Enantiomeric resolution of methylamphetamine and ephedrine: Does this affect the δ13 C, δ15 N, and δ2 H stable isotope ratios of the product?

The use of stable isotope ratio mass spectrometry (IRMS) as a profiling tool for methylamphetamine has evolved over the last decade. Stable isotope ratios of carbon (δ13 C), nitrogen (δ15 N), and hydrogen (δ2 H) of methylamphetamine are useful in determining the precursor used to manufacture methylamphetamine, and in many cases the synthetic origin of the methylamphetamine precursor. More recently, samples of seized methylamphetamine show that a resolution step is being employed in the manufacturing process. We sought to determine whether the δ13 C, δ15 N, and δ2 H values were affected by either a resolution performed on racemic methylamphetamine or a resolution on racemic ephedrine, a commonly used precursor to methylamphetamine. We found that for the types of resolution studied, IRMS is still able to provide useful information on the provenance of a methylamphetamine sample.

Complementary stable carbon isotope ratio and amount of substance measurements in sports anti-doping.

The detection of steroids originating from synthetic precursors against a background of their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). The complementary application of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) has been demonstrated to provide specific detection of endogenous steroid misuse for improved anti-doping analysis. Markers of synthetically derived steroids are reviewed on the basis of abnormal urinary excretions and low (13)C content. A combinatorial approach is presented for the interpretation of GC-MS and GC-C-IRMS data in the anti-doping context. This methodology can allow all relevant information concerning an individual's metabolism to be assessed in order to make an informed decision with respect to a doping violation.

The potential of urinary androstdiene markers to identify 4-androstenediol (4-ADIOL) administration in athletes.

Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require criteria that allow endogenous steroids to be distinguished from their synthetic analogues in urine. Methodology based on "looking outside the metabolic box" was used in this study to identify diagnostic urinary markers of 4-androstenediol (4-ADIOL) administration. Androst-2,4-diene-17-one and androst-3,5-diene-17-one are proposed to be formed in urine from acid-catalyzed hydrolysis of 4-ADIOL sulfoconjugate, a major phase II metabolic product of 4-ADIOL. The presence of these markers in the routine gas chromatography-mass spectrometry (GC-MS) steroid screen was suitable to identify samples requiring confirmation by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) - to measure the carbon isotope ratio (δ(13)C) of the androstdiene markers and confirm their likely synthetic origin based on depleted (13)C content.

Isolation and identification of three by-products found in methylamphetamine synthesized by the Emde route.

This article describes the isolation and structural elucidation of three compounds produced during the synthesis of methylamphetamine by the so-called "Emde" procedure. The "Emde" procedure involves the preparation of the intermediate chloropseudoephedrine or chloroephedrine from ephedrine or pseudoephedrine, respectively. The intermediates are then reduced to methylamphetamine with hydrogen under pressure in the presence of a catalyst. The by-product compounds were isolated from methylamphetamine by column chromatography and liquid chromatography (LC). Proton nuclear magnetic resonance spectroscopy ((1)H NMR), carbon nuclear magnetic resonance spectroscopy ((13)C NMR), and nanospray quadrupole-time of flight-mass spectrometry (Q-TOF-MS) were used to identify them as two stereoisomers of the compound N, N'-dimethyl-3,4-diphenylhexane-2,5-diamine and N-methyl-1-{4-[2-(methylamino)propyl]phenyl}-1-phenylpropan-2-amine.

Carbon isotope ratio (delta13C) values of urinary steroids for doping control in sport.

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (delta(13)C) profiling study (n=1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This delta(13)C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to delta(13)C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n=100; consisting of three countries) is also presented with comparison to delta(13)C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.

The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4-hydroxyandrostenedione metabolite.

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) - measured by gas chromatography/mass spectrometry (GC/MS) - in subjects with a normal ( approximately 1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C(6)-hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted (13)C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound-specific detection. C(4)-hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4-hydroxyandrostenedione (4OH-ADIONE). Complementary analysis of 4OH-ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration.

N-cyanomethyl-N-methyl-1-(3',4'-methylenedioxyphenyl)-2-propylamine: an MDMA manufacturing by-product.

This paper describes the structural elucidation of a compound produced during the synthesis of 3,4-methylenedioxymethylamphetamine (MDMA) via the reductive amination of 3,4-methylenedioxyphenyl-2-propanone (3,4-MDP-2-P) with methylamine and sodium cyanoborohydride. The compound was isolated from MDMA by column chromatography, proton and carbon nuclear magnetic resonance spectroscopy, LC/mass spectrometry, and total synthesis were used to identify the compound as N-cyanomethyl-N-methyl-1-(3',4'-methylenedioxyphenyl)-2-propylamine. This compound has been identified as a potential synthetic route marker for the reductive amination of 3,4-MDP-2-P with methylamine and sodium cyanoborohydride and as such it should prove valuable to forensic scientists engaged in profiling illicit drugs. Profiling MDMA can provide useful information to law enforcement agencies relating to synthetic route, precursor chemicals and reagents employed and may be used for comparative analyses of different drug seizures. This paper also describes the structural elucidation of the analogous methylamphetamine synthetic route marker compound, N-cyanomethyl-N-methyl-1-phenyl-2-propylamine, produced during the reductive amination of phenyl-2-propanone using methylamine and sodium cyanoborohydride.

Determination of urinary steroid sulfate metabolites using ion paired extraction.

The need for laboratories accredited by the World Anti-Doping Agency (WADA) to develop methods of analysis for steroids excreted primarily as their sulfate conjugates has faced significant analytical challenges. One of the issues relates to the extraction of these metabolites from urine in a relatively pure state. The use of (-)-N,N-dimethylephedrinium bromide as an ion pairing reagent was optimised to produce a method that is selective for the extraction of steroid sulfates prior to GC-MS or LC-MS analysis, with minimal contributions from the urine matrix. The recovery of androsterone from its sulfate conjugate was determined to be 67% with a relative quantitative uncertainty of +/-14% (k = 2).

Isotopic fractionation of endogenous anabolic androgenic steroids and its relationship to doping control in sports.

The use of gas chromatography (GC)-combustion (C)-isotope ratio mass spectrometry (IRMS) demonstrates that a single oral administration of dehydroepiandrosterone (DHEA, 100 mg) to a male subject significantly lowers the 13C content of etiocholanolone (Et) and androsterone (A) in the subject's urine. The difference in carbon isotope ratio (d13C per thousand) values between Et and A increases from 1.6 per thousand at the time of administration to 5.1 per thousand at 26 h post-administration, indicating preferential metabolism of administered DHEA to form Et in relation to A. Multiple oral administrations of DHEA to a male subject reveals lower d13C values during the excretion period of Et (-31.7 per thousand to -34.6 per thousand) and A (-31.4 per thousand to -33.0 per thousand) to that of the d13C value of the administered DHEA (-31.3 per thousand). Reference distributions of d13C Et and d13C A constructed from normal athlete populations within Australia and New Zealand show a small natural discrimination against 13C in the formation of Et relative to A (mean=0.3 per thousand, n=167, p=0.007). Amplified differences between d13C Et and d13C A, and in vivo 13C depletion measured by GC-C-IRMS are shown to be potentially useful for doping control.