Analysis of triacylglycerols and whole oils by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Analysis of Triacylglycerols and Whole Oilsby Matrix-Assisted LaserDesorption/Ionization Time of Flight MassSpectrometry

G. Reid Asbury, Khalid Al-Saad, and William F. SiemsDepartment of Chemistry, Washington State University, Pullman, Washington, USA

Richard M. HannanUSDA-ARS Regional Plant Introduction Station, Washington State University, Pullman, Washington, USA

Herbert H. Hill, Jr.Department of Chemistry, Washington State University, Pullman, Washington, USA

Since the introduction of matrix-assisted laser desorption/ionization (MALDI) mass spectrom-etry the majority of research has focused on developing analytical methods for the qualitativedetermination of water soluble biomolecules such as proteins, peptides, carbohydrates, andoligonucleotides. This paper, however, reports the use of MALDI for the analysis oftriacylglycerols and develops a new sample preparation method for nonpolar analytes.MALDI enables the rapid analyses of triacylglycerol (TAG) standards and mixtures of wholeoils. The new method provides excellent shot to shot reproducibility, making quantificationpossible. Detection limits were in the mid femtomole range and the resolution was around2000 which easily separated TAGs differing by one double bond. Sensitivity decreased withincreasing molecular weight, causing biased results when analyzing complex mixtures with asignificant range of molecular weight. In all cases only sodiated molecules and prompt lossesof a fatty acid sodium salt were observed in the spectra. From this information it was possibleto identify the three fatty acids on the glycerol backbone. Collision-induced dissociation wascarried out on a triacylglycerol which proved to be useful for additional structural information,including the corroboration of the fatty acid components. With MALDI the percent composi-tions of TAGs in a standard olive oil was accurately determined. Finally, MALDI was used toexamine the differences in lipid components between aged and fresh onion seeds, showing thepotential of the technique for observing changes in lipid components in seeds. (J Am SocMass Spectrom 1999, 10, 983–991) © 1999 American Society for Mass Spectrometry

Triacylglycerols (TAGs) are the major componentsof edible oils and hence are important sources ofcalories and nutrition in human beings. TAGs are

also stored in the bodies of animals as a future energysupply. In fact, over 90% of the weight of adipose tissuein animals is from TAGs. TAGs are also an importantsource of energy for plants. Of particular importance isthat degradation of TAGs in seeds can lead to poorviability and germination [1–3].

Traditional analysis has generally relied on saponi-fication and then methylation to form fatty acid methylesters (FAME) [4] which are then detected by gaschromatography (GC) or GC mass spectrometry(GCMS). This type of analysis does not allow detection

of the actual TAGs; only the total percentage of indi-vidual fatty acids can be determined. Enzymatic meth-ods have been employed for quantitative analysis ofTAGs [5]. They are excellent for quantification of totalconcentration, but give no information on the types oramounts of specific TAGs. TAG analysis has beenperformed by thin layer chromatography (TLC), bothargentation [6] and reversed phase [7]. Argentation TLCseparates TAGs based on their unsaturation, and there-fore is unable to separate saturated species. Further-more, determinations are generally based on rf valueswhich can lead to false identifications. In reversedphase TLC it is also difficult to identify individualcomponents. Supercritical fluid chromatography (SFC)[8] and high performance liquid chromatography(HPLC) [9] are both capable of partially separatingcomplex mixtures of TAGs, but both rely on retentiontime for identification which is difficult especially when

Address reprint requests to Herbert H. Hill, Jr., Department of Chemistry,Washington State University, Pullman, WA 99164-4630. E-mail: [email protected]

© 1999 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received February 3, 19991044-0305/99/$20.00 Revised April 30, 1999PII S1044-0305(99)00063-X Accepted May 18, 1999

peaks are only partially resolved. In order to solve theidentification difficulties, mass spectrometry of TAGshas been investigated. Traditional electron impact (EI)mass spectrometry yields a very weak molecular ion[10] and shows extensive fragmentation. Analysis ofcomplex mixtures would be extremely difficult consid-ering the extensive fragmentation. It was also foundthat significant memory effects occurred when analyz-ing TAGs by EI/MS. Chemical ionization (CI) massspectrometry does improve the signal from the molec-ular ion [11], but fragmentation is still quite prominent.Other mass spectrometric ionization techniques havebeen employed including fast-atom bombardment(FAB) [12], electrospray [13], and atmospheric pressurechemical ionization (APCI) [14]. Of these, APCI offersthe best sensitivity and quantification after separationby HPLC. More recently the ability to determine thecomplete structural characteristics of TAGs was per-formed with tandem sector mass spectrometry usingESI or FAB [15]. This work demonstrated the ability oftandem mass spectrometry to identify the location ofdouble bonds on the fatty acids chains of TAGs. Inaddition, matrix-assisted laser desorption/ionization(MALDI) mass spectrometry was shown to respond toTAGs using a liquid matrix [16], however no quantifi-cation or CID experiments were performed.

Since it was first introduced [17] MALDI has primar-ily been used for proteins [18, 19], carbohydrates [20],oligonucleotides [21], and synthetic polymers [22].Though MALDI is a very effective technique for tradi-tional biomolecules, it also is a sensitive detectionmethod for virtually all nonvolatile compounds. Wenow report the use of MALDI for the quantitative andqualitative determination of TAG standards and mixtures.

Experimental

Instrumentation and MALDI Analysis

All MALDI spectra were obtained using a PerSeptiveBiosystems (Framingham, MA) DE-RP time of flightmass spectrometer. The instrument used a nitrogenlaser at 337 nm to ionize and desorb the sample. It wasalso equipped with delayed extraction technology, areflectron, and a collision cell. All spectra were theaverage of 256 individual spectra. The laser was atten-uated to about 1/3 of its maximum power, depositingapproximately 100 mJ of energy per laser shot. Thevoltage applied was 25,000 V and a focusing guide wirewas held at a potential of 0.1% of the acceleratingvoltage. A delay time of 100 ns was used between thetime of the laser pulse and the turning on of theaccelerating voltage. The instrument was set in thereflector mode and spectra were taken from 0 to 5000m/z. The CID experiments were done by bleeding in asmall amount of air into the collision cell. The ion gaugeread about 5 3 1026 torr after the addition of the air asopposed to 5 3 1027 prior to adding air to the collisioncell. The default calibration file or a calibration file

based on matrix peaks was used. Mass measurementsand peak centroiding were done by the data system.

Seed Aging and Extraction

Onion (Allium cepa) seed, breeding line MSU 2399B Lot537879, was obtained from Dr. R. Watson, ARCO Seed(El Centro, CA). Until used, the seed was stored inplastic containers at 4 °C, 28%–30% relative humidity(%RH) at the USDA-ARS, Western Regional Plant In-troduction Station (WRPIS) seed storage facility inPullman, WA. Storage treatment conditions for the seedwere: (1) the control was held in standard seed storageat 4 °C, 28%–30% RH in the WRPIS facility. Samples ofseeds were put into small coin envelopes and stored indrawers, the same as normal procedure for the PlantIntroduction germplasm. (2) Seeds exposed to heattreatment with elevated oxygen (accelerated aging)were removed from the traditional seed storage and 6–7g of seed were put into 250 mL Erlenmeyer flasks. Thesewere placed into a warm water bath maintained atconstant 40–41 °C. The flasks were then flooded withpure oxygen for 2–3 min, and stoppered with rubberstoppers. A 95%–99% oxygen atmosphere at 40–41 °Cwas maintained by reflooding the flasks with oxygenevery 14 days.

Extractions were conducted using a high-pressureSoxhlet extractor (J & W Scientific, Folsom) and liquidcarbon dioxide as the extraction solvent. Seeds wereground dry in a 37-mL stainless steel container on astandard laboratory Waring blender for 20 s. Extrac-tions were run for 48 h at 650–700 lb/in.2 with atemperature differential of 38–41 °C at the base of thechamber to 4 °C on the cold finger. Aliquots from theextract were diluted into chloroform and stored in afreezer prior to MALDI analysis.

Reagents and Sample Preparation

All solvents used were reagent grade and were pur-chased from J. T. Baker (Phillipsburgh, NJ). The 2,5-dihydroxy benzoic acid (DHB) and the alpha-cyanocinnamic acid (CHCA) were both purchased from Al-drich Chemical (Milwaukee, WI). All of the TAG stan-dards and the dithranol (1,8 dihydroxy-9[10H] anthra-cenone) were obtained from Sigma (St. Louis, MO). TheK4[Fe(CN)6] and the glycerol were also purchased fromJ. T. Baker. In all cases the TAGs and the whole oilswere dissolved in chloroform. Four matrices were usedin these experiments including: DHB, CHCA, dithranol,and K4[Fe(CN)6]/glycerol. The K4[Fe(CN)6]/glycerolliquid matrix was prepared as previously described[16]. The CHCA was dissolved in a 50:50 (water:aceto-nitrile) solution containing 0.25% trifluoroacetic acid.The TAG standards were then mixed with this matrixon the sample plate itself or in a separate vial and thenspotted on the plate. The DHB was dissolved in eitheracetone or a 50:50 (water:acetonitrile) solution contain-ing 0.25% trifluoroacetic acid. The DHB in the water

984 ASBURY ET AL. J Am Soc Mass Spectrom 1999, 10, 983–991

mixture was used in the same way as the CHCA. TheDHB dissolved in acetone was spotted on the plate andallowed to dry (5 s) and then the TAGs dissolved inchloroform were spotted directly on top of the DHB.The dithranol was dissolved in chloroform and mixedwith the TAGs prior to spotting on the plate. In all cases1 mL of solution was used. The samples were spotted ona standard plate containing 100 small wells.

Results and Discussion

Matrix and Sample Preparation

A number of matrices were investigated for the MALDIanalysis of TAGs including alpha-cyano cinnamic acid(CHCA), dihydroxy benzoic acid (DHB), dithranol, andK4[Fe(CN)6]/glycerol. The K4[Fe(CN)6]/glycerol liquidmatrix was recently reported by Zollner et al. [16] to bean effective matrix for nonpolar compounds such asTAGs. Their method was to apply a small amount of a5% glycerol in methanol solution saturated withhexacyanoferrate to the MALDI plate. After allowingthe methanol to dry, the analyte was applied in chloro-

form on top of the glycerol layer. This sample prepara-tion would then give a thin film of UV absorbinghexacyanoferrate in glycerol with a thin film of analyteon top of the glycerol. Detection limits in the low fmolrange were reported for TAGs. Our attempts to use thissample preparation were very unsuccessful probablybecause our preparation seemed to destroy the thin filmof glycerol. After applying the chloroform solution tothe plate we were left with clumps of glycerol instead ofa thin film. These results were similar to those reportedby Zollner et al. [16] when water solutions were appliedto the thin UV absorbing layer.

Traditional MALDI matrices were also used such asCHCA and DHB. Both CHCA and DHB were preparedin 50% water : 50% acetonitrile with 0.25% trifluoroace-tic acid. The matrices were then mixed with the chloro-form solution of TAGs both in a separate vial anddirectly on the plate. The use of both matrices did leadto some sodiated molecules, but the shot to shot repro-ducibility and the detection limits were very poor.These results can be attributed to incompatibility of thesolvents. Dithranol, soluble in chloroform and firstreported by Juhasz and Costello [23], would seem tomake an ideal matrix for TAGs. However, the matrixonly achieved detection limits in the mid pmol rangeand did not show good shot to shot reproducibility.

A final sample preparation using DHB proved towork the best. The DHB was dissolved in acetone andthis solution was applied to the MALDI plate. Theacetone quickly evaporated leaving a thin layer of verysmall DHB crystals. The TAGs dissolved in chloroformwere then applied directly on top of this crystal layerand allowed to evaporate. This sample preparationproved to be much more sensitive than the others, with

Figure 1. Typical positive ion MALDI mass spectrum of a TAG. The standard in the above spectrumcontained two stearic acids and a lauric acid with a molecular weight of 835 u. The dominant peak inthe spectrum at m/z 857.7 is the sodiated molecular ion of the TAG. The spectrum also shows twoprompt fragment ions resulting from the loss of a sodium salt of a fatty acid.

Table 1. Summary of results from matrix studies

Matrix Solvent QuantificationDetection

limit

CHCA water/acetonitrile no mid pmolDHB water/acetonitrile no mid pmolDHB acetone yes mid fmolDithranol chloroform no mid pmolK4Fe(CN)6/glycerol methanol no ????a

aThe detection limit for this matrix was previously reported in the lowfmol range. We were however unable to produce any ions from TAGswith this matrix.

985J Am Soc Mass Spectrom 1999, 10, 983–991 TRIACYLGLYCEROLS BY MALDI-TOF

the detection limits in the fmol range. In addition, thismethod gave much better shot to shot reproducibility incomparison to the other sample preparations. Thissample preparation was then used for the remainder ofthe MALDI experiments with TAGs. The results of thematrix studies are summarized in Table 1.

MALDI Analysis of a TAG

Figure 1 shows the positive ion MALDI mass spectrumof a triacylglycerol standard composed of two stearic

acids and one lauric acid (C18:0/18:0/14:0) with amolecular weight of 834.7. The spectrum is dominatedby the sodiated molecule at 857.7 and shows no proton-ated molecule. The spectrum also shows two promptfragments associated with the loss of a RCOO2Na1

molecule. The peak associated with the loss of the C18fatty acid is approximately twice that of the C14 lossindicating that there is no preferential loss based on thesize of the fatty acid. This information can then quicklybe used to determine the three fatty acids in anyunknown TAG, however, it would be impossible to

Figure 2. The top spectrum (A) shows the positive ion MALDI mass spectrum of a mixture of sevenTAG standards: tricaprin (C10:0), trilaurin (C12:0), trimyristin (C14:0), tripalmitin (C16:0), tristearin(C18:0), triarachidin (C20:0), and tribehenin (C22:0) at similar concentrations. The bottom graph (B)shows a plot of the relative peak height of each standard. The graph clearly shows a decrease in signalintensity for the higher molecular weight TAGs.


determine the exact location of the individual fattyacids on the glycerol molecule. The loss of a sodiatedfatty acid seems reasonable because it corresponds to acharge-driven fragmentation process. All other stan-dards used resulted in similar ion formation andprompt losses.

MALDI Analysis of a TAG Mixture

Figure 2A shows the positive ion spectrum of a mixtureof seven TAGs: tricaprin (C10:0), trilaurin (C12:0),trimyristin (C14:0), tripalmitin (C16:0), tristearin (C18:0), triarachidin (C20:0), and tribehenin (C22:0). Therewas between 50 and 100 pmol of each TAG on thesample plate. All produced only sodiated moleculesand prompt fragmentation losses of a RCOO2Na1

molecule (data not shown). It was clear from the spec-trum that the peak height decreased with increasingmolecular weight. Figure 2b shows a graph of therelative peak height of the mixture after it was correctedfor the actual amount spotted on the plate. This graphfurther illustrates the loss in sensitivity of the highermolecular weight compounds in the mixture. This de-crease in sensitivity for higher molecular weight com-pounds may make quantification more difficult formixtures containing species with a wide range of mo-lecular weights.

Quantitative Analysis of TAGs

A calibration curve was constructed using the sodiatedpeak at m/z 662 of trilaurin (C12:0) in the range 1–20pmol. Above 20 pmol the increase in peak heightslowed relative to the amount of analyte on the plate,hence producing a nonlinear graph. The analysis wastaken by randomly moving the laser around within thesample spot to eliminate any operator bias. All pointswere the average of three analyses and gave rsd valuesin the 5%–15% range. Figure 3a shows the calibrationcurve based solely on peak heights using no form ofinternal standard. This graph shows a trend of in-creased peak height with increasing analyte; however,the linearity of the graph was not good with a correla-tion coefficient of 0.968. Although these results wereonly semiquantitative, it should be noted that thisanalysis is extremely easy, rapid, and sensitive com-pared to that done by saponification and chromato-graphic methods.

Figure 3b shows a calibration curve of the samestandards only this time the intensity of the analytepeak was divided by the intensity of a matrix peak (720m/z). This should help account for any random differ-ences in the surfaces of the samples. This graph wasmore linear than the one based solely on peak heights.The correlation coefficient in this case was 0.996. Fromthe data used to construct this calibration curve thedetection limit was determined to be about 100 fmol fortrilaurin. This was still a little higher than that reportedfor the K4[Fe(CN)6]/glycerol liquid matrix; however,

we feel our DHB sample preparation was easier andgave better reproducibility. Also the results indicatethat quantification was possible for TAGs using thismethod of sample preparation. It should be noted,however that samples containing isomeric compounds(identical m/z) would not be quantifiable individuallyby this method. The linear range was slightly more thanone order of magnitude, but represents a major im-provement considering the difficulty of quantificationusing MALDI.

CID of Trimyrstin

Figure 4a shows the CID spectrum for trimyristinacquired in the PSD mode. The CID spectrum shows theloss of the sodium salt of a fatty acid which alsooccurred as a prompt fragmentation to give the m/z 496ion. In addition, it shows the loss of a neutral fatty acidto give an ion of m/z 519. In fact, the loss of the neutralfatty acid is more prominent than the loss of thesodiated form. The rest of the CID spectrum is domi-nated by peaks resulting from charge-remote fragmen-tation. We can see a series of peaks separated by 14 u inthe high-mass region of the spectrum culminating witha prominent peak at m/z 590. These peaks are of ions

Figure 3. The top graph (A) shows the calibration curve fortrilaurin (C12:0) using the peak height without any internalstandard. The bottom graph (B) shows the same curve when aratio of the peak height for the analyte and a matrix peak is used.The bottom curve has a correlation coefficient better then 0.99.


resulting from the loss of CnH2n12 from one of the fattyacids. The prominence of the ion at m/z 590 can beattributed to the formation of a conjugated system.Similar results have recently been reported byDomingues et al. [24]. This can be seen more clearly byobserving Figure 4b. This figure gives an overview ofthe prominent fragmentation pathways seen for TAGsusing high energy MALDI-CID-MS.

Analysis of Standard Olive Oil by MALDI

The MALDI spectrum of a standard olive oil is shownin Figure 5. This standard olive oil contains five com-ponents: triolein (60%), 1,2 dioleoyl-3-palmitoyl-rac-

glycerol (30%), 1,2 dilinoleoyl-3-oleoyl-rac-glycerol(4%), 1,2 dioleoyl-3-stearoyl-rac-glycerol (4%), and trili-nolein (2%). The percent composition for the standardwas used as reported by Sigma Chemical. The m/z of thesodiated molecules for each of the above compoundsare 908, 882, 904, 910, and 902, respectively. Each ofthese ions is labeled in Figure 5. The figure also containssignificant isotope peaks for the large peaks at m/z 882and 908. From a visual inspection of the spectrum it wasobserved that the relative heights of each peak matchedthe expected values with the exception of the peak atm/z 910 which was clearly higher than expected. Itshould be noted that this peak (1,2 dioleoyl-3-stearoyl-rac-glycerol) also had a significant contribution from

Figure 4. The top spectrum (A) shows the CID spectrum for trimyristin (C14:0). The bottom graph(B) shows the major dissociative pathways for TAGs using high-energy CID.


the M 1 2 isotope from the m/z 908 peak (triolein).After subtracting the background and correcting forisotope contributions the results for the expected %compositions with the experimental % compositionsmatched very closely. These results are presented inTable 2. The range of percent differences was 2%–15%.This demonstrates the potential of MALDI to rapidlydetermine the compositions of simple unknown mix-tures of TAGs.

MALDI Analysis of Seed Oils Extracted fromFresh and Aged Seeds

MALDI spectra of both fresh and aged seeds fromonion (allium cepa) are shown in Figure 6. Both spectrashow many clusters of peaks. Within a cluster the peakwith the highest intensity is mass labeled. The twospectra are, however, dramatically different with theaged seeds showing considerably more high-masspeaks than the fresh seed. There also appears to be somemissing components from the aged seed in the m/z750–800 range. If one looks at the highest peak in eachcluster it appears that the additional high-mass peaks in

the aged seed were 16 mass units higher than theprevious cluster. This indicates that significant oxida-tion has taken place in the aging process. The mecha-nism and location of oxidation are unclear from thespectra alone, but by using simpler more controlledaging experiments and obtaining CID data, MALDImay provide useful information to help elucidate thisprocess.

Conclusions

Using the DHB/acetone sample method for TAGs givesdetection limits in the mid fmol range. This samplepreparation is very rapid and provides experimentalease. In addition, the shot to shot reproducibility ismuch better than traditionally seen in MALDI andyields quantitative results. MALDI also provided con-siderable qualitative information on TAGs includingthe three fatty acids on the glycerol backbone. MALDIwas also shown to give good agreement between ex-pected and experimental percent compositions for anolive oil standard. Finally, significant differences in thelipid fractions of fresh and aged onion seeds wereshown and MALDI appears to be an excellent tool forinvestigating the aging process in seeds.

AcknowledgmentThe authors would like to thank the Society of Analytical Chem-istry of Pittsburgh for sponsoring a summer ACS analyticalchemistry fellowship for G. Reid Asbury.

Figure 5. MALDI mass spectrum of an olive oil standard with the following composition: triolein60% (908 m/z), 1,2 dioleoyl-3-palmitoyl-rac-glycerol 30% (882 m/z), 1,2-dilinoleoyl-3-oleoyl-rac-glycerol4% (904 m/z), 1,2 dioleoyl-3-stearoyl-rac-glycerol 4% (910 m/z), and trilinolein 2% (902 m/z). Aftercorrecting the peak heights for isotope contributions, the experimental percentages differed from theknown percentages by 0.1%–2.4%.

Table 2. Experimental and expected % compositions of TAGsin standard olive oil

Mass(M 1 Na)1 Expected % Experimental % % Difference

882 30 28.1 6 0.9 26.3902 2 1.7 6 0.1 215.0904 4 3.6 6 0.1 210.0908 60 62.4 6 1.7 4.0910 4 4.1 6 0.8 2.5


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Figure 6. Shows the MALDI mass spectra of fresh (A) and aged (B) allium seed oil. The aged oilshows significantly greater amounts of higher mass components indicating extensive oxidation over time.


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Analysis of triacylglycerols and whole oils by matrix-assisted laser desorption/ionization time of flight mass spectrometry