Comparison of sulfo‐conjugated and gluco‐conjugated urinary metabolites for detection of methenolone misuse in doping control by LC‐HRMS,GC‐MS and GC‐HRMS

Methenolone (17β‐hydroxy‐1‐methyl‐5α‐androst‐1‐en‐3‐one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1‐methylene‐5α‐androstan‐3α‐ol‐17‐one) excreted conjugated with glucuronic acid using gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography mass spectrometry (LC‐MS) for the parent molecule, after hydrolysis with β‐glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC‐high resolution (HR)MS and the estimation of the long‐term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC‐HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti‐doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC‐HRMS using electrospray ionization in negative mode searching for [M‐H]^? ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1‐methylene‐5α‐androstan‐3α‐ol‐17‐one, 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one and 16β‐hydroxy‐1‐methyl‐5α‐androst‐1‐ene‐3,17‐dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC‐HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC‐MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction. Copyright © 2015 John Wiley & Sons, Ltd.     

Hyphenated mass spectrometric techniques-indispensable tools in clinical and forensic toxicology and in doping control

Hyphenated mass spectrometric techniques, particularly gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), are indispensable tools in clinical and forensic toxicology and in doping control owing to their high sensitivity and specificity. They are used for screening, library-assisted identification and quantification of drugs, poisons and their metabolites, prerequisites for competent expertise in these fields. In addition, they allow the study of metabolism of new drugs or poisons as a basis for developing screening procedures in biological matrices, most notably in urine, or toxicological risk assessment. Concepts and procedures using GC/MS and LC/MS techniques in the areas of analytical toxicology and the role of mass spectral libraries are presented and discussed in this feature article. Finally, perspectives of their future position are discussed.     

New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry (LC‐QTOFMS) for the first time. Liquid–liquid extraction was applied to processing urine samples, and dilute‐shoot analyses of intact metabolites were also presented. In LC‐QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M–H]^? as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography–mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α‐methyl‐5α‐androst‐3‐one‐17β‐sulfate) together with S2 (1α‐methyl‐5α‐androst‐17‐one‐3β‐sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse. Copyright © 2015 John Wiley & Sons, Ltd.     

Two new standard reference materials for the determination of drugs of abuse in human hair

Two new standard reference materials (SRM) for drugs of abuse in human hair have been developed. SRM 2379 consists of hair spiked with cocaine, benzoylecgonine, cocaethylene, phencyclidine, amphetamine, and methamphetamine. SRM 2380 consists of hair spiked with codeine, morphine, monoacetylmorphine, and tetrahydrocannabinol (THC). The SRMs were prepared by soaking the hair in a solution of the target analytes in water-dimethylsulfoxide. The concentration of each analyte was determined using two methods, one based upon gas chromatography/mass spectrometry (GC/MS) and one based upon liquid chromatography/mass spectrometry (LC/MS). Both methods used 0.1 M HCl for extraction of all the analytes from the hair, except for THC, which was extracted with 1 M NaOH. For isolation of the analytes from the extracts, the GC/MS-based methods used different clean-up procedures from those used for the LC/MS-based methods. The results from the two methods were in good agreement with mean differences for the analytes ranging from 4% to 16%. These materials will enable laboratories performing analyses of hair for drugs of abuse to test the accuracy of their methods.     

Doping control analysis of desmopressin in human urine by LC‐ESI‐MS/MS after urine delipidation

The World Anti‐Doping Agency (WADA) has recently added desmopressin, a synthetic analogue of the endogenous peptide hormone arginine vasopressin, to the Prohibited List, owing to the potential masking effects of this drug on hematic parameters useful to detect blood doping. A qualitative method for detection of desmopressin in human urine by high‐performance liquid chromatography–electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. Desmopressin purification from urine was achieved by means of delipidation with a 60:40 di‐isopropyl ether/n‐butanol and solid‐phase extraction with WCX cartridges. The lower limit of detection was 25 pg/mL. Extraction recovery was determined as 59.3% (SD 29.4), and signal reduction owing to ion suppression was estimated to be 42.7% (SD 12.9). The applicability of the method was proven by the analysis of real urine samples obtained after intravenous, oral and intranasal administration of desmopressin, achieving unambiguous detection of the peptide in all the cases. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of faropenem in human plasma and urine by liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed-phase column with 0.1% formic acid-methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r=0.9991 for plasma sample and r=0.9993 for urine sample) were obtained in the range 5-4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC-MS/MS.     

Determination of vitamin B5 in human urine by high-performance liquid chromatography coupled with mass spectrometry

In the present work, we have developed a simple and rapid liquid chromatography/mass spectrometry (LC/MS) method for the identification and quantification of vitamin B5 in human urine. Urine was spiked with vitamin B5 internal standard, hopantenic acid (HOPA), and then diluted with the LC mobile phase prior to its analysis by LC/MS. The quantification was performed in single ion monitoring mode. The calibration curve was linear (r2 = 0.999) between 0.25 to 10 microg/mL. With a limit of detection of 0.1 microg/mL the method was sensitive enough to determine low levels of vitamin B5 in urine. The overall quantitative efficiency of the method was evaluated by spiking urine samples with four different concentrations of vitamin B5; the intra-assay coefficient of variation was below 5% and the recoveries were between 96 to 108%. The results of the present study show that the proposed method is selective and sensitive enough for the quantification of vitamin B5 in urine.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

Screening and analysis of aconitum alkaloids and their metabolites in rat urine after oral administration of aconite roots extract using LC-TOFMS-based metabolomics

Aconite roots are popularly used in herbal medicines in China. Many cases of accidental and intentional intoxication with this plant have been reported; some of these are fatal because the toxicity of aconitum is very high. It is thus important to detect and identify aconitum alkaloids in biofluids. In this work, an improved method employing LC-TOFMS with multivariate data analysis was developed for screening and analysis of major aconitum alkaloids and their metabolites in rat urine following oral administration of aconite roots extract. Thirty-four signals highlighted by multivariate statistical analyses including 24 parent components and 10 metabolites were screened out and further identified by adjustment of the fragmentor voltage to produce structure-relevant fragment ions. It is helpful for studying aconite roots in toxicology, pharmacology and forensic medicine. This work also confirmed that the metabolomic approach provides effective tools for screening multiple absorbed and metabolic components of Chinese herbal medicines in vivo.     

Mass spectrometric characterization of urinary toremifene metabolites for doping control analyses

Toremifene is a selective estrogen receptor modulator included in the list of prohibited substances in sport by the World Anti-doping Agency. The aim of the present study was to investigate toremifene metabolism in humans in order to elucidate the structures of the most abundant urinary metabolites and to define the best marker to detect toremifene administration through the analysis of urine samples. Toremifene (Fareston) was administered to healthy volunteers and the urine samples were subjected to different preparation methods to detect free metabolites as well as metabolites conjugated with glucuronic acid or sulphate. Urinary extracts were analyzed by LC-MS/MS with triple quadrupole analyzer using selected reaction monitoring mode. Transitions for potential metabolites were selected by using the theoretical [M+H](+) as precursor ion and m/z 72 or m/z 58 as product ions for N,N-dimethyl and N-desmethyl metabolites, respectively. Toremifene and 20 metabolites were detected in excretion study samples, excreted free or conjugated with glucuronic acid or sulphate. Structures for most abundant phase I metabolites were proposed using accurate mass measurements performed by QTOF MS, based on fragmentation pattern observed for those metabolites available as reference standards. Several metabolic pathways including mono- and di-hydroxylation, N-desmethylation, hydroxymethylation, oxidation, dehalogenation and combinations were proposed. All metabolites were detected up to one month after toremifene administration; the most abundant metabolites were detected in the free fraction and they were metabolites resulting from dehalogenation. Several of the metabolites elucidated in this work have not been reported until now in the scientific literature.     

Development and validation of bioanalytical methods for imidafenacin (KRP-197/ONO-8025) and its metabolites in human urine by using liquid chromatography-tandem mass spectrometry

New bioanalytical methods have been developed for the determination of imidafenacin (KRP-197/ONO-8025, IM), a novel antimuscarinic drug developed for the treatment of overactive bladder, and its metabolites, M-2, M-3, M-4, M-6 and M-8 (method 1), M-5 and M-9 (method 2) in human urine by using liquid chromatography-tandem mass spectrometry. In each method, the urine sample was extracted by solid-phase extraction, separated on a semi-micro high-performance liquid chromatography column using gradient elution and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization or ionspray interface. Extraction recoveries of IM and metabolites were 81.4% or more. Calibration curves had good linearity in the concentration ranges 0.2-50 ng/mL for IM, M-2, M-3, M-4, M-6 and M-8 (method 1) and 1-250 ng/mL for M-5 and M-9 (method 2), respectively. The accuracy and precision in the intra-day and inter-day reproducibility tests were within +/-17.0 and 16.1% at the lowest concentrations, and within +/-12.8 and 11.1% at higher concentrations, respectively. Using these analytical methods, excretion profiles of IM and its metabolites in human urine were successfully determined after oral administration of IM at the therapeutic dosage of 0.1 mg.     

Development and validation of an LC–MS/MS Method for the quantitation of heparan sulfate in human urine

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC–MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC–MS/MS method for the quantitation of heparan sulfate in human urine using selected high‐abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid‐phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.     

Simultaneous quantification of atorvastatin and active metabolites in human plasma by liquid chromatography-tandem mass spectrometry using rosuvastatin as internal standard

A simple, sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of atorvastatin and its active metabolites ortho-hydroxyatorvastatin and para-hydroxyatorvastatin in human plasma using rosuvastatin as internal standard (IS). Following simple liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 559/440 for atorvastatin, m/z 575/466 for ortho-hydroxyatorvastatin, m/z 575/440 for para-hydroxyatorvastatin and m/z 482/258 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for atorvastatin and its two metabolites in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin and the IS from spiked plasma samples were 54.2 +/- 3.2, 50.1 +/- 3.8, 65.2 +/- 3.6 and 71.7 +/- 2.7%, respectively. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantitative determination of metformin,glyburide and its metabolites in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of an LC‐MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87–99%, and that from urine samples was 85–95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra‐day and inter‐day assays in plasma and urine samples, and the accuracy was 86–114% in plasma, and 94–105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Bioanalytical challenges and strategies for accurately measuring acyl glucuronide metabolites in biological fluids

Bioanalysis of unstable compounds such as acyl glucuronide metabolites represents a great analytical challenge owing to poor analyte stability in biological matrices. The primary goal for bioanalytical assay development is to minimize the breakdown of acyl glucuronide metabolite into its parent aglycone during sample collection, transportation, storage and analysis. Samples need to be stabilized ex vivo immediately after sample collection to minimize potential breakdown and thus to ensure accurate concentration measurement of both acyl glucuronide metabolite and its parent aglycone. In this review paper, formation of acyl glucuronide metabolites, the importance of establishing acyl glucuronide exposure measurement and safety coverage, optimization of sample pretreatment to stabilize the acyl glucuronide metabolites, current analytical strategy of assaying them as well as considerations for regulatory filings are discussed. It is important to identify acyl glucuronide metabolites that are capable of undergoing hydrolysis and pH-dependent intra-molecular migration as well as covalently binding to plasma and tissue proteins which can cause toxicity in vivo in the early stages of drug development. Carefully planning analytical experiments, identifying structures of acyl glucuronides and monitoring their concentrations in early drug development can help assess the risks associated with their exposures and potentially predict their concentrations in human circulation.     

New designer drug 1-(3-trifluoromethylphenyl) piperazine (TFMPP): gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry studies on its phase I and II metabolism and on its toxicological detection in rat urine

Studies are described on the phase I and II metabolism and the toxicological analysis of the piperazine-derived designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP) in rat urine using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). The identified metabolites indicated that TFMPP was extensively metabolized, mainly by hydroxylation of the aromatic ring and by degradation of the piperazine moiety to N-(3-trifluoromethylphenyl)ethylenediamine, N-(hydroxy-3-trifluoromethylphenyl)ethylenediamine, 3-trifluoromethylaniline, and hydroxy-3-trifluoromethylaniline. Phase II reactions included glucuronidation, sulfatation and acetylation of phase I metabolites. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of TFMPP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of TFMPP in human urine.     

Synthesis and bioanalytical evaluation of morphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma using LC-MS/MS

The aim of this work was to synthesize morphine‐3‐O‐sulfate and morphine‐6‐O‐sulfate for use as reference substances, and to determine the sulfate conjugates as possible heroin and morphine metabolites in plasma and urine by a validated LC‐MS/MS method. Morphine‐6‐O‐sulfate and morphine‐3‐O‐sulfate were prepared as dihydrates from morphine hydrochloride, in overall yields of 41 and 39% with product purities of >99.5% and >98%, respectively. For bioanalysis, the chromatographic system consisted of a reversed‐phase column and gradient elution. The tandem mass spectrometer was operated in the positive electrospray mode using selected reaction monitoring, of transition m/z 366.15 to 286.40. The measuring range was 5–500 ng/mL for morphine‐3‐O‐sulfate and 4.5–454 ng/mL for morphine‐6‐O‐sulfate in plasma. In urine, the measuring range was 50–5000 ng/mL for morphine‐3‐O‐sulfate and 45.4–4544 ng/mL for morphine‐6‐O‐sulfate. The intra‐assay and total imprecision (coefficient of variation) was below 11% for both analytes in urine and plasma. Quantifiable levels of morphine‐3‐O‐sulfate in authentic urine and plasma samples were found. Only one authentic urine sample contained a detectable level of morphine‐6‐O‐sulfate, while no detectable morphine‐6‐O‐sulfate was found in plasma samples.     

Quantification of ropivacaine and its major metabolites in human urine samples utilizing microextraction in a packed syringe automated with liquid chromatography-tandem mass spectrometry (MEPS-LC-MS/MS)

The determination of ropivacaine and its major metabolites in urine was performed using microextraction in a packed syringe as an on-line sample preparation method with LC and MS/MS. The sampling sorbent utilized was polystyrene polymer. [2H7]ropivacaine was used as the internal standard. The lower LOQ was 5.0 nmol/L. The calibration curves were obtained within the concentration range 5-2000 nmol/ L in urine. The regression correlation coefficients for urine samples were > or = 0.999 for all runs. The between-batch accuracy and precision values were determined from six replicates of quality control (QC) samples at three different concentrations in human urine. The mean accuracy values for the QC samples, reported as the percentage difference from the nominal value, were in the range of 99-115%. The precisions, given as the RSDs, were in the range 1.9-11%. The present method is miniaturized and fully automated and can be used for pharmacokinetic and pharmacodynamic studies.     

Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma,including metabolite concentrations at steady state

A novel, rapid and sensitive liquid chromatography tandem–mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi‐quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C₁₈ column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi‐quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5–100 μg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd.     

New clostebol metabolites in human urine by liquid chromatography time‐of‐flight tandem mass spectrometry and their application for doping control

In this study, clostebol metabolic profiles were investigated carefully. Clostebol was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadrupole time‐of‐flight mass spectrometry (MS) using full scan and targeted MS/MS techniques with accurate mass measurement for the first time. Liquid–liquid extraction and direct injection were applied to processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M–H]^? as target ion in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Fourteen metabolites were found for clostebol, and nine unreported metabolites (two free ones and seven sulfate conjugates) were identified by MS, and their potential structures were proposed based on fragmentation and metabolism pathways. Four glucuronide conjugates were also first reported. All the metabolites were evaluated in terms of how long they could be detected and S1 (4ξ‐chloro‐5ξ‐androst‐3ξ‐ol‐17‐one‐3ξ‐sulfate) was considered to be the long‐term metabolite for clostebol misuse detected up to 25 days by liquid–liquid extraction and 14 days by direct injection analysis after oral administration. Five conjugated metabolites (M2, M5, S2, S6 and S7) could also be the alternative biomarkers for clostebol misuse. Copyright © 2015 John Wiley & Sons, Ltd.