CHARACTERIZATION OF SYNTHETIC PHENETHYLAMINES USING LOW-

RESOLUTION AND HIGH-RESOLUTION MASS SPECTROMETRY

By

Alexandria Lynn Anstett

A THESIS

Submitted to

Michigan State University

in partial fulfillment of the requirements

for the degree of

Forensic Science – Master of Science

2017

ABSTRACT

CHARACTERIZATION OF SYNTHETIC PHENETHYLAMINES USING LOW-

RESOLUTION AND HIGH-RESOLUTION MASS SPECTROMETRY

By

Alexandria Lynn Anstett

Definitive identification and differentiation of synthetic designer drugs can be

challenging for forensic analysts due to the high structural similarities. The focus in this work

was the characterization of synthetic phenethylamines, a common class of designer drugs, using

mass spectrometry methods. A set of phenethylamine reference standards was analyzed using

both low-resolution and high-resolution mass spectrometry and the mass spectra were probed to

identify characteristic and distinguishing features. These features were integrated into a flow-

chart style characterization scheme for both low-resolution and high-resolution mass spectra.

The characterization scheme for low-resolution data utilizes retention index and neutral

losses to indicate phenethylamine structural subclass. Further, isotope patterns and characteristic

mass spectral features give a preliminary indication of the identity of substituent. This scheme is

immediately implementable into forensic practice because it exploits the instrumentation already

used for the identification of controlled substances. The high-resolution version of the

characterization scheme offers more robust characterization. From high-resolution mass analysis,

exact mass and mass accuracy of each ion were determined and mass defect filters were

developed. These mass defect filters were successful in characterizing compounds according to

structural subclass. Overall, this research provides tools for the characterization of synthetic

phenethylamines and highlights the potential for high-resolution mass spectrometry for forensic

applications, should this instrumentation become available in forensic laboratories.

iii

ACKNOWLEDGMENTS

Foremost, I would like to express my deepest appreciation, gratitude, and thanks to my

advisor, Dr. Ruth Smith for her guidance and willingness to share her knowledge, expertise,

excitement, and laughter throughout my graduate career at Michigan State University. I thank

her for challenging me to grow as a scientist and as a person. My gratitude knowns no bounds

and without her, this would not be possible. I would also like to thank my committee member

Dr. Victoria McGuffin, for her advice throughout this research and for always offering a

different perspective and asking questions that have challenged me and helped me to think

critically. I would also like to thank Dr. Steve Dow for serving on my committee on shorter

notice and agreeing to read my thesis over the Christmas holiday.

Further, I would like to thank those who helped facilitate this research, especially Scott

Smith and the staff of the MSU Mass Spectrometry and Metabolomics Core Facility, and David

Alonso and Joe Binkley from LECO Corp. for help with instrumentation and data collection. I

am also grateful to the National Institute of Justice who supported this research via grant number

2015-IJ-CX-K008. Points of view in this thesis are those of the author and do not necessarily

represent the official position or policies of the U.S. Department of Justice.

Additionally, I would like to thank current and past members of the Forensic Chemistry

group for their encouragement, guidance, patience, and support- especially sitting through

countless hours of AAFS and thesis defense practices. A special thank you to Fanny Chu,

Natasha Eklund, Cindy Kaeser, Amanda Setser, Barb Fallon, and Kristen Reese. An extra special

thank you to Trevor Curtis for being my “partner in crime” and making sure I always had a

friend to eat with. I would also like to thank my dearest friend and roommate Brianna Bermudez

iv

for taking Moose and I in, and always offering encouragement, advice, laughs, food, and love.

Further, to my “favorite” biologist, I would like to thank Alyssa Badgley for being there for me

as a best friend and shoulder to lean on throughout my entire graduate school journey. I honestly

couldn’t have done it without you, and wouldn’t have wanted too anyway. Thanks for being the

best “trace” partner, tailgater, and overall Spartan enthusiast I could have ever asked for

(“GREEEN”). Finally, I would like to thank my friends and family, especially my “moms” for

their support from across the country, Tristan Musser for his endless love, support, and patience

and my parents, Monica and Paul, for their unwavering, unconditional, encouragement and love

in everything I’ve ever done. This one’s for you guys. I am truly grateful to you all and couldn’t

have made it this far without you.

v

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................................ vii

LIST OF FIGURES ..................................................................................................................... viii

I. Introduction ................................................................................................................................. 1

1.1 Synthetic Designer Drugs...................................................................................................... 1

1.2 Current Methods of Analysis of Submitted Drug Samples and Limitations ........................ 3

1.3 Current Research of Synthetic Designer Drugs .................................................................... 4

1.4 Research Objectives and Goals ............................................................................................. 8

REFERENCES ............................................................................................................................. 12

II. Theory ...................................................................................................................................... 15

2.1 Chromatography Overview ................................................................................................. 15

2.2 Gas Chromatography Overview .......................................................................................... 15

2.2.1 Retention Index............................................................................................................. 19

2.3 Mass Spectrometry Overview ............................................................................................. 20

2.3.1 Mass Analysis: Single Quadrupole Mass Analyzer ..................................................... 21

2.3.2 Mass Analysis: Time-of-Flight Mass Analyzer............................................................ 24

2.3.3 Comparison of Low-Resolution and High-Resolution Mass Spectra .......................... 26

2.4 Mass Defect ......................................................................................................................... 28

2.4.1 Kendrick Mass Defect .................................................................................................. 29

REFERENCES ............................................................................................................................. 30

III. Materials and Methods ............................................................................................................ 32

3.1 Reference Standards ............................................................................................................ 32

3.2 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis ........................................... 35

3.3 Data Processing ................................................................................................................... 37

3.4 Mass Defect Filters.............................................................................................................. 38

3.4.1 Absolute Mass Defect Filters ....................................................................................... 38

3.4.2 Kendrick Mass Defect Filters ....................................................................................... 39

APPENDIX ................................................................................................................................... 41

IV. Characterization of Synthetic Phenethylamines by Low-Resolution Mass Spectrometry ..... 43

4.1 Retention Index ................................................................................................................... 43

4.2 Electron Ionization Mass Spectra of Synthetic Phenethylamine Subclasses ...................... 45

4.3 Neutral Losses from Molecular Ion to Distinguish 2C- from NBOMe-Phenethylamines .. 49

4.4 Distinction and Identification of Common Substituents for 2C- and NBOMe-

Phenethylamines........................................................................................................................ 53

4.4.1 Halogen Substitutions ................................................................................................... 53

4.4.2 Sulfur and Nitro Substitutions ...................................................................................... 59

4.5 Scheme for Characterization of Synthetic Phenethylamines using Low-Resolution Mass

Spectra ....................................................................................................................................... 62

4.6 Summary ............................................................................................................................. 70

vi

APPENDIX ................................................................................................................................... 71

REFERENCES ............................................................................................................................. 78

V. Characterization of Synthetic Phenethylamines by High-Resolution Mass Spectrometry ...... 80

5.1 Comparison of Low- and High-Resolution Mass Spectra .................................................. 80

5.2 Development of Mass Defect Filters ................................................................................... 84

5.2.1 Absolute Mass Defect Filters for Phenethylamines Based on Molecular Ions ............ 84

5.2.2 Absolute Mass Defect Filter for the APB-Phenethylamine Subclass ........................... 87

5.2.3 Absolute Mass Defect Filter for the 2C-Phenethylamine Subclass .............................. 88

5.2.4 Absolute Mass Defect Filter for the NBOMe-Phenethylamine Subclass ..................... 90

5.2.5 Kendrick Mass Defect Filters for Phenethylamines Based on Molecular Ions ............ 92

5.2.6 Kendrick Mass Defect Filters of the APB-Phenethylamine Subclass .......................... 93

5.2.7 Kendrick Mass Defect Filters of the 2C-Phenethylamine Subclass ............................. 94

5.2.8 Kendrick Mass Defect Filters of the NBOMe-Phenethylamine Subclass .................... 96

5.2.9 Kendrick Mass Defect Filters for Neutral Losses and Common Fragment Ions .......... 98

5.3 Scheme for Characterization of Synthetic Phenethylamines using High-Resolution Mass

Spectra ..................................................................................................................................... 108

5.4 Summary ........................................................................................................................... 115

APPENDICES ............................................................................................................................ 116

APPENDIX A: High-Resolution Mass Spectra ...................................................................... 117

APPENDIX B: Additional High-Resolution Characterization Scheme Examples ................. 125

REFERENCES ........................................................................................................................... 131

VI. Conclusions and Future Work .............................................................................................. 133

6.1 Conclusions ....................................................................................................................... 133

6.2 Future Work ...................................................................................................................... 134

vii

LIST OF TABLES

Table 2.1 Absolute mass defects of elements commonly used in this research ........................... 28

Table 3.1 Substituents for 2C-phenethylamine compound shown in Figure 3.1 ...........................33

Table 3.2 Substituents for NBOMe-phenethylamine compound shown in Figure 3.2 ..................34

Table A.1 Compound abbreviations and chemical names ............................................................ 42

Table 4.1 Retention index and molecular ion determinations of sample set compounds ............. 44

Table 5.1 Calculation of absolute mass defect molecular ion filter .............................................. 85

Table 5.2 Calculation of APB Kendrick mass defect filter .......................................................... 93

Table 5.3 Calculation of 2C Kendrick mass defect filter .............................................................. 95

Table 5.4 Calculation of NBOMe Kendrick mass defect filter .................................................... 97

Table 5.5 Ion table of 2C-H showing abundant ion elemental composition assignments and mass

accuracies ...................................................................................................................................... 99

Table 5.6 Table of remaining ions after common losses of all 2C compounds .......................... 100

Table 5.7 Kendrick mass defect filters associated with ion fragments after common neutral losses

..................................................................................................................................................... 102

Table 5.8 Ion table of 25H-NBOMe with elemental composition assignments and mass

accuracies of most abundant fragment ions above m/z 105 ........................................................ 104

viii

LIST OF FIGURES

Figure 1.1 Phenethylamine ............................................................................................................. 2

Figure 1.2 Phenethylamine analogs. (A) 2,5-dimethoxyphenethylamine (2C-H) (B) 4-ethyl-2,5-

dimethoxyphenyl-N-(2-methoxybenzyl) ethan-1-amine (25E-NBOMe) (C)

aminopropylbenzofuran (4-APB) (D) 1-(3, 5-dimethoxy-4-propoxyphenyl) propan-2-amine (3C-

P) ..................................................................................................................................................... 3

Figure 2.1 Schematic of gas chromatography (GC) instrument ................................................... 16

Figure 2.2 Example chromatogram of a multi-component gas chromatography separation ........ 19

Figure 2.3 Overall schematic of mass spectrometer (MS) ............................................................ 20

Figure 2.4 Electron ionization (EI) source .................................................................................... 21

Figure 2.5 Quadrupole mass analyzer showing two different ion trajectories occurring

simultaneously. The red ion is neutralized as it collides with one of the rods, is pumped away,

and not detected, while the blue ion has a stable trajectory through the analyzer and travels to the

detector .......................................................................................................................................... 22

Figure 2.6 Time-of-flight mass analyzer showing two different ion trajectories occurring

simultaneously. Both ions are accelerated in the pusher region with the same kinetic energy, but

the red ion penetrates deeper into the reflectron because it has larger mass, thus reaching the

detector after the blue ion ............................................................................................................. 25

Figure 2.7 Spectra and chemical structure of 2, 5- dimethoxyphenethylamine (2C-H) via (A)

low-resolution (QMS) and (B) high-resolution (TOFMS) mass spectrometry ............................ 27

Figure 3.1 Structures of the phenethylamines in the reference set (A) 4-(2-aminopropyl)

benzofuran (4-APB) (B) 5-(2-aminopropyl) benzofuran (5-APB) (C) 6-(2-aminopropyl)

benzofuran (6-APB) (D) 7-(2-aminopropyl) benzofuran and (E) the core structure of 2,5-

dimethoxyphenethylamine (2C-phenethylamines). The substituents at R1 and R2 and the

corresponding 2C compound are given in Table 3.1. ................................................................... 33

Figure 3.2 Structures of more of the phenethylamines in the reference set (A) 3,4,5-trimethoxy-

benzeneethanamine (mescaline), (B) 4-ethoxy-3,5-dimethoxy-benzeneethanamine (escaline)

both 3C-phenethylamines, (C) the core of N-benzyl phenethylamine analogs (NBOMe-

phenethylamines). The substituents at R1 and R2 corresponding NBOMe compound are given in

Table 3.2, and (D) 3,4,5-trimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine

(mescaline-NBOMe) ......................................................................................................................34

Figure 3.3 Structures of cathinones in the reference set (A) 4-methylmethcathinone

(mephedrone) and (B) 3-methylethcathinone (3-MEC) ............................................................... 35

ix

Figure 4.1 Representative spectra of (A) 6-APB, (B) 2C-H, and (C) 25H-NBOMe and proposed

structures for the most dominant fragment ions in each spectrum ............................................... 46

Figure 4.2 Mass spectra of (A) 25G-NBOMe and the cannabinoid (B) XLR-115 which both have

a molecular ion of m/z 330. NBOMes can be differentiated from cathinones using characteristic

peaks at m/z 91, 121, and 150. XLR-11 spectrum obtained from Cayman Chemical .................. 48

Figure 4.3 Mass spectrum of (A) 2C-H and (B) 2C-B showing characteristic 2C neutral losses of

29 and 60 Da and the structures of the fragment ions remaining after each loss ......................... 51

Figure 4.4 Mass spectrum of (A) 25H-NBOMe and (B) 25B-NBOMe showing characteristic

NBOMe neutral losses of 31 and 149 Da and the structures of the fragment ions remaining after

each loss, as well as common fragment ions (m/z 91, 121, 150) .................................................. 52

Figure 4.5 Characteristic isotope pattern in mass spectra of compounds containing bromine, (A)

2C-B and (B) 25B-NBOMe .......................................................................................................... 54

Figure 4.6 Characteristic isotope pattern in mass spectra of compounds containing chlorine (A)

2C-C and (B) 25C-NBOMe .......................................................................................................... 56

Figure 4.7 Full mass spectrum of (A) 2C-I and (B) expanded section of same spectrum to

highlight I+ and HI+ ions ............................................................................................................... 57

Figure 4.8 Full mass spectrum of (A) 25I-NBOMe and (B) expanded section of same spectrum to

highlight I+ and HI+ ions ............................................................................................................... 58

Figure 4.9 Mass spectrum of (A) 2C-T and (B) 25T-NBOMe indicating inconsistent sulfur

isotope pattern ............................................................................................................................... 60

Figure 4.10 Mass spectrum of (A) 2C-N and (B) 25N-NBOMe indicating M+ with an even mass

that suggests an even number of nitrogens present ....................................................................... 61

Figure 4.11 Characterization scheme for low-resolution mass spectra of synthetic

phenethylamines to distinguish APB, 2C, and NBOMe subclasses ............................................. 64

Figure 4.12 Characterization scheme for low-resolution mass spectra of synthetic

phenethylamines to determine substituents on 2C- or NBOMe-phenethylamines ....................... 65

Figure 4.13 Mass spectrum and structure of cathinone, 3-methylethcathinone (3-MEC) ............ 68

Figure 4.14 Mass spectrum of 3C phenethylamine, mescaline, which would be mischaracterized

as a 2C because of its loss of 29 Da (m/z 182) and 60 Da (m/z 151) ............................................ 69

Figure A.1 Low-resolution mass spectra of (A) 4-(2-aminopropyl)benzofuran (4-APB), (B) 5-(2-

aminopropyl)benzofuran (5-APB), and (C) 7-(2-aminopropyl)benzofuran ................................. 72

x

Figure A.2 Low-resolution mass spectra of (A) 2,5-dimethoxy-4-methylphenethylamine (2C-D),

(B) 2,5-dimethoxy-4-ethylphenethylamine (2C-E), (C) 3,4-dimethyl-2,5-

dimethoxyphenethylamine (2C-G), and (D) 2,5-dimethoxy-4-propylphenethylamine (2C-P) .... 73

Figure A.3 Low-resolution mass spectra of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2)

....................................................................................................................................................... 74

Figure A.4 Low-resolution mass spectra of (A) 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-

methyoxybenzyl)ethanamine (25D-NBOMe) and (B) 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-

methoxybenzyl)ethanamine (25E-NBOMe) ................................................................................. 75

Figure A.5 Low-resolution mass spectra of (A) 2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-4-

[(1-methylethyl)thio]-benzeneethanamine (25T-4-NBOMe), (B) 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-4-(propylthio)-benzeneethanamine (25T-7-NBOMe), and (C) 3,4,5-

trimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (mescaline-NBOMe) ............... 76

Figure A.6 Low-resolution mass spectra of (A) 4-ethoxy-3,5-dimethoxy-benzeneethanamine

(escaline) and (B) 4-methylmethcathinone (mephedrone) ........................................................... 77

Figure 5.1 Comparison of (A) low-resolution and (B) high-resolution mass spectra for 6-APB

(left), 2C-H (middle), and 25H-NBOMe (right) ........................................................................... 81

Figure 5.2 Comparison of (A) low-resolution and (B) high-resolution mass spectra for 2C-B.

Dominant fragment ions are labeled and in (B) assigned element formulae and mass accuracies

are given ........................................................................................................................................ 83

Figure 5.3 Absolute mass defect filter created using a training set of phenethylamines defined in

Table 5.1. The absolute mass defect filter was defined at 142.4 ± 54.1 mDa at a 99.9991%

confidence level. The horizontal lines represent the average (yellow), and the upper and lower

bounds of the mass defect filter (purple) ...................................................................................... 86

Figure 5.4 APB subclass absolute mass defect filter at 99.7 ± 1.6 mDa at a 90% confidence level.

The horizontal lines represent the average (black) and the upper and lower bounds of the mass

defect filter (red) ........................................................................................................................... 88

Figure 5.5 2C subclass absolute mass defect filter at 133.1 ± 32.2 mDa at a 95% confidence

level. The horizontal lines represent the average (light blue) and the upper and lower bounds of

the mass defect filters (dark blue) ................................................................................................. 90

Figure 5.6 NBOMe subclass absolute mass defect filter at 179.6 ± 20.5 mDa at a 95% confidence

level. The horizontal lines represent the average (light purple) and the upper and lower bounds of

the mass defect filter (dark purple) ............................................................................................... 91

Figure 5.7 APB subclass Kendrick mass defect filter at 95.9 ± 1.6 mDa at a 90% confidence

level. The horizontal lines represent the average (black) and the upper and lower bounds of the

mass defect filter (red) .................................................................................................................. 94

xi

Figure 5.8 2C subclass Kendrick mass defect filter at 92.2 ± 1.5 mDa at a 95% confidence level.

The horizontal lines represent the average (light blue) and the upper and lower bounds of the

mass defect filter (dark blue) ........................................................................................................ 96

Figure 5.9 NBOMe subclass Kendrick mass defect filter at 171.5 ± 7.7 mDa at a 99% confidence

level. The horizontal lines represent the average (light purple) and the upper and lower bounds of

the mass defect filter (dark purple) ............................................................................................... 97

Figure 5.10 Spectrum of 2C-H showing abundant ions ................................................................ 99

Figure 5.11 Proposed structures for fragment ions of 2C-H after their neutral losses ............... 100

Figure 5.12 Kendrick mass defect filters developed based on common losses of alkyl-substituted

2C compounds. Points represent KMD of fragment ions remaining after each respective loss.

The horizontal lines represent the average (lighter colors) and the upper and lower bounds of

each mass defect filter (darker colors) ........................................................................................ 102

Figure 5.13 Selected Kendrick mass defect filters representing losses of CH3N and C2H6NO for

all 2C fragments falling within said filters. Fragment shown outside the filter is from 2C-T. The

horizontal lines represent the average (light green and purple) and the upper and lower bounds of

each mass defect filter (dark green and purple) .......................................................................... 103

Figure 5.14 Spectrum of 25H-NBOMe and most abundant fragment ions above m/z 105 ........ 104

Figure 5.15 Proposed structures for fragment ions of 25H-NBOMe after their neutral losses .. 105

Figure 5.16 Proposed structural elucidation of 2C-N and 25N-NBOMe leading to the same

fragment (C9H11NO4) .................................................................................................................. 106

Figure 5.17 Selected Kendrick mass defect filter and corresponding NBOMe fragments falling

within the filter. Fragments shown outside the filter are from mescaline-NBOMe and 2C-T. The

horizontal lines represent the average (light green) and the upper and lower bounds of the mass

defect filter (dark green) ............................................................................................................. 106

Figure 5.18 Selected Kendrick mass defect filters and corresponding 3C fragments falling

outside the filters ........................................................................................................................ 107

Figure 5.19 Characterization scheme for high-resolution mass spectral data. M+adj is the mass of

the molecular ion adjusted for a halogen/sulfur/nitro substituent ............................................... 109 Figure 5.20 Mass spectrum and structure of cathinone, 3-methylethcathinone (3-MEC) showing

loss of C3H8O, which is uncharacteristic of the phenethylamine class....................................... 112

Figure 5.21 Mass spectrum of 3C-phenethylamine, mescaline and fragment ions remaining after

neutral losses, the KMD of which can be used to distinguish 2C from 3C-phenethylamines .... 114

xii

Figure A.1 High-resolution mass spectra of (A) 4-(2-aminopropyl)benzofuran (4-APB), (B) 5-(2-

aminopropyl)benzofuran (5-APB), and (C) 7-(2-aminopropyl)benzofuran ............................... 117

Figure A.2 High-resolution mass spectra of (A) 2,5-dimethoxy-4-methylphenethylamine (2C-D),

(B) 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and (C) 2,5-dimethoxy-4-

propylphenethylamine (2C-P) ..................................................................................................... 118

Figure A.3 High-resolution mass spectra of (A) 2,5-dimethoxy-4-chlorophenethylamine (2C-C),

(B) 2,5-dimethoxy-4-iodophenethylamine (2C-I), and (C) 2,5-dimethoxy-4-nitrophenethylamine

(2C-N) ......................................................................................................................................... 119

Figure A.4 High-resolution mass spectra of (A) 2,5 -dimethoxy-4-methylthiophenethylamine

(2C-T) and (B) 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) ........................................ 120

Figure A.5 High-resolution mass spectra of (A) 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-

methyoxybenzyl)ethanamine (25D-NBOMe), (B) 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-

methoxybenzyl)ethanamine (25E-NBOMe) and (C) 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-3,4-dimethyl-benzeneethanamine (25G-NBOMe) ............................. 121

Figure A.6 High-resolution mass spectra of (A) 4-bromo-2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe), (B) 4-chloro-2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-benzeneethanamine (25C-NBOMe), and (C) 4-iodo-2,5-dimethoxy-N-

[(2-methoxyphenyl)methyl]-benzeneethanamine (25I-NBOMe) ............................................... 122

Figure A.7 High-resolution mass spectra of (A) 2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-4-

(methylthio)-benzeneethanamine (25T-NBOMe), (B) 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-4-[(1-methylethyl)thio]-benzeneethanamine (25T-4-NBOMe), (C) 2,5-

dimethoxy-N-[(2-methoxyphenyl)methyl]-4-(propylthio)-benzeneethanamine (25T-7-NBOMe),

and (D) 3,4,5-trimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (mescaline-

NBOMe) ..................................................................................................................................... 123

Figure A.8 High-resolution mass spectra of (A) 4-ethoxy-3,5-dimethoxy-benzeneethanamine

(escaline) and (B) 4-methylmethcathinone (mephedrone) ......................................................... 124

Figure A.9 Mass spectrum of 2C-G and fragment ions remaining after neutral losses .............. 125

Figure A.10 Mass spectrum of 2C-B and fragment ions remaining after neutral losses ............ 128

Figure A.11 Mass spectrum of 25N-NBOMe and fragment ions remaining after neutral losses130

1

I. Introduction

1.1 Synthetic Designer Drugs

According to the 2015 National Drug Threat Assessment Summary, the abuse of

synthetic designer drugs has remained constant or increased since their popularity skyrocketed in

2008.1 These drugs are typically used by younger individuals, as they are marketed in packages

with bright colors and cartoons, and in a variety of fruit or candy flavors.1 By definition, a

designer drug is a synthetic version of a controlled substance that is produced with a slightly

altered molecular structure to avoid being classified as an already regulated compound.2

Consequently, users may experience the same psychoactive effects as controlled substances

without legal ramifications.

The 2013 National Drug Threat Assessment Summary defined seven classes of synthetic

designer drugs: cannabinoids, phencyclidines or arylcyclohexamines, tryptamines, piperazines,

pipradrols, tropane alkaloids, and phenethylamines, which are the focus of this research.3 The

Drug Enforcement Administration (DEA) has exercised emergency scheduling authority to

temporarily control over 20 synthetic drugs since the 2012 enactment of the Synthetic Drug

Abuse Prevention Act. As an amendment of the Controlled Substances Act, the Synthetic Drug

Abuse Prevention Act already had regulated 29 synthetic drugs as Schedule I substances. Despite

increased efforts in legislation and law enforcement, clandestine chemists are frequently two

steps ahead, because as soon as one designer compound is scheduled, a new analog appears on

the market. The new analog often differs only slightly in chemical structure or composition from

the regulated compound, for example, as an isomeric form or with different substitutions. The

core structure of phenethylamine, comprised of a benzene ring and amine side chain, is shown in

2

Figure 1.1, while some of its substituted analogs are shown in Figure 1.2. Within the

phenethylamine class, there are subclasses of 2,5-dimethoxyphenethylamines (2C), N-benzyl

phenethylamine analogs (NBOMe), aminopropyl benzofuran phenethylamines (APB), and 3,4,5-

trimethoxyphenethylamines (3C) compounds.4 Further, within each subclass, there are many

compounds available with varying substituents around the subclass-core structure. For example,

typical substitutions of varying alkyl chain length occur on the popular and well-known 2C-

phenethylamine core in the 3’ and 4’ positions, while non-alkyl substituents like halogens, nitro,

and sulfur groups occur in the 4’ position. Most compounds in the APB subclass do not have

additional substituents around the ring; instead, the location of the furan ring around the benzene

ring changes to create different isomers. Compounds in the NBOMe class have substituents in

the same locations as the 2C compounds, but also can be altered on the N-benzyl side of the

compound by adding substituents or changing the placement of the methoxy group. The

combinations of varying substituents and substitution positions to the different subclass core

structures is limitless. Thus, new, unscheduled “legal” highs are a challenge for law makers and

forensic analysts to identify, despite the legislation that is already in place.

NH2

Figure 1.1 Phenethylamine

3

Figure 1.2 Phenethylamine analogs. (A) 2,5-dimethoxyphenethylamine (2C-H) (B) 4-ethyl-2,5-

dimethoxyphenyl-N-(2-methoxybenzyl) ethan-1-amine (25E-NBOMe) (C)

aminopropylbenzofuran (4-APB) (D) 1-(3, 5-dimethoxy-4-propoxyphenyl) propan-2-amine (3C-

P)

1.2 Current Methods of Analysis of Submitted Drug Samples and Limitations

The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) has

recommendations for the analysis of controlled substances, with different analytical techniques

placed into categories based upon their maximum potential discriminating power.5 Currently, the

method of choice for the analysis of controlled substances in most forensic laboratories is gas

chromatography-mass spectrometry (GC-MS). This hyphenated technique separates components

in a given sample and the mass spectrum of each separated component is recorded. In these

instruments, electron ionization (EI) is used which, as a ‘hard’ ionization method, results in

substantial fragmentation of each separated component. As a result, the mass spectral

fragmentation patterns contain a significant amount of information from which structural

elucidation is possible to determine the identity of the compound. Gas chromatography-mass

spectrometry satisfies SWGDRUG’s recommendation that at least two analytical techniques be

used for identification. GC-MS is the preferred method for satisfying these recommendations

because of its reproducibility, cost effective nature, and the ability for high throughput of

samples.

(A) 2C (B) NBOMe (C) APB (D) 3C

O

O

3'

4'

NH2

H

3'

4'

O

O

N

O

O

NH2O

O

O

NH2

4

However, there are some limitations with using GC-MS for the identification of synthetic

designer drugs. Although there is extensive fragmentation of compounds using electron

ionization, this is often insufficient for definitive identification of synthetic designer drugs

because of the high structural similarity among compounds within the same class. Further, the

conventional instruments are equipped with a single quadrupole mass analyzer (GC-QMS),

which yields only nominal mass information for each fragment ion. This means that distinction

of isomeric compounds is very difficult. Isomeric compounds have the same molecular mass

and, therefore, exhibit similar mass spectra, with the same ions appearing only in different ratios.

Because new designer drug analogs appear on the market so quickly, an additional obstacle for

forensic laboratories to overcome is that oftentimes reference standards are not available. A lack

of reference standards is problematic because identification of drugs in forensic labs is based on

a visual comparison of the mass spectrum of the reference standard to the mass spectrum of the

questioned sample. Therefore, there is a need for improved methods for the identification and

characterization of synthetic designer drugs using the conventional GC-QMS instruments

available in forensic laboratories.

1.3 Current Research of Synthetic Designer Drugs

There has been extensive research of some designer drug compound classes, specifically

cathinones and cannabinoids, involving the identification and characterization of designer drugs

in street samples. However, the research has been performed primarily using high-resolution

mass spectrometry.6 - 10 High-resolution mass spectrometers are capable of acquiring accurate

mass data, from which the elemental composition of each ion can be determined with a high

degree of confidence.

5

An additional advantage of using high-resolution methods is that from the accurate mass,

the mass defect (defined as the difference between the exact and nominal mass) for each ion can

also be determined. These mass defects can be used to identify filters that are characteristic of a

given compound class and therefore, can be used for characterization of unknown synthetic

designer drugs. Grabenauer et al. used mass defect filters to characterize synthetic

cannabinoids.11 Compounds analyzed in the study had mass defects ranging from 0.13 and 0.23

Da. A filter was developed that was centered at 0.185 Da with a window of ±50 mDa and this

filter encompassed 75% of the known cannabinoids with the indole core structure. However, the

study used high-resolution mass spectrometry with liquid chromatography.

While liquid chromatography with high-resolution mass spectrometry has advantages in

the characterization of synthetic designer drugs (i.e., accurate mass data and mass defect filters),

there are limitations. These instruments use electrospray ionization, which is a soft ionization

method, resulting in little fragmentation. The outcome is a molecular ion which can allow for

molecular mass information, however, also results in less fragmentation and thus little structural

information. As structural information is especially important for unknowns, less fragmentation

can make definitive compound identification difficult. Another limitation, which is more

problematic, is that these instruments are not currently available in forensic laboratories. So,

while research is being conducted, the methods developed are neither practical nor

implementable for use in a forensic laboratory.

Zuba previously described a method for categorizing designer drugs into their compound

classes based on mass spectral data collected using the conventional GC-QMS instrument

configuration that is available in forensic labs. Zuba also used liquid chromatography-

electrospray ionization with quadrupole time of flight mass spectrometry (LC-ESI/QTOF-MS), a

6

more sophisticated instrument not readily available.4 The characterization of each compound was

based on molecular and fragment ions. However, with GC-QMS, only nominal mass data were

collected. Thus, only preliminary classification of known “legal highs” into general compound

class (e.g., phenethylamine versus cathinone) rather than subclass (e.g., APB versus 2C-

phenethylamine) was possible using Zuba’s characterization flow chart. Preliminary

classification into general compound class is problematic for large compound classes such as the

phenethylamines for which several subclasses exist, as discussed previously. Using the flow

chart, these compounds could be identified as phenethylamines, but no further sub-classification

would be possible. Because of the wide structural variation among subclasses of the

phenethylamine class, a more specific characterization is needed. Additionally, Zuba focused on

the investigation of fragmentation of compounds from only the cathinone class.

Other characterization methods for compounds in the phenethylamine and other classes

have been investigated. For example, Zuba and Sekula characterized the phenethylamine 3,4-

dimethyl-2,5-dimethoxyphenethylamine (2C-G); however, four different instruments utilizing

six analysis methods were needed to complete the characterization.7 These instruments included

GC-EI/MS and Fourier transform infrared spectroscopy (FTIR), both of which are commonly

available in forensic laboratories, LC-ESI/QTOF-MS, and two types of nuclear magnetic

resonance spectroscopy (NMR) which are not commonly used in forensic labs. Overall, using six

techniques to characterize one synthetic drug analog is strenuous, time-consuming, and

impractical for forensic analysts with large workloads. Similarly, Shevyrin et al. isolated,

identified, and characterized several indole-3-carboxylic acid synthetic cannabinoids by similar

methods (GC-QTOF-MS, ultra-performance LC-QTOF-MS, NMR, and FTIR)8 while Uchiyama

et al. identified fifteen designer drugs in street samples also using UPLC-ESI-MS and GC-EI/MS

7

along with NMR.9 In a different study, Uchiyama et al. characterized several cannabinoids and

NBOMe phenethylamines, but again using only one instrumental technique used in forensic

laboratories (GC-MS) and two techniques not used in forensic laboratories (LC-MS and NMR).6

The use of so many techniques for characterizing new analogs highlights the lack of a quick,

clear, concise, and practical method for characterization of these compounds.

Fornal characterized the cathinone compound class by subclass using high performance

LC-QTOF-MS.12 Based on structural features such as double-bond equivalency and the

characteristics of the amine group, nine different subclasses of cathinones were identified. The

fragmentation pathways for each class were proposed, and specific losses common to each class

were briefly discussed. However, because ESI was used for ionization, the proposed

fragmentation pathways developed will be different than those from the commonly used election

ionization method. Therefore, if a forensic analyst received a new analog belonging to one of the

nine cathinone compound classes presented, a direct characterization could not be made.

A DEA monograph authored by Casale and Hays describes the synthesis,

characterization, and differentiation of eleven NBOMe phenethylamines.13 Using GC-MS and

FTIR, both instruments used in a typical forensic lab, each NBOMe could be distinguished from

their corresponding 2C analog. However, each NBOMe was differentiated from one another by

relative ion abundances. If a reference standard is not available for comparison, using relative

abundances is problematic because ion abundances can vary from instrument to instrument as

well as sample run to run. While differentiation of NBOMes was reported, the method is still

somewhat subjective, therefore, an improved method of differentiation is needed.

Overall, there is a need for a rapid characterization scheme for synthetic designer drugs

that employs the conventional GC-QMS instrument configuration used by the majority of

8

forensic laboratories for controlled substance identification. Current characterization methods for

these compounds frequently do not use instrumentation available in forensic laboratories and,

therefore, the characterization schemes developed are not directly implementable into labs.

1.4 Research Objectives and Goals

This research focused on developing methods for the characterization of synthetic

designer drugs according to structural subclass. The focus in this initial work was the synthetic

phenethylamine compound class. More specifically, the objectives were:

1. To develop a characterization scheme based on data collected using common GC-

QMS instruments available in forensic laboratories.

2. To investigate the potential of high-resolution mass spectrometry and mass defect for

a more robust characterization of designer drugs.

The objectives were accomplished by achieving the following goals:

1. Developing a flow-chart style characterization scheme based on characteristic mass

spectral features obtained using GC-EI-QMS.

To do this, a range of phenethylamine standards encompassing different subclasses was analyzed

by both low- and high-resolution instruments (GC-QMS and GC-TOFMS). Because these

instruments use the same electron ionization method, their spectra are comparable. However, the

TOF mass analyzer provides accurate mass of each fragment ion. Accurate mass allows for the

confirmation of the elemental formula of each ion and the understanding of how these

compounds fragment under electron ionization conditions. The fragmentation pathways, mass

spectral features, and neutral losses that are characteristic of each phenethylamine subclass were

confirmed by the high-resolution spectra and are translatable to the GC-QMS data because the

9

same ionization method is used. Because GC-QMS is the same instrument configuration used in

forensic labs, the scheme is immediately implementable in forensic labs.

From a confirmed molecular ion, neutral losses were investigated for characterization. A

neutral loss is a fragment lost as a neutral molecule during ionization. By investigating what

common losses occur from compounds of each subclass, characteristic neutral losses can be

identified. Unknown compounds exhibiting those common losses may be able to be

characterized into specified subclasses.

Additionally, retention index can be calculated and used as another characterization

tool.14 In current forensic practices, it is the comparison of chromatographic as well as mass

spectral data of reference standards to questioned samples that allows for identification.

However, in the event that no reference sample is readily available to analyze on the laboratory

instrument, reference chromatograms and spectra from online sources such as the SWGDRUG

drug monographs or Cayman Chemical© can be downloaded.15, 16 The retention times from the

monographs may differ from that obtained experimentally in the lab because of variations in

temperature program, column length and diameter, stationary phase film thickness, or carrier gas

velocity and pressure. By calculating and using retention index, these variables are eliminated

and retention indices collected on two different instruments can be compared. Further, a range of

retention indices for each compound subclass was developed in this work and incorporated into

the characterization scheme. For example, a range of retention indices for the 2C-

phenethylamine class was determined, and if an unknown were to have a retention index that fell

within that range, it would increase the confidence in preliminarily characterizing that compound

as a 2C-phenethylamine.

10

2. Develop mass defect filters from the high-resolution data that can be used to enhance

characterization.

To do this, the exact mass data obtained were used to develop mass defect filters to use for more

confident characterization of phenethylamines into structural subclasses. The exact mass data

also highlight the utility of high-resolution mass spectrometry, should it ever become available to

forensic labs. Previous preliminary work had identified potential limitations in the development

of mass defect filters that this work overcomes.17 The first limitation is a lack of molecular ion.

Because molecules are being bombarded with such high energy in electron ionization mode, they

often do not remain intact and completely fragment. This means that some compounds do not

produce a molecular ion peak in their mass spectrum. This problem can be overcome by

determining the molecular ion using chemical ionization. Chemical ionization is a soft ionization

technique that most GC-QMS systems can be equipped to perform. A second limitation

associated with mass defect filters is determining how wide or narrow the filter should be. If the

filter is too wide, then compounds from different subclasses will be included. However, if the

filter is too narrow, then compounds from the same subclass may be excluded. These problems

are addressed by defining filters based on the Kendrick mass defect. A Kendrick mass defect is

an adjusted mass based on the conversion between exact mass of a methylene unit (CH2) and its

nominal mass. Kendrick mass is defined as the exact mass multiplied by this conversion and is a

way to normalize masses of a similar class of compounds. Thus, members of a homologous

series that differ only in the number of methylene groups will have the same Kendrick mass

defect. As a result, members of a given subclass will have the same Kendrick mass defects,

which will be different from the Kendrick mass defects of another subclass. The use of Kendrick

11

mass defect to overcome problems associated with the width of the filter are utilized in this

research.

The development of an “easy-to-follow” flow-chart style characterization scheme will be

easily and immediately implementable into forensic laboratories because it will have been

created using instrumentation and methodology that is already in place. The characterization

scheme will be used as an initial screening method to determine if further examination of a

submitted controlled substance sample is necessary. Additionally, the scheme will assist in

identification of new compounds in a constantly changing drug market, and allow for

characterization of unknowns for which no reference standard is available. Further, by

investigating the use of mass defect filters for a more robust characterization, this research

highlights the potential for high-resolution instrumentation for forensic applications. As

molecular ions are not always available for synthetic phenethylamines, mass defect filters are

developed for characteristic fragment ions of neutral losses. This, along with the Kendrick mass

defect and retention index, will provide sufficient information to distinguish synthetic

phenethylamines from different subclasses, and the utility of high-resolution will be highlighted,

should those instruments ever be made available.

12

REFERENCES

13

REFERENCES

1. National Drug Threat Assessment Summary. U.S Department of Justice Drug

Enforcement Administration, 2015.

https://www.dea.gov/docs/2015%20NDTA%20Report.pdf

2. Designer Drug. Merriam-Webster Dictionary. http://www.merriam-

webster.com/dictionary/designer%20drug

3. National Drug Threat Assessment. U.S. Department of Justice Drug Enforcement

Administration, 2013. http://www.dea.gov/resource-center/DIR-017-

13%20NDTA%20Summary%20final.pdf

4. Zuba, D. Identification of cathinones and other active components of ‘legal highs’ by

mass spectrometric methods. TrAC Trends in Analytical Chemistry. 2012 Feb; 32: 15-30.

5. Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG).

Recommendations.

http://www.swgdrug.org/Documents/SWGDRUG%20Recommendations%20Version%2

07-0.pdf

6. Uchiyama, N. Shimokawa, Y. Matsuda, S. Kawamura, M. Kikura-Hanajiri, R. Goda, Y.

Two new synthetic cannabinoids, AM-2201 benzimidazole analog (FUBIMINA) and (4-

methylpiperazin-1-yl) (1-pentyl-1H-indol-3-yl) methanone (MEPIRAPIM), and three

phenethylamine derivatives, 25H-NBOMe 3, 4, 5-trimethoxybenzyl analog, 25B-

NBOME, and 2C-N-NBOMe, identified in illegal products. Journal of Forensic

Toxicology 2013 Jan; 32(1): 105-15.

7. Zuba, D. Sekula, K. Identification and characterization of 2, 5-dimethoxy-3, 4-dimethyl-

β-phenethylamine (2C-G) - A new designer drug. Drug Testing and Analysis 2012 Jul;

5(7): 549-59.

8. Shevyrin, V. Melkozerov, V. Nevero, A. Eltsov, O. Shafran, Y. Analytical

characterization of some synthetic cannabinoids, derivatives of indole-3-carboxylic acid.

Forensic Science International. 2013 Oct; 232(1-3): 1-10.

9. Uchiyama, N. Matsuda, S. Kawamura, M. Shimokawa, Y. Kikura-Hanajiri, R. Aritake,

K. et al. Characterization of four new designer drugs, 5-chloro-NNEI, NNEI indazole

analog, α-PHPP and α-POP, with 11 newly distributed designer drugs in illegal products.

Forensic Science International. 2014 Oct; 243: 1-13.

10. Uchiyama, N. Kikura-Hanajiri, R. Ogata, J. Goda, Y. Chemical analysis of synthetic

cannabinoids as designer drugs in herbal products. Forensic Science International. 2012

May; 198(1-3): 31-38.

14

11. M. Grabenauer, W. L. Krol, J. L. Wiley, B. F. Thomas. Analysis of Synthetic

Cannabinoids using High-Resolution Mass Spectrometry and Mass Defect Filtering:

Implications for Nontargeted Screening of Designer Drugs. Journal of Analytical

Chemistry. 2012 June; 84(13): 5574-81.

12. Fornal, E. Study of collision-induced dissociation of electrospray-generated protonated

cathinones. Drug Testing and Analysis 2013 Nov; 6(7-8). 705-715.

13. Casale, J. Hays, P. Characterization of Eleven 2,5-Dimethoxy-N-(2-

methoxybenzyl)phenethylamine (NBOMe) Derivatives and Differentiation from their 3-

and 4- Methoxybenzyl Analogs – Part 1. U.S. Department of Justice Drug Enforcement

Administration Microgram Journal 9(2). 84 – 109.

14. Skoog, D. Holler, F. Crouch, S. Principles of Instrumental Analysis. 6th ed.; Thomas

Brooks/Cole: Belmont, CA, 2007.

15. Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG). Micrographs.

http://swgdrug.org/monographs.htm

16. Cayman Chemical. Product Search and Drug ID.

https://www.caymanchem.com/forensics/search

17. Chu, F. Improving Methods for the Analysis of Controlled Substances. Master’s Thesis,

Michigan State University, East Lansing, MI, 2015.

15

II. Theory

2.1 Chromatography Overview

Chromatography is an analytical technique used to separate chemical components of a

mixture, called analytes, by passing them through two phases in which the individual analytes of

the mixture, move at different rates based on their chemical properties. The two phases are called

the mobile phase and the stationary phase. The mobile phase is used to move the mixture through

a column, while the stationary phase is affixed inside the column. The separation of the different

analytes in the mixture is due to the transfer between the two phases. The two phases are

selected so that the analytes of the mixture distribute themselves between the mobile and

stationary phases to varying degrees.1 Typically, either gas or liquid chromatography can be

performed, depending on the matrix and the chemical composition of the analytes of the mixture

to be separated. For the purposes of this research, only gas chromatography will be discussed, as

it is the prevalent instrumentation used in forensic laboratories for controlled substance

identification.

2.2 Gas Chromatography Overview

In gas chromatography (GC) the sample to be analyzed is injected into the instrument for

separation (Figure 2.1). The sample then flows through the instrument by a carrier gas (the

mobile phase) to a capillary column within an oven. The mobile phase is an inert gas (e.g.,

helium) that does not chemically react with the sample. The inner wall of the column is coated

with the stationary phase, which can vary depending on the type of analytes to be separated. At

the end of the column is a detector, which generates a chromatogram. Each of these steps will

now be discussed in detail.

16

Figure 2.1 Schematic of gas chromatography (GC) instrument

The mobile phase is an inert gas, typically hydrogen or helium, which will not interact

with the sample, rather just flow through the system. The mobile phase is stored in a gas cylinder

near the GC instrument and flows into the injection port where samples are first injected.

Liquid and solid samples are first prepared by dissolving them in a suitable solvent for

GC analysis. A suitable solvent is one that is easily volatilized, non-reactive with the sample, and

will cause minimal degradation of the stationary phase inside the column. The sample is then

loaded into a syringe and injected into the injection port of the GC instrument. The injection port

must be hot enough (e.g., 250 °C) so that the sample is instantly volatilized. The injection port

also has mobile phase flowing through it at a set rate (e.g., 1 mL/min). Additionally, inside the

injection port there is a valve that can be used to deplete some of the sample to waste. This valve

is called the split valve and can be set to bleed off a specific ratio of the injected sample, as

defined by the user.1 A typical split ratio can be anywhere from 20:1 to 100:1, and the higher the

ratio, the less sample enters the column. A high split ratio would be used for concentrated

DetectorGas cylinder

Oven

Column

Syringe

Injection port

17

samples. In this work, a splitless injection was used. A splitless injection is advantageous

because all of the sample is introduced onto the column, which is useful for samples in which the

analyte is present in low concentrations. In forensic laboratories, many submitted controlled

substance samples contain both a cutting agent (e.g., caffeine) and the controlled substance.

Because the controlled substance in a submitted sample is at a lower concentration, a splitless

injection is typically used to ensure that the controlled substance is detected.

After injection, the vaporized sample enters the column. In gas chromatography, the

column is typically made of fused silica and can be anywhere from a few meters to 100 m in

length, with varying diameters (typically 0.25 mm), and differing stationary phase film

thicknesses (typically 0.25 µm). A column with the appropriate dimensions and stationary phase

must be chosen for efficient separation of components.

The stationary phase is adhered to the inside wall of the column and can vary in chemical

composition. The stationary phase is selected based on the chemistry of the analytes to be

separated (e.g., polar or non-polar molecules). In controlled substance analysis, typically a non-

polar stationary phase is used, meaning it is hydrophobic, (e.g., 5% diphenyl 95%

dimethylpolysiloxane). Analytes partition in and out of the stationary phase, and components that

are strongly retained, via strong intermolecular forces, by the stationary phase will move slowly

through the column, while those that have weak intermolecular force bonding with the stationary

phase travel rapidly. For example, in order to separate a mixture of non-polar compounds, the

analyst would choose a non-polar stationary phase because like compounds are attracted to one

another, via the intermolecular forces with the stationary phase. As a consequence of these

differences in migration rates, the chemical components of the mixture are separated into

different bands or peaks.1

18

To aid in separation of similar components, as well as to keep the sample in the gas

phase, the column is housed inside an oven that can be temperature programmed. Because the

retention of components is also dependent on their boiling points, the rate and how well they are

separated from one another can be manipulated by the temperature program of the oven. On a

non-polar stationary phase, the different components will interact with that phase based on their

polarity and affinity for it. The components will be released from the stationary phase based on

their boiling point as the temperature of the oven increases. A fast heating rate will allow a wider

range of compounds to be separated rapidly, but the compromise may be lower resolution.

Therefore, a compromise is needed to optimize the oven temperature program.

Once the various components of the sample have traveled through the column and

separated, they reach the detector. Various detectors such as flame ionization or electron-capture

devices can be used, depending on the application. In this research, a mass spectrometer (MS)

was used that was consistent with standard GC-MS use in forensic laboratories for controlled

substance identification. The detector’s primary purpose is to detect the separated components of

a mixture, and translate that information to a visual output. It is typically represented as a

chromatogram (Figure 2.2), where retention time is on the x-axis and signal abundance is on the

y-axis. Each peak represents a different separated component of the mixture. In forensic

laboratories, chromatograms are used for controlled substance identification by analyzing both

the questioned sample and a reference standard under the same conditions. The retention time

and peak shape of the reference standard are then compared to the suspected controlled

substance. If the retention times of the sample and reference standard are consistent (within ± 2

sec.), it is contributing evidence toward an identification of the substance.2

19

Figure 2.2 Example chromatogram of a multi-component gas chromatography separation

2.2.1 Retention Index

As previously discussed, retention time can be used as contributing information toward

the identification of an unknown compound, but it is dependent on many different variants, such

as the stationary phase, column length and diameter, and method parameters set by the user. For

example, if a sample was analyzed on two different instruments of the same make and model,

using the same method and program with the same stationary phase in the column, the retention

times may still vary slightly due to minor differences in the injection volume, injection speed,

syringe dwell time in the injection port, or flow rate of the carrier gas. However, the retention

time can be used to calculate the retention index (IT) of a compound. The IT is independent of

variables such as column dimensions, stationary phase thickness, flow rate, and temperature

program. By definition, the retention index of a compound is its retention time normalized to the

retention times of adjacently eluting normal-alkanes.3 Retention index is advantageous because it

allows the comparison of an adjusted retention time for a compound analyzed on different

instruments under varying conditions. Retention index is calculated using Equation 2.1

20

IT= 100 × [n + tr(unknown)- tr(n)

tr(n+1)- tr(n)] (2.1)

where IT is the retention index, n is the number of carbons in the smaller alkane (the one eluting

before the questioned sample), and tr is the retention time.

2.3 Mass Spectrometry Overview

For the detection of the samples, mass spectrometry was used in this research. Mass

spectrometry can give definitive identification of a compound by ionizing it and separating those

ions according to their mass-to-charge (m/z) ratios. This mass information can be used to identify

an unknown substance by comparing its information to a library of knowns, a reference standard,

or deducing its elemental composition. A mass spectrometer is comprised of three parts: the

ionization source, mass analyzer, and detector (Figure 2.3). All three of these components are

under vacuum (except the signal processor and readout) to ensure there are no unwanted

collisions between ions and to maintain free ion and electron flow. An under-vacuum system

ensures reproducible results and no contamination with air molecules.

Figure 2.3 Overall schematic of mass spectrometer (MS)

The ionization source is responsible for ionizing the gaseous output from the GC. From

the GC, separated components enter the ion source via a heated transfer line (e.g., 280 °C) and

are ionized. The transfer line must be sufficiently hot so that the separated components do not

condense out of the gas phase. There are many different types of ionization, however in this

research, electron ionization (EI) was used. Because samples are bombarded with a significant

Ionization source

Mass analyzer

Detector

Inlet from GC

Output

Vacuum pump

21

amount of energy, EI is classified as a “hard” ionization method and results in few intact

molecules and extensive fragmentation. Electrons are emitted from a heated tungsten filament in

the ionization source and are accelerated by applying 70 eV between the filament and anode

(Figure 2.4). As the sample gas travels through the repeller plate, it enters the path of electrons at

a 90° angle and through electrostatic repulsion, loses electrons to become positively charged

ions. These ions are then directed through the negatively charged focusing lens into the mass

analyzer. The mass analyzer is used to separate the newly created ions based on their m/z ratios

and send them to the detector. Two types of mass analyzers were used in this research, single

quadrupole and time-of-flight mass analyzers.

Figure 2.4 Electron ionization (EI) source

2.3.1 Mass Analysis: Single Quadrupole Mass Analyzer

A single quadrupole mass analyzer is one of the most common types of analyzers because

it is rugged and relatively inexpensive. It consists of four cylindrical rods that are parallel to one

another, as shown in Figure 2.5. Ions are filtered through the quadrupole based on the stability of

their trajectories as they travel through the oscillating electric fields that are applied to the rods.

22

A radio frequency (RF) voltage and a direct current (DC) offset voltage are applied between each

opposing rod pair and only ions of a specific m/z ratio will reach the detector for a given ratio of

voltages. Other ions will be unstable, collide with the rods, and be pumped away. The ratio of

voltages allows for selection of an ion with a particular m/z value, or allows the user to scan for a

range of m/z values by continuously varying the applied voltage while monitoring the RF/DC

ratio.

Figure 2.5 Quadrupole mass analyzer showing two different ion trajectories occurring

simultaneously. The red ion is neutralized as it collides with one of the rods, is pumped away,

and not detected, while the blue ion has a stable trajectory through the analyzer and travels to the

detector

Ions are then attracted to the detector, via its negative electric charge, which converts the

ions into an electrical signal that can be processed, stored in the memory of a computer, and

displayed.1 The most common detector is a continuous-dynode electron multiplier, which

collects, amplifies, and converts positive ions into electrical signal. The electron multiplier is a

cornucopia-shaped horn (known as a Woods horn), connected to a power source, with a

negatively charge entrance (i.e., -2 kV), and increasingly positively charged gradient walls. As

23

positively charged ions enter the horn, they collide with the wall and are converted to secondary

electrons. The secondary electrons are attracted along the positive electrical gradient farther

along the Woods horn, and each time they collide with the wall, additional secondary electrons

are ejected.1,4 The electrons are then converted to a voltage, the magnitude of which is indicative

of the abundance of each ion.

From quadrupole mass spectrometry (QMS), nominal mass information about the

original molecule is obtained because it is a low-resolution mass analyzer. Nominal mass is

defined as the integer mass of the most abundant, naturally occurring stable isotopes of a

molecule.4 The ability of the mass analyzer to distinguish between ions of similar m/z value is

defined by its resolving power.4 For example, low-resolution mass spectrometers are typically

only able to distinguish ions that are 1 mass unit (Da) apart. A limitation of nominal mass

resolution is that an elemental formula for each fragment ion cannot be confirmed. For example,

many formulae are possible for an ion at m/z 204 (i.e., C10H20O4 and C10H22NO3).

Resolution in mass spectrometry is defined by Equation 2.2 using two adjacent peaks in a

mass spectrum,

R = M

∆m (2.2)

where M is the mass of the first peak and Δm is the difference between the masses of the

adjacent peak. A larger resolution value indicates better separation of peaks. To measure the

minimum peak separation, Δm, and thus, the resolution, the peak width is measured at half of the

peak maximum (FWHM).4 In low-resolution mass analysis, resolution is typically on the order

of 102.

24

Higher resolution can be desirable as it indicates better discrimination between two

adjacent peaks, and allows for accurate mass measurement. Accurate mass measurement is the

mass measurement performed to a sufficient number of significant figures to allow for

unambiguous determination of an elemental composition, as is obtained with the time-of-flight

mass analyzer.4

2.3.2 Mass Analysis: Time-of-Flight Mass Analyzer

There are instances, such as within this research, where higher resolution is necessary and

can be achieved by high-resolution mass spectrometry. These instruments include mass analyzers

such as the time-of-flight (TOF) mass analyzer which yields exact mass to four decimal places.

Exact mass is defined as the most abundant naturally occurring stable isotope of an element, also

called its monoisotopic mass.4 Instruments like the TOF have resolution on the order of 103 - 105.

In TOFMS, the time required for an ion to travel from the ion source to the detector is

measured. Because all of the ions from the source are accelerated with the same energy, ions

travel at different velocities based on their differing m/z values. As the ions travel through the

analyzer, they are separated into different groups according to these velocities. For example, ions

of lower m/z value have higher velocity and reach the detector before ions of higher m/z value.

To increase resolving power of ions with similar m/z value, a reflectron-TOF was used in this

research. An ion mirror, in the form of an electric field with greater and opposite magnitude than

the electric field in the acceleration region, is positioned at an angle less than 180° to direct ions

toward the detector but not allow them to travel back toward the source.4 Ions with similar m/z

values but different energies take longer or shorter flight paths through the reflectron, thus

25

reaching the detector at different times. A schematic of a reflectron-TOF analyzer is shown in

Figure 2.6.

Figure 2.6 Time-of-flight mass analyzer showing two different ion trajectories occurring

simultaneously. Both ions are accelerated in the pusher region with the same kinetic energy, but

the red ion penetrates deeper into the reflectron because it has larger mass, thus reaching the

detector after the blue ion

After mass analysis, a spectrum is produced with resolution of ions that differ by less than 1 Da

and a mass accuracy, given in ppm, is assigned to each. Mass accuracy is a measure of the error

of measurement of the mass of the ion (Equation 2.3); therefore, good mass accuracy is

represented as a small, positive or negative value (i.e., the closer to zero, the better the mass

accuracy).5

Mass accuracy (ppm) = Theoretical exact mass-Measured exact mass

Theoretical exact mass × 10

6 (2.3)

Using the accurate mass information, the exact mass of each ion can be assigned an elemental

formula, leading to definitive identification of those ions. For example, an ion with the elemental

DetectorPusher

Reflectron

From ion source

26

formula assignment of C10H15NO2 would have a theoretical exact mass of 181.110279 Da. If the

measured mass of that ion was 181.1104 Da, the associated mass accuracy value would be -0.6

ppm via Equation 2.3. Exact mass from high-resolution mass spectrometry has the potential to

overcome limitations of nominal mass resolution data because of the definitive identification that

can be obtained.

2.3.3 Comparison of Low-Resolution and High-Resolution Mass Spectra

In mass spectrometry, after ionization, mass analysis, and detection of the ions, an output

is given in the form of a mass spectrum (Figure 2.7). The x-axis represents m/z value, while the

y-axis represents ion intensity. In both the low-resolution spectra, generated by QMS, and the

high-resolution spectra, generated by the TOFMS, the intact, positively charged molecule, called

the molecular ion (M+), is present at m/z 181 for the compound 2C-H. In the low-resolution

spectrum (Figure 2.7A), nominal mass values are associated with each ion (i.e., m/z 181, 152,

137, etc.). In the high-resolution spectrum (Figure 2.7 B), exact mass values are obtained to the

fourth decimal place (i.e., m/z 181.1104, 152.0833, 137.0601, etc.), along with a mass accuracy

(i.e., 0.6 ppm, 2.6 ppm, 1.5 ppm, etc.) (Section 2.3.2). From the exact and accurate mass, an

elemental formula can be discerned for each ion (i.e., C10H15NO2, C9H12O2, C8H9O2, etc.) and

structural arrangement can be proposed with confidence.

Each peak in the spectra represents a positively charged fragment of the molecular ion

after it has been bombarded by electrons during ionization (Section 2.3). The remaining,

uncharged part of the molecule that is pumped away is known as a neutral loss. In a spectrum,

the difference in mass between the molecular ion and a fragment ion corresponds to the neutral

loss. Elementally, the neutral loss can be identified by taking the chemical formula of the

27

molecular ion and subtracting the chemical formula of the fragment ion. For example, the

molecular ion of 2C-H (Figure 2.7) has a chemical formula of C10H15NO2 and the chemical

formula of the ion at m/z 152.0833 is C9H12O2, resulting in a neutral loss of CH3N. This research

investigates if compounds of similar structural classes fragment similarly and exhibit any

common neutral losses.

Figure 2.7 Spectra and chemical structure of 2, 5- dimethoxyphenethylamine (2C-H) via (A)

low-resolution (QMS) and (B) high-resolution (TOFMS) mass spectrometry

0 100 200 300

0

100

Abundance (

%)

m/z

A)

B)

M+

181.1104C10H15NO2

0.6 ppm

152.0833C9H12O2

2.6 ppm

137.0601C8H9O2

1.5 ppm

121.0645C8H9O

6.6 ppm

91.0543C7H7

5.5 ppm

M+

181

152

137

121

O

O

NH2

2C-H

91

O+

O

CH2

H

C+

O

O

O

CH2+

CH2+

0 100 200 300

0

100

Abundance (

%)

m/z

28

2.4 Mass Defect

Once the exact mass of an ion is obtained using high-resolution mass spectrometry, it can

be used to calculate other characteristics of the ions, such as mass defects. Different amounts of

energy are released by every elements’ isotope upon binding and stabilizing of its nucleus, called

nuclear binding energy. Absolute mass defect, commonly referred to as just mass defect, is the

difference in binding energy between every isotope to carbon-12, either positive or negative

(Table 2.1). Because each isotope has a different mass defect, each molecule of different

elemental composition will have a unique exact mass.6 Absolute mass defect is calculated by

Equation 2.4

Absolute mass defect = Exact mass - Nominal mass (2.4)

For example, the absolute mass defect of 2C-H would be its exact mass (181.1104 Da) minus its

nominal integer mass (181 Da) to yield a defect of 0.1104 Da.

Table 2.1 Absolute mass defects of elements commonly used in this research

ElementNominal

Mass (Da)

Most Abundant

Isotope Mass (Da)

Absolute Mass

Defect (Da)

C 12 12.00000 0.00000

H 1 1.007825 0.00782

N 14 14.003074 0.00307

O 16 15.994915 -0.00508

Cl 35 34.968853 -0.03115

Br 79 78.918336 -0.08166

F 19 18.998403 -0.00160

I 127 126.904477 -0.09552

S 32 31.972072 -0.02793

29

Mass defect can be a useful tool for the characterization of unknowns. Mass defect ranges

(called filters) can be created for classes of compounds in which the mass defect of an unknown

could fall within or outside of, indicating potential class characterization. However, the

limitation with absolute mass defect is that as mass increases, so too does the absolute mass

defect. Therefore, as compounds of increasing mass are added to the filter, it will continue to

become wider and less specific.

2.4.1 Kendrick Mass Defect

A second type of mass defect, called Kendrick mass defect, can be used for

characterizing compounds of the same homologous series. A Kendrick mass is first calculated by

normalizing exact mass to the mass of a methylene (CH2) group via Equation 2.5. The difference

between the new Kendrick mass and the nominal mass is calculated to obtain the Kendrick mass

defect (KMD) of the ion (Equation 2.6).6

Kendrick mass = Exact mass × 14.00000

14.01565 (2.5)

KMD = Nominal mass - Kendrick mass (2.6)

Theoretically, compounds of the same homologous series, which differ only in the number of

CH2 groups, will have the same Kendrick mass defect. For example, 2C-H has a theoretical

KMD of 91.95 mDa while 2C-P, which has the 2C-H structure with an additional propyl group,

also has a theoretical KMD of 91.95 mDa. KMD filters can be created for different classes of

compounds, and have more specificity than absolute mass defect filters for characterization of

unknowns. The filters are much narrower and based only on compounds within a homologous

series, so the risk of false positive characterization is reduced.

30

REFERENCES

31

REFERENCES

1. Skoog DA, Holler FJ, Crouch SR. Principles of Instrumental Analysis. 6th ed. Belmont,

CA: Thomas Brooks/Cole, 2007.

2. Virginia Department of Forensic Science. Controlled Substances Procedure Manual.

2016, 19, 64.

3. IUPAC Gold Book. https://goldbook.iupac.org/R05360.html (Accessed December 13,

2016).

4. Watson JT, Sparkman, OD. Introduction to Mass Spectrometry. 4th ed. Wiley, 2007.

5. Agilent Technologies. Mass Accuracy and Mass Resolution in TOF MS.

https://www.researchgate.net/file.PostFileLoader.html?id=567901175cd9e3a6cc8b4571&

assetKey=AS%3A309368938008576%401450770710216. (Accessed February 20,

2017).

6. Sleno, L. The use of mass defect in modern mass spectrometry. J. Mass Spectrom. 2012,

47, 226-236.

32

III. Materials and Methods

3.1 Reference Standards

Synthetic phenethylamine and cathinone reference standards spanning various structural

subclasses were purchased from Cayman Chemical (Ann Arbor, MI). These included four

aminopropyl benzofuran phenethylamines (APB) and eleven 2,5-dimethoxyphenethylamines

(2C) (Figure 3.1), two 3,4,5-trimethoxyphenethylamines (3C), twelve N-benzyl phenethylamine

analogs (NBOMe) (Figure 3.2), and two cathinones (Figure 3.3). Full chemical names for each

compound are given in the Appendix for this chapter. Throughout the remainder of this work, all

compounds will be referred to by their common abbreviations. All standards were prepared at a

concentration of 1 mg/mL of methanol (ACS grade, Sigma-Aldrich, St. Louis, MO).

For retention index determination, a mixture of normal (n-) alkanes was prepared using

alkanes ranging from C12 – C28, and C30 (Sigma-Aldrich, St. Louis, MO). Each alkane was

prepared to an approximate concentration of 13.5 mM in dichloromethane (ACS grade, EMD

Millipore, Darmstadt, Germany). Typically, to determine retention index, the n-alkanes are

spiked directly into the sample to be analyzed; however, this practice is not practical in a forensic

crime laboratory setting. Therefore, the alkane mixture was analyzed independently at the

beginning of the sample sequence, after every 10 sample injections, and at the end of the sample

sequence. Retention indices were calculated using Equation 2.1.

33

Figure 3.1 Structures of the phenethylamines in the reference set (A) 4-(2-aminopropyl)

benzofuran (4-APB) (B) 5-(2-aminopropyl) benzofuran (5-APB) (C) 6-(2-aminopropyl)

benzofuran (6-APB) (D) 7-(2-aminopropyl) benzofuran and (E) the core structure of 2,5-

dimethoxyphenethylamine (2C-phenethylamines). The substituents at R1 and R2 and the

corresponding 2C compound are given in Table 3.1.

Table 3.1 Substituents for 2C-phenethylamine compound shown in Figure 3.1

NH2O

NH2

O

O NH2NH2

O

4-APB 5-APB

6-APB 7-APB

A) B)

C) D)

NH2

O

O

R1

R2

E)

Compound R1 R2 Compound R1 R2

2C-H -H -H 2C-B -H -Br

2C-D -H -CH3 2C-C -H -Cl

2C-G -CH3 -CH3 2C-I -H -I

2C-E -H -CH2CH3 2C-N -H -NO2

2C-P -H -CH2CH2CH3 2C-T -H -SCH3

2C-T-2 -H -SCH2CH3

34

Figure 3.2 Structures of more of the phenethylamines in the reference set (A) 3,4,5-trimethoxy-

benzeneethanamine (mescaline), (B) 4-ethoxy-3,5-dimethoxy-benzeneethanamine (escaline)

both 3C-phenethylamines, (C) the core of N-benzyl phenethylamine analogs (NBOMe-

phenethylamines). The substituents at R1 and R2 corresponding NBOMe compound are given in

Table 3.2, and (D) 3,4,5-trimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine

(mescaline-NBOMe)

Table 3.2 Substituents for NBOMe-phenethylamine compound shown in Figure 3.2

NH2

O

O

O

NH2

O

O

O

A) B)Mescaline Escaline

NH

O

O

R1

R2

O

NH

O

O

OO

C) D) Mescaline-NBOMe

Compound R1 R2 Compound R1 R2

25H-NBOMe -H -H 25I-NBOMe -H -I

25D-NBOMe -H -CH3 25N-NBOMe -H -NO2

25G-NBOMe -CH3 -CH3 25T-NBOMe -H -SCH3

25E-NBOMe -H -CH2CH3 25T-4-NBOMe -H -SCHCH3CH3

25B-NBOMe -H -Br 25T-7-NBOMe -H -SCH2CH2CH3

25C-NBOMe -H -Cl

35

Figure 3.3 Structures of cathinones in the reference set (A) 4-methylmethcathinone

(mephedrone) and (B) 3-methylethcathinone (3-MEC)

3.2 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

Three different gas chromatography-mass spectrometry (GC-MS) systems were used to

analyze the reference set of standards: one low-resolution single quadrupole instrument (GC-

QMS) and two high-resolution time-of-flight instruments (GC-TOFMS).

The GC-QMS consisted of an Agilent 6890N gas chromatograph coupled to an Agilent

5975C mass spectrometer with an Agilent 7683B injector (Agilent Technologies, Santa Clara,

CA). The column was coated with a (5% diphenyl)-95% dimethylpolysiloxane (DB-5, Restek,

Bellefonte, PA) stationary phase with dimensions 30 m x 0.25 mm x 0.25 µm. The injection

temperature was 250 °C and a splitless injection was used. The injection volume was 1 µL. The

carrier gas was ultra-high purity helium (Airgas, Radnor Township, PA) at a nominal 1 mL/min

flow rate. The oven temperature program was as follows: 40 °C for 1 min, 20 °C/min to 280 °C

with a final hold of 7 min. The transfer line temperature was 280 °C. Electron ionization at 70 eV

was used, the ion source temperature was 230 °C and the mass analyzer temperature was 150 °C.

The mass scan range was 35 – 550 u, with a scan rate of 2.83 scans/s. Retention index data were

collected only using this instrument.

NH

O

A) B) 3-MECMephedrone

NH

O

36

The GC-TOFMS used to analyze all APB compounds, 2C-B, 2C-D, 2C-E, 2C-G, 2C-H,

2C-P, 2C-T, escaline, mescaline, 3-MEC, and mephedrone was a Waters Micromass GCT

Premier (Waters, Milford, MA), which consisted of an Agilent 6890N gas chromatograph

coupled to a Waters GCT mass spectrometer with an Agilent 7683B autosampler. The same

column dimensions and stationary phase (DB-5) as the GC-QMS analysis were used. The

injection temperature was 210 °C and an appropriate split ratio injection was used per sample,

ranging from splitless to 100:1. The injection volume was 1 µL. The carrier gas was ultra-high

purity helium at a flow rate of 1.3 mL/min. The oven temperature program was as follows: 50 °C

for 1 min, 15 °C/min to 280 °C with a final hold of 2 min. The transfer line temperature was 280

°C. Electron ionization at 70 eV was used and the ion source temperature was 180 °C while the

mass analyzer was held at 130 °C. The scan range was 35 – 300 u and the rate was 5.00 scans/s.

To ensure good mass accuracy, a constant infusion of perfluoro-tertbutylamine (PFTBA), a

calibrant, was used during each sample analysis. The resolution of the instrument was 7,000

FWHM.

The second GC-TOFMS analysis was used to analyze the remaining sample set

compounds (i.e., all of the NBOMe compounds, 2C-C, 2C-I, 2C-N, and 2C-T-2) on a LECO

Pegasus GC-HRT (LECO Corp., St. Joseph, MI) which consisted of an Agilent 7890N gas

chromatograph coupled to a LECO Pegasus HRT mass spectrometer with a Gerstel MPS2

(GERSTEL, Inc., Linthicum Heights, MD) autosampler. The column was coated with 1,4-

bis(dimethylsiloxy)phenylene dimethyl polysiloxane (Rxi-5sil ms) stationary phase and

dimensions of 20 m x 0.18 mm x 0.18 µm (Restek, Bellefonte, PA). The injection temperature

was 250 °C and a 100:1 split injection was used due to the high sensitivity of the instrument. The

injection volume was 1 µL. The carrier gas was ultra-high purity helium at a flow rate of 0.85

37

mL/min. The oven temperature program was as follows: 60 °C for 0.5 min, 36 °C/min to 340 °C

with a final hold of 4 min. The transfer line temperature was 300 °C. Electron ionization at 70 eV

was used and the ion source temperature was 250 °C. The scan range was 35 – 510 u and the rate

was 10 scans/s. To ensure good mass accuracy, again a constant infusion of PFTBA was used

during each sample analysis. The resolution of this instrument was 50,000 FWHM. Although a

different column and GC conditions were used for these samples, it does not affect the mass

spectra, which was used for all data processing and analysis.

3.3 Data Processing

Low-resolution mass spectra were generated by taking a single scan at the apex of the

chromatographic peak in the total ion chromatogram after GC-QMS analysis. All spectra were

exported from ChemStation (Agilent Technologies) into Microsoft Excel (Microsoft,

Albuquerque, NM). All low-resolution spectra were plotted in Origin (version 8.6, OriginLab

Corp., Northampton, MA) to generate spectra of publication quality.

The high-resolution spectra obtained from the Waters GCT Premier were generated by

taking scans within the peak in the total ion chromatogram and subtracting these from a range of

scans in the baseline region immediately before the peak using MassLynx (version 4.1, Waters).

The range of scans in the baseline region represented the baseline condition from that sample run

and contained background ions at m/z 281, 207, and 73 as well as ions from the calibrant at m/z

218, 131, and 69. The mass accuracy of each ion in the background-subtracted mass spectra was

assessed using the elemental composition algorithm in MassLynx. The elemental composition

function tabulates the mass accuracies of each ion against a list of potential elemental formulae

and assigns them with a given mass accuracy value (in ppm) according to the ion’s exact mass.

The potential formulae are restricted by a user-defined tolerance (i.e., 50 ppm mass accuracy)

38

and by user-defined values of the number of each possible element. For this work, the tolerance

was defined as 50 ppm mass accuracy and the reference standard’s known elemental formula

was used to define the number of carbon, hydrogen, nitrogen, oxygen, bromine, chlorine, iodine,

and sulfur elements. Mass spectra containing ions with mass accuracies within ± 20 ppm, were

considered acceptable and were exported to Microsoft Excel (version 12.0, Microsoft Corp.,

Redmond, WA) for further processing. In Microsoft Excel, the ion abundancies were normalized

to that of the base peak. The m/z and normalized abundancies were then plotted in Origin.

The high-resolution spectra obtained from the LECO Pegasus-HRT were generated from

the Peak True data processed files in the ChromaTOF (version 4.2.3.1, LECO Corp.) software.

Peak True files include data processing such as background and calibrant ion subtraction. The

spectra were generated by taking the scan at the apex of the total ion chromatographic peak in

the ChromaTOF software. In a similar manner as previously described, element formulae and

mass accuracies were assigned to each ion using the algorithm in the ChromaTOF software.

Mass spectra containing ions with mass accuracies ± 10 mDa were considered acceptable and

were also exported to Microsoft Excel for further processing. The abundance of each ion was

normalized to the relative abundance of the base peak and the spectra were plotted in Origin.

3.4 Mass Defect Filters

Only the high-resolution spectra were used to create mass defect filters. The exact

masses, nominal masses and mass accuracies for all ions were tabulated in Microsoft Excel.

3.4.1 Absolute Mass Defect Filters

The absolute mass defect of each compound was first calculated using Equation 2.4, and

expressed in mDa. The absolute mass defect of all the compounds in the training set were then

39

averaged to obtain the centroid of the filter. A confidence interval was then calculated by the

following equations:

CI = SEM × tCL (3.1)

SEM = σ

√n (3.2)

where CI is the confidence interval, SEM is the standard error of the mean, tCL is the t value for

the specified confidence level, σ is the standard deviation, and n is the number of mass defects in

the training set. Thus, the filter was represented as the centroid value with a given tolerance,

expressed in the form of a confidence interval.

All mass defect filters were calculated at commonly used confidence levels of 99.9, 99,

95 or 90%. As confidence levels increase, so does the width of the filter. The wider the filter, the

higher likelihood that a compound will incorrectly fall within it, as a false positive. However, a

filter that is too narrow will exclude compounds that should fall within it, as a false negative.

Therefore, the confidence level is chosen to maximize the specificity of each filter. The mass

defects of each test set compound were calculated in the same manner and plotted against the

calculated filter to investigate the success of the absolute mass defect for correctly characterizing

compounds according to structural subclass.

3.4.2 Kendrick Mass Defect Filters

Kendrick mass defect (KMD) filters of molecular ions and fragment ions were calculated

in a similar manner as described in Section 3.4.1. The Kendrick mass of each molecular or

fragment ion was first calculated using Equation 2.5. The Kendrick mass was then subtracted

from the nominal mass and expressed in mDa (Equation 2.6). Using the same method as

40

previously described, the average KMD of the training set was calculated as the centroid of the

filter and a confidence level was calculated as the associated tolerance. The KMD filters were

developed using the appropriate training set compounds and the test set compounds were used to

test the success of the filter in correctly characterizing compounds according to structural

subclass.

41

APPENDIX

42

APPENDIX: Compound abbreviations and full chemical names

Table A.1 Compound abbreviations and chemical names

Compound

Abbreviation

Full Chemical Name Compound

Abbreviation

Full Chemical Name

4-APB 4-(2-

aminopropyl)benzofuran

25H-NBOMe 2-(2,5-dimethoxyphenyl)-N-

(2methoxybenzyl)ethanamine

5-APB 5-(2-

aminopropyl)benzofuran

25D-NBOMe 2-(2,5-dimethoxy-4-methylphenyl)-N-

(2-methoxybenzyl)ethanamine

6-APB 6-(2-

aminopropyl)benzofuran

25G-NBOMe 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-3,4-dimethyl-

benzeneethanamine

7-APB 7-(2-

aminopropyl)benzofuran

25E-NBOMe 2-(4-ethyl2,5-dimethoxyphenyl)-N-(2-

methoxybenzyl)ethanamine

2C-H 2,5-

dimethoxyphenethylamine

25B-NBOMe 4-bromo-2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-

benzeneethanamine

2C-D 2,5-dimethoxy-4-

methylphenethylamine

25C-NBOMe 2-(4-chloro-2,5-dimethoxyphenyl)-N-

(2-methoxybenzyl)ethanamine

2C-G 3,4-dimethyl-2,5-

dimethoxyphenethylamine

25I-NBOMe 4-iodo-2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-

benzeneethanamine

2C-E 2,5-dimethoxy-4-

ethylphenethylamine

25N-NBOMe 2-(2,5-dimethoxy-4-nitrophenyl)-N-(2-

methoxybenzyl)ethanamine

2C-P 2,5-dimethoxy-4-

propylphenethylamine

25T-NBOMe 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-4-

(methylthio)-benzeneethanamine

2C-B 2,5-dimethoxy-4-

bromophenethylamine

25T-4-NBOMe 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-4-[(1-

methylethyl)thio]-benzeneethanamine

2C-C 2,5-dimethoxy-4-

chlorophenethylamine

25T-7-NBOMe 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-4-(propylthio)-

benzeneethanamine

2C-I 2,5-dimethoxy-4-

iodophenethylamine

Mescaline-

NBOMe

3,4,5-trimethoxy-N-[(2-

methoxyphenyl)methyl]-

benzeneethanamine

2C-N 2,5-dimethoxy-4-

nitrophenethylamine

Mescaline 3,4,5-trimethoxy-benzeneethanamine

2C-T 2,5-dimethoxy-4-

methylthiophenethylamine

Escaline 4-ethoxy-3,5-dimethoxy-

benzeneethanamine

2C-T-2 2,5-dimethoxy-4-

ethylthiophenethylamine

3-MEC 3-methylethcathinone

Mephedrone 4-methylmethcathinone

43

IV. Characterization of Synthetic Phenethylamines by Low-Resolution Mass Spectrometry

The fragmentation of various synthetic phenethylamines of different structural subclasses

(Section 1.1) is described in this chapter. Characteristic features in the low-resolution mass

spectra are identified that can be used for characterization. The two aims are (1) to understand

fragmentation of compounds within a structural subclass, and (2) determine which of these

fragments can be used to characterize unknowns, or more likely, new analogs of synthetic

phenethylamines. Phenethylamines will be analyzed by gas chromatography – quadrupole mass

spectrometry (GC-QMS) and chromatograms will be used to determine retention index, while

spectra will be probed for characteristic ions to be used to identify phenethylamines. The spectra

will be further probed to identify characteristic features of different structural subclasses.

Knowing these features, a characterization scheme will be developed and application for

characterization of “unknowns” will be demonstrated.

4.1 Retention Index

Differentiation of isomeric compounds oftentimes poses a challenge for forensic analysts,

particularly in cases where one isomer is controlled and the other is not. Mass spectra do not

contribute much to distinguish isomers except relative ion ratios. For example, compounds 2C-G

(not controlled) and 2C-E (controlled) are currently differentiated by the intensities of fragment

ions at m/z 165 and 180. In the mass spectrum of 2C-G, m/z 165 has a higher abundance than m/z

180 and vice versa for the spectrum of 2C-E. However, distinction based only on ion ratios is

challenging because instrument variability can affect the ratios. With GC-MS analysis, the

chromatographic retention time can be used for isomer differentiation because isomers have

different interactions with the stationary phase inside the GC column. Although not a current

practice in forensic laboratories, retention index determination is easily implementable. By

44

calculating the retention index for each compound, there is the potential to distinguish isomers.

Using Equation 2.1, the retention indices for each phenethylamine in this study were calculated

(Table 4.1). As discussed, 2C-E and 2C-G, have different retention indices at 1706 and 1751,

respectively, showing the utility of retention index in isomer differentiation. Additionally,

retention index ranges can be determined for each structural subclass. The aminopropyl

benzofuran (APB) subclass has a retention index range of 1499 to 1527. The 2,5-

dimethoxyphenethylamines (2C) subclass has a range of 1590 to 2000, while the N-benzyl

phenethylamine analog (NBOMe) subclass has a range of 2475 to 2839. Because these ranges do

not overlap, the retention index can be a useful first step in the characterization of a synthetic

phenethylamine. Retention index data were not collected for some of the compounds in the

sample set, as indicated by “-“ in Table 4.1.

Table 4.1 Retention index and molecular ion determinations of sample set compounds

- Indicates data not collected. A (5% diphenyl)-95%dimethylpolysiloxane (DB-5) stationary

phase was used for IT determination. Compound names can be found in Chapter III Appendix.

Compound Retention

Index

Molecular Ion Compound Retention

Index

Molecular Ion

CI

[M+H]+

EI

[M+]

CI

[M+H]+

EI

[M+]

4-APB 1505 176.12 175.1 25H-NBOMe 2475 302.23 301.1

5-APB 1527 176.11 175.1 25D-NBOMe 2519 316.25 315.1

6-APB 1527 176.12 175.1 25G-NBOMe 2619 330.26 329.1

7-APB 1499 176.08 175.1 25E-NBOMe 2569 330.26 329.2

2C-H 1590 182.15 181.1 25B-NBOMe 2746 - 379.0

2C-D 1653 196.19 195.1 25C-NBOMe 2649 336.19 335.1

2C-G 1751 210.18 209.1 25I-NBOMe - - Not detect.

2C-E 1706 210.19 209.1 25N-NBOMe 2839 347.30 346.1

2C-P 1774 224.21 223.1 25T-NBOMe 2816 348.28 347.0

2C-B 1856 260.08 259.1 25T-4-NBOMe - - 375.1

2C-C 1770 216.13 215.0 25T-7-NBOMe - - 375.2

2C-I 1961 308.08 307.0 Mescaline-

NBOMe

- - 331.2

2C-N 2000 277.14 226.1 Mescaline - - 211.1

2C-T 1958 228.14 227.1 Escaline - - 225.2

2C-T-2 - - 241.1 3-MEC - - 191.1

Mephedrone - - 177.1

45

4.2 Electron Ionization Mass Spectra of Synthetic Phenethylamine Subclasses

Representative low-resolution mass spectra collected using a single quadrupole mass

spectrometer (GC-QMS) for each of the three phenethylamine subclasses of interest in this work,

2C-, APB-, and NBOMe-phenethylamines, are shown in Figure 4.1. Based solely on the low-

resolution mass spectra, it is not possible to determine the exact fragmentation mechanism.

However, based on known fragmentation of phenethylamines, structures for the dominant ions

can be hypothesized.1-4

All phenethylamines are expected to have spectra with some degree of similarity. For

example, α-β bond cleavage occurs among all three subclasses, splitting the aromatic ring from

the amine chain. In the APB subclass, this results in a base peak of m/z 44 from the amine chain

(C2H6N+) (Figure 4.1 A), and in the 2C and NBOMe subclasses, this cleavage results in a

methoxy methylbenzene ion (C8H9O+) at m/z 121 (Figure 4.1 B and C). The fragmentation of

compounds in the NBOMe subclass further supports the hypothesis of α-β bond cleavage, by the

presence of a highly abundant ion at m/z 150, as the amine side of the compound after cleavage

(C9H12NO+). Additionally, a common ion among the three subclasses is m/z 77, which

corresponds to a positively charged benzene ring (C6H5+), however, this is not a phenethylamine-

specific ion and would be present in any aromatic compound spectrum.

46

Figure 4.1 Representative spectra of (A) 6-APB, (B) 2C-H, and (C) 25H-NBOMe and proposed structures for the most dominant

fragment ions in each spectrum

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

181

152

137

121270

150

121

91131

77

44

175

O

O

NH

O

6-APB 25H-NBOMe

O

O

NH2

2C-H

O NH2

O NH2

m/z 175

O CH2

+

m/z 131

C+

m/z 77

CH2

+NH2

m/z 44

O

O

NH2

m/z 181

O+

O

CH2

H

m/z 152

C+

O

O

m/z 137

O

CH2+

m/z 121

O

O

NH

O

m/z 301

O

O

NHC

+

CH2

+

NH

O

O

CH2

+

CH2

+

m/z 270

m/z 150 m/z 121

m/z 91

A) B) C)

301

47

Despite these similarities, the differences in the spectra are readily apparent due to

differences among the structural subclasses. The spectrum of 6-APB (Figure 4.1 A) exhibits a

base peak at m/z 44, a molecular ion at m/z 175, and prominent ions at m/z 131 and 77. Besides

the ion at m/z 44 previously discussed, α-β bond cleavage also results in an ion at m/z 131 that

consists of a benzofuran ring with a methyl group (C9H7O+). Compounds in the APB series are

traditionally isomers of 6-APB, differing only in the position of the furan ring around the

benzene ring. Therefore, the other APB compounds (4-APB, 5-APB and 7-APB) have very

similar spectra although three out of the four can be distinguished from one another based on

retention index (Section 4.1). Isomers 5-APB and 6-APB have the same retention indices,

however could be distinguished upon further optimization of the GC temperature program,

which was outside the focus of this work.

The spectrum of 2C-H (Figure 4.1 B) has a base peak at m/z 152, corresponding to a

positive radical dimethoxy-methylbenzene ion (C9H12O2+) without the amine chain, cleaved

between the α and β carbons. The ion at m/z 137 is a positively charged dimethoxy benzene ring

(C8H9O2+). As previously stated, the ion at m/z 121 (C8H9O

+) is also present in the spectrum of

25H-NBOMe (Figure 4.1 C) and is the base peak. Other predominant NBOMe ions include m/z

91 (charged methylbenzene, C7H7+) and m/z 150 (C9H12NO+). The three predominant ions at m/z

150, 121, and 91, with m/z 121 as the base peak, are very characteristic of the NBOMe class and

can be used to differentiate NBOMes from other compounds of similar mass. For example, 25G-

NBOMe and the popular cannabinoid XLR-11 have the same nominal mass of 330 Da, but can

be differentiated by the presence of the characteristic m/z 91, 121, 150 peaks (Figure 4.2). All

NBOMe compounds in the study exhibited these three peaks, with only slight variation in

48

Figure 4.2 Mass spectra of (A) 25G-NBOMe and the cannabinoid (B) XLR-115 which both have

a molecular ion of m/z 330. NBOMes can be differentiated from cathinones using characteristic

peaks at m/z 91, 121, and 150. XLR-11 spectrum obtained from Cayman Chemical

50 100 150 200 250 300

0

175000

Abundance

m/z

91

121

150

180

298

25G-NBOMe

N

F

O

O

O

NH

O

XLR-11

Abundan

ce

A)

B)

49

abundances between m/z 91 and 150 among the 12 compounds investigated. The molecular ion

of 25H-NBOMe is observed in very low abundance (0.2%) at m/z 301. Finally, the ion at m/z 270

is proposed to be the NBOMe molecule without one of its methoxy groups (C17H20NO2+).

Overall, the spectra are visually different and through mass spectral interpretation,

structural subclass can be determined relatively easily. Although the APBs are readily

distinguishable from the 2Cs and NBOMes, the class contains only isomeric compounds, so by

low resolution spectra it is difficult to determine exact ring position, and thus differentiate the

isomers within the class. Differentiation of 2C and NBOMe compounds is more challenging as

they have some common fragments and a whole series of compounds with different substituents.

Therefore, further investigation of the mass spectra must be done to distinguish these

compounds.

4.3 Neutral Losses from Molecular Ion to Distinguish 2C- from NBOMe-Phenethylamines

To distinguish the phenethylamine structural subclasses, particularly the 2C- and

NBOMe-phenethylamines, neutral losses from the molecular ion (M+) can be investigated. A

neutral loss is a fragment under ionization conditions that is lost as a neutral molecule. To look

for neutral losses in a spectrum, the mass of the neutral loss in question is subtracted from M+

(Section 2.3.3). Spectra of 2C-phenethylamines and NBOMe-phenethylamines were assessed for

neutral losses characteristic of each subclass.

All 2C-phenethylamines in the sample set exhibit losses of 29 and 60 Da from their M+.

A loss of 29 Da corresponds to the loss of CH3N, part of the amine side chain, and a loss of 60

Da corresponds to a loss of C2H6NO, part of the amine side chain and one of the methoxy

groups. Most of the 2Cs in this study had fragments remaining after a loss of 29 Da as their base

50

peak, otherwise it was a prominent peak. If the neutral loss of 29 Da (CH3N) corresponds to the

base peak or a highly prominent peak, this may be supporting evidence that an unknown is a 2C-

phenethylamine. Figure 4.3 shows example mass spectra of 2C-H and 2C-B exhibiting these

losses and shows how those losses occur structurally.

The NBOMe-phenethylamines also exhibit characteristic neutral losses from their

molecular ions. A loss of 31 Da corresponds to a loss of CH3O, one of the methoxy groups, and a

loss of 149 Da from the molecular ion corresponds to a loss of C9H11NO, the dimethoxy benzene

ring side of the structure after α-β cleavage. These losses may indicate an NBOMe compound as

preliminary characterization of an unknown. As discussed previously in Section 4.2, further

support of preliminary characterization is if the base peak is m/z 121, which is proposed to be a

methyl-dimethoxy benzene ring (C8H9O+), and could be formed several different ways, thus

causing that ion to be greater in abundance. Figure 4.4 shows the mass spectra of 25H-NBOMe

and 25B-NBOMe and ions from the characteristic neutral losses.

51

Figure 4.3 Mass spectrum of (A) 2C-H and (B) 2C-B showing characteristic 2C neutral losses of

29 and 60 Da and the structures of the fragment ions remaining after each loss

O

CH2+

0 100 200 300

0

100A

bundance (

%)

m/z

152 181

181121

-29 Da

-60 Da

A) 2C-HO

O

NH2

C10H15NO2

M+ = 181.1

C9H12O2

Loss of: CH3N

O+

O

CH2

H

C8H9O

Loss of: C2H6NO

M+

M+

CH2

+

O

Br

0 100 200 300

0

100

Abundance (

%)

m/z

B)

259199-60 Da

230 259-29 Da

2C-BC9H11BrO2

Loss of: CH3N

CH2

O+

Br

O

H

C8H8BrO

Loss of: C2H6NO

NH2

O

Br

O

C10H14BrNO2

M+ = 259.1

M+

M+

52

Figure 4.4 Mass spectrum of (A) 25H-NBOMe and (B) 25B-NBOMe showing characteristic

NBOMe neutral losses of 31 and 149 Da and the structures of the fragment ions remaining after

each loss, as well as common fragment ions (m/z 91, 121, 150)

0 100 200 300

0

100A

bundance (

%)

m/z

O

O

NH

OC18H23NO3

M+ = 301.1

152

301

301

270

-149 Da

-31 Da

A)

O

O

NHC

+

C17H20NO2

Loss of: CH3O

C9H12O2

Loss of: C9H11NO

O+

O

CH2

H

121

150

91

M+

M+

25H-NBOMe

0 100 200 300 400

0

100

Abundance (

%)

m/z

B)121

150

230

379

379

348

-149 Da

-31 Da

91

O

O

Br

NHC

+

C17H19BrO2

Loss of: CH3O

C9H11BrO2

Loss of: C9H11NOOH

+

O

Br

CH2

O

O

Br

NH

O

C18H22BrNO3

M+ = 379M+

M+

25B-NBOMe

53

4.4 Distinction and Identification of Common Substituents for 2C- and NBOMe-

Phenethylamines

Unlike the APB subclass, the 2C and NBOMe subclasses each contain a series of

compounds that differ in substituents, primarily on the aromatic ring. These substitutions often

include alkyl chains differing in number of carbons, sulfur or nitro groups, or halogens such as

bromine (Figure 4.3), chlorine, and iodine.

4.4.1 Halogen Substitutions

The presence of halogen substituents can often be determined by isotope ratios in the

mass spectrum. These ratios occur due to the naturally occurring abundance of halogen isotopes.

For example, Br has two naturally occurring isotopes: 79Br has 50.5% natural abundance and

81Br has 49.5% natural abundance, which means either isotope can occur in a molecule with

approximately equal probability.6 Spectra of molecules containing Br show characteristic

patterns consisting of doublets spaced 2 Da apart (Figure 4.5) in approximately a 1:1 ratio. For

example, in the spectrum of 2C-B (Figure 4.5 A), there are doublet peaks at m/z 259.1 and 261.1.

These peaks represent the same fragment ion (C10H14BrNO2+) but the ion at m/z 259.1 contains

79Br whereas the ion at m/z 261.1 contains 81Br. These doublets are observed for all fragment

ions that contain Br. In the spectrum of 2C-B doublets are observed at m/z 199, 215, and 230.

However, no doublets are observed for lower mass fragments (m/z 77.1, 91.1, or 105) because Br

has been cleaved and the remaining ion does not contain it. Similarly, the spectrum of 25B-

NBOMe (Figure 4.5 B) has bromine-containing doublets at m/z 346, 229, and 198.9 and

fragments that do not contain bromine at m/z 150, 121, or 91. Although in different structural

54

subclasses, 2C-B and 25B-NBOMe have similar isotope ratios due to the presence of Br,

enabling determination of the substituent by the characteristic isotope pattern.

Figure 4.5 Characteristic isotope pattern in mass spectra of compounds containing bromine, (A)

2C-B and (B) 25B-NBOMe

0 100 200 300 400

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

A)

259.1261.1

2C-B

25B-NBOMe

B)

346348

NH2

O

Br

O

O

O

Br

NH

O

230

215

19910591.1

77.1

229198.9

150

121

91

55

In a similar manner, chlorine can also be identified by its characteristic isotope pattern.

Chlorine has two naturally occurring isotopes: 35Cl has 75.7% natural abundance and 37Cl has

24.3% natural abundance, which is approximately a 3:1 ratio.6 Spectra of molecules containing

Cl show characteristic patterns consisting of doublets spaced 2 Da apart, in approximately the

3:1 ratio. For example, in the spectrum of 2C-C (Figure 4.6 A), the doublet of peaks at m/z 188

and m/z 186 represent the fragment ion C9H11ClO2+. However, the peak at m/z 186 includes 35Cl,

whereas the peak at m/z 188 includes 37Cl. The characteristic 3:1 ratio of these ions, with the

intensity of m/z 186 approximately 3 times that of m/z 188, is due to the natural abundance of Cl

isotopes observed. Other fragment ions containing Cl can be observed at m/z 215, 171, and 155.

Similarly, in the spectrum of 25C-NBOMe (Figure 4.6 B) the peak at m/z 348 has approximately

a 3:1 ratio of intensity with the ion at m/z 346.

Not all halogen substituents can be identified by isotope ratios. For example, iodine is

monoisotopic and, hence, its presence cannot be determined by isotope ratios. However, the

iodine in a compound can be identified by ions at m/z 126.9, corresponding to I+, and at m/z

127.9 corresponding to HI+. This may not be true in all cases, depending on the sensitivity of the

GC-MS instrument, as these ions are usually observed at relatively low abundances. For

example, the I+ and HI+ ions are observed in spectra of 2C-I and 25I-NBOMe (Figure 4.7 and

4.8, respectively), although the intensities of each ion is less than 1% of the base peak.

56

Figure 4.6 Characteristic isotope pattern in mass spectra of compounds containing chlorine (A)

2C-C and (B) 25C-NBOMe

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

A) 186

188

B)

302304

2C-C

25C-NBOMe

Cl

O

O

NH

O

O

O

NH2

Cl

155

171

215

346

348

57

Figure 4.7 Full mass spectrum of (A) 2C-I and (B) expanded section of same spectrum to

highlight I+ and HI+ ions

120 125 130 135

0

10

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

2C-I

126.9 127.9

A)

B)

O

O

NH2

I

58

Figure 4.8 Full mass spectrum of (A) 25I-NBOMe and (B) expanded section of same spectrum to

highlight I+ and HI+ ions

120 125 130 135

0

10

Abundance (

%)

m/z

0 100 200 300 400

0

100

Abundance (

%)

m/z

A)

B)

126.8 127.9

25I-NBOMe

O

O

NH

IO

59

4.4.2 Sulfur and Nitro Substitutions

Sulfur is also observed as a substituent on synthetic phenethylamines but is problematic

to identify in mass spectrometry. Although sulfur is not monoisotopic (32S occurs at 95% and 34S

occurs at 4% natural abundance), the isotope ratio is inconsistently observed. An [M+2]+ ion can

sometimes be observed at the 95:4 ratio, as is the case with 2C-T (Figure 4.9 A), where the

molecular ion is m/z 227 and there is a low-abundant ion at m/z 229. However, this isotope

pattern does not occur in all sulfur containing compounds, as seen in the mass spectrum of 25T-

NBOMe (Figure 4.9 B), where the molecular ion is m/z 347, but there is no corresponding ion at

m/z 349. Further, fragments containing 34S are at such low abundance, they may be mistaken as

noise, or attributed to isotope peaks from 13C. Additionally, sulfur ions would occur at m/z 32

and 34 which is below the typical scan range for mass spectrometry. Even if the mass scan range

was expanded, the sulfur isotopes are not likely to be observed ions in EI-MS by themselves.

Unfortunately, using low-resolution mass spectrometry, sulfur is not always identifiable as a

substituent.

Nitro (NO2) groups are also present as substituents on 2C and NBOMe compounds.

There is no specific isotope pattern but the common mass spectrometry “nitrogen rule” can be

used to indicate the presence of such a group. If a compound has an odd-mass M+, it contains an

odd number of nitrogens. If a compound has an even-mass M+, it contains an even number of

nitrogens. Phenethylamines typically contain one nitrogen, from the amine chain, meaning they

will have a M+ with an odd mass. For example, M+ in 2C-H is at m/z 181 and M+ for 25H-

NBOMe is at m/z 301 (Figure 4.1 B and C). However, the spectra of 2C-N and 25N-NBOMe

have even M+ of m/z 226 and m/z 346 (Figure 4.10), respectively, indicating an even number of

nitrogens on each, due to the NO2 substitution.

60

Figure 4.9 Mass spectrum of (A) 2C-T and (B) 25T-NBOMe indicating inconsistent sulfur

isotope pattern

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

2C-TA)

M+

227

25T-NBOMeB)

229

M+

347

O

S

O

NH2

O

S

O

NH

O

61

Figure 4.10 Mass spectrum of (A) 2C-N and (B) 25N-NBOMe indicating M+ with an even mass

that suggests an even number of nitrogens present

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

2C-NA)

226

25N-NBOMeB)

346M+

M+

O

O2N

O

NH2

O

O2N

O

NH

O

62

4.5 Scheme for Characterization of Synthetic Phenethylamines using Low-Resolution Mass

Spectra

From retention index and mass spectra interpretation, the APB structural subclass can be

distinguished from 2C-phenethylamines and NBOMe-phenethylamines (Section 4.1 and Section

4.2). Further, distinction of 2C-phenethylamines from NBOMe-phenethylamines is possible

based on characteristic neutral losses (Section 4.3). To some extent, identification of substituents

(i.e., specific compounds in 2C or NBOMe subclasses) can be determined based on isotope

patterns in the mass spectrum as well as the common “nitrogen rule” (Section 4.4).

To be more useful in laboratories for unknown identification, a flowchart style

characterization scheme was developed based on afore-mentioned features. The scheme is shown

in Figure 4.11 and examples follow. The scheme consists of two parts. Part A (Figure 4.11) is

designed to (1) distinguish APB from 2C and NBOMe subclasses and (2) distinguish 2C from

NBOMe compounds. The second part of the scheme (Part B, Figure 4.12) is designed to identify

a likely substituent (halogen, nitro) on 2C or NBOMe compounds.

To theoretically determine if the core structure of the unknown is either 2C-H or 25H-

NBOMe, the mass of the halogen should be subtracted (35, 79, or 126.9 Da for Cl, Br, or I,

respectively) and the mass of hydrogen (1 Da) should be added. If the new, adjusted, mass of M+

after subtraction of the substituent and addition of the hydrogen is 181 Da (i.e., M+ for 2C-H),

the compound may be a 2C-phenethylamine. If the new mass of M+ is 301 Da (i.e., M+ for 25H-

NBOMe), the compound may be an NBOMe-phenethylamine. Similar to the halogens, if there is

indication of a sulfur substituent, the mass of sulfur (32 Da) should be subtracted and the mass of

CH2 (14 Da) should be added. The mass of a methyl group is used instead of hydrogen because

of the position of sulfur within an alkyl chain. If the compound has been determined to have an

63

even M+, the mass of a nitro group (46 Da) should be subtracted and the mass of hydrogen

should be added.

64

Figure 4.11 Characterization scheme for low-resolution mass spectra of synthetic phenethylamines to distinguish APB, 2C, and

NBOMe subclasses

Is retention index available?IT between 1499 – 1527 suggests APB. IT between 1590 – 2000 suggests 2C. IT between 2475 – 2839 suggests NBOMe.

1

Yes

APB

Is there an ion at m/z 131 >10% abundance relative to the base peak?

Other

No No

No

Consistent with an APB-

phenethylamine

Not consistent with an APB-

phenethylamine

Yes

Yes

Does the spectrum have three dominant peaks at m/z 91, 121, and 150 with the base peak at m/z 121?

2

Is there a molecular ion? *Can be confirmed by CI data

3

Consistent with an NBOMe-phenethylamine. Continue to Step 3.

Not consistent with an NBOMe-phenethylamine. Continue to Step 3.

Is there a molecular ion? *Can be confirmed by CI data

3

NoYes

Does the compound have common losses of 31 and 149 Da from the molecular ion? Is m/z 121

the base peak?

4Does the compound lose 29 and 60 Da in

neutral losses from M+? Does it lose 29 Da from M+ as the base peak?

4See NOTE and Part B

See NOTE and Part B

Consistent with an NBOMe-phenethylamine

Continue to Part B.

NoYesNoYes

Not consistent with an NBOMe-

phenethylamine.

Consistent with a 2C-phenethylamine

Continue to Part B.

Not consistent with a 2C-phenethylamine.

Part A

65

Figure 4.12 Characterization scheme for low-resolution mass spectra of synthetic

phenethylamines to determine substituents on 2C- or NBOMe-phenethylamines

If Br, Cl, or I are present, subtract the mass of the halogen (79, 35, 126.9 Da) from the molecular ion and add the mass of hydrogen (1 Da).

If the compound has an even M+ subtract the mass of a nitro group (NO2) (46 Da) and add the mass of hydrogen (1 Da).

If S is present, subtract the mass of sulfur (34 Da) and add the mass of CH2 (14 Da).

Is the adjusted mass 181 Da?

Is there a halogen, sulfur, or nitro group present?**

5

Yes No

**If Br is present, double peaks (doublets) of similar abundance will be present, spaced two mass units apart for higher mass fragments

If Cl is present, a 3:1 abundance ratio will be present, spaced two mass units apart for higher mass fragments

If I is present, m/z 126.9 and m/z 127.9 should be present (I and HI, respectively)

If S is present, a low-abundant ion two mass units higher than M+ should be present

Nitrogen rule: If the mass of the molecular ion is even, there is an even number of nitrogens present, or none at all. Ex: The M+ for 2C-H has one nitrogen and its m/z 181 is odd, indicating an odd number of nitrogens, while M+ for 2C-N which has two nitrogens is m/z 226, an even number.

Consistent with an alkyl- or sulfur-substituted compound.

Yes

No

Is the adjusted

mass 301?

Yes

No

The unknown is consistent with a 2C-

phenethylamine.

The unknown is consistent with an NBOMe-phenethylamine.

The unknown is not consistent with an APB, 2C, or NBOMe-phenethylamine

NOTE: If no molecular ion is confirmed: only halogens can be identified. Cannot replace mass of halogen/sulfur/nitro with mass of

hydrogen/methyl

Part B

66

Example 1: 25B-NBOMe (Figure 4.5 B)

Part A:

1. Is retention index available? Yes, the retention index is 2746. This retention index

is within the retention index range identified for NBOMe-phenethylamines (2475 –

2839).

2. Does the spectrum have three dominant peaks at m/z 91, 121, and 150 with the

base peak at m/z 121? Yes. The spectrum has all three prominent peaks (m/z 91, 121,

150) and the base peak is m/z 121. Therefore, the unknown is consistent with an

NBOMe-phenethylamine.

3. Is there a molecular ion? Yes, the molecular ion was confirmed to be m/z 379 by EI-

MS.

4. Does the compound have common losses of 31 and 149 Da from the molecular

ion? Is m/z 121 the base peak? Yes, the compound has an ion at m/z 348 (379 – 31

Da) and at m/z 230 (379 – 149 Da). The base peak is at m/z 121. This indicates the

unknown is consistent with an NBOMe-phenethylamine.

Part B:

5. Is there a halogen, sulfur, or nitro group present? Yes, bromine doublets are

present. Doublets of similar intensity indicate the presence of Br.

a. Subtracting the mass of Br (79 Da) from the M+ (m/z 379) and adding the

mass of H (1 Da) equals a mass of 301 Da.

If treated as an unknown, 25B-NBOMe would be correctly characterized as an NBOMe-

phenethylamine with a bromine substituent.

67

Example 2: 3-methylethcathinone (3-MEC) (Figure 4.13)

Part A:

1. Is retention index available? No, the retention index of 3-MEC was not available.

2. Does the spectrum have three dominant peaks at m/z 91, 121, and 150 with the

base peak at m/z 121? No, the prominent peaks in the spectrum are at m/z 44.1, 72.1,

and 91.1.

3. Is there a molecular ion? Yes, a molecular ion was confirmed to be m/z 191.1 by EI-

MS.

4. Does the compound lose 29 and 60 Da as neutral losses from M+? Does it lose 29

Da from M+ as the base peak? No. No ion was observed at m/z 162 (191 – 29 Da)

and therefore, it was also not the base peak. There was a low abundant ion at m/z 131

(191 – 60 Da).

Part B:

5. Is there a halogen, sulfur, or nitro group present? No evidence of halogens, sulfur,

or nitro groups was observed.

If treated as an unknown, 3-MEC would not be characterized as an APB or NBOMe. It

cannot be determined if it would be characterized as a 2C compound.

The fragment after the loss of 60 Da cannot be confirmed to be from a loss of C2H6NO

(Section 4. 3) using the current instrumentation. High-resolution mass spectrometry would allow

elemental formula assignment for this fragment, along with an accurate mass measure of the

confidence in that elemental assignment. Using the current low-resolution flowchart, some

subjectivity still remains because it would be at the analysts’ discretion whether or not to

68

preliminarily characterize 3-MEC as a 2C-phenethylamine, as one of the two characteristic 2C

neutral losses is present.

Figure 4.13 Mass spectrum and structure of cathinone, 3-methylethcathinone (3-MEC)

Example 3: Mescaline (Figure 4.14):

Part A:

1. Is retention index available? No, the retention index of mescaline was not available.

2. Does the spectrum have three dominant peaks at m/z 91, 121, and 150 with the

base peak at m/z 121? No, these ions were not prominent in the mass spectrum.

3. Is there a molecular ion? Yes. The molecular ion was confirmed to be m/z 211.1 by

EI-MS.

0 50 100 150 200

0

225000

Ab

und

an

ce

m/z

3-MEC

NH

O

44.1

72.1

91.1119.1

69

4. Does the compound lose 29 Da and 60 Da in neutral losses from M+? Does it lose

29 Da from M+ as the base peak? The compound does lose both 29 and 60 Da, m/z

182 and 151, respectively, with the loss at 29 Da as the base peak. This indicates the

unknown is consistent with a 2C-phenethylamine.

Part B:

5. Is there a halogen, sulfur, or nitro group present? No evidence of halogens or

nitro groups was observed.

If treated as an unknown, mescaline would be incorrectly characterized as a 2C-

phenethylamine.

Figure 4.14 Mass spectrum of 3C phenethylamine, mescaline, which would be mischaracterized

as a 2C because of its loss of 29 Da (m/z 182) and 60 Da (m/z 151)

NH2O

O

O

Mescaline

0 100 200

0

500000

Ab

und

an

ce

m/z

211

182

151

70

4.6 Summary

A characterization scheme has been designed to be immediately implementable into

forensic laboratories as a “quick and easy” guide for preliminary characterization of unknowns.

Through retention index determination, mass spectral investigation, and neutral loss

determination, three phenethylamine structural subclasses can be differentiated. Additionally,

some substituent identification and isomer differentiation is possible. However, some limitations

have been highlighted using the current instrumentation. Without definitive identification of the

fragment element compositions, 2C- and 3C-phenethylamines cannot be differentiated, and some

subjectivity remains in differentiating cathinones from phenethylamine compounds.

71

APPENDIX

72

APPENDIX: Low- Resolution Mass Spectra

Figure A.1 Low-resolution mass spectra of (A) 4-(2-aminopropyl)benzofuran (4-APB), (B) 5-(2-

aminopropyl)benzofuran (5-APB), and (C) 7-(2-aminopropyl)benzofuran

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

NH2O NH2

O

4-APB 5-APB

A) B)

NH2

O 7-APBC)

73

Figure A.2 Low-resolution mass spectra of (A) 2,5-dimethoxy-4-methylphenethylamine (2C-D),

(B) 2,5-dimethoxy-4-ethylphenethylamine (2C-E), (C) 3,4-dimethyl-2,5-

dimethoxyphenethylamine (2C-G), and (D) 2,5-dimethoxy-4-propylphenethylamine (2C-P)

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

A) B)

C) D)

2C-D 2C-E

2C-G 2C-P

O

O

NH2

O

O

NH2

O

O

NH2

O

O

NH2

0 100 200 300

0

100A

bundance (

%)

m/z

74

Figure A.3 Low-resolution mass spectra of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2)

0 100 200 300

0

100

Abundance (

%)

m/z

2C-T-2O

O

NH2

S

75

Figure A.4 Low-resolution mass spectra of (A) 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-

methyoxybenzyl)ethanamine (25D-NBOMe) and (B) 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-

methoxybenzyl)ethanamine (25E-NBOMe)

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

A)

B)

25D-NBOMe

25E-NBOMe

O

O

NH

O

O

O

NH

O

76

Figure A.5 Low-resolution mass spectra of (A) 2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-4-

[(1-methylethyl)thio]-benzeneethanamine (25T-4-NBOMe), (B) 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-4-(propylthio)-benzeneethanamine (25T-7-NBOMe), and (C) 3,4,5-

trimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (mescaline-NBOMe)

0 100 200 300 400

0

100

Ab

und

an

ce (

%)

m/z

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

A) B)

C)

25T-4-NBOMe 25T-7-NBOMe

Mescaline-NBOMe

O

O

S

NH

O

O

O

NH

OS

O

NH

O

O

O

0 100 200 300 400

0

100

Ab

und

an

ce (

%)

m/z

77

Figure A.6 Low-resolution mass spectra of (A) 4-ethoxy-3,5-dimethoxy-benzeneethanamine

(escaline) and (B) 4-methylmethcathinone (mephedrone)

0 50 100 150 200

0

100

Ab

und

an

ce (

%)

m/z

A)

B)

Escaline

Mephedrone

O

O

O

NH2

NH

O

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

78

REFERENCES

79

REFERENCES

1. Chu, F. Improving Methods for the Analysis of Controlled Substances. Masters Thesis,

Michigan State University, East Lansing, 2015.

2. Zuba, D.; Sekula, K. Identification and characterization of 2,5-dimethoxy-3,4-dimethyl-

β-phenethylamine (2C-G) – A new designer drug. Drug Test. Analysis. 2013, 5, 549-559.

3. Chen, B. et. al. A general approach to the screening and confirmation of tryptamines and

phenethylamines by mass spectral fragmentation. Talanta. 2008, 74, 512-517.

4. Awad, T; DeRuiter, J.; Clark, C. R. GC-MS Analysis of Ring and Side Chain

Regioisomers of Ethoxyphenethylamines. J. Chromatogr. Science. 2008, 46, 675-679.

5. XLR-11. Cayman Chemical. https://www.caymanchem.com/product/11565 (accessed

December 1, 2016).

6. Reusch, William. Michigan State University.

https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/massspec/masspec1.h

tm (accessed October 5, 2016).

80

V. Characterization of Synthetic Phenethylamines by High-Resolution Mass Spectrometry

Limitations of the characterization scheme for low-resolution data were highlighted at the

end of Chapter IV, such as the inability to distinguish 2C- from 3C-phenethylamines (Section

1.1), and the inconclusive characterization of cathinones. Additionally, the elemental

composition of each fragment ion remaining after neutral losses could not be determined with a

high degree of certainty. Overall, nominal mass data were not sufficient for definitive

identification of structurally similar compounds, therefore a new approach is necessary. High-

resolution mass spectrometry measures the accurate mass of each ion, from which elemental

formulae can be assigned with a high degree of confidence. This leads to a better understanding

of the fragmentation of the phenethylamine compounds. High-resolution mass spectrometry also

enables the exploitation of the mass defect that can be investigated as a tool for characterizing

new analogs. In this chapter, the comparison of low-resolution and high-resolution spectra will

first be discussed, followed by the development of mass defect filters, and a discussion of their

implementation into a high-resolution version of the characterization scheme.

5.1 Comparison of Low- and High-Resolution Mass Spectra

The low- and high- resolution spectra of 6-APB, 2C-H, and 25H-NBOMe were compared

to ensure consistency in electron ionization (EI) between the ionization sources of the two

instruments (Figure 5.1). Although the high-resolution spectra have more peaks because they

were generated on a more sensitive instrument, both the low-resolution (Figure 5.1 A) and high-

resolution (Figure 5.1 B) spectra display the same peak patterns, molecular ions, and base peaks

for each compound. The same principles apply for mass spectral interpretation as discussed in

Chapter IV, such as characteristic NBOMe peaks at m/z 91, 121, and 150, and substituent

identification. However, with high-resolution mass spectrometry, the elemental formula for

81

Figure 5.1 Comparison of (A) low-resolution and (B) high-resolution mass spectra for 6-APB (left), 2C-H (middle), and 25H-NBOMe

(right)

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

0 100 200 300

0

100

Abundance (

%)

m/z

A)

B)

181.1104C10H15NO2

0.6 ppm

152.0833C9H12O2

2.6 ppm

137.0601C8H9O2

1.5 ppm

121.0645C8H9O

6.6 ppm

91.0543C7H7

5.5 ppm

O

O

NH

O

6-APB (IT = 1527) 25H-NBOMe (IT = 12475)O

O

NH2O NH2

2C-H (IT = 1590)

91

0 100 200 300

0

100

Abundance (

%)

m/z

270.1495C17H20NO2

2.55 ppm

150.0916C9H12NO1.79 ppm

121.0649C8H9O

0.68 ppm

91.0543C7H7

1.29 ppm

175.0986C11H13NO6.3 ppm

131.0506C9H7O

6.9 ppm77.0382

C6H5

11.7 ppm

44.0488C2H6N

27.2 ppm

181

152

137

121270

150

121

91131

77

44

175 301

0 100 200 300

0

100

Abundance (

%)

m/z

82

every ion can be determined, leading to more confidence of the identity of each fragment ion.

For example, 25H-NBOMe has the same three characteristic ions (m/z 91, 121 and 150, Figure

5.1 A) using high resolution mass spectrometry, at m/z 91.0453, 121.0649, and 150.0916 (Figure

5.1 B) but now the elemental formulae of each can be assigned as C7H7+, C8H9O

+, and

C9H12NO+, respectively, with high degrees of accuracy at 1.29, 0.68, and 1.79 ppm, respectively.

The formulae for these ions confirm the structural fragment elucidation proposed in Chapter IV

(Figure 4.1). The spectra of 2C-B (Figure 5.2) further highlight the similarities between low- and

high-resolution spectra, showing consistent fragmentation and doublets due to the presence of

bromine. The assigned elemental composition in the high-resolution spectrum confirmed the

presence of bromine. It should also be noted that the retention index (IT) for these compounds is

the same for high- and low-resolution instruments (Figure 5.1), as expected.

83

Figure 5.2 Comparison of (A) low-resolution and (B) high-resolution mass spectra for 2C-B.

Dominant fragment ions are labeled and in (B) assigned element formulae and mass accuracies

are given

0 50 100 150 200 250 300

0

100

Abundance (

%)

m/z

0 50 100 150 200 250 300

0

80000

Abundance

m/z

NH2

O

Br

O

2C-B (IT = 1856)

259.1

230

215

199

77.1

259.0183C10H14BrNO2

9.7 ppm

229.9938C9H11BrO2

1.7 ppm

214.9696C8H8BrO2

9.7 ppm

198.9772C8H8BrO6.5 ppm

77.0413C6H5

28.6 ppm

A)

B)

84

5.2 Development of Mass Defect Filters

Accurate mass data can be used not only to assign elemental formulae, but also to

calculate the mass defect of each ion. Compounds in the same structural class should

theoretically have similar mass defects because the core structure is consistent among analogs.

Because the mass defect of the core structure has a larger contribution to the overall mass defect,

the addition of various substituents should not change the overall mass defect substantially.

Therefore, mass defect was used as a tool to characterize compounds according to structural

class. To do this, mass defects of the molecular ions were calculated for phenethylamines and a

filter was developed as the mean mass defect ± a given tolerance. The efficacy of this filter to

characterize compounds as phenethylamines was then tested. Mass defects based on molecular

ions were calculated for a test set of compounds and tested to determine if the mass defect was

within the previously defined filter.

5.2.1 Absolute Mass Defect Filters for Phenethylamines Based on Molecular Ions

A training set contained 16 phenethylamines that were randomly selected from the full

sample set (Section 3.4.1). These included APB, 2C, and NBOMe compounds and the mass

defects of their molecular ions (M+) were calculated. Table 5.1 shows the exact masses, mass

accuracies, and mass defects for the M+ of all compounds in the training set. The mean mass

defect represented by the training set was 142.4 mDa and the tolerance was calculated as a

confidence interval at the 99.9991% confidence level. This confidence level was necessary to

encompass the range of mass defects in the training set. Thus, the filter was defined as 142.4 ±

54.1 mDa and is shown graphically in Figure 5.3, where the yellow line represents the average

85

mass defect and the purple lines represent the upper and lower bounds of the filter. All the mass

defects of the training set compounds fell within this filter.

Table 5.1 Calculation of absolute mass defect molecular ion filter

Compounds analyzed on Waters system that measures mass accuracy to one decimal place Compounds analyzed on LECO system that measures mass accuracy to two decimal places

(Section 3.3)

Training or Test Set

CompoundExact mass

(Da)

Nominal mass (Da)

Mass defect (mDa)

Mass accuracy

(ppm)

Filter (mDa)*

Training

2C-D 195.1243 195 124.3 8.2

142.4 54.1

2C-E 209.1404 209 140.4 5.72C-H 181.1104 181 110.4 0.62C-P 223.1573 223 157.3 0.42C-N 226.0956 226 95.6 3.53

2C-T-2 241.1134 241 113.4 1.14-APB 175.0999 175 99.9 1.1

25C-NBOMe 335.1188 335 118.8 28.2525D-NBOMe 315.1813 315 181.3 5.0125E-NBOMe 329.1962 329 196.2 7.2125G-NBOMe 329.1945 329 194.5 12.3825H-NBOMe 301.1654 301 165.4 5.9625N-NBOMe 346.1493 346 149.3 8.76

Mescaline-NBOMe

331.1755 331 175.5 6.84

Escaline 225.1351 225 135.1 6.2Mescaline 211.1207 211 120.7 0.5

* 99.9991% CL

Test

2C-G 209.1421 209 142.1 2.42C-B 259.0203 259 20.3 0.372C-C 215.0710 215 71.0 1.062C-I 307.0067 307 6.7 0.892C-T 227.0988 227 98.8 3.50

5-APB 175.1005 175 100.5 4.66-APB 175.0986 175 98.6 6.37-APB 175.0993 175 99.3 2.3

25B-NBOMe 379.0602 379 60.2 46.3325T7-NBOMe 375.1807 375 180.7 14.80

3-MEC 191.1310 191 131.0 0.0Mephedrone 177.1150 177 115.0 2.3

86

Figure 5.3 Absolute mass defect filter created using a training set of phenethylamines defined in

Table 5.1. The absolute mass defect filter was defined at 142.4 ± 54.1 mDa at a 99.9991%

confidence level. The horizontal lines represent the average (yellow), and the upper and lower

bounds of the mass defect filter (purple)

The filter was tested using the remaining compounds in the sample set, with the

exception of 25I-NBOMe, 25T-NBOMe, and 25T-7-NBOMe, which did not exhibit molecular

ions. The mass defect of 2C-G was 142.1 mDa and, hence, falls inside the filter. However, the

halogenated compounds in the test set pose problems because of the large mass defect associated

with the halogen (Section 2.4). For example, the mass defect associated with bromine is -81.6

mDa and, thus, has a significant impact on the mass defect of any fragment ion containing

bromine. As a result, compounds with halogens have smaller mass defects than similar

compounds that do not contain a halogen. For example, the mass defect of 2C-B is 20.3 mDa

compared to 110.4 mDa for 2C-H, where the only difference is the presence of Br. Therefore,

2C- and NBOMe-phenethylamines with halogens are not correctly characterized using this filter.

2C-B

2C-C

2C-I

25B-NBOMe

0

50

100

150

200

250

165 215 265 315 365

Ab

solu

te M

ass

Def

ect

(mD

a)

m/z

Training Set Phenethylamine Test Set Cathinone Test Set

87

More problematic, the absolute mass defects of the cathinone M+ from the test set also

fall within the filter (Figure 5.3). For example, 3-methylethcathinone (3-MEC) and mephedrone

have mass defects of 131.0 and 115.0 mDa, respectively. Although the cathinones are

structurally similar, differing from the core structure of phenethylamine by an addition of a

carbonyl group, there is a need to distinguish them from phenethylamines for a robust

characterization scheme.

The filter based on absolute mass defect of molecular ions shows potential, but the

tolerance is too wide, resulting in a filter that is not sufficiently specific to distinguish

phenethylamines from cathinones. Further, despite a large tolerance, phenethylamines containing

halogens are not successfully characterized due to the large mass defect contribution from the

halogen. In an effort to improve specificity of the filter, separate mass defect filters were

developed for the three different phenethylamine structural subclasses.

5.2.2 Absolute Mass Defect Filter for the APB-Phenethylamine Subclass

The training set for the APB mass defect filter contained 4-APB, 5-APB, and 6-APB.

From the data in Table 5.1, the APB filter based on absolute mass defect of molecular ions was

defined as 99.7 ± 1.6 mDa, at the 90% confidence level (Figure 5.4). This filter is very narrow

because it was defined with a set of isomeric compounds. Theoretically, the exact masses and

mass defects of isomers should all be the same, but because of the instrument variation, the

experimentally collected exact masses vary slightly, as represented by the mass accuracies. The

test set contained 7-APB, as well as the remaining 2C, NBOMe, 3C, and cathinone compounds

from the sample set. None of these fall inside the filter, indicating correct characterization.

88

Figure 5.4 APB subclass absolute mass defect filter at 99.7 ± 1.6 mDa at a 90% confidence level.

The horizontal lines represent the average (black) and the upper and lower bounds of the mass

defect filter (red)

5.2.3 Absolute Mass Defect Filter for the 2C-Phenethylamine Subclass

Because of large negative mass defect contribution of halogens to the mass defect of a

compound, only 2C-phenethylamines with alkyl side chains were used in the training set (i.e.,

2C-H, 2C-D, 2C-E, 2C-P). The 2C absolute mass defect filter was defined as 133.1 ± 32.2 mDa

at the 95% confidence level. This tolerance is narrower compared to the full set of

phenethylamines (Section 5.2.1), allowing for a more specific filter of the 2C subclass. The test

set contained the remaining 2C-phenethylamines and all APB, NBOMe, 3C, and cathinone

compounds. The compounds with a halogen, sulfur, or nitro group will fall outside the filter due

to the mass defect contribution from the substituent (Section 2.4). However, like the method

described in Section 4.4, halogens, sulfur, and nitro groups can be identified by isotope patterns

and mass spectral features in the mass spectrum. Further, the mass defect of the suspected

7-APB

95

99

103

165 175 185 195 205 215 225 235 245

Ab

solu

te M

ass

Def

ect

(mD

a)

m/z

APB Training Set Test Set

89

halogen and nitro group can be replaced with that of hydrogen and then tested against the filter

(Section 4.5). Although a sulfur substituent may not be able to be discerned from the mass

spectral features (Section 4.4.2), high-resolution mass spectrometry offers the advantage of

including sulfur during elemental formulae assignment for each fragment ion, and thus a sulfur

substituent can be identified and further replaced with a CH2 group (Section 4.5). For halogen,

nitro, and sulfur group replacement, the following exact masses are used to adjust the mass of the

molecular ion: 34.9689 Da for Cl, 78.9183 Da for Br, 126.9045 Da for I, 45.9929 Da for NO2,

31.9721 Da for S, 1.0078 Da for H, and 14.0157 Da for CH2.1 This adjusted mass, when

applicable, is used to calculate all mass defects.

After halogen/sulfur/nitro group replacement, all the mass defects of the 2C-

phenethylamines in the test set correctly fall within the 2C filter (Figure 5.5). However, the two

cathinone and two 3C-phenethylamine test compounds also fall within the 2C filter, highlighting

a lack of specificity despite the narrower tolerance associated with this filter. Furthermore,

because there is no limit to the m/z range the 2C filter extends, it encompasses the APB filter,

and significantly overlaps and encompasses many NBOMe compounds.

90

Figure 5.5 2C subclass absolute mass defect filter at 133.1 ± 32.2 mDa at a 95% confidence

level. The horizontal lines represent the average (light blue) and the upper and lower bounds of

the mass defect filters (dark blue)

5.2.4 Absolute Mass Defect Filter for the NBOMe-Phenethylamine Subclass

Similar to the 2C absolute mass defect filter (Section 5.2.3), only NBOMe-

phenethylamines with alkyl side chains were used in the training set (i.e., 25H-NBOMe, 25D-

NBOMe, 25E-NBOMe, and mescaline-NBOMe) to develop the filter. The NBOMe absolute

mass defect filter was defined as 179.6 ± 20.5 mDa at the 95% confidence level. The test set

contained the remaining NBOMe-phenethylamines, and all of the APB, 2C, 3C, and cathinone

compounds. After halogen/nitro/sulfur group replacement, all the mass defects of the test

NBOMe-phenethylamines correctly fell within the NBOMe filter except 25B-NBOMe and 25T-

7-NBOMe (Figure 5.6). The mass accuracy of the molecular ion of 25B-NBOMe was poor at

46.33 ppm, causing the mass defect (149.7 mDa) to fall outside the filter, despite substituting the

halogen with hydrogen. Absolute mass defect has a positive correlation with mass, so

75

105

135

165

195

225

150 200 250 300 350 400Ab

solu

te M

ass

Def

ect

(mD

a)

m/z

Training Set 2C Test Set APB Phenethylamines

NBOMe Phenethylamines Cathinone & 3C Test Set

91

compounds of higher mass will have higher mass defects, as is the case with 25T-7-NBOMe,

causing it to fall outside the filter, again despite replacing sulfur with CH2. As stated previously,

a limitation of this filter is that it overlaps with the 2C filter and lacks specificity. Additionally,

the theoretical mass defects of six of the most popular synthetic cannabinoids (JWH-018, JWH-

073, CP 47,497, AM-2201, UR-144, and XLR-11) were used to further test the specificity of the

NBOMe filter because cannabinoids have similar molecular masses as many NBOMe-

phenethylamines. Three of the six cannabinoids would fall within the filter.

Figure 5.6 NBOMe subclass absolute mass defect filter at 179.6 ± 20.5 mDa at a 95% confidence

level. The horizontal lines represent the average (light purple) and the upper and lower bounds of

the mass defect filter (dark purple)

Although a good starting point for differentiation, the absolute mass defect filters based

on the molecular ion have some limitations. The first is that based on mass defect alone, the

filters overlap if the m/z ranges have no limit. However, the use of retention index can be used to

overcome this limitation. Because the retention index ranges of APB, 2C, and NBOMe

25B-NBOMe

25T-7-NBOMe

75

115

155

195

235

150 200 250 300 350 400Ab

solu

te M

ass

Def

ect

(mD

a)

m/z

NBOMe Training Set NBOMe Test Set 2C Phenethylamines

APB Phenethylamines Cathinone & 3C Test Set Theoretical Cannabinoids

92

subclasses are distinctly different, this information can be used to determine which filter to test

the compound against.

Second, mass defects of all the 3C-phenethylamines and cathinones fall within the 2C

filter and many of the cannabinoid mass defects fall within the NBOMe filter. This highlights a

lack of specificity when using the absolute mass defects of a molecular ion. To further

investigate specificity, absolute mass defects of fragment ions and neutral losses common to each

subclass were also investigated; however, these filters were still not sufficiently specific and the

m/z and mass defect ranges overlapped. Because absolute mass defect filters were non-specific

for distinguishing the structural subclasses, Kendrick mass defect filters were investigated.

5.2.5 Kendrick Mass Defect Filters for Phenethylamines Based on Molecular Ions

To overcome the limitations of non-specific, overlapping, absolute mass defect filters,

Kendrick mass defect (KMD) filters were developed, again based on molecular ions. Only alkyl-

substituted phenethylamines were used in the subclass training sets to define the filters. Because

Kendrick mass defects are used to identify members of a homologous series, differing only in the

number of methyl (CH2) groups, compounds containing halogens, nitro groups, or sulfur are not

members of this homologous series, and therefore were not used to create the filters. All

compounds containing halogens or nitro groups had the masses of these substituents replaced

with hydrogen, while compounds containing sulfur had the masses replaced with CH2 similar to

Section 5.2.3, before calculating their KMD and being used to test the filter (Section 3.4.2).

93

5.2.6 Kendrick Mass Defect Filters of the APB-Phenethylamine Subclass

Table 5.2 shows the KMD and the associated filter for the APB-phenethylamine subclass

(Section 3.4.2).2 The APB KMD filter was determined to be 95.9 ± 1.6 mDa at the 90%

confidence level. This tolerance is less than those used in defining absolute mass defect filters

because the training set compounds should, theoretically, all have the same KMD. Thus, the

filter should be significantly more narrow, and further, more specific. The test set contained the

remaining APB-phenethylamines, all 2C, NBOMe, 3C, and cathinone compounds.

The APB test set compound, 7-APB, had a KMD that fell within the filter (96.2 mDa),

indicating correct characterization (Figure 5.7). The remaining test set compounds had KMD that

did not fall within the APB KMD filter, also indicating correct characterization. Further, the

APB and 2C KMD filters do not overlap, overcoming a limitation of the absolute mass defect

filters discussed in Section 5.2.4.

Table 5.2 Calculation of APB Kendrick mass defect filter

*90% CL

CompoundNominal

Mass (Da)

Kendrick

Mass (Da)

Kendrick Mass

Defect (mDa)

KMD Filter 2

(mDa)*

4-APB 175 174.9044 95.6

95.9 1.65-APB 175 174.9045 95.0

6-APB 175 174.9031 96.9

94

Figure 5.7 APB subclass Kendrick mass defect filter at 95.9 ± 1.6 mDa at a 90% confidence

level. The horizontal lines represent the average (black) and the upper and lower bounds of the

mass defect filter (red)

5.2.7 Kendrick Mass Defect Filters of the 2C-Phenethylamine Subclass

Table 5.3 shows the KMD and the associated filter for the 2C-phenethylamine subclass.

The 2C KMD filter was determined to be 92.2 ± 1.5 mDa at the 95% confidence level. The test

set contained the remaining 2C-phenethylamines, all APB, NBOMe, 3C, and cathinone

compounds.

After halogen/nitro group substitution, all the 2C-phenethylamines had KMD that fell

within the filter. The sulfur-containing compounds, 2C-T and 2C-T-2 have KMD around 155

mDa due to the contribution of sulfur to the KMD, which would cause these compounds to fall

outside the filter. However, because sulfur can be identified by including it in element

composition selection, the mass of sulfur is replaced with the mass of a methylene group

7-APB

90

95

100

165 175 185 195 205 215 225Ken

dri

ck M

ass

Def

ect (

mD

a)

m/z

Training Set APB Test Set

2C Phenethylamines 3C & Cathinone Test Set

95

(14.01565 Da), and compounds 2C-T and 2C-T-2 then correctly fall within the filter (Figure 5.8)

as members of the homologous series.

The 3C compounds (mescaline and escaline) have KMD that fall outside and above the

2C filter around 115 mDa, indicating correct characterization. Both 3C compounds have KMD

that fall near one another because they are members of their own homologous series, and thus

have similar KMD. The cathinone compounds (3-MEC and mephedrone) have KMD that fall

outside and below the 2C filter, around 82 mDa, and would also be correctly characterized. The

KMD of the APB- and NBOMe-phenethylamines did not fall within the 2C filter, further

indicating correct characterization.

Table 5.3 Calculation of 2C Kendrick mass defect filter

*95% CL

CompoundNominal

Mass (Da)

Kendrick

Mass (Da)

Kendrick Mass

Defect (mDa)

KMD Filter 2

(mDa)*

2C-H 181 180.9082 91.8

92.2 1.52C-D 195 194.9064 93.6

2C-G 209 208.9086 91.4

2C-P 223 222.9082 91.9

96

Figure 5.8 2C subclass Kendrick mass defect filter at 92.2 ± 1.5 mDa at a 95% confidence level.

The horizontal lines represent the average (light blue) and the upper and lower bounds of the

mass defect filter (dark blue)

5.2.8 Kendrick Mass Defect Filters of the NBOMe-Phenethylamine Subclass

The NBOMe KMD filter was defined using a training set of only alkyl-substituted

NBOMe compounds (Table 5.4). The NBOMe KMD filter was determined to be 171.5 ± 7.7

mDa at the 99% confidence level. The test set contained the remaining NBOMe

phenethylamines, all 2C, APB, 3C, and cathinone compounds, as well as the theoretical KMD of

six cannabinoids (Section 5.2.4). The cannabinoids are tested against the NBOMe KMD filter

because some were incorrectly characterized within the NBOMe filter when their absolute mass

defects were tested.

All the NBOMe test set compounds had KMD that fell inside the KMD filter with the

exception of 25B-NBOMe and mescaline-NBOMe (Figure 5.9). As discussed in Section 5.2.4,

2C-T-22C-T

3C-phenethylamines

cathinones80

100

120

170 180 190 200 210 220 230 240 250Ken

dri

ck M

ass

Def

ect (

mD

a)

m/z

Training Set 2C Test Set

APB Phenethylamines 3C & Cathinone Test Set

97

Table 5.4 Calculation of NBOMe Kendrick mass defect filter

*99% CL

Figure 5.9 NBOMe subclass Kendrick mass defect filter at 171.5 ± 7.7 mDa at a 99% confidence

level. The horizontal lines represent the average (light purple) and the upper and lower bounds of

the mass defect filter (dark purple)

the mass accuracy of the molecular ion of 25B-NBOMe is poor (46.33 ppm), causing the KMD

to fall outside the filter. Mescaline-NBOMe has a KMD that falls outside and above the NBOMe

KMD filter at 194.3 mDa because it is not a member of the same homologous series as the other

CompoundNominal

Mass (Da)

Kendrick

Mass (Da)

Kendrick Mass

Defect (mDa)

KMD Filter 2

(mDa)*

25H-NBOMe 301 300.8291 170.9

171.5 7.725D-NBOMe 315 314.8294 170.6

25G-NBOMe 329 328.8269 173.1

25B-NBOMeMescaline-NBOMe

2C-T-22C-T75

105

135

165

195

225

165 215 265 315Ken

dri

ck M

ass

Def

ect (

mD

a)

m/z

Training Set NBOMe Test Set2C Phenethylamines APB Phenethylamines3C & Cathinone Test Set Theoretical Cannabinoids

98

NBOMe compounds, much like its 3C counterparts’ relation to the 2C compounds. All the KMD

of the APB- and 2C-phenethylamine compounds fall outside the filter, indicating correct

characterization. The six theoretical KMD of the cannabinoids also fall outside the NBOMe

filter, illustrating KMD as a more specific and robust filter than the NBOMe absolute mass

defect filter.

Overall, KMD has the most specificity to differentiate and characterize unknown

compounds and give a preliminary indication of subclass. Further investigation of fragment ions,

neutral losses and common fragments was also performed to enhance the confidence of KMD

characterization as well as provide evidence toward characterization in the event that no

molecular ion is present or cannot be confirmed by chemical ionization.

5.2.9 Kendrick Mass Defect Filters for Neutral Losses and Common Fragment Ions

One of the limitations of using fragment ions in the low-resolution scheme was the lack

of elemental formulae assignment after a neutral loss. Using high-resolution mass spectrometry

this can be overcome by using exact mass for formulae assignment for each ion. Further, KMD

filters can be developed for the fragments remaining after common neutral losses. To investigate

KMD filters based on fragment ions, first the high-resolution spectra were probed and tables

were created for each 2C compound detailing the most prominent fragment ions, their mass

accuracies, and elemental compositions as shown in Figure 5.10 and Table 5.5 for 2C-H.

Knowing the elemental composition, the neutral losses from the molecular ion were then

determined, as shown in Figure 5.11 for 2C-H. Neutral losses were compiled for each 2C-

phenethylamine to identify common losses that may be characteristic of this subclass (Table 5.6).

99

Figure 5.10 Spectrum of 2C-H showing abundant ions

Table 5.5 Ion table of 2C-H showing abundant ion elemental composition assignments and mass

accuracies

0 50 100 150 200 250 300

0

100

Ab

und

an

ce (

%)

m/z

2C-HO

O

NH2

181.1104

152.0833

137.0601

121.0645

m/z Mass accuracy

(ppm)

Elemental composition

m/z Massaccuracy

(ppm)

Elemental composition

181.1104 0.6 C10H15NO2 121.0645 6.6 C8H9O

152.0833 2.6 C9H12O2 109.0643 9.2 C7H9O

137.0601 1.5 C8H9O2 105.0342 1.9 C7H5O

100

Figure 5.11 Proposed structures for fragment ions of 2C-H after their neutral losses

Table 5.6 Table of remaining ions after common losses of all 2C compounds

v

2C-H

Loss of: CH3N

Loss of: C2H6N

Loss of: C3H6NO

Loss of: C2H6NO

O

O

NH2

Molecular Formula: C10

H15

NO2

Monoisotopic Mass: 181.110279 Da

O+

O

CH2

H Molecular Formula: C9H

12O

2

Monoisotopic Mass: 152.083181 Da

C+

O

O

Molecular Formula: C8H

9O

2

Monoisotopic Mass: 137.059706 Da

O

CH2+

Molecular Formula: C8H

9O

Monoisotopic Mass: 121.064791 Da

O+ H Molecular Formula: C

7H

9O

Monoisotopic Mass: 109.064791 Da

LOSS

Molecular Formula

CH2N CH3N CH4N C2H6N C2H6NO

2C-H C10H15NO2 C9H13O2 C9H12O2 C9H11O2 C8H9O2 C8H9O

2C-D C11H17NO2 C10H15O2 C10H14O2 C10H13O2 C9H11O2 C9H11O

2C-G C12H19NO2 C11H17O2 C11H16O2 C11H15O2 C10H13O2 C10H13O

2C-E C12H19NO2 C11H17O2 C11H16O2 C11H15O2 C10H13O2 C10H13O

2C-P C13H21NO2 C12H19O2 C12H18O2 C12H17O2 C11H15O2 C11H15O

2C-B C10H14NO2Br C9H11O2Br C9H10O2Br C8H8O2Br C8H8OBr

2C-C C10H14NO2Cl C9H12O2Cl C9H11O2Cl C9H10O2Cl C8H8O2Cl

2C-I C10H14NO2I C9H12O2I C9H11O2I C8H8O2I C8H8OI

2C-N C10H14N2O4 C9H12NO4 C9H11NO4

2C-T C11H17NO2S C10H15O2S C10H14O2S C10H13O2S C9H11O2S C9H11OS

2C-T-2 C12H19NO2S C11H17O2S C11H16O2S C11H16O2S C10H13O2S C10H13OS

Ion present, but lower than 5% relative abundance Ion not present

101

The common losses were CH2N, CH3N, CH4N, C2H6N, and C2H6NO. From the five

alkyl-substituted 2C compounds, the KMDs of ion fragments remaining after each of these losses

were used to calculate Kendrick mass defect filters. The filter for each loss and their respective

confidence levels can be seen in Table 5.7. The five filters are shown in Figure 5.12 with the

KMDs of the alkyl-substituted 2C fragment ions used to define them.

Only one filter is distinctly separated from the rest: KMD of fragments resulting from a

loss of C2H6NO. This filter was selected as one to use in the characterization scheme. The

remaining filters (blue, orange, green and black) all had some degree of overlap because they all

were representing members of the same homologous series, where only the number of carbons

and hydrogens were different. Based on the structural elucidations of fragments, and

commonality of the loss among all 2C compounds, the filter representing the loss of CH3N was

also chosen to use as part of the characterization scheme. The KMDs of the fragments of non-

alkyl substituted 2Cs resulting from these losses were then calculated, replacing halogens and

nitro groups with hydrogen and sulfur groups with CH2 when appropriate (Section 5.2.7), and

plotted against the selected filters (Figure 5.13). After replacement, the KMD of fragments of

2C-B, 2C-C, 2C-I, 2C-N, and 2C-T-2 showing a loss of CH3N correctly characterized within the

CH3N loss filter (dark green). The KMD of fragments after a loss of C2H6NO of 2C-B, 2C-I, 2C-

N, 2C-T, and 2C-T-2 correctly fall within that respective filter (dark pink). Compound 2C-T also

showed a loss of CH3N, however because the remaining fragment ion had a poor mass accuracy

of 23.2 ppm, the KMD of the ion falls outside the corresponding filter. The incorrect

characterization of 2C-T highlights the importance of having good mass accuracy of fragment

ions.

102

Table 5.7 Kendrick mass defect filters associated with ion fragments after common neutral losses

Figure 5.12 Kendrick mass defect filters developed based on common losses of alkyl-substituted

2C compounds. Points represent KMD of fragment ions remaining after each respective loss.

The horizontal lines represent the average (lighter colors) and the upper and lower bounds of

each mass defect filter (darker colors)

Neutral Loss Filter (mDa) Confidence Level

CH2N 83.6 1.93 99%

CH3N 86.0 0.72 95%

CH4N 91.7 5.3 99%

C2H6N 92.8 0.16 99%

C2H6NO 69.6 5.16 99%

60

65

70

75

80

85

90

95

100

115 135 155 175 195 215

Ken

dri

ck M

ass

Def

ect (

mD

a)

m/z

Loss CH2N Loss CH3N Loss CH4N Loss C2H6N Loss C2H6NO

103

Figure 5.13 Selected Kendrick mass defect filters representing losses of CH3N and C2H6NO for

all 2C fragments falling within said filters. Fragment shown outside the filter is from 2C-T. The

horizontal lines represent the average (light green and purple) and the upper and lower bounds of

each mass defect filter (dark green and purple)

Common losses for NBOMes were investigated in the same way as the 2C compounds.

The high-resolution spectra were examined and ion tables were created (Figure 5.14, Table 5.8),

which facilitated structural elucidation of some of the fragments (Figure 5.15). In comparing

common neutral losses, it was observed that all NBOMe compounds lost one methoxy group

(CH3O) and exhibited a loss of C9H11NO, which is proposed to be a loss of the amine and

methoxy-phenyl chain. It was also observed that all compounds exhibited the same fragment of

m/z 121 as the base peak, m/z 150 and m/z 91 (methyl-benzene, not pictured).

[M-CH3N]+

2C-T

[M-C2H6NO]+

62

67

72

77

82

87

115 135 155 175 195 215

Ken

dri

ck M

ass

Def

ect (

mD

a)

m/z

Fragments after loss of CH3N Fragments after loss of C2H6NO

104

Figure 5.14 Spectrum of 25H-NBOMe and most abundant fragment ions above m/z 105

Table 5.8 Ion table of 25H-NBOMe with elemental composition assignments and mass

accuracies of most abundant fragment ions above m/z 105

25H-NBOMe

0 100 200 300

0

100

Abundance (

%)

m/z

270.1495

150.0916

121.0649

91.0543

O

O

NH

O

m/z Mass accuracy

(ppm)

Elemental composition

m/z Massaccuracy

(ppm)

Elemental composition

301.1654 1.8 C18H23NO3 150.0916 1.79 C9H12NO

270.1495 0.69 C17H20NO2 122.0684 34.28 C8H10O

152.0835 2.36 C9H12O2 121.0649 0.68 C8H9O

105

Figure 5.15 Proposed structures for fragment ions of 25H-NBOMe after their neutral losses

The NBOMe fragment remaining after the C9H11NO loss was observed to be the same as

the fragment remaining after a loss of CH3N in the 2C compounds (Figure 5.11). This

fragmentation is shown in Figure 5.16, using 2C-N and 25N-NBOMe as examples. Therefore,

the filter previously developed is applicable here – although it corresponds to a different neutral

loss, the same ion is remaining. The KMD of these NBOMe fragment ions was calculated

(halogens/nitro/sulfur groups replaced when applicable) and plotted against the 2C CH3N loss

filter. Except that from mescaline-NBOMe, all the fragments correctly characterized within the

filter (Figure 5.17) as shown in yellow. This helps to illustrate how NBOMe compounds

fragment, and their relationship with 2C compounds. No NBOMe-specific KMD neutral loss

filters were developed because NBOMes could be definitively identified by their characteristic

mass spectral features, presence of characteristic neutral losses, and the applicability of the 2C

CH3N loss filter.

O

O

NH

O CH2

+

NH

O

Molecular Formula: C9H

12NO

Monoisotopic Mass: 150.09134 Da

O

O

NHC

+

Molecular Formula: C17

H20

NO2

Monoisotopic Mass: 270.148855 Da

Molecular Formula: C18

H23

NO3

Monoisotopic Mass: 301.167794 Da

O

CH2

+

Molecular Formula: C8H

9O

Monoisotopic Mass: 121.064791 Da

O+

O

CH2

H

Molecular Formula: C9H

12O

2

Monoisotopic Mass: 152.083181 Da

25H-NBOMe

CH2

OH+

Molecular Formula: C8H

10O

Monoisotopic Mass: 122.072616 Da

v

Loss of: CH3O

Loss of: C9H11NO

Base peak

106

Figure 5.16 Proposed structural elucidation of 2C-N and 25N-NBOMe leading to the same

fragment (C9H11NO4)

Figure 5.17 Selected Kendrick mass defect filter and corresponding NBOMe fragments falling

within the filter. Fragments shown outside the filter are from mescaline-NBOMe and 2C-T. The

horizontal lines represent the average (light green) and the upper and lower bounds of the mass

defect filter (dark green)

O

O

O2N

NH2

O

O

O2N

NH

O

OH+

O

CH2

O2N2C-N

25N-NBOMe

Loss of CH3N

Loss of C9H11NO

2C-T

[M-CH3N]+

Mescaline-NBOMe

80

85

90

95

100

105

145 155 165 175 185 195 205 215

Ken

dri

ck M

ass

Def

ect (

mD

a)

m/z

2C fragments after loss of CH3N NBOMe fragments after loss of C9H11NO

107

To test the KMD fragment ion filters developed, the two 3C-phenethylamines and two

cathinone compounds were analyzed for fragment ions after common neutral losses. Neither

mephedrone nor 3-MEC exhibited losses of CH3N or C2H6NO. Mescaline and escaline did

exhibit losses of both CH3N and C2H6NO; however, when the KMD values of each of the four

remaining fragment ions were calculated and plotted, all four correctly characterized as being

outside both 2C fragment filters (Figure 5.18).

Figure 5.18 Selected Kendrick mass defect filters and corresponding 3C fragments falling

outside the filters

Developing KMD filters on common fragment ions is not possible because the

substituent on each compound causes different m/z in a spectrum, leading to a lack of ions in

common across a subclass. Further, some ions that are common across a subclass are not

necessarily characteristic, e.g., m/z 77, which is present in all spectra for aromatic compounds.

Developing the filters related to common neutral losses are more successful because members of

the 2C subclass have the same neutral losses, despite having different substitutions (Section 4.3).

2C-T

[M-C2H6NO]+

[M-CH3N]+

Mescaline-NBOMe

Mescaline

Escaline

MescalineEscaline

60

85

110

115 135 155 175 195 215

Ken

dri

ck M

ass

Def

ect (

mD

a)

m/z2C fragment ions after loss of CH3N 2C fragments after loss of C2H6NONBOMe fragment ions 3C fragment ions

108

5.3 Scheme for Characterization of Synthetic Phenethylamines using High-Resolution Mass

Spectra

With the addition of Kendrick mass defect filters based on molecular ions and Kendrick

mass defect filters based on fragment ions after neutral losses, the characterization scheme for

low-resolution data can be modified to create a characterization scheme based on high-resolution

data. Many of the same components of the low-resolution data scheme are retained, including

retention index determination, molecular ion confirmation, and substituent identification, and

where applicable, halogen/nitro/sulfur group replacement with the exact masses of hydrogen or

CH2 (Section 5.2.3). The order of the revised characterization scheme is slightly different such

that the substituent must be accounted for before the Kendrick mass defect filters can be applied.

The characterization scheme is presented in Figure 5.19 and two examples demonstrating

application of the scheme follow.

109

Figure 5.19 Characterization scheme for high-resolution mass spectral data. M+adj is the mass of the molecular ion adjusted for a

halogen/sulfur/nitro substituent

NoYes

Is there a molecular ion? *Can be confirmed by CI data

3

Continue to 3a.

Is there a halogen, sulfur, or nitro group

present? ***(next page)

Yes No

Yes No

Is retention index available?IT between 1499 – 1527 suggests APB. IT between 1590 – 2000 suggests 2C. IT between 2475 – 2839 suggests NBOMe.

1

Is there an ion at m/z 131 (C9H7O+) >10% abundance relative to the base peak?

2a

APB Other

Is there a molecular ion? *Can be confirmed by CI data

3

Yes No

Consistent with an APB-

phenethylamine

Not consistent with an APB-

phenethylamine

Consistent with an APB-

phenethylamine

Does the Kendrick mass defect** of the M+

adj. fall in the APB filter between 95.9 ±1.6 mDa (94.2 – 97.5 mDa)?

4

Yes

NoYes

See NOTE

Not consistent with an APB-

phenethylamine

No

Does the spectrum have three predominant peaks at m/z 91 (C7H7

+), 121 (C8H9O+) and 150 (C9H12NO+) with the base peak at m/z 121?

2b

Consistent with an NBOMe-phenethylamine. Continue

to Step 3.

Not consistent with an NBOMe-phenethylamine.

Continue to Step 3.

Is there a molecular ion? *Can be confirmed by CI data

3

See NOTE See NOTEIs there a halogen,

sulfur, or nitro group present? ***(next

page)

** Kendrick Mass defect is calculated by:

Exact mass * (14/14.01565) = Kendrick mass(Nominal mass - Kendrick Mass) * 1000 = Kendrick Mass defect in mDa

NoYesYes No

Continue to 3b. Continue to 3c. Continue to 3d.

NOTE: If no molecular ion is confirmed: only halogens can be identified.

110

Figure 5.19 (con’t)

***If Br is present, double peaks (doublets) of similar abundance will be present, spaced two mass units apart for higher mass fragments

If Cl is present, doublets in a 3:1 abundance ratio will be present, spaced two mass units apart for higher mass fragments

If I is present, m/z 126.9 and m/z 127.9 should be present (I and HI, respectively)

Nitrogen rule: If the mass of the molecular ion is even, there is an even number of nitrogens present, or none at all. Ex: The M+ for 2C-H has one nitrogen and its m/z 181 is odd, indicating an odd number of nitrogens, while M+ for 2C-N which has two nitrogens is m/z 226, an even number.

If S is present, there may be an [M+2]+ ion of low abundance

If Br, Cl, or I are present, subtract the mass of the halogen (78.9183, 34.9689, 126.9045 Da) from the molecular ion and add the mass of hydrogen (1.0078 Da).

If the compound has an even M+ subtract the mass of a nitro group (NO2) (45.9929 Da) from the molecular ion and add the mass of hydrogen (1.0078 Da).

If S is present, subtract the mass of sulfur (31.9721 Da) from the molecular ion and add the mass of CH2 (14.0157 Da).

This new adjusted M+ should be approximately 301 Da. Use it for Step 4.

3a 3b 3c 3d

Consistent with an alkyl-

or sulfur-substituted compound. Continue to

Step 4

If Br, Cl, or I are present, subtract the mass of the halogen (78.9183, 34.9689, 126.9045 Da) from the molecular ion and add the mass of hydrogen (1.0078 Da).

If the compound has an even M+ subtract the mass of a nitro group (NO2) (45.9929 Da) from the molecular ion and add the mass of hydrogen (1.0078 Da).

If S is present, subtract the mass of sulfur (31.9721 Da) from the molecular ion and add the mass of CH2 (14.0157 Da).

This new adjusted M+ should be approximately 181 Da. Use it for Step 4.

Consistent with an alkyl-

or sulfur-substituted compound. Continue to

Step 4

Does the Kendrick mass defect of the M+adj. fall in the NBOMe filter

between 171.5 ± 7.7 mDa (163.8 – 179.2 mDa)?

4 Does the Kendrick mass defect of the M+adj. fall in the 2C filter

between 92.2 ± 1.5 mDa (90.7 – 93.7 mDa)?

4

Consistent with an NBOMe-phenethylamine. Continue to Step 5.

Yes NoConsistent with a 2C-

phenethylamine. Continue to Step 5.

Yes NoNot consistent with an

NBOMe-phenethylamineNot consistent with a 2C-phenethylamine

Does the compound lose CH3N (approx. 29 Da) and C2H6NO (approx. 60 Da) in neutral losses from M+? Does it lose CH3N (approx. 29 Da) from M+

as the base peak?

5Does the compound have common loses of CH3O (approx. 31 Da) and C9H11NO (approx. 149 Da) from the molecular ion? Does the fragment

remaining after loss of C9H11NO fall within the CH3N KMD filter?*

5

Do the fragments remaining after the losses of CH3N and C2H6NO fall within the

KMD fragment filters?*Loss CH3N KMD filter = 86.0 ± 0.7 mDa

(85.2 – 86.7 mDa)Loss C2H6NO KMD filter = 69.6 ± 5.2 mDa

(64.5 – 74.8 mDa)

NoYesConsistent with an

NBOMe-phenethylamine. Not consistent with an

NBOMe-phenethylamine

No

Yes

Consistent with a 2C-

phenethylamine

Not consistent with a 2C-

phenethylamine

No

Yes

Not consistent with a 2C-phenethylamine.

If KMD falls between 95 – 110 mDa, it

is consistent with a 3C-phenethylamine

*Replace halogens/sulfur/nitro group when appropriate

111

Example 1: 3-methylethcathinone (3-MEC) (Figure 5.20)

1. Is retention index available? No, the retention index of 3-MEC was not available.

2a. Is there an ion at m/z 131 (C9H7O+) >10% abundance relative to the base peak? No,

there is an ion at m/z 131.0748 but the abundance is 1.1% relative to the base peak.

2b. Does the spectrum have three predominant peaks at m/z 91 (C7H7+), 121 (C8H9O+)

and 150 (C9H12NO+) with the base peak at m/z 121? No, these peaks are not present,

therefore this compound is not consistent with an NBOMe-phenethylamine.

3. Is there a molecular ion? Yes, the molecular ion was confirmed to be m/z 191.1310 with

a mass accuracy of 0.0 ppm.

a. Is there a halogen, sulfur, or nitro group present? No, no halogens, sulfur, or

nitro groups were determined to be present.

4. Does the Kendrick mass defect of M+adj fall in the 2C-phenethylamine filter between

92.2 ± 1.5 mDa (90.7 – 93.7 mDa)? No, the KMD of M+ (82.4 mDa) does not fall within

the 2C-phenethylamine KMD filter. This compound is not consistent with a 2C-

phenethylamine.

If treated as an unknown, 3-MEC would be not be characterized as an APB, NBOMe, or

2C-phenethylamine.

112

Figure 5.20 Mass spectrum and structure of cathinone, 3-methylethcathinone (3-MEC) showing

loss of C3H8O, which is uncharacteristic of the phenethylamine class

In the characterization scheme for low-resolution data, 3-MEC exhibited a loss of 60 Da,

which is the nominal mass of a loss of C2H6NO, a common neutral loss for 2C compounds.

However, with the advantage of high-resolution mass spectrometry, leading to elemental

formulae assignment, the loss of 60 Da from M+ of 3-MEC corresponds to a fragment at m/z

131.0748 and a formula assignment of C9H9N with a mass accuracy of 9.9 ppm. This would

equate to a loss of C3H8O, which also gives a nominal mass of 60 Da. Through the

characterization scheme for low-resolution data, it was determined that 3-MEC would not be

characterized as an APB or NBOMe, but it could not be discerned whether or not it was a 2C-

phenethylamine. However, using high resolution, 3-MEC would be correctly characterized as

being inconsistent with an APB-, NBOMe- and 2C-phenethylamine.

0 50 100 150 200

0

50

100

Abundance (

%)

m/z

3-MEC

NH

O

M+

191.13100.0 ppmC12H17NO

131.07489.9 ppm

C9H9N

Loss of C3H8O

113

Example 2: Mescaline (Figure 5.21)

1. Is the retention index available? No, the retention index of mescaline was not available.

2a. Is there an ion at m/z 131 (C9H7O+) >10% abundance relative to the base peak? No,

there is no ion at m/z 131.

2b. Does the spectrum have three predominant peaks at m/z 91 (C7H7+), 121 (C8H9O+)

and 150 (C9H12NO+) with the base peak at m/z 121? No, these peaks are not present,

therefore this compound is not consistent with an NBOMe-phenethylamine.

3. Is there a molecular ion? Yes, the molecular ion was confirmed to be m/z 211.1207 with

a mass accuracy of 0.5 ppm.

a. Is there a halogen, sulfur, or nitro group present? No, no halogens, sulfur, or

nitro groups were determined to be present.

4. Does the Kendrick mass defect of M+adj fall in the 2C-phenethylamine filter between

92.2 ± 1.5 mDa (90.7 – 93.7 mDa)? No, the KMD of M+ (115.0 mDa) does not fall

within the 2C-phenethylamine KMD filter. This compound is not consistent with a 2C-

phenethylamine.

5. Does the compound lose CH3N (approx. 29 Da) and C2H6NO (approx. 60 Da) in

neutral losses from M+? Does it lose CH3N from M+ as the base peak? Yes, the

spectrum has ions at m/z 182.0967 (211.1207 – 29.0240 = 182.0967) and m/z 151.0725

(211.1207 – 60.0482 = 151.0725). The ion at m/z 182.0967 has an elemental formula of

C10H14O3 with a mass accuracy of 13.2 ppm, corresponding to a neutral loss of CH3N

(C11H17NO3 – CH3N = C10H14O3). The ion at m/z 151.0725 has an elemental formula of

C9H11O2 with a mass accuracy of 22.5 ppm, corresponding to a neutral loss of C2H6NO

114

(C11H17NO3 – C2H6NO = C9H11O2). The loss of CH3N from M+ (m/z 182.0967) is the

base peak.

Figure 5.21 Mass spectrum of 3C-phenethylamine, mescaline and fragment ions remaining after

neutral losses, the KMD of which can be used to distinguish 2C from 3C-phenethylamines

a. Do the fragments remaining after the losses of CH3N and C2H6NO have

KMD that fall within the KMD fragment filters? [M-CH3N]+ KMD filter =

86.0 ± 0.7 mDa (85.2 – 86.7 mDa). [M-C2H6NO]+ KMD filter = 69.6 ± 5.2

mDa (64.5 – 74.8 mDa). No, the fragment remaining after a loss of CH3N (m/z

182.0967) has a KMD of 106.6 mDa and does not fall within the CH3N KMD

filter. The fragment remaining after a loss of C2H6NO (m/z 151.0725) has a KMD

of 96.2 mDa and does not fall within the C2H6NO KMD filter.

0 50 100 150 200 250

0

50

100

Ab

und

an

ce (

%)

m/z

Mescaline

M+

211.12070.5 ppm

C11H17NO3

182.096713.2 ppmC10H14O3

Loss of CH3N

O

O

O

NH2

151.072522.5 ppmC9H11O2

M+

211.12070.5 ppm

C11H17NO3

Loss of C2H6NO

OH+

O

O

CH2

O

O

CH2

+

115

b. Are the KMD of the fragments remaining after the neutral losses of CH3N

and C2H6NO between 95 – 110 mDa? Yes, the KMD of the fragments

remaining are 106.6 and 96.2 mDa. This is indicative of a 3C-phenethylamine.

If treated as an unknown, mescaline would be correctly characterized as a 3C-

phenethylamine.

In the characterization scheme for low-resolution data, mescaline was incorrectly characterized

as a 2C-phenethylamine. However, with the addition of mass defects filters, 2C- and 3C-

phenethylamines can be easily differentiated, and mescaline is correctly characterized.

5.4 Summary

High-resolution mass spectrometry can overcome the limitations of low-resolution mass

spectrometry by giving definitive identification of ions through elemental assignment and mass

accuracy measurements. Using exact mass measurements, mass defects can be explored for use

in characterization of unknown compounds to a specific designer drug class or subclass. This

allows for a more accurate preliminary characterization and a more detailed, descriptive

characterization scheme.

116

APPENDICES

117

APPENDIX A: High-Resolution Mass Spectra

Figure A.1 High-resolution mass spectra of (A) 4-(2-aminopropyl)benzofuran (4-APB), (B) 5-(2-

aminopropyl)benzofuran (5-APB), and (C) 7-(2-aminopropyl)benzofuran

0 50 100 150 200 250 300

0

50

100

Ab

undan

ce (

%)

m/z

0 50 100 150 200 250 300

0

50

100

Ab

und

an

ce (

%)

m/z

NH2O

NH2

O

4-APB 5-APB

A) B)

NH2

O 7-APBC)

0 50 100 150 200 250 300

0

50

100

Ab

und

an

ce (

%)

m/z

118

Figure A.2 High-resolution mass spectra of (A) 2,5-dimethoxy-4-methylphenethylamine (2C-D),

(B) 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and (C) 2,5-dimethoxy-4-

propylphenethylamine (2C-P)

0 50 100 150 200 250 300

0

100

Abundance (

%)

m/z

0 50 100 150 200 250 300

0

100

Abundance (

%)

m/z

0 50 100 150 200 250 300

0

100A

bundance (

%)

m/z

A) B)

C)

2C-D 2C-E

2C-P

O

O

NH2

O

O

NH2

O

O

NH2

119

Figure A.3 High-resolution mass spectra of (A) 2,5-dimethoxy-4-chlorophenethylamine (2C-C),

(B) 2,5-dimethoxy-4-iodophenethylamine (2C-I), and (C) 2,5-dimethoxy-4-nitrophenethylamine

(2C-N)

0 50 100 150 200 250 300

0

100A

bundance (

%)

m/z

O

O

NH2

Cl

O

O

NH2

I

0 50 100 150 200 250 300 350

0

100

Abundance (

%)

m/z

A)2C-C

B)2C-I

C)

2C-N

0 50 100 150 200 250 300

0

100

Abundance (

%)

m/z

O

O

NH2

O2N

120

Figure A.4 High-resolution mass spectra of (A) 2,5 -dimethoxy-4-methylthiophenethylamine

(2C-T) and (B) 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2)

O

O

NH2

S

A) 2C-T

B) 2C-T-2O

O

NH2

S

0 50 100 150 200 250 300

0

100

Ab

und

an

ce (

%)

m/z

0 50 100 150 200 250 300

0

100

Ab

und

an

ce (

%)

m/z

121

Figure A.5 High-resolution mass spectra of (A) 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-

methyoxybenzyl)ethanamine (25D-NBOMe), (B) 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-

methoxybenzyl)ethanamine (25E-NBOMe) and (C) 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-3,4-dimethyl-benzeneethanamine (25G-NBOMe)

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

A) B)25D-NBOMe 25E-NBOMeO

O

NH

O

O

O

NH

O

0 100 200 300

0

50

100

Abundance (

%)

m/z

O

O

NH

O

C) 25G-NBOMe

122

Figure A.6 High-resolution mass spectra of (A) 4-bromo-2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe), (B) 4-chloro-2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-benzeneethanamine (25C-NBOMe), and (C) 4-iodo-2,5-dimethoxy-N-

[(2-methoxyphenyl)methyl]-benzeneethanamine (25I-NBOMe)

A) B)

C)

25B-NBOMe 25C-NBOMe

25I-NBOMe

0 100 200 300 400

0

100

Ab

und

an

ce (

%)

m/z

0 100 200 300 400

0

100

Ab

und

an

ce (

%)

m/z

O

O

Br

NH

OCl

O

O

NH

O

O

O

NH

IO

0 100 200 300 400

0

100

Ab

und

an

ce (

%)

m/z

123

Figure A.7 High-resolution mass spectra of (A) 2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-4-

(methylthio)-benzeneethanamine (25T-NBOMe), (B) 2,5-dimethoxy-N-[(2-

methoxyphenyl)methyl]-4-[(1-methylethyl)thio]-benzeneethanamine (25T-4-NBOMe), (C) 2,5-

dimethoxy-N-[(2-methoxyphenyl)methyl]-4-(propylthio)-benzeneethanamine (25T-7-NBOMe),

and (D) 3,4,5-trimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (mescaline-

NBOMe)

A) B)

C) D)

25T-NBOMe 25T-4-NBOMe

25T-7-NBOMe Mescaline-NBOMe

O

O

S

NH

O

O

O

S

NH

O

O

O

NH

OS

O

NH

O

O

O

0 100 200 300 400

0

100

Abundance (

%)

m/z

0 100 200 300 400

0

100

Abundance (

%)

m/z

0 100 200 300 400

0

100

Abundance (

%)

m/z

0 100 200 300 400

0

100

Abundance (

%)

m/z

124

Figure A.8 High-resolution mass spectra of (A) 4-ethoxy-3,5-dimethoxy-benzeneethanamine

(escaline) and (B) 4-methylmethcathinone (mephedrone)

A)

B)

Escaline

Mephedrone

O

O

O

NH2

NH

O

0 100 200 300

0

50

100

Ab

und

an

ce (

%)

m/z

0 50 100 150 200

0

50

100

Ab

und

an

ce (

%)

m/z

125

APPENDIX B: Additional High-Resolution Characterization Scheme Examples

Example 1: 2C-G (Figure A.9)

1. Is retention index available? Yes, the retention index is 1751. This retention index falls

within the retention index range identified for 2C-phenethylamines (1590 – 2000).

2b. Does the spectrum have three predominant peaks at m/z 91 (C7H7+), 121 (C8H9O+)

and 150 (C9H12NO+) with the base peak at m/z 121? No, these peaks are not present,

therefore this compound is not consistent with an NBOMe-phenethylamine.

3. Is there a molecular ion? Yes, the molecular ion was confirmed to be m/z 209.1421 with

a mass accuracy of 2.4 ppm.

a. Is there a halogen, sulfur, or nitro group present? No, no halogens, sulfur, or

nitro groups were observed.

Figure A.9 Mass spectrum of 2C-G and fragment ions remaining after neutral losses

0 50 100 150 200 250 300

0

100

Ab

und

an

ce (

%)

m/z

O

O

NH2

2C-G209.14212.4 ppm

C12H19NO2

180.11521.1 ppmC11H16O2

209.14212.4 ppm

C12H19NO2

149.098512.7 ppmC10H13O

Loss ofCH3N

Loss ofC2H6NO

O+

O

CH2

H

O

CH2

+

126

4. Does the Kendrick mass defect of M+ fall in the 2C-phenethylamine filter between

92.2 ± 1.5 mDa (90.7 – 93.7 mDa)? Yes, the KMD of M+ (91.4 mDa) does fall within

the 2C-phenethylamine KMD filter. This compound is consistent with a 2C-

phenethylamine.

5. Does the compound lose CH3N (approx. 29 Da) and C2H6NO (approx. 60 Da) in

neutral losses from M+? Does it lose CH3N from M+ as the base peak? Yes, the

spectrum has ions at m/z 180.1152 (209.1421 – 29.0269 = 180.1152) and m/z 149.0985

(209.1421 – 60.0436 = 149.0985). The ion at m/z 180.1152 has an elemental formula of

C11H16O2 and a mass accuracy of 1.1 ppm, corresponding to a neutral loss of CH3N

(C12H19NO2 – CH3N = C11H16O2). The ion at m/z 149.0985 has an elemental formula of

C10H13O with a mass accuracy of 12.7 ppm, corresponding to a loss of C2H6NO

(C12H19NO2 – C2H6NO = C10H13O). The loss from CH3N from M+ (m/z 180.1152) is not

the base peak, but is a highly abundant ion.

a. Do the fragments remaining after the losses of CH3N and C2H6NO have

KMD that fall within the KMD fragment filters? [M-CH3N]+ KMD filter =

86.0 ± 0.7 mDa (85.2 – 86.7 mDa). [M-C2H6NO]+ KMD filter = 69.6 ± 5.2

mDa (64.5 – 74.8 mDa). Yes, the fragment remaining after a loss of CH3N (m/z

180.1152) has a KMD of 85.9 mDa and does fall within the CH3N KMD filter.

The fragment remaining after a loss of C2H6NO (m/z 149.0985) has a KMD of

68.0 mDa and falls within the C2H6NO KMD filter.

If treated as an unknown, 2C-G would be correctly characterized as a 2C-phenethylamine.

127

Example 2: 2C-B (Figure A.10)

1. Is retention index available? Yes, the retention index is 1856. This retention index falls

within the retention index range identified for 2C compounds (1590 – 2000).

2. Does the spectrum have three predominant peaks at m/z 91 (C7H7+), 121 (C8H9O+)

and 150 (C9H12NO+) with the base peak at m/z 121? No, these peaks are not present,

therefore this compound is not consistent with an NBOMe-phenethylamine.

3. Is there a molecular ion? Yes, the molecular ion was confirmed to be m/z 259.0203 with

a mass accuracy of 0.37 ppm.

a. Is there a halogen, sulfur, or nitro group present? Yes, bromine doublets are

present. Doublets of similar intensity indicate the presence of Br.

i. Subtracting the mass of Br (78.9182 Da) from the M+ (m/z 259.0203) and

adding the mass of H (1.0078 Da) yields an adjusted molecular ion (M+adj)

of m/z 181.1098.

4. Does the Kendrick mass defect of M+adj fall in the 2C-phenethylamine filter between

92.2 ± 1.5 mDa (90.7 – 93.7 mDa)? Yes, the KMD of M+adj (92.4 mDa) does fall within

the 2C-phenethylamine KMD filter. This is consistent with a 2C-phenethylamine.

5. Does the compound lose CH3N (approx. 29 Da) and C2H6NO (approx. 60 Da) in

neutral losses from M+? Does it lose CH3N from M+ as the base peak? Yes, the

spectrum has ions at m/z 229.9938 (259.0203 – 29.0265 = 229.9938) and m/z 198.9772

(259.0203 – 60.0431 = 198.9772). The ion at m/z 229.9938 has an elemental formula of

C9H11O2Br and a mass accuracy of 1.7 ppm, corresponding to a neutral loss of CH3N

(C10H14NO2Br – CH3N = C9H11O2Br). The ion at m/z 198.9772 has an elemental formula

of C8H8OBr with a mass accuracy of 6.7 ppm, corresponding to a loss of C2H6NO

128

(C10H14NO2Br – C2H6NO = C8H9OBr). The loss from CH3N from M+ (m/z 229.9938) is

the base peak.

a. Do the fragments remaining after the losses of CH3N and C2H6NO have

KMD that fall within the KMD fragment filters? [M-CH3N]+ KMD filter =

86.0 ± 0.7 mDa (85.2 – 86.7 mDa). [M-C2H6NO]+ KMD filter = 69.6 ± 5.2

mDa (64.5 – 74.8 mDa). Yes, after replacing the Br with a H on each fragment,

the fragment remaining after a loss of CH3N (m/z 152.0833) has a KMD of 86.5

mDa and does fall within the CH3N KMD filter. The fragment remaining after a

loss of C2H6NO (m/z 121.0667) has a KMD of 68.5 mDa and falls within the

C2H6NO KMD filter.

If treated as an unknown, 2C-B would be correctly characterized as a 2C-phenethylamine

with a bromine substituent.

Figure A.10 Mass spectrum of 2C-B and fragment ions remaining after neutral losses

0 50 100 150 200 250 300

0

100

Abundance (

%)

m/z

259.02030.37 ppm

C10H14BrNO2

198.97726.7 ppmC8H8OBr

Loss ofC2H6NO

229.99381.7 ppm

C9H11O2Br

Loss ofCH3N

NH2

O

Br

O

2C-B

CH2

O+

Br

O

H

CH2

+

O

Br

259.02030.37 ppm

C10H14BrNO2

129

Example 3: 25N-NBOMe (Figure A.11)

1. Is retention index available? Yes, the retention index is 2839. This retention index falls

within the retention index range identified for NBOMe compounds (2475 – 2839).

2b. Does the spectrum have three predominant peaks at m/z 91 (C7H7+), 121 (C8H9O+)

and 150 (C9H12NO+) with the base peak at m/z 121? Yes, the spectrum has prominent

peaks at m/z 91.0543, 121.0649, and 150.0915. These three peaks are consistent with the

NBOMe phenethylamine subclass.

3. Is there a molecular ion? Yes, the molecular ion was confirmed to be m/z 346.1493 with

a mass accuracy of -8.76 ppm.

a. Is there a halogen, sulfur, or nitro group present? Yes, an even-massed

molecular ion indicates the presence of more than one nitrogen. A nitro group is

present.

i. Subtracting the mass of NO2 (45.9929 Da) from the M+ (m/z 346.1493)

and adding the mass of H (1.0078 Da) yields an adjusted molecular ion

(M+adj) of m/z 301.1642

4. Does the Kendrick mass defect of M+adj fall in the NBOMe-phenethylamine filter

between 171.7 ± 7.7 mDa (163.8 – 179.2 mDa)? Yes, the KMD of M+adj (172.1 mDa)

does fall within the NBOMe-phenethylamine KMD filter. This is consistent with an

NBOMe-phenethylamine.

5. Does the compound lose CH3O (approx. 31 Da) and C9H11NO (approx. 149 Da) in

neutral losses from M+? Yes, the spectrum has ions at m/z 315.1284 (346.1493 –

31.0209 = 315.1284) and m/z 197.0686 (346.1493 – 149.0807 = 197.0686). The ion at

m/z 315.1284 has an elemental formula of C17H19N2O4 with a mass accuracy of -17.53

130

ppm, corresponding to a neutral loss of CH3O (C18H22N2O5 – CH3O = C17H19N2O4). The

ion at m/z 197.0686 has an elemental formula of C9H11NO4 with a mass accuracy of 1.74

ppm, corresponding to a neutral loss of C9H11NO (C18H22N2O5 – C9H11NO = C9H11NO4).

a. Does the KMD of the fragment remaining after the losses of C9H11NO fall

within the CH3N KMD filter (after replacement of the nitro group)? [M-

CH3N]+ KMD filter = 86.0 ± 0.7 mDa (85.2 – 86.7 mDa). Yes, after replacing

the NO2 with a H, the fragment remaining after a loss of C9H11NO (m/z 152.0835)

has a KMD of 86.3 mDa. This KMD does fall within the [M-CH3N]+ KMD filter.

If treated as an unknown, 25N-NBOMe would be correctly characterized as a NBOMe-

phenethylamine with a nitro group substituent.

Figure A.11 Mass spectrum of 25N-NBOMe and fragment ions remaining after neutral losses

0 100 200 300

0

100

Ab

und

an

ce (

%)

m/z

346.1493-8.76 ppmC18H22N2O5

346.1493-8.76 ppmC18H22N2O5

315.1284-17.53 ppmC17H19N2O4

197.06861.74ppmC9H11NO4

Loss ofCH3O

Loss ofC9H11NO

O

O

O2N

NH

OOH+

O

O2N

CH2

O

O

O2N

NHC

+

25N-NBOMe

121.0649

131

REFERENCES

132

REFERENCES

1. CRC Handbook of Chemistry and Physics, 89th ed.; Lide, D.R., Ed.; CRC Press:

Boca Raton, FL, 2008; Section 3, No. 339.

2. Chu, F. Improving Methods for the Analysis of Controlled Substances. MS Thesis,

Michigan State University. 2015

133

VI. Conclusions and Future Work

6.1 Conclusions

A sample set of designer drugs characteristic of the phenethylamine compound class was

analyzed by gas chromatography and both low- and high-resolution mass spectrometry. The

chromatographic data were used to develop characteristic retention index ranges for each

structural subclass. The spectral data were probed to identify characteristic spectral features to

identify compounds of similar subclasses. These features included the investigation of common

fragment ions, characteristic neutral losses, and substituent identification. The characteristic

subclass features were, in turn, used to develop a characterization scheme in the format of a flow

chart which crime laboratories can use as an initial screening method to determine if further

examination of a submitted controlled substance sample is necessary. This low-resolution

characterization scheme is immediately implementable in forensic laboratories because it has

been created using the gas chromatography-mass spectrometry (GC-MS) instrumentation already

in place and conventionally used for the identification of controlled substances. The

characterization scheme was successful in characterizing all APB-, 2C-, and NBOMe-

phenethylamines into their respective subclasses. However, some of the 3C-phenethylamines

and cathinone compounds used to test the scheme were mischaracterized or not characterized at

all. The lack of correct characterization means that while the low-resolution scheme is most

applicable in a forensic laboratory, there are some limitations, such as a lack of elemental

formulae assignment, and thus definitive identification, of the fragment ions in the mass spectra.

A high-resolution version of the same characterization scheme was developed for

increased confidence of a characterization and to overcome the limitations of the characterization

scheme for low-resolution data. This scheme exploits the use of accurate mass and mass defect

134

obtained from high-resolution mass spectrometry, with definitive identification of fragment ions.

Absolute and Kendrick mass defect filters were developed but only Kendrick mass defect filters

were implemented into the characterization scheme for structural subclass characterization due to

the greater specificity afforded. The characterization scheme for high-resolution data was

successful in characterizing all the phenethylamine and cathinone compounds, including those

mischaracterized and uncharacterized by the scheme for low-resolution data. Kendrick mass

defect filters offer a more specific characterization into structural subclass, and overcame many

limitations of mischaracterization using absolute mass defect. Overall, the utility of high-

resolution mass spectrometry for robust characterization of synthetic designer drugs was

highlighted, should that instrumentation ever be made available to forensic laboratories.

6.2 Future Work

Further investigation of the electron ionization-mass spectral features of sulfur and other

substituents should be performed. The presence of sulfur could not always be identified in the

mass spectra because it inconsistently exhibited characteristic features such as distinguishing

isotope patterns. Identifying other substituents such as fluorine, multiple nitrogens in a fragment

ion, or having several, differing substituents in a compound is an additional aim that could be

pursued. Another area of future direction would be the optimization of a GC-MS temperature

program for the differentiation of all retention indices of phenethylamine isomers. Some of the

isomers of the APB-phenethylamine subclass had the same calculated retention indices and thus

could not be distinguished from one another. However, if the gas chromatography temperature

program was further refined, this limitation could be overcome. Additionally, more research

should be conducted on how the substituent, such as a halogen, of a compound affects the

retention index. A third direction of future work should build on this work for the

135

characterization of compounds when a molecular ion is not available. Although the molecular

ion can be confirmed by chemical ionization, the ability to perform CI analysis may not be

possible. Therefore, more work is needed to identify characteristic features of the structural

subclasses based on fragment ions alone.

Because only reference standards were analyzed in this work, further experimentation

would be to first test the characterization schemes against a set of hypothetical unknowns, in

which the analyst does not know what they are. Following this, street samples would then be

tested, containing cutting agents and lower concentrations of unknown controlled substances.

Additionally, synthetic designer drugs of other compound classes, such as tryptamines, could be

tested against the current flow charts, or could be used to expand and refine the flow chart for

characterization of other compound classes.

While there are certain directions for future work and expansion, this research developed

two characterization schemes that will be able to assist in identification of compounds in a

constantly changing drug market, as well as allow characterization of unknowns for which no

reference standard is available.