A proposed approach to confirm heroin administration - Regional differences in heroin purity is a major factor.

In forensic toxicology, a marker of street heroin use is urgent especially in the absence of urinary 6-monoacetylmorphine. ATM4G, the Glucuronide of Acetylated product of Thebaine compound 4 Metabolite (ATM4), arising from byproducts of street heroin synthesis has been considered as a useful marker in some European studies. However, whether ATM4G is a universal marker particularly in Southeast Asia due to 'street' heroin with high purity, it's still unclear. To investigate putative markers for different regions, ATM4G and other metabolites including the Acetylated product of Thebaine compound 3 Metabolite (ATM3) and thebaol, also originated from thebaine were detected in 552 urine samples from heroin users in Taiwan. Results were compared with that from samples collected in the UK and Germany. Only a sulfo-conjugate of ATM4, ATM4S, was detected in 28 Taiwanese users using a sensitive MS3 method whilst out of 351 samples from the UK and Germany, ATM4G was present in 91. Thebaol-glucuronide was first time detected in 118. No markers were detected in urine following herbal medicine use or poppy seed ingestion. The presence of ATM4S/ATM4G might be affected by ethnicities and heroin supplied in regions. Thebaol-glucuronide is another putative marker with ATM4G and ATM4S for street heroin use.

Detection of mephedrone and its metabolites in fingerprints from a controlled human administration study by liquid chromatography-tandem mass spectrometry and paper spray-mass spectrometry.

The use of synthetic stimulants, including designer cathinones, remains a significant concern worldwide. Thus, the detection and identification of synthetic cathinones in biological matrices is of paramount importance for clinical and forensic laboratories. In this study, distribution of mephedrone and its metabolites was investigated in fingerprints. Following a controlled human mephedrone administration (100 mg nasally insufflated), two mass spectrometry-based methods for fingerprint analysis have been evaluated. The samples deposited on triangular pieces of chromatography paper were directly analysed under ambient conditions by paper spray-mass spectrometry (PS-MS) while those deposited on glass cover slips were extracted and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was 5-6 times more sensitive than PS-MS but required sample preparation and longer analysis time. Mephedrone was detected in 62% and in 38% of all post-administration samples analysed by LC-MS/MS and PS-MS, respectively. Nor-mephedrone was the only metabolite detected in 3.8% of all samples analysed by LC-MS/MS. A large inter- and intra-subject variation was observed for mephedrone which may be due to several factors, such as the applied finger pressure, angle and duration of contact with the deposition surface and inability to control the 'amount' of collected fingerprint deposits. Until these limitations are addressed, we suggest that the sole use of fingerprints can be a useful diagnostic tool in qualitative rather than quantitative analysis, and requires a confirmatory analysis in a different biological matrix.

Rapid Analysis of Anabolic Steroid Metabolites in Urine by Combining Field Asymmetric Waveform Ion Mobility Spectrometry with Liquid Chromatography and Mass Spectrometry.

The combination of field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry (LC-FAIMS-MS) has been developed for the analysis of glucuronide and sulfate metabolites of seven anabolic-androgenic steroids in urine. Separation by FAIMS-MS was investigated in positive ion mode for selected cationic adducts (H+, NH4+, Na+, K+, and Cs+). LC-FAIMS-MS analysis of the doubly sodiated adducts ([M + 2Na - H]+) of isobaric and coeluting steroid metabolites allowed their rapid (8 min) qualitative and quantitative determination in spiked urine using hydrophilic interaction liquid chromatography prior to FAIMS-MS separation, with discrimination >95% achieved between the steroids investigated. A quantitative evaluation of the LC-FAIMS-MS method was performed giving limits of detection in the range 1-6 ng mL-1, limits of quantification in the range 3-20 ng mL-1, with reproducibility (%RSD < 10%; n = 6) and linearity (R2 > 0.99). The LC-FAIMS-MS method demonstrates increases in signal-to-noise ratios for the doubly sodiated steroid metabolites in unspiked urine (>250%) by the reduction of isobaric interferences from the matrix. An alternative or additional tool for identification of the steroid metabolites is based on the observations of different patterns of sodium acetate clusters that are characteristic for each metabolite.

Excretion of mephedrone and its phase I metabolites in urine after a controlled intranasal administration to healthy human volunteers.

Mephedrone is a stimulant drug structurally related to cathinone. At present, there are no data available on the excretion profile of mephedrone and its metabolites in urine after controlled intranasal administration to human volunteers. In this study, six healthy male volunteers nasally insufflated 100 mg of pure mephedrone hydrochloride (Day 1). Urine was collected at different timepoints on Day 1 and then on Days 2, 3 and 30. Samples were analysed for the presence of mephedrone and its metabolites, namely, dihydro-mephedrone, nor-mephedrone (NOR), hydroxytolyl-mephedrone, 4-carboxy-mephedrone (4-carboxy) and dihydro-nor-mephedrone (DHNM), by a validated liquid chromatography-tandem mass spectrometry method. All analytes were detected in urine, where 4-carboxy (Cmax = 29.8 µg/ml) was the most abundant metabolite followed by NOR (Cmax = 377 ng/ml). DHNM was found at the lowest concentrations (Cmax = 93.1 ng/ml). Analytes exhibited a wide range of detection windows, but only 4-carboxy and DHNM were detectable in all samples on Day 3, extending the detection time of mephedrone use. Moreover, mephedrone had a mean renal clearance of 108 ± 140 ml/min, and 1.3 ± 1.7% of unchanged parent drug was recovered in urine in the first 6 h post administration. It is hoped that this novel information will be useful in future studies involving mephedrone and other stimulant drugs.

Pharmacokinetics of Mephedrone and Its Metabolites in Whole Blood and Plasma after Controlled Intranasal Administration to Healthy Human Volunteers.

Mephedrone is a popular synthetic cathinone, known for its psychostimulant effects. At present, there is no data available on the pharmacokinetics of mephedrone and its metabolites in concurrently collected whole blood and plasma samples after a controlled intranasal administration to healthy volunteers. In this study, six healthy male volunteers nasally insufflated 100 mg of pure mephedrone hydrochloride (Day 1). Whole blood and plasma samples were collected at different time points after the administration and were analyzed for the presence of mephedrone and its metabolites, dihydro-mephedrone (DHM), nor-mephedrone (NOR), hydroxytolyl-mephedrone (HYDROXY), 4-carboxy-mephedrone (4-CARBOXY) and dihydro-nor-mephedrone (DHNM), by validated liquid chromatography-tandem mass spectrometry methods. All analytes were detected in whole blood and plasma for 6 h post administration, with mephedrone and NOR also being detectable on Day 2 in some participants. 4-CARBOXY, followed by NOR, was the most abundant metabolite in both matrices. Compared to other psychostimulants, mephedrone showed rapid absorption (mean Tmax of 52.5 ± 20.7 min in plasma and 55.0 ± 18.2 min in whole blood) and elimination (mean t1/2 of 1.98 ± 0.30 h in plasma and 2.12 ± 0.33 h in whole blood). In addition, statistical analysis showed that median whole blood to plasma distribution ratios, reported here for the first time, were statistically different from 1 (unity) for mephedrone (median: 1.11), DHM (median: 1.30) and NOR (median: 0.765). It is hoped that the study will aid forensic and clinical toxicologists in detection, identification and interpretation of cases associated with mephedrone use.

Biochemical and physiological aspects of endogenous androgens.

This review attempts to give a synopsis of the major aspects concerning the biochemistry of endogenous androgens, supplemented with several facets of physiology, particularly with respect to testosterone. Testosterone continues to be the most common adverse finding declared by World Anti-Doping Agency accredited laboratories, such samples having an augmented testosterone to epitestosterone ratio. Knowledge regarding the precursors and metabolism of endogenous testosterone is therefore fundamental to understanding many of the issues concerning doping with testosterone and its prohormones, including the detection of their administration. Further, adverse findings for nandrolone are frequent, but this steroid and 19-norandrostenedione are also produced endogenously, an appealing hypothesis being that they are minor by-products of the aromatization of androgens. At sports tribunals pertaining to adverse analytical findings of natural androgen administration, experts often raise issues that concern some aspect of steroid biochemistry and physiology. Salient topics included within this review are the origins and interconversion of endogenous androgens, the biosynthesis of testosterone and epitestosterone, the mechanism of aromatization, the molecular biology of the androgen receptor, the hypothalamic-pituitary-testicular axis, disturbances to this axis by anabolic steroid administration, the transport (binding) of androgens in blood, and briefly the metabolism and excretion of androgens.

Pharmacokinetics of Mephedrone Enantiomers in Whole Blood after a Controlled Intranasal Administration to Healthy Human Volunteers.

Mephedrone, which is one of the most popular synthetic cathinones, has one chiral centre and thus exists as two enantiomers: R-(+)-mephedrone and S-(-)-mephedrone. There are some preliminary data suggesting that the enantiomers of mephedrone may display enantioselective pharmacokinetics and exhibit different neurological effects. In this study, enantiomers of mephedrone were resolved via chromatographic chiral recognition and the absolute configuration was unambiguously determined by a combination of elution order and chiroptical analysis (i.e., circular dichroism). A chiral liquid chromatography tandem mass spectrometry method was fully validated and was applied to the analysis of whole blood samples collected from a controlled intranasal administration of racemic mephedrone hydrochloride to healthy male volunteers. Both enantiomers showed similar kinetics, however, R-(+)-mephedrone had a greater mean Cmax of 48.5 ± 11.9 ng/mL and a longer mean half-life of 1.92 ± 0.27 h compared with 44.6 ± 11.8 ng/mL and 1.63 ± 0.23 h for S-(-)-mephedrone, respectively. Moreover, R-(+)-mephedrone had a lower mean clearance and roughly 1.3 times greater mean area under the curve than S-(-)-mephedrone. Significant changes in the enantiomeric ratio over time were observed, which suggest that the analytes exhibit enantioselective pharmacokinetics. Even though the clinical significance of this finding is not yet fully understood, the study confirms that the chiral nature, and consequently the enantiomeric purity of mephedrone, can be a crucial consideration when interpreting toxicological results.

Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control.

Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1-progesterone was also detected. Δ1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry.

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

Doping in sport--1. Excretion of 19-norandrosterone by healthy women, including those using contraceptives containing norethisterone.

19-Norandrosterone (19-NA) is the principal urinary metabolite of the anabolic steroid nandrolone and its prohormones. The administration of these 19-nor androgens is prohibited in sport by the World Anti-Doping Agency (WADA) but, even so, adverse findings for 19-NA continue to be commonly reported. Little is known about the urinary concentrations of 19-NA that can occur in women who are not using anabolic steroids, including those using oral contraceptives containing the 19-nor progestogen norethisterone. In 2004, WADA lowered the reporting threshold for 19-NA for females from 5 to 2ng/mL. The lack of any substantial data on 19-NA excretion in women prompted this large-scale investigation. In this investigation, single untimed urines collected from 1202 female volunteers, 38 of whom were taking norethisterone containing contraceptives, were analysed for 19-NA. None of the women was a competitive athlete and pregnancy had been excluded by a urinary test for human chorionic gonadotropin (hCG). Only one sample exceeded the 19-NA reporting threshold having a concentration of 4.1ng/mL. This sample was from a user of a norethisterone-containing contraceptive.

Doping in sport--2. Quantification of the impurity 19-norandrostenedione in pharmaceutical preparations of norethisterone.

The finding of measurable amounts of 19-norandrostenedione in norethisterone tablets prompted us to develop an assay to quantify this steroid. 19-Norandrostenedione is an anabolic steroid whose use in sport is prohibited by the World Anti-Doping Agency (WADA). The assay was developed using isotope dilution and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 19-norandrostenedione in norethisterone formulations, with [3,4-(13)C(2)]-19-norandrostenedione as the internal standard. The results showed amounts up to 1.01+/-0.01microg (mean+/-S.E.M.) per tablet in those containing 5mg of norethisterone or norethisterone acetate (0.02%, w/w) and up to 0.5+/-0.01microg (mean+/-S.E.M.) per tablet (0.05%, w/w) in oral contraceptive tablets containing 0.35-1.5mg of norethisterone or norethisterone acetate. No tablet tested exceeded the British Pharmacopoeia limit of 0.1% for this impurity.

Doping in sport: 3. Metabolic conversion of oral norethisterone to urinary 19-norandrosterone.

The detection of 19 norandrosterone (19-NA) in a competitor's urine sample is taken as prima facie evidence of administration of nandrolone or other 19-norsteroid but a potential problem is that administration of norethisterone, a progestogen used for menstrual disorders and for hormonal contraception, also results in the excretion of 19-NA that can exceed the laboratory reporting threshold of 2ng/mL. The contribution of norethisterone to urinary 19-NA with and without 19-norandrostenedione, a known norethisterone tablet impurity, requires evaluation. Preparations containing, either <2ng or 1microg 19-norandrostenedione impurity per 5mg of norethisterone, administered to female volunteers (n=10) in doses comparable to those used for menstrual disorders (5mg three times daily for 10 days), resulted in maximal 19-NA concentrations of 51 and 63ng/mL, respectively. The maximal concentration of 19-NA, 2h post-administration of a single 1microg dose of 19-norandrostenedione, was 2.4ng/mL. These results prove unequivocally that norethisterone is metabolized to 19-NA and that there is only a minor contribution from the impurity 19-norandrostenedione. Administration to women (n=30) of a single contraceptive tablet containing norethisterone (1mg) with one of the highest proportions of the impurity 19-norandrostenedione ( approximately 0.5microg, 0.05%, w/w) resulted in a urinary 19-NA concentration of 9.1ng/mL, with a maximum concentration ratio of 19-NA to the norethisterone metabolite 3alpha,5beta-tetrahydronorethisterone of 0.36. We provide data that should remove the need for time-consuming follow-up investigations to consider whether doping with 19-norandrogens has occurred.

Stability of mephedrone and five of its phase I metabolites in human whole blood.

Mephedrone is a new psychoactive substance known to be unstable in biological matrices stored at room temperature or refrigerated. While the instability of mephedrone has been investigated before, there is currently no data regarding the stability of mephedrone metabolites. In this study, a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of mephedrone and five of its phase I metabolites (dihydro-mephedrone, nor-mephedrone, hydroxytolyl-mephedrone, 4-carboxy-mephedrone and dihydro-nor-mephedrone) in human whole blood has been developed and validated. Samples were extracted by a mixed mode solid-phase extraction and analyzed on a pentafluorophenylpropyl column. The method was successfully validated for selectivity, linearity (0.2-2 to 10-100 ng/mL), limits of detection (50-500 pg/mL) and quantification (200-2000 pg/mL), precision (0.924-8.27%), accuracy (86.6-115%), carryover, recovery (32.5-88.3%), and matrix effects (71.0-108%). Analyte stability in human whole blood preserved with sodium fluoride/potassium oxalate was assessed at +4°C and -20°C after 24 hours, 48 hours, 4 days, and 10 days of storage. Instability was observed in samples stored at +4°C: nor-mephedrone and 4-carboxy-mephedrone lost 40.2 ± 6.7% and 48.1 ± 4.8%, respectively, of their initial concentration at low concentration level and 33.8 ± 4.2% and 44.6 ± 6.5%, respectively, at high concentration level after 10 days. All analytes were more stable at -20°C where the highest loss of 22.6 ± 6.9% was observed for 4-carboxy-mephedrone after 10 days. This is the first time stability of mephedrone metabolites in human whole blood has been assessed, indicating -20°C to be the recommended storage condition for all analytes in clinical settings.

Elucidation of the biosynthetic pathways of boldenone in the equine testis.

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatography tandem mass spectrometry (LC-MS/MS) and LC high resolution mass spectrometry (LC-HRMS). Samples were analysed both underivatised and derivatised to increase the certainty of identification. In addition to previously reported endogenous steroids, analysis of non-spiked testicular tissue samples demonstrated the presence of boldenone and boldienone at trace levels in the equine testis. Incubation of homogenates with deuterium or carbon isotope labelled testosterone and androstenedione resulted in the matching stable isotope analogues of boldenone and boldienone being formed. Additionally, deuterium and carbon labelled 2-hydroxyandrostenedione was detected, raising the possibility that this steroid is a biosynthetic intermediate. In conclusion, boldenone and boldienone are naturally present in the equine testis, with the biosynthesis of these steroids arising from the conversion of testosterone and androstenedione. However, additional work employing larger numbers of animals, further enzyme kinetic experiments and pure reference standards for 2-OH androstenedione isomers would be required to better characterize the pathways involved in these transformations.

Detection of cocaine and its metabolites in whole blood and plasma following a single dose, controlled administration of intranasal cocaine.

The disposition of drugs and their metabolites have been extensively described in the literature, based primarily on the analysis of plasma and urine. However, there are more limited data on their disposition in whole blood, which is often the only specimen available in forensic investigations and cases of driving under the influence of drugs. In this study, we have, for the first time, established pharmacokinetic properties of cocaine (COC) and its metabolites from concurrently collected whole blood and plasma samples, following a single 100 mg dose of cocaine hydrochloride administered via nasal insufflation to seven healthy volunteers. The median Cmax of COC and its major metabolites, benzoylecgonine (BZE) and ecgonine methyl ester (EME), were closely related in whole blood and plasma. The median Cmax for COC in plasma was 379.7 ng/mL (347.5-517.7) and 344.24 ng/mL (271.6-583.2) in whole blood. The median Cmax for BZE in plasma was 441.2 ng/mL (393.6-475. and 371.18 ng/mL (371.1-477.3) in whole blood, EME was 105.5 ng/mL (93.6-151.8) in plasma and 135.5 ng/mL (87.8-183) in whole blood. Calculated medians of the whole blood to plasma ratio of COC (0.76), BZE (0.98) and EME (1.02) of approximately 1, strongly suggesting that the erythrocyte cell wall presents no barrier to COC and its metabolites. Furthermore, whole blood and plasma concentrations of COC were strongly correlated (R2  = 0.0914 R = 0.956, p < 0.0001), as was BZE (R2  = 0.0932 R = 0.965, p < 0.0001) and EME (R2  = 0.0964R = 0.928, p < 0.0001). The minor oxidative metabolite norcocaine (NCOC) was detected in both whole blood and plasma at concentrations between 1 and 5 ng/mL within 60-180 minutes, suggesting that NCOC could be indicator of recent COC administration. Data from this study have shown for the first time that COC and its metabolites BZE and EME are evenly distributed between plasma and whole blood following controlled single-dose intranasal COC administration.

Ion trap MS/MS of intact testosterone and epitestosterone conjugates--adducts, fragile ions and the advantages of derivatisation.

In ion trap mass spectrometry, fragile ions may fragment under the application of resonance ejection during precursor mass isolation, reducing MS/MS spectral intensity. In this study the steroidal epimers testosterone glucuronide (TG) and epitestosterone glucuronide (EG) have been chosen as a model for exploring whether compound structure is linked to ion trap fragility. Both compounds form multiple adducts by ESI-MS, namely protonation, ammonium and sodium, however, the mass spectrum of EG displays a more intense ammonium adduct peak than TG. [TG+NH(4)](+), [EG+NH(4)](+) and [EG+H](+) were found to be fragile ions. To explain the differences in adduct formation and fragility, molecular modelling was employed. Ammonium adduction was localised to the glucuronide ring oxygens and while EG has eight possible adduction sites, only seven were located for TG explaining the increased ammonium adduct abundance with EG. In EG the bond between the steroid and the glucuronide was slightly longer and the oxygen in this bond was more basic than TG. This shows that the EG bond is weaker which may contribute to the fact that [EG+H](+) but not [TG+H](+) is fragile. To investigate whether stability could be restored by chemical means, EG was derivatised with tris(trimethoxyphenyl)phosphonium chloride or methylated on the carboxylic acid and Girard P or methoxylamine on the 3-keto group. Derivatisation of the steroid rather than the glucuronide eliminated fragility and using a charged derivative eliminated adduct formation. This work demonstrates the importance of carefully considering the nature of the derivative and the site of derivatisation.

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry.

Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.

Important considerations for the utilisation of methanolysis in steroid analysis.

The effective analysis of anabolic-androgenic steroids in urine usually requires a suitable deconjugation method for the analysis of phase II metabolites such as sulphates and glucuronides. Acid hydrolysis using methanolysis is one adopted method of deconjugation that efficiently and rapidly cleaves both sulphates and glucuronides contemporaneously. The formation of artefactual by-products is a known disadvantage of this harsh method. However, the possible promotion of deuterium-hydrogen exchange of isotopically labelled internal standards has received little attention in the literature. This report demonstrates a complete deuterium-hydrogen exchange from deuterium labelled D9 -progesterone to progesterone driven by the acidic conditions of the methanolysis. The likely mechanisms of this exchange reaction are postulated, and the results compared to other deuterated steroids. This finding highlights the importance for careful consideration when selecting labelled internal standards in a conjunction with methanolysis.

Monitoring dehydroepiandrosterone (DHEA) in the urine of Thoroughbred geldings for doping control purposes.

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.

An introduction to mass spectrometry based proteomics-detection and characterization of gonadotropins and related molecules.

This review introduces fundamental aspects of mass spectrometry (MS) based proteomics and illustrates how MS is an effective tool for the analysis of glycoprotein hormones. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) MS are complementary approaches that have been applied for the analysis of gonadotropins, e.g. to characterize differences in the oligosaccharide distribution of commercial human chorionic gonadotropin preparations, for isolated nicked beta-subunit, and identification of a metabolite of placental transforming growth factor in pharmaceutical hCG preparations. Immunoaffinity trapping and concentration of digested sample extract prior to MS analysis confers analytical sensitivity akin to immunoassay. A desirable objective would be to develop for clinical purposes a rapid procedure for MS detection and characterization of gonadotropins. Refinement of on-target immobilization and digestion for subsequent ionization by MALDI may eventually help to provide this capability. The advent of hybrid mass spectrometers will further advance the characterization of these complex molecules.