techniques for data processing are described in the
XMASS user manual.[187]
A significant feature of the XMASS program is that it facilitates the use of macro
procedures. These predefined macro functions are exhibited as icons in a menu-
driven user-friendly interface. Various pulse sequences for different mass
spectrometry experiments can be carried out simply by selecting the appropriate
icons. These macro routines are accessible to the user and are readily modified.
The mass spectra obtained by the XMASS program can be saved as ASCII files
(rather than binary files) and can then be imported into other software for subsequent
additional processing.
2.3.2- High Performance Liquid Chromatography
HPLC was used to collect various isomers in the methanol extract of various
macadamia nut oils prior to analysing using high mass accuracy FTICR mass
spectrometer. The system used in this research project included:
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 89
- Pump: Waters 600 Multisolvent Delivery System (Waters - division of
Millipore, Milford, MA 01757 USA).
- Injector: Waters U6K Fluid Unit (Waters - division of Millipore, Milford, MA
01757 USA). The injector was equipped with a micro-switch to send a trigger
signal to the control program on injection.
- Interface: The data was transferred to the PC via a Shimadzu CBM-101 2-
channel communication bus module interface (Shimadzu Corporation, Japan).
The data was collected and manipulated by LC10 chromatography software
(Shimadzu Corporation, Japan) running on a PC under Microsoft™ Windows™
98 SE operating system.
- Column: Reverse-phase C18 Altex™ Ultrasphere™ ODS, 4.6 mm× 25 cm×
5μm (internal diameter× length× particle diameter).
- Precolumn (guard-column): Nova-Pak® C18 Guard-pak™ precolumn cartridge
(Waters - division of Millipore, Milford, MA 01757 USA).
- Detector: Sedex 55 Evaporative Light Scattering Detector (ELSD) (by
SEDERE 94140 Alfortville France).
- Fraction collector: Isco Foxy 200 (Isco Inc. Lincol, NE, U.S.A.)
2.3.2.1- Gradient Elution
Gradient elution (solvent programming) as detailed in Table 2.2 was used to optimise
the macadamia oil separation efficiency. Methanol and water mobile phase contained
0.05% v/v in glacial acetic acid to keep the pH low to minimise free FA ionisation.
This method gave reproducible chromatograms; hence this enabled the fraction
collection to be performed without the ELSD being connected. Usually when
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 90
collecting fractions using an ELSD a splitter is inserted between the outlet of the
column and the ELSD, but this was not required because of excellent reproducibility
of the separation.
Table 2.2- Solvent programming for HPLC in the gradient elution of macadamia oil methanol extract.
Time min Flow Rate (ml/min)
% Acetone
% Water (with 0.05% acetic acid)
% Methanol (0.05% in acetic acid)
Initial 1 0 15 85
40 1 0 0 100
50 1 0 0 100
52 2 0 0 100
70 2 0 0 100
75 2 100 0 0
90 2 100 0 0
91 1 0 15 85
120 1 0 15 85
A solution of macadamia oil methanol-extract was prepared at a concentration of 2
mg/ml in methanol and 25 µl of this solution was injected into the chromatograph.
Fractions were collected over a period of 90 minutes at 1 fraction per minute using a
dedicated fraction collector. The fractions were then examined by ESI FTICR mass
spectrometry in both positive- and negative-ion modes as discussed in Chapter 7.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 91
2.3.3- Gas Chromatography- Mass Spectrometry
For the GC-MS experiments, the GC was a Hewlett-Packard HP 5890 Series II with
a J&W DB-Wax 60 m × 0.5 mm × 0.25 µm, and the mass spectrometer was a VG
QUATTRO triple quadrupole mass spectrometer operated under single quadrupole
conditions. A standard 70 eV EI source was used. A standard mixture of FA methyl
esters was used to calibrate the time component of the GC experiment prior to
injecting each set of samples. The spectra were analyzed using Waters Masslynx
software version 4.0 (Milford Massachusetts 01757 USA).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 92
Chapter 3
3. Validation of the ESI FTICR-MS Method Developed for the
Analysis of Plant Oils
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 93
3.1- Relation between Peak Intensities and Concentration of the Ions
A number of studies have been published on the relation of peak intensities to the ion
abundances in FTICR-MS.[152,188-191] The spectral peak heights are not reflective of
the true ion abundances in the ICR cell,[189,190] due to the selective z-axis ejection
artefacts particularly for ions far apart in mass,[192] and due to space charge
interactions for the close-lying peaks.[190]
Comisarow has established a direct relationship between the instantaneous charge
induced on a detection electrode and the number of excited ions in the ICR cell.[132]
There are reports on the effect of more abundant ions in the ICR cell on the peak
intensity and the mass measurement accuracy of the less abundant ions.[193]
Cech and Enke have achieved a linear relationship between the peak intensity and the
concentration of a particular ion in the FTICR-MS for a certain range of
concentration by calibrating the peak intensities using standard solutions of the same
species prior to the injection of unknown samples.[188]
As mentioned above, matrix effects such as salt content, high concentration of co-
existing species, different solvent affinities of the molecules in solution and general
ionization efficiencies are among the factors influencing the peak intensities in ESI
FTICR-MS. Further discrimination can occur during the accumulation in hexapole
and the transmission of ions into the ICR cell according to the hexapole delay and
ion-transfer optics settings.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 94
However, for the ions within comparable range of concentrations, with similar
structures in a close range of masses, using same set of hexapole delay and ion-
transfer optics parameters, a sensible estimate of the relative concentrations of the
ions is achievable from the intensities of the peaks in an FTICR mass spectrum.
An additional complication that should be taken into account in the negative-ion
mode FTICR-MS of the solutions containing free FAs is the degree of completeness
of the dissociation reactions of FAs in the solution and during the evaporation
processes (pKa for stearic acid is 10.15, for oleic acid is 9.85 and for linolenic acid is
8.28 in aqueous solution). In a solution containing stronger FAs, the dissociation of
the weaker FAs and hydroxyl groups can be suppressed, resulting in a decrease in the
relative peak intensity of the weaker FAs and the hydroxyl groups.
On the other hand, in the positive-ion ESI FTICR-MS of free FAs, the ions are
generated by attachment of a cation to non-bonding electrons on the oxygen atoms,
that is a simple ion-dipole interaction and so relative positive-charged adduct ion
affinities of the FA become very relevant to the ionization process.
To verify the effect of acid-base dissociation reactions on the peak intensities in
negative-ion mode ESI FTICR-MS, solutions of the oil and the oil extract in pure
methanol were compared to the solutions of the oil and the oil extract in methanol
containing 10-3 M triethylamine as a volatile organic base. The existence of a basic
molecule in the solution is expected to promote the deprotonation of the acidic
species, resulting in an immense increase in the peak intensities of the anions.
The observed relative intensities for most of the peaks throughout the spectrum
showed a 10-20 percent increase, but the relative peak intensities found to be similar
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 95
in two spectra. This is consistent with the previous observations regarding the effect
of an alkaline solution on the deprotonation reactions in negative-ion ESI MS.[194]
3.2- Reproducibility of the ESI Source and the FTICR Mass Spectrometer
A solution containing suberic (C8:0 dicarboxylic acid), palmitoleic (C16:1), palmitic
(C16:0), linoleic (C18:2), oleic (C18:1), eicosenoic (C20:1) and eicosanoic (C20:0)
acids at approximately 10-4 M was prepared from chemicals present in the School of
Chemistry at The UNSW with unknown purities. As in this method development
study we are not performing quantitative analysis or comparing the intensities as a
measure of concentrations, the purity of the chemicals used was not a concern.
This solution was used to validate the method in various experiments discussed in
this section. Figure 3.1 shows the ESI FTICR mass spectrum of the test solution in
negative-ion mode.
The intensities of the peaks assigned to free fatty acids are different due to the fact
that the purities of the used chemicals were unknown. It is also possible that the
difference in the intensities is partly due to the instrumental parameters and relative
ionisation efficiencies.
Table 3.1 lists the assignment of the observed peaks in the negative-ion FTICR mass
spectrum of the prepared test solution.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 96
173.0822
281.2486
369.1532
200 300 400 500 600 700 800 900m/z
255.2335
563.5091 639.5650 905.8527
Figure 3.1- Negative-ion ESI FTICR mass spectrum of the test solution.
Table 3.1- Assignment of mass spectral peaks in the FTICR mass spectrum of a solution of fatty acids mixture solution shown in Figure 3.1.
Formula Measured Mass (Da)
Exact Mass (Da)
Compound Class
C8H13O4‾ 173.0822 173.0820 Di-FA* C9H15O4‾ 187.0976 187.0976 Di-FA* C14H27O2‾ 227.2022 227.2017 FA C16H29O2‾ 253.2177 253.2173 FA C16H31O2‾ 255.2335 255.2330 FA C17H33O2‾ 269.2478 269.2486 FA C18H29O2‾ 277.2201 277.2173 FA C18H31O2‾ 279.2347 279.2330 FA C18H33O2‾ 281.2486 281.2486 FA C18H35O2‾ 283.2636 283.2643 FA C20H37O2‾ 309.2805 309.2799 FA C20H39O2‾ 311.2956 311.2956 FA C16H27O8‾ 347.1717 347.1711 FA dimer C18H25O8‾ 369.1532 369.1555 FA dimer C36H67O4‾ 563.5091 563.5045 FA dimer C36H69O4‾ 565.5217 565.5201 FA dimer C37H73O6‾ 613.5481 613.5413 DAG C39H75O6‾ 639.565 639.5569 DAG C57H109O7‾ 905.8299 905.8179 TAG
* Di-FA: Dicarboxylic fatty acid (HOOC-R-COOH).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 97
Among the assigned peaks in this spectrum, there are 10 FAs, 2 short-chain di-
carboxylic acids, 4 FA dimers, 2 DAGs and one TAG molecule assigned. This table
of chemical compounds clearly demonstrates that the fatty acids used to prepare the
test solution were not pure. The short-chain fatty dicarboxylic acids are possibly
formed by the oxidation of the double bond of unsaturated FAs such as oleic acid.
To investigate the reproducibility of the ESI source and to verify the mass accuracy
of the FTICR mass spectrometer for the free FAs, the solution (containing suberic,
palmitoleic, palmitic, linoleic, oleic, stearic, eicosenoic and eicosanoic acids) was
examined seventeen times on the ESI FTICR mass spectrometer in negative-ion
mode with oleate anion used as the internal calibrant. The observed peaks in the
negative-ion spectra of the solution range from 173.0822 Da for suberic acid at the
lower mass end to 565.5217 Da for oleic-stearic acid dimer at the high mass end. The
ion transfer parameters used in this experiment were optimized and used in the
analysis of the oil and the oil extract samples.
Figure 3.2 illustrates the relative intensities of palmitate anion (255.2330 Da) peak
versus the measured masses in 17 analyses of the test solution. The relative standard
deviation (RSD) of peak intensities is 7.0% and the RSD of mass measurement is
4×10-5 % or 0.4 ppm. Oleate anion (281.2486 Da) is used as the reference compound
for the relative intensities and the internal standard for the mass measurements.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 98
Relative Intensities of Palmitate Anion (Mass 255.2330 Da) in 17
Experiments
0.1
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
0.2
255.23365 255.2337 255.23375 255.2338 255.23385 255.2339 255.23395 255.234 255.23405Measured Mass (Da)
R.I.
Figure 3.2- Relative intensities of palmitate anion peaks versus measured masses in 17 consecutive FTICR mass spectra of the test solution (normalised versus oleate anion peak as 1).
Figure 3.3 illustrates the relative intensities of stearate anion (283.2643 Da) peak
versus measured masses in 17 analyses of the test solution. The RSD of the peak
intensities is 4.2% and the RSD of mass measurement is 7×10-5 % or 0.7 ppm. Oleate
anion (281.2486 Da) is used as the reference compound for the relative intensities
and the internal standard for the mass measurements.
Relative Intensities of Stearate Anion (Mass 283.2643) in 17 Experiments
0.20
0.21
0.21
0.22
0.22
0.23
0.23
0.24
283.2649 283.265 283.2651 283.2652 283.2653 283.2654 283.2655 283.2656
Measured Mass (Da)
R.I.
Figure 3.3- Relative intensities of stearate anion peaks versus measured masses in 17 consecutive FTICR analyses of the test solution (normalised versus oleate anion peak as 1).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 99
Figure 3.4 illustrates average mass deviation from exact masses for the assigned
peaks in the test solution in seventeen separate negative-ion FTICR-MS analyses.
Only external calibration is performed in this experiment. Mass deviations are
calculated using the following equation:
│ Exact mass – Measured mass │ Mass deviation (ppm) = ──────────────────── × 1000,000 Exact mass
Generally, higher mass deviations are observed at higher ionic masses (about 8.8
ppm at 563 Da) and lower mass deviations are observed at lower ionic masses (about
1.7 ppm at 173 Da).
Average Deviations from Exact Masses vs. Average Measured Masses for 17 ESI FTICR-MS Analyses of the Standard Solution
0
1
2
3
4
5
6
7
8
9
10
150 200 250 300 350 400 450 500 550 600
Average measured mass (Da)
Aver
age
Devi
atio
n fr
om E
xact
Mas
s (p
pm)
Figure 3.4- Average deviations from exact masses vs. average measured masses in 17 consecutive FTICR mass spectra of the test solution. Each point represents the average of 17 mass measurements of a particular FA in the test solution.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 100
3.3- Detection Limits of FTICR-MS in FA Measurement
To verify the detection limit of the FAs in FTICR mass spectrometer, approximately
10-4 M solutions of palmitic, palmitoleic, stearic, oleic, and linoleic acids were
prepared. The criterion was to include saturated and unsaturated FAs in the test
solutions (C16:0, C16:1, C18:0, C18:1, C18:2). Successive dilutions (1/10, 1/100,
1/1000, 1/10000, and so on) were prepared for each of the FAs in methanol. The
solutions were examined on the FTICR mass spectrometer starting with the most
dilute until a signal was observed for that particular acid. The experiment was carried
out in both positive- and negative-ion modes. The detection limits found to be
approximately 10-12 M in positive-ion mode and approximately 10-13 M in negative-
ion mode FTICR-MS (five times of the background noise).
3.4- Effect of the Hexapole Ion Trap Delay on the Peak Intensities
To examine possible contribution to the ions observed in the present analytical
macadamia oil experiments, the hexapole ion trap delay (typically 0-8 S) was
studied. In this experiment, a solution of the processed macadamia nut oil was
injected to the ESI FTICR mass spectrometer with the hexapole trap delay varied
from 0 s to 8 s in 0.5 s increments. At each hexapole delay setting, the spectrum was
recorded, and then the results were compared using the intensities of the observed
peaks in the spectrum.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 101
At hexapole delays longer than 2.5 s the peak intensities did not change dramatically,
while in hexapole delays shorter than 1 s, the intensities of the peaks diminished
drastically. Consequently, a hexapole delay of 2 s was selected for the FTICR-MS
experiments as the optimum setting.
3.5- Aging Stability of the Oil Samples
To investigate the stability of the oil samples, stability tests were performed on the
processed macadamia nut oil. The processed macadamia oil was left in the laboratory
in the daylight at room temperature for up to ten months. The oil was analysed on
ESI FTICR mass spectrometer during this period. Figure 3.5 shows the positive-ion
ESI FTICR mass spectra of neat macadamia oil on three different dates. Figure 3.5-a
shows the FTICR mass spectrum of neat macadamia oil analysed in December 2002,
Figure 3.5-b illustrates the FTICR mass spectrum of neat macadamia oil analysed in
July 2003 and Figure 3.5-c shows the FTICR mass spectrum of neat macadamia oil
analysed in September 2003. As the free fatty acid content of the oil is considered as
a measure of the stability of the oil, it was necessary to investigate the stability of the
macadamia oil sample. The duration of the stability investigation was the total time
spent on the experimental work in this study.
The observed variations in the three FTMS spectra are minor, suggesting that the oil
was stable during the period of the aging stability test.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 102
825.682845.606
853.711
869.715
879.734
897.746
907.765
923.754937.798
955.749 971.749
825.679837.587
853.708
865.625
879.725
907.756
923.743935.771
953.687 967.709
825.689837.601
851.713
879.746
895.747
907.778
923.763939.768
951.765 969.746
820 840 860 880 900 920 940 960 980 m/z
Figure 3.5- Positive-ion FTICR mass spectra of methanol solution of neat processed macadamia oil in (a) December 2002, (b) July 2003 and (c) September 2003.
3.6- Fragmentation during Ion Transfer
At high capillary-skimmer potential differences, ions are highly accelerated and
experience more collisions. Due to this higher number of impacts of the accelerated
ions in the capillary-skimmer region, unnecessary fragmentations may occur.
To investigate the possibility of fragmentation of FAs and the acylglycerols in
positive- and negative-ion modes in the capillary-skimmer and hexapole ion-trap
regions of the ion transfer optics, the test solution of FAs was injected to the FTICR
mass spectrometer while the voltage on the capillary was scanned from the lowest
signal-producing voltage up to the highest possible voltage (~±400 V).
(a)
(c)
(b)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 103
In the positive-ion mode increasing the capillary-skimmer potential to greater than
200 V resulted in the detachment of the attached sodium ion from the FA and
acylglycerol molecular ion complexes, resulting in a corresponding signal loss.
To decrease the possibility of molecular ion fragmentation occurring in both
positive- and negative-ion modes, the capillary-skimmer potential was set to the least
signal-producing voltage in both positive- and negative-ion modes. Typically this
voltage was set to about ± 40 V (10% of the scale) in positive and negative modes to
minimise the possibility of adventitious collision induced dissociation processes (see
Table 2.1).
Figure 3.6 illustrates the effect of capillary-skimmer voltage on the spectrum of the
FA solution in negative-ion mode. At a capillary-skimmer voltage of 300 V the
intensities of the main peaks diminish (approximately 20 times weaker than
intensities at 20 V) and some higher mass peaks appear in the spectrum. The peaks in
the higher mass region are assigned to compounds with high number of oxygen
atoms. At high capillary-skimmer voltages oxidation-reduction reactions are more
likely to occur, producing molecules with high number of oxygen atoms. In addition,
at high capillary-skimmer voltages, excessive CIDs occur resulting in breaking the
FA dimers and also detachment of Na+ from positive ions such as DAGs and MAGs,
causing weak peak intensities and loss of sensitivity in the obtained spectra (See
Figure 3.6 c). Note that in Figure 3.6 (c) the intensity of the spectrum is two orders of
magnitude lower than spectra (a) and (b) (107 vs. 109). If the three spectra in Figure
3.6 are superimposed using same intensity scale, the peaks in spectrum (c) would not
be observed.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 104
On the other hand, a drawback of applying a very low capillary-skimmer voltage is
that dimers of free FAs form in the gas phase in hexapole region. These molecules do
not exist in the oil and are generated in the instrument. However, the dimers peaks
are easily recognised as few peaks appearing only in the 400-600 Da region, and
readily disappear by applying higher capillary-skimmer voltages. The FA dimer
peaks could also be used as internal calibrants for the DAG region peaks.
140.6254
173.0821
212.0756
281.2485
369.1529
140.6250
173.0820
212.0756
281.2487
281.2482
347.1690 413.1159455.3403
509.4622565.5182
639.5568
851.7574907.8229
0.00
0.25
0.50
0.75
1.00
1.25
9x10
In ten s.
0.0
0.2
0.4
0.6
0.8
1.0
1.29
x10
0
2
4
6
8
7x10
200 300 400 500 600 700 800 900 m/z Figure 3.6- Negative-ion FTICR mass spectra of the FA test solution at three capillary-skimmer voltages, a) 20 V, b) 125 V and c) 300 V.
Since the ESI produces ions continuously and the FTICR experiment is gated, the
hexapole ion trap is used to build up the concentration of ions from the ESI source
prior to injection of a packet of ions into the ion optics (Figure 2.2). However, during
(a) 20 V
(b) 125 V
(c) 300 V
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 105
this ion trapping process it is possible for some ions to undergo ion molecule
interactions including association and/or CID processes. The longer the ions stay in
the hexapole, the higher the possibility of the CID fragmentation to occur.
3.7- Discussion of the Validity of the Mass Spectral Peak Assignments
The major parameters in assigning empirical formulae to the FTICR mass spectral
peaks in this study is based three parameters: high mass accuracy, high resolution
and knowledge of the chemical composition of macadamia oil obtained in previous
studies and different methods we used such as GC-MS.
High mass accuracy of <2 ppm enables us to assign possible empirical formulae to
the FTICR mass spectral peaks. This high accuracy minimises the number of
possible combinations of the free FAs in a particular ion.
High resolution of approximately 50,000 or higher enables us to differentiate the
isobars (molecules with same nominal masses but different elemental compositions).
Isotopic ratios could be used but in FTICR-MS the isotopic ratios can have an error
of about 10%.
The major FAs in macadamia oil are oleic, palmitoleic and linoleic acid. The MAGs,
DAGs and TAGs all contain various combinations of the above FAs. Each MAG,
DAG and TAG assignment may contain a different possible combination of FAs
giving the same molecular mass. For example, a TAG molecule assigned to C18:1,
C18:1, C16:0 could be assigned to C18:1, C18:0, C16:1. But we would expect the
intensity of the sodium adduct of the first combination to have the highest relative
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 106
intensity in the mass spectrum of neat macadamia oil. This is due to the fact that
C18:1 and C16:1 comprise the highest percentage of the oil constituents, while C16:0
and C18:0 are minor constituents of macadamia oil.
3.8- Normal Probability (Rankit) Test of the Peak Intensities and Measured Masses
To investigate possible existence of systematic error in the measurement of the peak
intensities and masses, normal probability (Rankit) tests were carried out. A normal
distribution of the measured peak intensities and masses contradicts the presence of a
systematic error in the measurements. As is explained elsewhere in this thesis
(section 1.4.1.9), the correlation coefficient (R) of the trend line is a measure of the
normality of the distribution of the measured peak intensities and masses. An R value
close to 1 denotes a normal distribution of the points, disagreeing with the existence
of a systematic error in the measurements.
The FA test solution was analysed on the FTICR mass spectrometer 17 consecutive
times in negative-ion mode. The intensities of the peaks and the measured masses
were examined using a Rankit test method. The Rankit test plot yielded an R value of
0.984 for the measured masses and an R value of 0.991 for the measured intensities,
suggesting a normal distribution of the intensities and the measured masses of the FA
peaks in negative-ion mode, ruling out the existence of systematic errors. Figures 3.7
and 3.8 show the rankit plot for the intensities and mass measurements of palmitate
anion in the FA test solution. Similar plots could be produced for other FAs in the
FA solution.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 107
Rankit plot of measured masses (palmitate anion, 255.2330 Da) for 17 FTICR-MS analyses of the FA standard solution in negative-ion mode
R2 = 0.9695
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
255.2336 255.2337 255.2338 255.2339 255.2340 255.2341
m / z (Da)
z
Figure 3.7- Rankit plot of the measured masses of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode.
Rankit plot of relative intensities (palmitate anion, 255.2330 Da) for 17 FTICR-MS analyses of the FA standard solution in negative-ion mode
R2 = 0.9824
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
0.13 0.135 0.14 0.145 0.15 0.155 0.16 0.165 0.17
Relative Intensity
z
Figure 3.8- Rankit plot of the peak intensities of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 108
Chapter 4
4. Positive-ion ESI FTICR-MS of Processed Macadamia Oil
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 109
4.1- Introduction
In this chapter the positive-ion ESI FTICR mass spectra of neat, methanol extract,
hydrolysed and esterified processed macadamia oil are discussed. For each spectrum,
only the peaks larger than 2% in intensity are assigned and tabulated, although some
interesting compounds with lower intensities are sometimes listed in the tables.
Comparisons between spectra are made to investigate the validity of the assignments
and various experiments.
For the purposes of this study we have assigned the peaks in the mass spectra
according to elemental analysis and exact-mass measurements in conjunction with
common compounds assigned by GC-MS in other studies undertaken on similar
macadamia nut oil[195,196] and the common knowledge of plant lipid chemistry. Here
however we are able to identify the parent molecular ion species in the neat and
methanol extract samples whereas in the earlier studies such compounds are reduced
to FA methyl esters by derivatization for GC-MS analysis.
4.2- Positive-ion ESI FTICR-MS of Processed Macadamia Oil
The positive-ion ESI-FTICR mass spectrum of neat processed macadamia oil is
shown in Figure 4.1.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 110
319.2249 393.2609
575.5055
603.5352
631.4933659.5219
825.6786
853.7187
879.7338
907.7650
937.7980
300 400 500 600 700 800 900 1000 m/z Figure 4.1- Positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil.
In all, we can identify at least 187 different ionic species in Figure 4.1 though many
of them occur at trace levels (<1%). Application of the Bruker elemental analysis
software, carbon-13 isotope analysis and high-resolution and high-accuracy mass
measurements allows a number of the peaks in Figure 4.1 to be assigned and these
are listed in Table 4.1. The extrapolation from the molecular formulae to the actual
components in the oil are only tentative because no structural analyses have been
carried out on the assigned ions.
TAG Region
DAG Region
MAG and FA Region
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 111
Table 4.1. Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass spectrum of neat macadamia oil shown in Fig. 4.1.
Observed Mass# m/z
Exact Mass m/z
Compound Class Assigned Sodium Adduct Compound Normalized
% 319.2249 319.2244 FA Hydroxylinoleic acid- C18:2 13.0 333.2405 333.2400 FAME Hydroxynonadecdienoic acid- C19:2 5.4 353.2665 353.2662 MAG Glycerol palmitate 5.7 367.2401 367.2455 MAG Glycerol hydroxypalmitoleate 5.8 379.2840 379.2819 MAG Glycerol oleate 2.6 393.2609 393.2611 MAG Glycerol-hydroxylinoleate 11.7 411.2654 411.2717 MAG Glycerol-dihydroxyoleate 5.4 575.5022 575.5010 FA Dimer Dimer of C17:0 and C18:1* 31.2 603.5310 603.5323 FA Dimer Dimer of C19:0 and C18:1** 49.5 615.4870 615.4959 DAG Glycerol oleate palmitoleate 9.1 617.5053 617.5115 DAG Glycerol oleate palmitate 13.1 631.4998 631.4908 DAG Glycerol hydroxyoleate palmitoleate 11.2 643.5212 643.5272 DAG Glycerol dioleate 13.7 659.5195 659.5221 DAG Glycerol hydroxyoleate oleate 19.5 675.5127 675.5170 DAG Glycerol dihydroxyoleate 16.9 691.5120 691.5119 DAG Glycerol trihydroxyoleate 10.3 823.6764 823.6786 TAG Glycerol tripalmitoleate 6.1 825.6786 825.6943 TAG Glycerol palmitate dipalmitoleate 9.1 851.7098 851.7099 TAG Glycerol oleate dipalmitoleate 29.3 853.7187 853.7256 TAG Glycerol stearate dipalmitoleate 34.4 879.7338 879.7412 TAG Glycerol dioleate palmitoleate 67.8 881.7537 881.7569 TAG Glycerol dioleate palmitate 61.5 907.7650 907.7725 TAG Glycerol trioleate 100.0 # Three known compounds, glycerol oleate (379.2819 m/z), glycerol dioleate (643.5272 m/z), and
glycerol trioleate (907.7725 m/z) are used as internal standards to correct for the mass errors. The observed masses in the table are before applying internal calibration.
* Could be dimer of C18:1 and methyl palmitate ** Could be dimer of C18:1 and methyl oleate
Three distinct regions are observed in the FTICR mass spectrum of macadamia nut
neat oil in Figure 4.1. The peaks in the region m/z 200-450 are assigned to sodiated
adducts of free FAs, MAGs and some FA dimers that form in the gas phase. The
peaks in the region m/z 500-700 are assigned to sodiated adducts of DAGs as well as
to sodiated adducts of several FA dimers; and the peaks in the region m/z 800-1000
are assigned to sodiated adducts of TAGs present in the oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 112
4.2.1- Free Fatty Acids and Monoacylglycerols Region, m/z 150-400
Figure 4.2 illustrates expanded FA region of macadamia oil FTICR mass spectrum.
181.7591
207.1000
221.1153
247.2408
265.2526
279.2312
319.2249
339.2910
353.2617
367.2402
381.2489
393.2609
411.2706
433.2917
175 200 225 250 275 300 325 350 375 400 425 m/z Figure 4.2- Expanded FA region of positive-ion ESI-FTICR mass spectrum of neat macadamia oil.
As mentioned in Section 1.2.7, the neutralising, deodorising and degumming
processes remove the majority of more polar unsaturated free fatty acids (such as
oleic and palmitoleic acids) and other polar compounds from the oil. One might
expect to observe traces of less polar saturated fatty acids such as palmitic acid in the
oil. The detection of free FAs in the neat oil is significant in the context of hydrolytic
rancidity and shows the value of the high-resolution ESI FTICR-MS technique.
Other studies in the literature typically use GC or GC-MS to analyze the FA
components in oils. In these latter cases the oil is first esterified before analysis; a
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 113
step that effectively reduces the free FAs and all of the acylglycerols in the oil to
their corresponding methyl esters.
Furthermore, the very high relative population of acylglycerol molecular ions in the
FTICR cell arising from neat oil, has an ion-suppression (cloud) effect on the peak
intensities of trace level ions in the cell, resulting in the observation of only two free
fatty acids in the neat oil spectrum at m/z 319.2249 (C18:2) and m/z 333.2405
(C19:2). The C18:2 cation is assigned to NaC18H31O3+ and could have been formed
from the hydration of the C18:3 linolenic acid. The C19:2 cation is assigned to
NaC19H34O3+ and could have been produced by the hydration of the C19:3
nonadectrienoic acid.
A number of unusual compounds are observed in the expanded FA region spectrum
in Figure 4.2 giving rise to sodiated adducts that can be assigned to acylglycerols
with acyl substituents with odd number of carbon atoms such as glycerol
pentadecenoate, C18H34O4Na+, m/z 337.2359. Alternatively, this sodiated adduct can
be assigned to dihydroxy oleic acid. When the neat oil was esterified, derivatives of
odd numbered fatty acids were not observed, but the hydroxy oleic acid was
observed instead. So we have assigned them in Table 4.1 to be sodiated hydroxy-
adducts. Additional chemical and tandem mass spectrometry experiments are
required to more precisely identify the molecular structure of these compounds. In
such cases peaks are assigned to hydroxy-species (e.g. hydroxy linoleic acid m/z
319.2249) because an ion of this mass is observed from both the neat oil as well as
from the esterified oil sample indicating that it is a hydroxy but not a peroxyacid
compound.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 114
4.2.2- Diacylglycerol Region, m/z 500-750
Figure 4.3 shows an expanded view of the DAG region of macadamia nut oil
spectrum in Figure 4.1.
575.5022
603.5310
617.5053
631.4998
643.5212
659.5195
675.5127
689.4995
707.5155
560 580 600 620 640 660 680 700 m/z Figure 4.3- Expanded DAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil.
Fatty acids produce dimers in the gas phase by forming a stable hydrogen-bonded
ring.[197] The assignment of two peaks in the expanded DAG region of macadamia oil
spectrum in Figure 4.3 is consistent with the dimerisation reaction of FAs in the gas-
phase. These are m/z 575.5022 which is assigned to a dimer of oleic acid and
heptadecanoic acid and m/z 603.5310 that is assigned to a dimer of oleic acid and
nonadecanoic acid respectively.
Several observed peaks in Figure 4.3 are assigned to DAGs such as m/z 631.4998,
glycerol oleate-palmitoleate with one additional oxygen atom and m/z 659.5195,
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 115
glycerol dioleate with one additional oxygen atom. The additional oxygen atom is
expected to be a hydroxyl group on the hydrocarbon chains of the oleate or
palmitoleate acyl groups. A hydroxyl group on the alkyl chain could form by
addition of a water molecule to a carbon double bond on the FA chain. This
“hydration reaction,” is known to occur in the presence of hydrated acids.[197]
Another possibility is the occurrence of electrochemical reactions in the ESI source
due to the electrical discharges at high voltages.[198,199] These reactions can generate
active intermediates during the electrospray ionisation process.[119,120,200] Further
structural investigation is required to more accurately identify the assigned hydroxy
fatty acid compounds, but this was not performed in this study.
4.2.3- Triacylglycerol Region, m/z 800-1000
As might be expected,[201] Figure 4.1 reveals that TAGs and DAGs are the major
constituents of neat macadamia oil. This observation assumes that the ionisation
efficiencies of each group of chemical compounds such as free FAs, MAGs, DAGs
and TAGs are similar within each group. In positive-ion ESI, the mechanisms of
sodium cation attachment to these molecules is similar in each group. No evidence
has been presented for this assumption in present study.
As FTICR-MS resolves compounds according to their masses only, structural
isomers are not resolved by FTICR-MS. Thus, each mass spectral peak can
potentially represent one compound or a mixture of isomers of that compound. In
Chapter 7 we show that such isomers can be isolated using HPLC and then analysed
using the ESI FTICR mass spectrometer in an offline mode.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 116
Figure 4.4 illustrates an expanded view of the TAG region of macadamia nut oil
spectrum in Figure 4.1. The thin vertical line underneath each peak is produced by
the peak-picking routine in Bruker software.
853.7187
879.7338
907.7650
850 860 870 880 890 900 910 m/z Figure 4.4- Expanded TAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil. The thin vertical line underneath each peak is produced by the peak-picking routine in Bruker software.
The five major peaks in the TAG region of Figure 4.4 are assigned to glycerol-
trioleate (m/z 907.7650), glycerol dioleate palmitate (m/z 881.7425), glycerol
dioleate palmitoleate (m/z 879.7338), glycerol palmitate palmitoleate oleate (m/z
853.7187) and glycerol dipalmitoleate oleate (m/z 851.7098). This result is consistent
with the previous report by Cavaletto where the major FAs associated with
macadamia nut oil are oleic acid 65%, and palmitoleic acid 18% and palmitic acid
7%,[196] and with Maguire where the same major FA components of macadamia oil
are reported as 65%, 17% and 8%respectively.[202] Our GC-MS study discussed in
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 117
Chapter 6 shows the major FA components as oleic 58%, palmitoleic 21% and
palmitic 11% methyl esters. The relative peak areas are rounded off to nearest whole
number for comparison purpose.
CID in tandem with a chromatography stage could provide more structural
information about the FAs present in the acylglycerols of the oil. For example, the
sodiated adduct at m/z 851.7098 in Table 4.1 has been assigned to glycerol oleate
dipalmitoleate; alternatively it could have been assigned to glycerol linoleate
palmitoleate palmitate. CID and chromatography experiments would help to refine
the assignments in such cases, but the CID experiments were not attempted in this
study.
4.3- Positive-ion ESI FTICR-MS of Methanol Extract of Processed Macadamia Oil
The purpose behind extraction of the oil in methanol is to eliminate the spectral
interference of triacylglycerols from the oil and thereby enhance the detection of free
FAs in the oil in mass spectra. The methanol extraction achieves this goal, however,
there are some factors observed which are less desirable and complicate
interpretation of the results.
Figure 4.5 shows the positive-ion FTICR mass spectrum of the methanol extract of
macadamia nut oil. The procedure for methanol extraction of macadamia nut oil is
described in section 2.2.1.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 118
Eight sodiated FAs, three MAG and seven DAG ions assigned to the peaks in this
spectrum are listed in Table 4.2.
277.2114
305.2418
351.2479
379.2776
449.1479
587.4582
615.4831
643.5163
907.7481
300 400 500 600 700 800 900 1000m/z
Figure 4.5- Positive-ion ESI-FTICR mass spectrum of the methanol extract of macadamia nut oil.
A comparison of Table 4.2 (extracted macadamia oil) with Table 4.1 (neat
macadamia oil) shows that methanol extraction process has removed the majority of
the acylglycerols such as TAGs from the extracted sample dramatically and
enhanced the concentration and hence improved the detection of free FAs.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 119
Table 4.2. Assignment of the mass spectral peaks (>2%) in positive-ion ESI-FTICR mass spectrum of the methanol extract of macadamia oil shown in Figure 4.5.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Sodium Adduct Compound Normalized
Intensity % 277.2114 277.2138 FA Palmitoleic acid- C16:1 28.8 279.2285 279.2294 FA Palmitic acid- C16:0 14.5 293.2449 293.2451 FA Heptadecanoic acid- C17:0 2.6 303.2288 303.2294 FA Linoleic acid- C18:2 7.0 305.2418 305.2451 FA Oleic acid- C18:1 47.1 307.2608 307.2607 FA Stearic acid- C18:0 3.9 319.2596 319.2608 FA Nonadecenoic acid- C19:1 10.4 333.2759 333.2764 FA Eicosenoic acid- C20:1 5.0 351.2479 351.2506 MAG Glycerol palmitoleate-C16:1 13.5 375.2499 375.2506 MAG Glycerol linolenate- C18:3 1.0 379.2776 379.2819 MAG Glycerol oleate- C18:1 53.7 449.1479 - - Unknown 23.1 587.4582 587.4646 DAG Glycerol dipalmitoleate- C16:1 30.0 615.4831 615.4959 DAG Glycerol oleate palmitoleate 87.6 617.5134 617.5115 DAG Glycerol oleate palmitate 37.9 633.5049 633.5065 DAG Glycerol hydroxyoleate palmitate 19.6 643.5163 643.5272 DAG Glycerol dioleate 100.0 659.5104 659.5221 DAG Glycerol hydroxyoleate oleate 12.4 675.5121 675.5170 DAG Glycerol dihydroxyoleate 5.7
Figure 4.5 demonstrates the importance of decreasing the ion suppression (cloud)
effect in the FTICR cell by extraction process of the oil, resulting in a significant
decrease in the number of acylglycerol ions in FTICR cell and improvement in the
free FA peak intensities. Eight peaks in Table 4.2 are assigned to free FAs in the
methanol extract of macadamia nut oil, compared to only one in the neat nut oil.
If this extract was to be analysed by GC-MS, all of the acid components would need
to be converted to esters prior to analysis. Consequently, the free FAs and FAMEs (if
present) would not be distinguishable. The alternative approach is to use HPLC
which can separate the ester and acid components. In a further attempt to resolve
such structural isomers, the macadamia oil methanol extract was analyzed by HPLC.
This is discussed later in Chapter 7.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 120
Arachidonic, lauric and myristic acids observed in small amounts (<3%) by previous
workers[196] were detected at trace levels (<1%) in this experiment.
The composition of the free FAs observed in methanol extract of macadamia oil in
Table 4.2 is representative of the free FA composition of macadamia nut oil.
However, it seems significantly different from the composition of FAMEs observed
in esterified macadamia oil in Table 4.4. This shows that the source of the observed
free FAs in the oil is not simple release of the FAs by hydrolysis of acylglycerols in
the oil, but the FAs could have been released selectively via biological routes such as
microorganism enzyme activities as well as possibly the actual processing of the oil.
For example, we compare the relative concentrations of C18:1 and C16:1 in the
FTICR mass spectrum of the methanol extract of the oil and GC mass spectrum of
the esterified oil. This ratio is found to be 47:29 using FTICR-MS in the methanol
extract of the oil, and 60:31 and 56:27 in two distinct GC-MS experiments on the
esterified oil samples. This ratio is reported as 69:20 in literature.[196] The GC
analysis requires an esterification process that releases majority of the FA
substituents from the acylglycerols present in the oil extract as their FAMEs, while
FTICR-MS directly measures the FFA content of the methanol extract of the oil.
This again demonstrates utility of FTICR-MS as a complementary technique in the
analysis of free FAs in such oils.
The mass spectra reveal that the quantity of acylglycerols (mainly TAGs) in the nut
oil (Fig. 4.1) is high compared to the amount of FFAs and the similar compounds
containing additional oxygen atoms in their acyl chains. Since the presence of the
latter compounds in the oil is evidence of oxidation of the glycerols, we take this
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 121
observation as evidence of the relative stability of the oil. The ESI FTICR analysis of
the neat and methanol extract of the macadamia oil highlights this point.
4.4- Positive-ion ESI FTICR-MS of Hydrolysed Processed Macadamia Oil
Figure 4.6 shows the positive-ion FTICR mass spectrum of hydrolysed macadamia
nut oil. Nineteen sodiated FAs, two MAGs, two DAGs, two FA dimers and one TAG
ions are assigned to peaks in this spectrum and are listed in Table 4.3. The procedure
for the hydrolysis experiment is described in Section 2.2.2.
The point behind performing the hydrolysis reaction on macadamia oil is to provide
more evidence to confirm the assigned peaks in the neat macadamia oil spectrum
(Figure 4.1) and esterified oil spectrum (Figure 4.7) but more importantly to
investigate the fatty acid composition of acylglycerols present in macadamia oil.
277.2140
305.2453
351.2521
617.5135643.5297
855.7443881.7609
300 400 500 600 700 800 900 m/z Figure 4.6- Positive-ion FTICR mass spectrum of hydrolysed macadamia nut oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 122
Table 4.3. Assignment of the major mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass spectrum of hydrolysed macadamia nut oil shown in Figure 4.6.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Sodium Adduct Compound Normalized
Intensity % 277.2140 277.2138 FA Palmitoleic acid- C16:1 28.2 279.2297 279.2295 FA Palmitic acid C16:0 8.5 301.2151 301.2138 FA Linolenic acid C18:3 2.7 303.2297 303.2295 FA Linoleic acid C18:2 12.9 305.2453 305.2451 FA Oleic acid C18:1 100 307.2593 307.2608 FA Stearic acid C18:0 2.6 319.2254 319.2244 FA Hydroxylinoleic acid C18:2 7.5 319.2615 319.2608 FA Nonadecenoic acid C19:1 5.2 321.2389 321.2400 FA Hydroxyoleic acid C18:1 3.3 333.2774 333.2764 FA Eicosenoic acid C20:1 3.7 337.2342 337.2349 FA Dihydroxyoleic acid C18:1 3.2 351.2521 351.2506 MAG Glycerol palmitoleate 3.2 353.2679 353.2662 MAG Glycerol palmitate 2.3 379.2828 379.2819 MAG Glycerol oleate 1.8 617.5135 617.5115 DAG Glycerol oleate palmitate 11.4 643.5297 643.5272 DAG Glycerol dioleate 8.3 855.7443 855.7412 TAG Glycerol dipalmitate oleate 41.0 881.7609 881.7569 TAG Glycerol dioleate palmitate 44.8 897.7499 897.7518 TAG Glycerol dioleate hydroxy oleate 34.5
A comparison of Table 4.3 with Table 4.4 shows consistency of the assignments. For
example, methyl palmitoleate in Table 4.4 that shows 33% intensity and is consistent
with palmitoleic acid in Table 4.3 which shows 28% relative intensity. The majority
of the FAMEs in Table 4.4 show consistent intensities with corresponding FAs in
Table 4.3.
Figure 4.6 also shows that ESI FTICR-MS is capable of examining whether the
hydrolysis reaction is complete, which in this case demonstrates the fact that the
reaction is partially incomplete as some DAGs and TAGs are observed in the
hydrolysate.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 123
4.5- Positive-ion ESI FTICR-MS of Esterified Processed Macadamia Oil Figure 4.7 shows the positive-ion FTICR mass spectrum of esterified macadamia nut
oil with nine sodiated FA methyl esters, five MAG and three DAG ions assigned to
the peaks in this spectrum that are listed in Table 4.2. The procedure for
esterification is described in section 2.2.3 and is similar to the literature procedures
used for esterification of animal fats and plant oils.[203]
Table 4.4 lists the assignment of the mass spectral peaks in the positive-ion ESI
FTICR mass spectrum of esterified macadamia oil shown in Figure 4.7.
291.2290
319.2599
351.2507
395.2758
617.5111643.5279
200 300 400 500 600 700 800 900 1000 m/z
Figure 4.7- Positive-ion ESI-FTICR mass spectrum of esterified macadamia oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 124
Table 4.4- Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass spectrum of esterified macadamia nut oil shown in Figure 4.7.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Sodium Adduct Compound Ratio of the
Intensity % 291.2290 291.2295 FAME Methyl palmitoleate- C16:1 32.7 293.2449 293.2451 FAME Methyl palmitate- C16:0 4.8 305.2443 305.2451 FAME Methyl heptadecenoate- C17:1 5.5 307.2606 307.2608 FAME Methyl heptadecanoate- C17:0 2.6 317.2450 317.2451 FAME Methyl linoleate- C18:2 10.8 319.2599 319.2608 FAME Methyl oleate- C18:1 100 333.2391 333.2400 FAME Methyl hydroxylinoleate- C18:2 7.2 337.2348 337.2349 FAME Methyl dihydroxyheptadecenoate 5.6 347.2922 347.2920 FAME Methyl eicosenoate C20:1 5.8 351.2507 351.2506 MAG Glycerol palmitoleate 9.0 367.2456 367.2455 MAG Glycerol hydroxypalmitoleate 2.3 379.2797 379.2819 MAG Glycerol oleate 19.5 393.2593 393.2611 MAG Glycerol hydroxylinoleate 6.2 395.2758 395.2768 MAG Glycerol hydroxyoleate 20.8 615.4940 615.4959 DAG Glycerol oleate palmitoleate 2.3 617.5111 617.5115 DAG Glycerol oleate palmitate 2.4 643.5279 643.5272 DAG Glycerol dioleate 4.1
As a result of the esterification process, most of the DAG and all of the TAG
molecules in the nut oil are converted to their corresponding methyl esters. The
esterification reaction appears incomplete because small amounts (<2%) of sodiated
MAG and DAG ions can be assigned to peaks in Figure 4.7 (e.g., glycerol oleate m/z
379.2797 and glycerol-dioleate m/z 643.5279).
Figure 4.8 shows an expanded FA region of the FTICR mass spectrum of esterified
neat macadamia oil.
The assignment of the base peak in Figure 4.8 at m/z 319.2599 to the sodiated
methyl-oleate adduct confirms the high percentage of oleic acid component of the
DAGs and the TAGs in macadamia nut oil.[196]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 125
277.1132
291.2290
297.2783 305.2443
319.2599
333.2391351.2507
365.2385
379.2797 395.2758
280 300 320 340 360 380 400 m/z
Figure 4.8- Expanded FA and FAME region of the positive-ion ESI-FTICR mass spectrum of esterified macadamia nut oil shown in Figure 4.7.
Other methyl ester ions assigned in this spectrum are eicosenoic (m/z 347.2922),
heptadecanoic (m/z 307.2606), heptadecenoic (m/z 305.2443), linoleic (m/z 317.2450),
palmitic (m/z 293.2449) and palmitoleic (m/z 291.2290) methyl esters. Myristic (m/z
265.2138) and stearic (m/z 321.2764) acid methyl esters were detected in trace level
(<1%) in the esterified macadamia oil. Arachidonic (m/z 341.2451) and lauric (m/z
237.1825) acid methyl esters observed by previous workers in small amounts (<3%)
are also detected at trace levels (<1%) in this experiment.
The relative concentrations of methyl oleate to methyl palmitoleate in the esterified
macadamia nut oil are measured 62:20 using FTICR mass spectrometry, and in the
GC-MS experiments (Chapter 6) the ratio is found as 58:22, whereas in the literature
this ratio is reported as 65:18 using GC-MS technique.[196] Given that the oils used in
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 126
these experiments were not identical, the results show a good consistency to within a
~10% interval.
As is expected from the assignments listed in Table 4.1, several odd-numbered
FAMEs (C17:0, C17:1 and C15:1) and one FAME containing an extra oxygen-
bearing functional group in the alkyl chain (methyl hydroxylinoleate, m/z 333.2391)
are assigned and listed in Table 4.4. The presence of hydroxy and methoxy
derivatives of FAs in oils has been reported in the literature previously[177,204-207] and
since any peroxy-, or epoxy-acid components present in the original nut oil would
have been destroyed by the esterification step, we have assigned this latter ion to the
hydroxylinoleate. No further work is undertaken in this thesis to identify the
chemical structure of the ions listed in Table 4.4 as this work was beyond the scope
of the present study.
The results of Table 4.4 which show the FA components of the esterified neat oil,
best correlate with the literature GC-MS results of Cavaletto[196] because they should
contain all the methyl esters produced from the esterification of the free FAs, MAGs,
DAGs and TAGs in the nut oil. In general the literature and present results correlate
well with only relatively small differences observed for the relative concentrations of
the various FA profiles. The principal components are oleic acid at 62% (cf.
65%[196]) and palmitoleic at 20% (cf. 18%[196]). Arachidonic and myristic acids are
observed by Cavaletto but they were observed at trace levels (<1%) in present work.
Eicosenoic and stearic acids were not detected in our GC-MS experiments but were
observed in the ESI FTICR-MS experiments as in the GC-MS experiments by
Cavaletto.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 127
High resolution of FTICR mass spectrometry allows us to resolve peaks with very
close masses. As an example, Figure 4.9 illustrates four peaks in the m/z 279.12 to
m/z 279.23 region resolved in the FTICR mass spectrum of esterified macadamia oil.
279.1924
279.1205
279.1591
279.2294
C14H24O4Na+
C13H20O5Na+
C15H28O3Na+
C16H32O2Na+
279.08 279.10 279.12 279.14 279.16 279.18 279.20 279.22 279.24 279.26 m/z
Figure 4.9- Positive-ion FTICR mass spectrum of esterified macadamia oil in m/z 279.12 to m/z 279.23 region. Peaks 0.03 Da apart are resolved.
Table 4.4 includes a number of compounds that are observed in ESI FTICR mass
spectrum of esterified macadamia oil but are not observed in the GC-MS analysis of
the esterified macadamia oil sample including MAG molecules such as glycerol
palmitoleate and glycerol oleate and DAG molecules such as glycerol oleate
palmitate and glycerol dioleate.
In addition, two hydroxy FAMEs and three hydroxy MAGs are assigned in Table 4.4
including methyl hydroxylinoleate, methyl dihydroxyheptadecenoate, glycerol
hydroxypalmitoleate, glycerol hydroxylinoleate and glycerol hydroxyoleate. Neither
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 128
of these compounds are observed in our GC-MS analysis of macadamia oil samples
or are reported in the published GC-MS studies on plant oils.
4.6- Positive-ion ESI FTICR-MS of Esterified Methanol Extract of Processed Macadamia Oil
Figure 4.10 shows the positive-ion FTICR mass spectrum of the esterified methanol
extract of the macadamia nut oil with twelve sodiated FA methyl esters and two
mono-acylglycerol ions assigned to peaks in this spectrum listed in Table 4.5. The
procedure for esterification[67] is described in Section 2.2.3. Performing the
esterification reaction on methanol extract of the oil is to compare the product
analysis using both GC-MS and FTICR-MS.
255.1568
291.2295
319.2597
337.2729
395.2756
445.3122
471.2924504.3099
532.3405
587.5014615.5254
250 300 350 400 450 500 550 600 650 m/z
Figure 4.10- Positive-ion FTICR mass spectrum of esterified methanol extract of macadamia nut oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 129
Table 4.5. Assignment of the mass spectral peaks (>1%) in the positive-ion ESI-FTICR mass spectrum of the esterified methanol extract of processed macadamia oil shown in Fig. 4.10.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Sodium Adduct Compound*
Normalized Intensity
% 291.2295 291.2295 FAME Methyl palmitoleate- C16:1 23.7 293.2448 293.2451 FAME Methyl palmitate- C16:0 2.2 305.2447 305.2451 FAME Methyl heptadecenoate- C17:1 1.4 307.2610 307.2608 FAME Methyl heptadecanoate- C17:0 1.0 315.2288 315.2294 FAME Methyl linolenate- C18:3 4.8 317.2448 317.2451 FAME Methyl linoleate- C18:2 22.1 319.2597 319.2608 FAME Methyl oleate- C18:1 100.0 321.2770 321.2764 FAME Methyl stearate- C18:0 1.9 335.2558 335.2557 FAME Methyl hydroxyoleate- C18:1 6.6 337.2729 337.2713 FAME Methyl hydroxy stearate- C18:0 11.4 347.2922 347.2920 FAME Methyl eicosenoate- C20:1 3.7 363.2504 363.2505 FAME## Methyl dihydroxy nonadecdienoate- C19:2 3.6 381.2608 381.2611 MAG Glycerol hydroxy heptadecenoate 9.3 395.2756 395.2768 MAG Glycerol hydroxyoleate 75.5 445.3122 445.3136 MAG Glycerol dihydroxy stearate + methanol 55.5
* The hydroxy-compounds alternately may be assigned to peroxy substituents. See text for further discussion. ## Could be MAG, glycerol heptadecdienoate.
Table 4.5 lists the assignments of the mass spectral peaks in the FTICR mass
spectrum of esterified methanol extract of macadamia oil.
Twelve FAME sodium adducts are assigned to the peaks in the esterified methanol
extract of macadamia oil spectrum in Table 4.5. Comparison of Table 4.5 (esterified
methanol extract of macadamia oil) and Table 4.2 (methanol extract of macadamia
oil) shows a higher relative intensity of oleic FAME in Table 4.5. This is consistent
with observing an intense peak for glycerol dioleate in Table 4.2 that will release
more oleate FAME during esterification process, resulting in an increase in the
relative intensity of methyl oleate in Table 4.5.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 130
Other FA sodium adducts assigned in the methanol extract of the oil in Table 4.2
such as heptadecenoic acid and eicosenoic acid also appear in the assignments of the
esterified methanol extract of the oil as FAME sodium adducts in Table 4.5.
4.7- A Comparison of the Fatty Acids Observed in the Positive-ion ESI FTICR Mass Spectra of the Neat, Methanol Extract, Hydrolysed, Esterified and Esterified Methanol Extract of Processed Macadamia oil
In this section, a summary of the occurrence of the common fatty acids (i.e.
unsubstituted), either as free fatty acids or as substituents on the acylglycerols, in the
processed macadamia oil is discussed.
Figure 4.11 shows a comparison of the fatty acid regions (m/z 175-450) positive-ion
ESI FTICR mass spectra of the (a) neat, (b) methanol extract, (c) hydrolysed, and (d)
esterified processed macadamia oil.
Figure 4.12 shows a graphical comparison of the unsubstituted FA or FA derivative
adducts assigned for the methanol extract (Table 4.2), the hydrolysed (Table 4.3), the
esterified (Table 4.4) and the esterified methanol extract (Table 4.5) of the processed
macadamia nut oil. For this figure, the FAs that show evidence of hydration, e.g.
NaC18H35O3+ are not included. Unlike the intensity values in the tables in this
chapter, the peak intensities in Figure 4.12 have been normalised to 100% for the FA
acid sodium adducts displayed. Table 4.6 lists the percentages of the fatty acid
components used in Figure 4.12.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 131
As discussed in Section 4.2, there are no (or trace levels only) unsubstituted free FA
adducts observed in the positive-ion ESI FTICR mass spectrum of the neat processed
macadamia oil. Free FAs are indeed present in the neat oil according to the negative-
ion results presented next in Chapter 5, but these are not observed as adducts in
Figure 4.11(a) presumably because of charge suppression of the minor components
by the large number of TAG ions observed in Figure 4.1. Also, when these
experiments were undertaken no attempt was made to dope the sample with
additional sodium cations. Since the oil is made up of >98% TAGs, it is also possible
the free FAs do not compete effectively with the larger number of TAG molecules
for the adventitious sodium cations available present in the oil to form the adduct
ions.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 132
181.7591
265.2526
319.2249339.2910
353.2617
393.2609
411.2706
m/z
277.2114
305.2418
319.2588351.2479
379.2776
395.2569413.2616
m/z
231.1143
277.2140
305.2453
319.2217 351.2521 413.2671
m/z
277.1132
291.2290
319.2599
351.2507379.2797
395.2758
413.2674175 200 225 250 275 300 325 350 375 400 425 m/z
(a)
(b)
(c)
(d)
Figure 4.11- A comparison of the ESI FTICR mass spectra of the FA region of (a) neat macadamia oil, (b) methanol extract of macadamia oil, (c) hydrolysed macadamia oil and (d) esterified macadamia oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 133
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C19:1
C20:0
C20:1
C20:4
0
5
10
15
20
25
30
35
40
45
50
55
60
65
A Comparison of the Fatty Acid Components Observed in Positive-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil
Peak
Rel
ativ
e In
tens
ity %
Fatty Acid
+ESI-MS Extract+ESI-MS Esterified Oil+ESI-MS Esterified Extract+ESI-MS Hydrolysed Oil
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C19:1
C20:0
C20:1
C20:4
0
5
10
15
20
25
30
35
40
45
50
55
60
65
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C19:1
C20:0
C20:1
C20:4
0
5
10
15
20
25
30
35
40
45
50
55
60
65
A Comparison of the Fatty Acid Components Observed in Positive-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil
Peak
Rel
ativ
e In
tens
ity %
Fatty Acid
+ESI-MS Extract+ESI-MS Esterified Oil+ESI-MS Esterified Extract+ESI-MS Hydrolysed Oil
+ESI-MS Extract+ESI-MS Esterified Oil+ESI-MS Esterified Extract+ESI-MS Hydrolysed Oil
Figure 4.12- A graphical comparison of the unsubstituted FA anions observed in the positive-ion FTICR mass spectra of the neat (Table 4.1), the methanol extract (Table 4.2), hydrolysed (Table 4.3) and the esterified (Table 4.4) processed macadamia nut oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 134
Table 4.6- A comparison of the fatty acid components observed in the positive-ion ESI-FT-ICR mass spectrometry experiments.
Fatty Acid Component
ESI FTICR-MS Neat oil
Positive-ion
ESI FTICR-MS Methanol Extract
Positive-ion
ESI FTICR-MS Esterified oil Positive-ion
ESI FTICR-MS Esterified Extract
Positive-ion
ESI FTICR-MS Hydrolysed Oil
Positive-ion
Literature GC-MS
Fatty Acids/ % C14:0 -* - - - - - C14:1 - - - - - - C15:0 - - - - - - C15:1 - - - - - - C16:0 - 12.2 3.0 1.4 5.2 7.4 C16:1 - 24.1 20.2 14.7 17.2 18.4 C17:0 - 2.2 1.6 0.6 - - C17:1 - - 3.4 0.9 - - C18:0 - 3.3 - 1.2 1.6 2.8 C18:1 - 39.5 61.7 62.2 61.1 64.9 C18:2 - 5.9 6.7 13.7 7.9 1.5 C18:3 - - - 3.0 1.6 - C19:1 - 8.7 - - 3.2 - C20:0 - - - - - - C20:1 - 4.2 3.6 2.3 2.3 2.3 C20:4 - - - - - 1.9
* Less than the limit of detection. Only fatty acid components are used in calculations and comparisons; fatty acids containing additional oxygen atoms are not listed. Peak intensities % is used for FTICR-MS and peak area % is used for literature Cavaletto[196] GC-MS results.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 135
Figure 4.11(b), the positive-ion spectrum of the methanol extract of the oil, shows a
dramatic improvement in S/N ratio over that of the neat oil for the free FAs as a
consequence of the removal of the majority of the TAG molecules in the methanol
extraction process. This extraction effectively concentrates the minor component free
FAs in the sample and effectively removes the ion suppression created by the larger
number of TAG ions.
The ratio of the relative intensities of the peaks assigned to palmitoleic/oleic acids
(m/z 277.2114 and 305.2418) in the methanol extract of the oil is approximately
0.6:1.0, while this ratio in the hydrolysed oil and esterified oil spectra, 4.11(b) and
4.11(c) respectively, appears to be approximately 0.25:1.0. The major source of free
FAs in hydrolysate and FAMEs in esterified oil samples are the TAG molecules and
to a lesser extent the MAGs and DAGs. These FAs originating from the
acylglycerides are commonly what are quoted and indeed measured in the more
standard GC-MS analyses of plant oils as the fatty acid composition of the oil.
However, in the case of the spectrum 4.11(b), the methanol extract of the oil, the
source of the free FAs are the biological reactions in the macadamia nuts and any
remaining active enzymes in the oil after processing, the effect of atmospheric
oxygen and auto-oxidation as well as the various oil temperatures used in the
processing and indeed the processing procedure itself. All of these factors may result
in the difference between the free FA ratio and the FA ratio measured using more
conventional method for the oil. It is the presence in the macadamia oil of these
“free” fatty acids, as distinct from the “bound” fatty acids in the acylglycerides that
contributes to the overall acidity and in some cases “character” of the oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 136
Figure 4.12 and Table 4.6 summarizes several interesting results from the positive-
ion ESI FTICR MS experiments discussed previously in this chapter. If one can
directly correlate the cation concentrations with those of the acids in the oil then it is
apparent that the C18:1 (oleic) and C16:1 (palmitoleic) with a smaller amount of
C18:2 (linoleic) acids are the most common acids both in the acylglycerides at
around 62%, 17% and 9% respectively. The free FA concentrations in the methanol
extract show a similar trend. In the latter case though there are increased
concentrations of the C19:1 (9%) and C16:0 (palmitic, 12%) observed. Palmitic acid
is observed in the hydrolysed and esterified oils but at a much lower concentration of
around 3%.
Interestingly we might expect that the FA concentrations deduced from the methanol
extract and the esterified methanol extract of the oil to show a similar FA profile
since the esterification reaction should essentially transform all of the FAs in the oil
extract into FA esters. The observation that they are indeed different and that the
latter more closely follows the trend of the hydrolysed neat oil and the esterified neat
oil indicates that derivatisation of the oil may lead to changes in the perceived oil
profile. The changes could occur because of the acylglycerides that have been
hydrated or the ones soluble in methanol thus being extracted along with the free
fatty acids and then undergoing hydrolysis in the transesterification reaction. It may
also to a lesser extent reflect the different sodium affinities of an ester versus a fatty
acid in the ESI spray process. The presence of DAGs and to lesser extent TAGs in
Figure 4.2 supports the former hypothesis.
Figure 4.12 highlights the presence of the other minor fatty acids present in the oil
such as the C20:1 and C20:4 as well as some of the odd numbered fatty acids C17:0
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 137
and C17:1. It is unexpected that the C18:3 (linolenic acid) is not observed in the
methanol extract and the esterified oil but is detected in the esterified extract and the
hydrolysed oil.
One interesting result which will be discussed again in Chapter 6 is the interesting
correlation between the observed profile of the major FA components in the oil and
that observed previously in GC-MS experiment by Cavaletto in 1980.[196] In their
results, they report concentrations for the major FA components in macadamia oil
which closely matches our results for the analysis of the acylglyceride FAs in our
processed macadamia oil. We observe 62%: 17%: 3% for the FAs C18:1, C16:1:
C16:0 acids compared to Cavaletto’s result of 65%: 18%: 7% respectively.
In brief, Figure 4.11 demonstrates that:
- Free FAs in the neat oil are not observed due to their low relative concentration
and high cloud effect of TAG positive ions in the FTICR cell that causes the ion-
suppression of the minor peaks of free FAs. To minimize ion suppression and to
improve the signal to noise ratio of minor constituents of the oil, solvent
extraction and chromatographic separations are carried out. Spectrum (b) in
Figure 4.11 shows a dramatic improvement in signal intensity of free FAs in the
methanol extract of the oil due to the removal of the majority of the TAG
molecules in the extraction process.
- The ratio of the relative intensities of the peaks assigned to palmitoleic/oleic
acids (m/z 277.2114 and 305.2418) in the methanol extract of the oil is
approximately 0.6:1.0, while this ratio in the hydrolysed oil and esterified oil
spectra, (b) and (c) respectively, appears to be approximately 0.25:1.0. The
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 138
major source of free FAs in the hydrolysed oil and FAMEs in esterified oil
are TAG molecules that comprise about 98% of the macadamia oil. However,
in the case of the spectrum (b) of the methanol extract of the oil, the source of
free FAs is the biological reactions, effect of atmospheric oxygen, light and
ambient temperature which result in a different ratio of relative intensities of
the assigned peaks.
- The signal to noise ratio of the peaks in the FA region of spectrum (a) is
significantly lower than that of the other three spectra in Figure 4.11. In
addition, peaks assigned to two major FAs oleic and palmitoleic acids are
observed in the methanol extract spectrum (b) but they are not observed in the
neat oil spectrum (a) due to the ion suppression effect of the TAG ions in the
FTICR cell. As a result, the observed peaks in the FA region of the neat oil
spectrum (a) basically arise from the background peaks due to the high signal
amplification applied by the Bruker software in FA region of spectrum (a).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 139
Chapter 5
5. Negative-ion ESI FTICR-MS of Processed Macadamia Oil
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 140
5.1- Introduction
In this chapter the results of the negative-ion ESI FTICR mass spectrometry
experiments on the processed macadamia nut oil samples are presented and
discussed. In particular the neat processed macadamia nut oil, the methanol extract of
the oil and hydrolysed processed macadamia nut oil are examined using this
technique.
In the negative-ion electrospray ionisation of the macadamia nut oil samples, the
singly-charged negative ions are formed by the deprotonation of acidic species such
as fatty acids and alcohols by transferring a proton to the solvent molecules; in this
case, methanol. MAGs and DAGs generate negative ions in the ESI source by
deprotonation of the unsubstituted hydroxyl group on glycerol.
Unsubstituted TAG molecules do not produce negative ions in ESI mass
spectrometry due to the fact that they do not possess an ionisable proton. However,
numerous peaks are observed in the TAG region of the negative-ion FTICR mass
spectra of the neat macadamia oil samples and these peaks are assigned to TAG
molecules containing additional oxygen bearing functional groups such as hydroxyl
groups or hydroperoxy group on the acyl substituents. These ions are observed
because they are produced from substituted TAG molecules that contain a detachable
proton.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 141
5.2- Negative-ion ESI FTICR Mass Spectra of Neat Processed Macadamia Oil
5.2.1- Introduction
In this section the results of negative-ion FTICR-MS analysis of neat processed
macadamia nut oil are presented and discussed. Peaks are assigned in three regions
of the spectra including free fatty acids and MAGs region (m/z 150-400), DAGs
region (m/z 500-750) and TAGs region (m/z 800-1000).
Figure 5.1 shows negative-ion ESI FTICR mass spectrum of the neat processed
macadamia nut oil. In this chapter only the peaks with intensities greater than 2% of
the base peak in each FTICR spectrum are discussed, unless otherwise stated.
The spectra presented in this chapter are also contained in digital form in the attached
DVD Appendix.
More than 120 peaks are distinguished in three mass regions in the FTICR mass
spectrum of neat macadamia oil in Figure 5.1. The region m/z 150-400 contains
peaks associated with anions of free FAs and MAGs. The region m/z 500-750
contains peaks assigned to the anions of FA dimers, DAGs and also DAGs with
additional oxygen bearing functional groups. The region m/z 800-1000 contains
peaks assigned to the anions of TAGs with additional oxygen bearing functional
groups such as hydroxyl functional group.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 142
157.1232
187.0977
227.2016
255.2327
281.2486
311.1687
537.4880 627.4762655.5031
682.5263 875.7683901.7849
931.7126
200 300 400 500 600 700 800 900 1000m/z
847.7396
212.0753
339.1999
325.1841
Figure 5.1- Negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil.
5.2.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400
Figure 5.2 shows an expanded fatty acid region (m/z 150-400) of the negative-ion
ESI FTICR mass spectrum of neat processed macadamia oil shown in Figure 5.1.
Table 5.1 lists the assignment of the associated mass spectral peaks in Figure 5.2.
The majority of the anions produced from the neat processed macadamia oil in this
region are associated with FAs and dicarboxylic fatty acids. The base peak in Figure
5.2 is at m/z 255.2327 and is assigned to C16H31O2¯ which is expected to be the
palmitate anion, C16:0 (<1 ppm). Another nearby peak at m/z 253.2173 (45%) is
FA Region
DAG Region TAG Region
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 143
assigned to C16H29O2¯ which is expected to be the palmitoleate anion, C16:1 (<0.1
ppm).
157.1232
173.0819187.0977
199.1702
227.2016241.2172
255.2327
269.2485
281.2486
297.1532
311.1687325.1841
339.1999
150 175 200 225 250 275 300 325 350 375 400m/z
212.0753
Figure 5.2- Fatty acid region of the negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil in Figure 5.1, m/z 150-400.
A related series of C18 anions is observed at m/z 283.2642 (17%), 281.2486 (74%)
and 279.2329 (6%). These are assigned to C18H35O2¯ (0.1 ppm), C18H33O2¯ (0.2
ppm) and C18H31O2¯ (<0.1 ppm) respectively. Based on the GC and HPLC results in
Chapters 6 and 7 respectively, we expect these anions to be stearate (C18:0), oleate
(C18:1) and linoleate (C18:2) respectively.
The peaks at nominal masses m/z 311, 325 and 339 might be considered to arise
from the next members of the homologous series C16 : C18 : C20 : C22 : C24,
however high resolution mass spectrometry throws some doubt on this assignment.
C20H39O2¯ has a mass of m/z 311.2956 that is 406 ppm above the observed mass at
m/z 311.1687. Similarly C21H41O2¯ has a mass of m/z 325.3112 that is 399 ppm
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 144
above the observed mass at 325.1841 and C22H43O2¯ has a mass of m/z 339.3269
which is 373 ppm above the observed mass at 339.1999. The existence of sulphur in
plant oils is reported by Wu et al. using the FTICR-MS technique,[106] therefore, we
assign these peaks to C17H27O3S¯ (<0.1 ppm), C18H29O3S¯ (0.7 ppm) and
C19H31O2S¯ (0.2 ppm) respectively.
Table 5.1- Assignment of the mass spectral peaks (>2%) in the expanded fatty acid region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.2.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Anion Normalized
% 157.1232 157.1234 FA C9H17O2‾, C9:0 9.5 171.1388 171.1391 FA C10H19O2‾, C10:0 2.3 173.0819 173.0819 Di-FA* C8H13O4‾ 2.5 187.0977 187.0976 Di-FA* C9H15O4‾ 4.8 199.1702 199.1704 FA C12H23O2‾, C12:0 2.9 212.0753 Unknown - - 31.3 213.1860 213.1860 FA C13H25O2‾, C13:0 2.0 225.1858 225.1860 FA C14H25O2‾, C14:1 2.9 227.2016 227.2017 FA C14H27O2‾, C14:0 16.4 239.2018 239.2017 FA C15H27O2‾, C15:1 2.9 241.2172 241.2173 FA C15H29O2‾, C15:0 13.1 253.2173 253.2173 FA C16H29O2‾, C16:1 45.4 255.2327 255.2330 FA C16H31O2‾, C16:0 100.0 267.2329 267.2330 FA C17H31O2‾, C17:1 4.0 269.2125 269.2122 FA C16H29O3‾ 2.3 269.2485 269.2486 FA C17H33O2‾, C17:0 4.7 279.2329 279.2330 FA C18H31O2‾, C18:2 6.0 281.2486 281.2486 FA C18H33O2‾, C18:1 74.3 283.2642 283.2643 FA C18H35O2‾, C18:0 17.4 295.2277 295.2279 FA C18H31O3‾ 4.3 297.2431 297.2435 FA C18H33O3‾ 8.0 311.1687 311.1686 S-FA# C17H27O3S‾ 36.3 313.2388 313.2384 FA C18H33O4‾ 3.1 323.1680 323.1711 FA C14H27O8‾ 3.3 325.1841 325.1843 S-FA# C18H29O3S‾ 32.7 337.1836 337.1868 FA C15H29O8‾ 2.2 339.1999 339.1999 S-FA# C19H31O3S‾ 26.6
* Di-FA: Dicarboxylic fatty acid # S-FA: Sulphur containing fatty acid
Table 5.1 indicates that peaks observed at m/z 227.2016, 241.2172, 267.2329 and
269.2485 in Figure 5.2 are assigned to C14H27O2¯ (0.3 ppm), C15H29O2¯ (0.6 ppm),
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 145
C17H31O2¯ (0.1 ppm) and C17H33O2¯ (0.5 ppm) respectively. These fatty acids are not
observed in positive-ion FTICR mass spectrum of macadamia oil in Figure 4.1.
Dicarboxylic acids can form by oxidative cleavage of the double bonds of olefinic
hydrocarbons.[208] Two dicarboxylic acids are assigned in Table 5.1. The peak at m/z
173.0819 is assigned to C8H13O4¯ (<0.1 ppm) and the peak at m/z 187.0976 is
assigned to C9H15O4¯ (0.7 ppm).
Dicarboxylic acid species might be expected to form dianions (such as ¯OOC–R–
COO¯) because of the possible loss of two acidic protons from the two carboxylic
acid groups. However, no peaks were observed in Figure 5.2 that could be assigned
to the double-charge anions of dicarboxylic acids.
5.2.3- Diacylglycerol Region, m/z 500-750
Figure 5.3 shows an expanded view of m/z 500-750 region of the negative-ion
FTICR mass spectrum of neat processed macadamia oil in Figure 5.1. Table 5.2 lists
the assignment of the associated mass spectral peaks observed in Figure 5.3.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 146
537.4880
563.5040
627.4762
655.5031
682.5263
500 525 550 575 600 625 650 675 700 725 750m/z
511.4740
Figure 5.3- DAG region of the negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil in Figure 5.1, m/z 500-750.
Table 5.2- Assignment of the mass spectral peaks (>0.2% of the base peak in Figure 5.1) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of processed macadamia oil shown in Figure 5.3.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Anion Normalized#
% 509.4566 509.4575 FA dimer C32H61O4‾ 1.8 511.4740 511.4732 FA dimer C32H63O4‾ 1.9 535.4734 535.4732 FA dimer C34H63O4‾ 1.9 537.4880 537.4888 FA dimer C34H65O4‾ 3.7 563.5040 563.5045 FA dimer C36H67O4‾ 2.9 565.5201 565.5201 FA dimer C36H69O4‾ 1.5 627.4762 627.4630 DAG C39H63O6‾ 3.6 629.4889 629.4787 DAG C39H65O6‾ 1.8 637.5016 637.4837 DAG C41H65O5‾ 1.4 655.5031 655.4943 DAG C41H67O6‾ 6.1 657.5103 657.5100 DAG C41H69O6‾ 2.1 665.5378 665.5362 DAG C40H73O7‾ 2.0 682.5263 682.5263 DAG C39H72O8N‾ 7.7 699.4532 699.4630 DAG C45H63O6‾ 0.2 701.4524 701.4787 DAG C45H65O6‾ 0.2
# Peak intensities normalized vs. base peak (palmitate anion, C16H31O2¯) in Figure 5.1.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 147
5.2.3.1- Free Fatty Acid Dimers
Spectral complications in the ESI mass spectrometry analysis of mixtures of polar
compounds often arise from the formation of dimer ions (clusters) in the electrospray
process. During the spray process of the macadamia oil solutions that contain free
fatty acids, two fatty acids molecules can form a dimer in the gas phase via hydrogen
bonding around the hydrogen atom of one of the fatty acid molecules. In positive-ion
mode, fatty acid dimers with the Na+ attached are observed. In negative-ion mode,
[FA2-H+]¯ dimers are observed. These anions could form from loss of a proton from
one of the fatty acids in the FA2 dimer molecule. Alternatively, the anion could form
from the association of a [FA-H+]¯ with a FA.
In Table 5.2 six such clusters are assigned. The peak at m/z 509.4566 is assigned to
C32H61O4¯ (1.8 ppm), the peak at 511.4740 is assigned to C32H63O4¯ (1.6 ppm), the
peak at 535.4734 is assigned to C34H63O4¯ (0.4 ppm), the peak at 537.4880 is
assigned to C34H65O4¯ (1.6 ppm), the peak at m/z 563.5040 is assigned to C36H67O4¯
(0.9 ppm) and the peak at 565.5201 is assigned to C36H69O4¯ (4.8 ppm).
The peak observed at m/z 509.4566 in Figure 5.3 can be assigned to the anion cluster
formed by the combination of palmitoleic acid (C16H30O2) and palmitate anion
(C16H31O2¯) to give C32H61O4¯. Alternatively, a cluster anion of palmitic acid
(C16H32O2) and palmitoleate anion (C16H29O2¯) will produce the same anion.
The peak observed at m/z 511.4740 in Figure 5.3 can be assigned to the cluster anion
of palmitic acid (C16H32O2) and palmitate anion (C16H31O2¯) to give C32H63O4¯.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 148
The peak observed at 535.4734 can be assigned to the anion cluster of oleic acid
(C18H34O2) and palmitoleate anion (C16H29O2¯) to give C34H63O4¯. Alternatively, the
combination of oleate anion (C18H33O2¯) and palmitoleic acid (C16H30O2) will
produce the same anion.
To assign chemical structures to these ions would require tandem mass spectrometry,
i.e. CID studies; however, we observe in Table 5.2 that the two most abundant free
fatty acids in the processed macadamia oil are palmitic (C16H32O2) and oleic
(C18H34O2) acids. An anion cluster formed from these two acids (C34H65O4¯) would
result in the peak observed at m/z 537.4880.
Figure 5.4 shows a comparison of the (a) experimental and (b) simulated mass
spectra of the C34H65O4¯ anion. The simulated spectrum was generated using the
Bruker software. The peak at m/z 537.4880 is chosen due to the fact that it shows the
highest intensity among the dimer peaks assigned in Table 5.2.
537.4880
537.4888
538.4923
539.4954
537 538 539 540 541 542 m/z
(a)
(b)
538.4926 539.4963
539.5066
540.5089
537.4880
537.4888
538.4923
539.4954
537 538 539 540 541 542 m/z
(a)
(b)
538.4926 539.4963
539.5066
540.5089
Figure 5.4- A comparison of the experimental (a) and simulated (b) isotopic distribution of the C34H65O4¯ fatty acid dimer anion.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 149
The base peak at m/z 537.4880 is assigned to the 12C341H65
16O4¯ isotopic anion (1.4
ppm). The adjacent peak at m/z 538.4926 is assigned to the 13C112C33
1H6516O4¯
isotope (0.6 ppm). The third isotope peak at m/z 539.4963 is assigned to the
13C212C32
1H6516O4¯ isotope (1.6 ppm). It is interesting to note that whilst the peak at
m/z 537.4880 and 538.4926 show a good intensity correlation (1:0.4), the
experimental intensity of the peak at nominal mass m/z 539 is unusually high. A high
resolution inspection of this peak (not shown here) shows that the peak at nominal
mass m/z 539 is composed of two partially overlaying peaks. A smaller peak at m/z
539.4963 that is assigned to 13C312C31
1H6516O4¯ isotope (1.7 ppm) and a larger peak
at m/z 539.5066 that could be assigned to fatty acid dimer 12C341H67
16O4¯ (3.9 ppm).
The latter peak at m/z 539.5066 (<0.1%) could be assigned to an anion cluster of
palmitic acid (C16H32O2) and stearic acid (C18H36O2) to give C34H67O4¯ by releasing
a proton. The intensity of this latter peak is less than 1% of the base peak and is not
reported in Table 5.2.
The excellent isotope pattern match of the experimental peaks and the simulated
peaks in Figure 5.4 strengthens the assignments.
Similarly, the peak observed at m/z 563.5040 in Figure 5.3 can be assigned to the
anion cluster formed by the combination of oleic acid (C18H34O2) and oleate anion
(C18H33O2¯) to give C36H67O4¯.
The peak observed at m/z 565.5201 in Figure 5.3 can be assigned to the anion cluster
formed by the combination of oleic acid and stearic acid less a proton to produce
C36H69O4¯.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 150
5.2.3.2- Diacylglycerols
The peak observed at m/z 627.4762 in Figure 5.3 is assigned to the DAG anion
(C39H63O6¯) containing additional oxygenated functional group. As the glycerol
group contains three carbon atoms, the remaining 36 carbon atoms could be of two
oleic acyl groups (C18:1). Other combinations of fatty acids can also be suggested
that will add up to 36 carbon atoms such as C20:1 with C16:1. As the most common
fatty acid in macadamia oil TAGs is known to be oleic acid, two C18:1 is the most
sensible assignment for fatty acids of this DAG anion. The location of the two oleic
acyl substituents on the glycerol chain could be on carbons 1 and 2 or on carbons 1
and 3. Without further tandem MS studies it is impossible to assign a conclusive
structure to this anion.
Similarly, DAGs containing additional oxygenated functional groups are observed at
m/z 629.4889, 655.5031, 657.5103, 665.5378, 682.5263, 699.4532 and 701.4524.
These molecules could bear an extra hydroxy group. Hydroxy acids form by
hydration of carbon double bonds. The oxidation of FAs and formation of hydroxy
FAs is discussed in detail by Hamilton et al.[209] and by Frankel.[210]
A related peak at m/z 629.4787 assigned to the DAG C39H65O6¯ (16 ppm) most
likely involves saturation of one of the double bonds of the fatty acid side chain of
the C39H63O6¯ DAG anion.
Similarly, the anion pair assigned to the peaks at m/z 655.5031 and 657.5103 also
differs by double bond saturation. These peaks are assigned to C41H67O6¯ (13 ppm)
and C41H69O6¯ (< 0.5 ppm) respectively.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 151
ESI FTICR mass spectrometry differentiates the ions according to their masses and is
not capable of resolving isomers. Thus, further chromatographic separations,
chemical derivatisation and spectroscopic studies are necessary to identify more
conclusively the oxygen bearing functional groups on these molecules.
5.2.4- Triacylglycerol Region, m/z 800-1000
Figure 5.5 shows an expanded TAG region (m/z 800-1000) of the negative-ion ESI
FTICR mass spectrum of neat processed macadamia oil shown in Figure 5.1. Figure
5.5 (b) shows an expansion of the region m/z 870-882 of this region. Table 5.3 lists
the assignment of the mass spectral peaks observed in Figure 5.5.
847.7396
875.7683
901.7849
917.6892
931.7126
945.7158959.7272
800 820 840 860 880 900 920 940 960 980 1000m/z
847.7396
875.7683
901.7849
917.6892
931.7126
945.7158959.7272
800 820 840 860 880 900 920 940 960 980 1000m/z Figure 5.5- (a) TAG region of the negative-ion ESI FTICR mass spectrum of the neat processed macadamia nut oil in Figure 5.1, m/z 800-1000, (b) expanded peaks in the TAG region, m/z 870-882.
873.7573
875.7683
870 872 874 876 878 880 882m/z
873.7573
875.7683
870 872 874 876 878 880 882m/z
(b)
(a)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 152
Table 5.3- Assignment of the mass spectral peaks (>2% of the base peak in Figure 5.1) in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.5.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Anion Normalized
% 847.7396 847.7396 TAG C53H99O7‾ 5.8 873.7573 873.7553 TAG C55H101O7‾ 6.1 875.7683 875.7709 TAG C55H103O7‾ 9.4 901.7849 901.7866 TAG C57H105O7‾ 6.3 917.6892 917.6876 TAG C58H93O8‾ 2.1 931.7126 931.7244 TAG C56H99O10‾ 4.0 945.7158 945.7036 TAG C53H101O13‾ 2.7 959.7272 959.7193 TAG C57H99O11‾ 2.5
In general, the peaks in TAG region appear less intense compared to the peaks in FA
and DAG regions. The most intense peak in this region is observed at m/z 875.7683
with an intensity of 9.4% of the base peak in Figure 5.1 (palmitate anion).
The peak at m/z 847.7396 is assigned to the TAG anion C53H99O7¯ (<0.1 ppm).
Removal of three carbon atoms for the glycerol backbone of the TAG leaves 50
carbon atoms for the fatty acid substituent groups. Given the abundance of palmitic
and oleic acid in these oils, a reasonable assignment for this anion is one which
contains two palmitic acids (C16:0) and one oleic acid (C18:1) side chain groups,
one of which contains an OH group. The OH group on the acyl chain could have
been generated by hydration reaction (addition of a water molecule) on the double
bond of a C16:1 acyl substituent to produce C16:0 with an OH substituent.
Figure 5.5 (b) shows an expansion of the region m/z 870-880 that contains two of the
most intense peaks in the TAG region of Figure 5.1.
The peak at m/z 873.7573 in Figure 5.5 (b) is assigned to the TAG anion C55H101O7¯
(2.3 ppm). This anion is likely to contain a glycerol substituted by three acyl groups
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 153
including two oleates and one palmitate, containing 52 carbon atoms in total (C18:1
+ C18:1 + C16:0) and an extra OH group that bears the negative charge by releasing
a proton.
The peak at m/z 875.7683 in Figure 5.5 (b) is assigned to the TAG anion C55H103O7¯
(2.9 ppm). This anion is likely to contain a glycerol backbone substituted by three
acyl groups including an oleate, a stearate and a palmitate, containing 52 carbon
atoms in total (C18:1 + C18:0 + C16:0) and an extra OH group that bears the
negative charge by releasing a proton.
Similarly, the peak at m/z 901.7849 is assigned to the TAG anion C57H105O7¯ (1.9
ppm). Again, a sensible assignment for the general structure of this TAG anion could
be two oleate and one stearate substituents (C18:1 + C18:1 + C18:0) on the glycerol
backbone and an extra OH group on one of the fatty acid side chain groups bearing
the negative charge.
The peak at m/z 917.6892 is assigned to C58H93O8¯ (1.7 ppm), this could correspond
to the TAG anion with two C18:2 and one C19:5 substituents. There are two hydroxy
groups on the fatty acid side chains.
The peak at m/z 931.7126 is assigned to C56H99O10¯ (13 ppm), the peak at m/z
945.7158 is assigned to C53H101O13¯ (9.5 ppm) and the peak at m/z 959.7272 is
assigned to C57H99O11¯ (8.2 ppm). These highly oxygenated TAG anions could be
clusters formed by the attachment of a number of methanol or glycerol molecules to
a TAG molecule. No further investigation to elucidate the structure of these TAG
anions was performed in this study because of the number of ions involved.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 154
It is noticeable that all of the assigned anions in the TAG region of negative-ion
FTICR mass spectrum in Figure 5.5 contain at least one saturated fatty acid
substituent and one extra hydroxyl group. This observation supports the idea that the
hydroxyl group is produced by hydration reaction on a double bond on the fatty acid
side chain.
These highly oxygenated fatty acids are observed in negative-ion mode due to the
fact that they are likely to bear a hydroxyl group on the side chains that can release a
proton to generate a negative charge on the fatty acid chain. These compounds are
not observed in the GC-MS analysis of these samples. In GC-MS analysis of these
samples it is unlikely that such TAGs would have been identified due to the fact that
the samples are esterified prior to the GC-MS analysis that can dramatically change
the structure of these highly oxygenated compounds.
5.3- Negative-ion ESI FTICR Mass Spectra of the Methanol Extract of Processed Macadamia Oil
5.3.1- Introduction In this section the results of the negative-ion FTICR-MS analysis of the methanol
extract of processed macadamia nut oil are presented and discussed. Peaks are
associated and discussed in three regions including free fatty acids region (m/z 150-
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 155
400), DAGs and fatty acid dimers region (m/z 500-750) and TAGs region (m/z 800-
1000).
Figure 5.6 shows negative-ion ESI FTICR mass spectrum of the methanol extract of
processed macadamia nut oil, m/z 120-980, and selected peaks are assigned in Table
5.4. In this section only peaks with intensities >2% of the base peak are tabulated and
assigned.
As an overview, three mass regions are observed in Figure 5.6. The region m/z 150-
400 contains peaks associated with anions of free FAs. The region m/z 500-750
contains peaks assigned to charged FA dimers, DAGs and also DAGs with additional
oxygen bearing functional groups. The region m/z 800-1000 contains peaks assigned
to TAGs with additional oxygen bearing functional groups. Only selected peaks in
each mass region are assigned in Table 5.4 as a summary of the assignment of all
three regions. More comprehensive tables can be found in sections 5.3.2 to 5.3.4.
The concentration of TAGs is very low in the methanol extract of the oil due to the
fact that TAGs have a low solubility in methanol at 0 °C.[66] As a consequence, the
relative concentrations of the minor oil constituents and the DAGs are increased
dramatically in the extracted solution.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 156
169.0875
253.2179
281.2493
391.2650
445.2677525.3814
563.5009
655.5151701.4695
901.7928
200 300 400 500 600 700 800 900 m/z
873.7773
Figure 5.6- Negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil.
FA Region
DAG Region TAG Region
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 157
Table 5.4- Assignment of selected mass spectral peaks (>2%) in the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Fig. 5.6.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Anion Normalized
% 169.0875 169.0870 FA C9H13O3‾, C9:2 8.0 253.2179 253.2173 FA C16H29O2‾, C16:1 51.7 255.2335 255.233 FA C16H31O2‾, C16:2 21.2 279.2350 279.2330 FA C18H31O2‾, C18:2 7.4 281.2493 281.2486 FA C18H33O2‾, C18:1 100.0 283.2650 283.2643 FA C18H35O2‾, C18:0 4.6 391.2650 391.2643 Wax* C27H35O2‾ 11.3 535.4720 535.4732 FA Dimer C34H63O4‾ 1.9 563.5009 563.5045 FA Dimer C36H67O4‾ 3.3 609.5162 609.5100 DAG C37H69O6‾ 5.2 611.5250 611.5256 DAG C37H71O6‾ 3.2 627.4822 627.4871 DAG C36H67O8‾ 8.6 629.4869 629.4998 DAG C36H69O8‾ 3.1 637.5436 637.5413 DAG C39H73O6‾ 9.2 655.5151 655.4943 DAG C38H71O8‾ 10.5 657.5139 657.5100 DAG C38H73O8‾ 3.1 671.4377 671.4317 DAG C43H59O6‾ 4.3 673.4334 673.4321 DAG C39H61O9‾ 4.1 699.4664 699.4630 DAG C45H63O6‾ 7.3 701.4695 701.4787 DAG C45H65O6‾ 7.5 845.7322 845.724 TAG C53H97O7‾ 4.3 847.7458 847.7396 TAG C53H99O7‾ 3.8 873.7773 873.7917 TAG C56H105O6‾ 10.2 875.7690 875.7709 TAG C55H103O7‾ 5.7 901.7928 901.7855 TAG C57H105O7‾ 10.5
* See Section 1.1 for definition of waxes
5.3.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400
Figure 5.7 shows an expanded view of the negative-ion FTICR mass spectrum of the
m/z 150-400 region of Figure 5.6 and Table 5.5 lists the corresponding assignment of
the mass spectral peaks in Figure 5.7.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 158
157.0879
169.0875187.0977
212.0756227.2007
253.2179
281.2493
297.2445 325.1949 339.2095
391.2650
150 175 200 225 250 275 300 325 350 375 400 m/z Figure 5.7- FA region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 150-400.
Table 5.5- Assignment of the mass spectral peaks (>2%) in the expanded FA region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.7.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Anion Normalized
% 169.0875 169.0870 FA C9H13O3‾, C9:2 8.0 171.1028 171.1027 FA C9H15O3‾, C9:1 3.4 187.0977 187.0976 Di-FA# C9H15O4‾ 4.3 227.2007 227.2017 FA C14H27O2‾, C14:0 3.9 253.2179 253.2173 FA C16H29O2‾, C16:1 51.7 255.2335 255.2330 FA C16H31O2‾, C16:0 21.2 279.2350 279.2330 FA C18H31O2‾, C18:2 7.4 281.2493 281.2486 FA C18H33O2‾, C18:1 100.0 283.2650 283.2643 FA C18H35O2‾, C18:0 4.6 295.2291 295.2279 FA C18H31O3‾, C18:2 3.9 297.2445 297.2431 FA C18H33O3‾, C18:1 4.6 311.1877 311.1864 FA C17H27O5‾, C17:3 3.6 313.2370 313.2384 FA C18H33O4‾, C18:1 2.3 325.2757 325.2748 FA C20H37O3‾, C20:1 2.0 391.2650 391.2643 Wax* C27H35O2‾ 11.3
# Di-FA: Dicarboxylic fatty acid * See Section 1.1 for definition of waxes
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 159
A comparison of the FA region of the ESI FTICR mass spectra of macadamia neat
oil and the methanol extract of the oil (Figures 5.2 and 5.6 respectively) shows that
extraction has increased the concentration of free FAs as larger number of peaks
assigned to free fatty acids are now observed, as well as increasing the S/N ratio.
Furthermore, the relative intensities of various free fatty acids also appear to change.
As an example, the relative intensity of the C16H29O2¯ / C18H33O2¯ peaks in Figure
5.2 is 61/100 (45/74) while this relative intensity is 52/100 in Figure 5.7.
The extraction appears to be working in different ways for different free FAs. As an
example, the absolute intensity of the peak at m/z 281.2493 (assigned to C18H33O2¯)
has increased by four times in the negative-ion extract spectrum compared to that in
the neat oil spectrum, while the absolute intensity of the peak at m/z 255.2440
(assigned to C16H31O2¯) shows a decrease in the methanol extract spectrum
compared to that in the neat oil spectrum. This difference in the relative
concentrations (and hence the relative negative-ion peak intensities) of the free FAs
in the oil and the methanol extract are likely to be due to the differences in the
solubility of these compounds in methanol at the extraction temperature. It is
expected that as the degree of unsaturation on the alkyl chain increases, the
corresponding solubility of the free fatty acid in methanol will increase at 0 °C.[66]
The most abundant free fatty acid observed in the methanol extract of the oil is at
m/z 281.2493 that is assigned to C18H33O2¯ (2 ppm). As stated in section 5.2.1 this is
expected to be the oleate anion (C18:1). The related peaks at m/z 279.2330 (7%) and
2832643 (5%) are assigned to C18H31O2¯ (2 ppm) and C18H35O2¯ (7 ppm) which are
the linoleate (C18:2) and stearate (C18:0) anions respectively.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 160
The next most abundant peaks are at m/z 253.2179 (52%) and 255.2335 (21%).
These are assigned to C16H29O2¯ (2 ppm) the palmitoleate anion C16:1 and
C16H31O2¯ (2 ppm) the palmitate anion C16:0. Interestingly this is the reverse order
to what was observed in neat oil in Figure 5.2, where the C16:0/C16:1 intensity ratio
is observed to be 50/23 compared to 21/52 for the oil extract. As stated earlier, we
can only attribute this difference to the different solubilities of the fatty acids in
methanol at 0 °C used in the extraction procedure.
As observed in the FTICR mass spectrum of neat processed macadamia oil in Figure
5.2, short-chain fatty acids, di-carboxylic acids and dihydroxy FAs are assigned to
the peaks in Figure 5.7.
Two short-chain fatty acids and one dicarboxylic acid are assigned in Table 5.5.
These are at m/z 169.0875 (8%), m/z 171.1028 (3%) and m/z 187.0977 (4%) and are
assigned to C9H13O3¯ (C9:2, 3 ppm), C9H15O3¯ (C9:1, 0.6 ppm) and C9H15O4¯ (C9:0
dicarboxylic acid, 0.5 ppm). As mentioned earlier in Section 1.5, Steenhorst has
separated and reported short-chain hydroxy fatty acids as a product of oxidation of
acylglycerols.[177] The oxidation cleavage of the carbon-carbon double bond on the
fatty acid chains produces hydroxy fatty acids and dicarboxylic acids. Oleic acid is
the most abundant fatty acid in macadamia oil with a double bond at carbon 9 that
can produce C9 hydroxy fatty acids and C9 dicarboxylic acids in oxidation cleavage.
A number of fatty acids with odd numbers of carbon atoms are assigned to the peaks
in this region but these are not listed in Table 5.5 due to the fact that their intensities
are lower than 2% of the base peak. These fatty acids include C15H27O2¯ (C15:1, m/z
239.2020, 0.5 ppm), C15H29O2¯ (C15:0, m/z 241.2158, 0.3 ppm), C17H31O2¯ (C17:1,
m/z 267.2333, 0.7 ppm) and C17H33O2¯ (C17:0, m/z 269.2484, 0.4 ppm).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 161
5.3.2.1- Kendrick Mass Defect (KMD) Values
As pointed out earlier in Section 1.6 the KMD values can be used to validate the
assignments of homologous series of compounds, in this case, fatty acids. In this
section we use the KMD values, in addition to high-resolution mass accuracy
measurements, to support the assignments of the homologous series of fatty acid
anions such as C15:1, C16:1, C17:1 and C18:1.
Table 5.6 lists the KMD values of the assigned anions in Table 5.5. A number of
fatty acid anions with an extra oxygen bearing functional group are assigned to the
peaks in this region and are included in Table 5.6.
Table 5.6- Calculated Kendrick mass defects for homologous series in the negative-ion ESI FTICR mass spectrum of methanol extract of macadamia nut oil.
Observed Mass m/z Assigned Anion Kendrick Mass Defect 157.0873 C8H13O3‾ 0.912 157.1237 C9H17O2‾ 0.948 171.1030 C9H15O3‾ 0.912 227.2021 C14H27O2‾ 0.948 239.2020 C15H27O2‾ 0.935 241.2158 C15H29O2‾ 0.947 253.2173 C16H29O2‾ 0.935 255.2330 C16H31O2‾ 0.949 269.2124 C16H29O3‾ 0.912 267.2333 C17H31O2‾ 0.935 269.2484 C17H33O2‾ 0.948 281.2486 C18H33O2‾ 0.935 283.2643 C18H35O2‾ 0.949 297.2437 C18H33O3‾ 0.912 309.2804 C20H37O2‾ 0.935 311.1693 C17H27O3S‾ 0.822 325.1851 C18H29O3S‾ 0.822 339.2013 C19H31O3S‾ 0.823
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 162
KMD values are expected to be similar for homologous series; for example, all fatty
acid anions with a saturated acyl chain show a KMD value of 0.948 ± 0.001 (such as
C9H17O2¯, C16H31O2¯ and C18H35O2¯), all free fatty acid anions with a
monounsaturated acyl chain show a KMD value of 0.935 ± 0.001 (such as
C15H27O2¯, C16H29O2¯ and C18H33O2¯), all fatty acid anions containing an additional
oxygen atom show a KMD value of 0.912 ± 0.001 (such as C8H13O3¯, C9H15O3¯,
C16H29O3¯ and C18H33O3¯) and all sulphur containing anions show a KMD value of
0.822 ± 0.001.
In Table 5.6 all of the members of each homologous series are shaded in the same
colour, for example, C15H27O2¯, C16H29O2¯, C17H31O2¯, C18H33O2¯ and C20H37O2¯
are mono-unsaturated fatty acid anions that show a KMD value of 0.935 and are
shaded in green colour. Similarly, C8H13O3¯, C9H15O3¯, C16H29O3¯ and C18H33O3¯
are assigned to fatty acid anions containing extra oxygen bearing functional groups
(such as hydroxyl group) and show a KMD value of 0.912 and are shaded in blue.
Similarly, C9H17O2¯, C14H27O2¯, C15H29O2¯, C16H31O2¯, C17H33O2¯ and C18H35O2¯
are assigned to saturated fatty acid anions with a KMD value of 0.948 ±0.001 and are
shaded in tan. Similarly, C17H27O3S¯, C18H29O3S¯ and C19H31O3S¯ are sulphur
containing anions that show a KMD value of 0.822 and are shaded in grey.
5.3.3- Diacylglycerol Region, m/z 500-750 Figure 5.8 shows an expanded view of the negative-ion FTICR mass spectrum of the
m/z 500-750 region of Figure 5.6 and Table 5.7 lists the assignment of selected mass
spectral peaks in this Figure.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 163
511.3516
525.3814
535.4720
553.4007
563.5009
583.5034
599.4459
609.5162
627.4822
637.5436
645.4739
655.5151
671.4377
682.5405
691.4627
701.4695
717.4638
731.4594747.5145
500 525 550 575 600 625 650 675 700 725 750 m/z Figure 5.8- DAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 500-750.
Two FA dimers and twelve DAGs are assigned in Table 5.7. Further investigation is
needed to identify the functional groups and their positions on the DAG molecules,
but that is not undertaken in this work because this would be beyond the scope of the
present thesis.
KMD values are also listed in Table 5.7 as they are used to confirm various
assignments of homologous anion series in Figure 5.7. Similar homologous series are
shaded in same colour in Table 5.7.
A comparison of the DAG regions of the FTICR mass spectra of processed
macadamia oil shown in Figure 5.3 (Table 5.2) and the methanol extract of the oil
shown in Figure 5.8 (Table 5.7) reveals that, in general, all peaks have higher S/N
ratios and are more intense relative to the base peak in the extract spectrum.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 164
Table 5.7- Assignment of the mass spectral peaks (>2%) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.8.
Observed Mass m/z
Exact Mass m/z
Compound Class
Assigned Anion
Normalized % KMD values
525.3814 525.3797 DAG C30H53O7‾ 4.4 0.797 535.4720 535.4732 FA Dimer C34H63O4‾ 1.9 0.875 563.5009 563.5045 FA Dimer C36H67O4‾ 3.3 0.875 609.5162 609.5100 DAG C37H69O6‾ 5.2 0.840 611.5250 611.5256 DAG C37H71O6‾ 3.2 0.847 627.4822 627.4871 DAG C36H67O8‾ 8.6 0.786 629.4969 629.4998 DAG C36H69O8‾ 3.1 0.789 637.5436 637.5413 DAG C39H73O6‾ 9.2 0.837 655.5151 655.4943 DAG C38H71O8‾ 10.5 0.783 657.5139 657.5100 DAG C38H73O8‾ 3.1 0.781 671.4377 671.4317 DAG C43H59O6‾ 4.3 0.694 673.4334 673.4321 DAG C39H61O9‾ 4.1 0.681 699.4664 699.4630 DAG C45H63O6‾ 7.3 0.692 701.4695 701.4787 DAG C45H65O6‾ 7.5 0.693
For example, the peak at m/z 699.4664 (C45H63O6¯) in the extract spectrum is 38
times higher in absolute intensity than the same peak in the neat oil spectrum. As in
the previous section, this observation illustrates the advantage of extracting minor
components from the TAG dominated oil prior to mass analysis if one is interested in
the composition of the minor components in the oil. It is noteworthy that such
components are not observed in a GC-MS analysis of such oils.
In Table 5.2, six fatty acid dimers are assigned, while in Table 5.7, only two fatty
acid dimers are assigned. This difference is due to the fact that the minimum reported
intensities in Table 5.7 are greater than 2% of the base peak, while in Table 5.2, the
minimum reported intensities are 0.2% of the base peak. The normalized intensities
of four of the assigned fatty acid dimers in Table 5.2 are lower than 2%. Two of the
assigned fatty acid dimers are reported in both Tables 5.2 and 5.7 include the peak at
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 165
m/z 535.4720 (2%) assigned to C34H63O4¯ (2 ppm) and the peak at m/z 563.5009
(3.3%) assigned to C36H67O4¯ (6 ppm). Since the most abundant fatty acids in
macadamia oil are oleic (C18:1), palmitic (C16:0) and palmitoleic (C16:1) acids, it is
expected that one C18:1 and one C16:1 can combine to produce the C34H63O4¯ dimer
anion. Similarly, it is expected that two C18:1 can combine to produce the C36H67O4¯
dimer anion. KMD values confirm that these latter two assignments belong to a
homologous series of dimers and possess same functional groups.
The peak at m/z 525.3814 (4%) is assigned to a DAG anion C30H53O7¯ (3 ppm). The
two fatty acid substituents on the glycerol could be C16 and C11. One of the fatty
acid substituents could be a short-chain hydroxy fatty acid.
Two peaks at m/z 609.5162 (5.2%) and 611.5250 (3.2%) are assigned to the DAG
anions C37H69O6¯ (10 ppm) and C37H71O6¯ (1 ppm) respectively. The peak at m/z
609.5162 could be assigned to a DAG anion that contains C18:1 and C16:0 side
chains, one of which has a hydroxyl group, possibly C16:0. The hydroxyl group
could have been produced in a hydration reaction of the double bond on a C16:1.
Alternatively, the two fatty acid substituents could be C18:0 and C16:1 and the
C18:0 could bear the hydroxyl group as a result of hydration reaction on the double
bond of C18:1. Further CID and MS/MS analysis is needed to elucidate the
substituents of this DAG molecule.
Branched side chains in TAG molecules, since TAGs are major components of
macadamia oil, are of interest because they may lead to different properties of the oil.
The detection and analysis of these groups and unsaturated carbon position can be
achieved by tandem MS and derivatisation. Careful HPLC and tandem MS could be
used to direct the acyl glycerol molecular ions to more completely define the exact
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 166
composition of the oil. The determination of the position of the side chains on
glycerol in acylglycerols are discussed in Section 1.1.3.1.
Similarly, the peak at m/z 611.5250 could be assigned to a DAG anion that contains
a C18:0 and a C16:0 side chains, one of which contains one hydroxyl substituent.
Given that the palmitic acid substituent is a major constituent of acylglycerols in
macadamia oil, the two fatty acid substituents on the glycerol in this TAG anion are
likely to be palmitic acid and hydroxy oleic acid acyl groups. The hydroxyl group on
the oleic acid acyl group could have been produced in hydration reaction on the
double bond of linoleic acid acyl substituent.
The anion at m/z 627.4822 in Table 5.7 could be assigned to two possible DAG
anion formulae, one of which contains a nitrogen atom, C39H65NO5¯ (7 ppm). The
second possible assignment is DAG anion C36H67O8¯ (3 ppm). Similarly, the peak at
m/z 629.4969 could be assigned to two DAG anions, one of which contains two
nitrogen atoms, C38H65N2O5¯ (3 ppm) and the other to C36H69O8¯ (5 ppm). To assess
the assignments, KMD values were calculated for the peaks at m/z 627.4822 (KMD=
0.782) and m/z 629.4869 (KMD= 0.782). The same KMD values suggest that these
two molecule anions belong to a homologous series. Consequently, the peak at m/z
627.4822 is assigned to DAG anion C36H67O8¯ and the peak at m/z 629.4869 is
assigned to DAG anion C36H69O8¯. The alternative nitrogen-bearing molecules have
different number of nitrogen atoms and cannot fit in same homologous series.
Similarly, the KMD value for the peak at m/z 655.5151 in Table 5.7 was selected as
0.783 for the DAG anion C38H71O8¯ (6 ppm). The DAG anion C41H67O6¯ (6 ppm)
which is another alternative assignment has a KMD value of 0.762 and was rejected.
Same calculations were carried out for the peak observed at m/z 657.5139 and the
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 167
KMD was found to be 0.783 for possible DAG anion C38H73O8¯ (6 ppm). The DAG
anion C41H69O6¯ (4 ppm) with a KMD value of 0.776 was rejected. These four latter
assigned anions at m/z 627.4822, 629.4869, 655.5151 and 657.5139 belong to the
same homologous series with a KMD value of 0.785 ± 0.004.
The peak at m/z 671.4377 is assigned to a DAG anion C43H59O6¯ (9 ppm) which
could be a glycerol with two C20H31O2¯ substituents, one of which has a hydroxyl
group substituent. Similarly, the peak at m/z 699.4664 is assigned to a DAG anion
C45H63O6¯ (5 ppm) that could be composed of a glycerol substituted with two
heneicosa hexaenoate (C21H29O2¯) substituents, one of which contains a hydroxyl
group. In a similar way, the peak at m/z 701.4695 is assigned to a DAG anion
C45H65O6¯ (13 ppm) that could contain a glycerol substituted with a heneicosa
hexaenoate (C21H29O2¯) and a heneicosa pentaenoate (C21H31O2¯), one of which
contains a hydroxyl group. The last three assigned anions at m/z 671.4377, 699.4664
and 701.4695 belong to a homologous series with a KMD= 0.693 ± 0.001.
As mentioned previously in Section 5.1, it is possible to observe DAG anions in
negative-ion mode because of the mobile proton on the hydroxyl substituent of the
glycerol. Such anions contain five oxygen atoms in the anion ionic formulae. It is
interesting that no such anions are observed in Table 5.7; in fact, most of the anions
observed contain six or eight oxygen atoms. No doubt the extensive hydration of the
unsaturated carbon double bonds on the acid side chain groups of the glycerol
enhances the ionisation efficiencies of the DAG molecules in the oil. It is noteworthy
that such species cannot be identified in the oil by the more traditional GC-MS
experiments. Whether such hydration of the acyl substituents occur naturally in the
oil or by the processing of the oil is not tested in this study.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 168
5.3.4- Triacylglycerol Region, m/z 800-1000
Figure 5.9 shows the m/z 800-1000 expanded view of the negative-ion FTICR mass
spectrum of the methanol extract of the processed macadamia oil (Figure 5.6). Table
5.8 lists the assigned peaks and the associated KMD values in this spectrum.
An important point in Table 5.8 is that all of the peaks are assigned to TAG anions
with additional oxygen atoms compared to standard TAG anions. The assigned
anions in Table 5.8 contain seven oxygen atoms or more, whereas the common TAG
molecules have six oxygen atoms.
The extra oxygen atom in the assigned anions in Table 5.8 is likely to be a hydroxy
group that is capable of releasing a proton and producing an anion. As stated
previously in Section 5.1, the common TAG molecules with three ester bonds (six
oxygen atoms) are unable to produce negative ions in ESI source due to the absence
of ionisable functional groups such as hydroxyl group.
Two most intense peaks assigned in the TAG region in Table 5.8 are at m/z 901.7868
and m/z 873.7559. The peak at m/z 901.7868 (10.6%) is assigned to C57H105O7¯ (0.2
ppm). Since the most abundant fatty acid substituent in the TAGs in macadamia oil is
C18:1, it is expected that this anion is composed of a glycerol with two C18:1
substituents and one C18:0 substituent. The C18:0 glycerol substituent could be the
one that contains the hydroxyl group that produces the negative charge by releasing
the proton. The hydroxyl group could have been generated by the hydration reaction
of a carbon double bond on a C18:1 substituent.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 169
845.7311
873.7559
891.7726
901.7868
919.7962
800 820 840 860 880 900 920 940 960 980 1000m/z
(b)
845.7322
873.7773
891.7837
901.7928
919.8176
(a)
845.7311
873.7559
891.7726
901.7868
919.7962
800 820 840 860 880 900 920 940 960 980 1000m/z
(b)
845.7322
873.7773
891.7837
901.7928
919.8176
(a)
845.7322
873.7773
891.7837
901.7928
919.8176
(a)
Figure 5.9- TAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 800-1000, (a) prior to internal calibration and (b) after internal calibration was carried out.
Table 5.8- Assignment of the mass spectral peaks (>2%) and KMD values in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.9.
Calibrated Mass m/z
Exact Mass m/z
Compound Class
Assigned Anion
Normalized %
KMD Value
845.7311 845.7240 TAG C53H97O7‾ 4.3 0.780 847.7437 847.7396 TAG C53H99O7‾ 3.8 0.793 873.7559 873.7553 TAG C55H101O7‾ 10.2 0.780 875.7681 875.7709 TAG C55H103O7‾ 5.7 0.793 891.7726 891.7658 TAG C55H103O8‾ 2.9 0.770 901.7868 901.7866 TAG C57H105O7‾ 10.6 0.780 919.7962 919.7971 TAG C57H107O8‾ 2.5 0.770
From this study it is not possible to determine the location of the various C18:1
substitutions on the glycerol backbone. Such issues are discussed in Chapter 8 under
Future Work.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 170
The peak at m/z 873.7559 (10.2%) is assigned to C55H101O7¯ (0.7 ppm). This ion
could be composed of a glycerol with two C18:1 and one C16:0 substituents. One of
these substituents would also contain a hydroxyl group, probably produced by the
hydration of a carbon double bond. The oil contains both C16:1 and C18:1
substituents on glycerol in the TAG molecule, and so both of these could be the part
of the hydration reaction.
The peak at m/z 875.7681 (5.7%) is assigned to C55H103O7¯ (3 ppm) that is expected
to be composed of a glycerol with one C18:1, one C18:0 and one C16:0 acyl
substituent groups on the glycerol. It also contains a hydroxyl group necessary to
produce the anion by providing a mobile proton. Whether the hydration of the carbon
double bond is on a C18:1 or a C16:1 to produce the C18:0 or C16:0 is unknown.
The peak at m/z 845.7311 (4.3%) is assigned to C53H97O7¯ (0.8 ppm) which could be
a hydroxy glycerol with the substituents C16:0, C16:1 and a C18:1 The C16:0 could
be the side chain that contains the hydroxyl group that produces the negative charge
by releasing a proton. The hydroxyl group could have been generated by the
hydration reaction of a carbon double bond on a C16:1, palmitoleate substituent.
The peak at m/z 847.7437 (3.8%) is assigned to C53H99O7¯ (4.8 ppm) which could be
a hydroxyglyceride with two C16:0 and one C18:1 substituents. One of the C16:0
substituents could contain the hydroxy group that provides the negative charge. The
hydroxyl group could have been generated by the hydration reaction of a carbon
double bond on a C16:1 substituent.
The peak at m/z 891.7726 (2.9%) is assigned to C55H103O8¯ (7.6 ppm) which could
be a hydroxyglyceride with C16:0, C18:0 and C18:1 substituents. There are two
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 171
hydroxyl groups on this anion, possibly with one on the C16:0 and one on the C18:0
substituents and produced by hydration of carbon double bond on a C16:1 and a
C18:1 substituent in a TAG such as C16:1; C18:1; C18:1. Such TAGs are common
in the oil (see Table 5.8).
The peak at m/z 919.7962 (2.5%) is assigned to C57H107O8¯ (<1 ppm). It is expected
that this anion is composed of a glycerol with one C18:1 substituent and two C18:0
substituents. Each of the C18:0 substituents could contain one hydroxyl group and
one of the hydroxyl groups could provide the negative charge by releasing a proton.
This anion could have been produced by a carbon double bond hydration reaction of
trioleate, the most common TAG in macadamia oil.
A comparison of Table 5.3 (TAGs region of neat oil) with Table 5.8 (TAGs region of
the methanol extract of the oil) reveals that peaks assigned to the TAG molecules
with higher molecular masses (such as peaks at m/z 931.7126, 945.7158 and
959.7272) are assigned in Table 5.3 but not in Table 5.8. This could be due to the
differences in the solubilities of TAG compounds in methanol at the extraction
temperature. In addition, one TAG molecule with relatively lower molecular mass
(m/z 845.7311) is assigned in Table 5.8 but not in Table 5.3.
5.3.4.1- KMD Values of the Assignments in the TAG Region
Additional support for the assignments in Table 5.8 are the calculated KMD values
for the assigned peaks. In general, the KMD values of the assigned anion TAG peaks
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 172
can be categorized into three groups. There are three anions with KMD= 0.780, two
anions with KMD= 0.793 and two anions with KMD= 0.770.
All of the three assigned TAG anions with KMD= 0.780 have two unsaturated fatty
acid substituents and one saturated fatty acid substituent. In particular, the peak
assigned at m/z 845.7311 has two C18:1 and one C16:0 substituents, the peak
assigned at m/z 873.7559 has two C18:1 and one C16:0 substituents and the peak
assigned at m/z 901.7868 has two C18:1 and one C18:0 substituents.
Both of the assigned TAG anions with KMD= 0.793 have two saturated substituents
and one unsaturated substituent. In particular, the peak assigned at m/z 847.7437 has
two C16:0 substituents and one C18:1 substituent and the peak assigned at m/z
875.7681 has C18:0, C16:0 and C18:1 substituents.
Both of the assigned TAG anions with KMD= 0.770 have two hydroxy groups and
both have two saturated fatty acid substituents and one unsaturated fatty acid
substituent on the glycerol backbone. The peak assigned at m/z 891.7726 has one
C16:0, one C18:0 and one C18:1 substituents on glycerol and the peak assigned at
m/z 919.7962 has two C18:0 and one C18:1 substituents.
5.3.5- Stability of the Methanol Extract of Processed Macadamia Oil
In this section it was decided to examine the possible change in the methanol extract
of processed macadamia oil over a period of time (576 days) by examining the
negative-ion FTICR mass spectra of the methanol extract of the oil over this period.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 173
The methanol extract of processed macadamia was stored under nitrogen gas
atmosphere in a freezer.
Figure 5.10 shows a comparison of the negative-ion ESI FTICR mass spectra of the
methanol extract of processed macadamia oil on two dates, 22/01/2003 and
19/08/2004.
161.9739
253.2173
281.2486
311.1687 391.2617477.3009 563.5056
599.4452627.4757
655.5071
901.7856
169.0875
253.2179
281.2493
391.2650445.2677
563.5009 655.5151701.4695 845.7514 901.7928
200 300 400 500 600 700 800 900m/z Figure 5.10- A comparison of the negative-ion ESI FTICR mass spectra of the methanol extract of macadamia oil on two dates: (a) 22/01/2003 and (b) 19/08/2004.
Although this is not a detailed study, the comparison reveals several useful points
regarding the ESI FTICR mass spectrometry of the methanol extract of processed
macadamia oil and the relative stability of the oil. It would appear that even though
the oil is stored at about -15 °C and kept under nitrogen gas atmosphere, there is still
some degradation occurring.
The most obvious change is the ratio of the C18:1/C16:1 peaks at m/z 281.2486 and
253.2173 respectively. In Figure 5.10 (a) this ratio is 19/5 where in Figure 5.10 (b) it
is 23/12. This is an increase in the relative intensity of the peak at m/z 253.2173 by
1.9 times in Figure 5.10 (b).
(a)
(b)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 174
Similarly, the fatty acid peak at m/z 311.1687 also appears to have diminished in
relative intensity. The ratio of the peak at m/z 281.2486 to the peak at 311.1687 in
Figure 5.10 (a) is 19/3 while this ratio in Figure 5.10 (b) is 23/0.8. This is a decrease
in the relative intensity of the peak at 311.1687 by 4 times in Figure 5.10 (b).
It should be noted that there are slight differences in the m/z values of the various
ions in Figures 5.10 (a) and (b). For example m/z 281.2468 and 281.2499. This arises
because only external calibration was used to mass calibrate the spectra. These
relative errors are taken into account here in comparing the spectra.
Furthermore the DAG and TAG regions show diminished relative ion intensities in
Figure 5.10 (b) compared to Figure 5.10 (a). For example, the relative intensity of the
peak at m/z 281.2486 to the peak at 655.5071 is 1.9 in Figure 5.10 (a), while this
ratio is 9.5 in Figure 5.10(b). This is 5 times decrease in the intensity of the peak at
655.5071 which is assigned to a DAG anion C38H71O8¯ (6 ppm). In the TAG region,
the relative intensity of the peak at m/z 281.2486 to the peak at m/z 901.7856 is 5.8
in Figure 5.10 (a), while this ratio is 9.5 in Figure 5.10 (b). This is 1.6 times decrease
in the intensity of the peak at 901.7856 which is assigned to the TAG anion
C57H105O7¯.
In general, even under storage of the oil under nitrogen gas in a freezer there is still
some activity in the oil chemistry. In particular, it appears that the oxygenated DAGs
and TAGs decrease in concentration producing increased quantities of free fatty
acids with an increase in the relative concentration of the C16:1 (palmitoleic acid) it
is possible that even after all the processing that occurred in the oil production there
are still some enzymes present in the oil which lead to its reactivity.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 175
A general conclusion in this section is that the methanol extract of macadamia oil
shows a reasonably good stability; the changes in the spectrum are minimal after
about 19 months. Few peaks have diminished in the TAG and DAG region and few
peaks have diminished in the fatty acid region. There are few peaks that are not
common in both spectra, such as m/z 212.0748 and m/z 701.4695, the source of
which is not clearly known, but could be due to a trace-level contamination in the
solvent used for sample preparations on two different dates. Furthermore, there are
some differences in the intensities of the free fatty acids that can be a result of the
enzymatic activities in the sample.
5.4- Negative-ion ESI FTICR-MS of the Hydrolysed Processed Macadamia Oil
5.4.1- Introduction
In this section the results of negative-ion FTICR-MS analysis of hydrolysed
processed macadamia nut oil are presented and discussed. Peaks are discussed in two
regions including free FAs region (m/z 150-400) and FA dimers region (m/z 500-
750).
The hydrolysis of the processed macadamia oil releases all of the acyl substituents as
fatty acids. This reaction allows analytical experiments to be undertaken which
provide detailed information about the FA profile of the oil. The hydrolysis reaction
takes place in alkaline solution as described in section 2.2.2 and all of the acyl
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 176
substituents on the TAG, DAG and MAG molecules in the oil are released.
Therefore, the ESI FTICR mass spectrum of hydrolysed macadamia oil is expected
to contain fewer peaks compared to the FTICR mass spectrum of the neat macadamia
oil (Figure 5.1) and the methanol extract of the oil (Figure 5.6).
5.4.2- FTICR Mass Spectrum of Hydrolysed Processed Macadamia Oil
Figure 5.11 shows the ESI FTICR mass spectrum of hydrolysed processed
macadamia oil and Table 5.9 lists the assigned peaks in this Figure.
Figure 5.11 shows a base peak at m/z 281.2486 which is assigned to C18H33O2¯ (<0.1
ppm). This anion is expected to be the oleate anion as oleate is reported to be the
major acyl substituent on the TAG molecules in macadamia oil using GC-MS.[196]
Our GC-MS analysis of esterified macadamia oil confirms that in Chapter 6 also the
oleate substituent on the glycerol is the main constituent of macadamia oil (Section
6.1) comprising 59.6% of the total area of the GC-MS measured peaks.
Associated with this peak at m/z 281.2486 are two less intense peaks (approximately
4%) at m/z 279.2439 and 283.2640. These peaks are assigned to the anions
C18H31O2¯ (0.8 ppm) and C18H35O2¯ (1 ppm) which are most likely the linoleate
(C18:2) and stearate (C18:0) anions respectively, based on GC-MS results.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 177
187.0975
253.2177
281.2486
357.2655
563.5039
100 200 300 400 500 600 700 800 900 1000m/z
535.4767
187.0975
253.2177
281.2486
357.2655
563.5039
100 200 300 400 500 600 700 800 900 1000m/z
535.4767
Figure 5.11- Negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil.
Table 5.9- Assignment of the mass spectral peaks (>2% of the base peak) in the negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil shown in Fig. 5.11.
Observed Mass m/z
Exact Mass m/z
Compound Class Assigned Anion Normalized
% 187.0975 187.0976 Di-FA* C9H15O4 ‾ 2.7 227.2020 227.2017 FA C14H27O2‾, C14:0 2.1 253.2177 253.2173 FA C16H29O2‾, C16:1 34.3 255.2331 255.2330 FA C16H31O2‾, C16:0 14.1 269.2133 269.2122 FA C16H29O3‾ 4.5 279.2439 279.2330 FA C18H31O2‾, C18:2 4.5 281.2486 281.2486 FA C18H33O2‾, C18:1 100 283.2640 283.2643 FA C18H35O2‾, C18:0 4.1 295.2278 295.2279 FA C18H31O3‾ 2.9 297.2439 297.2435 FA C18H33O3‾ 9.9 309.2807 309.2799 FA C20H37O2‾, C20:1 3.3 311.2231 311.2228 FA C18H31O4‾ 3.7 535.4767 535.4732 FA dimer C34H63O5‾ 9.3 537.4843 537.4888 FA dimer C34H65O5‾ 5.6 563.5039 563.5045 FA dimer C36H67O4‾ 30.9 565.5071 565.5201 FA dimer C36H69O4‾ 4.2
* Di-FA: Dicarboxylic fatty acid (HOOC-R-COOH)
Two other peaks in this region of the spectrum at m/z 295.2278 and 297.2439 are
assigned to the C18 anions C18H31O3¯ (0.4 ppm) and C18H33O3¯ (1 ppm)
FA Region
FA Dimers Region
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 178
respectively. These anions could arise from linolenic (C18:3) and oleic (C18:1) acids
respectively where one of the double bonds has been hydrated.
A further C18 fatty acid is assigned to the peak at m/z 311.2231 as C18H31O4¯ (1
ppm). This C18 anion could arise from the hydration of two of the carbon double
bonds in the C18:4, stearidonic acid (moroctic acid, C18H28O2) effectively adding
two water molecules to the acid anion. Whether these anions showing evidence of
hydrated carbon double bonds are present in the original macadamia oil sample or
are generated in the hydrolysis reaction of the TAG molecules is not tested in this
study.
The second most intense peak (34%) in Figure 5.11 is at m/z 253.2177 is assigned to
the C16:1 anion C16H29O2¯ (1.6 ppm). This anion most likely arises from palmitoleic
acid. A related C16 peak at m/z 255.2331 is assigned to C16H31O2¯ (0.4 ppm) and
most likely arises from C16:0 palmitic acid. As a confirmation of this assignment,
palmitoleic acid is detected and assigned at a similar spectral percentage (32.7%) in
the esterified macadamia oil FTICR mass spectrum in positive-ion mode (Table 4.4).
Furthermore, our GC-MS analysis of esterified macadamia oil has measured the
relative intensity of palmitoleic acid / oleic acid as 20.6/59.6 that is 34.6%. This ratio
in negative-ion ESI FTICR mass spectrum of hydrolysed macadamia nut oil is
34/100 that is 34.0%. The results of two mass spectrometric experiments appear
consistent and strongly confirm each other.
One other related C16 peak is observed at m/z 269.2133. This peak is assigned to the
C16H29O3¯ (4 ppm) anion which as with the C18 series is associated with hydration
of a carbon double bond in a C16:3 acid.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 179
Two other peaks are assigned to fatty acid anions in Figure 5.11. The first at m/z
227.2020 (2%) is assigned to C14H27O2¯ (2 ppm) and could arise from C14:0
myristic acid. The second peak at m/z 309.2807 (3%) is assigned to C20H37O2¯ (3
ppm) and could arise from C20:1 gadoleic acid.
The remaining peak in the FAs region (m/z 150-400) in Figure 5.11 is assigned to a
dicarboxylic acid. The dicarboxylic acid at m/z 187.0975 is assigned to C9H15O4¯
(0.5 ppm) and is most likely the HCO2-C7H14-CO2¯ anion.
Four odd numbered free FAs are observed in the negative-ion FTICR mass spectrum
of neat macadamia oil in Table 5.1; however, these FAs are not detected in the
FTICR mass spectrum of hydrolysed macadamia oil. This observation supports the
idea that the odd numbered free FAs in the neat macadamia oil are not released from
the TAG molecules, but they could be byproducts of possible biological or
biochemical activities in the oil by the microorganisms and fungi.[211,212]
In the FA dimer region (m/z 500-750) the most intense peak of the fatty acid dimers
is observed at m/z 563.5039 (30.9%) and is assigned to C36H67O4¯ (1 ppm). This
anion most likely forms from the combination of an oleic acid molecule with an
oleate anion (C18H34O2---C18H33O2¯).
Similarly, the peak at m/z 565.5071 (4.2%) is assigned to C36H69O4¯ (1 ppm) which
could be a combination of C18:1 and C18:0 acids less a proton.
The remaining two fatty acid dimers observed at m/z 535.4767 and 537.5039 are
assigned to the anions C34H63O4¯ (4 ppm) and C34H65O4¯ (8 ppm) respectively. The
latter anion could arise from the combination of oleic acid (C18:1) and palmitolenic
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 180
acid (C16:2) less a proton. These assignments are consistent with the observed
relative intensities of the fatty acids in Table 5.9.
As mentioned earlier (Section 4.1), fatty acids form dimers in gas phase. The dimers
are observed in negative-ion FTICR mass spectra of processed macadamia oil
samples. At high capillary-skimmer voltages, the FA dimer peaks disappear due to
an increase in the kinetic energy and collision induced dissociation of the dimer
anion species in the capillary-skimmer region. We have applied a relatively low
capillary-skimmer voltage in our FTICR-MS experiments to keep the peaks of the
dimers in our spectra for two main reasons:
1- Dimers are anions of interest with precisely known molecular masses in the
DAG region of the spectrum and are used as internal calibrants.
2- A low capillary-skimmer voltage ensures that the anions formed in the DAG
and TAG regions from the analysed samples are not removed from the
spectrum due to CID.
5.4.3- Comparison of FTICR Mass Spectra of Macadamia Oils
An interesting extension to this work as discussed in Chapter 8 as future works is to
examine the differences between processed and unprocessed (cold pressed)
macadamia oil, Preliminary studies were commenced in this present research but
only the results for this section are presented.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 181
Figure 5.12 shows a comparison of the negative-ion ESI FTICR mass spectra of
hydrolysed (a) processed macadamia oil (Figure 5.11), (b) cold pressed batch 12 and
(c) cold press batch 13 macadamia oils.
The hydrolysis was carried out as discussed in Section 2.2.2 and cold pressing is
discussed in Section 1.2.6.
187.0975
253.2177
281.2486
297.2439357.2569
535.4767
563.5039
591.5321
253.2178
281.2484
309.2807535.4749
563.5027
591.5369
253.2176
281.2483
535.4761563.5045
100 200 300 400 500 600 m/z
187.0975
253.2177
281.2486
297.2439357.2569
535.4767
563.5039
591.5321
253.2178
281.2484
309.2807535.4749
563.5027
591.5369
253.2176
281.2483
535.4761563.5045
100 200 300 400 500 600 m/z Figure 5.12- Comparison of the negative-ion ESI FTICR mass spectrum of (a) hydrolysed processed macadamia oil, (b) hydrolysed cold pressed oil batch 12 and (c) hydrolysed cold pressed oil batch 13.
Figure 5.12 (b) and (c) show the same mass distribution of peaks assigned to FAs
and FA dimers as discussed in Section 5.4. For example, the peak at m/z 253, 281,
297 and 309 are assigned to the fatty acids C16:1, C18:1, C18-OH and C20:1. The
peaks at m/z 535, 563 and 591 are assigned to FA dimers of the common FAs present
(a)
(b)
(c)
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 182
in the oil. As might be expected from the different samples there are slight
differences in the relative intensities of these various ions.
Based on the work presented in this thesis thus far, it is not surprising these spectra
are similar. This is because the hydrolysis reaction effectively reduces the TAG
molecules to glycerol and fatty acids. To detect the differences between processing
an oil and cold pressing an oil one needs to compare the minor species present in the
oil such as the free FAs and MAGs and DAGs. As well, careful attention needs to be
paid to what actual processes are involved in the processing (Section X.Y) and cold
pressing (Section V.W) of the oil.
The experiments thus far, indicate that the methanol extraction of the oil concentrates
the minor components by removal of the TAGs which typically make up 95% of
macadamia oil. This is where one might expect to see differences between the
different oils.
Such a detailed study of the effect of oil preparation and even cultivar differences
and also oil rancidity would form the basis of another complete research project as
discussed in Chapter 8 under future work.
The industrial refining processes carried out on the macadamia oil have not altered
the total FA profile of the oil. In other words, the processes have only removed the
low-concentration ingredients of the oil such as free fatty acids and polyunsaturated
fatty acids.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 183
5.5- A Summary of the Negative-ion ESI FTICR-MS of the Neat, Methanol Extract and Hydrolysed Processed Macadamia Oil
In this section, a summary of the occurrence of the common fatty acids either, as free
fatty acids or as substituents on the acylglycerols, in the processed macadamia oil is
discussed.
Figure 5.13 shows a comparison of the unsubstituted FA anions observed in
negative-ion FTICR-MS analysis of the neat (Table 5.1), the methanol extract of
(Table 5.4) and the hydrolysed (Table 5.9) processed macadamia nut oil. For this
figure, the anions that show evidence of hydration, e.g. C18H31O3¯ are not included.
Unlike the intensity values in the tables, the peak intensities in Figure 5.13 have been
normalised to 100% for the FA anions displayed.
Figure 5.13 highlights several interesting results from the negative-ion ESI FTICR-
MS experiments discussed previously in this chapter. If one can directly correlate the
anion concentrations with those of the acids then the hydrolysed oil, which contains
the fatty acids released from the acylglycerides, shows the highest concentration of
C18:1 oleic acid at 62% followed by the C16:1 palmitoleic acid at 21% and palmitic
acid at 9%. The presence of the C16 acids is important for the use of macadamia oil
in the cosmetic industry. The other minor acids (2 to 3%) such as the C14:0
(myristic), C18:0 (stearic) C18:2 (linoleic) and C20:1 (eicosenoic) are significant in
this experiment since for them to be observed they must be present in TAG side
chain groups in reasonable proportions. Their presences as such would help to give
the processed macadamia oil distinct chemical features.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 184
C14:0
C14:1
C15:0
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C19:1
C20:0
C20:1
0
5
10
15
20
25
30
35
40
45
50
55
60
65
A Comparison of the Fatty Acid Components Observed in Negative-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil
Peak
Rel
ativ
e In
tens
ity %
Fatty Acid
-ESI-MS Oil-ESI-MS Extract-ESI-MS Hydrolysed Oil
C14:0
C14:1
C15:0
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C19:1
C20:0
C20:1
0
5
10
15
20
25
30
35
40
45
50
55
60
65
A Comparison of the Fatty Acid Components Observed in Negative-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil
Peak
Rel
ativ
e In
tens
ity %
Fatty Acid
-ESI-MS Oil-ESI-MS Extract-ESI-MS Hydrolysed Oil
-ESI-MS Oil-ESI-MS Extract-ESI-MS Hydrolysed Oil
Figure 5.13- A graphical comparison of the unsubstituted FA anions observed in negative-ion FTICR mass spectra of the neat (Table 5.1), the methanol extract (Table 5.4) and the hydrolysed (Table 5.9) processed macadamia nut oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 185
It is equally striking in Figure 5.13 that the neat oil possesses a large concentration of
C16:0 (palmitic acid) at 35%, followed by 26% of C18:1 (oleic acid) and 16% of
C16:1 (palmitoleic acid). In the neat oil there are three other obvious components
with concentrations around 5 to 6%. These are the C14:0, C15:0 and C18:0 acids.
Also there are 5 other components with concentrations around 1 to 2% including the
C14:1, C15:1, C17:0, C17:1 and C18:2 acids.
It needs to be remembered that these acids are present in the processed oil as free
FAs and of course contribute to the overall acidity of the oil. In general, the free FAs
observed in the methanol oil extract tend to follow the concentrations of those
observed in the hydrolysed sample rather than those of the neat oil, given the slight
variations of intensity that are observed. This at first glance appears to be an unusual
result since the methanol extraction is expected to concentrate the free FAs in the
neat oil by removing the acylglycerides. The extraction does indeed improve the S/N
ratio of the experiment but it also appears to change the relative concentrations of the
free FAs in the oil sample. Our explanation for this unexpected result is that the
methanol extraction is more selective for the unsaturated free FAs resulting in a bias
in the free FA distribution in the methanol extract sampling of the processed
macadamia oil. The observed differences in fatty acid anion composition for
negative-ion ESI FTICR-MS experiments could also be due to the fact that FTICR-
MS is not a good quantitative tool as the errors in peak heights may vary between 5
to 15 percent, another source of observed differences could be due to the fact that the
averaging of the results for several experiments was not performed. However, the
agreement between the negative-ion ESI FTICR-MS results for the methanol extract
and the hydrolysed processed macadamia nut oil is reliable, whereas the results for
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 186
neat macadamia oil are totally different. Hence the results of the fatty acid anions for
the methanol extracted sample could be used to estimate the fatty acid composition
of the oil.
The TAGs account for >98% of the neat oil constituents. The similarity of the
hydrolysis and the esterification processes of neat macadamia oil is that the released
acyl substituents on the TAG molecules dominate the FTICR mass spectra. The
hydrolysate spectrum will contain free fatty acids and the esterified spectrum will
contain FAMEs. It is expected that the FA composition of the FTICR mass spectrum
of the hydrolysed neat oil in both positive and negative-ion modes show similarities
with the positive-ion FTICR mass spectrum of the esterified neat macadamia oil.
Figure 5.14 shows a comparison of the positive and negative-ion FTICR mass
spectra of hydrolysed neat macadamia oil and the positive-ion FTICR mass spectrum
of esterified neat macadamia oil. Table 5.10 lists the observed relative peak
intensities in the FTICR mass spectra of hydrolysed neat processed macadamia oil in
both positive and negative-ion modes and the positive-ion mode FTICR mass
spectrum of the esterified neat processed macadamia oil.
One final comparison can be made between the FAs observed in the hydrolysed
methanol extract negative-ion ESI FTICR spectrum and the literature positive-ion EI
GC-MS result obtained by Cavaletto in 1980.[196] This comparison shows that the six
common fatty acids in both experiments agree to within a few percent. For the most
common acids C16:0:C16:1:C18:1 the ratios are 9:21:62 for the FTICR MS and
7:18:65 for the GC-MS. However as should now be obvious, the negative-ion
FTICR MS provides considerably more detailed information with regard to the minor
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 187
and oxygenated compounds present in the oil than the GC-MS experiment.
Furthermore it is able to identify oxygenated MAGs, DAGs and TAGs in the oil.
In a comparison of the peak intensities in the positive-ion FTICR mass spectra of
esterified neat macadamia oil and the hydrolysed neat oil, the most intense peak in
both spectra is assigned to C18:1 with 61.1% and 61.7% for the hydrolysed and the
esterified oil respectively, followed by C16:1 with 17.2% and 20.2%. The peak
assigned to C18:2 shows a good consistency with 7.9% and 6.7% in hydrolysed and
esterified oil respectively, followed by the peaks assigned to C20:1 with 2.3% and
3.6%. The peaks assigned to C16:0 in both spectra show relative intensities of 5.2%
and 3.0% in hydrolysed and esterified oil respectively.
Comparison of Hydrolysed Oil (+ and -) and Esterified Oil (+) FTICR Mass Spectra
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
C16
:0
C16
:1
C17
:0
C17
:1
C18
:0
C18
:1
C18
:2
C18
:3
C19
:1
C20
:0
C20
:1
C20
:4
Fatty Acids
Rel
ativ
e Pe
ak In
tens
ity
Hydrol. +ve
Hydrol. -ve
Esterf . +ve
Figure 5.14- Comparison of the ESI FTICR mass spectrum of hydrolysed processed macadamia oil in positive and negative-ion modes with esterified neat oil in positive-ion mode
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 188
Table 5.10- Relative intensities of the assigned mass spectral peaks (>2% of the base peak) in the FTICR mass spectra of hydrolysed neat macadamia oil in both positive and negative-ion modes and the esterified oil in positive-ion mode.
Fatty Acid Component
ESI/FTICR-MS Hydrolysed Oil
Positive-ion
ESI/FTICR-MS Hydrolysed oil Negative-ion
ESI/FTICR-MS Esterified oil Positive-ion
C16:0 5.2 8.7 3.0 C16:1 17.2 21.1 20.2 C17:0 1.6 C17:1 3.4 C18:0 1.6 2.5 - C18:1 61.1 61.6 61.7 C18:2 7.9 2.8 6.7 C18:3 1.6 - - C19:1 3.2 - - C20:0 - - C20:1 2.3 2.0 3.6 C20:4 - - -
In the same way, we compare the relative intensities of the observed peaks in the
FTICR mass spectra of the hydrolysed neat macadamia oil in positive and negative-
ion modes. The most intense peak is assigned to C18:1 with 61.1% in positive and
61.6% in negative-ion modes, followed by the peak assigned to C16:1 with 17.2% in
positive and 21.1% in negative-ion modes. The peak assigned to C18:2 shows a
relative intensity of 7.9% in positive and 2.8% in negative-ion mode and the peak
assigned to C16:0 shows 5.2% in positive and 8.7% in negative-ion mode.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 189
Chapter 6
6. Gas Chromatography-Mass Spectrometry of Processed
Macadamia Oil
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 190
6.1- Introduction
In this chapter the GC mass spectra of the esterified neat and methanol extract of the
processed macadamia oil are measured and discussed. A comparison of these results
with the positive-ion and negative-ion ESI FTICR mass spectra of the esterified and
hydrolysed neat processed macadamia oil is also presented.
Due to funding and time limitations only the processed macadamia oil was used for
GC-MS analyses.
Free fatty acids, DAGs and MAGs are not separated well in GC column due to the
high polarity of the acid molecules which binds them to the stationary phase thereby
resulting in poor separation efficiency. Also the TAG molecules have high boiling
points which means they need to be analysed in the GC column at high temperatures
(280 to 360 °C) which can result in the decomposition of temperature-sensitive
molecules in the sample and/or bleeding in the GC column, hence shorter column
life. The answer to this problem of sample polarity and volatility in this type of
analysis is to transesterify the acylglycerols thereby reducing the FA acyl side-chains
to fatty acid methyl esters which are less polar and possess lower boiling points.
It should be noted that it is possible to directly analyse free fatty acids in the oil
samples using special GC columns especially designed for this purpose. In general
such columns are not used routinely due to the complications in the associated mass
spectrometry arising from high temperature such as column bleeding.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 191
The GC-MS results reported in this chapter are usually carried out on the basis of
retention indices, characteristic mass values and intensity ratio of mass peaks
compared to library spectra.
The GC-MS experiments reported in this chapter are to investigate the applicability
of the present ESI FTICR-MS experiments with those used in previous studies on
macadamia oil, in particular GC-MS. As well, we compare our measured oil
composition with other literature studies to provide an independent confirmation of
the actual fatty acid composition of the oil. For example, that the most common FA
in the oil is C18:1 (oleic acid). The chromatographic aspect of the GC-MS
experiment confirms this type of detail.
The GC-MS experiments were performed on a Hewlett-Packard HP 5890 Series II
with a J&W DB-Wax 60 m × 0.5 mm × 0.25 µm column and a VG QUATTRO mass
spectrometer that is discussed in section 2.3.3.
The 70 eV EI mass spectra were assigned from the GC retention indices and by
comparison of the GC-mass spectral m/z and peak intensity values with known
standards and library spectra.
The transesterification process for the oil has been discussed previously in Section
2.2.3.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 192
6.2- GC-MS Analysis of Esterified Processed Macadamia Oil
Figure 6.1 illustrates the GC-MS total ion chromatogram (TIC) of the esterified
processed macadamia oil over a GC retention time of 80 min. The assignments of the
peaks in Figure 6.1 are listed in Table 6.1.
For the sake of space and since only standard well known FAMEs are identified in
the individual EI mass spectra corresponding to the peaks in Figure 6.1, these EI
mass spectra are not reported in this chapter.
Retention time (min)Retention time (min) Figure 6.1- GC-MS TIC of esterified processed macadamia oil.
Table 6.1- Assignment of the peaks in the GC-MS TIC of esterified processed macadamia oil.
Retention time (min) FAME Suggested by the Library Search Routine Peak Area %
52.75 Tetradecanoic acid, C14:0 1.40 59.75 Hexadecanoic acid, C16:0 10.92 60.67 Hexadecenoic acid, C16:1 21.50 66.15 Octadecanoic acid, C18:0 4.67 66.92 Octadecenoic acid, C18:1 58.41 68.08 Octadecdienoic acid, C18:2 1.93 72.68 Eicosanoic acid, C20:0 1.17 73.56 Eicosenoic acid, C20:1 1.18
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 193
The observed peak at 10.8 min in Figure 6.1 is assigned to residual toluene
(esterification solvent). The peak at 49.3 min is assigned to the antioxidant butylated
hydroxytoluene (BHT; 2,6-bis (1,1-dimethylethyl)-4-methylphenol) added to the
esterified oil to stabilize it. Since these materials are not part of the oil sample, they
are not included in Tables 6.1 and 6.2.
The remaining eight peaks in Figure 6.1 are assigned to one C14, two C18 and two
C20 FAMEs. The tetradecanoic ME C14:0, eicosanoic ME C20:0 and eicosenoic ME
C20:1 all represent minor components at peak area percentages of around 1 to 2%.
The two saturated FAMEs hexadecanoic (palmitic) ME, C16:0 and octadecanoic
(stearic) ME, C18:0 are observed at peak area percentages of 11% and 5%
respectively. The largest peak in the trace is assigned to octadecenoic (oleic) ME,
C18:1 at 58%.
6.3- GC-MS Analysis of the Esterified Methanol Extract of Macadamia Oil
Figure 6.2 shows the GC-MS TIC of the esterified methanol extract of processed
macadamia oil. The assignments of the peaks in Figure 6.2 are listed in Table 6.2.
For the sake of space and since only standard well known FAMEs are identified in
the individual EI mass spectra corresponding to the peaks in Figure 6.2, these EI
mass spectra are not reported in this section.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 194
Retention time (min)Retention time (min) Figure 6.2- GC-MS TIC of esterified methanol extract of macadamia oil. Table 6.2- Assignment of the peaks in the GC-MS TIC of esterified methanol extract of processed macadamia oil.
Retention time (min) FAME Suggested by the Library Search Routine Peak Area % 52.66 Tetradecanoic, C14:0 0.73 59.55 Hexadecanoic, C16:0 8.38 60.45 Hexadecenoic, C16:1 26.80 63.15 Hydroxy octadecanoic#, C18:0 6.66 65.84 Heptadecanoic, C17:0 0.88 66.57 Octadecanoic, C18:0 1.98 66.70 Octadecenoic, C18:1 52.03 67.88 Octadecdienoic, C18:2 2.19 70.04 Octadectrienoic, C18:3 0.36
# See text for more discussion The observed peak at 49.3 min is assigned to the antioxidant BHT added to the
esterified oil. The remaining nine peaks in Figure 6.2 are assigned to one C14, two
C16, one C17, four C18 and one hydroxy C18 FAMEs. The tetradecanoic ME C14:0,
the heptadecanoic ME, C17:0, octadecanoic (stearic) ME, C18:0, octadecdienoic
(linoleic) ME, C18:2 and octadectrienoic ME, C18:3 (linolenic) ME, all represent
minor components at peak area percentages of around 1-2%. The two saturated MEs
hexadecanoic (palmitic) ME, C16:0 and hydroxy-octadecanoic (hydroxy-stearic)
ME, HO-C18:0 are observed at peak area percentages of 8% and 7% respectively.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 195
The two largest peaks in the trace are assigned to octadecenoic (oleic) ME, C18:1 at
52% and hexadecenoic (palmitic) ME, C16:1 at 27%.
The EI mass spectrum observed for the peak in Figure 6.2 at a retention time of
63.15 min shows a good match with both that of hydroxy-octadecanoic as well as for
the hydroxy-FAME of this acid. The GC-MS library search routine suggests the
candidates are hydroxy stearic acid with a confidence level of 89% and hydroxy
stearic acid methyl ester with 87% confidence level. However, since a FA is not
expected to be observed in the esterified sample nor operate in the GC column, we
assign it to the FAME.
Interestingly, in the ESI FTICR mass spectrum of the esterified extract of macadamia
oil in Figure 4.10 methyl hydroxy stearate has been assigned with a relative intensity
of 11.4%.
6.4- A Summary of the Positive-ion and Negative-ion ESI FTICR-MS and GC-MS of the Hydrolysed and Esterified Neat Processed Macadamia Oil
In this section, a summary of the common fatty acid substituents on the acylglycerols
as determined by positive-ion and negative-ion ESI FTICR-MS and GC-MS of the
hydrolysed and esterified neat processed macadamia oil is presented and discussed.
We have deliberately chosen these above experiments and samples as they all
involve removal and identification of the FA side-chains from the acylglycerides; the
TAGs in particular. A comparison, with say the esterified extract of the oil, is not
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 196
necessarily expected to reflect the FA composition of the TAG molecules in the oil
sample and this is what is measured in the literature GC-MS experiment.[196]
Figure 6.3 shows a comparison of the FA ions observed in the positive-ion (Tables
4.3 & 4.4) and negative-ion (Table 5.9) ESI FTICR-MS as well as the GC-MS
analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia oil.
Table 6.3 lists the percentages of the fatty acid components used in Figure 6.3. These
results are also compared with the GC-MS measurement by Cavaletto on macadamia
oil reported in 1980.[196]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 197
Comparison of FTICR of Hydrolysed Oil (+ and -), Esterified Oil (+)and GC-MS TIC of the Present Study and the Literature
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0C
14:0
C14
:1
C15
:0
C15
:1
C16
:0
C16
:1
C17
:0
C17
:1
C18
:0
C18
:1
C18
:2
C18
:3
C19
:1
C20
:0
C20
:1
C20
:4
Fatty Acids
Rel
ativ
e Pe
ak In
tens
ity %
Hydrolysed PositiveHydrolysed NegativeEsterfied PositiveGC-MS Present StudyGC-MS Literature
Figure 6.3- A comparison of the FA ions observed in the positive-ion (Tables 4.3 & 4.4) and negative-ion (Table 5.9) FTICR-MS as well as the GC-MS analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia nut oil. GC-MS literature values are from reference 190.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 198
Table 6.3- A comparison of the FA ions observed in the positive-ion (Tables 4.3 & 4.4) and negative-ion (Table 5.9) FTICR-MS as well as the GC-MS analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia nut oil.
Fatty Acid Component
ESI/FTICR-MS Hydrolysed Oil
Positive-ion
ESI/FTICR-MS Hydrolysed oil Negative-ion
ESI/FTICR-MS Esterified oil Positive-ion
GC-MS Esterified oil‡
GC-MS Literature‡
C14:0 Not observed* 2.1 Not observed 1.4 Not observed C14:1 Not observed Not observed Not observed Not observed Not observed C15:0 Not observed Not observed Not observed Not observed Not observed C15:1 Not observed Not observed Not observed Not observed Not observed C16:0 5.2 8.7 3.0 10.9 7.4 C16:1 17.2 21.1 20.2 21.5 18.4 C17:0 Not observed Not observed 1.6 Not observed Not observed C17:1 Not observed Not observed 3.4 Not observed Not observed C18:0 1.6 2.5 Not observed 4.7 2.8 C18:1 61.1 61.6 61.7 58.4 64.9 C18:2 7.9 2.8 6.7 1.9 1.5 C18:3 1.6 Not observed Not observed Not observed Not observed C19:1 3.2 Not observed Not observed Not observed Not observed C20:0 Not observed Not observed Not observed 1.2 Not observed C20:1 2.3 2.0 3.6 Not observed 2.3 C20:4 Not observed Not observed Not observed Not observed 1.9
* Less than the limit of detection Peak intensities % is used for FTICR-MS and peak area % is used for GC-MS Lit. (Cavaletto, Ref. 190) results. ‡ Only fatty acid components are used in calculations and comparisons; fatty acids containing additional oxygen atom are not included.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 199
For this figure, any ions that show evidence of hydration, e.g. C18H31O3¯ are not
included. Unlike the intensity values for the FAs presented the mass spectrometry
tables, the peak intensities in Figure 6.3 have been normalised to 100% for the
various FAs and FAMES.
Figure 6.3 highlights several interesting results from the ESI FTICR MS and GC-MS
experiments discussed previously in this and preceding chapters. If one can directly
correlate the ion concentrations with those of the corresponding acids in the oil then
there is an excellent correlation observed for the FA composition of the oil using
these experiments; all of which involve oil derivatisation. All five experiments show
the major FA component of the processed macadamia oil as oleic acid C18:1 at 62%
±2%, follow by palmitoleic acid C16:1 at 20% ±2% and palmitic acid C16:0 at 7%
±3% where the error is one standard deviation. This is quite a remarkable result
given the literature measurement was nearly 30 years ago.
The minor acids observed such as the C14:0 (myristic), C17:0, C17:1, C18:0 (stearic)
C18:2 (linoleic), C19:1 and C20:0 (eicosanoic), C20:1 (eicosenoic) and C20:4 are
significant in Figure 6.2 since for them to be observed they must be present in the
TAG molecules in reasonable concentrations. The minor acid components C18:0,
C18:2 and C20:1 are detected in several experiments but some acids, e.g. C17:1 and
C20:4 are only observed in a single mass spectrometry experiment.
A comparison of the percentage of the FAME components resulting from GC-MS
analysis of the esterified macadamia oil experiments (Table 6.1) with those obtained
in the ESI FTICR-MS experiment on the esterified macadamia oil (Table 4.4) shows
a remarkably good correlation. For example, the ESI FTICR-MS ratio of oleic /
palmitoleic methyl esters is 100:33 and in the GC-MS experiment it is 100:37
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 200
(58:22). This result gives confidence in the use of the high resolution ESI FTICR
mass spectrometer for the direct semi-quantitative analysis of the macadamia nut oil
(excluding isomers).
One important difference between the GC-MS and FTICR-MS results is that the
former does not indicate the presence of the FA components bearing one or more
additional oxygen components on the acyl chain of the acylglycerols or the free FAs.
To observe these compounds, a higher temperature GC column and/or derivatisation
using trimethylsilane would need to be incorporated into the GC-MS experiment.
Interestingly free FAs are readily observed in the ESI FTICR-MS experiments that
demonstrates the simplicity of the sample preparation in the latter technique for
detailed semi-quantitative analysis of such plant oils.
6.5- Conclusions
GC-MS provides useful information regarding the overall fatty acid composition of
the acylglycerols of the macadamia oil samples through esterification and analysis of
the neat oil. A similar experiment carried out on the methanol extract of the oil can
reveal information about the FA composition of the MAGs and DAGs in the oil.
Finally, a careful GC-MS examination of the methanol extract of the oil could reveal
limited information about the composition of the free FAs in the oil. The
chromatography and the availability of large mass spectral libraries is a major
advantage of this method as it allows the direct confirmation of FAME isomers and
hence the FAs present in the TAG molecules in the oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 201
In general the results of this chapter demonstrate that high resolution ESI FTICR-MS
is a much more sensitive technique than GC-MS which allows the observation of all
the pristine trace components in the oil as well as the production of an accurate FA
profile of the oil when the correct oil samples are analysed. In the latter case, this
involves sampling the hydrolysed or esterified neat oil in either positive-ion or
negative-ion mode. Because the pristine oils can be analysed on ESI FTICR-MS
experiment whereas only the esterified oil can be examined by GC-MS, the former
technique reveals the presence of many more minor components in the macadamia
oil; In particular, the observation of free FAs, MAGs and DAGs and hydrated
derivatives of these materials.
The results reported in this chapter give confidence in the use of the high resolution
ESI FTICR mass spectrometer for the direct semi-quantitative analysis of the FAs in
macadamia nut oil and indeed other plant oils. All five experiments in Figure 6.3
show a very similar fatty acid profile for the processed macadamia oil and this is
quite a remarkable result given the GC-MS literature measurement was obtained
nearly 30 years ago.
However it should be remembered that the ESI FTIC-MS experiments as presented
to date do not distinguish FA isomers and for this chromatography is required. In
Chapter 7, off-line ESI FTICR-MS of HPLC fractions of the methanol extract of
processed macadamia oil is presented and discussed.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 202
Chapter 7
7. Off-line ESI FTICR-MS of HPLC Fractions of the Methanol Extract
of Processed Macadamia Oil
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 203
7.1- Introduction
In this chapter the results of the HPLC separation and the associated FTICR mass
spectra of the methanol extract of the processed macadamia oil fractions are
presented and discussed.
As discussed earlier in section 1.5, previous studies have been carried out on the
application of HPLC in the separation and identification of the plant oil
ingredients,[79,179] particularly in combination with mass spectrometry.[213-215]
There are two major reasons behind the fraction collection experiment performed in
this present study.
First, online HPLC ESI FTICR-MS was not practical with the FTICR instrument
used. As Figure 2.11 shows, a FTICR-MS pulse sequence to obtain a broadband
mass spectrum takes about 5 seconds to complete. For this present analysis it was
found that an accumulation time in the hexapole ion guide of 3 to 4 seconds was
needed to obtain sufficient signal intensity for the analysis of the macadamia oil
samples. Furthermore, the scan is repeated 50 times to increase the signal/noise ratio
in the FT mass spectra. Overall, the accumulation of a spectrum could take up to 5
minutes. This time is too long for online HPLC ESI FTICR mass spectrometry
because during the 5 minute period, more than one compound might elute out of the
HPLC column and this is contrary to the degree of separation expected for this
experiment. The only practical option in this case was to collect the fractions and
analyse each fraction on the ESI FTICR mass spectrometer at a later time (off-line
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 204
mode). Newer instruments have been developed and used to perform online HPLC
FTICR-MS in the analysis of biochemical samples.[216]
Secondly, to overcome possible ion charge cloud effects in the FTICR cell the
methanol extract was fractionated to reduce the total number of ions with a wide
range of concentration (peak intensities) in the FTICR cell. The ion charge cloud
effect can result in a low signal to noise ratio for the low-concentration ions in the
FTICR cell (signal suppression).[193,217,218]
In addition, possible isomers could be isolated and collected using HPLC technique.
The isomers of unsaturated fatty acids and acylglycerols can arise from different
double bond locations on the FA chain or different substitution positions of FA
substituents on the glycerol molecule.
For the experiments described in this chapter, 90 fractions of one minute duration of
the methanol extract of processed macadamia oil were collected using a dedicated
fraction collector (Section 2.3.2). The Majority of the collected fractions were
subsequently analysed in the ESI FTICR mass spectrometer in both negative- and
positive-ion modes without dilution. The mass spectra obtained are discussed in
conjunction with the HPLC chromatograms in this chapter. A complete set of
positive- and negative-ion FTICR-MS data files can be found in Appendix DVD.
Note that the assignments in the tables are postulations and no further CID or
MS/MS analysis has been conducted to elucidate the chemical structures of the ions.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 205
7.2- HPLC of the Methanol Extract of Processed Macadamia Oil
The HPLC chromatogram of the methanol extract of processed macadamia oil is
shown in Figure 7.1. The specific ion assignments for the peaks shown in Figure 7.1
are based on the ESI FTICR mass spectra discussed in more detail in section 7.3 to
7.7 and listed in Tables 7.1 to 7.6. The HPLC experiment was carried out seven times
to verify the reproducibility of the separation. The retention times were found to be
reproducible to 0.1%.
Considerable time was invested in the HPLC method development in an attempt to
separate the large number of compounds in plant oils. In fact this method enables
FFAs and MAGs to be separated from DAGs. This method allows oil samples from
different batches of the same oil to be clearly differentiated, but no chromatographic
technique will separate all the components in plant oil samples. In order to improve
the separation of compounds in plant oils several different sample preparation
methods and HPLC techniques would have to be used, even then it is not possible to
separate all of the compounds into single peaks.
Assuming that the eluted components possess similar solubilities in the mobile phase
and separation occurs exclusively according to the hydrophobicities, it is expected
that the relatively less hydrophobic molecules such as free FAs, highly unsaturated
esters and MAGs will elute earlier on a C18 reversed-phase column, followed by the
more hydrophobic DAGs and TAGs. A number of peaks, assigned to residual TAG
molecules, appear in the chromatogram at retention times 78-84 minutes; the
methanol extraction procedure (section 2.2.1) removes the majority of the TAGs
from the processed oil sample. On further examination of the peak shapes when the
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 206
chromatogram was expanded, it was observed that each major peak did contain in
general more than one chemical compound, in fact several compounds.
Three distinct regions are visible in Figure 7.1, containing free FAs and MAGs,
DAGs and TAGs. Each region is discussed in more detail in sections 7.3 to 7.7.
0 20 40 60 80Retention time (min)
29.33
54.4458.16
52.01
62.61
80.83
23.91
0 20 40 60 80Retention time (min)
0 20 40 60 80Retention time (min)
29.33
54.4458.16
52.01
62.61
80.83
23.91
Figure 7.1- HPLC chromatogram of the methanol extract of processed macadamia oil.
The first four eluted HPLC fractions are considered as blanks for the ESI FTICR
mass spectrometry analyses. The HPLC solvent system (methanol-water, 0.05% in
acetic acid) contains a number of compounds which contribute to the peaks in the
blank FTICR mass spectra (Figures 7.16 and 7.17). These background peaks are
excluded from the mass spectra of the macadamia oil samples. The positive-ion and
Glycerol Palmitoleate-oleate Glycerol dioleate
Oleic acid
Glycerol Oleate
Glycerol dipalmitoleate
Triacylglycerols Free fatty acids and MAGs
Diacylglycerols
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 207
negative-ion FTICR mass spectra of the blank fractions are shown in Figures 7.16
and 7.17 in Section 7.8.
7.3- ESI FTICR-MS of the HPLC Fractions 19 to 31 of the Methanol Extract of Processed Macadamia Oil
Figure 7.2 shows an expansion of the HPLC chromatogram of the methanol extract
of processed macadamia oil (Figure 7.1) at retention times 19 to 31 minutes.
20.90
22.51
23.90
25.02
29.30
Retention time (min)20 25 30
20.90
22.51
23.90
25.02
29.30
Retention time (min)20 25 30
Figure 7.2- Expansion of HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times of 19 to 31 minutes.
The more intense peaks in Figure 7.2 are observed at retention times 20.9 minutes
(fractions 20 and 21), 23.9 minutes (fractions 23 and 24) and 29.3 minutes (fractions
29 and 30). Figure 7.3 shows the positive-ion FTICR mass spectra and Figure 7.4
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 208
shows the negative-ion FTICR mass spectra of the HPLC fractions 19 to 31
(retention times 19 to 31 minutes).
407.3152327.2286305.2468
379.2834
353.2668
277.2144 299.1956
377.2651
m/z270 280 290 300 310 320 330 340 350 360 370 380 390 400
Fraction 19
Fraction 31
Fraction 25
407.3152327.2286305.2468
379.2834
353.2668
277.2144 299.1956
377.2651
m/z270 280 290 300 310 320 330 340 350 360 370 380 390 400
Fraction 19
Fraction 31
Fraction 25
Figure 7.3- Positive-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 209
Fraction 31
281.2486
253.2173
Fraction 19
m/z250 300 350270260 290280240 320310 330 340 360
317.2264
Fraction 31
281.2486
253.2173
Fraction 19
m/z250 300 350270260 290280240 320310 330 340 360
m/z250 300 350270260 290280240 320310 330 340 360
317.2264
Figure 7.4- Negative-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil. Table 7.1 lists the assignments of the observed peaks in the positive-ion FTICR mass
spectra of HPLC fractions 19 to 31 and Table 7.2 lists the assignments of the
observed peaks in the negative-ion FTICR mass spectra of HPLC fractions 19 to 31.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 210
Table 7.1. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.3.
Fraction Observed Mass m/z
Assigned Sodium Adduct Cation
19 377.2655 C21H38O4 Glycerol linoleate 20 277.2127 C16H30O2 Palmitoleic acid 20 299.1956 C18H28O2 Octadecatetraenoic acid 21 277.2142 C16H30O2 Palmitoleic acid 21 353.2668 C19H38O4 Glycerol palmitate 21 379.2820 C21H40O4 Glycerol oleate 22 353.2665 C19H38O4 Glycerol palmitate 22 379.2809 C21H40O4 Glycerol oleate 23 379.2809 C21H40O4 Glycerol oleate 24 379.2823 C21H40O4 Glycerol oleate 25 379.2830 C21H40O4 Glycerol oleate 26 379.2834 C21H40O4 Glycerol oleate 27 305.2457 C18H34O2 Oleic acid 27 379.2840 C21H40O4 Glycerol oleate 28 305.2455 C18H34O2 Oleic acid 28 349.2928 C21H40O2 Heneicosenoic acid 28 361.2720 C21H38O3 Hydroxy heneicosadienoic acid 28 379.2840 C21H40O4 Glycerol oleate 29 305.2455 C18H34O2 Oleic acid 29 361.2726 C21H38O3 Hydroxy heneicosadienoic acid 29 381.2993 C21H42O4 Glycerol stearate 30 381.3001 C21H42O4 Glycerol stearate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 211
Table 7.2. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.4.
Fraction Observed Mass m/z
Assigned Anion
19 277.2169 C18H29O2‾, Linolenate, C18:3 20 253.2173 C16H29O2‾, Palmitoleate, C16:1 20 277.2177 C18H29O2‾, Linolenate, C18:3 21 253.2173 C16H29O2‾, Palmitoleate, C16:1 22 253.2173 C16H29O2‾, Palmitoleate, C16:1 22 279.2329 C18H31O2‾, Linoleate, C18:2 23 279.2341 C18H31O2‾, Linoleate, C18:2 24 279.2328 C18H31O2‾, Linoleate, C18:2 25 255.2329 C16H31O2‾, Palmitate, C16:0 26 255.2328 C16H31O2‾, Palmitate, C16:0 27 255.2329 C16H31O2‾, Palmitate, C16:0 27 281.2486 C18H33O2‾, Oleate, C18:1 28 255.2328 C16H31O2‾, Palmitate, C16:0 28 281.2486 C18H33O2‾, Oleate, C18:1 29 281.2486 C18H33O2‾, Oleate, C18:1 30 281.2486 C18H33O2‾, Oleate, C18:1
Figure 7.2 shows a peak at retention time 20.9 minutes. Analogous peaks are
observed in the positive-ion FTICR mass spectra of fractions 20 and 21 at m/z
277.2127 and m/z 277.2142 in Figure 7.3 that are assigned to the palmitoleic acid
sodium adduct cation in Table 7.1. In addition, analogous peaks are observed in the
negative-ion FTICR mass spectra of fractions 20 and 21 at m/z 253.2184 in Figure
7.4 that are assigned to palmitoleate anion in Table 7.2.
The peak observed at retention time 23.9 minutes in the expanded HPLC
chromatogram in Figure 7.2 shows an analogous peak in fractions 23 and 24 in the
positive-ion FTICR mass spectra in Figure 7.3 at m/z 379.2834 that is assigned to
glycerol oleate in Table 7.1.
The peak at retention time 29.3 minutes in the chromatogram Figure 7.2 shows an
analogous peak in the positive-ion FTICR mass spectrum of fraction 29 at m/z
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 212
305.2455 in Figure 7.3 that is assigned to oleic acid sodium adduct cation in Table
7.1. In addition, in the negative-ion FTICR mass spectra in Figure 7.4, a peak
appears in fractions 28 and 29 at m/z 281.2492 that is assigned to oleate anion in
Table 7.2.
7.4- ESI FTICR-MS of the HPLC Fractions 32 to 50 of the Methanol Extract of Processed Macadamia Oil
Figure 7.5 shows an expansion of the high performance liquid chromatogram of the
methanol extract of processed macadamia oil (Figure 7.1) at retention times 32 to 50
minutes.
30 35 40 45 50
32.94
37.2738.85
42.6744.29
46.79
47.71
Retention time (min)
30 35 40 45 50
32.94
37.2738.85
42.6744.29
46.79
47.71
Retention time (min) Figure 7.5- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times 32 to 50 minutes.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 213
7.4.1- Fractions 32 to 35
In the positive-ion FTICR mass spectra of the fractions 32 to 35 all the observed
peaks appear weaker than the background peaks.
In the negative-ion FTICR mass spectra of HPLC fractions 32 to 35, a weak peak
appears at m/z 283.2653 in fractions 34 and 35 that is assigned to stearic acid anion.
Figure 7.6 shows the negative-ion FTICR mass spectra of fractions 34 and 35.
223.0198 255.2330
283.2642
305.0243 325.1846 339.2007365.2676
223.0197255.2332
283.2642309.2804
319.2403 345.2577 377.0852
220 240 260 280 300 320 340 360 380m/z
(a)
(b)
223.0198 255.2330
283.2642
305.0243 325.1846 339.2007365.2676
223.0197255.2332
283.2642309.2804
319.2403 345.2577 377.0852
220 240 260 280 300 320 340 360 380m/z
(a)
(b)
Figure 7.6- Negative-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 34 and (b) fraction 35 of the methanol extract of processed macadamia nut oil.
The peak at m/z 283.2642 is assigned to stearic acid anion, C18H35O2¯ (C18:0), and
the peak at m/z 309.2804 is assigned to eicosenoic acid anion, C20H37O2¯ (C20:1).
7.4.2- Fractions 36 to 38
Figure 7.7 shows the positive-ion FTICR mass spectra of the HPLC fractions 36 to
38 of the methanol extract of processed macadamia oil and Table 7.3 lists the
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 214
assignments of the observed peaks in the positive-ion FTICR mass spectra of HPLC
fractions 36 to 38.
603.4734 619.4605
643.4551
671.4958
705.5375
721.5297
601.4527
619.4606647.4999
673.5103
689.5142
705.5376 721.5298
601.4527
605.4836
629.4817 647.5010
673.5110
689.5150
705.5380721.5301
600 620 640 660 680 700 720 740m/z
(a)
(c)
(b)
603.4734 619.4605
643.4551
671.4958
705.5375
721.5297
601.4527
619.4606647.4999
673.5103
689.5142
705.5376 721.5298
601.4527
605.4836
629.4817 647.5010
673.5110
689.5150
705.5380721.5301
600 620 640 660 680 700 720 740m/z
603.4734 619.4605
643.4551
671.4958
705.5375
721.5297
601.4527
619.4606647.4999
673.5103
689.5142
705.5376 721.5298
601.4527
605.4836
629.4817 647.5010
673.5110
689.5150
705.5380721.5301
600 620 640 660 680 700 720 740m/z
(a)
(c)
(b)
Figure 7.7- Positive-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 36, (b) fraction 37 and (c) fraction 38 of the methanol extract of processed macadamia nut oil.
Table 7.3. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 36 to 38 in Figure 7.7.
Fraction Observed Mass m/z
Assigned Sodium Adduct Cation
36 619.4605 C35H64O7 Glycerol dihydroxy dipalmitoleate 36 671.4958 C39H68O7 Glycerol dihydroxy dilinoleate 37 601.4527 C35H62O6 Glycerol hydroxy palmitoleate palmitolenate 37 673.5063 C39H70O7 Glycerol dihydroxy oleate linoleate 37 705.5376 C40H74O8 Glycerol trihydroxy oleate nonadecenoate 38 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 215
7.4.3- Fractions 38 to 40 Figure 7.8 shows the positive-ion FTICR mass spectra of the HPLC fractions 38 to
40 of the methanol extract of processed macadamia oil and Table 7.4 lists the
assignments of the observed peaks in Figure the positive-ion FTICR mass spectra of
HPLC fractions 38 to 40.
601.4563
605.4836
629.4817 647.5030
673.5110
689.5150
705.5334721.5311
605.4872
629.4836 647.5011
673.5119
689.5131
605.4874
627.4689631.5015
647.5023
655.5054
673.5119
689.5134
600 620 640 660 680 700 720 740m/z
(a)
(c)
(b)
601.4563
605.4836
629.4817 647.5030
673.5110
689.5150
705.5334721.5311
605.4872
629.4836 647.5011
673.5119
689.5131
605.4874
627.4689631.5015
647.5023
655.5054
673.5119
689.5134
600 620 640 660 680 700 720 740m/z
(a)
(c)
(b)
(a)
(c)
(b)
Figure 7.8- Positive-ion ESI FTICR mass spectra of the HPLC fractions, (a) fraction 38, (b) fraction 39 and (c) fraction 40 of the methanol extract of processed macadamia nut oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 216
Table 7.4. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 38 to 40 in Figure 7.8.
In the negative-ion FTICR mass spectra of HPLC fractions 38 to 40, only fraction 40
contains peaks with intensities above the background level. Figure 7.9 shows the
negative-ion FTICR mass spectrum of the HPLC fraction 40 of the methanol extract
of processed macadamia oil.
Table 7.5 lists the assignments of the observed peaks in the negative-ion FTICR
mass spectrum of HPLC fraction 40.
Table 7.5. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectrum of the HPLC fraction 40 in Figure 7.9.
Fraction Observed Mass m/z
Assigned Sodium Adduct Cation
38 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate 38 629.4817 C37H66O6 Glycerol hydroxy oleate palmitate 38 689.5150 C43H70O5 Glycerol diarachidonate 39 673.5063 C39H70O7 Glycerol dihydroxy oleate linoleate 39 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate 40 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate 40 655.5054 C39H68O6 Glycerol hydroxy dilinoleate 40 671.4959 C39H68O7 Glycerol dihydroxy dilinoleate 40 673.5063 C39H70O7 Glycerol dihydroxy oleate linoleate
Fraction Observed Mass m/z
Assigned Negative Ion
40 617.4314 C37H61O7 Glycerol dihydroxy linolenate palmitolenate 40 683.4381 C41H63O8 Glycerol trihydroxy linolenate arachidonate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 217
617.4314
683.4381
600 620 640 660 680 700 720 740m/z
617.4314
683.4381
600 620 640 660 680 700 720 740m/z
Figure 7.9- Negative-ion ESI FTICR mass spectrum of the HPLC fraction 40 of the methanol extract of processed macadamia oil.
7.4.4- Fractions 44 to 48
Figure 7.10 shows the positive-ion FTICR mass spectra of the HPLC fractions 44 to
48 of the methanol extract of processed macadamia oil.
661.5551
657.5162
607.5044
633.5147
673.5124
m/z600 650640630620610 670 690680660 700
659.5363
Fraction 44
Fraction 48
661.5551
657.5162
607.5044
633.5147
673.5124
m/z600 650640630620610 670 690680660 700
659.5363
661.5551
657.5162
607.5044
633.5147
673.5124
m/z600 650640630620610 670 690680660 700
m/z600 650640630620610 670 690680660 700
659.5363
Fraction 44
Fraction 48
Figure 7.10- Positive-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 218
Table 7.6 lists the assignments of the observed peaks in the positive-ion FTICR mass
spectra of HPLC fractions 44 to 48.
Table 7.6. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 44 to 48 in Figure 7.10.
Figure 7.11 shows the negative-ion FTICR mass spectra of the HPLC fractions 44 to
48 of the methanol extract of processed macadamia oil and Table 7.7 lists the
assignments of the observed peaks in Figure 7.11.
Fraction Observed Mass m/z
Assigned Sodium Adduct Cation
44 629.4752 C37H66O6 Glycerol hydroxy oleate palmitate 44 657.5202 C39H70O6 Glycerol hydroxy oleate linoleate 44 673.5124 C39H70O7 Glycerol dihydroxy oleate linoleate 45 631.4987 C37H68O6 Glycerol hydroxy oleate palmitoleate 45 629.4752 C37H66O6 Glycerol hydroxy oleate palmitate 45 657.5199 C39H70O6 Glycerol hydroxy oleate linoleate 45 659.5360 C39H72O6 Glycerol hydroxy oleate oleate 45 673.5124 C39H70O7 Glycerol dihydroxy oleate linoleate 45 675.5281 C39H72O7 Glycerol dihydroxy dioleate 46 631.4987 C37H68O6 Glycerol hydroxy oleate palmitoleate 46 633.5185 C37H70O6 Glycerol hydroxy oleate palmitate 46 655.4984 C39H68O6 Glycerol hydroxy linoleate linoleate 46 657.5185 C39H70O6 Glycerol hydroxy oleate linoleate 46 659.5360 C39H72O6 Glycerol hydroxy dioleate 46 675.5244 C39H72O7 Glycerol dihydroxy dioleate 47 631.4987 C37H68O6 Glycerol hydroxy oleate palmitoleate 47 635.5287 C37H72O6 Glycerol hydroxy stearate palmitate 47 657.5162 C39H70O6 Glycerol hydroxy oleate linoleate 47 659.5218 C39H72O6 Glycerol hydroxy dioleate 47 661.5550 C39H74O6 Glycerol hydroxy oleate stearate 48 635.5304 C37H72O6 Glycerol hydroxy stearate palmitate 48 661.5550 C39H74O6 Glycerol hydroxy oleate stearate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 219
673.5209
697.5669
647.4976
695.5555711.5540
645.4973
669.5363
680 690 700 710 720m/z670660650640
Fraction 44
Fraction 48
673.5209
697.5669
647.4976
695.5555711.5540
645.4973
669.5363
680 690 700 710 720m/z670660650640
Fraction 44
Fraction 48
Figure 7.11- Negative-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.
Table 7.7. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 44 to 48 in Figure 7.11.
Fraction Observed Mass m/z
Assigned Anion
44 645.4973 C39H65O7 Glycerol dihydroxy linoleate linolenate 44 647.4976 C39H67O7 Glycerol dihydroxy dilinoleate 44 669.5363 C39H73O8 Glycerol hydroxy oleate dihydroxy stearate 45 645.4950 C39H65O7 Glycerol dihydroxy linoleate linolenate 45 669.5396 C39H73O8 Glycerol hydroxy oleate dihydroxy stearate 45 695.5555 C41H75O8 Glycerol trihydroxy arachidate linoleate 45 711.5540 C41H75O9 Glycerol tetrahydroxy arachidate linoleate 46 669.5312 C39H73O8 Glycerol hydroxy oleate dihydroxy stearate 46 695.5453 C41H75O8 Glycerol trihydroxy arachidate linoleate 47 673.5213 C41H69O7 Glycerol dihydroxy arachidonate oleate 47 697.5624 C41H77O8 Glycerol trihydroxy arachidate oleate 48 647.5013 C39H67O7 Glycerol dihydroxy dilinoleate 48 673.5209 C41H69O7 Glycerol dihydroxy arachidonate oleate 48 697.5669 C41H77O8 Glycerol trihydroxy arachidate oleate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 220
7.5- ESI FTICR-MS of the HPLC Fractions 50 to 60 of the Methanol Extract of Processed Macadamia Oil
Figure 7.12 shows an expansion of the HPLC chromatogram of the methanol extract
of processed macadamia oil (Figure 7.1) at retention times 50 to 60 minutes.
Retention time (min)
6050 55
50.65
51.96
54.45 58.05
Retention time (min)
6050 55
Retention time (min)
6050 55 6050 55
50.65
51.96
54.45 58.05
Figure 7.12- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 50 to 60 minutes.
Four major peaks are observed in the HPLC chromatogram in Figure 7.12 at
retention times approximately 51, 52, 54 and 58 minutes.
Figure 7.13 shows the positive-ion FTICR mass spectra of the HPLC fractions 50 to
60 corresponding to Figure 7.12 and Table 7.8 lists the assignments of the observed
peaks in Figure 7.13.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 221
561.4554
661.5547
643.5359
615.5078
587.4741
689.5858
560 580 600 620 640 660 680 700m/z
Fraction 50
Fraction 60
561.4554
661.5547
643.5359
615.5078
587.4741
689.5858
560 580 600 620 640 660 680 700m/z
Fraction 50
Fraction 60
Figure 7.13- Positive-ion ESI FTICR mass spectra of the HPLC fractions 50 to 60 of the methanol extract of processed macadamia oil. Four distinct peaks are observed in the FTICR mass spectra in Figure 7.13 in
fractions 50, 51, 54 and 57 that is consistent with the observed peaks in the
chromatogram in Figure 7.12.
According to the assignments in Table 7.8, most of the observed peaks at retention
times 50 to 60 minutes are assigned to DAGs such as glycerol dioleate (m/z
643.5354) and few have been assigned to hydroxy DAGs such as Glycerol hydroxy
dioleate (m/z 659.5326).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 222
Table 7.8. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 50 to 60 in Figure 7.13.
7.6- ESI FTICR-MS of the HPLC Fractions 60 to 63 of the Methanol Extract of Processed Macadamia Oil
A peak is observed in Figure 7.1 at retention time 62.6 minutes. Figure 7.14 shows
the positive-ion FTICR mass spectra of the HPLC fractions 60 to 63 of the methanol
Fraction Observed Mass m/z
Assigned Sodium Adduct Cation
50 561.4554 C33H62O5 Glycerol tetradecanoate palmitoleate 50 587.4738 C35H64O5 Glycerol dipalmitoleate 50 661.5547 C39H74O6 Glycerol hydroxy stearate oleate 51 561.4575 C33H62O5 Glycerol tetradecanoate palmitoleate 51 587.4741 C35H64O5 Glycerol dipalmitoleate 51 659.5363 C39H72O6 Glycerol hydroxy oleate oleate 52 587.4733 C35H64O5 Glycerol dipalmitoleate 52 613.4888 C37H66O5 Glycerol palmitoleate linoleate 52 639.5086 C39H68O5 Glycerol dilinoleate 52 659.5326 C39H72O6 Glycerol hydroxy oleate oleate 52 663.5629 C39H76O6 Glycerol hydroxy stearate stearate 52 689.5754 C41H78O6 Glycerol hydroxy oleate arachidate 53 589.4900 C35H66O5 Glycerol palmitate palmitoleate 53 615.5064 C37H68O5 Glycerol palmitoleate linoleate 53 639.5086 C39H68O5 Glycerol dilinoleate 54 589.4889 C35H66O5 Glycerol palmitate palmitoleate 54 615.5078 C37H68O5 Glycerol palmitoleate oleate 54 641.5207 C39H70O5 Glycerol oleate linoleate 55 615.5066 C37H68O5 Glycerol palmitoleate oleate 55 641.5191 C39H70O5 Glycerol oleate linoleate 56 617.5202 C37H70O5 Glycerol palmitate oleate 56 643.5354 C39H72O5 Glycerol dioleate 57 617.5226 C37H70O5 Glycerol palmitate oleate 57 643.5359 C39H72O5 Glycerol dioleate 58 617.5208 C37H70O5 Glycerol palmitate oleate 58 643.5355 C39H72O5 Glycerol dioleate 59 643.5340 C39H72O5 Glycerol dioleate 60 643.5337 C39H72O5 Glycerol dioleate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 223
extract of processed macadamia oil. Table 7.9 lists the assignment of the observed
peak in Figure 7.14.
645.5562
671.5712
685.5493
643.5337
630 640 650 660 670 680 690 700m/z
Fraction 60
Fraction 63
645.5562
671.5712
685.5493
643.5337
630 640 650 660 670 680 690 700m/z
630 640 650 660 670 680 690 700m/z
Fraction 60
Fraction 63
Figure 7.14- Positive-ion ESI FTICR mass spectra of the HPLC fractions 60 to 63 of the methanol extract of processed macadamia oil.
Table 7.9. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 60 to 63 in Figure 7.14.
In the negative-ion FTICR mass spectra of the HPLC fractions 60 to 63 of the
methanol extract of macadamia oil, only background peaks are observed.
Fraction Observed Mass m/z
Assigned Sodium Adduct Cation
60 643.5337 C39H72O5 Glycerol dioleate 61 645.5562 C39H74O5 Glycerol oleate stearate 61 671.5712 C41H76O5 Glycerol arachidate linoleate 61 685.5493 C41H74O6 Glycerol hydroxy eicosenoate linoleate 62 671.5712 C41H76O5 Glycerol arachidate linoleate 63 645.5562 C39H74O5 Glycerol oleate stearate 63 671.5712 C41H76O5 Glycerol arachidate linoleate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 224
7.7- ESI FTICR-MS of the HPLC Fractions 77 to 83 of the Methanol Extract of Processed Macadamia Oil
Figure 7.15 shows an expansion of the HPLC chromatogram of the methanol extract
of processed macadamia oil (Figure 7.1) at retention times 77 to 83 minutes.
Retention time (min)
75 80 85
79.50
80.22
80.76
81.30
82.67
Retention time (min)
75 80 85
79.50
80.22
80.76
81.30
82.67
Figure 7.15- Expanded HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 77 to 83 minutes.
Four major peaks are observed in the chromatogram in Figure 7.15 at retention times
approximately 79, 80 and 81 minutes. We examine the FTICR mass spectra of the
HPLC fractions 77 to 83 for possible corresponding peaks.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 225
Figure 7.16 shows the positive-ion FTICR mass spectra of the HPLC fractions 77 to
83 of the methanol extract of processed macadamia oil and Table 7.10 lists the
assignment of the observed peak in Figure 7.16.
851.7301 879.7475
923.7823
871.7582
897.7740
921.7902
m/z850 860 870 880 890 900 910 920 930
Fraction 77
Fraction 83
851.7301 879.7475
923.7823
871.7582
897.7740
921.7902
m/z850 860 870 880 890 900 910 920 930
m/z850 860 870 880 890 900 910 920 930850 860 870 880 890 900 910 920 930
Fraction 77
Fraction 83
Figure 7.16- Positive-ion ESI FTICR mass spectra of the HPLC fractions 77 to 83 of the methanol extract of processed macadamia oil.
Table 7.10. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 77 to 83 in Figure 7.16.
Fraction Observed Mass m/z
Assigned Sodium Adduct Cation
77 871.7582 C53H100O7 Glycerol dipalmitate hydroxy oleate 77 897.7740 C55H102O7 Glycerol dioleate hydroxy palmitate 77 921.7902 C58H106O6 Glycerol dioleate nonadecenoate 78 871.7582 C53H100O7 Glycerol dipalmitate hydroxy oleate 78 897.7740 C55H102O7 Glycerol dioleate hydroxy palmitate 78 921.7902 C58H106O6 Glycerol dioleate nonadecenoate 79 897.7740 C55H102O7 Glycerol dioleate hydroxy palmitate 79 923.7823 C57H104O7 Glycerol dioleate hydroxy oleate
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 226
In the negative-ion FTICR mass spectra of the HPLC fractions 77 to 83, the intensity
of the observed peaks are lower than the background peaks.
All of the observed peaks in the positive-ion FTICR mass spectra of the HPLC
fractions 77 to 83 are assigned to TAG molecules in Table 7.10. Additionally, a
number of TAG molecules with additional oxygen bearing functional groups are
assigned to the peaks in the HPLC fractions 77 to 83.
7.8- Positive- and Negative-ion FTICR Mass Spectra of the HPLC Blank Fractions of the Methanol Extract of Processed Macadamia Oil Figure 7.17 shows the positive-ion FTICR mass spectrum of the HPLC blank
fraction.
268.9991301.1401
312.0576
344.9841
413.2671
441.2145455.2234
525.1366
550.6269
250 300 350 400 450 500 550 600m/z
268.9991301.1401
312.0576
344.9841
413.2671
441.2145455.2234
525.1366
550.6269
250 300 350 400 450 500 550 600m/z Figure 7.17- Positive-ion FTICR mass spectrum of the HPLC blank fraction. See text for more discussion.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 227
The peak at m/z 413.2671 in the blank spectrum could be assigned to C24H38O4Na+
cation (2 ppm). The absolute intensity of the peak at m/z 413.2671 is about 1.2×107
which is considered a weak peak.
The background noise is visible in Figure 7.17 due to the fact that the peaks are weak
and the auto-scale function of the software has automatically expanded the intensities
to the full-scale.
Figure 7.18 shows the negative-ion FTICR mass spectrum of the HPLC blank
fraction.
223.0213
255.2343
283.2657
305.0247
387.0281463.0191
545.0138 627.0305
250 300 350 400 450 500 550 600 650m/z
223.0213
255.2343
283.2657
305.0247
387.0281463.0191
545.0138 627.0305
250 300 350 400 450 500 550 600 650m/z Figure 7.18- Negative-ion FTICR mass spectrum of the HPLC blank fraction. The intensity of the main peak at m/z 255.2343 is about 5×107 that is considered a weak peak.
The peak at 255.2343 could be assigned to C16H31O2¯ (5 ppm) that could be
palmitate anion. As the solvent contains 0.05% (v/v) acetic acid, the source of the
palmitate anion could be the impurities in the acetic acid.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 228
7.9- General Discussion:
Table 7.11 lists a summary of the assignments of the major FTICR-MS peaks
observed in HPLC fractions of the methanol extract of processed macadamia oil in
both positive- and negative-ion modes. Since the HPLC peaks are broad, some of the
assigned peaks are observed in more than one fraction. Furthermore, as each fraction
contains one minute elution off the column, all of the fractions contain more than one
compound. It should be noted that due to the complexity of the chemical composition
of the oil samples, it is not plausible to separate all of the components in the oil
samples using reversed-phase HPLC. The lipid classes (such as FAs, MAGs, DAGs
and TAGs etc.) could be separated as groups. These groups could be separated and
analysed later using more sophisticated chromatography techniques. Time limitations
did not allow us to perform all of the possible separations in the present study.
Using acetic acid in the HPLC mobile phases suppresses the ionization of the free
FAs, hence assists in obtaining sharper peaks in the chromatograms. However, the
stationary phase of the Beckman C18 Ultrasphere reversed-phase column is not fully
end-capped, thus residual silanol groups are present on the surface of the stationary
phase material. These residual silanol groups will interact with the less hydrophobic
(more polar) molecules and cause significant peak broadening observed in this study.
In the negative-ion FTICR-MS, the anion is generated in a deprotonation reaction.
The proton is transferred from the neutral oil species to a solvent molecule in an
equilibrium reaction (affected by pH). As the HPLC solvent is 0.05% (v/v) in acetic
acid, the ionization equilibria of the weaker acids (such as hydroxyl groups on the
glycerol molecule) are pushed to the left. This suppresses the ionisation reactions of
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 229
the species with a proton on hydroxyl groups. As a result, in the negative-ion FTICR-
MS of the methanol extract of processed macadamia oil, only a few free fatty acids
are observed in Table 7.11.
In the positive-ion FTICR-MS, on the other hand, the attachment of sodium cation to
the non-bonding electrons on the oxygen atoms of free fatty acids and acylglycerols
generates the positive ion sodium adduct. As the attachment of sodium ion is a
simple dipole-dipole interaction and is not affected by the presence of acetic acid in
the solvent, positive ions are observed for free fatty acids and acylglycerols in Table
7.11.
According to Table 7.11, most of the compounds occur in more than one fraction. As
an example, the peak assigned to glycerol palmitate (m/z 353.2668) appears in
fractions 20 to 22 in Table 7.11. This is due to the fact that, in general, each peak
contains more than one compound, due to the isomers and co-eluting peaks[172]
(compounds with different chemical structure but with same retention times) in one
minute fraction collecting time.
Each fraction contains more than one compound due to the complex composition of
the macadamia oil samples. Some of the compounds have different structures but
similar retention times. In addition, some isomers are eluted at the same retention
times (fractions). For example, the peak assigned to glycerol oleate (m/z 379.2834)
appears in fractions 20 to 28 (19 to 28 minutes) in Table 7.11 that is an indication of
the existence of isomers. Several isomers of this compound could arise from different
double bond or side chain locations on the FA chain or different substitution sites on
carbon 1 or carbon 2 of the glycerol backbone (MAG with the substituent on carbon
3 is identical to the one with the substituent on carbon 1).
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 230
Table 7.11. Assignment of the major mass spectral peaks in the ESI-FTICR mass spectra of the HPLC fractions of the methanol extract of macadamia nut oil.
* See text for more discussion
The FTICR-MS analysis of the HPLC fractions 19 to 31 in positive-ion mode shows
five free FAs, MAGs and one hydroxy fatty acid (Table 7.1). In negative-ion mode
FTICR-MS of the fractions 19 to 31 shows only free fatty acids, mostly anions of
palmitic, palmitoleic, linolenic, linoleic and oleic acids (Table 7.2).
Observed in Fraction Numbers*
Assigned Compound
Assigned Positive-ion Mass
m/z
Assigned Negative-ion Mass
m/z Positive-
ion Negative-
ion
Palmitoleic acid C16:1 277.2144 253.2184 20, 21 20 to 22 Palmitic acid C16:0 279.2295 255.2341 - 25 to 28 Linolenic acid C18:3 301.2134 277.2169 19, 20 19, 20 Linoleic acid C18:2 303.2288 279.2340 22 to 24 22 to 24 Oleic acid C18:1 305.2468 281.2492 27, 28 27 to 32 Stearic acid C18:0 307.2627 283.2653 - 34, 35 Glycerol palmitoleate 351.2490 - 15 to 18 - Glycerol palmitate 353.2668 - 20 to 22 - Glycerol linoleate 377.2651 - 17 to 21 - Glycerol oleate 379.2834 - 20 to 28 - Glycerol eicosenoate 407.3145 - 29 to 32 - Glycerol heneicosapentaenoate 413.2668 - 27, 28 - Glycerol dipalmitoleate 587.4738 - 50 to 52 - Glycerol palmitoleate-palmitate 605.4872 - 39 to 41 - Glycerol palmitoleate-oleate 615.5078 - 53 to 55 - Glycerol palmitate-oleate 617.5202 - 56 to 58 - Glycerol hydroxy oleate-palmitoleate 631.4987 - 46, 47 - Glycerol hydroxy oleate-palmitate 633.5147 - 43 to 45 - Glycerol dioleate 643.5359 - 56 to 59 - Glycerol oleate-stearate 645.5562 - 61 to 63 - Glycerol linoleate-hydroxy oleate 657.5199 - 45 to 47 - Glycerol linoleate-hydroxy stearate 659.5360 - 45 to 47 - Glycerol oleate-hydroxy stearate 661.5534 - 47 to 50 - Glycerol eicosenoate-oleate 671.5712 - 61 to 63 - Glycerol hydroxy oleate-hydroxy linoleate 673.5119 - 39, 40 -
Glycerol palmitate-heptadecanoate- oleate 869.7438 - 69 to 72 - Glycerol hydroxy palmitoleate-oleate-linoleate 893.7718 - 68 to 70 -
Glycerol hydroxy palmitoleate-oleate-oleate 895.7503 - 72 to 76 -
Glycerol hydroxy palmitate-oleate-oleate 897.7740 - 77 to 79 - Glycerol hydroxy oleate dioleate 923.7823 - 79 -
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 231
The FTICR-MS analysis of the HPLC fractions 35 to 50 in both positive- and
negative-ion modes results in important points. First, most of the compounds
assigned to the FTICR peaks of fractions 35 to 50 (see Tables 7.2 to 7.6), contain
functional groups with additional oxygen atoms such as hydroxyl group.
Second, being able to separate a group of acylglycerols containing additional oxygen
atoms using HPLC is evidence that the redox reactions in the ESI chamber are not
the main origin of these compounds. It appears that the redox reactions in the ESI
source were not responsible for the observation of the acylglycerols with additional
oxygen atoms.
Third, as has been discussed elsewhere,[172] molecules with higher number of
hydroxy group are eluted faster on a C18 reversed-phase column due to their lower
hydrophobicity. As an example, the peak at m/z 711.5540 is observed in fraction 45
along with peaks at m/z 669.5312.
As explained in section 7.1, the separation in C18 column is based on the
hydrophobicity of the compounds being eluted. The less hydrophobic molecules
elute earlier and the more hydrophobic molecules elute later. This is evident in
Figure 7.11 and Table 7.7; peaks assigned to less hydrophobic molecules are
observed in earlier fractions and peaks assigned to more hydrophobic molecules are
observed in later fractions. As an example, the peak assigned to C33H62O5 (m/z
561.4554) appears in fraction 51, while the peak assigned to C39H72O5 (m/z
643.5337) is observed in fraction 60. Also, molecules containing hydroxyl groups
(such as C39H74O6, glycerol hydroxy stearate oleate, m/z 661.5547) elute faster on a non-
polar C18 column due to their lower hydrophobicity (more polar). Similar correlation
between HPLC retention times and the chemical structure of saturated, unsaturated
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 232
and oxygenated (hydroxy, keto and epoxy) fatty acids and their methyl esters on C18
reversed-phase HPLC column is reported by Lin et al.[172]
Similarly, as evident in Figure 7.14 and Table 7.9, DAG molecules are eluted in
accordance with their hydrophobicity. Less hydrophobic molecules (such as
C39H72O5 m/z 643.5337) are eluted in earlier fractions while more hydrophobic
molecules (such as C41H76O5 m/z 671.5712) are eluted in later fractions. Another
example is the peak assigned to C41H74O6 m/z 685.5493 that is eluted earlier in
fraction 61 due to the additional oxygen bearing functional group (such as hydroxy
group) that make the molecule less hydrophobic (more polar).
A comparison of the assigned peaks in the positive-ion FTICR mass spectrum of the
methanol extract of macadamia oil (Table 4.2) and the assigned peaks in the positive-
ion FTICR mass spectra of the HPLC fractions of the methanol extract of macadamia
oil (Table 7.11), shows that, as expected, most of the peaks observed in the FTICR
mass spectrum of the methanol extract of macadamia oil are observed in the FTICR
mass spectra of the HPLC fractions as well.
Few of the peaks that are assigned in Table 4.2 are not assigned in Table 7.11. This is
due to the fact that the minimum acceptable level of intensity of peaks in the FTICR
mass spectra of the HPLC fractions is elevated to exclude the peaks from the
background.
Fatty acids with odd number of carbon atoms have been observed in the methanol
extract of macadamia oil in low levels (see Table 4.2); however, they are not
assigned in Table 7.11 due to the fact that the intensities of the peaks assigned to
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 233
odd-numbered fatty acids are lower than the intensities of the peaks in the
background fractions.
A comparison between the assigned peaks in the negative-ion FTICR mass spectrum
of the methanol extract of macadamia oil (Table 5.5) with the assigned peaks in the
negative-ion FTICR mass spectra of the HPLC fractions of the methanol extract of
macadamia oil (Table 7.11) shows that, generally, in negative-ion mode, the peaks
generated by free fatty acids are strong. On the other hand, the peaks generated by
the acylglycerols in negative-ion mode are weak. In Table 7.11, none of the
acylglycerol peaks are assigned in negative-ion mode. This is a consequence of the
blank fraction peaks that are subtracted from the sample spectra. The minimum
acceptable intensity of the peaks was about 2% in the FTICR mass spectra of the
methanol extract of macadamia oil (Table 5.5), whereas it is elevated to about 10%
(absolute intensity of about 5×107) for the FTICR mass spectra of the HPLC
fractions (Table 7.11), resulting in no acylglycerol peaks being assigned, as their
peaks are weaker than 5×107.
In a previous study of HPLC of free FAs and FA methyl esters on the same C18
reversed-phase column used in the present study, Lin and coworkers have reported
the retention times for standard mixtures of free FAs and FA methyl esters in a range
of solvent systems and chromatographic conditions.[172]
Table 7.12 shows a comparison of the retention times of the free FAs obtained in
present study and the previous study by Lin and coworkers.[172]
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 234
Table 7.12. A comparison of the retention times of the HPLC analysis of the methanol extract of macadamia nut oil with standard FA solutions analysed on same column in a previous study.
The fraction numbers obtained in the present study are in excellent agreement with
the retention times reported in a study carried out on standard FA mixtures on a
similar column approximately 8 year earlier.[172] For example, the previously
reported retention times for oleic acid and palmitoleic acid are 27.1 and 20.6 minutes
respectively, whereas in Table 7.11 oleic acid is assigned to the peak observed in
fractions 27 and 28 minutes that includes timeframe of 26 to 28 minutes and
palmitoleic acid is assigned to the peaks observed in fractions 20 to 21 minutes that
includes the timeframe of 19 to 21 minutes.
It should be noticed that the fraction numbers in this present study do not reflect the
exact retention times, as the fractions are collected within a period of one minute.
The fatty acids elute in this period of time and their actual retention time could lay
anywhere within the measured fraction collection time frame.
Fatty Acid Retention Times Obtained in
Present Study (minutes)
Retention Times Reported in Previous Study
(minutes)
Palmitoleic acid C16:1 19 to 22 20.6 Palmitic acid C16:0 24 to 28 25.4 Linolenic acid C18:3 18 to 20 18.4 Linoleic acid C18:2 21 to 24 22.4 Oleic acid C18:1 26 to 28 27.1 Stearic acid C18:0 33 to 35 34.0
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 235
Chapter 8
8. Conclusions and Future Work
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 236
8.1- General Conclusion for this Study
The results from this study indicate that important analytical results can be obtained
by researchers in non-specialised Lipidomics laboratories. With the development of
new high resolution mass spectrometry instrumentation such as the “Orbitrap” one
might expect that studies such as the one reported in this thesis will become more
common.
In the food science and health industries, plant oils are viewed to be important. We
have shown in this study that our simple and straightforward analysis of such
material is readily achieved by ESI FTICR-MS. Research into biosynthesis and
metabolism of such seed oils will be stimulated by analytical results such as those
presented in this thesis.
This study shows that high-resolution ESI FTICR-MS can be used to postulate the
chemical composition of the various free fatty acids and mono-, di- and tri-
acylglycerols in plant oils. ESI FTICR-MS analysis of the methanol extract of plant
oil allows a profile of the free fatty acids in the oil to be readily and clearly produced
with minimal sample preparation. Methanol extract of plant oils could contain
various fatty acids that are not necessarily generated by simple hydrolysis of the
acylglycerols present in the oil. They could be generated through multistep biological
reactions carried out by enzymes such as lipoxygenase from microorganisms
including bacteria and fungi residing in or on the surface of the macadamia nut.
Furthermore, FTICR-MS analysis of the methanol extract of macadamia oil,
esterified oil, hydrolysed oil and the esterified methanol extract of the oil permits
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 237
clear assessments of the efficiency of extraction, esterification and hydrolysis
procedures respectively.
In addition, in the FTICR spectrum of the oil, various constituents of the oil are
observed in the same spectrum. These constituents include FAs, MAGs, DAGs and
TAGs. Although the intensities of the FTICR-MS peaks are not very well related to
the concentration of the species in the sample, the intensities of similar compounds in
macadamia oil are used to estimate the relative concentrations of the respective
species.
We observe for the first time, using this technique, the presence of fatty acids and
acylglycerols containing one or more additional oxygen atoms which may be in the
form of hydroxy, hydroperoxy, peroxy, oxo (ketone or aldehyde functionalities),
hydroxy and/or epoxy substituents in the macadamia oil. We also observe for the
first time the presence of a number of unusual free fatty acids, mono- and di-
acylglycerols containing an odd number of carbon atoms in the oil. The origin of the
odd-numbered free fatty acids could be the enzymes activities in the oil or the
industrial refining processes.
In this study, compound containing elements other than C, H and O are reported in
macadamia oil samples, including compounds containing nitrogen, phosphorus and
sulphur, using molecular formula generating routines in Bruker software that
calculates and suggests the possible molecular formulas combined with a particular
observed peak.
This study demonstrates the higher sensitivity of ESI FTICR-MS compared to GC-
MS for analysing low level compounds in plant oils. In FTICR-MS of plant oils
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 238
many more compounds are observed compared to GC-MS analysis. The simplicity of
the sample preparation when combined with the results from the high resolution high
mass accuracy ESI FTICR-MS experiments on the macadamia oil indicate that this
method is a powerful tool that can be used for the analysis of trace compounds in
lipids including plant oils and animal fats.
Compared to GC-MS technique, FTICR-MS requires simpler sample preparation
steps in the analysis of plant oils. The sample would need to be dissolved in preferred
solvent and introduced to the FTICR mass spectrometer using ESI source. However,
analyzing isomers in FTICR-MS needs application of a chromatographic separation
technique such as HPLC.
The continuous capillary-skimmer potential control feature enabled us to observe the
effect of the kinetic energy on the structure and the FTICR mass spectra of the
generated molecular ions. By varying the applied capillary-skimmer potential
difference, various fragments of the investigated molecules were generated. The
observed fragments provided additional information regarding the molecular formula
of the ions.
Positive-ion and negative-ion FTICR mass spectrometry produce valuable
complementary information in regard to the molecular formula of the chemical
content of the analysed lipid samples. The obtained information was used in
conjunction with the Kendrick mass tables to reject or accept the molecular formulas
proposed by the molecular formula generation routine of Bruker software.
HPLC fraction collection combined with FTICR-MS improves the sensitivity,
selectivity and signal to noise levels due to the lower number of compounds in each
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 239
HPLC fraction, resulting in lower ion-suppression and cloud effects in the FTICR
cell when analyzing fractions compared to the whole oil sample and the methanol
extract of macadamia oil.
The difficulties in the application of FTICR mass spectrometry in the analysis of
lipids include the inability of the technique to resolve isomers such as structural and
stereo-isomers.
8.2- Future Work
We show in this present work, for the first time, that off-line HPLC when combined
with ESI FTICR-MS is readily able to distinguish several isomeric fatty acids, mono-
and diacylglycerols in a methanol extract of the macadamia oil. To differentiate and
structurally characterize isomeric species some form of chromatography (CE, GC or
HPLC) and chemical derivatization must be used prior to any tandem CID or photo-
dissociation studies.
A possible development of application of FTICR-MS of plant oils is to produce high-
resolution libraries of the plant oil finger prints due to the high resolution and high
sensitivity of this technique. These libraries could be used in food and oil industry to
monitor the quality of plant oils and assess possible adulterations or contaminations.
Due to the applied hexapole delay in Bruker APEX II used in this study, the HPLC
separation and fraction collection was carried out in offline mode. New FTICR-MS
instruments with shorter hexapole delays or with different accumulation techniques
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 240
could be used to reduce the duration of each single analysis. This could lead to
application of online HPLC ESI FTICR-MS experiments.
A future project in this field could be the application and development of separation
techniques in conjunction with FTICR-MS technique to elucidate the structure of the
wide range of the compounds in plant oils including free fatty acids, fatty acid esters,
MAGs, DAGS, TAGs, waxes and trace level compounds in the oils. This could
involve the development of on-line HPLC-FTICR-MS techniques to aid in the
identification of chemical compounds in the oils. HPLC techniques with optimized
higher separation capabilities could be used to separate the isomers further apart.
Another wide range of future study could be aimed to separate acylglycerols present
in the oil and analyse the various acyl groups substituted on the glycerol backbone.
This includes identification of the acyl substituents and location of the acyl
substituents on the glycerol backbone (1,2 or 1,3 substitution) and steric
configuration of the chiral acylglycerols (R and S configuration), location of the
possible double bonds and branches on the acyl substituents.
A possible future application of high resolution FTICR mass spectrometry in this
field could be the online quality control of the trace level (potentially harmful)
byproducts produced during the refining processes in oil processing industries. The
final products could be routinely analysed for the level of undesirable compounds in
the oil such as trans fatty acids or oxidation products. The industrial oil processing
systems could then be optimised based on the feedbacks received from the high
resolution FTICR mass spectrometry laboratory. This application could lead to an
improvement in the quality of the oil processing industries that would enhance the
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 241
consumable products in terms of trace level potentially harmful compounds in a wide
range of food products.
The high resolution FTICR mass spectrometry could be applied in future study of the
products of the hydration reaction of carbon double bonds on the fatty acid chains of
acylglycerols. Such studies could investigate whether the hydroxyl groups are
produced in the hydration reaction on the carbon double bond or could be a
byproduct of the hydrolysis reaction we perform on the oil samples.
Another future study in the field of GC-MS analysis of plant oil samples could be
separation, derivatisation and analysis of the highly oxygenated acylglycerols that
can provide the food and oil industries with valuable information regarding the
reactions and products of plant oils oxidation and rancidity.
The oxidation of lipids plays an important role in the food industries, as the oxidation
of lipids can produce unpleasant compounds in the food product. By preparing
libraries of high resolution FTICR mass spectra of lipids and their oxidation
products, one can determine the level of oxidation and rancidity occurred in the lipid
sample in a particular set of conditions. This could lead to the design of optimized
and more environmentally friendly transport, storage and refrigeration systems that
could lead to higher quality products and could save the food industries time, energy
and money.
A potential future study would be the investigation of the possibility of oxidation-
reduction reactions occurring in the electrospray source which could be responsible
for some of the observed acylglycerol molecules with additional oxygen atoms or
oxygen bearing functional groups in this present study. Various separation and
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 242
spectrometric techniques such as GC, HPLC, CID, MS/MS and MSn, FTIR,
FTNMR, ESR and other methods could be used to elucidate the chemical structures
of these unusual compounds in the lipid samples such as animal fat and plant oils.
Applying higher magnetic fields such as 15 T FTICR-MS would increase the
resolution, mass accuracy and sensitivity (S/N ratio). This would enable high-
resolution (broadband) spectra to be obtained routinely. Achieving high resolution
(broadband) would enable one to observe the isotopes in more detail.
Further future work can include development of specialised instruments to carry out
all the necessary separations and spectrometric analyses of the lipids including plant
oils and animal fats. The development of Lipidomics methods could benefit the food
and health industries by investigating possible adulterations or contaminations of the
lipids prior to use.
In the field of organic and bioorganic research of lipids, assessment of the degree of
reaction completion, determination of the reaction products under a particular set of
physical conditions, effect of various catalysts on the kinetics, route and efficiency of
the reactions are among the possible applications of the FTICR mass spectrometry
technique. The optimum physical conditions and the most suitable catalysts to
achieve the highest efficiency of the organic synthesis and bioorganic reactions could
be applied using the information obtained in the FTICR-MS investigations.
Lipidomics techniques could be used in forensic and criminal investigations by
setting up a library of human lipids. Very high sensitivity and very low detection
limits of the FTICR-MS technique allows forensic and crime-scene investigators to
analyse human body fat left at the crime scene on a single human hair or in a finger
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 243
print. These evidences could be used in conjunction with proteomics, DNA analysis
and other forensic methods to provide additional information and evidence for
complicated crime scenes.
Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 244
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