synthesis and analytical profiles for regioisomeric and isobaric amines related to mdma, mdea

The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report:

Document Title: Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of Mass Spectral Equivalence

Author: C. Randall Clark, Ph.D. Document No.: 236243

Date Received: October 2011 Award Number: 2006-DN-BX-K016

This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally-funded grant final report available electronically in addition to traditional paper copies.

Opinions or points of view expressed are those

of the author(s) and do not necessarily reflect the official position or policies of the U.S.

Department of Justice.

1

Final Technical Report

Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of

Mass Spectral Equivalence

Award Number: 2006-DN-BX-K016

Submitted By: C. Randall Clark, Ph.D. Auburn University

Abstract: This project has focused on issues of resolution and discriminatory capabilities in

controlled substance analysis providing additional reliability for forensic evidence and

analytical data on regioisomeric and isobaric phenethylamines of forensic interest. The broad

objective of this research is improved specificity in the analytical methods used to identify

MDMA, MBDB, MDEA, and related phenethylamine controlled substances. This improved

specificity comes from methods which allow the forensic analyst to eliminate non-drug

imposter substances as the source of analytical data matching that of the controlled drug

substance. This project has developed methods to discriminate between the

methylenedioxyphen-ethylamine drugs and those regioisomeric and isobaric molecules having

the same molecular weight and major fragments of equivalent mass (i.e. identical mass

spectra). The work has emphasized those analytical methods commonly employed in forensic

laboratories for confirmation of drug identity: gas chromatography, mass spectrometry,

infrared spectrometry. When compounds exist which produce the same mass spectrum (same

This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.

2

MW and fragments of equivalent mass) as the drug of interest, the identification by GC-MS

must be based entirely upon the ability of the chromatographic system to resolve these

substances.

The initial phase of this work involved the organic synthesis of the direct

regioisomeric substances related to MDMA, MBDB, and MDEA. We also prepared

representative samples of the indirect regioisomeric molecules (such as the 2, 3, and 4-

methoxymethcathinones) and the isobaric substances, compounds of the same nominal mass

but different elemental composition (for example 2, 3, and 4-ethoxyphenethylamines and the

10 different ring substitution patterns for the methoxy-methyl-phenethylamines). Additional

compounds were prepared in related categories to further refine the developed analytical

methods and confirm/challenge experimental observations. A number of deuterium labeled

analogs has been prepared to establish mass spectral fragmentation patterns. Other isomeric

series were prepared such as the methoxymethylene-substitution phenyl ring pattern for the

MDMA and the MBDB side chain series and the 2-, 3-, and 4-ethoxyphenyl compounds for

the MBDB series. Overall, in this project more than 90 phenethylamines having a

regioisomeric or isobaric relationship to MDMA or MBDB have been synthesized and

evaluated.

The chromatographic retention properties for each series of isomers have been

evaluated by gas chromatographic techniques on a variety of stationary phases. These studies


3

established structure-retention relationships (elution orders) for the side chain regioisomers and

the isobaric amines on a number of stationary phases.

Mass spectrometry studies have focused on the underivatized free amines and have

clearly established the significant similarity in the mass spectrum for the side chain and ring

regioisomers. Derivatization studies have established methods for differentiation of side chain

substitution patterns yielding unique fragment ions of significant abundance in the EI mass

spectrum for these compounds. The perfluoroacyl derivatives provide maximum mass spectral

information for compound individualization and in a number of regioisomeric subsets the HFBA

derivatives provide more data for molecular individualization than PFPA or TFA derivatives. A

detailed background study established the advantages of perfluoroacyl derivatives over other

alkyl or aryl amide derivatives. A second detailed background study provided the scientific

evidence for the mechanism of fragmentation producing the unique m/z 110, 160 and 210 ions

for the methamphetamine side chain in the TFA, PFPA and HFBA derivatives respectively. The

mass spectral studies clearly show that the individual side chain isomers of m/z58 and m/z72 can

be identified by specific fragment ions. Additionally unique ions help to point the direction of

investigation for ring substitution pattern. For example the unique fragment ions at m/z107 in the

spectrum of ethoxy-substituted phenethylamines allows its’ differentiation from

methylenedioxyphenethylamines. Vapor phase infrared spectrometry following separation by gas

chromatography (GC-IRD) provided confirmation of the nature and position of aromatic ring

substituent.


4

TABLE OF CONTENTS

Abstract……………………………………………………………………………………1

Excutive Summary……………………………………………………………………….9

I.Introduction…………………………………………………………………………….16

I.1 Statement of the Problem ……………………………………………………….....16

I.2 Literature Citations and Review……………………………………………………20

I.3 Statement of Hypothesis or Rationale for the Research……………………………...47

II. Methods (Narrative Descriptions of the Experimental Design) ……………………...48

II.1 Synthesis Design ……………………………………………………………………48

II.2 Methods (Synthetic materials, and procedures) …………………………………..57

II.2.1 Materials……………………………………………………………………….57

II.3 Analytical Methods……………………………………………………………...…58

II.3.1 Instruments……………………………………………….………………….…58

II.3.2 GC- Columns………………………………………………………………..…59

III. Results………………………………………………………………………………61

Chapter 1…………………………………………………………………………….62

Mass Spectrometry and Gas Chromatographic Studies on Direct Regioisomers to 3,4-MDMA…………………………………………….…………..62 Mass spectral studies of direct regioisomers of 3,4-MDMA (methylenedioxyphenyl substituted side-chain regioisomers) …… ….……………62


5

Mass spectral studies of perfluroacyl derivatives of 3,4- MDMA and their direct regioisomers………………………………………67

Gas chromatographic separation of perfluroacyl derivatives of 3,4- MDMA and their direct regioisomers………………………………………83

Conclusions of Chapter 1…………………………………………………………88

Chapter 2……………………………………………………………………………89

Mass Spectrometry and Gas Chromatographic Studies of Indirect Regioisomers Related to 3,4-MDMA, the Methoxymethcathinones. …………89

Mass spectral studies of the undreivatized and the perfluroacyl derivatives of the methoxymethcathinones…………………………………………………90

Gas chromatographic Separation of 2,3-MDMA , 3,4-MDMA from the Methoxymethcathinnes………………………………………………………...…101

Conclusion of Chapter 2……………………………………………………….…106

Chapter 3……………………………………….…………………………………107

Mass Spectrometry and Gas Chromatographic Studies on Acylated Derivatives of a Series of Side Chain Regioisomers of 2-Methoxy-4-Methyl Phenethylamines…………………………………………………………………107

Mass Spectral Studies on Chain Regioisomers of 2-Methoxy-4-Methyl Phenethylamines…………………………………………………………………107

Gas Chromatographyic separation of the perflouroacyl derivatives of 2- methoxy-4-methylphenethylamines from 2,3- and 3,4-MDMA……………123


6

Conclusion of Chapter 3………………………………………..……………………127

Chapter 4…….……………………………………………………………………128

GC-MS Analysis of Acylated Derivatives of the Side Chain Regioisomers of 4-Methoxy-3-Methyl Phenethylamines Related to Methylenedioxymethamphetamine………..128

Mass Spectrometral studies of 4-methoxy-3-methyl phenethylamines along with 2,3- and 3,4- MDMA……………………………………….…………………….128 Gas Chromatography of the regioiosmeric4-methoxy-3-methyl Phenethylamines and 2,3- and 3,4-MDMA………………………………………146 Conclusions of Chapter 4………………………………………………….……...149

Chapter 5……………………………………………………….…………………....150

Gas Chromatographic and Mass Spectral Studies on Acylated Side Chain Regioisomers of 3-Methoxy-4-methyl-phenethylamine and 4-Methoxy-3-methylphenethylamine…150

Mass Spectral Studies on Side Chain Regioisomers of 3-Methoxy-4-methyl-phenethylamine and 4-Methoxy-3-methylphenethylamine……………………...150

Gas Chromatography of 3-Methoxy-4-methyl-phenethylamine, 4-Methoxy-3-methyl-phenethylamine, 2,3- and 3,4-MDMA………………………………….173

Conclusions of Chapter 5…………………………………………………………..176

Chapter 6……………………………….…………………………………………177

Mass Spectral and Gas Chromatographic Studies of Methoxy Methyl Methamphetamines Isobaric to 3, 4-MDMA. ……………………………………………………………177 Mass Spectral Studies of Methoxy Methyl methamphetamines Isobaric to 3,4-MDMA………………………………………………….……….177


7

Mass Spectral Studies of Perfluroacyl Derivatives Methoxymethyl Methamphetamines compared to 2,3- and 3,4-MDMA………………………………………………183 Gas chromatographic separation of the perfluroacyl derivatives of ring substituted methoxy methyl methamphetamines, 2,3- and 3,4- MDMA. ………200

Conclusions of Chapter 6………………………………………………………..211

Chapter 7…………………………………………………………………………….213

GC–MS Analysis of Ring and Side Chain Regioisomers of Ethoxyphenethylamines………………………………………………………….213 Mass spectrometry of Ring and Side Chain Regioisomers of Ethoxyphenethylamines………………………………………………………….213 Gas chromatography of Ring substituted Ethoxy Phenethylamines, 2,3- and 3,4-MDMA. …………………………………………………………….234

Conclusions of Chapter 7…………………………………………………………238

Chapter 8……………………………………………………………………...239

GC-MS Studies on Direct Side Chain Regioisomers Related to Substituted Methylenedioxyphenethylamines; MDEA, MDMMA and MBDB……………...239 Mass Spectral Studies……………………………………………………………239 Gas chromatography of Side Chain Regioisomeric Phenethylamines Related to the Controlled Substances MDEA, MDMMA, and MBDB. ………………….257

Conclusion of Chapter 8………………………………………………………...260

Chapter 9…………………………………………………………………...260

GC-MS Evaluation of a series of Acylated Derivatives of 3,4-Methylenedioxymethamphetamine .......................................................................260


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Mass Spectrometry of Acylated Derivatives of 3,4-Methylenedioxymethamphetamine………………………………………………261

Gas Chromatography………………………………….………………………….275

Conclusions of Chapter 9…………………………………………………………..277

IV. Conclusions……………………………………………………………………278

IV.1 Discussion of findings. …………………………………………………………278

IV.2 Implications for policy and practice. …………………………………………...278

IV.3 Implications for further research………………………………………………..279

V. References………………………………………………………………………….281

VI. Dissemination of Research Findings………………………………………………293

VI.1 Scientific Publications Related to this Project…………………………………….293

VI.2 Scientific Presentations Related to this Project…………………………..……….298


9

Executive Summary: In this project, we have synthesized over 90 regioisomeric and isobaric

phenethylamines of mass spectral equivalence and evaluated the analytical profiles of these

amines. The work has emphasized those analytical methods commonly employed in forensic

laboratories for confirmation of drug identity: gas chromatography, mass spectrometry,

infrared spectrometry. The synthetic efforts focused on those regioisomeric and isobaric

substances most likely to appear in future forensic samples. In some cases compounds were

prepared to complete a set of all the possibilities in a series when some of the regioisomers

have already appeared in clandestine samples. In other cases the compounds prepared were

available directly or indirectly from commercially available precursor substances. We have

collected, cataloged and categorized the mass spectra for the free amines and several

derivatives of each of the 90 amines; studied the gas chromatographic retention properties of

various subsets of the amines and their derivatives; collected and evaluated vapor phase

infrared spectra for these compounds. As of the date of this writing (January, 2011) twenty

scientific publications resulting from this area of research are in print or accepted for

publication in Forensic Science International and Journal of Chromatographic Science. A few

other manuscripts related to this project are yet to be written. Additionally 12 presentations

have been made at national meetings during this project period.

The overall goal of this project was a comprehensive study of those regioisomeric

and isobaric amines capable of producing mass spectra equivalent to those of MDMA,

MBDB and MDEA. The availability of all the necessary compounds to establish and


10

prove the structure-retention, structure-fragmentation and other structure-property

analytical experiments was the starting point for this research. The mass spectra of

phenethylamines are characterized by a base peak formed from an amine initiated alpha-

cleavage reaction of the carbon-carbon bond of the ethyl linkage between the aromatic

ring and the amine group. In methamphetamine and ring substituted methamphetamines

(for example 3,4-methylenedioxymethamphetamine), the alpha-cleavage reaction yields

the substituted imine fragment at m/z 58 and the benzyl fragment or substituted benzyl

fragment at the appropriate mass.

C N

C N C N

C N C N

H

CH3

H

H

CH3

CH3

H

HH

CH2CH3

HH

H

H3C

H3C

H3C

H

HCH3CH2

H

m/z=58, C4H8N+

Regioisomeric Forms of m/z 58

In this project the substituted benzyl fragment for 3,4-MDMA and its

regioisomeric and isobaric equivalents occurs at m/z 135. There are four side chain

regioisomers of methamphetamine with the potential to yield mass spectra essentially

equivalent to methamphetamine yielding regioisomeric imine fragment ions in their

electron ionization mass spectra at m/z 58. Thus, for every individual ring substitution


11

pattern there are five side chains yielding equivalent m/z 58 fragment ions (the

methamphetamine side chain and four regioisomeric equivalents). The ring substitution

pattern in methylenedioxymethamphetamine (MDMA) doubles the number of isomers to ten,

the five side chain isomers in which the methylenedioxy-ring is substituted in a 3,4-manner

and the five in which the methylenedioxy-ring is substituted in a 2,3-pattern. Thus, there are

nine other amines with the potential to produce a mass spectrum essentially the same as that of

3,4-MDMA. Most of these potential designer analogues have the strong possibility to be

misidentified as 3,4-MDMA based on mass spectrometry. In addition to the ten amines

containing the methylenedioxy-group, other substitution patterns have the potential to produce

mass spectra with fragments of equivalent mass to those of 3,4-MDMA.

O

O

58

H3C

H3CO

58

EtO

58 58

H3CO

H3CO

58O

H3CO

58H3C

Example of Regioisomeric and Isobaric Compounds in this Study


12

For example, the reported stimulant methoxy-methcathinones are uniquely isomeric

with the MDMAs having the same MW, elemental composition and expected major fragment

ions of equal mass to those of the MDMAs. Additionally isobaric substitution patterns

especially those likely to undergo few if any unique/characteristic fragmentations such as

methoxy-methyl disubstitution of the methamphetamine aromatic ring, ethoxy-

monosubstitution as well as methoxymethylene- monosubstitution are compounds with the

potential to produce El mass spectra essentially equivalent to that of 3,4-MDMA. Each

methoxy-methyl, methoxymethylene- or ethoxy-substitution pattern would produce 5-side

chain isomers analogous to methamphetamine with the potential to yield mass spectra

equivalent to 3,4-MDMA. The o, m, and p-ethoxy substituents would yield a total of 15-

isomers, 15 for the methoxymethylenes and the methoxy-methyl disubstituted aromatic ring

system (10 unique disubstitution patterns) can result in over 50 isomers.

Representative samples have been prepared in this study for each aromatic ring substitution

pattern and each side chain regioisomer. A complete set of all side chain regioisomers was

prepared in enough categories to confirm mass spectral methods for specific side chain

identification. The number of side chain regioisomers for the MBDB series doubles to 20 for

each ring substitution pattern and the base peak for these compounds is m/z72. In this project

efforts were concentrated on preparing compounds necessary to confirm methods developed

for the MDMA isomers (m/z58). When the number of mass spectral equivalent isomeric

substances is relatively small, chromatographic separation and reference standard


13

availability is not a major concern. However, the continued designer exploration of some

drug categories will likely produce even greater numbers of regioisomeric and isobaric

substances especially among the phenethylamines. As the number of compounds having

mass spectral equivalence increases so does the challenge of specific identification via

chromatographic resolution and other analytical methods. In this project we have

systematically examined the analytical profiles of compounds from each structural

category. The results of the work will allow the forensic analyst to differentiate among

most of the regioisomeric and isobaric equivalents of MDMA without the need for

reference standards of all these compounds.

The mass spectra in almost all the compounds prepared in this study showed the

expected m/z 58 and 135 ions with no features for specific differentiation. Upon

derivatization with perfluoroacyl reagents such as trifluoroacetyl, pentafluoropropionyl

and heptafluorobutryl characteristic and distinguishing ions were obtained in many cases.

These are specific ions which occur for differentiation and not just changes in the relative

abundance of common ions. In every case specific ions to identify the side chains were

obtained. The individualization is the result of fragmentation of the alkyl carbon-nitrogen

bond yielding hydrocarbon fragments at m/z 148, 162 and 176 as well as other unique

fragments from these regioisomeric amides. The N-ethylamides show a specific ion to

identify the ethyl group by loss of mass 28 and the methamphetamine side chain yields a

unique ion at m/z110, 160 and 210 for the TFA, PFPA and HFBA derivatives


14

respectively. Thus, following chemical derivatization the variations in regioisomeric

m/z58 side chains can be specifically identified. This is possible due to specific ions

identifying the number of carbons attached directly to the aromatic ring in an

uninterrupted manner and the nature of the substituent attached to nitrogen. During the

course of this work the mass spectral fragmentation mechanisms and proof of structure

for these diagnostic ions have been confirmed by homologation experiments and

deuterium labeling studies. These additional compounds were synthesized in order to

confirm the details of the observed mass spectral fragmentation pathways.

In an additional fundamental study, a series of acylation reagents were evaluated to

find the reagent yielding maximum specific fragmentation information as well as appropriate

chromatographic properties. The perfluoroacyl derivatives of MDMA show excellent

chromatographic properties and unique mass spectral fragment ions. The perfluoroalkyl

amides of TFAA, PFPA and HFBA yield a unique series of mass spectral fragments at

m/z 110, 160 and 210 respectively. Additionally, the base peaks in these mass spectra

occur at relatively higher masses at m/z 154, 204 and 254 respectively. These ions are the

perfluoroacylimines resulting from loss of 135 mass units (3,4-methylenedioxybenzyl)

from the molecular ion. This (M-135)+ species also occurs for the acetyl, propionyl and

butyryl amides however these ions rearrange through loss of the acyl group to yield a

common ion at m/z 58. Thus, the base peak for the non-fluorinated hydrocarbon

derivatives is the same as that observed for the underivatized MDMA, m/z 58. The


15

formyl, d3-acetyl, d5-benzoyl and pentafluorobenzoyl derivatives provided

chromatographic and confirmatory mass spectral evidence for fragmentation pathways.

In many cases the ring substituent can be identified following perfluoroacylation

as well. In some cases the position of ring substitution is not available from the mass

spectral data. In all cases compounds having identical mass spectra in the derivatized and

underivatized form, can be differentiated by vapor phase infrared spectrometry. GC-IRD

studies yield individual spectra to differentiate primarily between ring substituents. Thus

these techniques in concert provide molecular individualization for most of these

compounds. The differentiation by IRD of the methcathinones is straight forward due to

the carbonyl absorption in this set of compounds.

Similar studies for the precursor substances synthesized in this study have helped

to provide analytical details for the substituted phenylacetones and 2-butanones and

related ketones. Additionally the tertiary amines cannot yield stable amide derivatives for

side chain or ring substituent identification. These compounds can only be differentiated

by underivatized mass spectra, vapor phase infrared spectrometry and other techniques.


16

Main Body of the Final Technical Report

I. Introduction

I.1 Statement of the Problem

3,4-Methylenedioxymethamphetamine, MDMA, is a Schedule I drug under the

Controlled Substances Act (CSA). Primarily illicitly manufactured in and trafficked from

Europe, MDMA is the most popular of the club drugs. DEA reporting indicates widespread

abuse of this drug within virtually every city in the United States. The mass spectrum is often

the confirmatory piece of evidence in the identification of drugs in the forensic laboratory.

While the mass spectrum is often considered a specific "fingerprint" for an individual

compound, there are other substances which produce very similar or almost identical mass

spectra. Many of these compounds which yield the same mass spectrum are positional isomers

of side-chain or aromatic ring substituents. Such compounds having MS-equivalency and

similar elution properties, perhaps coelution, represent a serious analytical challenge for the

forensic chemist. The ability to distinguish between these isomers directly enhances the

specificity of the analysis for the controlled substance. If other compounds exist which have

the potential to produce the same mass spectrum as the drug of interest then the identification

by GC-MS must be based entirely upon the ability of the chromatographic system to separate

the isomeric substance from the actual drug. Those substances which coelute with the drug of

abuse will be misidentified. The ultimate concern then is "if the laboratory has never analyzed

the counterfeit substances, how can the analyst be sure that these compounds would not coelute


17

with the controlled drug?" The courts expect forensic drug chemistry to identify a substance

as an individual compound, not report it as an unknown member of a large group of

regioisomeric/isobaric substances. Methamphetamine is an excellent example which illustrates

the possibility of counterfeit amines based on side chain isomerism. The El mass spectrum for

methamphetamine is dominated by the m/z 58 fragment and the m/z 91 fragment with very

little molecular ion at m/z 149. There are four other side chain isomers of methamphetamine

having a molecular weight of 149 which yield an imine fragment of m/z58 and an

unsubstituted benzyl fragment of m/z 91.

NH2

H3C CH3

PhentermineC10H15N, MW=149

MethamphetamineC10H15N, MW=149

NH

H CH3

CH3

NH2

1-Phenyl-2-aminobutaneC10H15N, MW=149

N

N,N-DimethylphenethylamineC10H15N, MW=149

NH

N-EthylphenethylamineC10H15N, MW=149

C2H5

CH3

CH3

C2H5

Methamphetamine and Regioisomeric Side Chains Yielding m/z 58.

However, the high resolution capabilities of capillary GC coupled with the limited number of

compounds (a total of five for the methamphetamines having an unsubstituted aromatic ring)

can solve the issue in this case. Aromatic ring substitution makes this issue much more


18

challenging in forensic drug analysis. When the number of mass spectral equivalent

isomeric substances is relatively small, chromatographic separation and reference

standard availability is not a major concern. However, the continued designer exploration

of some drug categories will likely produce even greater numbers of regioisomeric and

isobaric substances especially among the phenethylamines. As the number of compounds

having mass spectral equivalence increases so does the challenge of specific

identification via chromatographic resolution and other analytical methods.

Major Fragment Ions for Substituted Methamphetamines


19

The results of this project allow the forensic analyst to differentiate among most

of the regioisomeric and isobaric equivalents of MDMA and minimizes the need for a

large number of reference standards of all these compounds.

I.2 Literature Citations and Review

I.2.1 Introduction

Designer drug exploration of the methylenedioxyphenethylamines has produced

several drugs of abuse in recent years. These derivatives include 3,4-

methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA)

and 3,4-methylenedioxyethyl-amphetamine (MDEA), 3,4-methylenedioxyphenyl-2-

butanamine (BDB) and 2-methylamino-1-(3,4-methylenedioxyphenyl)butane (MBDB).

The methylenedioxy-derivatives of amphetamine and methamphetamine represent the

largest group of designer drugs (Figure I.2-1).


20

O

O

R1

N

R3R2

MDA: R1 = CH3, R2 = R3 = H

MDMA: R1 = CH3, R2 = CH3, R3 = H

MDEA: R1 = CH3, R2 = C2H5, R3 = H

BDB: R1 = C2H5, R2 =R3 = H

MBDB: R1 = C2H5, R2 = CH3, R3 = H

Figure I.2-1: Chemical structures of methylenedioxyphenalkyl amine derivatives.


21

MDMA, also known as eccstcy, with its stimulant and hallucinogenic effects in humans

is the most commonly used derivative of this series and has become a major drug of

abuse in recent years. In 2003, the estimated annual production of ecstacy worldwide was

100-125 tons and the estimated retail price for that drug alone was 63.74 billion US

dollars for 1.4 billion tablets. In the same year eight million people were reported to

abuse Ecstacy alone [UNODCCP, 2003]. In 2005, the reported consumption of ecstacy

among US students indicated that 1.7 % of the eight grade students in the US had

consumed the drug and this increased to 2.6% among tenth grade students and as high as

3.0 % among students in grade twelve [MTF, 2005]. About 0.1 % of the global

population (age 15 and above) consume ecstasy and significantly higher ratios, 0.5 –

2.4%, have been reported from countries in the Oceania region, Western Europe and

North America. West Europe and North America together account for almost 85% of

global consumption. Use of ecstasy, however, is increasingly spreading to developing

countries as well.

Tablets or capsules are the most common way to administer MDMA, however the

powder also can be snorted, smoked or—in rare cases—injected (TCADA 2002). Drug

effects may last up to six hours and are known to include the production of profoundly

positive feelings, empathy for others, elimination of anxiety, and extreme relaxation.

MDMA is also said to suppress the need to eat, drink, or sleep, enabling users to endure

two- to three-day parties. For a while, it has been clear that many tablets sold as ecstasy

do not always contain MDMA as an active substance and in many cases these tablets are

notoriously impure [Ecstasy Data, 1996-2006]. Ecstasy tablets are prepared in clandestine

laboratories and, during most of the last decade; Western Europe has been the world’s


22

major manufacturing region. The most frequently mentioned country of origin was

Belgium followed by Germany, the UK, Spain and the USA. The most frequently

mentioned source countries located in Eastern Europe were the Baltic countries, Poland

and Belarus. China, Indonesia and Thailand were the most frequently reported source

countries located in Asia. In Africa, the Republic of South Africa, and in South America,

Colombia, were identified as source countries for ecstasy [UN ODCCP, 2003].

The goal of clandestine manufacturers is often to prepare substances with

pharmacological profiles that are sought after by the user population. Clandestine

manufacturers are also driven by the desire to create substances that circumvent existing

laws. In Europe, as a result of the substance-by-substance scheduling approach, the

appearance of new substances cannot be immediatley considered as illicit drugs. This

offers room for clandestine experimentation into individual substances within a class of

drugs with similar pharmacological profiles, perhaps yielding substances of increased

potency. In the USA, continued designer exploration has resulted in legislation

(Controlled Substances Analog Act) to upgrade the penalties associated with clandestine

use of all compounds of a series. Thus, identification of new MDA derivatives and other

designer drugs is essential and a significant task for forensic laboratories.

I.2.2 Analytical Methods Used to Identify and Separate 3,4-MDMA

I.2.2.1 Spectroscopy

I.2.2.1.1 Mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) is the main tool used for the

detection and identification of unknown drugs in forensic and other drug screening

laboratories. The mass spectra of 3,4-methylenedioxyamphetamines are characteristic and


23

can be used for differentiation from other phenethylamines [Noggle et al., 1988]. The

electron impact (EI) mass spectra for 3,4-methylenedioxyamphetamines exhibit weak to

extremely weak molecular ions which, in some cases, may not be evident without

computer enhancement. The base peaks result from the alpha cleavage involving the

carbon-carbon bond of the ethyl linkage between the aromatic ring and the amine

nitrogen producing the products shown in Scheme I.2.2-1. Furthermore a peak at m/z

135, which corresponds to methylenedioxybenzyl cation, is characterstic for these

compounds.

Unfortunately, the EI technique is often insufficient, alone, to discriminate

between structurally similar phenethylamines. The amine dominated fragmentation

reactions in these compounds often yields low mass fragments of similar mass and little

(if any) molecular ion species. The differentiation of these compounds is only achieved

by means of derivatization and chromatographic methods. Borth et al., reported

regioisomeric differentiation of 2,3- and 3,4-methylenedioxyphenalkylamines by using

collision-induced dissociation (CID) mass spectrometry under EI and chemical ionization

(CI) [Borth et al., 2000].

In a recent study, [Sachs and Woo, 2007] described the use of the low mass

region between mass 39 and 58 for differentiation between methamphetamine and its four

possible regioisomers. All five regioisomeric phenethylamines yield m/z 58 as the imine

base peak therefore the additional two carbons of the side chain are bonded to the

fragment imine species. The reported method [Sachs and Woo, 2007] for differentiation

between the compounds uses m/z 39 as a reference ion and compares its relative

abundance to the ions at m/z 41, m/z 42 and m/z 56. The relative abundance of each of


24

the three ions (m/z 41, 42 and 56) was characteristic for each of the five side chain

regioisomers. The use of the m/z 39 as a pseudo-internal standard was platform

independent and reported to be consistent among numerous published reports on similar

compounds [Sachs and Woo, 2007].

O

OHN

R

O

OHN

R

EI

70ev NH

R

O

O

m/z = 163

O

O

CH2

m/z = 135

R m/z H 44CH3 58C2H5 72C3H7 86

Scheme I.2.2-1: Mass fragments (EI) of 3,4-methylenedioxyamphetamines.

Methamphetmaine was the only one of the five regioisomeric amines which

showed both m/z 41 and m/z 56 in greater abundance than m/z 39. The mechanistic

fragmentation process for the formation of each of these diagnostic low mass ions was

described in detail. However, the disadvantage of the method is the relatively low

intensity of these low mass ions and the lack of a unique and characteristic ion for each of

the regioisomeric side chains (i.e. all five compounds show fragment ions at m/z 39, 41,

42 and 56 and differentiation is based on the relative abundance of these ions).


25

I.2.2.1.2 Nuclear magnetic resonance (NMR)

NMR is a nondestructive flexible technique that can be used for the simultaneous

identification of pure compounds and even mixtures of compounds in one sample. Its

advantages, compared to GC-MS techniques, include stereochemical differentiation and

the capability to analyze nonvolatile compounds. However, the lack of use in forensic

laboratories can be attributed to the high cost of instrumentation and the poor sensitivity

of NMR. The chemical shift data with proton assignments, appropriate proton-proton J-

coupling as well as 13C-NMR shift assignments for MDMA as the free base and

hydrochloride salt in deuterochloroform has been reported [Dal Cason et al., 1997].

Solid state NMR also can be used for analytical purposes in much the same way as

solution NMR. The observed chemical shifts however differ in the solution and solid

states because of conformational freezing and packing effects. Lee et al. reported the

differences in chemical shifts of the solid state NMR and solution NMR for MDMA [Lee

et al., 2000]. This study described the differences between chemical shifts of

MDMAHydrochloric acid in ecstasy tablets and pure crystals.

I.2.2.1.3 Infrared (IR) spectroscopy

The absorption of IR radiation is also considered one of the non-destructive

techniques that can be used for the identification of organic molecules. The region from

1250 to 600 cm-1 is generally classified as the “fingerprint region” and is usually a result

of bending and rotational energy changes of the molecule as a whole. However since the

clandestine samples are usually impure, overlapping absorptions of different molecules


26

present in the sample becomes a possibility. Hence, this region is not useful for

identifying functional groups, but can be useful for determining whether or not samples

are chemically identical. Common absorptions for 3,4-methylenedioxyamphetamines

were reported [Young, 2000]. Because of the polymorphic crystalline structure of the

hydrochloride salt compared to the base form, different infrared spectra were determined

for MDMA hydrochloride alone [Shulgin, 1986]. Near infrared (NIR) spectroscopy

(1100-2500 nm) offers a possibility for fast and nondestructive screening of ecstasy

tablets. Modern NIR equipment is portable and easy to handle so that confiscated samples

can be measured on the spot and in real time. It has been shown that differentiation of

placebo, amphetamine and ecstasy samples is possible [Sonderman and Kovar, 1999].

Infra red detector coupled with GC also had significant application in the

identification of controlled substances in presence of their isomers. Generally isomers

other than optical isomers yield different IR spectra. Diastereomers resulting from two or

more chiral centers also exhibit different infrared spectra [Lanig et al, 2003].

Polymorphism in the solid phase can yield variations in the infrared spectrum of an

individual compound. Analytes in the solid phase can crystallize in different ways,

allowing for different crystal forms and different intermolecular interactions resulting in

significant variability in the IR spectrum of an individual compound [Lanig et al, 2003].

GC-FTIR spectroscopy is characterized by scanning quickly enough to obtain IR spectra

of peaks eluting from the capillary columns. Thus, this technique combines the separation

power of GC with the identification power of IR and is not affected by polymorphism

since the IR spectra are obtained in the vapor phase. GC-IR and MS have been

successfully used for the identification of some amphetamine isomers [Duncan and


27

Soine, 1988]. The GC-IR Spectra of methamphetamine and phentermine have been

previously reported [Kempfert, 1988] along with other non-phenethylamine forensic

compounds.

I.2.2.1.4 Raman spectroscopy

Raman spectroscopy also has been used to study ecstasy tablets [Bell et al., 2000a].

The use of far-red (785 nm) excitation reduces interfering background luminescence and

therefore the level of fluorescence background, even in untreated samples, is sufficiently

low which makes it possible to obtain good quality data in reasonable times. It was

shown that Raman methods can be used to distinguish between ecstasy analogs and the

compounds can be identified even in a mixture with bulking agents in ecstasy tablets. The

spectra can also be used to identify bulking agents, the relative concentration of drug to

bulking agent and the degree of hydration of the active compounds. Bell et al. found that

composition profiling by Raman methods is a fast and effective method of discriminating

between ecstasy tablets manufactured in different ways and with different drug feed

stocks [Bell et al., 2000b]. Recently a surface-enhanced Raman scattering (SERS)

spectroscopy method was developed for a rapid determination of MDMA in illicit

samples [Sagmuller B. et al., 2001].

I.2.2.1.5 Ultra violet (UV) spectroscopy

The 3,4-methylenedioxyphenyl group is a strong chromophore in the UV range

with two major absorption bands at 285 nm and 235 nm range with the absorptivity

slightly higher at 285 nm [Noggle et al., 1987]. Because of their common chromophore, a


28

variety of 3,4-methylenedioxyamphetamines and 3,4-methylenedioxyphenyl-2-

butanamines show similar UV absorption properties and therefore are not discriminated

by UV-spectroscopy [Clark et al., 1995].

I.2.2.2 Chromatography

Chromatography has played a key role in forensic drug separation and identification

over the past 35 years. Gas chromatography (GC) coupled with several detection methods

such as flame ionization (FID), nitrogen phosphorus (NPD), electron capture (EC), and

mass spectrometry (MS) has been used for the analysis of different N-substituted

analogues of 3,4-methylenedioxyamphetamines. The determination of MDMA, MDA

and MDEA in biological samples has mainly been carried out using GS-MS [Clauwaert

et al., 2000; Peters FT et al., 2005]. Actually, GC-MS is considered the method of choice

in forensic laboratories. High-performance liquid chromatography (HPLC) with different

detectors is another often applied technique in the determination of N-substituted analogs

of 3,4-methylenedioxyamphetamines. Most of the literatures describe the identification

and quantitative determination of MDA, MDMA, and MDEA, and their metabolites in

biological samples. Derivatization is mainly required in the analysis of biological

samples to improved sensitivity and selectivity, while such a technique is not usually

applied in the analysis of non-biological samples such as drug forms.


29

I.2.2.2.1 High-performance liquid chromatography (HPLC)

I.2.2.2.1.1 Analysis of biological samples.

The determination of MDA, MDMA and MDEA in biological samples has been

done by HPLC with various detectors, such as ultraviolet (UV), diode array detection

(DAD), fluorescence (FL), electrochemical (ED), mass spectrometry (MS), and mass

spectrometry –mass spectrometry (MS-MS) and these studies are reviewed in Table I.2-1.

Table I.2-1: HPLC methods to separate 3,4-methylenedioxyamphetamines from

biological matrices.

Separated compounds Matrix Column Detector Reference

MDA, MDEA, MDMA, MBDB

Oral fluids Kromasil 100 C8

FL Concheiro et al. 2005

MDA, MDEA, MDMA urine octadecyl C18 FL De Costa JL et al., 2004

R-MDA, S-MDA, R-HME, S-HME, R-MDE, S-MDE

Plasma urine

chiral-CBH FL Buechler J et al. 2003

MDA, MDMA, MDEA, amphetamine, methamphetamine, ephedrine

plasma oral fluids

Hypersil BDS C18

MS-MS Wood M. et al. 2003

MDMA, MDA, amphetamine

Blood, urine, postmortem tissue

Hypersil BDS phenyl

MS Mortier KA et al., 2002

R-MDA, S-MDA, R-MDMA, S-MDMA, R-MDEA, S-MDEA

plasma ChiralDex, LiCrospher 60 RP-select B

FL Brunnenberg and Kovar, 2001

MDA, MDMA, MDEA

blood, vitreous

Hypersil BDS C18

FL Clauwaert et al., 2000


30

humor, urine

MDA, MDEA, MDMA, MBDB, 2-CB and some phenethylamines

serum Superspher 100 RP 18

APCI-MS Bogusz et al., 2000

MDA, MDMA, MDEA

hair PLRP-S FL Tagliaro et al., 1999

amphetamine, methamphetamine, MDA, MDMA, MDEA

urine Silica column (APEX)

UV-visible

Talwar et al., 1999

MDA, MDMA, MDEA, MBDB

urine, serum, saliva

LiChrocart-Li-Crospher 100 RP-18

FL Mancinelli et al., 1999

amphetamine, methamphetamine, MDA, MDMA, MDEA

serum Superspher Select B a ECOcart

APCI-MS, DAD/UV

Bogusz et al., 1997

MDA, MDMA

plasma, urine

Spherisorb ODS-1

DAD Helmlin et al., 1996

MDA, MDMA, MDEA

whole blood Whatman silica Partisphere

ED Michel et al., 1993

I.2.2.2.1.2 Analysis of non-biological samples.

Reversed phase LC is the main technique used for separation and quantitation of the

active substances, usually 3,4-methylenedioxy-amphetamines, in ecstasy tablets. Usually

the column of the choice is conventional C18 or a base-deactivated C18 stationary phase

(Table I.2-2). Mancinelli et al. have developed a HPLC-fluorimetric procedure suitable

for different matrices, dosage forms and biological samples, to determine MDA, MDMA,

MDEA, and MBDB [Mancinelli et al., 1999]. This procedure was carried out in basic


31

isocratic conditions and because of the high pH (11.4) the analytes are non-ionized. The

analysis time is less than 10 min and the first eluting compound is MDA (4.94 min)

followed by MDEA (6.77 min), MBDB (7.53 min) and last eluting one is MDMA (9.50

min).

Sadeghipour and Veuthey also reported the use of fluorimetric detection in

determination of MDA, MDMA, MDEA, and MBDB [Sadeghipour and Veuthey, 1997].

The selectivity of the method was verified not only with common substances, which can

appear in seized tablets, but also with some drugs of abuse such as cocaine, morphine,

amphetamine, methamphetamine, etc. The selectivity of the method is based on that only

methylenedioxylated amphetamines are natively fluorescent and therefore detectable. The

optimal mobile phase condition was determined as 20 mM NaH2PO4 solution (adjusted to

pH 3.8)-acetonitrile (85:15, v/v). Under these conditions, the analytes were ionized and

analysis time was less than 6 min. The elution order was MDA, MDMA, MDEA and the

last eluting compound was MBDB.

In addition, Sadeghipour et al. have developed a reversed-phase LC method with

UV detection for separation and quantitation of five amphetamines (amphetamine,

methamphetamine, MDA, MDMA, and MDEA) and ephedrine in the presence of

adulterants in illicit drugs [Sadeghipour et al., 1997]. The comparison of a regular

reversed phase column (RP18 Nucleosil 100) and a base-deactivated column (RP18-AB

Nucleosil 100) was demonstrated and as a result, the base-deactivated phase gave higher

efficiency and lower peak asymmetry and capacity factors which allowed a rapid

separation of amphetamines with good resolution. The mobile phase composition was

optimized by studying the influence of pH, buffer composition and the organic solvent


32

type. The best results were obtained with acetonitrile concentrations between 7 and 10%

and with phosphate buffer (pH adjusted to between 3.4 and 3.8).

The reversed phase separation of N-alkyl MDAs has been achieved on a C18 stationary

phase and a ternary mobile phase [Noggle et al., 1987 and 1988]. The ternary mobile

phase consisted of pH 3 phosphate buffer, acetonitrile, and methanol containing

triethylamine. The use of triethylamine as a competing base (silanophile) was necessary

on Bondapak C18 stationary phase to prevent peak tailing.

Table I.2-2: HPLC methods to separate 3,4-methylenedioxyamphetamines from

non-biological matrices.

Separated compounds Column Detector Reference


LiChrocart-LiCrospher 100 RP-18

FL Mancinelli et al., 1999

MDA, MDMA; MDEA; MBDB

FL Sadeghipour and Veuthey, 1997

MDA, MDMA, MDEA, and other phenethylamines

RP18 Nucleosil 100, RP18-AB Nucleosil 100

FL Sadeghipour et al., 1997

MDEA, MDMMA, MBDB, MDP-3-MB

Bondclone C18 UV Clark et al., 1996

MDA, MDMA, MDEA, MDMMA

Bondclone C18 UV Clark et al., 1995a

BDB, MBDB, MDP-2-EB, MDP-2-MMB, MDP-2-OHB


MDP-3-B, MDP-3-MB MDP-3-EB, MDP-3-MMB, MDP-3-OHB


MDMA, BDB, MDP-3-B



33

MDA, MDMA, NOHMDA

Nucleosil C18 UV Valaer et al., 1990

MDA, MDMA, NOHMDA

Deltabond C8 UV Valaer et al., 1990

MDA, MDMA, MDMMA, MDEA, N-isopropyl MDA, N-n-propyl MDA, NOHMDA

Bondapak C18 UV Noggle et al., 1988

MDA, MDMA, MDEA, N-isopropyl MDA, N-n-propyl MDA, N-isobutyl MDA, N-n-butyl MDA

Bondapak C18 UV Noggle et al., 1987

The elution order of the N-alkyl MDAs is based on the relative lipophilicity of the

derivatives and thus retention increases with the size of the alkyl chain on the nitrogen.

The N,N-dimethyl MDA (MDMMA) elutes before N-ethyl MDA (MDEA), and the

branched isopropyl derivative elutes before N-n-propyl MDA [Noggle et al., 1988].

Under these conditions the N-hydroxy MDA (NOHMDA) has the highest retention (25

min) which has been explained by higher polarity and lower basicity of the compound

compared to the other N-alkyl MDAs. The pKa value for NOHMDA has been determined

by titration to be 6.22, which is much lower than the pKa values of other N-alkyl MDAs

(cirka 10) [Valaer et al., 1990]. The separation of NOHMDA from MDA and MDMA

was improved by using a Deltabond C8 stationary phase which enabled the separation

without the need for competing base and in a shorter analysis time (10 min) [Valaer et al.,

1990].

Clark et al. reported the separation studies of N-substituted MDAs, N-substituted-2-

butanamines and N-substituted-3-butanamines [Clark et al., 1995a]. The separation of the

compounds was accomplished using a C18 stationary phase (Bondclone C18) with an


34

acidic mobile phase. The elution order under these conditions was according to the size

of the N-substituent. The N-substituted MDAs eluted in the same order as previously;

MDA (5.08 min), MDMA (5.87 min) and MDEA (6.68 min). The elution order of N-

substituted-2-butanamines and N-substituted-3-butanamines was similar. The three-

carbon side chain propanamines (MDAs) had lower capacity factors than the 2-and 3-

butanamines when comparing compounds with identical N-substituents. In addition, 3-

butanamines display higher capacity factors than the 2-butanamines of the same N-

substituent in every case. When compounds with the same molecular weight as MDMA

were compared, it was found that MDMA eluted first, followed by BDB and the last

compound to elute was MDP-3-B [Clark et al., 1995a]. The separation of compounds

with the same molecular weight as MDEA was also studied under the same conditions

and the results are shown in Table I.2-3 [Clark et al., 1996].

Table I.2-3: Reversed phased LC separation of MDEA and its regioisomers.

Compound Rt (min)

MDMMA 7.93

MDEA 8.77

MBDB 10.91

MDP-3-MB 12.60


35

I.2.2.2.2 Gas chromatography (GC)

I.2.2.2.2.1 Analysis of biological samples.

Publications on GC procedures with different detectors used for the determination

of 3,4-methylenedioxyamphetamines from biological samples are reviewed in Table I.2-

4. A mass spectrometer is the most specific detector for drug testing. Several manuscripts

describe drug testing using GC with less specific detectors. For example Drummer et al.

identified amphetamine, methamphetamine, MDMA, MDA, and other drugs of forensic

interest in blood using GC-NPD [Drummer et al., 1994]. In addition, the analysis has

been performed by a dual channel GC combined with a NP and an EC detector [Lillsunde

et al., 1996]. Ortuño et al. stated that when analyzing plasma samples with GC-NPD,

good chromatographic separation and adequate sensitivity of underivatized compounds

can be achieved but the same approach is not applicable for urine [Ortuño et al., 1999].

When GC-MS is used for screening blood samples, the selected ion monitoring (SIM)

mode is often applied [Marquet et al., 1997].

Table I.2-4: GC procedures for the identification and/or quantification of 3,4-

methylene-dioxy amphetamines from biological samples.

Separated compounds Matrix Column Detector Reference

Enantiomers of MDEA, MDMA, MDA

blood HP5-MS MS(EI) Peters FT et al., 2005

Enantiomers ofMDA, MDMA, MDEA, amphetamine, methamphetamine

urine MS(EI) Paul BD et al., 2004

MDMA, MDEA hair DB5-MS MS (SIM) Girord and Staub, 2000


serum DB5-MS MS (SIM) Weinman et al., 2000


36

MDMA and its metabolites (MDA, HMMA, HMA)

plasma, urine

Ultra-2 NPD Ortuño et al., 1999

R and S-MDMA and its chiral metabolites (MDA, HMMA, HMA)

urine DB5-MS MS (EI, PCI) deBoer et al., 1997

MDA, MDMA, MDEA, amphetamine, methamphetamine

blood HP5-MS MS (EI, SIM)

Marquet et al., 1997

MDA, MDMA, MDEA, amphetamine, methamphetamine

urine SPB-5 MS (EI, CI, SIM)

Dallakian et al., 1996


urine FSC HP-5 MS (EI, SIM)

Kronstrand, 1996

MDA, MDMA, amphetamine, methamphetamine, etc.

blood FSC HP-5 NPD, ECD Lillsunde et al., 1996

MDA, MDMA, MDEA, BDB, MBDB

urine HP1 MS (SIM) Maurer, 1996

MDA, MDMA, amphetamine, methamphetamine

blood FSC BP-5 NPD Drummer et al., 1994

MDA, MDMA, MDEA urine FSC DP-5 MS (CI, SIM)

Lim et al., 1992

I.2.2.2.2.2 Analysis of non-biological samples.

O’Connell and Heffron established a GC-MS procedure to determine the principal

amphetamines, MDMA, MDEA, MDA, cocaine and pharmacologically active impurities

in ecstasy tablets [O’Connell and Heffron, 2000]. The tablets were ground into powders

and dissolved in ethanol. The column used was a HP-1 fused-silica capillary column

column (60 m x 0.25 mm id) with a 1 m film thickness of methylsilicone.

Lillsunde and Korte have reported a method for analyzing 12 ring and N-substituted

amphetamine-derivatives in body fluids or seized materials by GC combined either with


37

MS, EC or NP detector [Lillsunde and Korte, 1991]. GC-MS was used for identification

with a packed column, 2% SP-2110 / 1% SP-2510. GC-ECD and GC-NPD was used for

quantitation with a fused silica capillary column, SE-54. Derivatization was done with

heptafluorobutyric anhydride and then most of the 12 amphetamines examined were

separated and their retention times are listed in Table I.2-5. Only MDEA and 2,5-

dimethoxy-4-ethylamphetamine as well as 3,4,5-trimethoxyamphetamine and 3-methoxy-

4,5-methylenedioxyamphetamine co-eluted. On the other hand, the mass spectra are

characteristic and can be used to distinguish these compounds from each other.

Table I.2-5: Retention times (min) of HFBA derivatives of amphetamines on packed

and capillary columns [Lillsunde and Korte, 1991].

Compound (HFBA-derivative) Rt

(packed column) Rt

(capillary column)

amphetamine 3.3 2.74

methamphetamine 3.8 3.61

N-ethylamphetamine 4.1 3.95

4-methoxyamphetamine 5.8 4.77

MDA 7.5 5.77

2,5-dimethoxyamphetamine 6.9 6.24

MDMA 7.5 6.87

2,5-dimethoxy-4-ethylamphetamine 7.8 7.23

MDEA 7.8 7.25

3,4,5-trimethoxyamphetamine 8.8 7.76

3-methoxy-4,5-methylenedioxyamphetamine 8.8 7.77

4-bromo-2,5-dimethoxyamphetamine 10.2 8.70


38

Gas chromatographic separation of MBDB, MDEA, N,N-dimethyl-3,4-

methylenedioxy-amphetamine (MDMMA) and N-methyl-1-(3,4-methylenedioxyphenyl)-

3-butanamine (HMDMA) has been achieved on a 12 m x 0.20 mm id methylsilicone

column (HP-1) [Noggle et al., 1995]. These compounds have the same molecular weight

and similar retention properties (Table I.2-6). HMDMA has the highest retention time

and the other three compounds elute over approximately 0.25 minutes with MDEA and

MDMMA eluting before MBDB. In this system compounds having the C3 carbon side

chain attached to the aromatic ring elute before the two aryl-C4 butanamines.

The use of mass spectrometry as a detector does not provide significant data for

differentiation among MDEA, MBDB and MDMMA since these regioisomeric

compounds also yield regioisomeric fragment ions of equal mass. HMDMA can be easily

distinguished from the other three compounds because the difference in mass spectrum

produced by substitution of the methylamino group at the 3-position of the butanamine

side chain. Pentafluoropropionylamide (PFPA) derivativatization of MDEA, HMDMA,

and MBDB, was used to improve mass spectrometric differentiation [Clark et al., 1996].

The GC analysis of the three PFPA derivatives showed slightly higher retention for

derivatized compounds and they eluted in the same order as underivatized compounds.

Tertiary amines such as MDMMA do not form stable PFPA-derivatives and therefore it

can be easily identified since the mass spectrum remains unchanged after acylation with

PFPA.


39

Table I.2-6: GC separation of MDEA, MDMMA, MBDB and HMDMA.

Compound Structure Rt

MDEA HN

O

O

7.23

MDMMA N

O

O

7.35

MBDB O

OHN

7.50

HMDMA O

O

N

H

7.68

I.2.3 General Problems for Identification and Separation.

Regioisomerism at the aromatic ring and the alkyl side-chain of the

methylenedioxyalkylamines produces a variety of compounds that have very similar

analytical properties. The methylenedioxy ring can be fused to the aromatic in a 2,3- or

3,4-pattern, yielding regiosiomerism of the aromatic portion of the molecule.

Identification of 2,3-methylenedioxy-phenalkylamines is of importance to forensic

chemists, although those compounds are not as likely to appear on the clandestine

market. Alkyl side-chain regioisomerism is most significant when imine fragments of

equivalent mass and similar abundance appear in the mass spectra of these compounds.

Some of the alkyl side-chain regioisomers can be differentiated by derivatization [Clark

et al., 1995b], but for example 2,3- and 3,4-methylenedioxyphenalkyl-amines cannot be

differentiated by derivatization [DeRuiter et al., 1998].


40

Further more there are other compounds that do not necessarily contain the

methylenedioxy ring, however they constitute indirect regioisomeric and isobaric

substances related to the drug of abuse 3,4-MDMA. These substances have the same

molecular weight and are capable of producing mass spectra similar to 3,4-MDMA. The

co-elution of one or more of these substances with MDMA remains a possibility.

Additionally the lack of reference materials for these substances complicates the

identification procedure.

I.2.3.1 Differentiation of regioisomeric 2,3- and 3,4-methylenedioxy phenalkyl

amines and isobaric substances related to MDMA and MDEA by

chromatographic methods.

The analytical properties of the 2,3-MDAs, such as 2,3-MDA, 2,3-MDMA, 2,3-

MDEA, and 2,3-MDMMA, has been compared to the corresponding 3,4-MDAs [Casale

et al., 1995]. The EI mass spectra of 2,3- and 3,4-MDAs showed fragments of the same

mass with only slight differences in relative intensity. The distinguishable difference is

the relative abundance of ions at m/z 135 and m/z 136. Such differences cannot be

considered significant from the analytical point of view, in particular when the analytes

must be detected in a chromatographic run and the mass spectra of the compounds of

interest show interferences from co-chromatographing substances. Gas chromatographic

studies of the regioisomers on the methylsilicone stationary phase showed that all four

2,3-MDAs were easily resolved from its respective 3,4-regiosiomers and their retention

times were significantly less than the corresponding 3,4-substituted MDA. Naturally, the

regioisomeric MDAs could each be differentiated by proton NMR via variances in both

chemical shifts and overall peak patterns. However, NMR is not a technique, which is


41

commonly used in the forensic laboratories and therefore the differentiation has usually

depended on chromatographic and mass spectrometric methods.

DeRuiter et al. studied liquid chromatographic and mass spectral methods to

differentiate 2,3- and 3,4-methylenedioxyphenyl ring substitution regioisomers of

MDMA, BDB, MDEA, and MBDB [DeRuiter et al., 1998]. The derivatization of the

side-chain regioisomers to pentafluoropropionylamide (PFPA) derivatives enabled

differentiation by mass spectroscopy. The fragmentation of the bond between the alkyl

side-chain and the nitrogen in the PFPA-derivatized amines yielded prominent ions that

identified the nature of the hydrocarbon chain attached directly to the aromatic ring. The

reversed-phase LC system consisting of a C18 stationary phase (Hypersil Elite C18) and a

mobile phase of 30% methanol in pH 3 phosphate buffer gave an excellent separation of

regioisomeric MDMA and BDBs. The first eluting compound was 3,4-MDMA followed

by 2,3-MDMA and then 3,4-BDB and last eluting compound was 2,3-BDB. A similar

elution order was obtained when regioisomeric MDEA and MBDBs were separated on

the same stationary phase but by using a different mobile phase composition. The

optimum isocratic separation was achieved when the mobile phase was 10% acetonitrile

in pH 3 phosphate buffer. The two compounds with the C3 alkyl chain attached to the

aromatic ring eluted first; the 3,4-MDEA eluted before the 2,3-MDEA, and the two

compounds with C4 alkyl group showed greater retention. The observed elution order was

the same; first are eluting compounds with shorter alkyl chains attached to the aromatic

ring and secondly, the 3,4-regioisomers elute before the 2,3-regioisomers, for several C18

stationary phases in both methanol- and acetonitrile-modified acidic aqueous systems. In

addition, four regioisomers of 3,4-MDMA and 3,4-MDEA were analyzed by GC on a


42

methylsilicone stationary phase under temperature-programmed conditions. 3,4-MDMA

and its regioisomers eluted over a 0.6 min time window from 6.7 to 7.3 min and 3,4-

MDEA and its regioisomers produced an elution range of 0.5min (7.0 to 7.5 min). The

elution order of the compounds was not mentioned.

Aalberg et al. reported the use of Dry Lab software for the separation of direct

and indirect regioisomers related to the drug of abuse 3,4-MDMA and 3,4 MDEA using

LC. The Dry lab software uses initial four runs under identical conditions using two

different gradient times and two different temperatures. By entering the collected

retention data along with column dimensions, mobile phase composition, flow rate and

column efficiency, the software will generate three dimensional resolution map and

suggest the best conditions for separating the desired compounds in a physical mixture.

Aalberg reported the 10 direct side chain and ring regioisimers of MDMA

including N-ethyl-3,4-methylenedioyphenylethanamine, N,N-dimethyl-3,4-methylene-

dioxyphenethanamine, 3,4-methylenedioxyphetramine, 3,4-methylenedioxyBDB and

their 2,3- ring regioisomers. The HPLC study showed the rentention time increase with

side chain length of the uninterrupted hydrocarbon attached to the aromatic ring. Base

line separation of these compounds was carried out on an XTerra column at 40C with tG

of 90 min (initial 3% methanol, final 30% methanol in pH 3 phosphate buffer). On

Supelcosil ABZ+Plus column a better sepration was obtained at 41C with a tG of 230

min (initial 3% methanol, final 40% methanol in pH 3 phosphate buffer). Dry lab helped

in determining the optimum isocratic separation of these compounds on a Supelcosil

ABZ+Plus with 2% methanol and pH 3 phosphate buffer at 45C [Aalberg et al. 2003].


43

The indirect regioisomers of 3,4-MDMA included in the Aalberg study were p-

ethoxymethamphetamine, 1-p-ethoxyphenyl)-2-butanamine, 4-methoxy-3-methylmeth-

amphetamine, 1-(3-methyl-4-methoxyphenyl)-2-butanamine, ,-dimethyl-1-(3-methyl-

4-methoxyphenyl)-2-ethanamine, N-methyl-1-(2-methoxyphenyl)-1-methyl-2-propan-

amine, N-methyl-1-(3-methoxyphenyl)-1-methyl-2-propanamine, N-methyl-1-(4-

methoxyphenyl)-1-methyl-2-propanamine and p-methoxymethcathinone. The sepration

of these substances was only achieved on Supelcosil ABZ+Plus and XTerra columns,

when acetonitrile was used as an organic modifier in the mobile phase. Separations

conditions were gradient mobile phase with a gradient time of 20.90 min at 40C, starting

from 14% and ending at 26% acetonitrile in pH 3 phosphate buffer. It was very hard to

get a base line separation for both N-methyl-1-(3-methoxyphenyl)-1-methyl-2-

propanamine and N-methyl-1-(4-methoxyphenyl)-1-methyl-2-propanamine because of

the similarity of their retention properties. The Supelcosil ABZ+Plus offered the best

resolution of all the 19 compounds with the gradient mobile phase (initial mobile phase

consisted of 3% acetonitrile and pH 3 phosphate buffer, and the final composition, 9.6%

acetonitrile, was reached in 35 min) at room temperature [Aalberg et al. 2003].

Aalberg et al. also reported gas chromatographic separations for the above

described substances. For the 2,3- and 3,4- regioisomers directly related to the drug of

abuse MDMA, optimum separation was obtained using a 35% phenylmethylsilicone

phase (DB35MS) and temperature programming of 1800C as intial temperature and an

increase in rate of 0.3 0C/ minute. These conditions were determined by the retention

modeling software, Dry Lab [Aalberg et al., 2004].


44

Other indirect ring and side chain regioisomers and isobaric substances related to

MDMA were separated on non polar Ultra 2 column (25 m, 0.2 mm, 0.33m) with a

temperature program rate of 7.3C/min. The analysis time was decreased on a narrow

pore column (Hp-5) by optimizing the temperature program through segmented

temperature ramp using Dry Lab software. A base line separation of all the 19 direct,

indirect and isobaric substances related to MDMA was not obtained in the study [Aalberg

et al., 2004].

I.2.3.2 Differentiation of regioisomeric 2,3- and 3,4-methylenedioxyphen-

alkylamines and isobaric substances by mass spectroscopic methods.

The underivatized methylenedioxyphenalkylamines give virtually identical EI mass

spectra containing mainly intense immonium ions as mentioned earlier. The

differentiation of some methylenedioxyphenalkylamines can be achieved by means of

derivatization using various chromatographic methods. The use of tandem mass

spectrometry (MS-MS) is reported to give additional information contained in the

collision induced dissociation (CID) mass spectra of molecular ions using EI and

especially methane CI [Borth et al., 2000a]. CID mass spectra are obtained by parent ions

colliding with an inert gas during the passage through the reaction chamber of a tandem

mass spectrometer.

In their study Borth et al. were able to differentiate 18 regioisomeric methylene-

dioxyphenyl-2-propanamines and methylenedioxyphenyl-2-butanamines (Figure I.2-2).

The mixture of compounds was analyzed by GC on a methylsilicone stationary phase and


45

an insufficient separation was obtained. Several compounds (3a and 1c or 4a, 2c and 3b

or 4b, 3c and 3d or 4c and 4d) co-eluted.

In general, the molecular ion CID mass spectra using EI were distinct, except in the

case of compounds 4e and 4f the molecular ions did not have sufficient intensity for

recording daughter ion mass spectra. The EI-CID mass spectra of all 2,3-

methylenedioxyphenethylamines derivatives (1a-d and 3a-d) are dominated by

immonium base peak ions with the general formula [CnH2n+nN]+ (m/z 44, 58, 72, etc.)

resulting from an -cleavage reaction. In contrast the EI-CID mass spectra of 3,4-

methylenedioxy isomeric compounds show significant or base peaks ions at m/z 136.

This signal was explained to be due to a rearrangement of nitrogen H atoms to the

aromatic ortho position eliminating an imine or by a specific six-center H-rearrangement

of a -H-atom of the alkyl side chain to the aromatic ring eliminating a neutral enamine

[Borth et al., 2000a].

The CI-CID spectra of all ring substituted regioisomers generate a very intense or

base peak ion at m/z 135 via cleavage of the benzyl bond by the charge of ammonium

cation [Borth et al., 2000b]. The 2,3-methylenedioxy isomeric compounds formed a

significant [C7H7O2]+ ion at m/z 123 with the mass of a protonated

methylenedioxybenzene. The 3,4-methylenedioxy ring-substituted compounds do not

show this ion to a significant extent, but they show a significant homologous [C8H9O2]+

ion at m/z 137 by a formally benzylic bond cleavage. Therefore, the study of Borth et al

using CI-CID mass spectrometry suggests that, the ion at m/z 123 indicates the 2,3-ring-

substituted phenethylamine isomers and the ion at m/z 137 the 3,4-ring-substituted

isomers.


46

NR2R1

OO

NR2R1

O

O

a: R1 = H; R2 = H a: R1 = H; R2 = H

b: R1 = H; R2 = CH3 b: R1 = H; R2 = CH3

c: R1 = H; R2 = C2H5 c: R1 = H; R2 = C2H5

d: R1 = CH3; R2 = CH3 d: R1 = CH3; R2 = CH3

1 2

NR2R1

OO

NR2R1

O

O

a: R1 = H; R2 = H a: R1 = H; R2 = H

b: R1 = H; R2 = CH3 b: R1 = H; R2 = CH3

c: R1 = H; R2 = C2H5 c: R1 = H; R2 = C2H5

d: R1 = CH3; R2 = CH3 d: R1 = CH3; R2 = CH3

e: R1 = H; R2 = n-C3H7

f: R1 = H; R2 = i-C3H7

3 4

Figure I.2-2: Chemical structures of 18 regioisomeric methylenedioxyphenyl-2-

propanamines and methylenedioxyphenyl-2-butanamines [Borth et

al., 2000a].


47

I.3 Statement of Hypothesis or Rationale for the Research

Analytical specificity in forensic drug analysis requires knowledge of all possible

isomers in order to identify an individual compound and eliminate all other possibilities.

Knowledge of the properties of all the possible regioisomeric/isobaric substances is

essential in order to specifically identify any one of these compounds. The precursor

chemicals are available to synthesize any of these compounds and several have already

appeared in clandestine drug samples. It is perhaps a matter of time until one appears

which has GC retention properties and MS equivalence to MDMA or MBDB. The goal

of this work is to have the data and methods for differentiation available to prevent

misidentification at that future time.


48

II. Methods (Narrative Descriptions of the Experimental Design)

II.1 Synthesis Design

Synthetic Methods Not Available


49



50



51



52



53



54



55



56




57


II.2 Methods (Synthetic materials):



58

II.3 Analytical Methods II.3.1 Instruments

GC-MS analysis was performed on two diggerent GC-MS. the first GC-MS is an

HP-5890 GC coupled with a HP-5970 mass selective detector (Hewlett Packard, Palo

Alto , CA) using Helium (grade 5.0) as carrier gas. The mass spectrometer was operated

on the electron impact (EI) mode using ionization voltage of 70 ev and a source

temperature of 230 oC [Instrument 1]. Samples were dissolved in HPLC grade

acetonitrile (Fisher Scientific NJ, USA) and manually introduced (1 µL), individually and

in a physical mixture using a 10µL Hamilton syringe (Hamilton Co., Reno Nevada,

USA).

The Second GC-MS is GC–MS analysis was performed using an Agilent

Technologies (Santa Clara, CA) 7890A gas chromatograph and an Agilent 7683B auto

injector coupled with a 5975C VL Agilent mass selective detector. The mass spectral

scan rate was 2.86 scans/s. The GC was operated in splitless mode with a helium (grade

5) flow rate of 0.7 mL/min and the column head pressure was 10 psi (68.946 kPa). The


59

MS was operated in the electron impact (EI) mode using an ionization voltage of 70 eV

and a source temperature of 230 8C. The GC injector was maintained at 250 8C and the

transfer line at 280 0C [ Instrument 2]

GC–IRD studies were carried out on a Hewlett-Packard 5890 Series II gas

chromatograph and a Hewlett-Packard 7673 auto injector coupled with a Hewlett-

Packard 5965B infrared detector obtained from Analytical Solutions and Providers,

Covington, Kentucky. The vapor phase infrared detector (IRD) spectra were recorded in

the range of 4000–550 cm_1 with a resolution of 8 cm_1 and a scan rate 1.5 scans/s. The

IRD flow cell temperature as well as the transfer line was 280 8C and the GC was

operated in the splitless mode with a carrier gas (helium grade 5) flow rate of0.7 mL/min

and a column head pressure of 10 psi[ Instrument 3].

H1NMR for the identification of raction intermediates were collected for samples

dissolved in deuterated chloroform using a Brucker Avance 250 (250MHz) instrument

with the chemical shifts being reported as ppm downfield from tetramethylsilane

[Instrument 4]

II.3.2 GC- Columns

Different capillary GC columns were evaluated throughout the course of this

work, however only columns showed best compromises between resolution and

analysis time are illustrated in Table II.1. All columns used were purchased from

Restek Corporation (Bellefonte PA, USA) and have the same dimensions, 30m x

0.25mm-I.d. column coated (fd) with o.25 µm. Inlet pressure was converted

according to the constant flow mode and the total flow was 60 ml/min. The


60

injection was in the split mode with an injector temperature at 250C except for

Rt-βDEXcst-TM, the injector temperature was adjusted to 200C as

recommended by the manufacturer.

Table II.1: List of columns used and their compostion.

Column Name Column Composition

Rtx-1 100% Dimethyl polysiloxane

Rtx-5 95% dimethyl-5%diphenyl polysiloxane

Rtx-35 65% dimethyl-35%diphenyl polysiloxane

Rxi-50 50% phenyl to 50% methyl polysiloxane

Rtx-200 trifluoropropyl methyl polysiloxane

Rt-βDEXcst-TM 14% cyanopropyl phenyl – 86 %

dimethylpolysiloxane


61

III. Results During the course of this project we have prepared a large number of ring and side-chain

substituted phenethylamines and compared and evaluated the analytical properties of

these compounds. This work has included the 5 side chain isomers of 3,4-

methylenedioxy ring system yielding the m/z 58 base peak in the EI-mass spectrum (3,4-

MDMA and the 4 side chain isomers as well as the equivalent 2,3-methylenedioxy series

of compounds. Additional studies have characterized the 2-, 3-, and 4-methoxymeth-

cathinones which can be considered indirect isomers since these compounds have the

same elemental composition as the MDMA series.

A number of individual synthetic and analytical studies have evaluated the 2-, 3-, and 4-

ethoxyphenethylamines as well as the 10 different ring substitution patterns for the

methoxy-methyl-phenethylamines. Furthermore the 2-, 3-, and 4-methoxy-alpha-methyl-

methamphetamines were prepared and evaluated as well.

The methamphetamine side-chain isomer of every ring substitution pattern was prepared

and evaluated in this study.

A series of all ten of the MBDB (m/z 72) side chain isomers was prepared and evaluated

as well as of the more common side chains for several other ring substitution patterns.

Well over 90 compounds were evaluated in this project which allowed us to confirm

specific structure property relationships for differentiation among most of these isomers.

The next chapters provide the details of specific studies as an example of each category

of compounds evaluated in this project.


62

Chapter 1 Mass Spectrometry and Gas Chromatographic Studies on Direct

Regioisomers to 3,4-MDMA.

There are nine other methylenedioxy-substituted phenethylamines with the

potential to produce a mass spectrum essentially the same as 3,4-MDMA, five of them

being 2,3-methylenedioxy ring substituted regioisomers.

Mass spectral studies of direct regioisomers of 3,4-MDMA (methylenedioxyphenyl substituted side-chain regioisomers).

The mass spectra of the phenethylamine drugs of abuse including 3,4-MDMA are

characterized by a base peak formed by an -cleavage reaction involving the carbon-

carbon bond of the ethyl linkage between the aromatic ring and the amine. In 3,4-MDMA

(MW=193) the -cleavage reaction yields the 3,4-methylenedioxybenzyl fragment at

mass 135/136 (for the cation and the radical cation, respectively) and the substituted

imine fragment at m/z 58. Thus, the mass spectrum for 3,4-MDMA contains major ions

at m/z 58 and 135/136 as well as other ions of low relative abundance.

There are nine other methylenedioxy substituted regioisomers (a total of ten

compounds) of the 3,4-MDMA molecule (MW=193) which yield -cleavage fragments

at m/z 58 and 135/136 during analysis by mass spectrometry (Scheme 1.1). The mass

spectra in Figure 1.1 are for the ten possible direct regioisomers of the MDMA molecule.

The first five side chain regioisomers show the methylenedioxy-group fused to the

aromatic ring in a 3,4-manner while in compounds 6 through10 the substitution is in the

2,3-manner. All compounds show the expected fragments (m/z 58 and 135), and in


63

addition, most of the compounds show the molecular ion at m/z 193. In a direct

comparison of the 3,4-regioisomers versus the 2,3-regioisomers with the identical side

chain, the major difference is the greater relative abundance of the radical cation at m/z

136 for the 3,4-substitution pattern in most cases.

Clark et al [1996, 1998] and Aalberg et al. [2000, 2003] in previous work have

described the analytical properties of these unique direct regioisomeric equivalences to

the drug of abuse 3,4-methylenedioxymethamphetamine. These results illustrated that the

mass spectrum alone cannot be used to identify an individual compound within this group

to the exclusion of all others. Their observations [Aalberg et al., 2000 and 2004]

illustrated that 3,4-MDMA (3) and N-ethyl-2,3-methylenedioxyphenyl-2-ethanamine (7)

co-eluted using some common gas chromatographic stationary phases and conditions.

However, additional studies [Aalberg et al., 2004] have identified capillary gas

chromatographic phases and conditions for the complete resolution of compounds 1-10.

Optimum separation was obtained using a 35% phenylmethylsilicone phase (DB35MS)

and temperature programming conditions determined by retention modeling software

(Dry Lab).

While these studies have shown that all ten compounds can be resolved, the lack

of mass spectral specificity makes the specific identification of MDMA (with the

exclusion of all other regioisomers) a significant challenge. The lack of available

reference samples for all ten of these regioisomeric molecules further complicates the

individual identification of any one of these substances. When other compounds exist

with the potential to produce the same or nearly identical mass spectrum as the drug of

interest, the identification by gas chromatography-mass spectrometry (GC-MS) must be


64

based primarily upon the ability of the chromatographic system to separate the

“counterfeit substance” from the actual drug of interest. If not, those substances co-

eluting with the target drug in chromatographic systems could be misidentified as the

target drug. Without the appropriate standards a thorough method validation is not

possible, and thus co-elution of drug and non-drug combinations would remain a

possibility. Furthermore, the ability to distinguish between these regioisomers directly

enhances the specificity of the analysis for the target drugs of interest

O

O

H2C C N

C2H8

O

O

CH2

C N

C2H8

m/z = 135

m/z = 58

70ev

MW = 193

Scheme 1.1: General mass spectral fragmentation for the methylenedioxy regioisomers

of MDMA (compounds 1-10).


65

Figure 1.1: Structures and mass spectra of methylenedioxy substituted side-chain

regioisomers.

Structure of regioisomers Mass spectrum

N

O

O

1

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

m/z-->

Abundance

Average of 10.543 to 10.576 min.: 00050920.D (-)58

135775142 88 17810565 193117 163148

NH

O

O

2

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

5000

10000

15000

20000

25000

30000

35000

m/z-->

Abundance


136

7751 9165 19339 105 149119 163 176

O

O NH

CH3 3

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

m/z-->

Abundance


7751 13591 19365 14939 105 119 163 176


66

O

O NH2

4

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

20000

40000

60000

80000

100000

120000

140000

160000

m/z-->

Abundance


42 7751 1359165 193147119105 177162

O

O NH2

5

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

5000

10000

15000

20000

25000

30000

m/z-->

Abundance


136

7741 51164106 19365 91 121 147 176

N

OO

6

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000

m/z-->

Abundance


77 1355142 1788810565 117 147 161

NH

OO

7

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

10000

20000

30000

40000

50000

60000

70000

80000

90000

m/z-->

Abundance


1357751

8842 10565 178148 162 19111895


67

NH

CH3

OO

8

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

m/z-->

Abundance


77 1355142 89 105 17865 192162118 14895

NH2

OO

9

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

5000

10000

15000

20000

25000

m/z-->

Abundance


42 77 9151 65 193148135105 119 160

NH2

OO

10 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

0

2000

4000

6000

8000

10000

12000

14000

16000

m/z-->

Abundance


7741 13551 16419310565 91 116 147

Mass spectral studies of perfluroacyl derivatives of 3,4- MDMA and its direct regioisomers

The perfluoroacylated derivatives of the eight primary and secondary amines

(Compounds 2-5 and 7-10) were prepared and evaluated for their ability to individualize

the GC-MS properties of the compounds in this uniquely regioisomeric series and to

maintain or improve chromatographic resolution. Of course, the two tertiary amines,

compounds 1 and 6, would not form stable amide derivative.


68

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

m/z-->

AbundanceScan 530 (9.772 min): 110903-1.D

42

51

77

91

119

135

148

160

176

204

220 244252 272 294 309 324

339

Acylation of the amines significantly lowers the basisty of nitrogen and can allow

other fragmentation pathways to play a more prominent role in the mass spectrum [F. W.

McLafferty et al, 1993]. The mass spectra for the eight pentaflouropropionyl and

heptflourobutryl amides are shown in Figures 1.2 and 1.3, respectively

From these spectra a common peak occurs at m/z 204 and 254 which corresponds

to the loss of 135 mass units from the molecular ions at 339 and 389 for PFPA and HFBA

amides, respectively.

Figure 1.2: Mass Spectra of the PFPA derivatives of compounds (2-5) and (7-10).

O

O

N C2F5

O

a


69

O

O

N C2F5

O

b

O

O

HN C2F5

O

c

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

500000

1000000

1500000

2000000

m/z-->


42

51

77

91

119

135

147

162

176

204

220 241 259269282 300 324

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

20000

40000

60000

80000

100000

120000

140000

m/z-->


41

59

77

91

119

135

145

164

176

204

324

339


70

O

O

NH

C2F5O

d

O

O

N C2F5

O

e

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

m/z-->


41

51

77

89

119

135

147161

176

204

220 292310

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

20000

40000

60000

80000

100000

120000

140000

160000

180000

m/z-->


4151

77

91119

135

148

160

176

204

220

339


71

O

O

N C2F5

O

f

O

O

HN C2F5

O

g

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

50000

100000

150000

200000

250000

m/z-->


42

51 7791

119 135

147

162

177

204

220

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

m/z-->


41

59

77

91

119

135

146

164

176

204

324

339


72

O

O

HN C2F5

O

h

Figure 1.3: Mass Spectra for the HFBA derivatives of compounds (2-5) and (7-

10).

O

O

N c3F7

O

a

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

2000

4000

6000

8000

10000

12000

14000

16000

18000

m/z-->


41

5179

105

135

161

176

204

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

m/z-->


42

6977

105119

148

169

178197

226

254

279 302 322 360374

389


73

O

O

N C3F7

O

b

O

O

HN C3F7

O

c

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

m/z-->


42

6977

105

131

135

162

192

210

220

254

263 291 310 332 350 374

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

70000

75000

80000

85000

m/z-->


41

59

77

105119

135

169

176

214

254

374

389


74

O

O

NH

C3F7O

d

O

O

N C3F7

O

e

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

m/z-->


41

69

77

105 131

135

161

176

214

226

254

296302 330360361

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

m/z-->


42

6977

105119

148

169

178197

226

254

279 302 322 360374

389


75

O

O

N C3F7

O f

O

O

HN C3F7

O

g

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

m/z-->


42

6977

105

119

135

162

192

210

220

254

263 291 313 339350 374

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

m/z-->


42

59

77

105119

135

169

176

214

226

254

277 295305320 350374

389


76

O

O

HN C3F7

O h

These ions at m/z 204 and 254 is the PFPA and HFBA imine species likely

formed from the alpha cleavage of the amide nitrogen to eliminate the 2, 3- and 3, 4-

methylenedioxybenzyl radical, thus the m/z 204 and 254 ions in PFPA and HFBA amides

are analogous to m/z 58 in the underivatized species because all these ions represent the

(M-135)+ species. The general fragmentation pattern and structures for the m/z 204 and

254 ions are shown in Scheme 1.2. The methylenedioxybenzyl cation at m/z 135 is a

fragment common to all the spectra in Figures 1.2 and 1.3. The decreased role for alpha

cleavage reaction in the fragmentation of these amides allows the formation of ions more

diagnostic of each individual side chain isomer. Acylation, and in particular the

perflouroacylation, weakens the bond between nitrogen and the alpha-carbon of the

substituted methylenedioxyphenethyl group, allowing the formation of charged

hydrocarbon species of increased relative abundance. These hydrocarbons of varying

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

m/z-->


41

6977

105 131

135

161

176

214

226

254

265280 311 330

360

370

389


77

mass significantly individualize the mass spectra and provide specific structure

information.

Scheme 1.2: Formation of the (M-135)+ ions in the perfluoroacyl-derivatives of the

regioisomeric amines.

The mass spectra in Figures 1.2 and 1.3 illustrate the role of hydrocarbon

fragments at m/z 148, 162 and 176 in the electron impact mass spectral differentiation

among these side chain regioisomers.

The spectra for the N-ethyl derivatives in Figures 1.2a, 1.2e and 1.3a, 1.3e) show

a base peak at m/z 148 corresponding to the alkane radical cation which occurs from

hydrogen rearrangement and subsequent fragmentation of the alkyl carbon to nitrogen

bond of the phenethylamine side chain (Scheme 1.3).


78

O

O

CH C

R1

R2

N R3

R4

H

CO

O

O

HC C

R1

R2

N R3

R4

CHO

O

O

HC C

R1

R2

R1=R2=H, m/z=148R1=H, R2=CH3, m/z = 162R1=R2=CH3, m/z = 176R1=H, R2=C2H5, m/z = 176

Scheme 1.3: Mechanism for the formation of the alkane radical cation in the

perfluoroacyl-derivatives of the regioisomeric amines.

This ion at m/z 148 would only occur for the N-ethyl regioisomer. The spectra in

Figures 1.2b, 1.2f and 1.3b, 1.3f show the 2,3- and 3,4-methylenedioxyphenylpropane

hydrocarbon ion at m/z 162, identifying this molecules as the PFPA and HFBA

derivatives of 2,3- and 3,4-methyelenedioxymethamphetamines, respectively. The

proposed mechanism for the formation of the hydrocarbon fragment is illustrated in

Scheme 2.3. The spectra for the PFPA and HFBA derivatives of the primary amines 4, 5,

9 and 10 show ions at m/z 176 from the corresponding 2,3- or 3,4-methylenedioxyphenyl

alkyl radical cation. This m/z 176 results from hydrogen rearrangement and subsequent

fragmentation of alkyl carbon to nitrogen bond. The lower abundance of m/z 176 for the

2, 3- and 3, 4-methylenedioxyphentramines (compounds 4 and 9) may be attributed to

steric inhibition of hydrogen transfer in the alpha, alpha-dimethyl substitution pattern.


79

While the alkene ions at 148, 162, and 176 help to identify the side chain

regioisomers, one complicating factor in the PFPA derivatives for the N-

ethylphenethylamines (Figures 1.2a and 1.2e) is the appearance of an ion at m/z 176 in

addition to the base peak at m/z 148. The 176 ion suggests a four carbon chain directly

attached to the aromatic ring as occurs for the alpha-ethyl- and alpha, alpha-dimethyl-

phenethylamines (Figures 1.2 c, d, g, h and 1.3 c, d, g, h). The m/z 176 ion in the spectra

for the PFPA derivatives of the N-ethyl regioisomers (Figures 1.2a and 1.2e) is a

rearrangement of the m/z 204 ion resulting in the loss of mass 28 (the N-ethyl group) via

hydrogen transfer (Scheme 1.4). This coincidental common mass from two different

fragmentation pathways is confirmed by examining the mass spectra for the HFBA

derivatives of the N-ethyl-phenethylamines shown in Figure 1.3a and 1.3e. The loss of 28

mass units from the acylimine fragment at m/z 254 yields the equivalent fragment ion at

m/z 226. Thus, the HFBA derivatives may offer more unique characteristic ions for

individualization of these regioisomeric substances.

Scheme 1.4: Mechanism for the loss of mass 28 from the perfluoroacyl-N-ethyl-

imine cation.


80

A comparison of the PFPA derivatives for 3,4- and 2,3-

methylenedioxymethamphetamine (Figures 2.2b and 2.2f) with the HFBA derivatives

(Figures 1.3b and 1.3f) indicated unique ions at m/z 160 and m/z 210. This mass

difference of 50 (CF2) suggests these ions contain the perfluoroalkyl group for each

derivative, C2F5 and C3F7, respectively the suggested mechanism of forming these masses

can be illustrated in Scheme 1.5. Additional information about these ions can be obtained

by a comparison of the mass spectra for the PFPA and HFBA derivatives of 3,4-MDMA

and NCD3-3,4-MDMA (MDMA-d3) in Figure 1.4. The corresponding ions in these

spectra occur at m/z 163 and 213 and the equivalent ions for the derivatives of d5-MDMA

also occur at m/z 163 and 213. Thus, an analysis of the masses of the components which

make up the fragment at m/z 160 for example include C2F5 (119 mass units) and CH3 (15

mass units) leaving only a mass of 26 available for the total of 160. The mass 26 would

correspond to CN and the proposed mechanism for the formation of (C2F5CNCH3)+ is

shown in Scheme 1.5. An equivalent fragmentation pathway has been reported [C.R.

Clark et al, 1995] for methamphetamine.

R2CNH3C

m/z = 160 (R2 = C2F5)m/z = 210 (R2 = C3F7)

H3C N

CHO

R2

CH3

H3C N

CH

C

O

R2

CH3

m/z = 204 (R2 = C2F5)m/z =254 (R2 = C3F7)

Scheme 1.5: Formation of m/z 160 and 210 from the PHPA and HFBA derivatives of 2,3- and 3,4-MDMA


81

Figure 1.4: Mass spectra of the PFPA and HFBA derivatives for the d3 and d5- MDMA.

O

O

N

CD3

C2F5

O

a

O

O

N

CD3

C2F5

OD

H D

b

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m/z-->


45

5177

89

119

135

147

162

179

207

223 269 303

342

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41

5178

119

136

163

208

344


82

O

O

N

CD3

C3F7

O

C

O

O

N

CD3

C3F7

OD

H D

d

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45

6977

105

131

135

162

194

213

223

257

263 313 332 353 373

392

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70000

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110000

m/z-->


46

6978

107 133

136

164

196

213

258

315

394


83

The spectra for the derivatives of d3- and d5-MDMA in Figure 2.4 also lend

support to the proposed mechanism for the formation of the alkene fragment at m/z 162

illustrated in Scheme 1.3. The exact structure for the d5-MDMA is shown in Scheme 1.6,

the benzylic position contains one hydrogen and one deuterium and the transfer

fragmentation can occur to remove either species to form the alkene radical cation. Thus,

the resulting alkene can yield ions at m/z 163 or 164 depending on the probability of

transfer.

Scheme 1.6: Formation of m/z 163 and 164 in the perfluoroacyl-derivatives of d5-

MDMA.

Gas chromatographic separation of perfluroacyl derivatives of 3,4- MDMA and their direct regioisomers

The PFPA and HFBA derivatives of the eight primary and secondary amines were

compared on two stationary phases using capillary columns of the same dimensions, 30m

x 0.25mm and 0.25um depth of film using instrument 1. Previous studies on the


84

chromatographic properties of the underivatized compounds [Aalberg et al., 2000 and

2004 ] have shown that other compounds in this series co-eluted with MDMA using some

common gas chromatographic stationary phases and conditions.

The stationary phases compared were the relatively nonpolar phases, 100%

dimethyl polysiloxane (Rtx-1) and 95% dimethyl-5% diphenyl polysiloxane (Rtx-5).

Several temperature programs were evaluated and the best compromises between

resolution and analysis time were used to generate the data in Table 8 and the

chromatograms in Figures 1.5 and 1.6

The two chromatograms for the PFPA derivatives in Figure 19 were generated

using two temperature programs. The first program used was to hold the column

temperature at 100 oC for 1 minute, ramped to 180 oC at 9 oC/minute, hold at 180 oC for 2

minutes ramp to 200 oC at 10 oC/minute [TP-1]. This program was used to separate

compounds 2-5 and 7-10 in their HFBA and PFPA forms on Rtx-5. The same program

was also used to separate the PFPA derivatives of the same compounds on Rtx-1. The

second temperature program used to resolve the HFBA derivatives of compounds 2-5 and

7-10 on Rtx-1. The program was set up to hold the column temperature at 70 oC for 1

minute, ramped to 150 oC at 7.5 oC/minute, hold at 150 oC for 2 minutes ramp to 250 oC

at 10 o /minute[ TP-2].

The resulting elution order and resolution are quite similar. In fact the elution

order is the same for all the chromatograms in Figures 1.5 and 1.6. The chromatograms

show that the 2,3-isomer elutes before the corresponding 3,4-isomer for all the side chain

regioisomers. For example, 2,3-MDMA elutes before 3,4-MDMA, and this pattern holds

for all side chain regioisomers. When the ring substitution pattern is held constant (ie 2,3-


85

or 3,4-) and the side chain elution order is evaluated the two secondary amides elute

before the two tertiary amides. Additionally, in every case in this limited set of

compounds the branched side chain elutes before the straight chain isomer when the ring

substitution pattern and the degree of amide substitution are constant. Therefore, the 2,3-

phentermine-PFPA elutes first followed by 2,3-BDB-PFPA (both secondary amides),

then 2,3- MDMA-PFPA, and N-ethyl 2,3-methylenedioxyphenethylamine-PFPA the two

tertiary amides. Perhaps the most useful information in these chromatograms is the

relative elution of the derivatized controlled substance MDMA and its closest eluting

regioisomeric equivalents. Both the PFPA and HFBA derivatives of MDMA elute

between the N-ethyl-2,3- and 3,4-methylenedioxyphenethylamine PFPAs and HFBAs,

both the N-ethyl regioisomers show very distinct mass spectra with several characteristic

ions to differentiate these compounds from the drug of abuse MDMA. Thus,

derivatization methods coupled with both chromatographic and mass spectral procedures

can allow for the complete characterization of the side chain substitution pattern for these

ten uniquely isomeric substances, however, the HFBA derivatives offer more unique

fragment ions for additional discrimination among these regioisomeric substances.


86

Abundance

Time (minutes)

Figure 1.5: Capillary gas chromatographic separation of the PFPA derivatives of

compounds (2-5) and (7-10). Columns used: A Rtx-1; B Rtx-5.

10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.500

10000

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60000

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80000

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110000

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130000

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Time-->

AbundanceTIC: 111103-4.D

9.00 10.00 11.00 12.00 13.00 14.00 15.000

500000

1000000

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4000000

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Time-->


9

4

10

8

5

2

9

4

10

8

7

3

A

2

5

B

7 3


87

Abundance

Time (minutes) Figure 1.6: Capillary gas chromatographic Separation of HFBA derivatives of

compounds (2-5) and (7-10). Columns used: A Rtx-1; B Rtx-5

20.00 21.00 22.00 23.00 24.00 25.00 26.000

100000

200000

300000

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600000

700000

800000

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1000000

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8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.000

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9

9

4

4

10

10

8

8

5

5

7

7

3

3

2

2

A

B


88

Conclusions of Chapter 1

3,4-MDMA and nine other methylenedioxyphenethylamines are a unique subset

of regioisomeric molecules; each compound has a molecular weight of 193 and yields a

base peak at m/ z 58 in the MS from the loss of the corresponding methylenedioxybenzyl

group. Thus, the traditional EI MS provides little stru ctural information for diff e

rentiating among these 10 compounds. Because of the unique similarity of these

compounds by MS, the specific identification of a compound such as 3,4-MDMA

requires methods to eliminate any of the other nine isomers.

This elimination process may be accomplished on the basis of chromatography

alone but would ultimately require the analyst to use reference samples of each of the 10

amines. The reference samples would be necessary to determine if any of the isomeric

methylenedioxyphenethylamines coeluted with 3,4-MDMA. Derivatization of the eight

primary and secondary amines with various acylating agents yields amides with similar

resolution to the underivatized amines by capillary GC on RTX-1 and RTX- 5 stationary

phases. However the perfluoroacyl derivatives significantly individualize the mass

spectra for these amides and allow for specific identification. The individualization is the

result of fragmentation of the alkyl carbon–nitrogen bond yielding hydrocarbon

fragments at m/ z 148, 162, and 176, as well as other unique fragments from these

regioisomeric amides. The PFPA and HFBA derivatives are essentially equivalent for

chromatographic purposes. However, the HFBA derivatives offer more unique fragment

ions for additional discrimination among these regioisomeric substances.


89

Chapter 2

Mass Spectrometry and Gas Chromatographic Studies of Indirect Regioisomers Related to 3,4-MDMA, the

Methoxymethcathinones.

The methoxymethcathinones constitutes a set of indirect regioisomers of the

controlled drug substance 3,4-methylenedioxymethamphetamine. The various isomeric

forms of the methoxymethcathinones have mass spectra essentially equivalent to 3, 4-

MDMA, all have molecular weight of 193 and major fragment ions in their electron

ionization mass spectra at m/z 58 and 135/136. For these individual regioisomers the m/z

135/136 ion is the methoxybenzoyl carbocation (C8H7O2) + not the methylenedioxybenzyl

carbocation (C8H7O2)+. The specific identification and differentiation between these

compounds and the drug of abuse 3,4-MDMA must be based on a combination of mass

spectral data as well as chromatographic resolution of these regioisomeric substances.

The mass spectra of the underivatized methoxymethcathinones as well as their

perfluroacetyl derivatives will be compared with 3,4- and 2,3- MDMA. The

chromatographic separation of the underivatized and derivatized methoxymethcathinones

from 2,3- and 3,4-methylenedioxymethamphetamines will be discussed.


90

Mass spectral studies of the underivatized and the perfluoroacyl derivatives of the methoxymethcathinones

Methoxymethcathinones represent a potentially significant challenge for

analytical drug chemistry. All the regioisomeric methoxymethcathinones have the same

molecular weight (193) and the same side chain as the drug of abuse 3,4-MDMA.

The mass spectra for the three methoxymethcathinone regioisomers (Figure 2.1)

show a base peak at m/z 58 as seen for 2,3- and 3,4-MDMA (Figure 2.1). The major

fragmentation pattern for the methoxymethcathinones is shown in Scheme 2.1. The

methoxybenzoyl (C8H7O2)+ fragment has the same mass and empirical formula as the

methylenedioxybenzyl (C8H7O2)+ cation occurring at m/z 135. Furthermore the m/z 58

ion in the methoxymethcathinones is the same imine structure as that obtained in the

mass spectra of both 3,4- and 2,3-MDMA.

O

O

HN

OCH3

HN

O

EI70ev

EI70 ev

HC NH

MW = 193MW=193

m/z = 58EI70 ev

eI70 ev

O

O

CH2

OCH3

O

m/z = 135 m/z = 135

C8H7O2+ C8H7O2

+

Scheme 2.1: EI fragmentation pattern of the underivatized 2,3- ,3,4-MDMAs and

the methoxymethcathinones


91

Figure 2.1: Mass spectra of the underivatized methoxymethcathinones. Structure of regioisomers Mass spectrum

1

HN

O

OCH3

2

OCH3

HN

O

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58

63

77

80

92

105108 121

135

146147 160 175178 193

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42

58

6477

86

92107108 121

135136 148 162164 178 191


92

HN

O

H3CO

3

Since the mass spectra of the methoxymethcathinones is almost identical to the

drug of abuse 3,4-MDMA, perfluoroacylated derivatives of 3,4- and 2,3-

methylenedioxymethamphetamines and their regioisomeric secondary amines, ortho,

meta and para-methoxymethcathinones, were prepared and evaluated in an effort to

individualize their mass spectra and to improve chromatographic resolution. Acylation

of the amines significantly lowered the basicity of nitrogen and allowed other

fragmentation pathways to play a more prominent role in the mass spectrum. The mass

spectra for the five pentaflouropropionyl and heptflourobutryl amides are shown in

Figures 2.2 and 2.3, respectively.

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

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42

58

64

77

79

92

107108 121

135

137 150 162165 178 191


93

Figure 2.2: Mass spectra of the PFPA derivatives of 3,4-MDMA (a); 2,3-MDMA

(b) ;ortho (c); meta (d) and para (e)-methoxymethcathinones.

O

O

N C2F5

O

a

O

O

N C2F5

O

b

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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1000000

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42

51

77

91

119

135

147

162

176

204

220 241 259269282 300 324

339

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42

51 7791

119 135

147

162

177

204

220

339


94

OCH3

N

O

C2F5O

c

N

O

C2F5O

OCH3 d

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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41

56

77

92107

135

160

204

339

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4256

77

92 119

135

137

160

176

204

220 300 339


95

N

O

C2F5O

H3CO

e

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30000

40000

50000

60000

m/z-->


41

6477

92 107

135

160176

204

339


96

Figure 2.3: Mass Spectra for the HFBA derivatives of 3,4-MDMA (a); 2,3-MDMA

(b); ortho (c); meta (d) and para (e)-methoxymethcathinones.

O

O

N C3F7

O

a

O

O

N C3F7

O

b

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42

6977

105

131

135

162

192

210

220

254

263 291 310 332 350 374

389

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42

6977

105

119

135

162

192

210

220

254

263 291 313 339350 374

389


97

OCH3

N

O

C3F7O

c

N

O

C3F7O

OCH3

d

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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42

69

77

92

120

135

169176

210

220

254

289 313322 350 370 389

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42

69

77107

119

135

169

176

210

220

254

285 346 370

389


98

N

O

C3F7O

H3CO

e

From these spectra a common peak occurs at m/z 204 and 254 which

corresponds to the loss of 135 mass units from the molecular ions at 339 and 389 for

PFPA and HFPA amides. This ion at m/z 204 and 254 is the PFPA and HFPA imine

species likely formed from the alpha cleavage of the amide nitrogen to eliminate the 2, 3-

and 3, 4-methylenedioxybenzyl and methoxybenzoyl radicals. Thus the m/z 204 and 254

in PFPA and HFPA amides are analogous to m/z 58 in the underivatized species because

all these ions represent the (M-135) + species. The general fragmentation pattern and

structures for the m/z 204 and 254 ions are shown in Scheme 2.2

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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m/z-->


42

69

77

92

119

135

169176

210

220

254

285 313 358374389


99

The relative abundances for the m/z 204 and 254 ions are always higher in the 3,4- and

2,3-methylenedioxymethamphetamines than in the methoxymethcathinones The

methylenedioxybenzyl and the methoxybenzoyl cations at m/z 135 are fragments

common to all the spectra (Figures 3.2 and 3.3). The relative abundance of m/z 135 in

perfluoroacyl derivatives of methoxymethcathinones is higher than that observed for 3,4-

and 2,3-methylenedioxymethamphetamines. The m/z 135 ion is the base peak in the mass

spectra for the derivatives of the methoxymethcathinones likely due to the additional

carbonyl site for initial radical cation formation in these compounds.

HC N

CH3

CH3

O

R

m/z = 204 (R = C2F5)m/z =254 (R = C3F7)

O

O

N

CH3

CH3

R

O

NR

CH3

CH3O

O

H3CO

EI/ 70evEI/ 70ev

Scheme 2.2: Formation of m/z 204 and m/z 254 for the PFPA and HFBA derivatives

of 2,3-, 3,4-MDMAs and methoxymethcathinones

The decreased role for alpha cleavage reaction in the fragmentation of these

amides as a result of perflouroacylation which weakens the bond between nitrogen and

the alpha-carbon of the substituted methylenedioxyphenethyl group, allowing the


100

formation of charged hydrocarbon species of increased relative abundance. These

hydrocarbons of varying mass significantly individualize the mass spectra and provide

specific structural information. The mass spectra in Figures 2.2 and 2.3 illustrate the role

of the hydrocarbon fragment at m/z 162 in the electron impact mass spectral

differentiation among these regioisomeric compounds. The spectra in Figures 2.2a, 2.2b,

2.3a and 2.3b show the 2,3- and 3,4-methyelenedioxyphenylpropene radical cation at m/z

162, identifying these molecules as the PFPA and HFBA derivatives of 2,3- and 3,4-

methyelenedioxy methamphetamines, respectively. The formation of the m/z 162 ion has

been described chapter 1 and requires the transfer of hydrogen from the benzylic carbon.

This fragmentation mechanism does not take place in the PFPA and HFBA derivatives of

the methoxymethcathinones due to the absence of benzylic hydrogen (Scheme 2.3). One

can conclude that the presence of alkene ions at m/z 162, can be used to identify the side

chain of 3,4- and 2,3-methyelenedioxymethamphetamines and exclude the regioisomeric

methoxymethcathinones. Conversely, the base peak at m/z 135 as well as the absence of

the m/z 162 ion would identify one of these substances as a methoxymethcathinone

regioisomer.

A comparison of the PFPA derivatives between the 3,4- and 2,3-

methylenedioxymethamphetamine (Figures 2.2a and 2.2b) with their HFBA derivatives

(Figures 3.3a and 3.3b) indicates unique ions at m/z 160 and m/z 210. This mass


derivative, C2F5 and C3F7, respectively. These unique ions have been fully characterized

in chapter 1 using deuterated analogs of 3,4-MDMA and methamphetamine. These ions

at m/z 160 and 210 are the result of a rearrangement decomposition of ions 204 and 254


101

respectively (Scheme 1.2). The m/z 204 and 254 ions have the same structure whether

generated from derivatives of the MDMAs or the methoxymethcathinones. Therefore

these unique ions do not provide any information to differentiate between the two groups

of substances.

O

O

C N R

O

HHO

O

N R

OH

H

O

O

CH

H

m/z 162

N

O

RO

H3CO R = C2F5, PFPA derivativeR= C3F7, HFBA derivative

Scheme 2.3: Benzylic hydrogen transefer to form m/z 162 occur only in MDMA

perfluroaceyl derivatives but not for methoxymethcathinones.

Gas chromatographic separation of 2,3-MDMA and 3,4 MDMA from

the methoxymethcathinones

Gas chromatographic properties of the PFPA and HFBA derivatives of the 2,3-

and 3,4-methylenedioxymethamphetamines and 2-, 3-, and 4-methoxymethcathinones

were compared on two stationary phases using capillary columns of the same dimensions,

30m x 0.25mm and 0.25um depth of film. The stationary phases compared in this study


102

were the relatively nonpolar phases, 100% dimethyl polysiloxane (Rtx-1) and 95%

dimethyl-5% diphenyl polysiloxane (Rtx-5). The underivatized compounds were not

completely resolved with 2-methoxymethcathinone co-eluting with 3-

methoxymethcathinone using these common gas chromatographic stationary phases and

some common temperature programming conditions (Figure 2.4).

The PFPA and HFBA derivatives showed improved resolution when compared to

the underivatized amines. Several temperature programs were evaluated and the best

compromise between resolution and analysis time were used to generate the

chromatograms in Figures 2.5 and 2.6.

The two chromatograms for the PFPA derivatives (Figure 2.5) were generated

using two different temperature programs; the resulting elution order and resolution are

quite similar. In fact the elution order is the same for all the chromatograms shown in

Figures 2.5 and 2.6. In each case the derivatized 2-methoxymethcathinone (compound 1)

eluted first followed closely by the 3-methoxymethcathinone derivative (compound 2).

The third compound to elute is the 2,3-MDMA derivative (compound 4) and the

derivatized 4-methoxymethcathinone (compound 3) is the forth peak in each

chromatogram. The derivatized form of 3,4-MDMA (compound 5) showed the greatest

retention in each chromatogram.


103

Time (minutes)

Figure 2.4: Capillary gas chromatogram for a physical mixture of underivatized

3,4 MDMA, 2,3-MDMA and methoxymethcathinones.

11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.000

100000

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1+2

43

5


104

Abundance

Time (minutes)

Figure 2.5: Capillary gas chromatographic separation of PFPA derivatives of

compounds 1-5. Columns used: A Rtx-1; B Rtx-5.

1 2

3

5

8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.000

500000

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2

4 3 5

A

B

7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.500

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4


105

Abundance

Time (minutes) Figure 2.6: Capillary gas chromatographic separation of HFBA derivatives of

compounds 1-5. Columns used; A: Rtx-1; B: Rtx-5.

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.000

500000

1000000

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B

1

5

3

4

2

1

2

4

3

5


106

Conclusion of Chapter 2

3,4- and 2,3-MDMA and 2-, 3-, and 4-methoxymethcathinone are a unique subset of

regioisomeric molecules; each compound has a Mw of 193 and yields a base peak for the

N-methyl imine of acetaldehyde at m/z 58 in the mass spectrum from the loss of the

corresponding C8H7O2 methylenedioxybenzyl and methoxybenzoyl groups, respectively.

Thus, the traditional EI-MS provides little structural information for differentiating

among these five compounds. Because of the unique similarity of these compounds by

MS, the specific identification of a compound such as 3,4-MDMA requires methods to

eliminate the other four isomers. This elimination process may be accomplished on the

basis of chromatography alone but would ultimately require the analyst to use reference

samples of each of the five amines. Derivatization of these amines with various acylating

agents yields amides with improved resolution compared with the underivatized amines

by capillary GC on Rtx-1 and Rtx-5 stationary phases. These perfluoroacyl derivatives

significantly individualize the mass spectra for these amides. The individualization is the

result of fragmentation of the alkyl carbon–nitrogen bond, yielding hydrocarbon

fragments at m/z 162 as well as other unique fragments from the MDMA amides that

were not formed for the methoxymethcathinones. The PFPA and HFBA derivatives are

essentially equivalent for chromatographic purposes; however, the HFBA derivatives

offer more unique fragment ions for additional mass spectral discrimination among these

regioisomeric substances.


107

Chapter 3

Mass Spectrometry and Gas Chromatographic Studies on Acylated

Derivatives of a Series of Side Chain Regioisomers of 2-Methoxy-4-

Methyl Phenethylamines

In this chapter GC-MS studies are described for a series of side chain isomers of 2-

methoxy-4-methylphenethylamines. These isomers have an isobaric relationship (equal

mass, different elemental composition) to the controlled substance 3,4-MDMA.

Mass Spectral Studies on Chain Regioisomers of 2-Methoxy-4-Methyl

Phenethylamines

Mass spectrometry is the primary method for confirming the identity of drugs and

other substances of abuse in forensic samples. The five side chain regioisomers of 2-

methoxy-4-methylphenethylamine (Figure 3.1, Compounds 1-5) have the potential to

yield mass spectra essentially equivalent to 3,4-MDMA (and 2,3-MDMA). All have

molecular weight of 193 and major fragment ions in their electron ionization mass

spectra at m/z 58 and 135/136 (Figure 3.2). The m/z 58 ion in the methoxy methyl

phenethylamine is regioisomeric with that obtained in the mass spectra of both 2,3 and

3,4-MDMA. Additionally, the isobaric methoxy methyl benzyl (C9H11O)+ fragments

have the same nominal mass as the methylenedioxybenzyl (C8H7O2)+ cation occurring at

m/z 135 (Scheme 3.1). The individual mass spectra for 2,3- and 3,4-MDMA are also


108

presented in Figure 3.2 (Compounds 6 and 7). The data in Figure 3.2 show that the mass

spectra do not provide any major fragment ions to differentiate among these compounds.

Figure 3.1: Structures of the side chain regioisomers of 2-methoxy-4-methylphenethylamines, and 2,3- and 3,4-MDMA.

H3C

N

OCH3

1

H3C

HN

OCH3

2

H3C

HN

OCH3 3

H3C

NH2

OCH3

4

H3C

NH2

OCH3

5

HN

O

O

6

O

O

HN

7 (3,4-MDMA)


109

HC

N

CH3

CH3

m/z = 135

m/z = 58

MW = 193

H2C

CH2

CH

CH3

HN

CH3

O

O

H2C

O

O

CH2

EI 70ev

H

H3C

H3C

C N

C2H8

OCH3

OCH3

CN

C2H8

m/z = 135

Scheme 3.1: EI fragmentation pattern for compounds 1-7. Figure 3.2: Mass Spectra of the underivatized amines 1-7.

H3C

N

OCH3 1

40 60 80 100 120 140 160 180 2000

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m/z-->

AbundanceAverage of 10.725 to 10.848 min.: 71707--6.D

41 51

58

65 77

91

92 103 121122

136

149162163 176 191193


110

H3C

HN

OCH3 2

H3C

HN

OCH3

3

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

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AbundanceAverage of 9.175 to 9.253 min.: 110606-1.D

42

58

597778

91

103 115120 133135 149 160162

176188

191

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

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m/z-->


42

58

657779

91 105

115121 131136

146 160164 178 189192


111

H3C

NH2

OCH3

4

H3C

NH2

OCH3

5

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

100000

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m/z-->


41

58

6577

81

91 105

115121136

146148 161 176178

193

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

10000

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m/z-->


41

58

65

7779

91 105

117

121

136

137

149

164

176 193


112

HN

O

O

6

O

O

HN

7

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

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42

58

63

77

7991 105

117120

135

146148 163 174178 192

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42

58

63

77

8991 105

117120

135

137 148 162 174178 191


113

OCH3H3C

N

R1 R2 X

R3

O

R1+R2+R3 = C2H7 C2F5= X,, MW = 339 C3F7= X, MW= 389

OCH3H3C

m/z = 135

N

R1

R2

R3

X

O

m/z = 204, X= C2F5m/z = 254, X= C3F7

OCH3H3C

R1

R2

m/z = 148, R1=R2=Hm/z = 162, R1= CH3; R2=Hm/z = 176, R1= CH3; R2= CH3m/z = 176,R1= C2H5; R2= H

H3C

m/z = 105

Scheme 3.2: Mass spectral fragmentation products for the acylated 2-methoxy-4-methyl phenethylamines.

In the next phase of this study various perfluoroacylated derivatives of the

regioisomeric primary and secondary amines were prepared and evaluated in an effort to

individualize their mass spectra and provide unique marker ions for specific

identification. Generally, acylation of the amines significantly lowers the basicity of

nitrogen and can allow other fragmentation pathways to play a more prominent role in the

resulting mass spectrum.

The mass spectra for the pentafluoropropionyl and heptfluorobutyryl amides are

shown in Figures 3.3 and 3.4, respectively. From these spectra a common peak occurs at

m/z 204 and 254 which corresponds to the loss of 135 mass units from the molecular ions


114

at 339 and 389 for PFPA and HFBA amides. The m/z 204 and 254 ions in the PFPA and

HFBA amides are analogous to m/z 58 in the underivatized species because all these ions

represent the (M-135)+ species. The general fragmentation pattern and structures for the

m/z 204 and 254 ions are shown in Scheme 3.2. The 2-methoxy-4-methylbenzyl cation

(m/z 135) and the methylenedioxybenzyl cation (m/z 135) are fragments common to all

spectra in Figures 3.3 and 3.4. However, the 2-methoxy-4-methylbenzyl cation at m/z

135 in the perfluoroacyl derivatives shows a very high relative abundance. Indeed the m/z

135 ion is the base peak in all the PFPA derivatives of compounds 2,4,5 and in the HFBA

derivatives of compounds 4 and 5. The remaining two HFBA derivatives of compounds 2

and 3 and the PFPA derivative of compound 3 show the m/z 135 ions as a major

fragment of at least 90% relative abundance. This would suggest that the perfluoroacyl

derivatives of compounds 2, 3, 4 and 5 offer a distinct discrimination between the

methylenedioxy and the 2-methoxy-4-methyl substitution patterns based on the

difference in relative abundances of the substituted benzyl cation at m/z 135.


115

Figure 3.3: Mass Spectra of the PFPA derivatives of compounds 2-7.

H3C

N C2F5

O

OCH3

2

H3C

N C2F5

O

OCH3 3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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m/z-->


42 65 77

91

119

135148

162

176

204

220 237 272 294 309 324

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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m/z-->


42

56 79 91

105

135

147

162

190

204

220 237 269282 308 324339


116

H3C

NH C2F5

O

OCH3

4

H3C

NH C2F5

O

OCH3

5

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42

59

79 91

105

135

146164

176

204

215 257 282 306 324

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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m/z-->


41

65

79 91

105

135

145

161

176

204

220 237246 278292 310

339


117

N

O

O C2F5

O

6

O

O

N C2F5

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

m/z-->


42

56 77

91

119 135

147

162

177

204

220233 259263 300 324

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

50000

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m/z-->


42

51

77

91

119

135

147

162

176

204

220 237 259269282 300 324

339


118

Figure 3.4: Mass spectra of the HFBA derivatives of compounds 2-7.

H3C

N C3F7

O

OCH3 2

H3C

N C3F7

O

OCH3 3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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m/z-->


42

69

91

92 119

148

169

178197

226

254

322 344 370

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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m/z-->


42

6991

105

115

135

162

192

210

220

254

309 358374389


119

H3C

NH C3F7

O

OCH3 4

H3C

NH C3F7

O

OCH3

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

20000

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200000

220000

240000

260000

m/z-->


41

59

91

105

121

135

169

176

214

226

254

273 313 350 374

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

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m/z-->


41

6979

105

121

135

161

176

214

226

254

266 296313328360361

389


120

N

O

O C3F7

O

6

O

O

N C3F7

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

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42

6977

105131

135

162

192

210

220

254

263 291 309 339350 370

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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42

6977

105

131

135

162

191

210

220

254

263 293309 350 370

389


121

The decreased role for the alpha cleavage reaction in the fragmentation of these

amides allows the formation of more diagnostic ions of each individual isomer. Acylation

weakens the bond between nitrogen and the alkyl carbon of the phenethyl side chain,

allowing the formation of charged side chain specific hydrocarbon species of increased

relative abundance. These hydrocarbons of varying mass significantly individualize the

mass spectra and provide specific structure information. The spectra for the N-ethyl

isomer (compound 2) in Figures 3.3-2 and 3.4-2 show a base peak at m/z 148

corresponding to the alkene radical cation which occurs from hydrogen rearrangement

and subsequent fragmentation of the alkyl carbon to nitrogen bond of the phenethylamine

side chain (see Scheme 3.2). This ion at m/z 148 would only occur for the N-ethyl

regioisomer. The spectra in Figures 3.3-3, 3.3-6, 3.3-7, 3.4-3, 3.4-6 and 3.4-7 show the

substituted phenylpropene hydrocarbon radical cation at m/z 162, identifying these

molecules as the PFPA and HFBA derivatives of 2-methoxy-4-methylmethamphetamine

and the 2,3-, 3,4-methylenedioxymethamphetamines respectively. The spectra for the

PFPA and HFBA derivatives of the primary amines (compounds 4 and 5) show ions at

m/z 176 from the corresponding substituted phenylbutene radical cation. The lower

abundance of m/z 176 for the 4-methoxy-3-methylphentramine (compound 4) may be the

result of steric inhibition of hydrogen transfer in the alpha, alpha-dimethyl substitution

pattern.



ethylphenethylamines (Figure 3.3-2) is the appearance of an ion at m/z 176 in addition to

the base peak at m/z 148. The 176 ion suggests a four carbon chain directly attached to


122

the aromatic ring as occurs for the alpha-ethyl- (compound 5) and alpha, alpha-dimethyl-

(compound 4) phenethylamines (Figures 3.3-4, 3.3-5 and 3.4-4, 3.4-5). The m/z 176 ion

in the spectra for the PFPA derivatives of the N-ethyl regioisomers (Figure 3.3-2) is a


hydrogen transfer This coincidental common mass from two different fragmentation

pathways is confirmed by examining the mass spectra for the HFBA derivatives of the N-

ethyl-phenethylamines shown in Figure 3.4-2. The loss of 28 mass units from the

acylimine fragment at m/z 254 yields the equivalent fragment ion at m/z 226. Thus, the

HFBA derivatives may offer more characteristic ions for individualization of these

regioisomeric substances.

A comparison of the mass spectra for the PFPA and HFBA derivatives of all three

ring substituted methamphetamines (Compounds 3, 6 and 7) indicates unique ions at m/z

160 and m/z 210 (see Figures 3.3-3, 3.3-6, 3.3-7; 3.4-3, 3.4-6, and 3.4-7). This mass


derivative, C2F5 and C3F7 respectively. This ion has been described in previous chapters

as the cationic methylated nitrile C2F5CNCH3 or C3F7CNCH3. The equivalent

fragmentation further supported by the analysis of the mass spectra of the PFPA and

HFBA derivatives of d3- and d5-MDMA in previous studies [Chapter 1]. This

fragmentation pathway appears unique to the acylated N-methyl C-methyl pattern

(methamphetamine side chain).

The presence of relatively high abundances of the m/z 105 ion in the spectra of

the PFPA and HFBA derivatives of compound 3 allows for significant discrimination of

this 2-methoxy-4-methylmethamphetamine from the two isobaric methylenedioxy-


123

methamphetamines isomers (compounds 6 and 7). This ion at m/z 105 represents the loss

of 30 mass units (formaldehyde, CH2O) from the methoxymethylbenzyl cation at m/z

135. Previous reports suggested that the m/z 105 ion is characteristic of all o-methoxy

substitution patterns regardless the position of the methyl group on the aromatic ring

[Chapter 6]. However, the absence of this fragment from the spectra of the derivatives of

compound 2 suggests the necessity of further structure fragmentation studies for these

compounds.

Gas Chromatographyic separation of the perflouroacyl derivatives of 2- methoxy-4-

methylphenethylamines from 2,3- and 3,4-MDMA

The PFPA and HFBA derivatives of the six primary and secondary amines were

compared on two stationary phases using two GC-MS systems, The relatively nonpolar

100% dimethyl polysiloxane (Rtx-1) and the more polar trifluoropropyl methyl

polysiloxane (Rtx-200). Several temperature programs were evaluated and one program

showing the best compromise between resolution and analysis time was used to generate

the chromatograms in Figures 3.5 and 3.66. The elution order was the same for both

stationary phases and all temperature programs evaluated in this study. The

chromatograms of the perfluoroacyl compounds show that the 2,3-MDMA elutes before

the corresponding 3,4-isomer, which elutes last. When the ring substitution pattern is held

constant (2-methoxy-4-methyl), the side chain elution order is secondary amides before

the tertiary amides and in this limited set of examples branched isomers elute before the

more linear ones. Therefore, the amides of compound 4 elute first followed by the amide


124

of compound 5 (both secondary amides), then the amides of compound 3 (the

methamphetamine side chain) and finally the amides of compound 2 (the more linear of

these two tertiary amides). The amides of 2,3-MDMA elute between the secondary and

tertiary amides in this group of compounds.

Perhaps the most useful information in these chromatograms is the relative elution of the

derivatized controlled substance 3,4-MDMA and its closest eluting regioisomeric and

isobaric equivalents. Both the PFPA and HFBA derivatives of 3,4-MDMA elute last and

the N-ethyl-2-methoxy-4-methylphenethylamine PFPAs and HFBAs are the closest

eluted compounds in the 2-methoxy-4-methyl phenethylamine series. The isobaric

acylated N-ethyl amides show very distinct mass spectra with several characteristic ions

to differentiate it from the corresponding amides of the drug of abuse 3,4-MDMA. Thus,


can allow for the complete differentiation of the side chain substitution pattern of the 2-

methoxy-4-methylphenethylamines from 3,4-MDMA and its regioisomer, 2,3-MDMA.

Additionally in this limited study, the Rtx-200 stationary phase provided the greatest

resolution of the amides of compound 7 (3,4-MDMA) from all the other isomers.


125

Abundance

Time (minutes) Abundance

Time (Minutes) Figure 3.5: Chromatograms of the PFPA (A) and HFBA (B) derivatives of

compounds 2-7. GC-MS system 1, Rtx-200 column.

4

4

5

3

3

2

2

7

7

65

6

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Time-->


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A

B


126

Abundance

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Time-->

Abundance

TIC: 0801207-14.D\data.ms

Time (minutes)

Abundance

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

Time-->

Abundance


Time (minutes)

Figure 3.6: Chromatograms of the PFPA (A) and HFBA (B) derivatives of

compounds 2-7. GC-MS system2, Rtx-1 column.

4

5

3

6 2

7

72

6

3

5

4

A

B


127


Differentiation of the side chain substitution pattern of the 2-methoxy-4-

methylphenethylamines from 3,4-MDMA and its regioisomer, 2,3-MDMA was

accomplished using a combination of gas chromatography and mass spectrometry.

Derivatization of the primary and secondary amines with various acylating agents yields

amides with improved resolution compared to the underivatized amines by capillary gas

chromatography on Rtx-1 and Rtx-200 stationary phases.

Additionally, the perfluoroacyl derivatives significantly individualize the mass spectra for

these amides and allow for unambiguous identification. The individualization results

from fragmentation of the alkyl carbon-nitrogen bond yielding characteristic hydrocarbon

fragments at m/z 105, 148, 162, and 176 as well as other unique fragments.


128

Chapter 4

GC-MS Analysis of Acylated Derivatives of the Side Chain

Regioisomers of 4-Methoxy-3-Methyl Phenethylamines Related to

Methylenedioxymethamphetamine

In this chapter all the side chain regioisomeric possibilities of 4-methoxy-3-

methyl phenethylamines, as a representative of the m/z58 side chain regioisomers of the

methoxy group in the 4 position, will be compared from the mass spectral and

chromatographic aspects to 2,3- and 3,4-MDMA.

Mass Spectral studies of 4-methoxy-3-methyl phenethylamines along with 2,3- and

3,4- MDMA


other substances of abuse in forensic samples. The mass spectra of phenethylamines are

characterized by a base peak formed from an amine initiated alpha-cleavage reaction

involving the carbon-carbon bond of the ethyl linkage between the aromatic ring and the

amine. In 3,4-methylenedioxymethamphetamine (MW=193) the alpha-cleavage reaction

yields the substituted imine fragment at m/z 58 and the 3,4-methylenedioxybenzyl

fragment at mass 135/136 (for the cation and the radical cation, respectively). Thus, the

mass spectrum for 3,4-methylenedioxymethamphetamine contains major ions at m/z 58

and 135/136 as well as other ions of low relative abundance. There are a total of five

regioisomeric forms of the m/z 58 imine species.


129

The five side chain regioisomers of 4-methoxy-3-methylphenethylamine

(Compounds 1-5) have the potential to yield mass spectra essentially equivalent to 3,4-

MDMA and 2,3-MDMA ( structures shown in Figure 4.1). All have molecular weight of

193 and major fragment ions in their electron ionization mass spectra at m/z 58 and

135/136 (Figure 4.2). The isobaric methoxy methyl benzyl (C9H11O)+ fragments have the

same mass as the methylenedioxybenzyl (C8H7O2)+ cation occurring at m/z 135.

Furthermore the m/z 58 ion in the methoxy methyl phenethylamine is regioisomeric with

that obtained in the mass spectra of both 2,3 and 3,4-MDMA (Scheme 4.1). The

individual mass spectra for 2,3- and 3,4-MDMA are also presented in Figure 4.2

(Compounds 6 and 7).

Figure 4.1: Structures of side chain regioisomers of methamphetamine

N

1

N,N-Dimethylphetheylamine

HN

2

N-ethylphenethylamine

HN

3

Methamphetamine

NH2

4

α, α-Dimethylphenethylamine

NH2

5

1-Phenyl-2-butanamine


130

Figure 4.2: Mass Spectra of the undreivatized amines:

H3CO

CH3

N

1

H3CO

CH3

HN

2

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42

58

597778

91

103 115120 133135 149 160162

176188

191

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41

58

65 77

80

91

103 117121

136

137 149163 174176 191


131

H3CO

CH3

HN

3

H3CO

CH3

NH2

4

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42

58

65 7782

91

103 115121

136

137 147 162 174178 191

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41

58

65 7782

91

103 115121

135

137 148 161 175178

193


132

H3CO

CH3

NH2

5

HN

O

O

6

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41

58

59 747891

103105121

132

136

148 159164 176 193

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42

58

63

77

7991 105

117120

135

146148 163 174178 192


133

O

O

HN

7

HC

N

CH3

CH3

m/z = 135

m/z = 58

MW = 193

H2C

C

N

CH2

CH

CH3

HN

CH3

O

O

H2C

m/z = 135

O

O

CH2

EI 70ev

H

H3CO

CH3

CH3

H3CO

C2H8

C N

C2H8

Scheme 4.1: EI fragmentation pattern of the underivatized compounds 1-7

This lack of mass spectral specificity in addition to the possibility of

chromatographic co-elution, with 3,4-MDMA, could result in misidentification of the

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

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42

58

63

77

8991 105

117120

135

137 148 162 174178 191


134

target drug. Furthermore, the lack of available reference samples could complicate the

individual identification of any one of these substances. This constitutes a significant

analytical challenge where the specific identification by gas chromatography-mass

spectrometry (GC-MS) must be based primarily upon the ability of the chromatographic

system to separate the regioisomeric/isobaric non-drug substance from the actual drug of

interest. Additionally, the ability to distinguish between these regioisomers directly

enhances the specificity of the analysis for the target drugs of interest.

In the next phase of this study various perfluoroacylated derivatives of the

regioisomeric primary and secondary amines were prepared and evaluated in an effort to


identification. Acylation of the amines significantly lowers the basicity of nitrogen and

can allow other fragmentation pathways to play a more prominent role in the resulting

mass spectrum.

The mass spectra for the twelve pentafluoropropionyl and heptfluorobutryl

amides are shown in Figures 4.3 and 4.4, respectively. From these spectra a common

peak occurs at m/z 204 and 254 which corresponds to the loss of 135 mass units from the

molecular ions at 339 and 389 for PFPA and HFPA amides. This ion at m/z 204 and 254

is the PFPA and HFPA imine species likely formed from the alpha cleavage of the amide

nitrogen to eliminate the 4-methoxy-3-methylbenzyl radical as well as the 2, 3- and 3, 4-

methylenedioxybenzyl radical. Thus the m/z 204 and 254 ions in the PFPA and HFPA

amides are analogous to m/z 58 in the underivatized species because all these ions


m/z 204 and 254 ions are shown in Scheme 4.2. The 4-methoxy-3-methylbenzyl cation


135

and the methylenedioxybenzyl cation (m/z 135) are fragments common to all spectra in

Figures 4.3 and 4.4. However, the 4-methoxy-3-methylbenzyl cation at m/z 135 in the

perfluoroacyl derivatives shows a very high relative abundance. Indeed the m/z 135 ion is

the base peak in all the PFPA derivatives of compounds 2-5 and in the HFBA derivatives

of compounds 4 and 5. The remaining two HFBA derivatives of compounds 2 and 3

show the m/z 135 ions as a major fragment of at least 90% relative abundance. This

would suggest that the pentafluoroacyl derivatives offer a distinct discrimination between

the methylenedioxy and the 4-methoxy-3-methyl substitution patterns based on the

difference in relative abundances of the substituted benzyl cation at m/z 135.


amides allows the formation of ions more diagnostic of each individual isomer. Acylation

weakens the bond between nitrogen and the alkyl carbon of the phenethyl side chain,

allowing the formation of charged hydrocarbon species of increased relative abundance.

These hydrocarbons of varying mass significantly individualize the mass spectra and

provide specific structure information. The mass spectra in Figures 4.3 and 4.4 illustrate

the role of hydrocarbon fragments at m/z 148, 162 and 176 in the electron impact mass

spectral differentiation among these regioisomers.


136

Figure 4.3: Mass Spectra of the PFPA derivatives of compounds 2-7.

H3CO

CH3

N C2F5

O

2 3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 3600

100000

200000

300000

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700000

m/z-->


42 65 77

91

119

135148

176

191

204

220 272281294 324

339

355

H3CO

CH3

N C2F5

O

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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m/z-->


42

5677

91

119

135

147

162

176

204

220228 259269282 300 324

339


137

H3CO

CH3

NH C2F5

O

4

H3CO

CH3

NH C2F5

O

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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m/z-->


42

59

77

91

119

135

146164

176

204

220 237 257263 282 306 324

339

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m/z-->


41

65 77

91

119

135

147

161

176

204220 237246 263 294 310320

339


138

N

O

O C2F5

O

6

O

O

N C2F5

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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42

56 77

91

119135

147

162

177

204

220 242 259263 282 300 324

339

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

m/z-->


42

5177

91

119

135

147

162

191

204

220 243 259269282 300 324

339


139

Figure 4.4: Mass spectra of the HFBA derivatives of compounds 2-7

H3CO

CH3

N C3F7

O

2

H3CO

CH3

N C3F7

O

3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

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42

69

77

91

119

148

169

178197

226

254

271281 313322 341 370

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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150000

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m/z-->

AbundanceAverage of 12.605 to 12.795 min.: 11130610.D

42

69

77

91

120

135

162

191

210

220

254

256 281 313319 341 374

389


140

H3CO

CH3

NH C3F7

O

4

H3CO

CH3

NH C3F7

O

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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m/z-->


42

59

77

91

121

135

169

176

214214

254

256 281 313330 356374

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

100000

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700000

m/z-->


41

69

77

91

120

135

161

176

207226

254

255 281 313328344360

389


141

N

O

O C3F7

O

6

O

O

N C3F7

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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m/z-->


42

6977

105131

135

162

192

210

220

254

256 281 313327 350 374

389

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

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700000

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1000000

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m/z-->


42

6977

105131

135

162

191

210

220

254

263 293310 332 350 370

389


142

H3CO

CH3

N

H3CO

CH3

N

R

O

O

N

N

O

O

R

R

O

O

O

R

O

H2C

N R

O

N R

O

HN R

O

N R

O

N R

O

R=C2F5 , m/z =339R=C3F7 , m/z =389

R=C2F5 , m/z =204R=C3F7 , m/z =254

H3CO

CH3

NH R

O

H3CO

CH3

NH R

O

HN R

O

Scheme 4.2: Formation of the (M-135)+ ions in the perfluoroacyl derivatives of the

regioisomeric 4-methoxy-3-methylphenethyl amines, 2,3- and 3,4-MDMA.

The spectra for the N-ethyl isomer (compound 2) in Figures 4.3-2 and 4.4-2 show

a base peak at m/z 148 corresponding to the alkene radical cation which occurs from


143

hydrogen rearrangement and subsequent fragmentation of the alkyl carbon to nitrogen

bond of the phenethylamine side chain (see Scheme 4.3). This ion at m/z 148 would only

occur for the N-ethyl regioisomer. The spectra in Figures 4.3-3, 4.3-6, 4.3-7, 4.4-3, 4.4-6

and 4.4-7 show the substituted phenylpropane hydrocarbon ion at m/z 162, identifying

these molecules as the PFPA and HFBA derivatives of 4-methoxy-3-

methylmethamphetamine and the 2,3-, 3,4-methylenedioxymethamphetamines

respectively. The proposed mechanism for the formation of the hydrocarbon fragment is

illustrated in Scheme 4.3.

CH

R5

H

R1

R2

N R3

O

R4

HC

R5

R1

R2

N R3

O

R4H

HC

R5

C

R1

R2

R3 = C2H5 (Compound 2) CH3 (Compounds 3,6,7) H2 (Compound 4,5)R4 = C2F5, PFPA Derivatives C3F7, HFBA DerivativesR5 = 4-Methoxy-3-methyphenyl (Compounds 2-5) = 2,3-Methylenedioxyphenyl (Compound 6) =3,4-Methylenedioxyphenyl (compound 7)

R1=R2 = H, m/z= 148 (Compound 2)R1= H, R2= CH3, m/z =162 (Compounds 3,6,7)R1=R2 = CH3, m/z =176 (Compound 4)R1=H, R2= C2H5, m/z=176 (Compound 5)

Scheme 4.3: Mechanism for the formation of the alkene radical cation in the

perfluroacyl derivatives ofthe regioisomeric 4-methoxy-3-methylphenethyl amines,

2,3- and 3,4-MDMA.

However the base peak of m/z 135 in the spectra of the PFPA derivative of

compound 3 offer a significant discrimination of 4-methoxy-3-methylmethamphetamines

over its two isobaric methylenedioxymethamphetamines isomers (compounds 6 and 7) .

The spectra for the PFPA and HFBA derivatives of the primary amines (compounds 4


144

and 5) show ions at m/z 176 from the corresponding substituted phenylbutene radical

cation. This m/z 176 results from hydrogen rearrangement and subsequent fragmentation

of alkyl carbon to nitrogen bond. The lower abundance of m/z 176 for the 4-methoxy-3-

methylphentramine (compound 4) may be attributed to steric inhibition of hydrogen

transfer in the alpha, alpha-dimethyl substitution pattern.



ethylphenethylamines (Figure 4.3-2) is the appearance of an ion at m/z 176 in addition to

the base peak at m/z 148. The 176 ion suggests a four carbon chain directly attached to

the aromatic ring as occurs for the alpha-ethyl- (compound 5) and alpha, alpha-dimethyl-

(compound 4) phenethylamines (Figures 4.3-4, 4.3-5 and 4.4-4, 4.4-5). The m/z 176 ion

in the spectra for the PFPA derivatives of the N-ethyl regioisomers (Figure 4.3-2) is a


hydrogen transfer (see Scheme 4.4). This coincidental common mass from two different


derivatives of the N-ethyl-phenethylamines shown in Figure 4.4-2. The loss of 28 mass

units from the acylimine fragment at m/z 254 yields the equivalent fragment ion at m/z

226. Thus, the HFBA derivatives may offer more characteristic ions for individualization

of these regioisomeric substances.

A comparison of the mass spectra for the PFPA and HFBA derivatives of all three

ring substituted methamphetamines (Compounds 3, 6 and 7) indicates unique ions at m/z

160 and m/z 210 (see Figures 4.3-3, 4.3-6, 4.3-7; 4.4-3, 4.4-6, and 4.4-7). This mass



145

derivative, C2F5 and C3F7 respectively. An analysis of the masses of the components

which make up the fragment at m/z 160 for example include C2F5 (119 mass units) and

CH3 (15 mass units) leaving only a mass of 26 available for the total of 160. The mass 26

would correspond to CN and the proposed mechanism for the formation of

(C2F5CNCH3)+ is shown in Scheme 4.5. An equivalent fragmentation pathway has been

further supported by the analysis of the mass spectra of the PFPA and HFBA derivatives

of d3- and d5-MDMA in chapter 1.

H2C N

CH2

CH2H

RC

O

H2C N

H

R

O

R=C2F5, m/z = 204R=C3F7, m/z = 254

R=C2F5, m/z = 176R=C3F7, m/z = 226

Scheme 4.4: Mechanism for the loss of mass 28 from the perfluoroacyl-N-ethyl-

imine cation.


146

N

H3C

CHH3C O

C

R

N C

OCH

RH3C

H3C

N C RH3C

R= C2F5, m/z= 204R= C3F7, m/z= 254

R= C2F5, m/z= 160R= C3F7, m/z= 210

Scheme 4.5: Mechanism for the formation of the m/z 160 and m/z 210 ions in the

mass spectra of the perfluoroacyl derivatives of the ring substituted

methamphetamines (Compounds 3, 6 and 7).

Gas Chromatography of the regioisomeric 4-methoxy-3-methylphenethyl amines,

2,3- and 3,4-MDMA.

The PFPA and HFBA derivatives of the six primary and secondary amines were

compared on two stationary phases of the same dimensions, 30 m x 0.25 mm and 0.25

µm depth of film. The stationary phases compared in this study were the relatively

nonpolar phase 100% dimethyl polysiloxane (RTX-1) and the more polar trifluoropropyl

methyl polysiloxane (Rtx-200). Several temperature programs were evaluated and one

program showing the best compromise between resolution and analysis time was used to

generate the chromatograms in Figure 4.5. The chromatograms of the perfluoroacyl

compounds show that the 2,3-MDMA elutes before the corresponding 3,4-isomer, which

elutes last. When the ring substitution pattern is held constant (4-methoxy-3-methyl), the

side chain elution order is secondary amides before the tertiary amides and in this limited

set of examples branched isomers elute before the more linear ones. Therefore, the


147

amides of compound 4 elute first followed by the amide of compound 5 (both secondary

amides), then the amides of compound 3 (the methamphetamine side chain) and finally

the amides of compound 2 (the more linear of the tertiary amides). The amides of 2,3-

MDMA elute between the secondary and tertiary amides in this group of compounds.

Perhaps the most useful information in these chromatograms is the relative elution of the

derivatized controlled substance 3,4-MDMA and its closest eluting regioisomeric and

isobaric equivalents. Both the PFPA and HFBA derivatives of 3,4- MDMA elute last and

the N-ethyl-4-methoxy-3-methylphenethylamine PFPAs and HFBAs are the closest

eluted compounds in the 4-methoxy-3-methyl phenethylamine series. The isobaric N-

ethyl amides show very distinct mass spectra with several characteristic ions to

differentiate it from the corresponding amides of the drug of abuse 3,4-MDMA. Thus,


can allow for the complete differentiation of the side chain substitution pattern of the 4-

methoxy-3-methylphenethylamines from 3,4-MDMA and its regioisomer, 2,3-MDMA.


148

Abundance

Time (minutes) Abundance

Time (Minutes) Figure 4.5: GC Chromatograms of the PFPA (A) and HFBA (B) derivatives of

compounds 2-7. Rtx-200 column.

12.00 14.00 16.00 18.00 20.00 22.00 24.000

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400000

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800000

1000000

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1400000

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1800000

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2600000

2800000

3000000

3200000

Time-->


12.00 14.00 16.00 18.00 20.00 22.000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

Time-->


4

4

5

5

6

6

3

3

2

2

7

7


149


3,4-MDMA , 2,3-MDMA and the five side chain regioisomers of 4-methoxy-3-

methyl phenethylamines are a unique subset of regioisomeric and isobarc molecules;

each compound has a molecular weight of 193 and yields a base peak at m/z 58 in the

mass spectrum from the loss of the corresponding methylenedioxybenzyl and 4-

methoxy-3-methylbenzyl groups respectively. Thus the traditional electron impact mass

spectrum provides little structural information for differentiating among these seven

compounds. Because of the unique similarity of these compounds by mass spectrometry,

the specific identification of a compound such as 3,4-MDMA requires methods to

eliminate other regioisomeric and isobaric substances.

This elimination process could be accomplished on the basis of chromatography

alone but ultimately would require the analyst to use reference samples of the other

substances. Derivatization of the primary and secondary amines with various acylating

agents yields amides with improved resolution compared to the underivatized amines by

capillary gas chromatography on TRx-1 and TRX-200 stationary phases. Additionally,

the perfluoroacyl derivatives significantly individualize the mass spectra for these amides

and allow for specific identification. The individualization is the result of fragmentation

of the alkyl carbon-nitrogen bond yielding characteristic hydrocarbon fragments at m/z

148, 162 and 176 as well as other unique fragments.


150

Chapter 5

Gas Chromatographic and Mass Spectral Studies on Acylated Side

Chain Regioisomers of 3-Methoxy-4-methyl-phenethylamine and 4-

Methoxy-3-methyl-phenethylamine

This chapter describes the MS and GC behavior of a set of selected compounds

having nonequivalent ring substituents at the 3- and 4-position of the aromatic ring. Thus,

these ten compounds represent all the possible regioisomeric methoxy methyl

phenethylamines having the same 3,4 substitution pattern as 3,4-MDMA.

Mass Spectral Studies on Side Chain Regioisomers of 3-Methoxy-4-methyl-

phenethylamine and 4-Methoxy-3-methyl-phenethylamine


related substances in forensic samples. The mass spectra of phenethylamines are

characterized by a base peak formed from an amine initiated alpha-cleavage reaction

involving the carbon-carbon bond of the ethyl linkage between the aromatic ring and the

amine. In 3,4-methylenedioxymethamphetamine (MW=193) the alpha-cleavage reaction

yields the substituted imine fragment at m/z 58 and the 3,4-methylenedioxybenzyl

fragment at mass 135/136 (for the cation and the radical cation, respectively). Thus, the

mass spectrum for 3,4-methylenedioxymethamphetamine contains major ions at m/z 58

and 135/136 as well as other ions of low relative abundance [ Chapter 1].

The side chain regioisomers of 3-methoxy-4-methyl phenethylamine and 4-

methoxy-3-methyl phenethylamine (Compounds 1-10, Figure 5.1) have the potential to


151

yield a mass spectrum essentially equivalent to 3,4-MDMA. All have molecular weight

of 193 and major fragment ions in their electron ionization mass spectra at m/z 58 and

135/136 (Figure 5.2). The individual mass spectra for 2,3- and 3,4-MDMA are also

presented in Figure 5.2 (Compounds 11 and 12). The isobaric methoxy-methyl-benzyl

(C9H11O)+ fragments have the same mass as the methylenedioxybenzyl (C8H7O2)+ cation

occurring at m/z 135. Furthermore the m/z 58 ion in the ring substituted methoxy-methyl

phenethylamine is regioisomeric with that obtained in the mass spectra of both 2,3 and

3,4-MDMA (Scheme5.1). This lack of mass spectral specificity for the isomers shown in

Figure 5.2, in addition to the possibility of chromatographic co-elution with 3,4-MDMA,

could result in misidentification in this series of drugs and drug-like substances

Figure 5.1: Structures of Side Chain Regioisomers of 3-Methoxy-4-methyl-

phenethylamine, 4-Methoxy-3-methyl-phenethylamine, 2,3- and 3,4-MDMA

H3C

OCH3

N

N,N-Dimethyl-3-methoxy-4-methyl

phenethylamine 1

H3CO

CH3

N

N,N-Dimethyl-4-methoxy-3-methyl

phenethylamine 6

H3C

OCH3

HN

N-Ethyl-3-methoxy-4-methyl

phenethylamine 2

H3CO

CH3

HN

N-Ethyl-4-methoxy-3-methyl

phenethylamine 7


152

H3C

OCH3

HN

3-Methoxy-4-methyl methamphetamine

3

H3CO

CH3

HN

4-Methoxy-3-methyl methamphetamine

8

H3C

OCH3

NH2

3-Methoxy-4-methyl phentramine

4

H3CO

CH3

NH2

3-Methoxy-4-methyl phentramine

9

H3C

OCH3

NH2

3-Methoxy-4-Methyl BDB

5

H3CO

CH3

NH2

4-Methoxy-3-Methyl BDB

10

HN

O

O

11

(2,3-MDMA)

O

O

HN

12

(3,4-MDMA)

.


153

HC

N

CH3

CH3

m/z = 135 m/z = 58

MW = 193

H2C

CH2

CH

CH3

HN

CH3

O

O

H2C

O

O

CH2

EI 70ev

H

C N

C2H8

CN

C2H8

m/z = 135

CH3

CH3

H3CO

H3CO

Scheme 5.1: Structures of the major EI fragments for 3-Methoxy-4-methyl-

phenethylamine, 4-Methoxy-3-methyl-phenethylamine, 2,3- and 3,4-MDMA

Figure 5.2: Mass Spectra of 3-Methoxy-4-methyl-phenethylamine, 4-Methoxy-3-

methyl-phenethylamine, 2,3- and 3,4-MDMA.

H3C

OCH3

N

1

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

m/z-->

Abundance

Average of 12.281 to 12.316 min.: 071022-01.D\data.ms58.1

91.142.1 77.0103.1 135.1 149.1115.0 193.1176.0161.9


154

H3C

OCH3

HN

2

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

70000

m/z-->

Abundance


136.1

91.177.044.0 103.1 149.1121.1 191.1176.1161.1

H3C

OCH3

HN

3

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

100000

200000

300000

400000

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600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

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2000000

2100000

2200000

m/z-->

Abundance


91.1135.1

77.1103.142.1 120.1 178.1162.1147.1 192.1


155

H3C

OCH3

NH2

4

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

900000

950000

1000000

m/z-->

Abundance


91.1135.1

77.1 178.142.1 103.1 121.1 161.1148.1 191.1

H3C

OCH3

NH2

5

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

320000

340000

m/z-->

Abundance


136.1

91.1

41.1 77.1121.1 164.1103.1

149.1 193.1176.1


156

H3CO

CH3

N

6

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

900000

950000

1000000

1050000

1100000

1150000

1200000

1250000

m/z-->

Abundance


91.142.1 135.177.1 149.1 193.2103.1 120.1 176.1162.1

H3CO

CH3

HN

7

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200

4400

4600

m/z-->

Abundance


136.144.0

91.0

77.0121.0 191.1149.0

103.0 176.0


157

H3CO

CH3

HN

8

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

900000

950000

1000000

1050000

1100000

1150000

m/z-->

Abundance


136.191.1

77.142.1 121.1103.1 162.1 178.1150.1 192.2

H3CO

CH3

NH2

9

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

m/z-->

Abundance


135.191.1

42.1 178.177.0 121.1103.1 161.1148.1 193.1


158

H3CO

CH3

NH2

10

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000

32000

34000

36000

38000

40000

42000

44000

46000

48000

50000

m/z-->

Abundance


136.1

91.1

41.1 121.074.5

164.1103.1 149.1

176.1 191.1

HN

O

O

11

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

m/z-->

Abundance


77.1 135.042.1 88.6 105.1 178.1 192.1118.1 163.1147.1


159

O

O

HN

12

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

m/z-->

Abundance


135.077.042.1 88.6 105.0 178.1148.1 193.1163.1120.1

The second phase of this study involved the preparation and evaluation of various

perfluoroacylated derivatives of the regioisomeric primary and secondary amines, in an

effort to individualize their mass spectra via formation of unique marker ions and

improved chromatographic resolution. Acylation of the amines generally lowers the

basicity of nitrogen and can allow other fragmentation pathways to play a more

prominent role in the resulting mass spectrum. Of course the tertiary amine (compounds 1

and 6) do not form a stable amide derivative.

The mass spectra for the twenty pentafluoropropionyl and heptfluorobutryl

amides are shown in Figures 5.3 and 5.4, respectively. From these spectra a common

peak occurs at m/z 204 and 254 which corresponds to the loss of 135 mass units from the

molecular ions at 339 and 389 for PFPA and HFPA amides. This ion at m/z 204 and 254

is the PFPA and HFPA imine species likely formed from the alpha cleavage of the amide


160

nitrogen to eliminate the methoxy-methyl-benzyl or methylenedioxybenzyl radical. Thus

the m/z 204 and 254 in PFPA and HFPA amides are analogous to m/z 58 in the

underivatized species because all these ions represent the (M-135)+ species (Scheme 5.2).

The 3-methoxy-4-methylbenzyl cation, 4-methoxy-3-methylbenzyl cation and the

methylenedioxybenzyl cation (m/z 135) are fragments common to all spectra in Figures

5.3 and 5.4.

Figure 5.3: Mass spectra of the PFPA derivatives of compounds 2-5 and 7-12

H3C

OCH3

N C2F5

O

2

40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

m/z-->

Abundance


176.0

204.1

339.191.1 119.0

56.1272.1 310.1229.1 251.0


161

H3C

OCH3

N C2F5

O

3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

m/z-->

Abundance


162.1

135.1

42.1 91.1339.1

65.1 300.1228.1 269.0113.0

H3C

OCH3

NHC2F5

O

4

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

m/z-->

Abundance


136.1

176.1

59.1 339.1

91.1

306.1237.1 282.1260.1114.0


162

H3C

OCH3

NH C2F5

O

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

m/z-->

Abundance


135.1

204.1

339.1

91.141.1

310.165.1 237.1 282.1113.0 260.1

H3CO

CH3

N C2F5

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

m/z-->

Abundance


91.1176.0

339.1204.165.142.1 272.1 294.0229.1113.0


163

H3CO

CH3

N C2F5

O

8

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

m/z-->

Abundance


162.1

204.1

91.142.1

339.165.0

269.0228.0 308.0

H3CO

CH3

NHC2F5

O

9

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

m/z-->

Abundance


204.0

176.1

91.059.0 339.1

263.1 306.1237.1113.1


164

H3CO

CH3

NH C2F5

O

10

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

m/z-->

Abundance


176.1

91.1 339.1

41.1 204.165.1 310.1263.1237.0113.0 288.1

N

O

O C2F5

O

11

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

m/z-->

Abundance


162.1

339.1119.042.1 77.1

300.1263.1241.1


165

O

O

N C2F5

O

12

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

m/z-->

Abundance


162.1

135.1

339.177.142.1105.1

269.0 300.1243.1

Figure 5.4: Mass Spectra of the HFBA derivatives of compounds 2-5 and 7-12

H3C

OCH3

N C3F7

O

2

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

m/z-->

Abundance


226.0

254.1

389.191.1

117.156.1 178.0

322.1 360.1282.0


166

H3C

OCH3

N C3F7

O

3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

m/z-->

Abundance


162.1

210.0

135.1

91.142.1

389.1

350.1293.1 319.0

H3C

OCH3

NHC3F7

O

4

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

m/z-->

Abundance


136.1176.1

59.1

389.191.1 214.0

307.1 356.1280.0


167

H3C

OCH3

NH C3F7

O

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

m/z-->

Abundance


135.1

254.1

389.1

91.1226.041.1

360.1307.1 334.1280.0

H3CO

CH3

N C3F7

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

m/z-->

Abundance


91.1

226.0

169.0 254.0 389.1

65.1196.9 322.140.1 359.1279.1


168

H3CO

CH3

N C3F7

O

8

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

2100000

2200000

2300000

m/z-->

Abundance


162.1

254.1

210.0

91.1

42.1

389.1

319.0293.1 350.1

H3CO

CH3

NHC3F7

O

9

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

3600000

m/z-->

Abundance


254.1

176.1

91.159.1 389.1

214.0313.1 356.1288.0


169

H3CO

CH3

NH C3F7

O

10

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

3600000

3800000

m/z-->

Abundance


176.1

91.1389.1

254.141.1226.0 360.166.0 313.1288.0201.0

N

O

O C3F7

O

11

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

m/z-->

Abundance

Scan 2013 (16.320 min): 071023-09.D\data.ms254.1

162.1210.1

135.1389.142.1 77.1

105.1350.1291.0 320.1


170

O

O

N C3F7

O

12

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

2100000

2200000

2300000

m/z-->

Abundance


162.1

135.1210.0

77.142.1 389.1

105.1

310.0 350.1282.9

N

R1 R2 X

R3

O


N

R1

R2

R3

X

O

m/z = 204, X= C2F5m/z = 254, X= C3F7

R1

R2

CH3

XCNH3C

m/z = 160 (X = C2F5)m/z = 210 (X = C3F7)Cpds ,3,8,11 and 12m/z = 148, R1=R2=H,cpd 2 and 7

m/z = 162, R1= CH3; R2=H,cpds ,3,8, 11 and 12m/z = 176, R1= CH3; R2= CH3, cpds 4 and 9m/z = 176,R1= C2H5; R2= H, cpds 5 and 10

H3CO

H3CO

CH3

Scheme 5.2: Structures of the major EI fragments for the perfluoroacylated 3-

Methoxy-4-methyl-phenethylamines and 4-Methoxy-3-methyl-phenethylamines


171

Indeed the m/z 135 ion is the base peak in all the PFPA and HFBA derivatives of

compounds 7-10 and this increased relative intensity may serve as an indicator ion for

discrimination of the 4-methoxy-3-methyl ring substitution pattern from other ring

substitution patterns in this study. The decreased role for alpha cleavage reaction in the

fragmentation of these amides allows the formation of ions more diagnostic of each

individual isomer. Acylation weakens the bond between nitrogen and the alpha-carbon


These hydrocarbons of varying mass identify the number of carbons attached directly to

the aromatic ring. The mass spectra in Figures 5.3 and 5.4 show hydrocarbon fragments

at m/z 148, 162 and 176 for a two-carbon, three-carbon and four-carbon chain attached

directly to the aromatic ring.

The spectra for the N-ethyl derivatives in Figures 5.3-2, 5.-7, 5.4-2 and 5.4-7

show a base peak at m/z 148 corresponding to the two-carbon alkene radical cation which

occurs from hydrogen rearrangement and subsequent fragmentation of the alkyl carbon to

nitrogen bond of the phenethylamine side chain. This ion at m/z 148 would only occur for

the N-ethyl regioisomer. The relative abundance of both m/z 148 and 135 offer a clear

discrimination of compound 2 from its direct regioisomer (compound 7) as well as from

the other isomers. The spectra in Figures 5.3-3, 5.3-8, 5.3-11, 5.3-12, 5.4-3, 5.4-8, 5.4-11

and 5.4-12 show the ring substituted methoxy-methyl or the

methylenedioxyphenylpropene hydrocarbon ion at m/z 162 (the three-carbon alkene

radical cation), identifying this molecules as the PFPA and HFBA derivatives containing

the methamphetamine side chain, compounds 3, 8, 11 and 12 respectively. The spectra

for the PFPA and HFBA derivatives of the primary amines 4, 5, 9 and 10 show ions at


172

m/z 176 from the four-carbon alkene radical cation of the 3-methoxy-4-methyl- and 4-

methoxy-3-methyl-phenethylamines. This m/z 176 results from hydrogen rearrangement

and subsequent fragmentation of the alkyl carbon to nitrogen bond yielding the

methoxymethyl-phenylbutene radical cation. The lower abundance of m/z 176 for

compounds 4 and 9 may be attributed to steric inhibition of hydrogen transfer in the

alpha, alpha-dimethyl substitution pattern.



ethylphenethylamines (Figures 5.3-2 and 5.3-7) is the appearance of an ion at m/z 176 in

addition to the base peak at m/z 148. Based on the previous discussion, the m/z 176 ion

suggests a four carbon chain directly attached to the aromatic ring as occurs for the alpha-

ethyl- and alpha, alpha-dimethyl-phenethylamines (Figures 5.3-4, 53-5, 5.3-9, 5.3-10 and

5.4-4, 5.4-5, 5.4-9, 5.4-10). The m/z 176 ion in the spectra for the PFPA derivatives of

the N-ethyl regioisomers (Figures 5.3-2 and 5.3-7) is a rearrangement of the m/z 204 ion

resulting in the loss of mass 28 (the N-ethyl group) via hydrogen transfer. This

coincidental common mass from two different fragmentation pathways is confirmed by

examining the mass spectra for the HFBA derivatives of the N-ethyl-phenethylamines

shown in Figures 5.4-2 and 5.4-7. The loss of 28 mass units from the acylimine fragment

at m/z 254 yields the equivalent fragment ion at m/z 226. Thus, the HFBA derivatives

may offer more characteristic ions for individualization of these regioisomeric substances

compared to the PFPA derivatives.

A comparison of the PFPA derivatives for compounds 3, 8, 11 and 12 (Figures

5.3-3, 5.3-8, 5.3-11 and 5.3-12) with their HFBA derivatives (Figures 5.4-3, 5.4-8, 5.4-


173

11 and 5.4-12) indicates unique ions at m/z 160 and m/z 210. This mass difference of 50

(CF2) suggests these ions contain the perfluoroalkyl group for each derivative, C2F5 and

C3F7 respectively. Additional information about these ions were obtained in previous

deuterium labeling studies which confirmed that methyl group on nitrogen is a part of this

resulting fragment. The remaining mass 26 would correspond to CN and the proposed

structures of m/z 160 and 210 are shown in Scheme 5.2.

Gas Chromatography of 3-Methoxy-4-methyl-phenethylamine, 4-Methoxy-3-

methyl-phenethylamine, 2,3- and 3,4-MDMA

The PFPA and HFBA derivatives of the primary and secondary amine side chain

regioisomers of the ring substituted methoxy methyl phenethyl amines, 2,3-MDMA and

3,4-MDMA were compared on a 100% dimethyl polysiloxane (Rtx-1) stationary phase.

Several temperature programs were evaluated and one program showing the best

compromise between resolution and analysis time was used to generate the

chromatograms in Figure 5.5. The chromatograms show that when the ring substitution

pattern is held constant (i.e. 3-methoxy-4-methyl- or 4-methoxy-3-methyl-) the two

secondary amides elute before the two tertiary amides. Additionally, in every case in this

limited set of compounds the branched side chain elutes before the straight chain isomer

when the ring substitution pattern and the degree of amide substitution are constant,

regardless of the derivatizing agent. Therefore, the alpha, alpha-dimethyl isomer elutes

first followed by the alpha-ethyl isomer (both secondary amides), then the

methamphetamine, and N-ethyl phenethylamines (the two tertiary amides).


174

When the side chain is held constant, the 3-methoxy-4-methyl ring substitution

pattern elutes before the 4-methoxy-3-methyl ring substitution pattern and this elution

order is the same for both the PFPA and the HFBA derivatives. Perhaps the most useful

information in these chromatograms is the relative elution of the derivatized controlled

substance 3,4-MDMA and its closest eluting regioisomeric equivalents. Both the PFPA

and HFBA derivatives of 3,4-MDMA elute after the N-ethyl-3-methoxy-4-methyl

phenethylamine and 4-methoxy-3-methylphenethylamine PFPAs and HFBAs, both the

N-ethyl regioisomers show very distinct mass spectra with several characteristic ions to

differentiate these compounds from the drug of abuse 3,4-MDMA. Thus, derivatization

methods coupled with chromatographic and mass spectral procedures can allow for the

characterization and differentiation of these ten uniquely isomeric substances. The

individualization is possible without the need for reference samples of all these uniquely

similar substances.


175

11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

Time-->

Abundance


11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

Time-->

Abundance


Figure 5.5: GC Separation of the PFPA (A) and HFBA (B) derivatives of

compounds 2-5 and 7-12; Rtx-1 column.

4

9

5

10 3

11 8

2 12

4

9

5 10 3

11

8

2

7 12

A

B


176


3,4-MDMA , 2,3-MDMA and ten side chain regioisomers of 3-methoxy-4-

methyl-phenethylamine and 4-methoxy-3-methyl-phenethylamine are a unique subset of

regioisomeric and isobaric molecules; each compound has a molecular weight of 193 and

yields a base peak at m/z 58 in the mass spectrum from the loss of the corresponding

methylenedioxybenzyl or the mass equivalent isobaric ring substituted methoxy methyl

benzyl groups. Thus the traditional electron impact mass spectrum provides little

structural information for differentiating among these ten compounds.

Derivatization of the eight primary and secondary amines with various acylating

agents yields amides which significantly individualize the mass spectra for these amides

and allow for specific identification. The individualization is the result of fragmentation

of the alkyl carbon-nitrogen bond yielding hydrocarbon fragments at m/z 148, 162 and

176 as well as other unique fragments from these regioisomeric amides. The PFPA and

HFBA derivatives are essentially equivalent for chromatographic purposes however; the

HFBA derivatives offer more unique fragment ions for additional discrimination among

these regioisomeric substances. Chromatographic resolution of the acylated amines was

achieved on relatively non polar stationary phase Rtx-1 (100% dimethyl polysiloxane).


177

Chapter 6

Mass Spectral and Gas Chromatographic Studies of Methoxy Methyl

Methamphetamines Isobaric to 3, 4-MDMA.

Isobaric substances are compounds of the same nominal mass but with different

elemental composition. There are isobaric substances, which do not contain the

methylenedioxy substitution pattern in the aromatic ring, yet they are able to yield the

same major fragments in their mass spectra as the controlled drug substance MDMA.

Among these MDMA isobaric substances are the ring substituted methoxy methyl

methamphetamines.

Mass Spectral Studies of Methoxy Methyl methamphetamines Isobaric to 3,4-MDMA

The mass spectrum of 3,4-MDMA is characterized by a base peak formed by an

-cleavage reaction involving the carbon-carbon bond of the ethyl linkage between the

aromatic ring and the amine. In 3,4-MDMA (MW=193) and its direct regioisomer, 2,3-

MDMA, the -cleavage reaction yields the 3,4-methylenedioxybenzyl and 2,3-

methylenedioxybenzyl fragments at mass 135/136 (for the cation and the radical cation,

respectively) and the substituted imine fragment at m/z 58. Thus, the mass spectrum for

3,4-MDMA contains major ions at m/z 58 and 135/136 as well as other ions of low

relative abundance.

The various methoxy methyl ring substitution patterns of methamphetamine have

the potential to yield mass spectra essentially equivalent to 3,4-MDMA and 2,3-MDMA,


178

all have molecular weight of 193 and major fragment ions in their electron ionization

mass spectra at m/z 58 and 135/136 (Figure 6.1). The isobaric methoxy methyl benzyl

(C9H11O)+ fragments have the same mass as the methylenedioxybenzyl (C8H7O2)+ cation

occurring at m/z 135. Furthermore the m/z 58 ion in the methoxymethamphetamine is the

same imine structure as that obtained in the mass spectra of both 3,4 and 2,3-MDMA

(Scheme 6.1).

HC

NH

CH3

CH3

m/z = 135

m/z = 58

70ev

MW = 193

HN

H3CO

H3C

CH2H3CO

H3C

Scheme 6.1: General mass spectral fragmentation for the ring substituted methoxy

methyl methamphetamines.


179

Figure 6.1: Structures and mass spectra of ring substituted methoxy methyl

methamphetamines

Structures

Mass spectra

HN

CH3OCH3

CH3 1

HN

CH3OCH3H3C

2

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

m/z-->


42

58

6577

82

91105

115121 135 146147 163 174178 192

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

500000

1000000

1500000

2000000

2500000

3000000

3500000

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HN

CH3OCH3

H3C

3

HN

CH3OCH3

CH3

4

HN

CH3CH3

OCH3

5

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HN

CH3H3C

OCH3

6

HN

CH3

H3C

OCH3

7

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CH3

cH3

OCH3

8

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182

HN

CH3H3CO

CH3

9

HN

CH3CH3H3CO

10

The mass spectra for the ten ring substituted methoxy methyl methamphetamines

in Figure 6.1 show only the major fragment ion at equivalent masses. This lack of mass

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183

spectral specificity in addition to the possibility of chromatographic co-elution, with 3,4-

MDMA, could result in misidentification of the target drug. Furthermore, the lack of

available reference samples for all ten of these isobaric molecules complicates the

individual identification of any one of these substances. This constitutes a significant

analytical challenge, where the use of gas chromatography-mass spectrometry (GC-MS)

must be based primarily upon the ability of the chromatographic system to separate the

“counterfeit substance” from the actual drug of interest. Additionally, the ability to

distinguish between these regioisomers directly enhances the specificity of the analysis

for the target drugs of interest.

Mass Spectral Studies of Perfluroacyl Derivatives Methoxymethyl Methamphetamines compared to 2,3- and 3,4-MDMA

The perfluoroacylated derivatives of the ten methoxy methyl methamphetamines

were prepared and evaluated for their ability to individualize the mass spectral properties

of these compounds and to maintain or improve chromatographic resolution. Acylation of

the amines significantly lowers the basicisty of nitrogen and can allow other

fragmentation pathways to play a more prominent role in the mass spectrum. These

perfluoroacyl derivatives allowed the unique fragmentation reactions which characterized

and served to individualize the side chain regioisomers. However, in these substances the

perfluoroacyl derivatives were less successful at differentiating the 2,3- from the 3,4- ring

substitution pattern of the identical side chain. Since all ten of the methoxy methyl

methamphetamines have the same side chain and have ten different substitution patterens,

perfluroacylation may not allow for complete compound individualization based only on


184

the observed mass spectrum. The mass spectra for the ten pentafluoropropionyl (PFPA)

and heptflourobutryl (HFBA) amides are shown in Figures 6.2 and 6.3. For comparative

purposes in this chapter 2,3- and 3,4-MDMA will be given the compounds number 11

and 12.

Figure 6.2: Mass spectra of the PFPA derivatives of methoxy methyl

methamphetamines, 2,3- and 3,4-MDMA

N

CH3

C2F5

O

OCH3

CH3

a

N

CH3

C2F5

O

H3COCH3

b

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135

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162

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339


185

N

CH3

C2F5

O

H3C

OCH3

c

N

CH3

C2F5

O

CH3

OCH3 d

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56 7791

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204

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339

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339


186

N

CH3

C2F5

O

CH3

OCH3 e

N

CH3

C2F5

O

OCH3

H3C

f

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339

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187

N

CH3

C2F5

O

OCH3

H3C

g

N

CH3

C2F5

O

OCH3

CH3

h

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162

176

204

339

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339


188

N

CH3

CH3

C2F5

O

CH3

H3CO

i

j

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77

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339

N

CH3

CH3

C2F5

O

CH3H3CO

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56 77

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220

339


189

O

O

N C2F5

O

k

O

O

N C2F5

O

l

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91

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339

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51 7791

119 135

147

162

177

204

220

339


190

Figure 6.3: Mass spectra of the HFBA derivatives of methoxy methyl

methamphetamines, 2,3- and 3,4-MDMA

N

CH3

C3F7

O

OCH3

CH3 a

N

CH3

C3F7

O

H3COCH3

b

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69 91

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256 342 389

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69 91

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115

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162

192

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220

254

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389


191

N

CH3

C3F7

O

H3C

OCH3 c

N

CH3

C3F7

O

CH3

OCH3 d

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220

254

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389

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162

192

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220

254

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389


192

N

CH3

C3F7

O

CH3

OCH3 e

N

CH3

C3F7

O

OCH3

H3C

f

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69 91105

131135

162

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220

254

293310 350 374

389

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69 91

105 131

135

162

191

210

220

254

278 310 332 350 374

389


193

N

CH3

C3F7

O

OCH3

H3C

g

N

CH3

C3F7

O

OCH3

CH3

h

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69 91105 131

135

162

191

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220

254

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389

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105115

135

162

192

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220

254

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389


194

N

CH3

CH3

C3F7

O

CH3

H3CO

i

N

CH3

CH3

C3F7

O

CH3H3CO j

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69

91

92 120

135162

192

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220

254

278 319

389

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105119

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162

192

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220

254

313330 372

389


195

O

O

N C3F7

O

k

O

O

N C3F7

O

l

All these spectra show major high mass fragment ion at m/z 204 or 254

corresponding to the loss of 135 mass units from the molecular ion 339 and 389 from the

PFPA and HFBA amides, respectively. The ions at m/z 204 and 254 are the PFPA and

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6977

105

131

135

162

192

210

220

254

263 291 310 332 350 374

389

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119

135

162

192

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220

254

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389


196

HFBA imine species likely formed from the alpha cleavage of the amide nitrogen to

eliminate the methoxymethylbenzyl radical. Thus the m/z 204 and 254 ions in PFPA and

HFBA amides are analogous to m/z 58 in the underivatized species because all these ions


m/z 204 and 254 ions are shown in Scheme 6.2. The methoxymethylbenzyl cation and

radical cation at m/z 135/136 is also a common fragment in most of the spectra in Figures

6.2 and 6.3. The identical fragmentation pathways for the PFPA and HFBA derivatives of

2,3- and 3,4-MDMA produced ions of the same structure at m/z 204 and 254 and isobaric

ions at equivalent masses for the benzylic species at m/z135/136.

N R

O

H3C

H3CO

EI 70 ev

N R

O

H3CO

H3C Scheme 6.2: Formation of m/z 204 (R=C2F5) and m/z 254 (R= C3F7) from

perfluoroaceyl derivatives of methoxy methyl methamphetamines.

Acylation, and in particular the perflouroacylation, weakens the bond between

nitrogen and the alpha-carbon of the substituted methoxy methyl phenethyl group,


The mass spectra in Figures 6.2 and 6.3 illustrate the role of hydrocarbon fragments at


197

m/z 162, 105 and 210 in the electron impact mass spectral differentiation among these

isobaric compounds.

The mass spectra for all derivatives (Figures 6.2 and 6.3) show a common peak at

m/z 162 corresponding to the alkene radical cation which occurs from hydrogen

rearrangement and subsequent fragmentation of the alkyl carbon to nitrogen bond of the

phenethylamine side chain (Scheme 6.3). The isobaric methylenedioxyphenylpropene

radical cation is observed in the mass spectrum of the PFPA and HFBA derivatives of

2,3- and 3,4-MDMA.The presence of the m/z 162 ion (as described previously) indicates

that a 3-carbon chain is attached directly to the aromatic ring in an uninterrupted manner.

The companion ion identifying the substituent on nitrogen as the N-methyl group occurs

at m/z160 in the PFPA derivatives (Figure 6.2) and at m/z 210 in the HFBA derivatives

(Figure 6.3)

Additionally the PFPA and HFBA derivatives of d3- and d5-MDMA, in Figure 1.4

and discussed earlier in chapter 1, also lend support to the proposed structure for the

formation of the alkene fragment at m/z 162 illustrated in Schemes 1.5 and 1.6 in chapter

1. A comparison of the PFPA derivatives (Figure 6.2) with the HFBA derivatives (Figure

6.3) indicated unique ions at m/z 160 and m/z 210. This mass difference of 50 (CF2)

suggests these ions contain the perfluoroalkyl group for each derivative, C2F5 and C3F7

respectively. The PFPA and HFBA derivatives of d3- and d5-MDMA, (Figure 1.4;

chapter 1) showed that the m/z 160 and m/z 210 ions contain the N-methyl group and

supports the proposed structure of the characteristic nitrile fragment.The suggested

mechanism of forming these masses was illustrated previously in Scheme 1.5; chapter 1.


198

H3CO

H3C

CH

CH3

N

R

H

O

CH3

H3CO

H3C

HC

CH3

N

RHO

CH3

H3CO

H3C

HC

CH3

R = C2F5 PFPA derivatives R= C3F7 HFBA Derivatives

m/z= 162

Scheme 6.3: Formation of m/z 162 from perfluoacyl derivatives of methoxy methyl

methamphetamines.

The mass spectra of the 2-methoxy-substituted methyl methamphetamines

derivatives shown in Figure 6.2 a, b, c, and d for the PFPA derivatives and Figure 6.3 a,

b, c and d for the HFBA derivatives show a more prominent m/z 105 ion than the other

substitution patterns. This ion at m/z 105 represents the loss of 30 mass units

(formaldehyde, CH2O) from the methoxymethylbenzyl cation at m/z 135. The further

loss of formaldehyde (CH2O) from those benzylic cations having an ortho-methoxy

group can be attributed to a 1,6-hydride shift from the carbon of the methoxy group to the

methylene of the methoxymethyl benzyl cation followed by another hydride

rearrangement and loss of formaldehyde to give the methyl benzyl cation (Scheme 6.4).


199

CH2

OCH2

H3CH

CH2

OCH2

H3C

CH2

H3C

H

H2C

H3C

H

m/z = 105 Scheme 6.4: Formation of m/z 105 form methoxymethylbenzyl radical.

As expected in this study, acylation of the side chain nitrogen in these isobaric

methamphetamines did not individualize the resulting mass spectra. Thus, differentiation

among these compounds and differentiation from 3,4-MDMA remains a significant

challenge for chromatographic studies. However, the mass spectra obtained for the PFPA

and HFBA derivatives do provide information which could allow these ten

methoxymethylmethamphetamines to be divided into three subsets based on the position

of the methoxy-group ring substitution.

The mass spectra of the PFPA and HFBA derivatives of the ortho-methoxy subset

(compounds 1-4) all show m/z 105 ion formed through the mechanism described in

Scheme 6.4. This ion does not occur to any significant extent in the derivatives of the

meta-methoxy of para-methoxy subsets. The m/z 105 ion is not observed in the mass

spectrum of the PFPA and HFBA derivatives of 2,3- and 3,4-MDMA. Thus the m/z 105

ion may distinguish this ortho-methoxy subset from MDMA but no specific ions were

observed to distinguish among the members of this subset, compounds 1-4.

The PFPA and HFBA derivatives of compounds 5-8 (methoxy group in the meta

postion of the aromatic ring) can be differentiated from the PFPA and HFBA derivatives


200

of compounds 1-4 by the absence of m/z 105. Perfluroacylation did not offer an

advantage in distinguishing these compounds from each other and from the MDMAs.

The para-methoxy subset, the PFPA and HFBA derivatives of compounds 9 and

10, shows a very different distribution of ions than that observed for either of the other

two subsets. The low mass ions at m/z 135 and m/z 162 show a very high relative

abundance and actually appear as the base peak in several spectra. These are the only

derivatives not showing the perfluoroacylimine at m/z 204 or m/z 254 as the base peak.

In summary, perfluoroacylation did not allow mass spectrometry to individualize

these compounds. The presence of the m/z 105 ion suggests the methoxy group is in the

ortho-position of the aromatic ring while the significant abundance of the low mass ions

at m/z 135 and m/z 162 indicates the methoxy group is substituted at the para-position of

the aromatic ring. The meta-substituted aromatic ring methoxy group isomers did not

show any unique fragments to distinguish them from 2,3- and 3,4-MDMA.

Gas chromatographic separation of the perfluroacyl derivatives of ring substituted

methoxy methyl methamphetamines, 2,3- and 3,4- MDMA.

The underivatized and PFPA and HFBA derivatives of 2,3- and 3,4-MDMA and

their isobaric substituted methoxy methyl methamphetamines were compared on four

stationary phases using capillary columns of the same dimensions, 30m x 0.25mm and

0.25um depth of film.

Several temperature programs were evaluated, however only one program

yielding the best compromises between resolution and analysis time was used to generate

the twelve chromatograms for the PFPA and HFBA derivatives in Figures 6.5-6.10,


201

respectively. The program (TP-2) was set up to hold the column temperature at 70 oC for

1 minute, ramped to 150 oC at 7.5 oC/minute, hold at 150 oC for 2 minutes and finally

ramped to 250 oC at 10 oC/minute. Only chromatograms generated on Rtx-1 and Rtx-35

are shown in Figures 6.5-6.10.

The co-elution of some underivatized, PFPA and HFBA derivatives of 2,3-, 3,4-

MDMA and their isobaric substances varied from one stationary phase to another.

Underivatized compound 2 co-elutes with compound 4 while underivatized compound 6

co-elutes with underivatized compounds 7 and 9 on Rtx-1 stationary phase. Each of the

PFPA and HFBA derivatives of compounds 8 and 9 co-elute, also the HFBA derivatives

of compounds 4 and 6 co-elute on the Rtx-1 stationary phase.

Four sets of underivatized compounds were found to co-elute on Rtx-5, 2,3-

MDMA co-elutes with compound 6, compound 2 co-elutes with compound 4, compound

5 co-elutes with compound 8 and finally compound 7 co-elutes with compound 9. In case

of the PFPA derivatives of the 2,3- , 3,4-MDMA and their isobaric substituted methoxy

methyl methamphetamine, there are two sets of compounds co-elute namely compound

11 co-elutes with compound 7 and compound 8 co-elutes with compound 9. Three sets of

the HFBA derivatives were found to co-elute, compound 11 co-elutes with compound 7,

compound 1 co-elutes with compound 3 and finally compound 5 co-elutes with

compound 8on the same column.

Three sets of underivatized compounds were found to co-elute on Rtx-35,

compound 4 co-elutes with compound 6, compound 5 co-elutes with compound 8 and

finally compound 7 co-elutes with compound 9. In case of the PFPA derivatives of the

2,3-, 3,4-MDMA and their isobaric substituted methoxy methyl methamphetamine, there


202

are two sets of compounds which co-elute where compound 5 co-elutes with compound 9

and compound 4 co-elutes with compound 6. The HFBA derivatives of compounds 2, 8

and 9 co-elute on the same column.

The best resolution, among the evaluated columns, was achieved on Rtx-200 where in a

physical mixture of the 12 PFPA derivatized compounds yielded 11 peaks with

compounds 7 and 9 co-elute (Figure 6.4). The PFPA derivatives allow more distinctive

differentiation between compounds 7 and 9 based on mass spectrometry. Compound 9

has a base peak at m/z 135 compared to m/z 204 base peak for compound 7. Thus a

sample containing compound 7 or 9 could be identified based on mass spectrometry. The

similarity in chromatographic properties among these regioisomeric and isobaric

molecules in the derivatized and underivatized form provides for a significant

chromatographic challenge. However, all four of the stationary liquid phases evaluated in

this study successfully resolved 3,4-MDMA from the other isomers. The variation in

chromatographic selectivity among the phases resulted in various coelutions within the

isobaric methoxy methyl methamphetamines. Since mass spectrometry of the

perfluoroacyl derivatives of the isobaric methoxymethyl methamphetamines (compounds

1-10) successfully divided these compounds into subsets based on the ring position of the

methoxy group, the chromatographic properties were evaluated using the same subsets.

The chromatographic properties of each subset of compounds was compared to 2,3- and

3,4-MDMA.


203

Abundance

Time (minutes)

Figure 6.4: Capillary gas chromatograph of a physical mixture of the PFPA

derivatives of methoxy methyl methamphetamines (Compounds 1-10), 2,3- (11) and

3,4-MDMA (12) . Column used: RTX-200.

In all the chromatographic studies 3,4-MDMA elutes last in every subset and in

every form (derivatized and underivatized). In the first subset (the ortho-methoxy

substituted aromatic ring), compounds 1-4, along with 2,3-MDMA and 3,4-MDMA, the

elution order of the perfluroacyl derivatives of these compounds was compound 1

followed by 3, 2, 4, 2,3-MDMA- and finally 3,4-MDMA (compound 12). The elution

order, on Rtx-1 and Rtx-35, was the same for this subset of compounds. ( Figures 6.5 and

6.6). This subset of isobaric amines was identified as having a significant m/z 105 ion in

their mass spectra.

In the second subset (the meta-methoxy substituted aromatic ring), compounds 5-

8 along with 2,3-MDMA and 3,4-MDMA, the PFPA derivative of compound 6 elutes

12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.000

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3 2

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8

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12


204

first followed by 2,3-MDMA-PFPA, 7-PFPA, 8-PFPA, 5-PFPA and finally 3,4-MDMA-

PFPA(compound 12); (Figure 6.7A). The elution order is the same for the HFBA

derivatives of the same compounds on Rtx-1 (Figures 6.8A). This elution order has

changed on Rtx-35, where compound 7 elutes before 2,3-MDMA, and most significantly

2,3-MDMA (compound 11) and 8 co-elute. This change in elution order takes place for

the PFPA and HFBA of these compounds (Figures 6.7b and 6.8b) which suggests that

Rtx-35 is not the best column for resolving this subset of compounds. It is this subset of

isobaric amines that showed no distinguishing characteristics (neither unique fragment

ions nor unique relative abundance of fragment ions) in their mass spectra.

In the third subset (the para-methoxy substituted aromatic ring), compounds 9, 10,

2,3-MDMA and 3,4-MDMA, the elution order of the perfluroacyl derivatives was 2,3-

MDMA followed by 9, 10 and finally 3,4-MDMA. The elution order was the same on

both Rtx-1 and Rtx-35 (Figures 6.9 and 6.10). It is this subset of compounds which

showed mass spectra most easily distinguished from the other subsets and from 2,3- and

3,4-MDMA.


205

Abundance

Time (minutes)


compounds 1-4, 2,3-MDMA and 3,4-MDMA. Columns used: A RTX-1;

B RTX-35.

15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

Time-->


1

3

2 4

11

12

15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

Time-->


1

3

2 4

11

12

A

B


206

Abundance

Time (minutes)

Figure 6.6: Capillary gas chromatographic separation of HFBA derivatives of

compounds 1-4, 2,3-MDMA and 3,4-MDMA. Columns used: A RTX-

1; B RTX-35.

14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.000

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

Time-->


1

3

2 11

12

16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.500

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

Time-->


1

3 2 4

11

12

A

B

4


207

Abundance

Time (minutes)


compounds 5-8, 2,3-MDMA and 3,4-MDMA. Columns used: A

RTX-1; B RTX-35.

14.00 15.00 16.00 17.00 18.00 19.00 20.000

200000

400000

600000

800000

1000000

Time-->


6

8

5 1

12

6

7

8 + 11

5

A

B

15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

Time-->


7

11


208

Abundance

Time (minutes) Figure 6.8: Capillary gas chromatographic separation of HFBA derivatives of

compounds 5-8, 2,3-MDMA and 3,4-MDMA. Columns used: A

RTX-1; B RTX-35.

6

11 7

5

12

17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.500

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

Time-->


6

7

8+11

5

12

16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.000

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

Time-->


A

B

8


209

Abundance

.

Time (minutes)


compounds 9-10, 2,3-MDMA (11) and 3,4-MDMA(12) . Columns

used: A RTX-1; B RTX-35.

15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

Time-->


9

10

12

17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.500

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

Time-->


11

9

10

12

A

B

11


210

Abundance

Time (minutes)

Figure 6.10: Capillary gas chromatographic separation of HFBA derivatives of

compounds 9-10, 2,3-MDMA(11) and 3,4-MDMA(12). Columns

used: A RTX-1; B RTX-35.

16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500

100000

200000

300000

400000

500000

600000

700000

800000

900000

Time-->


11

9

10

12

16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.500

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

Time-->


11

9

10

12

A

B


211


The methoxy methyl methamphetamines have an isobaric relationship to the 2,3-

and 3,4-methylenedioxymethamphetamines and represent a unique analytical challenge

in forensic drug chemistry. Each of these compounds has a molecular weight of 193 and

yields a base peak for the N-methyl imine of acetaldehyde at m/z 58 in the mass

spectrum. This ion represents the loss of 135 mass units from the molecular ion

corresponding to the substituted benzyl species, C8H7O2 (methylenedioxybenzyl) and

C9H11O (methoxy methyl benzyl). Thus, the traditional electron impact mass spectrum

provides little structural information for differentiating among these 12 compounds.

Because of the unique similarity of these compounds by MS, the specific identification of

a compound such as 3,4-MDMA requires methods to eliminate the other isomers. The

ring-substituted methoxy methyl methamphetamines were evaluated for their

chromatographic and mass spectral properties compared to 2,3- and 3,4-MDMA. The

mass spectra studies showed almost identical mass spectra for all 10 methoxy methyl

methamphetamines and 2,3- and 3,4-MDMA.

The perfluoroacylated derivatives of the 10 methoxy methyl methamphetamines

were prepared and evaluated for their ability to individualize the mass spectra of these

compounds and to maintain or improve chromatographic resolution. The

perfluoroacylation process did not produce unique mass spectra characteristic of each

compound. However, derivatization did subdivide the methoxy methyl

methamphetamines into subgroups based on the position of the methoxy group on the

aromatic ring relative to the alkylamine side-chain. The presence of the m/z 105 ion

suggests the methoxy group is in the 2-position relative to the alkylamine side-chain, and


212

the significant abundance of the low mass ions at m/z 135 and m/z 162 indicates the

methoxy group is substituted at the 4-position of the aromatic ring. The 3-substituted

aromatic ring methoxy group isomers did not show any unique fragments to distinguish

them from 2,3- and 3,4-MDMA as the perfluoroacylated derivatives.

Different stationary phases and temperature programs were used in an effort to

separate the methoxy methyl methamphetamines from 2,3- and 3,4-MDMA. The best

resolution among the evaluated columns was achieved on Rtx-200, where a mixture of

the 12 compounds gave 11 peaks and only the PFPA derivatives of methoxy methyl

methamphetamines 7 and 9 coelute. The perfluoroacyl derivatives of the methoxy methyl

methamphetamines were divided into three subsets based on the ring substitution pattern

of the methoxy group. The derivatives of the individual subsets were each resolved on

Rtx-1.


213

Chapter 7

GC–MS Analysis of Ring and Side Chain Regioisomers of

Ethoxyphenethylamines

The target compounds of this chapter are a series of ring substituted ethoxy

phenethylamines with molecular weight 193 and the potential to produce mass spectra

with major fragment ions at m/z 58 for the imine and m/z 135/136 for the substituted

benzyl fragment. The major fragment ions observed in the EI mass spectrum for 3,4-

MDMA (MW = 193) occur at equivalent masses.

Mass spectrometry of Ring and Side Chain Regioisomers of Ethoxyphenethylamines

MS is the primary method for confirming the identity of drugs and related

substances in forensic samples. The mass spectra of phenethylamines are characterized

by a base peak formed from an amine initiated alpha-cleavage reaction involving the

carbon–carbon bond of the ethyl linkage between the aromatic ring and the amine. In 3,4-

methylenedioxymethamphetamine (MW = 193) the alpha-cleavage reaction yields the

substituted imine fragment at m/z 58 and the 3,4-methylenedioxybenzyl fragment at mass

135/136 (for the cation and the radical cation, respectively). Thus, the mass spectrum for

3,4-methylenedioxymethamphetamine contains major ions at m/z 58 and 135/136 as well

as other ions of low relative abundance. The ring substituted ethoxy methamphetamines

(Compounds 1–3, Figure 7.1) have the potential to yield a mass spectrum essentially

equivalent to 2,3- and 3,4-MDMA. There are five possible side chain regioisomers for

each one of these ring substitution patterns.


214

The five side chain regioisomers of 2-ethoxyphenethylamine included in this study are

shown in Figure 7.1 (Compounds 1, 4–7). All five side chain regioisomers have

molecular weight of 193 and major fragment ions in their electron ionization mass

spectra at m/z 58 and 135/136 (Scheme 7.1). The individual mass spectra for 2,3- and

3,4-MDMA are also presented in Scheme 7.1 (Compounds 8 and 9). The isobaric ethoxy

benzyl (C9H11O)+ fragments have the same mass as the methylenedioxybenzyl (C8H7O2)+

cation occurring at m/z 135. Furthermore the m/z 58 ion in the 2-ethoxyphenethylamines

is regioisomeric with that obtained in the mass spectra of both 2,3 and 3,4-MDMA

(Figure 7.2).

HC

N

CH3

CH3

m/z = 135

m/z = 58

MW = 193

H2C

CH2

CH

CH3

HN

CH3

O

O

H2C

O

O

CH2

EI 70ev

H

C N

C2H8

CN

C2H8

m/z = 135

OC2H5

OC2H5

Scheme 7.1: EI fragmentation pattern for ring substituted ethoxy phenethylamines. Figure 7.1: Structures of the ring substituted ethoxymethamphetamines, side chain regioisomerics of 2-ethoxyphenethylmethamphetamines, and 2,3- and 3,4-MDMA.


215

HN

OC2H5 1

HN

OC2H5 2

HN

C2H5O

3

N

OC2H5

4

HN

OC2H5 5

NH2

OC2H5

6

NH2

OC2H5 7

HN

O

O

8

O

O

HN

7 (3,4-MDMA)


216

This lack of mass spectral specificity for the isomers shown in Figure 7.2, in addition to

the possibility of chromatographic co-elution with 3,4-MDMA, could result in

misidentification in this series of drugs and drug-like substances.

Figure 7.2: Mass Spectra of compounds ring substituted ethoxy Phenethylamines,

2,3- and 3,4-MDMA. HN

OC2H5 1

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

m/z-->

Abundance


77.1 91.1 107.142.1 135.1 178.1119.1 149.1 192.1163.1


217

HN

OC2H5 2

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

m/z-->

Abundance


77.0107.142.1 91.0 135.1

178.1120.1 148.1 192.2162.1

HN

C2H5O 3

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

5000

10000

15000

20000

25000

30000

35000

m/z-->

Abundance


107.044.077.0 135.091.0 121.0 148.0 178.1162.1 191.1


218

N

OC2H5 4

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

m/z-->

Abundance


91.142.1 77.1121.1107.1 193.1133.1 146.1 162.1 178.1

HN

OC2H5 5

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

m/z-->

Abundance


136.191.177.0107.1 121.142.1 193.2149.1 178.1163.1


219

NH2

OC2H5 6

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

900000

950000

1000000

1050000

1100000

1150000

1200000

1250000

1300000

1350000

1400000

1450000

1500000

1550000

m/z-->

Abundance


77.091.042.1 107.1

178.1135.1149.1119.0 161.1 193.1

NH2

OC2H5 7

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

50000

100000

150000

200000

250000

300000

350000

400000

450000

m/z-->

Abundance


91.077.0 136.141.1 107.1164.1121.0 150.1 193.1178.1


220

HN

O

O

8

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

m/z-->

Abundance


77.1 135.042.1 88.6 105.1 178.1 192.1118.1 163.1147.1

O

O

HN

9

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

m/z-->

Abundance


135.077.042.1 88.6 105.0 178.1148.1 193.1163.1120.1


221

In the next phase of this study, various Acylated derivatives of the ring substituted

ethoxy methamphetamines and the primary and secondary amines of the side chain

regioisomers of 2-ethoxyphenethylamine were prepared and evaluated in an effort to


identification. Acylation of the amines significantly lowers the basicity of nitrogen and

can allow other fragmentation pathways to play a more prominent role in the resulting

mass spectrum. Of course the tertiary amine (compound 4) does not form a stable amide

derivative. The mass spectra for the pentafluoropropionyl and heptafluorobutyryl amides

are shown in Figures 7.3 and 7.4, respectively. Perfluroacyl derivatives of the ring

substituted ethoxy methamphetamines (Compounds 1–3) have almost identical mass

spectra with the derivatized 2,3- and 3,4-MDMA (Compounds 8 and 9) except for a

unique ion at m/z 107. This ion at m/z 107 represents the loss of 28 mass units (ethylene,

C2H4) fromthe ethoxybenzyl cation at m/z 135 (Scheme 7.2). Although the relative

abundance varies significantly, them/z 107 ion is present in all mass spectra of the PFPA

and HFPA of compounds (1–3 and 5–7) and offers a unique fragment ion to discriminate

these compounds from 3,4- and 2,3-MDMA. The m/z 107 ion is also present in relatively

low abundance in the mass spectra of the underivatized ethoxy amines shown in Figure

7.2.

Figure 7.3: Mass Spectra of the HFBA derivatives of ring substituted ethoxy

Phenethylamines, 2,3- and 3,4-MDMA.


222

N

OC2H5

C3F7

O

1

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

2100000

2200000

2300000

2400000

m/z-->

Abundance


162.1

210.0

135.1

91.1

42.1

389.1342.1309.0283.1

N C3F7

O

OC2H5 2

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

m/z-->

Abundance


162.1

210.0

42.1135.1107.177.1

389.1350.1309.0282.0


223

N C3F7

O

C2H5O 3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

m/z-->

Abundance


254.0

135.1

107.1

210.0

42.1 77.0

389.1319.0 360.1291.1

N C3F7

OC2H5

O

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

900000

950000

m/z-->

Abundance


226.091.1

254.1

120.1389.1

56.1 178.0342.1295.1


224

NH

OC2H5

C3F7

O

6

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

m/z-->

Abundance


136.1

59.1 107.1176.1

214.0

389.1

350.1302.0

NH

OC2H5

C3F7

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

m/z-->

Abundance


176.1

107.1

254.1

77.1

41.1

389.1226.0

360.1324.1285.0


225

N

O

O C3F7

O

8

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

m/z-->

Abundance


162.1

210.0

135.042.1 389.177.0

105.0

350.0308.9282.9

O

O

N C3F7

O

9

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

m/z-->

Abundance


162.1

135.0

210.0

77.042.1

389.1105.0

318.9 350.1292.9


226

Figure 7.4: Mass Spectra of the PFPA derivatives of the ring substituted ethoxy

Phenethylamines, 2,3- and 3,4-MDMA.

N

OC2H5

C2F5

O

1

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

m/z-->

Abundance


162.1

135.1

91.142.1

339.165.0 294.1233.0113.1 258.9

N C2F5

O

OC2H5 2

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

m/z-->

Abundance


162.1

119.042.1 77.0339.1

300.1254.0 278.0232.0


227

N C2F5

O

C2H5O 3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

m/z-->

Abundance

Average of 15.912 to 15.993 min.: 092607-29.D\data.ms162.1 204.0

135.1107.0

42.1 77.0

339.1268.9 309.9240.9

N C2F5

OC2H5

O

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

m/z-->

Abundance


91.1

176.0

204.0

119.0

339.156.0

292.1244.9


228

NH

OC2H5

C2F5

O

6

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

50000

100000

150000

200000

250000

300000

350000

400000

450000

m/z-->

Abundance


136.1

59.1 107.1176.1

339.184.0 282.1245.0 311.0

NH

OC2H5

C2F5

O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

m/z-->

Abundance


176.1

107.1

204.077.041.1

339.1310.1274.0250.1


229

N

O

O C2F5

O

8

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

100000

200000

300000

400000

500000

600000

700000

800000

900000

m/z-->

Abundance


162.1

119.042.1 339.177.0

262.9 300.1240.9140.7

O

O

N C2F5

O

9

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

m/z-->

Abundance


162.1

135.0

77.042.1 339.1

105.0268.9 300.1242.9


230

OH2C

H3C

O HO

CH2

CH2

H2C

H

CH2CH2

OH2C

H3C

CH2R

HO

CH2

R

HO

CH2

and / or

Scheme 7.2: mass spectral fragmentation of ethoxyphenethylamines yielding cation

at m/z 107

These spectra in Figures 7.3 and 7.4 show a common peak at m/z 204 and 254,

which corresponds to the loss of 135 mass units from the molecular ions at 339 and 389

for the PFPA and HFBA amides. This ion at m/z 204 and 254 is the PFPA and HFBA

imine species formed from the alpha cleavage of the amide nitrogen to eliminate the

ethoxy benzyl radical or themethylenedioxybenzyl radical. Thus the m/z 204 and 254

ions in the PFPA and HFBA amides are analogous to m/z 58 in the underivatized species

because all these ions represent the (M-135)+ species. The general fragmentation pattern

and structures for the m/z 204 and 254 ions are shown in Scheme7.3. The ethoxy benzyl

cation (m/z 135) and the methylenedioxybenzyl cation (m/z 135) are fragments common

to all spectra in Figures 7.3 and 7.4. However, the ethoxy benzyl cation in the

perfluoroacyl derivatives shows a very high relative abundance in some of these isobaric

compounds.

Indeed them/z 135 ion is the base peak in the PFPA andHFBA derivatives of Compound

7. This would suggest that the perfluoroacyl derivatives offer some level of


231

discrimination between the methylenedioxy and ethoxy ring substitution patterns based

on the difference in relative abundances of the substituted benzyl cation at m/z 135.

N

R1 R2 X

R3

O


N

R1

R2

R3

X

O

m/z = 204, X= C2F5m/z = 254, X= C3F7

OC2H5

R1

R2

OC2H5

XCNH3C

m/z = 160 (X = C2F5)m/z = 210 (X = C3F7)Cpds 1,2,3,8 and 9m/z = 148, R1=R2=H,cpd 5

m/z = 162, R1= CH3; R2=H,cpds 1,2,3,8 and 9m/z = 176, R1= CH3; R2= CH3, cpd 6m/z = 176,R1= C2H5; R2= H, cpd 7 Scheme7.3: Mass spectral fragmentation products for the acylated ethoxy

phenethylamines.


amides allows the formation of ions more diagnostic of individual side chain isomers.

Acylation weakens the bond between nitrogen and the alkyl carbon of the phenethyl side

chain, allowing the formation of charged hydrocarbon species of increased relative

abundance. These hydrocarbons of varying mass significantly individualize the mass

spectra and provide specific structural information. The mass spectra in Figures 7.3 and

7.4 illustrate the role of hydrocarbon fragments at m/z 148, 162, and 176 in the electron

impact mass spectral discrimination among the side chain regioisomers. The spectra for


232

the N-ethyl isomer (compound 5) in Figures 7.3-5 and 7.4-5 show a base peak at m/z 148

corresponding to the alkene radical cation which occurs from hydrogen rearrangement

and subsequent fragmentation of the alkyl carbon to nitrogen bond of the phenethylamine

side chain (see Scheme 7.3). This ion at m/z 148 would only occur for the N-ethyl

regioisomer. The spectra in Figures 7.3-1, 7.3-2, 7.3-3, 7.3-4,7.3-9, 7.4-1, 7.4-2, 7.4-3,

7.4-4 and 7.4-9 show the substituted phenylpropane hydrocarbon ion at m/z 162,

identifying these molecules as the

PFPA and HFBA derivatives of 2-, 3-, 4-ethoxymethamphetamines and the 2,3-, 3,4-

methylenedioxymethamphetamines, respectively. However, the base peak of m/z 162 and

relatively high abundance m/z 107 in the spectra of the perfluroacyl derivatives of

compound 3 offer a significant discrimination of 4-ethoxymethamphetamines over the 2-,

3-ethoxymethamphetamines and the two isobaric methylenedioxymethamphetamines

isomers (Compounds 1, 2, 8, and 9 in Figures 7.3 and 7.4). The spectra for the PFPA and

HFBA derivatives of the primary amines (compounds 6 and 7 in Figure 7.3 and 7.4)

show ions at m/z 176 from the corresponding substituted phenylbutene radical cation.

The lower abundance of m/z 176 for the 2-ethoxy phentermine (Compound 6) may be

attributed to steric inhibition of hydrogen transfer in the alpha, alpha-dimethyl

substitution pattern.



ethylphenethylamines [Figure 7.4-5] is the appearance of an ion at m/z 176 in addition to

the base peak at m/z 148. The 176 ion might suggest a four carbon chain directly attached

to the aromatic ring as occurs for the alpha-ethyl- (Compound 7) and alpha, alpha-


233

dimethyl- (Compound 6) phenethylamines [Figures 7.3-6, 7.3-7, and 7.4-6, 7.4-7]. The

m/z 176 ion in the spectra for the PFPA derivatives of the N-ethyl regioisomers [Figure

7.4-5] is a rearrangement of the m/z 204 ion resulting in the loss of mass 28 (the N-ethyl

group) via hydrogen transfer. This coincidental common mass from two different


derivatives of the N-ethyl-phenethylamines shown in Figure 7.3-5. The loss of 28 mass

units from the acylimine fragment at m/z 254 yields the equivalent fragment ion atm/z

226. Thus, the HFBA derivatives may offer more characteristic ions for individualization

of these regioisomeric substances.

A comparison of the mass spectra for the PFPA and HFBA derivatives of all ring

substituted methamphetamines (Compounds 1, 2, 3, 8, and 9) indicates unique ions at m/z

160 and m/z 210 [see Figures 7.3-1, 7.3-2, 7.3-3, 7.3-8, 7.3-9, 7.4-1, 7.4-2, 7.4-3, 7.4-8

and 7.4-9]. This mass difference of 50 (CF2) suggests these ions contain the

perfluoroalkyl group for each derivative, C2F5 and C3F7, respectively. An evaluation of

the masses of the components which make up the fragment at m/z 160 for example

include C2F5 (119 mass units) and CH3 (15 mass units).

Previous deuterium labeling studies have confirmed that methyl group on nitrogen is a

part of this resulting fragment (Chapter 1). The remaining mass 26 would correspond to

CN and the proposed structures of m/z 160 and 210 are shown in Scheme 7.3. An

equivalent fragmentation pathway has been reported for methamphetamine and further

supported by the analysis of the mass spectra of the PFPA and HFBA derivatives of d3-

and d5-MDMA in a previous study (Chapter1).


234

Gas Chromatography of Ring substituted Ethoxy Phenethylamines, 2,3- and 3,4-

MDMA.

The PFPA and HFBA derivatives of the ring substituted ethoxy

methamphetamines and the primary and secondary side chain regioisomers of the 2-

ethoxyphenethylamine were compared on the relatively non polar 100% dimethyl

polysiloxane (Rtx-1).

Four physical mixtures were prepared, two containing the HFBA and the PFPA

derivatives of 2-, 3-, and 4-ethoxymethamphetamine along with 2,3- and 3,4 MDMA,

while the other two contained the side chain regioisomers of 2-ethoxyphenethylamine

(Compounds 1, 5, 6 and 7) with the isobaric 2,3- and 3,4-MDMAs. Several temperature

programs were evaluated and one program showing the best compromise between

resolution and analysis time was used to generate the retention data in Table I and the

chromatograms in Figures 7.5 and 7.6. Chromatograms shows that 3,4- MDMA has the

highest affinity for the stationary phase among this set of compounds both in the free

amine form and the two amide forms investigated in this study.

The chromatograms in Figure 7.5 show the separation of the PFPA (Figure 7.5A)

and HFBA (Figure 7.5B) derivatives of the five compounds having the methamphetamine

side chain. This common side chain allows for a comparison of relative stationary phase

affinity as a function of ring substituents in this set of compounds. The 2-ethoxy

substitution pattern shows the least retention and compound 1 has the lowest retention

time. The 3-ethoxymethamphetamine (Compound 2) elutes second followed by 2,3-

MDMA (Compound 8) then the 4-ethoxy substituent compound 3. The compound

showing the highest retention time and eluting last is 3,4-MDMA (Compound 9). Among


235

the ethoxy substituents the 2-isomer shows the least retention followed by the 3-isomer,

and the 4-ethoxy isomer has the greatest retention on the Rtx-1 phase. The elution order

is the same for both the PFPA and HFBA derivatives with the HFBA derivatives having

slightly greater retention under identical chromatographic conditions.

The chromatograms in Figure 7.6 show the separation of the side chain regioisomers as

the ring substitution pattern is held constant (2-ethoxy). The side chain elution order is

secondary amides (Compounds 6 and 7) before the tertiary amides (Compounds 1 and 5)

and in this limited set of examples branched isomers elute before the more linear ones.

Therefore, the amides of compound 6 elute first followed by the amide of compound 7

(both secondary amides), then the amides of compound 1 (the methamphetamine side

chain) and finally the amides of compound 5 (the more linear of the tertiary amides).

The methylenedioxy isomers (Compounds 8 and 9) elute later than any of the side chain

isomers of the 2-ethoxy ring substitution pattern. Thus, these chromatographic results

coupled with the mass spectral data allow for the individualization of each member of

this series. The derivatized side chain isomers separated in Figure 7.6 can be

differentiated by unique fragment ions in their mass spectra in addition to the well

resolved chromatographic results. The compounds having a common side chain are well

resolved in Figure 7.5 and them/z 107 fragment ion allows for the identification of those

compounds having the ethoxy ring substituent.


236

1

2

8

3

9

1

3

8

9

2

A

B

11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

Time-->

Abundance


11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

Time-->

Abundance



compounds 1-3,8 and 9; Rtx-1 column


237

6

7 1

5

8

9

6

7

1 5

8 9

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

Time-->

Abundance


10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

6500000

7000000

7500000

8000000

8500000

Time-->

Abundance



compounds 1, 5,6,7,8 and 9; Rtx-1 column


238


3,4-MDMA, 2,3-MDMA, and the three ring substituted ethoxy

methamphetamines are a unique subset of regioisomeric and isobaric molecules; each

compound has a molecular weight of 193 and yields a base peak at m/z 58 in the mass

spectrum from the loss of the corresponding methylenedioxybenzyl and ring substituted

ethoxybenzyl groups, respectively. The traditional electron impact mass spectrum

provides little structural information for differentiating among these seven compounds.

Derivatization with acylating agents yields amides with improved resolution compared to

the underivatized amines by capillary gas chromatography on the Rtx-1 stationary phase.

Additionally, the perfluoroacyl derivatives significantly individualize the mass

spectra for these amides and allow for specific identification. The ring substituted ethoxy

methamphetamines are characterized from 2,3- and 3,4-MDMA by the presence of m/z

107. Side chain regioisomers yield unique hydrocarbon fragment ions at m/z 148, 162

and 176.


239

Chapter 8

GC-MS Studies on Direct Side Chain Regioisomers Related to

Substituted Methylenedioxyphenethylamines; MDEA, MDMMA and

MBDB

There are an additional seven direct side chain regioisomeric phenethylamines

related to the controlled substances MDEA, MDMMA, and MBDB. The structures of this

set of compounds are shown in Figure 8.1.

A previous report from our research group included the synthesis and mass spectral

evaluation of these ten regioisomeric compounds. The mass spectra of the underivatized

compounds provided very little structural information for the specific differentiation

among these regioisomers even though these regioisomers were separated by capillary

gas chromatography.

In this chapter, derivatization of the primary and seconday amines was carried out in

an effort to obtain more specific ions that would help discriminating among the members

of this set of compounds.

Mass Spectral Studies

Mass spectrometry is the primary method for confirming the identity of drugs in

forensic samples. Figure 8.2 shows representative electron impact mass spectra of the

three drugs of abuse; MDEA, MBDB and MDMMA, involved in the study (Compounds

6, 7, 9). The MS spectra of all ten underivatized regioisomers were previously reported.

The mass spectra of the ten regioisomeric compounds (MW=207) are characterized by a


240

base peak at m/z 72 formed by α-cleavage reaction involving the carbon–carbon bond of

the ethyl linkage between the aromatic ring and amine nitrogen.

Figure 8.1: Structures of the side chain regioisomeric phenethylamines related to

MDEA, MDMMA, and MBDB.

O

O

NH2

1

1-(3,4-Methylenedioxyphenyl)-2-

pentanamine

O

O

NH2

2

1-(3,4-Methylenedioxyphenyl)-3-

methyl-2-butanamine

O

O

NH2

3

1-(3,4-Methylenedioxyphenyl)-2-methyl-2-butanamine

O

O

NH

5 N-propyl -1-(3,4-

Methylenedioxyphenyl)-2-ethanamine

O

O

NH

5 N-isopropyl-1-(3,4-

Methylenedioxyphenyl) -2-ehanamine

O

O

NH

6

MDEA 3,4-Methylenedioxy-N-

ethylamphetamine


241

O

O

NH

7

MBDB

N-methyl-1-(3,4-Methylenedioxyphenyl)-2-butanamine

O

O

HN

8

N-methyl-3,4-methylenedioxyphentermine

O

O

N

9

MDMMA 3,4-Methylenedioxy-N,N-

dimethylamphetamine

O

O

N

10

N-ethyl-N-methyl-3,4-

methylenedioxyphenyl)-2-ethanamine

Other less abundant peaks were observed at m/z 135/136 from the 3,4-

methylenedioxybenzyl cation and radical cation fragments, respectively as well as other

ions of low relative abundance. The EI mass spectra of these regioisomers show some

variation in the relative intensity of the major ions with only one or two minor ions that

might be considered side-chain specific fragments. Thus, the ultimate identification of

any one of these amines with the elimination of the other nine regioisomeric substances

can not depend on their mass spectra alone.

This lack of mass spectral specificity in addition to the possibility of

chromatographic co-elution with any of the drugs of abuse; MDEA, MBDB and

MDMMA could result in misidentification of the target drug. Furthermore, the lack of

available reference samples for the seven regioisomeric 3,4-methylenedioxy-


242

phenethylamines compounds complicates the individual identification of any one of these

substances. This constitutes a significant analytical challenge, where the specific

identification by GC–MS must be based primarily upon the ability of the

chromatographic system to separate the regioisomeric substance from the actual drug of

interest. Additionally, the ability to distinguish between these regioisomers directly

enhances the specificity of the analysis for the target drugs of interest.

Figure 8.2: EI Mass Spectra of MDEA (compound 6) MBDB (compound 7) and

MDMMA (compound 9).

O

O

NH

6

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 2100

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

m/z-->

Abundance

Scan 1404 (12.270 min): 080506-27.D\data.ms72.1

44.1

135.1

105.1163.1 192.191.1 148.1 206.2121.157.1 176.1


243

O

O

NH

7

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

m/z-->

Abundance


135.1

57.1 88.742.1 178.1105.1 120.1 148.1 163.1 206.2191.2

O

O

N

9

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

3600000

3800000

4000000

4200000

4400000

m/z-->

Abundance

Scan 659 (7.678 min): 090223-01.D\data.ms72.1

135.042.1 56.1

105.091.1 147.1 163.1 192.1121.0 177.1 204.1

Various perfluoroacyl derivatives of the regioisomeric primary and secondary

amines (Compounds 1-8) were prepared and evaluated in an effort to individualize their


244

mass spectra and maintain or improve chromatographic resolution. Acylation of the

amines significantly lowers the basicity of nitrogen and can allow other fragmentation

pathways to play a more prominent role in the mass spectrum. The trifluoroacetyl,

pentafluoropropionyl and heptafluorobutryl derivatives of compounds 1-8 were evaluated

for their ability to individualize the mass spectra through the formation of specific

fragments. Compounds 9 and 10 are tertiary amines and do not form a stable amide

derivative.

The mass spectra for the eight pentaflouropropionyl (PFPA) amides are shown in

Figure 8.3. The spectra for the PFPA derivatives are representative of all the

perfluoroacyl amides in this study. From these spectra, a common peak occurs at m/z 218

which corresponds to the loss of 135 mass units from the molecular ion at 353. This ion is

the PFPA imine species, likely formed from the α-cleavage of the amide nitrogen to

eliminate the 3,4-methylenedioxybenzyl fragment at m/z 135. Thus this ion is analogous

to m/z 72 in the underivatized species because it represents the (M–135)+ species.

The decreased role for the α-cleavage fragmentation of these amides allows the

formation of more diagnostic ions for each individual isomer. Acylation, and in particular

the perflouroacylation, weakens the bond between nitrogen and the α-carbon of the

substituted phenethyl group, allowing the formation of charged hydrocarbon species of

increased relative abundance. These alkenes (radical cations) of varying mass are formed

due to the transfer of a benzylic hydrogen to the ionized carbonyl oxygen followed by the

loss of a neutral amide species. The resulting alkene radical cations (3,4-

methylenedioxyphenylalkenes) significantly individualize the mass spectra and provide

specific structural information. The mass spectra in Scheme 8.1 illustrate the role of the


245

alkene fragments at m/z 148, 162, 176 and 190 in identification of these regioisomers.

These ions identify the number of carbons in the hydrocarbon chain attached directly to

the aromatic ring in an uninterrupted manner.

Figure 8.3: Mass spectra of the PFPA derivatives of compounds 1-8.

O

O

HN C2F5

O

1

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

m/z-->

Abundance


190.1

353.177.1 161.1

55.1105.0 218.1

310.1280.1252.1


246

O

O

HN C2F5

O2

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

m/z-->

Abundance


190.1

353.1

77.155.1218.1

164.0105.1310.1280.1252.1

O

O

NH C2F5

O3

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

m/z-->

Abundance


135.0

55.1

164.0 190.1 353.177.1

105.0 324.1296.1241.1 269.0


247

O

O

N

O C2F5

4

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

6500000

m/z-->

Abundance


176.0353.1

77.1 119.043.1218.1

244.1 324.1294.1266.1

O

O

N

O C2F5

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

500000

1000000

1500000

2000000

2500000

3000000

3500000

m/z-->

Abundance


176.0

353.143.1 77.1 119.0

218.1 244.1 309.1280.1


248

O

O

N

O C2F5

6

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

6500000

m/z-->

Abundance


162.1

190.0

135.1

77.1353.1

105.151.0

309.1273.1243.1

O

O

N

C2F5O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

m/z-->

Abundance


176.1

135.0

42.177.1 353.1

105.1324.1269.1 293.0244.1


249

O

O

N

C2F5O

8

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

m/z-->

Abundance


160.0

135.0

56.1

353.1103.179.1 190.1 282.1 310.1251.9

Further examination of the mass spectra of the PFPA derivatives for the eight

regioisomers (Figure 8.3) indicates unique ions at m/z 160 for both compounds 7 and 8.

An analysis of the masses of the components, which make up the fragment at m/ z 160

include for example C2F5 (119 mass units) and CH3 (15 mass units), leaving only a mass

of 26 available for the total of 160. The mass 26 would correspond to CN and the

proposed mechanism for the formation of (C2F5CNCH3)+ is shown in Scheme 8.2. The

analogous ion occurs at m/z 110 and m/z 210 for the TFA and HFBA derivatives,

respectively. An equivalent fragmentation pathway has been previously reported and this

fragment is characteristic of N-methyl substituted phenethylamine such as compound 7

and 8.


250

O

O

CH

H

C

R1

N

R2

R3

X

CO

O

O

HC

C

R1

N

R2

R3

X

CHO

O

O

HC

C

R1

R2

R1=H, R2= C3H7, m/z=190, cpd 1R1=H, R2= CH(CH3)2, m/z=190, cpd 2R1=CH3, R2= C2H5, m/z= 190, cpd 3R1=H, R2=H, m/z=148, cpds 4 and 5R1=H, R2= CH3, m/z=162, cpd 6R1=H, R2= C2H5, m/z= 176, cpd 7R1=CH3, R2= CH3, m/z= 176, cpd 8

X= CF3, TFA C2F5, PFPA C3F7, HFBA

Scheme 8.1: Mechanism of the formation of the alkene radical cation in the perfluoroacyl derivatives of the primary and secondary regioisomeric amines

N

R1

CH3

m/z = 168, X = CF3

= 218, X = C2F5

=268, X = C3F7

X

O

N

O

X

CH3

R1N C XH3C

m/z = 110, X = CF3

= 160, X = C2F5

= 210, X = C3F7

N X

CH3

OR1

O

OR2

R1 = H, R2 = C2H5, cpd 7R1 = CH3, R2 = CH3, cpd 8X = CF3, TFA C2F5, PFPA

C3F7, HFBA

R2

R2

Scheme 8.2: Formation of m/z 110, 160, 210 for compounds 7 and 8

An important fragmentation pathway characteristic to the PFPA derivative of

compound 6 (the only N-ethyl compound in this limited series) and compounds 4, 5,

which are N-propyl and isopropyl amines is illustrated in Scheme 8.3. This results in the

formation of fragment ions at m/z 190 (Compound 6) or m/z 176 (Compounds 4, 5) for

their PFPA amides. These ions came from the imine fragment at m/z 218 through

hydrogen rearrangement and subsequent cleavage of the N-alkyl group on the nitrogen.

This occurs only with the N-alkyl group of ethyl or larger.


251

N

H3C

CH2

m/z = 168, X = CF3 = 218, X = C2F5 =268, X = C3F7

X

O

Compound 6 m/z = 140, X = CF3 = 190, X = C2F5 = 240, X = C3F7Compound 4, 5 m/z = 126, X = CF3 = 176, X = C2F5 = 226, X = C3F7

N X

CH2

OCH3

O

O

R1=H, R2=H Compound 6R1=H, R2=CH3 Compound 4R1=CH3, R2=CH3 Compound 5

H

CHR2

H

R1

NH

H3C X

O

H

R2

R1

Scheme 8.3 Formation of m/z 140, 190 and 240 for compound 6 and m/z 126, 176 and 226 for compounds 4 and 5.

Derivatization categorized this limited set of compounds into three subset groups

depending on the mass recorded for base peak. The first group includes compounds 1 and

2 having m/z 135 as the base peak in their PFPA derivatives. The second group comprises

compounds 4 and 5 having a base peak at m/z 148. Finally, the third group includes

compounds 3, 6, 7 and 8 which are characterized by having the fragment ions at m/z 218

as the base peak.

Moreover, derivatization also enables the discrimination among some members

within the same group. For the second group, compounds 4 and 5 could be differentiated

by the difference in the relative abundance of the fragment ions at m/z 176. Among the

members of the third group, compounds 7 and 8, which are the only two N-methyl

compounds in this series, could be characterized by the fragment ions at m/z 160. In

addition, they can also be differentiated by inverse intensities of the relative abundances

of fragment ions at m/z 160 and 176.


252

The fragment ion m/z 190 in the mass spectra of the PFPA derivative of compound 6

(Scheme 8.3) can be confused with the same mass fragment ion found in the mass spectra

of the PFPA derivatives of compounds 1-3 formed by another fragmentation pathway

(Scheme 8.1). The TFA and HFBA derivatives eliminated such confusion through the

observance of analogous ions at m/z 140 and 240 for both derivatives respectively formed

through the same fragmentation pathway shown in Scheme 11.3. Figure 11.4 shows

representative mass spectra of the TFA derivatives of compounds 1-3 and 6.

Figure 8.4: Mass spectra of the TFA derivatives of compounds 1-3 and 6.

O

O

HN CF3

O

1

40 60 80 100 120 140 160 180 200 220 240 260 280 3000

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

m/z-->

Abundance


190.1

303.177.0161.055.1

105.1242.1222.0 264.1 284.1


253

O

O

NH CF3

O

2

40 60 80 100 120 140 160 180 200 220 240 260 280 3000

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

m/z-->

Abundance


190.1

303.1

77.055.0

168.1105.0

259.0228.1 281.0

O

O

NH CF3

O

3

40 60 80 100 120 140 160 180 200 220 240 260 280 3000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

m/z-->

Abundance

Scan 766 (8.302 min): 090219-03.D\data.ms168.1

135.1

55.1

303.1

190.1114.077.1

274.1232.0211.0 254.0


254

O

O

N

O CF3

6

40 60 80 100 120 140 160 180 200 220 240 260 280 3000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

m/z-->

Abundance


140.1

77.1303.1

51.1 105.1

205.1 264.1236.1 284.1

The ion at m/z 176 can be formed through two different fragmentation pathways

(Scheme 8.1 and 8.3) for the PFPA derivatives of compounds 4, 5, 7 and 8. The use of

TFA and HFBA as derivatizing agents resolved this problem by providing characteristic

peaks at m/z 126 and 226 for the TFA and HFBA derivatives of compounds 4 and 5,

respectively. Figure 8.5 shows representative mass spectra of the HFBA of these four

compounds. Hence, TFA and HFBA derivatives provide more structure specific fragment

ions than the PFPA analog.


255

Figure 8.5: Mass spectra of the HFBA derivatives of the compounds 4, 5, 7 and 8.

O

O

N

O C3F7

4

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m/z-->

Abundance


226.0

403.177.143.1

268.1119.0177.1 374.1344.1294.1

O

O

N

O C3F7

5

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000

500000

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5000000

m/z-->

Abundance


226.0

43.1403.177.1

105.1268.1194.1 294.1 359.1330.0


256

O

O

N

C3F7O

7

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000

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Abundance


176.1

135.0

210.0

42.0

69.0

403.1

240.0100.0

373.9

O

O

N

C3F7O

8

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Abundance


210.0

176.1

135.056.1

103.1 403.1239.9 331.9 360.1306.0


257

Gas Chromatography of Side Chain Regioisomeric Phenethylamines Related to the

Controlled Substances MDEA, MDMMA, and MBDB.

GC-MS is a powerful tool that combines the identification power of mass

spectrometry with the separation power of gas chromatography. The separation of a

physical mixture of the compounds 1-10 in the underivatized form was the subject of a

previous report from our laboratory.

Several stationary phases and different temperature programs were evaluated in

an effort to resolve the TFA, PFPA and HFBA derivatives in this study. The Rtx-200

phase provided adequate resolution for these compounds of closely related retention

properties. However, different temperature programs were necessary for resolving each

set of perfluoroacyl derivatives.

The separation was performed using three temperature programs for each

derivative studied. Program one was used to separate the trifluoroacetyl (TFA)

derivatives and consisted of an initial hold at 100°C for 1.0 min, ramped up to 180°C at a

rate of 12°C min-1, held at 180°C for 2.0 min then ramped to 200°C at a rate of 10°C min-

1 and held at 200°C for rest of the run duration. The second program was used to resolve

the pentafluoropropionic anhydride (PFPA) derivatives. It started at 100°C for 1.0 min,

ramped up to 180°C at a rate of 7.5°C min-1, held at 180°C for 2.0 min then ramped to

200°C at a rate of 10°C min-1 and held at 200°C. The third program utilized to separate

the heptafluorobutyric anhydride (HFBA) derivatives had an initial oven temperature at

100°C for 1.0 min, followed by temperature increase at a rate of 9°C min-1 to reach


258

180°C, held at 180°C for 2.0 min then ramped to 200°C at a rate of 10°C min-1 and held

at 200°C for the remaining of the run.

Figure 8.6 shows the chromatographic separation of all derivatized forms (TFA,

PFPA and HFBA) of compounds 1-8. The chromatogram shows that the derivatives of

the primary amines (compounds 1-3), the most branched side chain (compound 3) elutes

first and the least branched (compound 1) elutes last. For the secondary amines

(compounds 4-8), the perfluroamides elute in a similar pattern. Thus the compound of

greatest side chain branching (compound 8) elutes first followed by compound 6, 7, 5 and

the most linear tertiary amide (compound 4) elutes last. The resolution of the PFPA and

HFBA derivatives was better than that of the TFA derivatives. However both TFA and

HFBA derivatives provide more structurally specific ions in the mass spectrum to help in

discriminating among these compounds. Hence, the HFBA derivatives are considered the

best choice to discriminate by mass spectrometry and resolve by gas chromatography this

set of regioisomeric phenethylamine derivatives.

18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00

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2 8 3

1

6

7 5

4

A


259

18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00

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Abundance


17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00

100000

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Abundance


Figure 8.6 Gas chromatographic separation of A: TFA; B: PFPA and C: HFBA derivatives of compounds (1-8), Column used; Rtx-200.

3 8 2 6 1

7 5

4 B

3

6

8

21

7

5

4

C


260


Three regioisomeric methylenedioxyphenethylamines (MDEA, MDMMA, and

MBDB), having the same molecular weight and major mass spectral fragments of

equivalent mass, have been reported as drugs of abuse. Seven additional

phenethylamines have a side chain regioisomeric relationship with the controlled

substances. Derivatization of the eight primary and secondary amines with various

perfluroacylating agents yields amides which significantly individualized their mass

spectra and allowed for specific side chain identification. The individualization is the

result of formation of unique marker ions or as a result of differences in the relative

abundances of some common ions. The trifluoroacetyl and heptafluorobutryl derivatives

offer more unique fragment ions over pentafluoropropionyl derivatives. Chromatographic

resolution of the perfluroacyl amides was achieved on a relatively polar stationary phase

Rtx-200.

Chapter 9

GC-MS Evaluation of a Series of Acylated Derivatives of 3,4-


The aim of this chapter is to study and compare the mass spectral properties of

MDMA after derivatization with different reagents commonly used for amine acylation.

The second task is to evaluate the retention properties of each acyl derivative under

chromatographic conditions regularly used by forensic chemists. The derivatizing agents


261

used in this study included trifluoroacetic anhydride (TFAA), pentafluoropropionic

anhydride (PFPA), heptafluorobutric anhydride (HFBA), pentafluorobenzoyl chloride,

acetic anhydride, propionic anhydride , butyric anhydride and benzoyl chloride.

Additionally, the formyl, d3-acetyl and d5-benzoyl derivatives were prepared to confirm

mass spectral fragmentation pathways.

Mass Spectrometry of Acylated Derivatives of 3,4-


Mass spectrometry is the principal method used for confirming the identity of

drugs and related substances in forensic samples. Figure 9.1 shows the structure of 3,4-

MDMA and the mass spectrum for the underivatized amine. The mass spectrum for 3,4-

MDMA is presented here primarily for comparison with the various acylated derivatives

reported in this study. The fragmentation of phenethylamines is generally characterized

by a base peak from an amine initiated alpha-cleavage reaction breaking the carbon-

carbon bond of the ethyl linkage between the aromatic ring and the nitrogen atom of the

amine group. With regards to 3,4-MDMA, initial ionization of nitrogen followed by

cleavage of the alpha bond yields the m/z 58 imine cation, the base peak in the MDMA

mass spectrum.

Figure 9.1: Mass Spectrum of the underivatized MDMA


262

O

O

HN

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900

100000

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800000

900000

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m/z-->

Abundance


135.077.042.1 88.6 105.0 178.1148.1 193.1163.1120.1

Formation of the molecular ion via ionization of the aromatic pi-electrons

followed directly by alpha-cleavage yields the benzylic cation at m/z 135 and hydrogen

rearrangement from the equivalent molecular ion followed by alpha-cleavage initiated at

the distant radical site yields the radical cation at m/z 136 in Figure 9.1. The structures for

these fragment ions are shown in Scheme 9.1

The initial group of derivatives evaluated in this study were the simple alkyl

amides of 3,4-MDMA obtained by derivatization with acetic, propionic and butyric

anhydrides. Acylation of the amine results in significantly lower nitrogen basicity and

this often results in the formation of unique and characteristic fragment ions.


263

.

HC

N

CH3

CH3

m/z = 58

O

O

O

O

CH2

H

m/z = 135

HN

EI 70eV

O

O

CH2

m/z = 136

H

H

Scheme 9.1. EI fragmentation pattern for the underivatized MDMA.

Figure 9.2 (A, B and C) shows the mass spectra for the homologous acetyl,

propionyl and butyryl amides. These three spectra show molecular ions of low relative

abundance at m/z 235, 249 and 263 respectively. Additionally, a second homologous

series of fragment ions (M-135)+ occurs at m/z 100, 114 and 128 suggestive of the

acylated imine fragment for each of the amides. The mass spectrum in Figure 9.2D

provides evidence to support these structural assignments and shows the equivalent ion at

m/z 103 for the d3-acetamide. The structures for the acylated imine fragments included in

this study are shown in scheme 9.2


264

.

N

X

O

X = CF3, 289 = C2F5, 339 = C3F7, 389 = C6F5, 387 = H, 221 = CH3, 235 = C2H5, 249 = C3H7, 263 = C6H5, 297 = CD3, 238 = C6D5, 302

N

X

O

m/z = 154, X = CF3 = 204, X = C2F5 = 254, X = C3F7 = 252, X = C6F5 = 86, X = H = 100, X = CH3 = 114, X = C2H5

= 128, X = C3H7 = 162, X = C6H5

= 103, X = CD3 = 167, X = C6D5

O

O

Scheme 9.2: Imine fragmentation pattern of acylated derivatives of MDMA.

The spectra in Figure 9.2A, B and C show two prominent ions occurring at the

same mass for all three derivatives. The base peak for these derivatives is the m/z 58 ion

and this ion is equivalent to the base peak in the underivatized amine whose mass

spectrum is shown in Figure 9.1.

The m/z 58 ion in the spectrum of these derivatives is likely the result of

hydrogen rearrangement from the alkyl group of the acyl imine fragments at m/z 100,

114 and 128 to yield the common imine fragment at m/z 58. This fragmentation pathway

is illustrated in Scheme 9.3. Furthermore, the mass spectrum in Figure 9.2D for the d3-

acetamide shows the deutrated imine species at m/z 59 providing support for the

structural assignment and fragmentation pathway


265

Figure 9.2. Mass spectra of acetyl, propanyl, butryl and d3-Acetyl amides of MDMA

O

O

N CH3

O

A

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 2400

100000

200000

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700000

800000

m/z-->

Abundance


162.1

100.1

135.177.143.1

235.1192.1118.1 177.1 220.1

O

O

N C2H5

O

B

40 60 80 100 120 140 160 180 200 220 2400

100000

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300000

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600000

700000

800000

900000

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1300000

m/z-->

Abundance


162.1

114.1

135.1

77.1

42.1249.1192.198.1 220.1


266

O

O

N C3H7

O

C

40 60 80 100 120 140 160 180 200 220 240 2600

100000

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400000

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700000

800000

900000

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m/z-->

Abundance

Scan 4487 (30.740 min): 080204-10.D\data.ms58.1

162.1

128.1

77.1

41.1 105.1263.1192.1 220.1146.1 246.1

O

O

N CD3

O

D

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 2400

50000

100000

150000

200000

250000

300000

350000

400000

450000

m/z-->

Abundance


162.1

103.1

135.177.1

42.1 238.1193.1119.1 178.1 220.1


267

Figure 9.3. Mass Spectra of the formyl, benzoyl and d5-benzoyl amides of MDMA.

O

O

N H

O

A

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 2300

20000

40000

60000

80000

100000

120000

140000

160000

180000

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220000

m/z-->

Abundance

Average of 21.735 to 21.997 min.: 071121-04.D\data.ms162.186.1

58.1

135.0

105.142.1 221.1121.0 191.1177.0 206.972.0

O

O

N

O

B

40 60 80 100 120 140 160 180 200 220 240 260 280 3000

20000

40000

60000

80000

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240000

m/z-->

Abundance


162.1

77.1

135.051.1297.1191.1 251.9


268

167

O

O

N

O

D

D

D

D

DC

40 60 80 100 120 140 160 180 200 220 240 260 280 3000

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m/z-->

Abundance

Scan 10447 (65.479 min): 071126-20.D\data.ms110.1

162.1

82.1

135.154.1

191.1 302.1

The formamide (N-formyl) and benzamide (N-benzoyl) derivatives were prepared

and evaluated to determine the scope of the hydrogen rearrangement in the further

decomposition of the acyl imine species. The mass spectra of these compounds are shown

in Figure 9.3A-B. As shown in Scheme 9.3, the hydrogen of the formyl group does

transfer in significant abundance; however the resulting m/z 58 fragment is not the base

peak for this compound. The decreased likelihood of hydrogen transfer through the 3-

membered ring transition leaves the formyl imine species (m/z 86) as the more prominent

ion in its mass spectrum. The benzamide in Figure 9.3B does not show any indication of

a m/z 58 ion demonstrating that the aromatic hydrogens do not migrate in the same

manner as the hydrogen of the alkyl groups in the previously described spectra.


269

N

C

O

R

H

H

R=H, m/z = 100R= CH3, m/z =114R= C2H5, m/z = 128

N

H

m/z = 58

N

H

O

m/z = 86

Scheme 9.3 Formation of m/z 58 from the N-formyl, acetyl propionyl and butyryl

imine fragments

NO

O

XO

HH

NO

O

XHO

H

O

O

H

m/z= 162 Scheme 9.4 Formation of m/z 162 in all amide derivatives of MDMA

The other common fragment seen in all the mass spectra in Figure 9.2 and those

in Figure 9.3A and B is the m/z 162 ion. This fragment ion is the 3,4-

methylenedioxyphenylpropene radical cation occurring through hydrogen transfer from


270

the propyl group to the carbonyl oxygen followed by loss of the neutral amide species.

This mechanism for formation of the m/z 162 radical cation is shown in Scheme 9.4. An

interesting uniqueness was observed in the mass spectrum of the benzamide derivative in

Figure 9.3B. Two separate fragmentation pathways can yield ions at m/z 162, the 3,4-

methylenedioxyphenylpropene radical cation and the benzoylimine cation. Figure 9.3C

shows confirmation of this point with the mass spectrum for the d5-benzamide. The d5-

benzoylimine is shifted to m/z 167 as expected while the 3,4-

methylenedioxyphenylpropene fragment remains at m/z 162. Additionally, the benzoyl

fragment (C6H5CO+) at m/z 105 in Figure 9.3B is shifted to m/z 110 for the d5-benzoyl

derivative.

The mass spectra for all the derivatives described to this point have some

disadvantages for the identification of amines such as 3,4-

methylenedioxymethamphetamine. The derivatives formed from alkanoic carboxylic acid

anhydrides (acetic, propionic and butyric) fragment to yield base peaks at m/z 58

equivalent to the underivatized amines. Thus without the aid of chromatographic

separation, the completeness of the derivatization process itself could be questioned. The

loss of significant ion current as m/z 58 is a drawback to the use of these derivatives for

forensic analysis and the only unique major ion for identification of 3,4-MDMA is the

m/z 162 ion and this ion appears regardless of the acylating species. The benzamide

derivative yields significant ion current at unique masses such as m/z 105 and 162.

However the benzamide derivative of 3,4-MDMA was observed to have quite high GC

retention properties as will be described later in this study. These relatively high retention

properties may limit this derivative utility in some applications.


271

The mass spectra for a series of perfluoroacyl derivatives of 3,4-MDMA are

shown in Figure 9.4. One of the major features of these spectra is the absence of m/z 58

ion. This ion does not occur since no hydrogen is available for migration from the acyl

portion of these derivatives. The spectra in Figure 9.4A, B, C and D further show

common ions at m/z 135 and m/z 162. These ions are the 3,4-methylenedioxybenzyl

cation and the 3,4-methylenedioxyphenylpropene radical cation respectively. The

formation of these ions occurs in an analogous manner to that described earlier in this

report. The three perfluoroalkyl derivatives, trifluoroacetyl, pentafluoropropionyl and

heptafluorobutyryl amides show several ions in a homologous series separated by 50

mass units (CF2). The m/z 154, m/z 204 and m/z 254 ions in these three spectra represent

the perfluoroacylimine species (see Scheme 9.2). These ions are the base peaks in the

three spectra and without hydrogen atom to migrate from the acyl group, no m/z 58

decomposition ion is formed. Additionally, these spectra do not show any evidence of

analogous fluorine transfer decomposition products.

The second homologous series of ions in Figure 9.4A, B and C occur at m/z 110,

160 and 210 respectively. This series of ions differing by 50 mass ions (CF2) indicates

that the perfluoro fragment is a component of these ions. Previous studies in chapter 1

using d3-3,4-MDMA in which the three deuterium atoms are bonded to the N-methyl

group (N-CD3) have shown that all three deuterium labels are contained in the m/z 160

ion for the PFPA derivative. This leaves only 26 mass units (CN) to account for all the

mass of the m/z 160 ion. The mechanism and account for these ions is shown in Scheme

17.5.


272

Figure 9.4. Mass spectra of the fluorinated acyl derivatives of MDMA.

O

O

N CF3

O

A

40 60 80 100 120 140 160 180 200 220 240 260 2800

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320000

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Abundance


135.0

110.0

77.1289.142.1

204.0 250.1176.0 222.1 269.9

O

O

N C2F5

O

B

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

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340000

m/z-->

Abundance


162.1

135.0

77.142.1 339.1

105.1

269.9 300.0242.9


273

O

O

N C3F7

O

C

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

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1000000

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2200000

2300000

m/z-->

Abundance


162.1

135.1210.0

77.142.1 389.1

105.1

310.0 350.1282.9

O

O

N

O

F

F

F

F

FD

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

5000

10000

15000

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30000

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45000

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m/z-->

Abundance

Scan 6747 (43.913 min): 080204-11.D\data.ms195.0

162.1

252.1

135.144.077.1

105.0 225.1 387.1281.0


274

N

m/z = 154, X = CF3

= 204, X = C2F5 = 254, X = C3F7

X

O

N

O

X N C X

m/z = 110, X = CF3 = 160, X = C2F5 = 210, X = C3F7

Scheme 9.5: Formation of m/z 110, 160 and 210 in the spectra of the TFAA, PFPA

and HFBA derivatives of MDMA.

Based on our studies of numerous phenethylamines, this mechanism appears to be

specific for the perfluoroacyl derivatives of N-methyl substituted compounds. Next

chapter will address the scope of this mechanism.

While this project did not examine any perfluoroacyl derivatives in this

homologous series beyond the heptafluorobutyramide, the perfluorooctanoyl (PFO)

derivatives of 3,4-MDMA and methamphetamine have been reported. These derivatives

show a major fragment ion at m/z 410 which extends this series of rearrangement ions

(see Scheme 9.5) to higher chain homologues. The additional four CF2 groups in the PFO

derivative beyond the HFBA derivative account for the 200 additional mass units. The

work of Westphal et al further described the PFO derivative of d5-MDMA and clearly

showed that only three deuterium labels were incorporated into the analogous fragment

ion at m/z 413. The base in the mass spectrum of the PFO derivative of MDMA occurs at

m/z 454 which is the perfluoroacylimine species and analogous to the base peaks in

Figure 9.4A, B and C. Thus these data are consistent with the experimental observations

reported in this study.


275

The remaining spectrum in Figure 9.4D is for the pentafluorobenzamide and the

base peak at m/z 195 is the pentafluorobenzoyl cation. This ion is analogous to the m/z

105 and m/z 110 ions for the benzoyl and d5-benzoyl cations respectively (Scheme 9.6).

These ions would be major fragments in the benzamide of most amines and thus do not

provide ions characteristic of 3,4-MDMA.

N

X

O

X = C6F5, 387 = C6H5, 297 = C6D5, 302

X

O

m/z = 195, X = C6F5 = 105, X = C6H5

= 110, X = C6D5

O

O

Scheme 9.6. Formation of m/z 195, 105 and 110 from the pentafluorobenzoyl,

benzoyl and d5-benzoyl derivatives of MDMA.

Gas Chromatography The chromatogram in Figure 9.5 provides a comparison of the acyl group

structure and relative GC retention properties on a nonpolar stationary phase, 100%

dimethylpolysiloxane, Rtx-1. Several temperature programs were evaluated in this

project and one program showing the best compromise between resolution and analysis

time was used to generate the chromatogram in Figure 9.5.

The perfluoroalkylamides are the first group of derivatives to elute on the

dimethylpolysiloxane stationary phase. These amides elute much earlier than their

corresponding non-fluorinated hydrocarbon counterparts. Both the fluorinated and non-

fluorinated alkyl derivatives elute before the aromatic amides. In every case of direct

comparison, the perfluoro species eluted much earlier than its non-fluorinated


276

hydrocarbon counterpart. The most obvious example of this effect can be seen for the

benzamides in the last two peaks in the chromatogram (Figure 9.5). The high relative

retention for the benzamides coupled with the lack of major fragment ions could limit

their value in MDMA analysis and identification. The alkyl hydrocarbon derivatives

provide significantly increased chromatographic retention yet show the m/z 58 ion as the

base peak as does the derivatized amine, MDMA. Thus, the perfluoroalkyl derivatives

offer excellent chromatographic properties as well as a significant number of

characteristic fragment ions for MDMA identification.

10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00

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40000

60000

80000

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140000

160000

180000

200000

220000

240000

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300000

320000

340000

Time-->

Abundance


Figure 9.5. Gas chromatographic separation of MDMA derivatives. Peaks: 1, PFPA; 2, TFAA; 3, HFBA; 4, N-formyl; 5, acetyl; 6, propoinyl; 7, butyrl; 8, pentaflurobenzoyl and 9, benzoyl amides.

1

2

3 4

5

7

8

9

6


277


The perfluoroacyl derivatives of MDMA show excellent chromatographic

properties and unique mass spectral fragment ions. The perfluoroalkyl amides of TFAA,

PFPA and HFBA yield a unique series of mass spectral fragments at m/z 110, 160 and

210 respectively. Additionally, the base peaks in these mass spectra occur at relatively

higher masses at m/z 154, 204 and 254 respectively. These ions are the

perfluoroacylimines resulting from loss of 135 mass units (3,4-methylenedioxybenzyl)

from the molecular ion. This (M-135)+ species also occurs for the acetyl, propionyl and

butyryl amides however these ions rearrange through loss of the acyl group to yield a

common ion at m/z 58. Thus, the base peak for the non-fluorinated hydrocarbon

derivatives is the same as that observed for the underivatized MDMA, m/z 58. The

formyl, d3-acetyl, d5-benzoyl and pentafluorobenzoyl derivatives provided

chromatographic and confirmatory mass spectral evidence for fragmentation pathways.


278

IV. Conclusions

IV.1 Discussion of Findings.

The compounds investigated in this project are a series of closely related

regioisomeric and isobaric amines having mass spectra essentially equivalent to that of

MDMA, MDEA and MBDB. Mass spectral equivalence in the underivatized amines

shows that MS methods do not provide confirmation of drug identity and molecular

individualization. Additional scientific data for molecular individualization can be

obtained following simple chemical derivatization with perfluoroacylating reagents.

Perfluoroacylamines provide specific and unique mass spectral fragments

for side chain and some ring substituent identification. Vapor phase infrared spectrometry

(GC-IRD) provides additional confirmation for other ring substituents and for those

compounds not forming stable acylation products. Thus the results of this research have

provided improved specificity in the analytical methods used to identify these

phenethylamine controlled substances. This improved specificity comes from methods

which allow the forensic analyst to eliminate non-drug imposter substances as the source

of analytical data matching that of the controlled drug substance.

IV.2 Implications for policy and practice:

The results of this project allow the forensic analyst to differentiate among most

of the regioisomeric and isobaric equivalents of MDMA. This can be accomplished

without having access to a large number of reference standards of these compounds.


279

IV.3 Implications for further research:

Perhaps the first implication from the results of this work is an educational

program to efficiently and effectively transfer the findings to practicing forensic analysts.

While we have published extensively on this subject in the scientific literature, a one or

two day short course would allow us to put these data in the hands of forensic drug

chemists in an organized and effective manner. The course would include background

educational material on mass spectra and infrared interpretation as well as

chromatographic theory. The course could be offered in conjunction with regional

meetings of forensic scientists over a period of a year to 18 months and reach numerous

individual forensic scientists.

Retention index studies for all the ring substituted amines of the same side-chain,

especially the methamphetamine side-chain compounds. Since the methamphetamine

side chain is perhaps the most likely to appear in new designer drugs, retention indexing

studies on standardized GC columns and stationary phases could further decrease the

need for reference standards in identification of these regioisomeric and isobaric amines.

Such studies should be done in cooperation with collaborators in forensic science

laboratories.

Liquid Chromatographic studies on unique stationary phases to provide

retention/separation/resolution data on as large a subset of these compounds as possible.

Again concentrating on those compounds having the same side chain structure. These are


280

the compounds for which mass spectrometry provides the least information for molecular

individualization.

In concert with LC studies MS-MS techniques can be explored for evidence of

specific fragment ions identifying aromatic ring substitution patterns. The initial phase of

such a study would be to compare the fragment patterns for all the possible regioisomeric

substitution patterns in each series. Since we have all the compounds available already,

our group is well positioned to begin this study immediately.


281

V. References

Aalberg, L.; DeRuiter, J.; Noggle, F.T.; Sippola, E.; Clark,C. R. Chromatographic and

mass spectral methods of identification for the side-chain and ring regioisomers of

methylenedioxymethamphetamine. J. Chromatogr. Sci., 2000, 38, 329 – 337.

Aalberg, L.; DeRuiter, J.; Noggle, F.T.; Sippola, E.; Clark,C. R. Chromatographic and

Spectroscopic Methods of Identification for the Side Chain Regioisomers of 3,4-

Methylenedioxyphenethylamines Related to MDEA, MDMMA and MBDB. J.

Chromatogr. Sci., 2003, 41, 227-233.

Aalberg, L.; DeRuiter, J.; Sippola, E.; Clark,C. R. Gas Chromatographic Optimization

Studies on the Side Chain And Ring Regioisomers of Methylenedioxymetham-

phetamine. J. Chromatogr. Sci., 2004, 42, 293-298.

Bell, S.E.J.; Burns, D.T.; Dennis, A.C.; Speers, J.S. Rapid analysis of ecstasy and related

phenethylamines in seized tablets by Raman spectroscopy. Analyst 2000a, 125, 541-544.

Bell, S.E.J.; Burns, D.T.; Dennis, A.C.; Matchett, L.J.; Speers, J.S. Composition profiling

of seized ecstasy tablets by Raman spectroscopy. Analyst 2000b, 125, 1811-1815.

Bogusz, M.J.; Krüger, K.-D.; Maier, R.-D. Analysis of underivatized amphetamines and


282

related phenethylamines with high-performance liquid chromatography-atmospheric

pressure chemical ionization mass spectrometry. J. Anal. Toxicol. 2000, 24, 77-84.

Borth, S.; Hänsel, W.; Rösner, P.; Junge, T. Synthesis of 2,3- and 3,4-methylenedioxy-

phenalkylamines and their regioisomeric differentiation by mass spectral analysis using

GC-MS-MS. Forensic Sci. Int. 2000a, 114, 139-153.

Borth, S.; Hänsel, W.; Rösner, P.; Junge, T. Regioisomeric differentiation of 2,3- and 3,4-

methylenedioxy ring-substituted phenylalkylamines by gas chromatography / tandem

mass spectrometry. J. Mass Spectrom. 2000b, 35, 705-710.

Braga, S.; Paz, F. A. ; Pillinger, M. ; Seixas, J. ; Romao, C. and Goncalves, I. Structural

studies of β-cyclodextrin and permethylated β-cyclodextrin inclusion compounds of

cyclopentadienyl metal carbonyl complexes. Eur. J. Inorg. Chem. 2006, 10, 1662-1669.

Brunnenberg, M.; Kovar, K.-A. Stereospecific analysis of ecstasy-like N-ethyl-3,4-

methylenedioxyamphetamine and its metabolites in humans. J. Chromatogr. B 2001, 751,

9-18.

Buechler J, Schwab M, Mikus G, Fischer B, Hermle L, Marx C, Gron G, Spitzer M,

Kovar KA. Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-

3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine.


283

J. Chromatogr B Analyt Technol Biomed Life Sci., 2003, 793(2), 207-22.

Burns, D.T.; Lewis, R.J.; Stenson, P. Determination of 3,4-methylenedioxyamphetamine

analogues (“ecstasy”) by proton nuclear magnetic resonance spectrometry. Anal. Chim.

Acta 1997, 339, 259-263.

Casale, J.F.; Hays, P.A.; Klein, R.F.X. Synthesis and characterization of the 2,3-

methylenedioxyamphetamines. J. Forensic Sci. 1995, 40, 391.

Clark, C.R.; Valaer, A. K.; F.T. Noggle, F. T.; DeRuiter, J. GC-MS analysis of acylated

derivatives of methamphetamine and regioiosomeric Phenethylamines. J. Chromatogr.

Sci., 1995, 33, 485

Clark, C.R.; DeRuiter, J.; Valaer, A.; Noggle, F.T. Gas chromatographic-mass

spectrometric and liquid chromatographic analysis of designer butanamines related to

MDMA. J. Chromatogr. Sci. 1995a, 33, 328-337.

Clark, C.R.; DeRuiter, J.; Valaer, A.; Noggle, F.T. GC-MS analysis of acylated

derivatives of methamphetamine and regioisomeric phenethylamines. J. Chromatogr. Sci.

1995b, 33, 485-492.


284

Clark, C.R.; DeRuiter, J.; Noggle, F.T. Chromatographic and mass spectrometric

methods for the differentiation of N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine

from regioisomeric derivatives. J. Chromatogr. Sci. 1996, 34, 230-237.

Clauwaert, K.M.; van Bocxlaer, J.F.; de Letter, E.A.; van Calenbergh, S.; Lambert, W.E.;

de Leenheer, A.P. Determination of the designer drugs 3,4-methylenedioxy-

methamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxy-

amphetamine with HPLC and fluorescence detection in whole blood, serum, vitreous

humor, and urine. Clin. Chem. 2000, 46, 1968-1977.

Concheiro M, de Castro A, Quintela O, Lopez-Rivadulla M, Cruz A. Determination of

MDMA, MDA, MDEA and MBDB in oral fluid using high performance liquid

chromatography with native fluorescence detection. Forensic Sci Int. 2005, 150(2-3),

221-6.

da Costa JL, da Matta Chasin AA. Determination of MDMA, MDEA and MDA in urine

by high performance liquid chromatography with fluorescence detection. J Chromatogr B

Analyt Technol Biomed Life Sci. 2004, 811(1), 41-5.


285

Dal Cason, T.A.; Young, R.; Glennon, R.A. Cathinone: an investigation of several N-

alkyl and methylenedioxy-substituted analogs. Pharmacol. Biochem. Behav. 1997, 58,

1109-1116.

Dallakian, P.; Budzikiewicz, H.; Brzezinka, H. Detection and quantitation of

amphetamine and methamphetamine: electron impact and chemical ionization with

ammonia--comparative investigation on Shimadzu QP 5000 GC-MS system. J. Anal.

Toxicol. 1996, 20, 255-261.

de Boer, D.; Tan, L.P.; Gorter, P.; van de Wal, R.M.A.; Kettenes-van de Bosch, J.J.; de

Bruijn E.A.; Maes, R.A.A. Gas chromatographic/mass spectrometric assay for profiling

the enantiomers of 3,4-methylenedioxymetamphetamine and its chiral metabolites using

positive chemical ionization ion trap mass spectrometry. J. Mass Spectrom. 1997, 32,

1236-1246.

DeRuiter, J.; Holston, P.L.; Clark, C.R.; Noggle, F.T. Liquid chromatographic and mass

spectral methods of identification for regioisomeric 2,3- and 3,4-methylenedioxy-

phenalkylamines. J. Chromatogr. Sci. 1998, 36, 131-138.

Drummer, O.H.; Horomidis, S.; Kourtis, S.; Syrjanen, M.L.; Tippett, P. Capillary gas

chromatographic drug screen for use in forensic toxicology. J. Anal. Toxicol. 1994, 18,


286

134-138.

Duncan, W; Soine, W.H. Identification of Amphetamine Isomers by GC/IR/MS, J.

Chromatogr. Sci.1988, 26, 521-526

Ecstasy Data, independent laboratory pill testing program co-sponsored by Erowid,

Dancesafe, MAPS, & the Promind Foundation http://www.ecstasydata.org/

Girod, C.; Staub, C. Analysis of drugs of abuse in hair by automated solid-phase

extraction, GC/EI/MS and GC ion trap/CI/MS. Forensic Sci. Int. 2000, 107, 261-271.

Helmlin, H.J.; Bracher, K.; Bourquin, D.; Vonlanthen, D.; Brenneisen, R.; Styk, J.

Analysis of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in

plasma and urine by HPLC-DAD and GC-MS. J. Anal. Toxicol. 1996, 20, 432-440.

Kempfert, k. Forensic Drug Analysis by GC/FT-IR, Applied Spectroscopy 42 (1988)

845-849

Kronstrand, R. Identification of N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine

(MBDB) in urine from drug users. J. Anal. Toxicol. 1996, 20, 512-516.


287

Laing,R.R; Beyerstein, B.L; Siegel, J. A. Hallucinogens. A Forensic Drug Handbook,

Academic Press (2003) 191-192, 197

Lee, G.S.H.; Taylor, R.C.; Dawson, M.; Kannangara, K.; Wilson, M.A. High-resolution

solid state 13C nuclear magnetic resonance spectra of 3,4-methylenedioxyamphetamine

hydrochloride and related compounds and their mixture with lactose. Solid State Nucl.

Magn. Reson. 2000, 16, 225-237.

Lillsunde, P.; Korte, T Determination of ring- and N-substituted amphetamines as

heptafluorobutyryl derivatives. Forensic Sci. Int. 1991, 49, 205-213.

Lillsunde, P.; Michelson, L.; Forsström, T.; Korte, T.; Schultz, E.; Ariniemi, K.; Portman,

M.; Sihvonen, M.-L.; Seppälä, T. Comprehensive drug screening in blood for detecting

abused drugs potentially hazardous for traffic safety. Forensic Sci. Int. 1996, 77, 191-

210.

Lim, H.K.; Foltz, R.L. In vivo formation of aromatic hydroxylated metabolites of 3,4-

(methylenedioxy)methamphetamine in the rat: Identification by ion trap tandem mass

spectrometric (MS/MS and MS/MS/MS) techniques. Biol. Mass Spectrom. 1991, 20,

677-686.


288

Lim, H.K.; Zeng, S.; Chei, D.M.; Foltz, R.L. Comparative investigation of disposition of

3,4-(methylenedioxy)methamphetamine (MDMA) in the rat and the mouse by a capillary

gas chromatography-mass spectrometry assay based on perfluorotributylamine-enhanced

ammonia positive ion chemical ionization. J. Pharm. Biomed. Anal. 1992, 10, 657-665.

Mancinelli, R.; Gentili, S.; Guiducci, M.S.; Macchia, T. Simple and reliable high-

performance liquid chromatography fluorimetric procedure for the determination of

amphetamine-derived designer drugs. J. Chromatogr. B 1999, 735, 243-253.

Marquet, P.; Lacassie, E.; Battu, C.; Faubert, H.; Lachâtre, G. Simultaneous

determination of amphetamine and its analogs in human whole blood by gas

chromatography-mass spectrometry. J. Chromatogr. B 1997, 700, 77-82.

Maurer, H.H. On the metabolism and the toxicological analysis of

methylenedioxyphenylalkylamine designer drugs by gas chromatography-mass

spectrometry. Ther. Drug. Monit. 1996, 18, 465-470.

Michel, R.E.; Rege, A.B.; George, W.J. High-pressure liquid chromatography /

electrochemical detection method for monitoring MDA and MDMA in whole blood and

other biological tissues. J. Neurosci. Methods 1993, 50, 61-66.


289

Mortier KA, Dams R, Lambert WE, De Letter EA, Van Calenbergh S, De Leenheer AP.

Determination of paramethoxyamphetamine and other amphetamine-related designer

drugs by liquid chromatography/sonic spray ionization mass spectrometry. Rapid

Commun Mass Spectrom. 2002, 16(9), 865-70.

MTF (Monitoring the Future Study): Trends in Prevalence of Various Drugs for 8th-

Graders, 10th-Graders, and 12th-Graders, 2005,

http://www.drugabuse.gov/drugpages/MTF.html

Noggle, F.T. Jr.; Clark, C.R.; Andurkar, S.; DeRuiter, J. Methods for the analysis of 1-

(3,4-methylenedioxyphenyl)-2-butanamine and N-methyl-1-(3,4-methylenedioxyphenyl)-

2-propanamine (MDMA). J. Chromatogr. Sci. 1991a, 29, 103-106.

Noggle, F.T. Jr.; Clark, C.R.; DeRuiter, J. Gas chromatographic and mass spectrometric

analysis of samples from a clandestine laboratory involved in the synthesis of ecstasy

from sassafras oil. J. Chromatogr. Sci. 1991b, 29, 168-173.

Noggle, F.T. Jr.; Clark, C.R.; Valaer, A.K.; DeRuiter, J. Liquid chromatographic and

mass spectral analysis of N-substituted analogues of 3,4-methylenedioxyamphetamine. J.

Chromatogr. Sci. 1988, 26, 410-415.


290

Noggle, F.T. Jr.; DeRuiter, J.; Coker, S.T.; Clark, C.R. Synthesis, identification and acute

toxicity of some N-alkyl derivatives of 3,4-methylenedioxyamphetamines. J. Assoc. Off.

Anal. Chem. 1987, 70, 981-986.

Noggle, F.T.; Clark, C.R.; DeRuiter, J. Chromatographic and mass spectrometric analysis

of N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine and regioisomeric derivatives.

Microgram 1995, 28, 321-328.

O’Connell, D.; Heffron, J.J.A. Rapid analysis of illicit drugs by mass spectrometry:

Results from seizures in Ireland. Analyst 2000, 125, 119-121.

Ortuño, J.; Pizarro, N.; Farre, M.; Mas, M.; Segura, J.; Cami, J.; Brenneisen, R.; de la

Torre, R. Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in

plasma and urine by gas chromatography with nitrogen-phosphorus detection. J.

Chromatogr. B 1999, 723, 221-232.

Paul BD, Jemionek J, Lesser D, Jacobs A, Searles DA. Enantiomeric separation and

quantitation of (+/-)-amphetamine, (+/-)-methamphetamine, (+/-)-MDA, (+/-)-MDMA,

and (+/-)-MDEA in urine specimens by GC-EI-MS after derivatization with (R)-(-)- or

(S)-(+)-alpha-methoxy-alpha-(trifluoromethy)phenylacetyl chloride (MTPA). J Anal

Toxicol. 2004, 28(6), 449-55.


291

Peters FT, Samyn N, Lamers CT, Riedel WJ, Kraemer T, de Boeck G, Maurer HH. Drug

testing in blood: validated negative-ion chemical ionization gas chromatographic-mass

spectrometric assay for enantioselective measurement of the designer drugs MDEA,

MDMA, and MDA and its application to samples from a controlled study with MDMA.

Clin. Chem. 2005, 51(10), 1811-22.

Sachs, S.B., Woo, F. A detailed mechanistic fragmentation analysis of methamphetamine

and select regioisomers by GC/MS, J. Forensic Sci. 2007, 308-319.

Sagmuller B, Schwarze B, Brehm G, Schneider S. Application of SERS spectroscopy to

the identification of (3,4-methylenedioxy)amphetamine in forensic samples utilizing

matrix stabilized silver halides. Analyst. 2001, 126(11), 2066-71.

Schneiderman, E.and Stalcup, A. Cyclodextrins: a versatile tool in separation science. J.

Chromatogr. B. 2000, 745, 83-102.

Shulgin, A.T. The background and chemistry of MDMA. J. Psychoact. Drugs 1986, 18,

291-305.

Soine, W.H.; Shark, R.E.; Agee, D.T. Differentiation of 2,3-methylenedioxyamphetamine

from 3,4-methylenedioxyamphetamine. J. Forensic Sci. 1983, 28, 386-390.


292

Sondermann, N.; Kovar, K.A. Screening experiments of ecstasy street samples using near

infrared spectroscopy. Forensic Sci. Int. 1999, 106, 147-156.

Szejtli, J. and Osa, T. Comprehensive supramolecular Chemistry, Cyclodextrins, vol 3,

pergamon, Elsevier, Oxford, 1996.

Tagliaro, F.; De Battisti, Z.; Groppi, A.; Nakahara, Y.; Scarcella, D.; Valentini, R.;

Marigo, M. High sensitivity simultaneous determination in hair of the major constituents

of ecstasy (3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and

3,4-methylenedioxyethylamphetamine) by high-performance liquid chromatography with

direct fluorescence detection. J. Chromatogr. B 1999, 723, 195-202

Talwar, D.; Watson, I.D.; Stewart, M.J. Routine analysis of amphetamine class drugs as

their naphtaquinone derivatives in human urine by high-performance liquid

chromatography. J. Chromatogr. B 1999, 735, 229-241.

TCADA. 2002. A dictionary of slang drug terms: stimulants (online). Texas Commission

on Alcohol and Drug Abuse. http://www.tcada.state.tx.us/research/slang/stimulants

[Accessed 27 Sep. 2002].

UNODCCP (United Nations Office of Drug and Crime), Ecstasy and Amphetamine

global survey 2003, http://www.unodc.org/pdf/publications/report_ats_2003-09-23_1.pdf


293

Weinman, W.; Renz, M.; Vogt, S.; Pollak, S. Automated solid-phase extraction and two-

step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS.

Int. J. Legal Med. 2000, 113, 229-235.

Wood M, De Boeck G, Samyn N, Morris M, Cooper DP, Maes RA, De Bruijn EA.

Development of a rapid and sensitive method for the quantitation of amphetamines in

human plasma and oral fluid by LC-MS-MS. J Anal Toxicol. 2003, 27(2), 78-87.

Young, K.A. Attenuated Total Reflectance – Fourier Transform Infrared Spectroscopy

(ATR-FTIR) for the Analysis and Profiling of Seized Ecstasy Tablets, Masters Thesis,

University of Strathydrochloric acidyde, Glasgow, UK, 2000.


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VI. Dissemination of Research Findings VI.1 Scientific Publications Related to this Project:

Ashley L Thigpen, Jack DeRuiter and C Randall Clark, “GC-MS Studies on the

Regioisomeric 2,3- and 3,4-Methylenedioxyphenethylamines Related to MDEA,

MDMMA and MBDB,” J. Chromatogr. Sci., 45, 229 (2007).

Tamer Awad, Jack DeRuiter and C. Randall Clark, “Chromatographic and Mass Spectral

Studies on Methoxy Methyl Methamphetamines Related to 3,4-Methylenedioxy-

methamphetamine,” J. Chromatogr. Sci., 45, 466 (2007).

Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS Analysis of Regioisomeric

Ring Substituted Methoxy Methyl Phenylacetones,” J. Chromatogr. Sci., 45, 458 (2007).

Tamer Awad, C. Randall Clark and Jack DeRuiter, GC-MS Analysis of Acylated

Derivatives of the Side Chain Regioisomers of 4-Methoxy-3-methyl-phenethylamines

Related to Methylenedioxymethamphetamine,” J. Chromatogr. Sci., 45, 477 (2007).

Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS Analysis of Acylated

Derivatives of a Series of Side Chain Regioisomers of 2-Methoxy-4-Methyl-

Phenethylamines,” J. Chromatogr. Sci., 46, 375-380 (2008).


295

Tarek Belal, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS Studies on

Acylated Derivatives of 3-Methoxy-4-methyl- and 4-Methoxy-3-methyl-

phenethylamines: Regioisomers Related to 3,4-MDMA,” Forensic Science International,

178, 61-82 (2008).

Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC- MS Analysis of Ring and Side

Chain Regioismoers of Ethoxyphenethylamines,” J. Chromatogr. Sci., 46, 671-679

(2008).

Ashley L. Thigpen Tamer Awad, Jack DeRuiter and C. Randall Clark

“GC-MS Studies on the Regioisomeric Methoxy-Methyl-Phenethylamines Related to

MDEA, MDMMA and MBDB,” J. Chromatogr. Sci., 46, 900-906 (2008).

C. Randall Clark, Tamer Awad and Jack DeRuiter, “GC-IRD Analysis of Regioisomeric

Substituted Phenethylamines of Mass Spectral Equivalence,” Chromatography Today, 1

#4, 27-30 (2008).

Tamer Awad, Jack DeRuiter, Tarek Belal and C. Randall Clark, “Gas Chromatographic

and Mass Spectral Studies on the Acylated Side Chain Regioisomers of 3-Methoxy-4-

Methyl Phenethylamine and 4-Methoxy-3-Methyl Phenethylamine,” J. Chromatogr. Sci.,

47, 279-286 (2009).


296

Tarek Belal, Tamer Awad, C. Randall Clark and Jack DeRuiter, “GC-MS Evaluation of a

series of Acylated Derivatives of 3,4-Methylenedioxy-methamphetamine,” J.

Chromatogr. Sci., 47, 359-364 (2009).

Tamer Awad, Tarek Belal, Jack DeRuiter, Kevin Kramer, and C. Randall Clark,

“Comparison of GC-MS and GC-IRD methods for the differentiation of

methamphetamine and regioisomeric substances,” Forensic Science International, 185,

67–77 (2009).

Tarek Belal, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-IRD Methods for

the Identification of Isomeric Ethoxyphenethylamines and Methoxymethcathinones,”

Forensic Science International, 184, 54–63 (2009).

Hadir M. Maher, Tamer Awad and C. Randall Clark, “Differentiation of the

Regioisomeric 2, 3, and 4-Trifluoromethylphenylpiperazines (TFMPP) by GC-IRD and

GC-MS,” Forensic Science International, 188, 31-39 (2009).

Hadir M. Maher, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC–MS Studies on

Acylated Derivatives of Dimethoxyamphetamines (DMA): Regioisomers Related to 2,5-

DMA,” Forensic Science International, 192, 115-125 (2009).


297

Tamer Awad, Tarek Belal, C. Randall Clark, Jack DeRuiter, “GC-IRD Studies on

Regioisomeric Ring Substituted Methoxy Methyl Phenylacetones Related to 3,4-

Methylenedioxyphenylacetone,” Forensic Science International, 194, 39-48 (2010).

Hadir M. Maher, Tamer Awad, Jack DeRuiter and C. Randall Clark

“GC–IRD Methods for the Identification of Some Tertiary Amine Regioisomers of

MDMA,” Forensic Science International, 199, 18–28 (2010).

Abdullah M. Al-Hossaini, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS

and GC-IRD Analysis of Ring and Side Chain Regioisomers of Ethoxyphenethylamines

Related to the Controlled Substances MDEA, MDMMA and MBDB,” Forensic Science

International, 200, 73-86 (2010).

Tamer Awad, Tarek Belal, Hadir M. Maher, C. Randall Clark and Jack DeRuiter. “GC-

MS Studies on Side Chain Regioisomers Related to Substituted Methylenedioxy-

phenethylamines; MDEA, MDMMA and MBDB” J. Chromatogr. Sci., 48, 726-732

(2010).


298

VI.2 Scientific Presentations Related to this Project:

Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to

MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of Mass

Spectral Equivalence—Progress Presentation

C. Randall Clark, 2007 General Forensics R&D Grantees Meeting, , San Antonio, Texas,

February 2007.



Spectral Equivalence—Progress Presentation

C. Randall Clark, 2007 General Forensics R&D Advisory Group, Scottsdale, Arizona,

October 2007.

GC-MS Analysis of the Ring and Side Chain Regioisomers of Ethoxyphenethylamines

C. Randall Clark, Tamer Awad and Jack DeRuiter. American Chemical Society National

Meeting, New Orleans, Louisiana, USA; April 10, 2008.



Spectral Equivalence—Progress

C. Randall Clark, 2009 General Forensics R&D Grantees Meeting, Denver, Colorado.

February 2009.


synthesis and analytical profiles for regioisomeric and isobaric amines related to mdma, mdea

Documents