Specific characterization of non‐steroidal selective androgen peceptor modulators using supercritical fluid chromatography coupled to ion‐mobility mass spectrometry: application to the detection of enobosarm in bovine urine

Currently under development for therapeutic purposes in human medicine, non‐steroidal selective androgen receptor modulators (non‐steroidal SARMs) are also known to impact growth associated pathways. As such, they present a potential for abuse in sports and food‐producing animals as interesting alternative anabolic substances. Forbidden since 2008 by the World Anti‐Doping Agency (WADA) these compounds are however easily available and could be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies have to be developed for specific and sensitive detection of these compounds in biological matrices. Using an innovative analytical platform constituted of supercritical fluid chromatography coupled to ion mobility‐mass spectrometry, the present study enabled efficient separation and identification in urine of 4 of these drugs (andarine, bicalutamide, hydroxyflutamide, and enobosarm) in accordance with European Union criteria (Commission Decision 2002/657/EC). Besides providing information about compounds structure and behaviour in gas phase, such a coupling enabled reaching low limits of detection (LOD < 0.05 ng.mL⁻¹ for andarine and limits of detection < 0.005 ng.mL⁻¹ for the three others) in urine with good repeatability (CV < 21 %). The workflow has been applied to quantitative determination of enobosarm elimination in urine of treated bovine (200 mg, oral). Copyright © 2016 John Wiley & Sons, Ltd.     

Urinary detection of conjugated and unconjugated anabolic steroids by dilute‐and‐shoot liquid chromatography‐high resolution mass spectrometry

Anabolic androgenic steroids (AAS) are an important class of doping agents. The metabolism of these substances is generally very extensive and includes phase‐I and phase‐II pathways. In this work, a comprehensive detection of these metabolites is described using a 2‐fold dilution of urine and subsequent analysis by liquid chromatography‐high resolution mass spectrometry (LC‐HRMS). The method was applied to study 32 different metabolites, excreted free or conjugated (glucuronide or sulfate), which permit the detection of misuse of at least 21 anabolic steroids. The method has been fully validated for 21 target compounds (8 glucuronide, 1 sulfate and 12 free steroids) and 18 out of 21 compounds had detection limits in the range of 1–10 ng mL^?1 in urine. For the conjugated compounds, for which no reference standards are available, metabolites were synthesized in vitro or excretion studies were investigated. The detection limits for these compounds ranged between 0.5 and 18 ng mL^?1 in urine. The simple and straightforward methodology complements the traditional methods based on hydrolysis, liquid‐liquid extraction, derivatization and analysis by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS). Copyright © 2014 John Wiley & Sons, Ltd.     

In vitro metabolism study of a black market product containing SARM LGD‐4033

Anabolic agents are often used by athletes to enhance their performance. However, use of steroids leads to considerable side effects. Non‐steroidal selective androgen receptor modulators (SARMs) are a novel class of substances that have not been approved so far but seem to have a more favourable anabolic/androgenic ratio than steroids and produce fewer side effects. Therefore the use of SARMs has been prohibited since 2008 by the World Anti‐Doping Agency (WADA). Several of these SARMs have been detected on the black market. Metabolism studies are essential to identify the best urinary markers to ensure effective control of emerging substances by doping control laboratories. As black market products often contain non‐pharmaceutical‐grade substances, alternatives for human excretion studies are needed to elucidate the metabolism. A black market product labelled to contain the SARM LGD‐4033 was purchased over the Internet. Purity verification of the black market product led to the detection of LGD‐4033, without other contaminants. Human liver microsomes and S9 liver fractions were used to perform phase I and phase II (glucuronidation) metabolism studies. The samples of the in vitro metabolism studies were analyzed by gas chromatography‐(tandem) mass spectrometry (GC‐MS(/MS)), liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐(HR)MS/MS). LC‐HRMS product ion scans allowed to identify typical fragment ions for the parent compound and to further determine metabolite structures. In total five metabolites were detected, all modified in the pyrrolidine ring of LGD‐4033. The metabolic modifications ranged from hydroxylation combined with keto‐formation (M1) or cleavage of the pyrrolidine ring (M2), hydroxylation and methylation (M3/M4) and dihydroxylation (M5). The parent compound and M2 were also detected as glucuronide‐conjugates. Copyright © 2016 John Wiley & Sons, Ltd.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes

Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side‐effects. This makes them possible substances of abuse and the World Anti‐Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in vivo metabolite pattern and thus possible analytical targets have not been previously studied in this species. In this study, the urinary metabolites of the SARMs S1, S4, and S22 in horses were studied after intravenous injection, using ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry (UHPLC‐QToF‐MS). Eight different metabolites were found for SARM S1, nine for SARM S4, and seven for SARM S22. The equine urinary metabolite profiles differed significantly from those of humans. The parent compounds were only detected for SARMs S4 and S22 and only at the first sampling time point at 3 h post administration, making them unsuitable as target compounds. For all three SARMs tested, the metabolite yielding the highest response had undergone amide hydrolysis, hydroxylation and sulfonation. The resulting phase II metabolites (4‐nitro‐3‐trifluoro‐methyl‐phenylamine sulfate for SARMs S1 and S4 and 4‐cyano‐3‐trifluoro‐methyl‐phenylamine sulfate for SARM S22) are proposed as analytical targets for use in equine doping control. Copyright © 2015 John Wiley & Sons, Ltd.     

Sulfate metabolites as alternative markers for the detection of 4‐chlorometandienone misuse in doping control

Sulfate metabolites have been described as long‐term metabolites for some anabolic androgenic steroids (AAS). 4‐chlorometandienone (4Cl‐MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl‐MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl‐MTD metabolism by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to establish potential long‐term metabolites valuable for doping control purposes. 4Cl‐MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and gas chromatography‐tandem mass spectrometry (GC‐MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. Copyright © 2016 John Wiley & Sons, Ltd.     

HepG2 as promising cell-based model for biosynthesis of long-term metabolites: Exemplified for metandienone

In order to detect the abuse of substances in sports, the knowledge of their metabolism is of undisputable importance. As in vivo administration of compounds faces ethical problems and might even not be applicable for nonapproved compounds, cell-based models might be a versatile tool for biotransformation studies. We coincubated HepG2 cells with metandienone and D₃-epitestosterone for 14 days. Phase I and II metabolites were analyzed by high-performance liquid chromatography (HPLC)–tandem mass spectrometry and confirmed by gas chromatography–mass spectrometry (GC–MS). The metandienone metabolites formed by HepG2 cells were comparable with those renally excreted by humans. HepG2 cells also generated the two long-term metabolites 17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-androst-1,4,13-trien-3-one used in doping analyses, though in an inverse ratio compared with that observed in human urine. In conclusion, we showed that HepG2 cells are suitable as model for the investigation of biotransformation of androgens, especially for the anabolic androgenic steroid metandienone. They further proved to cover phase I and II metabolic pathways, which combined with a prolonged incubation time with metandienone resulted in the generation of its respective long-term metabolites known from in vivo metabolism. Moreover, we showed the usability of D₃-epitestosterone as internal standard for the incubation. The method used herein appears to be suitable and advantageous compared with other models for the investigation of doping-relevant compounds, probably enabling the discovery of candidate metabolites for doping analyses.     

Determination of GnRH and its synthetic analogues' abuse in doping control: Small bioactive peptide UPLC–MS/MS method extension by addition of in vitro and in vivo metabolism data; evaluation of LH and steroid profile parameter fluctuations as suitable biomarkers

Gonadotropin‐releasing hormone (GnRH) and its small peptide synthetic analogues are included in Section S2 of the World Anti‐Doping Agency (WADA) Prohibited List as they stimulate pituitary luteinizing hormone (LH) and testicular testosterone (T) secretion. Both the following approaches can be applied for determination of abuse of these peptides: direct identification of intact compounds and their metabolites in athletes' biofluids and evaluation of LH and T concentrations as mediate markers of drug intake. To develop an effective concept for GnRH and its analogues determination in anti‐doping control, in vitro and in vivo studies were conducted. A new method was applied to the evaluation of the slow‐release profile of buserelin, goserelin, and leuprolide biodegradable microspheres after the intramuscular injection in male volunteers. Eight metabolites of 10 GnRH analogues were identified after incubation with human kidney microsomes, most of them were leuprolide degradation products. Obtained data were added into ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for GnRH analogues determination. The detection time windows for administered peptides and their metabolites in urine samples were evaluated with 2 sample preparation techniques: dilute‐and‐shoot and solid‐phase extraction. To support the second hypothesis, the measurement of LH and the main parameters of the steroid profile were performed in urine samples. Just 1 compound among those investigated resulted in the LH concentration dropping to non‐physiological levels. Thus, for doping‐control purposes, monitoring of hormone levels fluctuations could be applied only together with longitudinal passport steroid profile data.     

Development of a multi‐residue high‐throughput UHPLC–MS/MS method for routine monitoring of SARM compounds in equine and bovine blood

Selective androgen receptor modulators (SARMs) are a group of anabolic enhancer drugs posing threats to the integrity of animal sports and the safety of animal‐derived foods. The current research describes for the first time the development of a semi‐quantitative assay for the monitoring of SARM family compounds in blood and establishes the relative stability of these analytes under various storage conditions prior to analysis. The presented screening method validation was performed in line with current EU legislation for the inspection of livestock and produce of animal origin, with detection capability (CCβ) values determined at 0.5 ng/mL (Ly2452473), 1 ng/mL (AC‐262536 and PF‐06260414), 2 ng/mL (bicalutamide, GLPG0492, LGD‐2226, ostarine, S‐1, S‐6, and S‐23), and 5 ng/mL (andarine, BMS‐564929, LGD‐4033, RAD140, and S‐9), respectively. The applicability of the developed assay was demonstrated through the analysis of blood samples from racehorses and cattle. The developed method presents a high‐throughput cost‐effective tool for the routine screening for a range of SARM compounds in sport and livestock animals.     

A pilot wastewater‐based epidemiology assessment of anabolic steroid use in Queensland,Australia

Anabolic‐androgenic steroids are synthetic compounds prohibited due to their performance‐enhancing characteristics. The use of these substances is known to cause health‐related issues, which highlights the importance of being able to evaluate the scale of consumption by the general population. However, most available research on the analysis of anabolic steroids is focused on animals and athletes in connection with doping. The potential of wastewater‐based epidemiology as an intelligence tool for the assessment of community level use of anabolic steroids is presented herein. A liquid chromatography tandem mass spectrometry method was developed for the analysis of 10 anabolic‐androgenic steroids and 14 endogenous hormones in influent wastewater. The validated method was applied to sixteen 24‐hour composite wastewater influent samples that were collected over a period of five years from two wastewater treatment plants in Queensland, Australia. Nine investigated compounds were found to be present at concentrations between 14 and 611 ng L^?1 which translated into 3–104 mg excreted per 1000 individuals per day. It was concluded that the developed analytical method is suitable for the analysis of AAS in wastewater matrix. Additionally, both the inclusion of metabolites and further investigation into deconjugation by enzymatic hydrolysis would aid in understanding and evaluating community anabolic steroid use. For the first time, this study presents the application of wastewater‐based epidemiology on anabolic‐androgenic steroids in Australia.     

气相色谱/燃烧炉/同位素比值质谱方法对尿中去氢表雄酮及其代谢物的检测

目的测定服用去氢表雄酮制剂后人尿中去氢表雄酮及其代谢物的同位素比值(¹³C与¹²C含量比值的相对值，以δ表示)进行测定，以检测尿中类固醇的来源。方法采用固相萃取、酶解、薄层色谱等方法对尿样中内源性类固醇激素进行提取、酶解游离型、分离，再经气相色谱/燃烧炉/同位素比值质谱方法测定去氢表雄酮、其代谢物内源性类固醇激素、内源性类固醇参照物的同位素比值的相对值(δ值)。将被检测物与参照物δ值之比，与判别标准比较进行来源判定。结果服用去氢表雄酮制剂后人尿中内源性类固醇激素代谢物δ值比未使用去氢表雄酮制剂正常值降低，并提示有外源性类固醇摄入。结论本法可以检测尿中去氢表雄酮或其代谢物的来源。     

Investigation of the metabolism of the selective androgen receptor modulator LGD‐4033 in equine urine,plasma and hair following oral administration

LGD‐4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD‐4033 in the horse following oral administration, in order to identify the most appropriate analytical targets for doping control laboratories. LGD‐4033 was orally administered to two Thoroughbred horses and urine, plasma and hair samples were collected and analysed for parent drug and metabolites. LC‐HRMS was used for metabolite identification in urine and plasma. Eight metabolites were detected in urine, five of which were excreted only as phase II conjugates, with the longest detection time being observed for di‐ and tri‐hydroxylated metabolites. The parent compound could only be detected in urine in the conjugated fraction. Seven metabolites were detected in plasma along with the parent compound where mono‐hydroxylated metabolites provided the longest duration of detection. Preliminary investigations with hair samples using LC–MS/MS analysis indicated the presence of trace amounts of the parent compound and one of the mono‐hydroxylated metabolites. In vitro incubation of LGD‐4033 with equine liver microsomes was also performed for comparison, yielding 11 phase I metabolites. All of the metabolites observed in vivo were also observed in vitro.     

In-depth metabolic study of nonsteroidal selective androgen receptor modulator GSK2881078 in thoroughbred horses and horse liver microsomes for doping control

Nonsteroidal selective androgen receptor modulators (SARMs) are a novel class of compounds that have not yet been clinically approved; however, they appear to have a better anabolic/androgenic ratio than steroids and cause slighter side effects. Sports drug testing laboratories are required to maintain continuously updated doping control analytical methods in light of the widespread misuse of SARMs in elite and amateur sports. This paper describes the metabolic conversion of SARM GSK2881078 in thoroughbred horses following oral administration and in vitro with equine liver microsomes. A liquid chromatography–high-resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. A total of five (M1–M5) in vivo metabolites and six (M1–M6) in vitro metabolites were detected under experimental conditions. Phase I metabolites mainly result from hydroxylation. Methoxylated and side-chain dissociated metabolites were also detected. Neither sulfonic acid nor glucuronic acid conjugated metabolites were observed in this study. Data reported here could aid in the detection of nonsteroidal SARM GSK2881078 and reveal its illicit use in competitive sports.     

UHPLC‐MS/MS and UHPLC‐HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination

Zolpidem and zopiclone (Z‐compounds) are non‐benzodiazepine hypnotics of new generation that can be used in drug‐facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z‐compounds and their metabolites by ultra‐high‐pressure liquid chromatography/tandem mass spectrometry (UHPLC‐MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC‐HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy‐ and three hydroxy‐ metabolites, that could be also detected by gas chromatography/mass spectrometry (GC‐MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N‐demethyl and N‐oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC‐MS/MS method for the determination of Z‐compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC‐MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP‐1, GHRP‐2, GHRP‐6, Hexarelin,and Ipamorelin

Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti‐Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti‐doping control we have investigated metabolites of GHRP‐1, GHRP‐2, GHRP‐6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid‐phase extraction on weak cation exchange cartridges and analyzed by means of nano‐liquid chromatography ‐ high resolution mass spectrometry. Six metabolites of GHRP‐1 were identified. GHRP‐1 in the parent form was not detected. GHRP‐1 (2‐4) free acid was detected in urine up to 27 h. GHRP‐2, GHRP‐2 free acid and GHRP‐2 (1‐3) free acid were detected in urine up to 47 h after administration. GHRP‐6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1‐3) free acid and Ipamorelin (1‐4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra‐pressure liquid chromatography‐tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration. Copyright © 2015 John Wiley & Sons, Ltd.     

Searching for new long‐term urinary metabolites of metenolone and drostanolone using gas chromatography–mass spectrometry with a focus on non‐hydrolysed sulfates

Sulfated metabolites have been shown to have potential as long‐term markers of anabolic–androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography–mass spectrometry (GC–MS) with non‐hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC–MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long‐term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC–chemical ionization–triple quadrupole MS (GC–CI–MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli–derived β‐glucuronidase, Helix pomatia–derived β‐glucuronidase/arylsulfatase and non‐hydrolysed sulfated steroids). For metenolone, a non‐hydrolysed sulfated metabolite, 1β‐methyl‐5α‐androstan‐17‐one‐3ζ‐sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non‐hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α‐hydroxy‐2α‐methyl‐5α‐androstan‐17‐one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).     

Liquid chromatography–mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes

Intact phase II steroid metabolites have poor product ion mass spectra under collision-induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17β-isomers of the sulfate conjugates, but limited differentiation for 17α/17β-isomers of glucuronide conjugates and for 3α/3β- and 5α/5β-stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed—according to the World Anti-Doping Agency (WADA) TD2015IDCR—the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC–ESI–MS/(MS).     

Toxicokinetics and analytical toxicology of the abused opioid U‐48800 — in vitro metabolism,metabolic stability,isozyme mapping,and plasma protein binding

Due to the risk of new synthetic opioids (NSOs) for human health, the knowledge of their toxicokinetic characteristics is important for clinical and forensic toxicology. U‐48800 is an NSO structurally non‐related to classical opioids such as morphine or fentanyl and offered for abuse. As toxicokinetic data of U‐48800 is not currently available, the aims of this study were to identify the in vitro metabolites of U‐48800 in pooled human liver S9 fraction (pS9), to map the isozymes involved in the initial metabolic steps, and to determine further toxicokinetic data such as metabolic stability, including the in vitro half‐life (t_1/2), and the intrinsic (CL_int) and hepatic clearance (CL_h). Furthermore, drug detectability studies in rat urine should be done using hyphenated mass spectrometry. In total, 13 phase I metabolites and one phase II metabolite were identified. N‐Dealkylation, hydroxylation, and their combinations were the predominant metabolic reactions. The isozymes CYP2C19 and CYP3A4 were mainly involved in these initial steps. CYP2C19 poor metabolizers may suffer from an increased U‐48800 toxicity. The in vitro t_1/2 and CL_int could be rated as moderate, compared to structural related compounds. After administration of an assumed consumer dose to rats, the unchanged parent compound was found only in very low abundance but three metabolites were detected additionally. Due to species differences, metabolites found in rats might be different from those in humans. However, phase I metabolites found in rat urine, the parent compound, and additionally the N‐demethyl metabolite should be used as main targets in toxicological urine screening approaches.     

Investigations into the analysis of intact drug conjugates in animal sport doping control – Development and assessment of a rapid and economical approach for screening greyhound urine

Animal sport doping control laboratories are constantly reviewing ways in which they can improve their service offering whilst ensuring that they remain economically viable. This paper describes the development and assessment of a rapid and economical method for the detection of intact glucuronide conjugates of three anabolic steroids and their metabolites along with three corticosteroids in canine urine. The analysis of intact drug conjugates for animal sport doping control is generally not performed routinely as it presents a number of analytical challenges, not least of which is the lack of availability of appropriate reference standards. Here, we report the development of a UHPLC–MS/MS method using APCI in the negative ion mode for the detection of intact phase II conjugates, including the importance of in vitro incubations in order to provide appropriate reference materials. Cross‐validation of the developed method demonstrated that the detection capability of the intact phase II conjugates of stanozolol, boldenone, nandrolone, and their metabolites along with the corticosteroids dexamethasone and methylprednisolone was equivalent to that achieved in routine race‐day screens. The new process has been in operation for approximately 2 years and has been used to analyze in excess of 13500 canine urine samples, resulting in a number of positive screening findings. To the best of our knowledge, this is the first reported use of a routine screen for intact drug conjugates within animal sport doping control.