Enantiomeric Composition of Abused Amine Drugs -- Chromatographic Methods of Analysis and Data Interpretation

Enantiomeric composition of abused amine drugs:

chromatographic methods of analysis and

data interpretation

Ju-Tsung Liu

, Ray H. Liu

*

a

Department of Forensic Education, Army Force of Military Police School, Wugu, Taipei, Taiwan, ROC b

Graduate Program in Forensic Science, University of Alabama at Birmingham, Birmingham, AL 35294-2060, USA

Keywords: Enantiomeric composition; Amine drugs; Chromatographic methods

1. Introduction

1.1. Enantiomers

Enantiomers are pairs of nonsuperimposable mirror-image compounds, typically include

one or more asymmetric (chiral) carbons (carbon bonded to four different groups). The two

individual components of an enantiomer-pair have identical chemical and physical

proper-ties, differing only in the way they react with other chiral compounds and the direction in

which they rotate plane-polarized light, and therefore, cannot be resolved by conventional

chromatographic methodologies.

The configuration of the groups bonded to the chiral atom is designated as S or R, while

D

/+ and

/

are used to describe the direction in which a polarized light is rotated by the

molecule. A left-handed or levorotatory (

/

) compound rotates polarized light to the left

while a right-handed or dextrorotatory (

/+) compound rotates polarized light to the right.

Configuration alone does not predict the light-rotating direction, a full description of a

compound often includes designations for the configuration (S/R) and the light-rotating

characteristic (

or +/

). When the quantities of two enantiomers in a solution are exactly

equal, it becomes optically inactive and is called a racemate.

0165-022X/02/$ - see front matterD 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X ( 0 2 ) 0 0 1 3 6 - 7

* Corresponding author. Fax: +1-205-934-2067. E-mail address: [email protected] (R.H. Liu).

Biological systems are typically chiral; thus, drugs derived from natural sources

commonly exhibit optical activity while synthetic products—unless specifically

synthe-sized for optical purity—exist as racemates. For the same reason, biological systems often

react differently toward each component of an enantiomer-pair. For example, the

-isomers

of some barbiturates act as depressants while the

-isomers act as convulsants.

1.2. Significance of enantiomeric analysis in the pharmaceutical industry

‘‘Chirality’’ is now a topic at the forefront of academic research and drug development.

For example, the 2001 Noble Prize in Chemistry has been awarded to ‘‘three scientists who

devised techniques for catalytic asymmetric synthesis—the use of chiral catalysts to

accelerate the production of single-enantiomer compounds for pharmaceutical use and a

wide range of other applications.’’

[1]

On the other hand, US Food and Drug

Admin-istration’s regulations place major emphases on the characterizations of enantiomeric

compositions of pharmaceutical products

[2]

. Drug firms are also actively involved in

developing new chiral drugs as single enantiomers and in carrying out ‘‘racemic

switches’’—redeveloping racemic mixture drugs as single enantiomers

[3]

. As a result,

approximately 40% of all dosage-form drug sales in 2000 was of single enantiomers, in

contrast to approximately one-third in 1999

[3]

.

1.3. Significance of enantiomeric analysis in forensic science laboratories

Enantiomeric characterization and composition determination are also important issues

in forensic laboratories involving in the analysis of abused drugs. Enantiomeric analyses are

often carried out to: (a) provide information for sentencing guidance for certain drug-related

offenses; (b) assist in drug-related investigations; and (c) determine whether the drug of

concern derives from a controlled substance.

Although enantiomeric specifications have long been an important factor in the

scheduling of commonly abused drugs, current US Federal drug codes are so worded that

both optical isomers are included. For example, both optical isomers of amphetamine,

methamphetamine, and cocaine and both natural and synthetic cocaine are Schedule II

drugs

[2]

as shown below:



_{US Code 1308.12 (b) (4): ‘‘Coca leaves (9040) and any salt, compound, derivative or}

preparation of coca leaves (including cocaine (9041) and ecgonine (9180) and their salts,

isomers, derivatives and salts of isomers and derivatives), and any salt, compound,

derivative, or preparation thereof which is chemically equivalent or identical with any of

these substances, . . .’’



_{US Code 1308.12 (d) (1): ‘‘Amphetamine, its salts, optical isomers, and salts of its}

optical isomers’’



_{US Code 1308.12 (d) (2): ‘‘Methamphetamine, its salts, isomers, and salts of its}

isomers’’

However, the United States Sentencing Commission guidelines manual distinguishes

between the enantiomers of methamphetamine for the purpose of sentencing. One gram of

L

-methamphetamine is considered to be the equivalent of 40 g of marijuana, while 1 g of

-methamphetamine or racemic methamphetamine is equivalent to 10 kg of marijuana

[4]

.

Enantiomeric analysis can also assist the investigation process. For example, the source

of cocaine samples—a question often asked in the investigation process or in the court—

may be elucidated by enantiomeric analysis

[5,6]

. Cocaine samples derived from the

extraction of coca leaves contain only

-cocaine, while completely synthetic cocaine

samples usually contain both

- and

-cocaine and diastereomers. Enantiomeric analysis

can also provide valuable information concerning clandestine conversions of norephedrine

and norpseudoephedrine to amphetamine and ephedrine and pseudoephedrine to

meth-amphetamine

[7 – 11]

. The single enantiomer starting materials tend to produce single

enantiomer products—single enantiomer S(

)-methamphetamine (

-methamphetamine)

from

-ephedrine or

-pseudoephedrine and R(

)-methamphetamine (

-methamphetamine)

from

-ephedrine or

-pseudoephedrine. It should be cautioned, however,

-methamphet-amine has also been extracted from the Vicks InhalerR

[12]

and

-methamphetamine-based

prescription drugs

[13]

.

Enantiomeric composition determinations of amphetamine and methamphetamine in

biological specimens serve well for confirming or contradicting the stated amphetamine

and methamphetamine sources of these compounds

[14,15]

. This is possible because of

the following enantiomeric characteristics of certain

amphetamine/methamphetamine-containing substances: (a)

-methamphetamine is used in Vicks InhalerR

[12]

; (b)

prescription methamphetamine is in

-form; and (c) amphetamine and methamphetamine

resulting from the use of licit medicine, such as deprenyl (selegiline), an antiparkinsonian,

are

-isomers

[13]

.

Enantiomeric composition alone, however, does not always lead to a definite conclusion.

For example, the detection of a racemic mixture of methamphetamine may indicate illicit

use; the same observation may also derive from (a) prescription use of a racemic precursor

molecule such as furfenorex

[16]

; or (b) legitimate use of

-methamphetamine along with

the concurrent use of

-methamphetamine (Vicks InhalerR)

[12]

or deprenyl

[13]

.

Detection of single enantiomer

-methamphetamine, however, does not necessarily prove

use of licit prescription either. As mentioned above, single enantiomer

-methamphetamine

can be manufactured from

-ephedrine or

-pseudoephedrine

[10]

.

1.4. Approaches in chromatographic separation of enantiomers and coverage of this

review

A substantial number of studies have been conducted on the use of capillary

electro-phoresis (CE) for enantiomeric composition determinations. CE may even become the

method of choice when an effective and robust interface (between the CE and the mass

spectrometric detecting device) is developed and become conveniently available. Since CE

approaches have been covered by an earlier issue of this journal, they will be excluded in

this review. Only chromatographic methods will be addressed.

This review will be limited to various chromatographic methods for enantiomeric

determination of amine drugs, with emphases on amphetamine and methamphetamine,

their precursor drugs, and structurally closely related compounds; and significance

pertaining to the observed enantiomeric composition data.

There have been a number of reviews on topics hereby addressed. For examples ‘‘Chiral

stationary phases for gas – liquid chromatographic separation of enantiomers’’

[17]

,

‘‘Determination of methamphetamine enantiomer ratios in urine by GC – MS’’

[16]

,

‘‘Amphetamine and methamphetamine determinations in biological samples by high

performance liquid chromatography’’

[18]

, ‘‘Liquid chromatographic analysis of

enantio-meric composition of abused drugs’’

[19]

, ‘‘Determination of amphetamine,

methamphet-amine and amphetmethamphet-amine-derived designer drugs or medicaments in blood and urine’’

[21]

,

‘‘Methamphetamine—properties and analytical methods of enantiomer determination’’

[20]

, and ‘‘Illegal and legitimate use? Precursor compounds to amphetamine and

meth-amphetamine’’

[22]

have been published in the 1983 – 2000 period. Relevant information in

these reviews will be summarized along with newer information.

2. Chromatographic approaches in the determination of enantiomeric compositions

Enantiomeric analysis of amphetamine and related drugs can be accomplished by a

variety of analytical techniques, including nuclear magnetic resonance (NMR)

approaches

[23]

. Among methods available, gas chromatography (GC) and

high-pressure liquid chromatography (HPLC), in combination with various detecting

devices, are most commonly used. Interest in enantiomeric purity, metabolic studies,

and pharmaceutical applications has lead to the development and use of a variety of

chiral derivatizing reagents and chiral stationary phases greatly facilitating GC and

HPLC applications.

2.1. Chiral derivatization and gas chromatographic analysis

Chiral derivatization, followed by GC separation of the resulting diastereomeric pairs, is

the most common approach adapted for enantiomeric analysis of amphetamine and related

drugs and metabolites

[16]

. Thanks to its commercial availability and effectiveness,

trifluoroacetyl-

-prolyl chloride (

-TPC,

Structure 1 in Fig. 1

) is widely used and has been

thoroughly studied. Many of these studies are summarized in the front section of

Table 1

.

In addition to trifluoroacetyl, other fluorinated groups, such as heptafluorobutyryl

[46 – 49]

have also been adapted. For example, Srinivas et al.

[49]

used

heptafluor-obutyryl-

-prolyl chloride (

-HPC,

Structure 2 in Fig. 1

) for the derivatization of

several racemic amphetamines under aqueous-alkaline condition (pH 9.5). The

result-ing diastereomeric pairs can be satisfactorily resolved under properly selected

iso-thermal conditions. This reagent has also been used for enantiomeric analysis of

methylenedioxymethamphetamine (MDMA) and its metabolites,

methylenedioxyam-phetamine (MDA), 3-methoxymethammethylenedioxyam-phetamine (HMMA), and

4-hydroxy-3-methoxyamphetamine (HMA), in rat

[50]

and human

[51]

specimens. In this latter

study, selected derivatization was achieved through a reaction with

-HPC, followed

by a reaction with N-methyl-N-trimethylsilyltrifluoroacetamide, resulting N-

-HPC-O-TMS (trimethylsilyl) derivatives. Preliminary results of the analysis of urine samples

indicated an enantioselective metabolism in the N-demethylation pathway for MDMA

in humans

[51]

.

Fig. 1. Structures of commonamphetamines chiral derivatizing reagents: N-trifluoroacetyl-L-prolyl chloride (1), N-heptafluorobutyryl-L-prolyl chloride (2), N-pentafluorobenzoyl-L-prolyl imidazolide (3), a-methoxy-a-(tri-fluoromethyl)phenylacetyl chloride (4), menthyl chloroformate (5), 1-phenylethyl isocyanate (6), 4-nitrophenyl-sulfonyl-L-prolyl chloride (7), (1S, 2S) N-[(2-isothiocyanato)-cyclohexyl]-pivalinoyl amide (8), 1-phenylethyl isothiocyanate (9), 9-fluorenylethyl chloroformate (10), 2,3,4,6-tetra-O-acetyl-h-D-glucopyranosyl isothiocyanate (11), 2,3,4-tri-O-acetyl-a-D-arabinopyranosyl isothiocyanate (12), o-phthaldialdehyde/N-acetyl-L-cysteine (13),

D-camphor-10-sulfonyl chloride (14), N-( p-toulenesulfonyl) prolyl isocyanate (15),

Table 1

Chiral derivatization and GC procedures for enantiomeric analysis of amphetamine and related drugs and metabolites

Reagenta Compound analyzedb Specimen Stationary phase Detection Reference

1 AM Std SP-2100

(Hewlett-Packard, Avondale, PA)

EI-MS [24]

1 AM, MAM Forensic sample SP-2100 EI-MS [25]

1 AM, MAM Human urine Nonpolar

(95% dimethyl – 5% phenyl methyl-polysiloxane) fused silica

EI-MS [26]

1 AM, MAM Human urine HP-5MS

(Hewlett-Packard)

EI-MS [27] 1 AM, a-phenylethylamine Forensic sample Ultra-1; Ultra-2

(Hewlett-Packard, Palo Alto, CA)

FID, FTIR [28] 1 AM Rat liver microsome HP-1 (Hewlett-Packard) FID [29]

1 AM, MAM Human urine HP-1; DB-17

(J&W Scientific, Rancho Cordova, CA)

EI-MS [30 – 33]

1 AM, MAM Human urine Cross-linked methylsilicone (Hewlett-Packard)

EI-MS [34]

1 AM, MAM Standard Ultra-1 EI-MS [35]

1 AM, MAM Human urine,

serum

HP-5MS EI-MS [36]

1 AM, MAM Human urine DB-5 (J&W Scientific) FID, EI-MS [37]

1 AM, MAM Human urine,

blood

Crosslinked 5% phenyl methyl silicone

EI-MS [38]

1 Methcathinone Standard HP-1 EI-MS [39]

1 AM, MAM, MDA, MDMA, MDEA

Human urine DB-17, HP-1; ZB-50 (Phenomenex)

EI-MS [40] 1 MDA, MDMA Blood Methylsilicone column

(Hewlett-Packard)

EI-MS [41,42] 1 MDMA, MDA Rat blood Methylsilicone column EI-MS [43] 1, 2 MDEA, MDA Rat brain 5% phenyl methyl

silicone (Hewlett-Packard, Palo Alto, CA)

EI-MS [44]

1 MDMA, MDA Blood, tissue HP-1 EI-MS [45]

2 Methylphendiate Blood, urine 1.5% OV-7/1.5% OV210 on Chromosorb-AW DMCS (Supelco, Cokville, Ont., Canada)

NPD, FID [46]

2 Methoxyphenamine, 2-OH-MAM, 2-OMe-AM, 2-OMe-5-OH-AM,

Human urine Cross-linked dimethyl silicone fused-silica (Hewlett-Packard: NJ)

NPD [47]

2 Norfenfluramine, fenfluramine

Human plasma Capillary OV-225 (Terochem Labs, Edmonton, Alberta, Canada)

Various combinations of fluorinated groups and amino acids have been explored to create

series of chiral derivatizing reagents. For example, Souter

[62]

evaluated the utilizations of

a variety of amino acids, including

-proline,

-leucine,

-valine and

-alanine, coupled

with trifluoroacetyl, pentafluoropropionyl, and heptafluorobutyryl groups.

Table 1 (continued)

Reagenta Compound analyzedb Specimen Stationary phase Detection Reference 2 Norfenfluramine, fenfluramine amphetamine, methamphetamine, 2-OMe-AM, p-OMe-AM, 3,4-dimethoxyamphetamine, methylphendiate, norephedrine, ephedrine

Standard Capillary OV-225 ECD [49]

2 MDMA, MDA, HMMA, HMA Rat urine, brain DB-5 coupled to DB 1301 (J&W Scientific, Rancho Cordova, CA) NCI-MS [50]

2 MDMA, MDA, HMMA, HMA

Urine DB-5MS (J&W Scientific, Folsom, CA)

PCI-MS [51] 3 AM Human plasma, saliva 5% OV-275 coated on Chromosorb W-AW (Supelco, Bellefonte, PA) CI-MS [52] ( )-4 AM, p-OH-AM, p-OMe-AM, 2,5-dimethoxy-4-methylamphetamine

Rat urine 3% phenyl methyl silicone on dimethylchloro-silane-treated diatomite support

MS [53]

( )-4 AM Rat urine 3% OV-17 coated

on Gas Chrom Q support

MS [54]

(+)-4 AM, MAM Human blood, gastric

Shimadzu CBP-5 (Shimadzu)

FID, MS [55] (+)-4 AM, MAM, p-OH-AM,

norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine,

Human urine Cross-linked 5% phenylmethylsilicone (SE-54)(Hewlett-Packard) NPD, MS [56 – 58] (+)-4 MAM, ephedrine, pseudoephedrine, methcathinone Standard DB-5 MS [59]

( )-4 MDMA, MDA Human urine, plasma

DB-17, HP Ultra 1 MS [60]

( )-5 AM, MAM Human urine DB-5, DB-17 MS [61]

a_{Only structure designations are listed. SeeFig. 1for the structures and names of these chiral derivatization} reagents.

b_{AM: amphetamine; MAM: methamphetamine; MDMA: methylenedioxymethamphetamine; MDA:} methylenedioxyamphetamine; MDEA: methylenedioxyethylamphetamine; HMMA: 4-hydroxy-3-methoxyme-thamphetamine; HMA: 4-hydroxy-3-methoxyamphetamine.

The use of N-pentafluorobenzoyl-S-

-prolyl-1-imidazolide (PFBPI,

Structure 3 in Fig.

1

) by Matin et al.

[52]

exemplified further deviation from this basic structural framework.

This reagent has also been studied by others as summarized in the middle section of

Table 1

.

L

- and

-a-Methoxy-a-(trifluoromethyl)phenylacetyl chloride (

- and

-MTPA,

Structure 4 in Fig. 1

) is a chiral pair of derivatization reagent that has also been

extensively studied. For example,

-MTPA was used to determine the enantiomeric

compositions of amphetamine and methamphetamine

[53,54]

; however, poor

deriva-tization yields for methamphetamine was reported. (This difficulty was overcome by

overnight incubation in a study using

-MTPA

[55]

.) Fallon et al.

[60]

used

-MTPA

to derivatize MDMA and MDA in human plasma and urine, while Shin and Donike

[56]

used

-MTPA to separate the enantiomers of amphetamines, phenol alkylamines,

and hydroxyamines. In this latter study, prior to N-acylation, amine salts were

converted to free bases and the hydroxy group was protected by TMS, TES

(triethylsilyl), or the t-BDMS (t-butyldimethylsilyl) groups in forming O-silyl ether

through the reaction to N-methyl-N-silylamide,

N-methyl-N-(trimethylsilyl)trifluoroace-tamide, N-methyl-N-(triethylsilyl)trifluoroaceN-methyl-N-(trimethylsilyl)trifluoroace-tamide, or

N-methyl-N-(t-butyldimethylsi-lyl)trifluoroacetamide, respectively. All resulting N-MTPA and O-silylated

diastereomeric pairs were well separated. This same approach has also been applied

to investigate the stereoselective metabolism of famprofazone

[56,57]

and selegiline

[58]

in humans. A slightly different version of this reagent,

-a-methoxy-a-(trifluor-omethyl)phenyl acetic acid, was used by LeBelle et al.

[59]

to analyze ephedrine,

pseudoephedrine, methamphetamine, and methcathinone.

Other chiral derivatizing reagents with different structural features are also shown in

Fig. 1

, while their major applicational characteristics are summarized in the later

section in

Table 1

. For example, Hughes et al.

[61]

analyzed amphetamine and

methamphetamine in urine as carbamate derivatives following reaction with

-menthyl

chloroformate

(Structure 5 in Fig. 1)

. With this approach,

-methamphetamine was

well resolved from illicit

-methamphetamine using a commonly used achiral

sta-tionary phase (DB-5).

2.2. Chiral derivatization and high-pressure liquid chromatographic analysis

Although not as convenient as GC, LC-based methodologies allow for (a) the

selection of larger and hopefully more effective derivatizing groups and (b) the use of

an ‘‘active’’ mobile phase (see Section 2.5), which introduces an additional parameter

not available to GC-based approaches.

Table 2

summarizes the use of various chiral

derivatization reagents to form diastereomers which were typically resolved by

reverse-phase C18 columns from various manufacturers.

In an early study, Miller et al.

[63]

compared four chiral reagents,

-MTPA,

2,3,4,6-tetra-O-acetyl-h-

-glucopyranosyl isothiocyanate (GITC,

Structure 11 in Fig. 1

),

(R)-

-1-phenylethyl isocyanate (PEIC,

Structure 6 in Fig. 1

), and

-arabinopyranosyl isothiocyanate (AITC,

Structure 12 in Fig. 1

), for their

effective-ness in resolving amphetamine enantiomers. Derivatization reactions were

accom-plished under mild conditions (25 – 75 jC) and were complete for all substrates within

60 min. The diastereomeric derivatives were separated by a C18 column with

Table 2

Chiral derivatization and HPLC procedures for enantiomeric analysis of amphetamine and related drugs and metabolites

Reagenta _{Compound analyzed}b _{Specimen Stationary phase Detection mode Reference} ( )-4, (+)-6, 11, 12 AM, p-Cl-AM, 2,5-dimethoxy-4-methyl-AM, 2,5-dimethoxy-4-thiomethyl-AM, 2,4-dimethoxy-5-methyl-AM Standard C18 (IBM Instruments, Danbury, CT) UV (220 or 254 nm) [63]

(+)-9 DOM, ephedrine Standard ODS (Beckmann, Berkeley, CA)

UV (254 nm) [64] 8 AM, MAM Standard ODS (Hypersil) UV (254 nm) [65] ( )-10 AM, MAM Rat serum C18 (Alltech

Associated, Deerfield, IL)

Fluorescence, FAB-MS

[66]

( )-10 AM, p-OH-AM Rat serum, urine C18 (Alltech Associated)

Fluorescence [67] ( )-10 MAM, p-OH-MAM,

AM, p-OH-AM

Human urine C18 (Alltech Associated)

Fluorescence [68] ( )-10 AM, p-OH-AM Rat liver C18 Fluorescence [69] (+)-10 Ephedrine, MAM,

pseudoephedrine

Forensic sample 5C18-AR (Waters Associates, Nacalai, Tesque) Fluorescence [70] Polymeric FMOC-L-proline

AM Spiked urine C18 (EM Science, Cherry Hill, NJ)

Fluorescence, UV – VIS, polarimeter

[71]

Spiked plasma C18-DB (Supelco, Bellefonte, PA) Fluorescence [72] 11 AM, MAM, ephedrine, pseudoephedrine, norephedrine, pseudonorephedrine,

Forensic sample C18 (Waters Associates)

UV (254 and 280 nm)

[8,10,11]

7 AM, MAM Standard Zorbax-Sil (Du Pont, Wilmington, DE) Supelcosil LC-Si (Supelco)

UV (254 nm) [73,74]

15 AM Standard Silica gel (Waters

Associates, Tianjing, China) UV (254 nm) [75] 14 AM, ephedrine, pseudoephedrine Standard C18; C8 (Perkin-Elmer) UV (260 nm) [76] OPA/ homochiral thiol AM, p-OH-AM, p-Cl-AM Standard C18 (Waters Associates) Fluorescence [77]

13 AM Human urine 100 RP18 (Merck) Fluorescence [78] 16 AM, MAM Human urine C18 (Alltech

Associates)

UV (340 nm) [79] a

Only structure designations are listed. SeeFig. 1for the structures and names of these chiral derivatization reagents.

b

methanol – water mobile phase. In general, diastereomeric pairs resulting from the

reaction with GITC, AITC, or MTPA were better resolved than those derived from

PEIC.

Noggle et al.

[8,10,11]

separated the enantiomers of ephedrine, pseudoephedrine,

amphetamine, and methamphetamine using GITC as the chiral derivatization agent.

The resulting amphetamine diastereomeric pair was not well resolved. This difficulty

was also reported when 4-nitrophenylsulfonyl-

-prolyl chloride (NPSP,

Structure 7 in

Fig. 1

) was used

[73,74]

. Kleidernigg and Lindner

[65]

synthesized chiral derivatizing

reagent (1S, 2S) N-[(2-isothiocyanato)-cyclohexyl]-pivalinoyl amide ((S,S)-PDITC,

Structure 8 in Fig. 1

) for the resolution of amphetamine and methamphetamine. The

resulting diastereomeric thioureas was reportedly better separated than the

correspond-ing GITC diastereomeric derivatives.

Gal and Sedman

[64]

evaluated

-1-phenylethyl isothiocyanate (PEITC,

Structure 9

in Fig. 1

) as a chiral derivatizing agent for the derivatization of enantiomeric

2,5-dimethoxy-4-methylamphetamine (DOM) and ephedrine, followed by the separation by

a C18 column.

Hutchaleelaha et al.

[66,67]

and Sukbuntherng et al.

[68]

from the same group used

-(9-fluorenyl)ethyl chloroformate (

-FLEC,

Structure 10 in Fig. 1

) to react with

amphet-amine, methamphetamphet-amine, and their hydroxy metabolites in urine (after h-glucuronidase

enzymatic cleavage of conjugates) for the formation of fluorescent diastereomers to

facilitate fluorescence detection. This same reagent was used by Gunaratna and Kissinger

[69]

to investigate the stereoselective metabolism of amphetamine in rat liver microsomes

by the cytochrome P-450 enzymes.

-FLEC was used by Chen et al.

[70]

for the analysis

of methamphetamine enantiomers in forensic samples.

Gao and Krull

[71]

developed an on-line solid-phase derivatization approach using

UV-fluorescence detection for the determination of amphetamine enantiomers in urine.

Polymeric 9-fluorenylmethyl chloroformate-

-proline (FMOC-

-proline) was used in this

study which was applied to the analysis of

,

-amphetamine in human plasma

[72]

.

Desai and Gal

[77]

described an enantiomeric analytical method for amphetamine

based on the reaction with o-phthaldialdehyde (OPA) and homochiral thiols. The

resulting highly fluorescent isoindole diastereomeric derivatives were resolved using a

C18 column. Pastor-Navarro et al.

[78]

developed a two-dimensional column-switching

method for on-line quantitation of amphetamine enantiomers in pharmaceuticals and

urine. The method used a C18 material for purification and a mixture of OPA and

N-acetyl-

-cysteine

(Structure 13 in Fig. 1)

as the derivatization reagent.

2.3. Chiral gas chromatographic methods

Chiral stationary phases (CSP) in GC columns allow for the analysis of

enantio-meric compositions without using prior formation of diastereoenantio-meric derivatives.

(However, achiral derivatizations are often used to improve the analytes’

chromato-graphic behavior and mass spectrometric characteristics

[80]

.) Peptide, diamide, and

ureide are used for the preparation of chiral stationary phases. Representative

structures of these phases are shown in

Fig. 2

. Further description of these phases

and their performance characteristics is available in a review by Liu and Ku

[17]

,

Fig. 2. Structures of monopeptide solvent – solute interaction (17), interactions of an N-TFA dipeptide ester (solvent) withD- andL-N-TFA amino acid esters (solute) (18), structure of the chiral stationary phase Chirasil-Val (19), general formula of ureide phases (20).

while their applications to the analysis of amphetamine and related drugs are

summarized in

Table 3

. Representative applications are further reviewed below.

In 1988, Ko¨nig et al.

[81]

resolved enantiomers of N-trifluoroacetylamphetamine and

N,O-di-(trifluoroacetyl)-4-hydroxyamphetamine on a capillary GC column which was

coated with 2,6-di-O-pentyl-3-O-acetyl-h-cyclodextrin stationary phase.

Jin and Beesley

[82]

compared the effectiveness and the elution orders of capillary GC

columns coated with three different chiral phases,

di-O-pentyl-3-O-trifluoroacetyl-h-cyclodextrin (B-TA), di-O-pentyl-3-O-trifluoroacetyl-g-di-O-pentyl-3-O-trifluoroacetyl-h-cyclodextrin (G-TA) and

2,6-di-O-pentyl-3-O-propionyl-g-cyclodextrin (G-PN) for the resolution of amphetamine and

methamphetamine enantiomers. Following the derivatization by acetic anhydride or

trifluoroacetic anhydride, separation was carried out isothermically. All enantiomers of

amphetamine and methamphetamine were separated within 30 min with separation factor

ranging between 1.02 and 1.06.

Armstrong et al.

[83]

used Chiraldex G-PN column to resolve trifluoroacetylated

derivatives of amphetamine, methamphetamine, ephedrine, and pseudoephedrine

enan-tiomers. Unexpectedly, trifluoroacetylated amphetamine has the opposite enantiomeric

elution order as acetylated amphetamine.

Wan et al.

[84]

used a mixed G-TA and OV-7 mixed phases for the analysis of

underiva-tized amphetamine and claimed improvement in thermal stability and shorter analysis time.

Hasegawa et al.

[85]

adapted Chirasil Dex-CB column to differentiate the metabolites

derived from the use of selegiline from the abuse of methamphetamine. Urine and plasma

Table 3

Chiral GC procedures for enantiomeric analysis of amphetamine and related drugs and metabolites Chiral stationary phase Compound

analyzeda Specimen Derivatizing reagentb Detection Reference Heptakis(3-O-acetyl-2,6-di-O-pentyl)-h-cyclodextrin

AM, p-OH-AM Standard TFA FID [81] 2,6-di-O-pentyl-3-O-trifluoroacetyl-g-cyclodextrin; 2,6-di-O-pentyl-3-O- trifluoroacetyl-g-cyclodextrin; 2,6-di-O-pentyl-3-O-propionyl-g-cyclodextrin

AM, MAM Standard TFA, AC FID [82]

Chiraldex G-PN; Chiraldex h-DM (Advanced Separation Technologies) AM, MAM, ephedrine, pseudoephedrin, deprenyl

Standard TFA, AC FID [83]

2,6-di-O-pentyl-3-O- trifluoroacetylated-h-cyclodextrin/OV 17

AM Standard FID [84]

Chirasil-Val (Applied Science) AM Standard L-TPC EI-MS [24] Chirasil-Val AM, MAM Forensic sample L-TPC EI-MS [25] Chirasil Dex-CB (Chrompack) Demethylselegiline,

AM, MAM

Urine, plasma PFP FID, EI-MS

[85] a_{AM: amphetamine; MAM: methamphetamine.}

b

specimens collected from selegiline users were derivatized with pentafluoropropionic

anhydride (PFPA) and the observed demethylseligiline, methamphetamine, and

amphet-amine were characterized as

-isomers.

2.4. Chiral high-pressure liquid chromatographic methods

Chiral stationary phases used for HPLC applications are grouped into five categories

[86]

as outlined in

Table 4

. Representative structures of these phases are illustrated in

Fig.

3

. Further description of these phases and their performance characteristics are available

in a review by Sellers et al.

[19]

, while their applications to the analysis of amphetamine

and related drugs are summarized in

Table 5

. Representative applications are further

reviewed below.

S-

-Naphthylurea column combined with UV detection was used for the analysis of

3,5-dinitrobenzoyl chloride (3,5-DNB) derivatized amphetamine enantiomers

[87,88]

.

Bourque and Krull

[89]

developed an analytical approach for on-line solid-phase

derivatization using a polymeric 3,5-DNB derivatizing reagent for the determination of

D

,

-amphetamine in urine.

The enantiomers of primary, secondary, and some tertiary amines were derivatized as

carbamate derivatives formed by reaction with h-naphthyl chloroformate. The enantiomeric

carbamates are resolved on an available Pirkle-type HPLC chiral stationary phase

consist-ing of (R)-N-(3,5-dinitrobenzoyl)-phenylglycine covalently bonded to silica, by usconsist-ing a

mobil phase consisting a mixture of isopropanol in hexane

[90]

.

Nagai et al.

[91]

assayed the enantiomeric compositions of amphetamine and

methamphetamine in human hair using a chiral cellulose-base column. In this application,

the analysts were derivatized as acetamide. Methamphetamine isomers and its metabolites

excreted in rat urine

[92]

were derivatized as benzoyl derivatives and analyzed by the

combined Chiralcel OB-H and OJ columns, which offered good peak resolution and

/

ratio data. Mixtures of amphetamine and methamphetamine could be separated

simulta-neously within 25 min. Faster metabolism of

-isomers occurred after the administration

of racemic methamphetamine and amphetamine, which confirms the metabolic

stereo-specificity. This approach also reported for simultaneous determination of optical isomers

of methamphetamine, amphetamine, p-hydroxymethamphetamine, and

p-hydroxyamphet-amine in rats

[93]

and humans urine

[94]

. Nagai et al.

[95 – 97]

investigated the

time-lapse changes of racemic ethylamphetamine in rats and humans urine base on the similar

procedure. In 1998, Matsushima et al.

[98]

using Chiralcel OB-H and Finepack SIL

Table 4

Category of chiral stationary phase for HPLC application Type Force for stereoisomeric retention

Type I Hydrogen bonding, pi – pi and dipole stacking

Type II Primarily attractive interaction; inclusion complexes also play an important role Type III Formation of inclusion complexes in chiral cavities

Type IV Formation of diastereoisomeric metal complexes with a selector ligand bound to the stationary phase

columns for identification of the optical activity and simultaneous analysis of racemates

MDA, MDMA and MDEA, and the urinary excretion of their optical isomers in rats was

investigated.

Fig. 3. Structures of HPLC chiral stationary phases. Type I: (R)-N-(3,5-dinitro-benzoyl)phenylglycine (covalent) (21), (R)-N-(3,5-dinitrobenzoyl)phenylglycine (ionic) (22), N-(3,5-dinitrobenzoyl)leucine (covalent) (23), (S)-N-(3,5-dinitrobenzoyl) leucine (ionic) (24), Supelcosil-LC-(S)-naphthyl urea (25); Type II: Chiracel OB (26), Chiracel OJ (27); Type III: h-Cyclodextrin (28).

Katagi et al.

[99]

determined the optical isomers methamphetamine and metabolites of

amphetamine and p-hydroxymethamphetamine in human urine directly using HPLC

thermospray – mass spectrometry with chiral h-CD phenylcarbamate-bonded silica column

(ULTRON ES-PhCD by Shinwa Chemical Ind., Kyoto, Japan).

Al-Dirbashi el al. derivatized methamphetamine and its metabolites in human urine

and hair (100 – 103) by fluorescent reagent, 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl

chloride (DIB). The resulting enantiomeric pair was separated using a semi-micro

Chiracel OD-RH column (Daicel Chemical Ind., Tokyo, Japan).

h-Cyclodexdrin chiral stationary phase has been used in several studies involving

amphetamine or related drugs. For example, Rizzi et al.

[104]

adapted this chiral

phase, ChiraDex (Merck, Darmstadt, Germany), to compare the efficiency in resolving

the enantiomers of amphetamine, methamphetamine, and various ring-substituted

amphetamines using two different approaches: (a) without derivatization and (b)

derivatization with phenyl isothiocyanate (PITC), naphthyl isothiocyanate (NITC),

and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). This approach is

further compared with chiral derivatization approach (Marfey’s reagent) using a C18

column. Lemr et al.

[105]

used a h-cyclodexdrin stationary phase (ChiraDex) to study

the influence of various parameters (mobil phase composition, pH, organic solvent,

salt nature and concentration, flow rate, injection amount, and temperature) on

enantiomeric separation of ephedrine, methamphetamine, and selegiline. Furthermore,

Sadeghipour and Veuthey

[106]

separated the enantiomers of MDA, MDMA, MDEA,

and MBDB on h-cyclodexdrin packed columns (Astec Cyclobond I 2000 and Astec

Cyclobond I 2000 RSP by Advanced Separation Technology, Whippany, NJ, USA)

coupled to a fluorimetric detector.

Makino et al.

[107]

developed a direct assay system for urine specimens. This approach

included two separation processes. A strong cation-exchange precolumn was used to

remove neutral anionic substances in urine; methamphetamine enantiomers trapped in the

column are then transferred to and separated by a phenyl-h-cyclodexdrin-bonded

semi-microcolumn (Chiral Drugk by Shiseido: Tokyo, Japan).

Chiral stationary phases derived from crown ethers have also been applied to the

analysis of amphetamine and related drugs. For example, DAICEL CROWNPACK CR

(+) (Daicel Chemicals, Japan) was used by Makino et al.

[108]

for the determination of

methamphetamine and amphetamine enantiomers. As little as 0.1%

-amphetamine in

bulk methamphetamine could be determined.

2.5. Enantiomeric separation by active mobile phases in high-pressure liquid

chromato-graphic methods

Transient diastereomeric complex formation is the fundamental mechanism underlying

chiral separation of enantiomers. Although not a widely adapted approach; nevertheless,

transient complexes can be formed between the analyte and a chiral component in the mobile

phase. The variance in elution times is a result of the rate of complex formation as well as the

difference in the affinities of the resulting complexes for the mobile phase or for the

stationary phase. Wainer

[86]

summarized enantiomer resolutions involving the addition of

chiral additives to the mobile-phase through the formation of three types of transient

Table 5

Chiral stationary phase for HPLC procedures for enantiomeric analysis of amphetamine and related drugs and metabolites

Chiral stationary phase Compound analyzeda Specimen Derivatizing reagent Detectionb _Reference Supelcosil LC-(S)-naphthylurea (Supelco UK, Poole, UK)

AM Human urine 3,5-DNB LCD-6AV [87]

Supelcosil LC-(S)-naphthylurea (Supelco, Bellefonte, PA) AM, MAM, ephedrine, pseudopehedrine, norephedrine

Human urine 3,5-DNB DAD-UV [88]

Supelcosil LC-(S)-naphthylurea

AM, norephedrine Human urine 3,5-DNB UV – VIS [89]

(R)-N-(3,5- dinitrobenzoyl)-phenylglycine

covalently bonded (Regis)

AM, MAM, ephedrine, pseudopehedrine, norephedrine, norpseudopehedrine, p-OH-AM, benzphetamine Standard h-naphthyl chloroformate UV – VIS [90] Chiralcel OB, OJ column (Diacel Ind., Tokyo, Japan)

AM, MAM Human hair Acetyl UV (220 nm) [91]

Chiralcel OB, OJ column AM, MAM Rat urine Benzoyl UV (220 nm) [92] Chiralcel OB, OJ column AM, MAM,

p-OH-MAM, p-OH-AM

Rat urine Benzoyl UV (220 nm) [93]

Chiralcel OB-H column AM, MAM Human urine Benzoyl UV (220 nm), OR-2 polarimeter (450 nm)

[94]

Chiralcel OB-H column EAM, AM Rat urine Benzoyl UV (220 nm) [95] Chiralcel OB-H,

OJ column

EAM, AM, p-OH-EAM, p-OH-AM

Rat urine Benzoyl UV (220 nm) [96]

Chiralcel OB-H column EAM, AM Human urine Benzoyl UV (220 nm) [97] Chiralcel OB-H,

Finepack SIL (Jasco, Tokyo, Japan)

MDA, MDMA, MDEA

Rat urine None UV (220 nm), polarimeter (450 nm)

[98]

Ultron ES-PhCD, ES-CD (Shinwa Chemical Ind.)

MAM, AM, p-OH- MAM

Human urine None TSP-MS, UV (220 nm) [99] Chiralcel OD-RH (Diacel Ind.) AM, MAM, p-OH- MAM Human urine, hair DIB Fluorescence [100 – 103] ChiraDex (Merck) AM, MAM,

p-OH-AM, p-OMe-AM, MDMA, MDEA, DOB, DOET, 2,5-dimethoxy-MAM DMA, Forensic sample PITC, NITC, AQC UV [104]

ChiraDex Ephedrine, MAM, selegiline

diastereomeric complexes: inclusion complexes, ion pairs, and transition metal ion

com-plexes.

This approach has been applied to the analysis of amphetamine-related drugs. For

example, Brunnenberg and Kovar

[109]

incorporated h-cyclodexdrin in the mobile phase

for the analysis of MDEA and MDA and a chiral protein phase (chiral-CBH) for

N-ethyl-4-hydroxy-3methoxyamphetamine (HME) to investigate the enantioselective metabolism of

MDEA and its metabolites (MDA and HME) in human plasma.

2.6. Combined application of chiral derivatization reagent and chiral stationary phase

Approaches involving combined use of chiral derivatization and chiral stationary phase

GC and HPLC analysis have also been explored. For example, Liu and Ku

[24]

and Liu et

al.

[25]

combined

-TPC derivatization and Chirasil-Val (Applied Sciences, State College,

PA) chiral stationary phase GC for the analysis of amphetamine enantiomers. With this

approach, the two minor diastereomeric pair derived from

-TPC impurity were also

resolved, resulting in the observation of four chromatographic peaks; the optical purity of

the chiral derivatization reagent

-TPC can thus be determined.

In a parallel HPLC/MS study using an ionic N-3,5-(dinitrobenzoyl)phenylglycine chiral

phase (Regis, Morton Grove, IL)

[111]

, enantiomers of amphetamine and

methamphet-amine were resolved. Another study

[112]

conducted by the same group involved the use of

Table 5 (continued)

Chiral stationary phase Compound analyzeda Specimen Derivatizing reagent Detectionb Reference Astec Cyclobond I 2000, I 2000 RSP (Advanced Separation Technologies) MDA, MDMA, MDEA, MBDB

Standard None Fluorescence [106]

Chiral Drugk (Shiseido) AM, MAM Human urine None DAD (210 nm) [107] DAICEL CROWNPACK

CR (+) (Diacel)

MAM, AM Human urine None DAD [108]

ChiraDex; Chiral CHB (Chrom Tech: Ha¨ngersten, Sweden) MDEA, MDA, HME Human plasma None Fluorescence, electrochemical [109] (+)-(18-Crown-6)-2,3,11, 12-tetracarboxylic acid bonded to silica gel

AM Standard None Absorbance [110]

Regis Pirkle covalent Phenylglycine; Regis Pirkle Ionic Phenylglycine

AM, MAM Standard L-TPC UV (254 nm), MS

[111]

a

AM: amphetamine; MAM: methamphetamine; MDMA: methylenedioxymethamphetamine; MDA: methylenedioxyamphetamine; MDEA: methylenedioxyethylamphetamine; DOB: 4-bromo-2,5-dimethoxyam-phetamine; DOET: 2,5-dimethoxy-4-ethylam4-bromo-2,5-dimethoxyam-phetamine; DMA: 2,5-dimethoxyam4-bromo-2,5-dimethoxyam-phetamine; MBDB: N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine; HME: N-ethyl-4-hydroxy-3-methoxyamphetamine.

b

3,5-DNB: 3,5-dinitrobenzoyl chloride; DIB: 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride; PITC: phenyl isothiocyanate; NITC: naphthyl isothiocyanate; AQC: 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; L-TPC: trifluoroacetyl-L-prolyl.

L

-TPC and GITC chiral derivatization reagents and achiral (C18) and chiral stationary

(SUPELCOSILR LC-(S) Naphthyl Urea phases (Supelco, Bellefonte, PA)) for the analysis

of ephedrine and pseudoephedrine. The following observations were reported:



_{Differences in the elution orders of the resulting}

-TPC and GITC derivatives. The order

for the resulting

-TPC derivatives is:

-ephedrine,

-ephedrine,

-pseudoephedrine,

and

-pseudoephedrine, while the order for the GITC derivatives is:

-pseudoephedrine,

-pseudoephedrine,

-ephedrine, and

-ephedrine.



_{With the HPLC parameters investigated, the C18 column does not provide adequate}

base-line resolution for the four components of ephedrine and pseudoephedrine.

With optimal HPLC parameters, the chiral column achieved base-line separation for

L

-TPC derivatives (with the exception of

-ephedrine and

-pseudoephedrine) and

similar base-line separation for GITC derivatives (with the exception of

-ephedrine

and

-pseudoephedrine). Sequential analysis of

-TPC and GITC derivatives, with

the naphthyl urea chiral column using THF/water compositions of 20:80 and 30:70,

respectively, will allow for base-line separation and quantitation of four enantiomeric

compositions—

-/

-ephedrine and

-/

-pseudoephedrine.



_{The detector’s (254 nm) responses toward the GITC derivatives were approximately 60}

times greater than that for the corresponding

-TPC derivatives.

Fig. 4. Structures of amphetamine-generating precursor compounds: amphetaminil (29), clobenzorex (30), ethyl-amphetamine (31), fenethylline (32), fenproporex (33), mefenorex (34), mesocarb (35), prenylamine (36).

3. Stereospecific metabolic processes and the effect of urine pH condition

Metabolic fates of many amphetamine-related drugs have been thoroughly studied. The

most important chemical processes are: aromatic hydroxylation at the 4-position, aliphatic

hydroxylation at the h-carbon position, N-dealkylation, oxidative deamination,

N-oxida-tion, and conjugation of the nitrogen.

N-demethylation reaction of methamphetamine was reportedly stereospecific with

-enantiomer being more rapidly proceeded. Thus, 16 h after the administration of racemic

methamphetamine, S-

-amphetamine was shown to be predominant

[113]

. On the other

hand,

-amphetamine was known to proceed with the h-hydroxylation at a faster rate

[114,115]

. Since the percentage of

-amphetamine was reportedly higher than the relative

percentage of

-methamphetamine

[14]

, the preferential conversion of

-methamphet-amine to

-amphetamine is apparently more significant than the preferential

h-hydrox-ylation of

-amphetamine.

Urine pH conditions can significantly affect the excretion patterns of amphetamines.

Since amphetamines are basic drugs (with pK

approximately 9.9), reabsorption at the

kidneys is insignificant under acidic urine conditions, resulting in the excretion of

more parent drugs. Under alkaline urine conditions, more significant reabsorption

occurs, with a net effect of increased drug half-lives. Increased metabolic degradation

will be observed.

Although urine pH conditions do not appear to cause differential excretion of

- and

-enantiomers

[116]

, its effect on the reabsorption rate will have a secondary effect on the

observed enantiomeric composition.

Fig. 5. Structures of amphetamine and methamphetamine-generating precursor compounds: benzphetamine (37), deprenyl (38), dimethylamphetamine (39), famprofazone (40), fencamine (41), furfenorex (42).

4. Amphetamine and methamphetamine metabolic precursor drugs

It is now well known that the use of a number of drugs can result in metabolic production

of amphetamine (alone) or methamphetamine and amphetamine

[16,21,22,117]

. Many of

these precursor compounds belong to the category of anorectics but are often used for

treating obesity; they are used because of their own therapeutic activities and are not

administered for the purpose of producing amphetamine or methamphetamine. Drugs that

have been studied and reportedly producing amphetamine are: amphetaminil, clobenzorex,

ethylamphetamine, fenethylline, fenproporex, mesocarb, prenylamine, and mefenorex

(Fig.

4)

. Those are known to produce methamphetamine and amphetamine are: benzphetamine,

deprenyl (selegiline), dimethylamphetamine, famprofazone, fencamine, and furfenorex

(Fig. 5)

.

The review of these compounds in this article will be limited to (a) methods used to

determine the enantiomeric compositions of these compounds and their metabolites; (b)

enantiomeric composition characteristics of these drugs and their metabolites, especially

amphetamine and methamphetamine; and (c) interpretative value of the observed

enantio-meric composition. Thus, the focus of the review is placed in analytical methodology and

data interpretation that may be helpful to differentiating amphetamine or

methamine produced by these precursor drugs from those derived from the abuse of

amphet-amine or methamphetamphet-amine. Most significant information in these aspects is summarized

in

Tables 6 and 7

.

Table 6

Precursors for amphetamine

Name Structurea Medical use Urinary specific metabolites Chirality Reference Amphetaminil 29 Psychostimulant; anorectic – –b [118 – 120] Clobenzorex 30 Anorectic; psychostimulant; sympathominetic 4-Hydroxyclobenzorex; clobenzorex (+) [121 – 127] Ethylamphetamine 31 Psychostimulant; anorectic; sympathominetic Ethylamphetamine; p-OH-ethylamphetamine Racemic [95 – 97, 128,129] Fenethylline 32 Psychostimulant Theophylline metabolites –b _{[130 – 134]} Fenproporex 33 Anorectic; psychostimulant Fenproporex Racemic [31,130, 135 – 137] Mefenorex 34 Psychostimulant; anorectic Mefenorex; 4-hydroxymefenorex –b [138,139] Mesocarb 35 Psychostimulant Mono- and

di-hydroxymesocarb

–b [140]

Prenylamine 36 Coronary vasodilator Diphenylpropylamine Racemic [141 – 149] a

Only structure designations are listed. SeeFig. 4for the structures and names of these drugs. b

4.1. Amphetamine-generating drugs

Metabolic products of Amphetaminil include amphetamine, hydrocyanic acid, and

benzaldehyde. Following administration to humans or rats, the unchanged parent drug was

not detectable in the urine sample

[118 – 120]

. The enantiomeric composition of this drug

has not been reported in the open literature.

Clobenzorex was first reported as a racemic drug

[121]

, but was later identified as

S-

-enantiomer

[122]

. This drug has been used as an adulterant in traditional Chinese

medicine

[123 – 125]

. A series of recent studies conducted by Valtier and Cody also

confirmed the chirality of this drug

[126]

. In these studies, chiral derivatization reagent,

L

-TPC, and HP-1 column were used for enantiomeric determination.

Although the use of this drug can result in the observation of amphetamine, it is rather

easy to differentiate the source of the observed amphetamine, thanks to the presence of

4-hydroxyclobenzorex (a metabolite of clobenzorex) which is detectable for at least as long as

amphetamine

[127]

.

The chirality of Ethylamphetamine has long been established as racemic mixture

[128]

.

Time-lapse changes of

- and

-enantiomers observed in human

[97]

and rat

[95,96]

urine

have also been studied. HPLC methods using Chiralcel OB-H and Chiral OJ columns

(Daicel Chemical Ind.) columns were found effective for enantiomeric determination

[99]

.

Under controlled conditions, the S-

-enantiomer was metabolized more rapidly than the

-enantiomer

[128]

. The time-lapse changes of

- and

-enantiomers of racemic

ethyl-amphetamine in the urine of rats and humans has been studied by Nagai et al.

[95 – 97]

. In

humans, the excreted dose of parent R-

-ethylamphetamine was found in higher

percent-age than that of S-

-ethylamphetamine, while the percentage of the excreted

-amphet-amine metabolite was found larger than that of

-amphetamine. The

/

ratios ranged

between 1.22 and 1.29. Rat studies produced different results, in where higher levels of

Table 7

Precursors for methamphetamine and amphetamine

Name Structurea _{Medical use Urinary specific} metabolites

Chirality Reference Benzphetamine 37 Anorectic

1-(4-Hydroxyphenyl)-2-(N-benzylamino)-propane

(+) [32,150 – 156] Deprenyl (selegiline) 38 Antiparkinsons;

MAO-B inhibitor

Desmethyldeprenyl; deprenyl-N-oxide

( ) [34,85, 157 – 161] Dimethylamphetamine 39 Illicit drug

Dimethylamphetamine-N-oxide; dimethylamphetamine –b [162 – 165] Famprofazone 40 Antipyretic; analgesic Famprofazone; p-hydroxydemethyl-famprofazone Racemic [33,36, 166 – 168] Fencamine 41 Psychostimulant Fencamine Racemic [153] Furfenorex 42 Anorectic

1-Phenyl-2-(N-methyl-N-g-valerolactonyl-amino) propane

–b _[170,171]

a_{Only structure designations are listed. SeeFig. 5for the structures and names of these drugs.} b_{Not reported.}

D

-enantiomers of ethylamphetamine and amphetamine were excreted than the

-enan-tiomers. The ratio of

/

was 0.51.

Presence of the parent drug and the hydroxylated metabolites, which can be detected at

a higher concentration and longer period after use, can be used to confirm the use of this

drug

[129]

.

Fenethylline is used medically as a psychostimulant for the treatment of children with

attention deficient disorders and has been used for the treatment of narcolepsy

[130]

or as

an antidepressant

[131]

. Enantiomeric composition of this drug has not been reported in

the open literature.

In a study using radiolabeled fenethylline, a relatively small amount (3.6%) of the parent

drug was found to excrete intact, while the percentages of the amphetamine, hippuric acid,

and 4-hydroxyamphetamine metabolites were found to be 24.5, 27.2, and 6.6%, respectively

[132]

. Theophylline, the N-dealkyl metabolite, was also found in urine. During the first 24 h,

the measured amount was approximately 13.7% of the dose given

[132]

.

Human studies of three volunteers (30-mg oral administration) conducted by Yoshimura

et al.

[133]

showed that carboxymethyl-theophylline and amphetamine were major

metabolites in the urine. Carboxymethyl-theophylline was found to disappear faster than

amphetamine, with only trace levels at 24 – 48 h. Amphetamine was found in higher

con-centration in the same time interval.

Kikura and Nakahara

[134]

investigated the detection of fenethylline and its metabolites

in human hair, following single-dose (50 mg/day, 1 day, n = 1) and multiple-dose (50 mg/

day, 3 days, n = 5) oral administration. In the proximal 1-cm segments, the concentrations of

fenethylline and amphetamine detected from subjects with multiple- and single-dose were

0.51 F 0.23 and 0.35 F 0.12 and 0.25 and 0.11 ng/mg, respectively. With the exception of

one sample, the concentration of fenethylline in human hair was found to range from 1.2 to

2.7 times greater than that of amphetamine. This is in contrast to rapid disappearing of

fenethylline in urine. Thus, hair samples may be valuable for confirming the use of

fenethylline.

Fenproporex is classified as a nonstimulant anorectic drug used for short-term treatment

of moderate to severe obesity

[121,130]

. Tognoni et al.

[135]

demonstrated that

admin-istration of fenproporex leads to formation of considerable amounts of amphetamine in the

body via cleavage of the nitrogen-cyanoethyl bond. A small amount of the parent drug is

excreted for a period of approximately 3-h postdose, whereas the metabolite amphetamine

is detectable for days

[136]

.

Amphetamine was detected in urine specimens collected from all five subjects studied.

The peak concentration appeared at approximately 6 – 20-h postdose and ranged from

approximately 1200 to 2100 ng/ml. It was detected (>5 ng/ml) in the urine for up to 119 h

[31]

. Metabolic amphetamine was a mixture of

- and

-enantiomers.

In a later study

[137]

, fenproporex was detected for longer periods than previously

reported. All urine samples with amphetamine higher than 500 ng/ml were also found to

contain detectable amounts of the parent drug for longer than 20-h postdose. The presence

of the parent drug and its relative concentration provides conclusive evidence for the

involvement of this drug.

Early study has shown the excretion of minimal (1%) intact mefenorex in rat and human

including 4-hydroxymefenorex, 4-hydroxyamphetamine, and 4-hydroxynorephedrine, in

urine. The metabolite 4-hydroxymefenorex is a useful indicator and is found in much higher

concentrations than the parent drug. After a single dose of 80 mg mefenorex, the parent

drug could only be found for 16 – 20 h and 4-hydroxymefenorex for about 32 h.

Amphet-amine could be detected from 32 to 68 h after ingestion. No study on the enantiomeric

composition of this drug was found in the open literature.

Since the parent drug and specific metabolites are not detectable for as long as the

metabolite amphetamine, misinterpretation of positive amphetamine tests is possible.

Rat study

[140]

on the metabolism of Mesocarb showed minimal excretion (1%) of the

parent drug, while the dihydroxy, hydroxy, and amphetamine metabolites showed 60%,

22%, and 4%, respectively. No study on the enantiomeric composition of this drug was

found in the open literature.

Prenylamine is marketed as a racemic mixture. The metabolism of this drug has been

well studied

[141 – 149]

. R-

-Naphthylethyl isocyanate was used as the chiral derivatization

reagent to study the enantioselective characteristic on the metabolism of this drug

[144]

.

Metabolism of S-

-prenylamine was faster than R-

-prenylamine, with S-

-metabolites

excreted more rapidly. In plasma, the amount of R-

-prenylamine and S-

-prenylamine was

found in 4:1 ratio

[146]

.

In addition to amphetamine, 4-hydroxyamphetamine, norephedrine,

4-hydroxynorephe-drine, and diphenylpropylamine were identified as metabolites

[147 – 149]

.

4.2. Methamphetamine and amphetamine-generating drugs

Benzphetamine is synthesized from S-

-methamphetamine as a pure S-

-enantiomer. It

is prescribed as an anorectic in the treatment of obesity as adjunct to a reduced caloric diet

[150,151]

. The metabolism of benzphetamine is nearly complete with little, if any, excreted

as unchanged drug

[152,153]

. 1-(4-Hydroxyphenyl)-2-(N-benzylamino)propane, an

aro-matic hydroxylation and N-demethylation product, has been identified as the major

metabolite in urine

[154,155]

. Typically, samples tested for amphetamine and

methamphet-amine are not hydrolyzed. Hydrolysis is needed for full recovery of

1-(4-hydroxyphenyl)-2-(N-benzylamino)propane. Recently, Sato et al.

[156]

developed a h-glucuronidase

hydrol-ysis, solid-phase extraction, and LC – MS procedure for the analysis of benzphetamine and

its five metabolites in rat urine.

With respect to the excretion of metabolites in the urine, Cody and Valtier

[32]

have

reported two distinctly different results following a single 50-mg oral dose of

benzphet-amine – HCl. It was shown that the metabolic production of amphetbenzphet-amine derived from

methamphetamine and, to a substantial degree, also from demethylbenzphetamine. Thus,

several subjects studied excreted substantially higher level of methamphetamine, while

others excreted more amphetamine than methamphetamine.

The following analytical findings resulting from the use of benzphetamine may help

identify the source of amphetamine and methamphetamine: (a) potential identification of

1-(4-hydroxyphenyl)-2-(N-benzylamino)propane; (b) methamphetamine and amphetamine

exist in S-

-enantiomeric forms; and (c) amphetamine/methamphetamine >1 for those

individuals with metabolic characteristic in generating higher amphetamine level than

methamphetamine.

Deprenyl (selegiline), in combination with levodopa, is used primarily for treating

Parkinson’s disease

[157,158]

. Deprenyl was shown to be extensively metabolized to form

desmethyldeprenyl, methamphetamine, amphetamine, and their conjugated p-hydroxy

derivatives

[159,160]

. Desmethyldeprenyl can be detected in urine for less than 12 h after

a single oral dose of 2.5 – 10 mg deprenyl

[34,85]

. Recently, Katagi et al.

[161]

identified

deprenyl-N-oxide in two deprenyl users’ urine, with abundance higher than three times of

desmethyldeprenyl.

Deprenyl serves as a good example for using enantiomer data to differentiate the sources

of amphetamine and methamphetamine derived from the use of licit drugs from abused

substances. Deprenyl is manufactured as R-

-enantiomer; thus generating

-amphetamine

and

-methamphetamine metabolites, while those derived from abused substances exists as

racemates or

-enantiomers. In cases in which

-methamphetamine is administered,

differentiation can be made based on the identification of unique deprenyl metabolites,

such as deprenyl-N-oxide or desmethyldeprenyl.

Dimethylamphetamine is not known as a prescription drug. Dimethylamphetamine

showed similar metabolic pattern in rats and humans, with the exception of more

pronounced aromatic hydroxylation level in rats

[162 – 164]

. Major metabolic products in

humans include the parent drug, dimethylamphetamine-N-oxide, methamphetamine, and

amphetamine. Recently, Katagi et al.

[165]

developed a simple and sensitive LC – MS

procedure for the simultaneous determination of dimethylamphetamine and its metabolites

dimethylamphetamine-N-oxide, methamphetamine, and amphetamine in urine.

Since dimethylamphetamine-N-oxide in urine can be satisfactorily detected in urine

even 3 – 5 days after the intake of dimethylamphetamine, it can serve as an effective

indicator for identifying dimethylamphetamine use.

Famprofazone is a component of the multi-ingredient medication Gewodin used as an

antipyretic and analgesic (with some slight sympathomimetic properties)

[130]

. Each tablet

contains 25 mg famprofazone, 250 mg paracetamol (acetaminophen), 75 mg

isopropyl-phenazone and 30 mg caffeine. This drug has been demonstrated to metabolically produce

methamphetamine and amphetamine and has been shown to be the cause of positive urine

drug testing

[166]

.

Cody

[33]

investigated the enantiomeric composition of metabolic amphetamine and

methamphetamine following a single 50-mg dose of famprofazone and reported higher

concentration of

-methamphetamine than

-methamphetamine in each sample.

-Meth-amphetamine in the first sample was 67% which increases to 100% in the last few sample.

The composition of amphetamine enantiomers were much closer, with

-amphetamine

beginning at approximately 50% and rising only to approximately 55% in the later samples.

These enantiomeric composition characteristics are of reference value for determining the

source of amphetamine and methamphetamine.

The identification of 3-hydroxymethylpyrazolone metabolite provides better proof of

famprofazone administration. However, it should be noted that

3-hydroxymethylpyrazo-lone is also a metabolite of propylphenazone. Intake of famprofazone can be proved

through the identification of the unchanged parent drug or the longer-lasting metabolite,

p-hydroxydemethylfamprofazone in urine

[167 – 169]

.

Mallol et al.

[169]

studied the metabolism of Fencamine in rats and humans and

reported the detection of intact fencamine in humans for 48 h following a 50-mg oral dose

of the drug. Approximately 32% of the dose was excreted as the intact drug in 48 h and

26.6% in the first 24 h in humans.

Fencamine is manufactured as a racemic mixture. Enantiomeric composition of the

resulting metabolites can be of assistance in confirming whether fencamine is ingested.

Administration of Furfenorex to human resulted in nearly complete conversion with

very little of the parent drug excreted in urine. Of various metabolites identified,

1-phenyl-2-(N-methyl-N-g-valerolactonylamino)propane was found to be unique and characteristic

of the use of furfenorex

[170]

. This metabolite was also excreted at higher levels than

amphetamine, methamphetamine, and their hydroxylated compounds, the sum of which

represents only about 6% of the dose

[153,171]

.

Acknowledgements

The preparation of this manuscript is facilitated by a visiting appointment granted to

Ray H. Liu by the (Taiwanese) National Science Council (NSC90-2811-B-043D-001).

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