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.     

Synthesis and characterization of 6β‐hydroxyandrosterone and 6β‐hydroxyetiocholanolone conjugated with glucuronic acid

The detection of testosterone (T) misuse by doping control laboratories is mainly based on monitoring urinary T phase I metabolites released after enzymatic hydrolysis of the corresponding phase II glucuronide metabolites by gas chromatography (tandem) mass spectrometry (GC‐MS(/MS)) methods. However, this strategy fails to properly determine two recently reported phase II metabolites of T conjugated with glucuronic acid that remained mostly conjugated after the hydrolysis step. These metabolites were identified as glucuronides of 6β‐hydroxyandrosterone (6β‐OH‐And) and 6β‐hydroxyetiocholanolone (6β‐OH‐Etio) but their exact conjugation site remained undetermined. In this study, the four possible glucuronides of 6β‐OH‐And and 6β‐OH‐Etio were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy. Moreover, their chromatographic properties and MS spectra were compared to those obtained for the urine samples collected after administration of T. Results confirmed that the recently reported metabolites were the 3α‐glucuronides of 6β‐OH‐And and 6β‐OH‐Etio. The synthesis and the elucidation of the exact structure of the metabolites presented in this study are crucial steps for the development of analytical methods in order to explore their role in T metabolism and their potential usefulness as biomarkers of T misuse. Copyright © 2014 John Wiley & Sons, Ltd.     

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography–high resolution mass spectrometry

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography–high‐resolution tandem mass spectrometry (LC–HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC–HRMS/MS system consisted of a YMC‐UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β‐glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono‐hydroxy metabolites, and mono‐hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono‐hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2–19‐fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.     

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.     

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).     

Metabolite profiling of guanfacine in plasma and urine of healthy Japanese subjects after oral administration of guanfacine extended‐release tablets

Guanfacine is used for the treatment of attention‐deficit/hyperactivity disorder (ADHD). Using liquid chromatography–tandem mass spectrometry (LC–MS/MS), metabolite profiling of guanfacine was performed in plasma and urine collected from healthy Japanese adults following repeated oral administration of guanfacine extended‐release formulation. Unchanged guanfacine was the most abundant component in both plasma and urine (from the MS signal intensity). In plasma, the M3 metabolite (a sulfate of hydroxy‐guanfacine) was the prominent metabolite; the M2 metabolite (a glucuronide of a metabolite formed by monooxidation of guanfacine), 3‐hydroxyguanfacine and several types of glucuronide at different positions on guanfacine were also detected. In urine, the M2 metabolite and 3‐hydroxyguanfacine were the principal metabolites. From metabolite analysis, the proposed main metabolic pathway of guanfacine is monooxidation on the dichlorobenzyl moiety, followed by glucuronidation or sulfation. A minor pathway is glucuronidation at different positions on guanfacine. As the prominent metabolites in plasma were glucuronide and sulfate of hydroxyguanfacine, which have no associated toxicity concerns, further toxicity studies of the metabolites, for example in animals, were not deemed necessary.     

Metabolites of lorazepam: Relevance of past findings to present day use of LC-MS/MS in analytical toxicology

The advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the sensitivity it confers, permits the analysis of both phase I and II drug metabolites that in the past would have been difficult to target using other techniques. These metabolites may have relevance to current analytical toxicology employing LC-MS/MS, and lorazepam was chosen as a model drug for investigation, as only the parent compound has been targeted for screening purposes. Following lorazepam administration (2 mg, p.o.) to 6 volunteers, metabolites were identified in urine by electrospray ionization LC-MS/MS, aided by the use of deuterated analogues generated by microsomal incubation for use as internal chromatographic and mass spectrometric markers. Metabolites present were lorazepam glucuronide, a quinazolinone, a quinazoline carboxylic acid, and two hydroxylorazepam isomers, one of which is novel, having the hydroxyl group located on the fused chlorobenzene ring. The quinazolinone, and particularly the quinazoline carboxylic acid metabolite, provided longer detection windows than lorazepam in urine extracts not subjected to enzymatic hydrolysis, a finding that is highly relevant to toxicology laboratories that omit hydrolysis in order to rapidly reduce the time spent on gas chromatography-mass spectrometry (GC-MS) analysis. With hydrolysis, the longest windows of detection were achieved by monitoring lorazepam, supporting the targeting of the aglycone with free drug for those incorporating hydrolysis in their analytical toxicology procedures.     

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.     

Phase I metabolism of the highly potent synthetic cannabinoid MDMB‐CHMICA and detection in human urine samples

Among the recently emerged synthetic cannabinoids, MDMB‐CHMICA (methyl N ‐{[1‐(cyclohexylmethyl)‐1H ‐indol‐3‐yl]carbonyl}‐3‐methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB‐CHMICA was investigated using liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS) and liquid chromatography‐electrospray ionization‐quadrupole time‐of‐flight‐mass spectrometry (LC‐ESI‐Q ToF‐MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC‐ESI‐MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB‐CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB‐CHMICA at the CB₁ receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of the in vitro metabolic profile of amlodipine in rat using liquid chromatography-mass spectrometry.

In the present study, the metabolic profile of amlodipine, a well-known calcium channel blocker, was investigated employing liquid chromatography-mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers - a triple-quadrupole (QqQ) and a quadrupole time-of-flight (Q-TOF) mass spectrometer - were utilized to acquire structural information on amlodipine metabolites. The metabolites were produced by incubation of amlodipine with primary cultures of rat hepatocytes. Incubations from rat hepatocytes were analyzed with LC-MS/MS, and 21 phase I and phase II metabolites were detected. Their product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites and thus to confirm the proposed structures of these metabolites. Mainly phase I metabolic changes were observed including dehydrogenation of the dihydropyridine core, as well as reactions of side chains, such as hydrolysis of ester bonds, hydroxylation, N-acetylation, oxidative deamination, and their combinations. The only phase II metabolite detected was the glucuronide of a dehydrogenated, deaminated metabolite of amlodipine. We propose several in vitro metabolic pathways of amlodipine in rat, based on our analysis of the metabolites detected and characterized.     

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).     

Detection and characterization of betamethasone metabolites in human urine by LC‐MS/MS

Glucocorticosteroids are prohibited in sports when administered by systemic routes and allowed using other administrations for therapeutic reasons. Therefore, markers to distinguish between routes of administration through the analysis of urine samples are needed in anti‐doping control. As a first step to achieve that goal, the metabolism of betamethasone (BET) was investigated in the present work. Urine samples obtained after BET intramuscular injection were hydrolyzed with β‐glucuronidase and subjected to liquid‐liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Common open screening methods for fluorine containing corticosteroids (precursor ion scan method of m/z 121, 147, 171, and neutral loss (NL) scan methods of 20 and 38 Da in positive ionization, and 46 and 76 Da in negative ionization) were applied to detect BET metabolites. Moreover, an NL method was applied to detect A‐ring reduced metabolites of BET, which are ionized as [M+NH₄]⁺ (NL of 55, 73, and 91 Da, corresponding to the consecutive losses of NH₃, HF and one, two and three water molecules, respectively). BET and 24 metabolites were detected. Six metabolites were identified by comparison with standards, and for ten, feasible structures were proposed based on mass spectrometric data. Eleven of the characterized metabolites had not been previously reported. Metabolites resulting from 11‐oxidation, 6‐hydroxylation, C20 or 4‐ene‐3‐one reduction and combination of some of them were detected. Moreover one metabolite resulting from cleavage of the side chain with subsequent oxidation of carbon at C17 was also detected. Copyright © 2014 John Wiley & Sons, Ltd.     

LC-MS/(MS) confirmatory doping control analysis of intact phase II metabolites of methenolone and mesterolone after Girard's Reagent T derivatization

In the present study, the application and evaluation of Girard's Reagent T (GRT) derivatization for the simultaneous detection and significantly important identification of different phase II methenolone and mesterolone metabolites by LC-MS/(MS) are presented. For the LC-MS analysis of target analytes two complementary isolation methods were developed; a derivatization and shoot method in which native urine is diluted with derivatization reagent and is injected directly to LC-MS and a liquid–liquid extraction method, using ethyl acetate at pH 4.5, for the effective isolation of both sulfate and glucuronide metabolites of the named steroids as well as of their free counterparts. For the evaluation of the proposed protocols, urine samples from methenolone and mesterolone excretion studies were analyzed against at least one sample from a different excretion study. Retention times, along with product ion ratios, were evaluated according to the WADA TD2021IDCR requirements, in order to determine maximum detection and identification time windows for each metabolite. Established identification windows obtained after LC-MS/(MS) analysis were further compared with those obtained after GC-MS/(MS) analysis of the same samples from the same excretion studies, for the most common analytes monitored by GC-MS/(MS). Full validation was performed for the developed derivatization and shoot method for the identification of methenolone metabolite, 3α-hydroxy-1-methylen-5α-androstan-17-one-3-glucuronide (mth3). Overall, the GRT derivatization presented herein offers a tool for the simultaneous sensitive detection of free, intact glucuronide and sulfate metabolites by LC-MS/(MS) that enhance significantly the detection and identification time windows of specific methenolone and mesterolone metabolites for doping control analysis.     

Fatal intoxication by 5F–ADB and diphenidine: Detection,quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q‐TOFMS

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F–ADB (methyl 2‐[1‐(5‐fluoropentyl)‐1H–indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC/Q‐TOFMS) in the information‐dependent acquisition mode only detected diphenidine. Further urinary screening using an in‐house database containing NPS and metabolites detected not only diphenidine but also possible 5F–ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F–ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F–ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F–ADB. Mono‐ and di‐hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N‐alkyl position are known to show higher cannabinoid receptor affinity relative to their non‐fluorinated analogues; 5F–ADB is no exception with high CB₁ receptor activity and much greater potency than Δ⁹‐THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F–ADB may be attributed to enzymatic and/or non‐enzymatic degradation, and further investigation into these possibilities is underway.     

A rapid and simple ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the detection of glucuronic acid-conjugated steroid metabolites

In the present study, we effectively detected 10 steroids and glucuronic acid-conjugated steroid metabolites in 12 min by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Steroids testosterone (T), 5α-dihydrotestosterone (DHT), androsterone (ADT), etiocholanolone (ETIO), estradiol (E₂) and their glucuronide conjugates were well-separated on an Eclipse Plus C₁₈ column (2.1 mm×50 mm, RRHD 1.8 µm). The mobile phase consisted of a mixture of methanol and ultrapure water (containing 1 mM ammonium formate) at a ratio of 60:40 (v/v), and the flow rate was set at 0.25 mL/min. The LC eluate was detected by electrospray ionization (ESI) source in both positive and negative ion modes. Neutral loss (NL of 176, 194, 211 and 229 Da in positive mode) and precursor ion (PI of m/z 141, 159 and 177 in positive mode and 75, 85 and 133 in negative mode) methods were applied for the detection of steroid glucuronides. The multiple reaction monitoring (MRM) transitions were m/z 289.3→97.1, 291.3→105, 291.3→199.2, 273.2→145.4 and 255.2→159.1 for T, DHT, ADT, ETIO and E₂ in positive mode, respectively; as well as m/z 463.3→85 for T glucuronide (T-G), m/z 465.3→75 for DHT glucuronide (DHT-G), ADT glucuronide (ADT-G), ETIO glucuronide (ETIO-G) and m/z 447.3→271 for E₂ glucuronide (E₂-G) in negative mode. In addition, the analytical method was also applied for the detection of steroid glucuronides in pooled human liver microsomes (HLM), which might serve as a basis for further investigation of steroid metabolism in vivo and in vitro.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Signal enhancement of glucuronide conjugates in LC‐MS/MS by derivatization with the phosphonium propylamine cation tris(trimethoxyphenyl) phosphonium propylamine,for forensic purposes

Although chemical derivatization for signal enhancement in drug testing is most often associated with gas chromatography, it also has the potential to improve the detection of analytes poorly ionized by atmospheric pressure ionization techniques, such as electrospray ionization used in liquid chromatography‐mass spectrometry. A number of acidic compounds, namely drug glucuronides (e.g. conjugates of temazepam, oxazepam, lorazepam, morphine, testosterone, epitestosterone, 5‐α‐dihydrotestosterone, androsterone, p‐nitrophenol, and paracetamol) were successfully derivatized with tris(trimethoxyphenyl) phosphoniumpropylamine to introduce a quaternary cation functionality to the analytes. Benzodiazepine glucuronides were more specifically investigated, and following positive mode electrospray ionization mass spectrometry, average improvements to peak areas as a result of derivatization were 67‐, 6‐, and 7‐ fold for temazepam, oxazepam, and lorazepam glucuronides. Average improvements to the signal‐to‐noise ratios for temazepam, oxazepam, and lorazepam glucuronides were 1336‐, 371‐ and 217‐fold, respectively. The values obtained for the derivatized conjugate were also typically higher than those for the underivatized parent drug. Urine containing benzodiazepine glucuronides was also successfully derivatized. The data indicates potential for the use of charge derivatization to improve the detection of molecules with acidic functionalities by liquid chromatography‐mass spectrometry (LC‐MS) techniques in certain scenarios. Copyright © 2014 John Wiley & Sons, Ltd.     

Biosynthesis of drug glucuronides for use as authentic standards

INTRODUCTION: Glucuronidation by the uridine diphosphate glucuronosyltransferases (UGTs) plays a pivotal role in the clearance mechanism of both xenobiotics and endobiotics. The detection of glucuronides at low micromolar concentrations is required to accurately model in vitro enzyme kinetics and in vivo pharmacokinetics. However, relatively few glucuronides are currently available as standards for developing liquid chromatography and mass spectroscopy (LC/MS) bioanalytical methods. METHODS: The glucuronidation capacity of hepatic microsomes prepared from rat (RLM), dog (DLM), monkey (MLM), and human (HLM) was examined for five xenobiotic substrates. In each case, glucuronide standards were produced using the enzyme source most efficient for the production of that specific glucuronide. RESULTS: Dog hepatic microsomes were used to produce glucuronides for anthraflavic acid (yield: 14 mg), buprenorphine (yield: 14 mg), and octylgallate (total yield: 13 mg), whereas propofol glucuronide (yield: 20 mg), and ethinylestradiol glucuronide (yield: 8 mg) were prepared using HLM. All glucuronides were characterized by LC/MS/MS and nuclear magnetic resonance (NMR) spectroscopy. DISCUSSION: The multimilligram quantities of glucuronide standards produced by this method have many applications throughout drug discovery and toxicology. In addition to allowing the quantification of glucuronide formation from in vitro and in vivo studies, the authentic standards produced could also be used to assess potential pharmacological or toxicological effects of metabolites.     

LC/MS/MS与GC/MS法分析鉴定小鼠尿中沙美特罗的代谢产物

目的:鉴定沙美特罗在小鼠尿中的主要代谢产物.方法:ig给药后,收集小鼠尿液,经固相提取,葡萄糖醛酸酶水解,进行LC/MS/MS分析和硅烷化后进行GC/MS分析同时分离鉴定沙美特罗代谢产物.结果和结论:在给药后尿样中发现沙美特罗原型和4种代谢产物M1～M4,其结构推测为19-羟基沙美特罗(M1)、2-羰基沙美特罗(M2)、19-羰基沙美特罗(M3)和19-羟基-8-甲氧基沙美特罗(M4).