characterization of synthetic phenethylamines ......alonso and joe binkley from leco corp. for help...
TRANSCRIPT
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
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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
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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
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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
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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
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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
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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.