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Rationale: High-resolution mass spectrometry (HRMS) has been demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for the elucidation of chemical structures. This work describes a method to detect 13 esters of testosterone (T) and 5 biomarkers in 0.1 mL of human serum using gas chromatography (GC) coupled to HRMS., Methods: Analytes were extracted from serum after deproteinization and liquid-liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full-scan mode was applied with a resolving power of 30 000 at m/z 400. Linearity, lower limit of quantitation, and measurement uncertainty were assessed. Precision and accuracy were assessed at 0.5 and 2 ng/mL, respectively. Mass accuracy (MA) and mass extraction window (MEW) were also evaluated., Results: T esters showed a linear response between 0.25 and 10 ng/mL (except for undecanoate, enanthate, and propionate that showed lineal responses between 0.5 and 10 ng/mL and isocaproate between 2 and 10 ng/mL); detection limits remained between 0.1 and 0.5 ng/mL and accuracy between 81% and 119%. The MA (MEW = 10 ppm) was maintained between -2.4 and 4.8 ppm. The biomarkers (T, androstenedione, dehydroepiandrosterone [DHEA], estradiol, and 17-OH-progesterone) showed a linear response within the evaluated range; quantification limits remained between 0.1 and 0.5 ng/mL (except for DHEA), the accuracy between 88% and 99%, and precision between 3.5% and 10.8%. Measurement uncertainties were found between 5.6% and 17.2%. MA (MEW = 3 ppm) was maintained between -0.47 and 0.12 ppm., Conclusions: The method to detect T esters and five endogenous biomarkers in serum using GC coupled to HRMS showed linear responses up to 10 ng/mL with adequate precision, accuracy, and uncertainties. It was possible to distinguish cholesterol from T-isocaproate based on the MEW of 10 ppm, preventing false positives. In addition, this method allows searching for other biomarkers and/or unknown metabolites and other ester forms not included here but at a later stage if necessary., (© 2024 John Wiley & Sons Ltd.)

Rationale: The proposed metabolomic workflow, based on coupling high-resolution mass spectrometry with computational tools, can be an alternative strategy for metabolite detection and identification. This approach allows the extension of the investigation field to chemically different compounds, maximizing the information obtainable from the data and minimizing the time and resources required., Methods: Urine samples were collected from five healthy volunteers before and after oral administration of 3β-hydroxyandrost-5-ene-7,17-dione as a model compound and defining three excretion time intervals. Raw data were acquired in both positive and negative ionization modes using an Agilent Technologies 1290 Infinity II series HPLC coupled to a 6545 Accurate-Mass Quadrupole Time-of-Flight. They were then processed to align peak retention times with the same accurate mass, and the resulting data matrix was subjected to multivariate analysis., Results: Multivariate analysis (PCA and PLS-DA models) demonstrated high similarity between samples belonging to the same collection time interval and clear discrimination between different excretion intervals. The blank and long excretion groups were distinguished, suggesting the presence of long excretion markers, which are of remarkable interest in anti-doping analyses. The correspondence of some significant features with metabolites reported in the literature confirmed the rationale and usefulness of the proposed metabolomic approach., Conclusions: The presented study proposes a metabolomics workflow for the early detection and characterization of drug metabolites by untargeted urinary analysis to reduce the range of substances still excluded from routine screening. Its application has detected minor steroid metabolites, as well as unexpected endogenous alterations, proving to be an alternative strategy that can allow gathering a more complete range of information in the antidoping field., (© 2023 John Wiley & Sons Ltd.)

Rationale: This work demonstrates the high potential of combining high-resolution mass spectrometry with chemometric tools, using metabolomics as a guided tool for anti-doping analysis. The administration of 7-keto-DHEA was studied as a proof-of-concept of the effectiveness of the combination of knowledge-based and machine-learning approaches to differentiate the changes due to the athletic activities from those due to the recourse to doping substances and methods., Methods: Urine samples were collected from five healthy volunteers before and after an oral administration by identifying three time intervals. Raw data were acquired by injecting less than 1 μL of derivatized samples into a model 8890 gas chromatograph coupled to a model 7250 accurate-mass quadrupole time-of-flight analyzer (both from Agilent Technologies), by using a low-energy electron ionization source; the samples were then preprocessed to align peak retention times with the same accurate mass. The resulting data table was subjected to multivariate analysis., Results: Multivariate analysis showed a high similarity between the samples belonging to the same collection interval and a clear separation between the different excretion intervals. The discrimination between blank and long excretion groups may suggest the presence of long excretion markers, which are particularly significant in anti-doping analysis. Furthermore, matching the most significant features with some of the metabolites reported in the literature data demonstrated the rationality of the proposed metabolomics-based approach., Conclusions: The application of metabolomics tools as an investigation strategy could reduce the time and resources required to identify and characterize intake markers maximizing the information that can be extracted from the data and extending the research field by avoiding a priori bias. Therefore, metabolic fingerprinting of prohibited substance intakes could be an appropriate analytical approach to reduce the risk of false-positive/negative results, aiding in the interpretation of "abnormal" profiles and discrimination of pseudo-endogenous steroid intake in the anti-doping field., (© 2021 John Wiley & Sons Ltd.)

Rationale: Systematic electron ionization fragmentation studies of steroids have been performed to elucidate and trace their characteristic fragmentation patterns. However, the electron ionization source setting at 70 eV electron energy is much higher than the ionization potential (7-15 eV) of most organic compounds, leading to extensive fragmentation. We present a multifactorial study on optimizing a low-energy electron ionization source to maximize molecular ion formation while minimizing the extent of fragmentation to improve the analytical sensitivity of steroids, especially the more thermolabile ones, while preserving the information that can be extracted from the data., Methods: Twenty-seven steroid reference materials, chosen to cover four main classes of urinary steroids, were considered; gas chromatography/quadrupole time-of-flight (GC/qTOF) analyses were carried out using an Agilent Technologies model 8890 gas chromatograph coupled to an Agilent Technologies model 7250 accurate-mass quadrupole time-of-flight (GC/qTOF) instrument. The effects of electron energy, emission current, and source temperature, as well as their potential interactions on steroid fragmentation pathways, have been assessed in full factorial experimental designs., Results: Three parameters were specifically evaluated to improve the chromatographic/spectrometric response of the selected steroids: (i) degree of fragmentation; (ii) relative abundance of the molecular ion; and (iii) peak width. The first two were evaluated by screening designs that highlighted collision energy and source temperature as the most influential factors on the analytical responses of the considered steroids, while emission current always showed a non-significant influence. Then, an optimization design was performed to select the final source setting by searching for the combination of factors that minimize peak tailing., Conclusions: The proposed analytical approach permits a faster selection of optimal experimental conditions for steroidomics analysis using low-energy electron ionization and high-resolution mass spectrometry. The development of these designs of experiments (DoE) in full factorial design (FFD) allowed multiple inputs to be monitored at the same time, highlighting the possible interactions and estimating the effects of a factor in the different levels of the other factors considered., (© 2021 John Wiley & Sons Ltd.)

Rationale: The metabolism of arimistane (Arim) was first described in 2015, and androst-3,5-diene-7β-ol-17-one was proposed as the main metabolite excreted in urine. Recently, a more detailed study describing the findings in urine after the administration of Arim has been published. This study corroborated the previously described metabolite but also described several phase I and II metabolites, analyzing trimethylsilylated urinary extracts using accurate mass spectrometry coupled to gas chromatography (GC/qTOF). The present communication is an extension of this late investigation aiming to implement the results of Arim metabolism using either accurate mass spectrometry and/or triple quadrupole tandem mass spectrometry, both coupled to liquid chromatography (LC/qTOF and LC/QqQ)., Methods: The samples used in this study were the same as previously studied using GC/qTOF. One single oral dose of Arim was administered to three volunteers, and samples collected before and up to 10 h after the Arim administration were analyzed. The unconjugated fraction of urine was removed, and the hydrolysis was performed with β-glucuronidase from Escherichia coli. The extracts were reconstituted in water:acetonitrile before the LC/qTOF and LC/QqQ analysis., Results: The presence of the proposed metabolites studied using GC was verified by accurate mass measurements. Twelve metabolites not found in the blank urine samples were identified by the accurate mass spectra with acceptable errors between -7.5 and 8.1 ppm: 4 reduced metabolites, 4 monohydroxylated metabolites, and 4 with an additional hydroxylation (bis-hydroxylated metabolites). Unlike in the study carried out using GC/qTOF, Arim itself was found in the samples of the three volunteers., Conclusions: Twelve metabolites were identified, and specific transitions were proposed. Despite the good results, some limitations remain. As for GC/qTOF, the α- or β configuration of hydroxy groups, as well as the exact position for some unsaturation, cannot be assigned with certainty. Because certified reference materials of these metabolites are not yet available, the molecular structures were hypothesized considering the previous study using GC., (© 2021 John Wiley & Sons Ltd.)

Rationale: The aromatase inhibitor formestane (4-hydroxyandrost-4-ene-3,17-dione) is included in the World Anti-Doping Agency's List of Prohibited Substances in Sport. However, it also occurs endogenously as do its 2-, 6- and 11-hydroxy isomers. The aim of this study is to distinguish the different isomers using gas chromatography/electron ionization mass spectrometry (GC/EI-MS) for enhanced confidence in detection and selectivity for determination., Methods: Established derivatization protocols to introduce [ 2 H 9 ]TMS were followed to generate perdeuterotrimethylsilylated and mixed deuterated derivatives for nine different hydroxy steroids, all with 3-keto-4-ene structure. Formestane was additionally labelled with H 2 18 O to obtain derivatives doubly labelled with [ 2 H 9 ]TMS and 18 O. GC/EI-MS spectra of labelled and unlabelled TMS derivatives were compared. Proposals for the generation of fragment ions were substantiated by high-resolution MS (GC/QTOFMS) and tandem mass spectrometry (MS/MS) experiments., Results: Subclass-specific fragment ions include m/z 319 for the 6-hydroxy and m/z 219 for the 11-hydroxy compounds. Ions at m/z 415, 356, 341, 313, 269 and 267 were indicative for the 2- and 4-hydroxy compounds. For their discrimination the transition m/z 503 → 269 was selective for formestane. In 2-, 4- and 6-hydroxy steroids loss of a TMSO radical takes place as cleavage of a TMS-derived methyl radical and a neutral loss of (CH 3 ) 2 SiO. Further common fragments were also elucidated., Conclusions: With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. 18 O-labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS 3 may be suitable in future bioanalytical applications requiring discrimination of the 2- and 4-hydroxy compounds., (© 2020 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Rationale: Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s using different techniques, the aim of this study was to investigate the hydroxylation in positions C2, C4 and C6 after in vitro experiments and in vivo excretion studies using gas chromatography time-of-flight (GC/TOF) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The results could be influenced by the mass spectrometric analyser used., Methods: Incubations were carried out with human liver microsomes and six enzymes belonging to the cytochrome P450 family using MT as a substrate. The trimethylsilyl derivatives of the samples were analysed using GC/TOF and GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6-hydroxymethyltestosterone (6-OH-MT) to avoid artefact interferences. A urinary excretion study was then performed after the administration of a 10 mg single oral dose of MT to a volunteer., Results: The formation of hydroxylated metabolites of MT in the C6, C4 and C2 positions after both in vitro and in vivo experiments was observed. Sample evaluation using GC/TOF showed an interference for 6-OH-MT that could only be resolved in GC/MS/MS by monitoring specific transitions. The transitory detection of these hydroxylated metabolites in urine agrees with previous investigations that had described this metabolic route as being of little significance., Conclusions: In doping analysis, the formation of 4-hydroxymethyltestosterone (oxymesterone) from MT cannot be underestimated. Although it is only detected as a minor and short-term excretion metabolite, it cannot be overlooked as it was found in both in vitro and in vivo experiments. The use of a combination of different mass spectrometric instruments allowed reliable conclusions to be reached, and it was shown that special attention must be given to artefact formation., (© 2020 John Wiley & Sons, Ltd.)

Several authors have described the generation of androsta-3,5-diene-7-one structures from androst-5-ene-3,7-dione or androst-5-ene-3β-ol-7-one under acidic conditions and/or at high temperatures. The goal of this study was to observe and to describe the results obtained after the chromatographic analysis of the trimethylsilyl derivatives of reference materials of 7-oxo-DHEA, 7α-hydroxy-DHEA, 7β-hydroxy-DHEA, and androsta-3,5-diene-7,17-dione known as arimistane., Methods: The purity of the analyte reference materials was verified by liquid chromatography/quadrupole mass spectrometry. The trimethylsilyl derivatives obtained using several mixtures with MSTFA (N-methyl-N-trimethylsilyl trifluoroacetamide) in comparison with solely MSTFA were analyzed by gas chromatography coupled to a time-of-flight detector equipped with a multimode inlet or to a simple quadrupole detector with a split/splitless inlet., Results: The study showed that the formation of arimistane from 7-oxo-DHEA occurs using common derivatization reagents used for the analyses by gas chromatography (GC). In addition, the formation of the enolized TMS derivative of 7-oxo-DHEA was observed in considerable amount when it was reacted with MSTFA. The analysis of 7α-hydroxy-DHEA resulted in the detection of ~1% of arimistane. The formation of unexpected artifacts from derivatization is influenced by the reagent itself, the reaction temperature, the inlet used and its configuration., Conclusions: The derivatization reagent, instrumental conditions (inlet), as well as the chemical structures of the analytes present in the matrix, can influence the results. So, before describing a new feature as a potential "new" metabolite, special caution must be taken since we could actually be dealing with an artifact., (© 2020 John Wiley & Sons, Ltd.)

Rationale: The selection of the most appropriate metabolites of the substances included in the Prohibited List of the World Anti-Doping Agency (WADA) is fundamental for setting up methods allowing the detection of their intake by mass spectrometric methods. The aim of this work is to investigate the metabolism of arimistane (an aromatase inhibitor included in the WADA list) in order to improve its detection capacity among the antidoping community., Methods: Urinary samples collected after controlled single administration of arimistane in three healthy volunteers were analysed using the common routine sample preparation in antidoping laboratories to determine the steroid profile parameters considered in the steroid module of the Athlete Biological Passport by gas chromatography coupled to tandem mass spectrometry (GC/MS/MS). For the elucidation of the proposed metabolites, GC coupled to high-accuracy MS (GC/qTOFMS) was used. Both mass spectrometers were operated in electron ionization mode. Non-conjugated (free), glucuronated and sulfated fractions were analysed separately., Results: No relevant effects on the steroid profile could be detected after a single oral dose (25 mg). Up to 15 metabolites, present only in the post-administration samples, were detected and some structures were postulated. These metabolites are mainly excreted as glucuro-conjugated into urine and only minor amounts of two metabolites are also excreted unconjugated or as sulfates., Conclusions: Arimistane itself was not observed in the free or glucuronated fractions, but only in the sulfate fraction. The peaks showing mass spectra in agreement with hydroxylated metabolites did not match with those for 7-keto-DHEA, 7α- or 7β-hydroxy-DHEA. This suggests that the first hydroxylation did not occur on C3, but on C2. These newly described metabolites allow the specific detection of arimistane misuse in sports., (© 2019 John Wiley & Sons, Ltd.)

Rationale: Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens., Methods: The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS)., Results: Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation., Conclusions: When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis., (© 2019 John Wiley & Sons, Ltd.)

Rationale: Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13 C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers., Methods: Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)-IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11-keto-etiocholanolone and 11β-hydroxyandrosterone. The obtained 13 C delta values were statistically analyzed to evaluate their inter- and intra-individual distribution., Results: The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra-individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway., Conclusions: For the selected ERCs of non-sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13 C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts., (© 2018 John Wiley & Sons, Ltd.)

A method for the toxicological screening of the new designer drug methylenedioxypyrovalerone (MDPV) is described; with an emphasis on its application for anti-doping analysis. The metabolism of MDPV was evaluated in vitro using human liver microsomes and S9 cellular fractions for CYP450 phase I and uridine 5'-diphosphoglucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II metabolism studies. The resulting metabolites were subsequently liquid/liquid extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) derivatives. The structures of the metabolites were further confirmed by accurate mass measurement using a liquid chromatography/quadrupole time-of-flight (LC/QTOF) mass spectrometer. The studies demonstrated that the main metabolites of MDPV are catechol and methyl catechol pyrovalerone, which are in turn sulfated and glucuronated. The method for the determination of MDPV in urine has been fully validated by assessing the limits of detection and quantification, linearity, repeatability, and accuracy. This validation demonstrates the suitability for screening of this stimulant substance for anti-doping and forensic toxicology purposes., (2010 John Wiley & Sons, Ltd.)

A rapid sample treatment procedure for the gas chromatography/mass spectrometry (GC/MS) determination of anabolic steroids in human urine has been developed. The new procedure makes use of ultrasonic energy to reduce reaction times and increase the overall sensitivity. The following variables affecting the performance of the ultrasonic treatment were optimised: (i) time, (ii) device, (iii) frequency, and (iv) temperature. It was found that, under an ultrasonic field, the hydrolysis of conjugated steroids with beta-glucuronidase from Escherichia coli K12 was possible with a treatment time of 10 min. The accuracy and precision of the ultrasonic method were found to be in agreement with those achieved with the conventional thermal conductivity procedure (Student's t-test; p = 0.05, n = 10). After the enzymatic hydrolysis, the derivatisation of the target compounds with trimethylsilyl (TMS) reagent, methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)/NH(4)I/dithioerythritol (DTE) (1000:2:4, v/w/w), was also accelerated using ultrasonic energy. In order to test the applicability of the use of ultrasonic energy in the acceleration of the derivatisation reaction with TMS, the classic method of thermal conductivity was applied for comparative purposes to a pool of 35 androgenic anabolic steroids (AAS) and/or their metabolites. The results demonstrated that after 3 min of sonication in a Sonoreactor device (50% amplitude), 19 of the 35 compounds studied showed similar reaction yield to those obtained with the classic procedure requiring 30 min (Student's t-test; p = 0.05, n = 5); 13 increased to higher silylation yields; and for the steroids 1-testosterone, danazol and etiocholanolone-D5, the same results were obtained using a sonication time of 5 min.The overall applicability of the ultrasonic-based sample treatment method is shown by the analysis of five urine samples. The results are similar to those achieved by the routine procedure. The new method is fast, robust, and allows high sample throughput., (2010 John Wiley & Sons, Ltd.)

Sildenafil (SDF), vardenafil (VDF) and tadalafil (TDF) are phosphodiesterase type 5 enzyme inhibitors (PDE5Is), used in the treatment of erectile disorders and to improve breathing efficiency in pulmonary hypertension. The increasing incidence of their use among young athletes has drawn the attention of the anti-doping authorities to the possible abuse of PDE5Is by athletes due to their pharmacological activities. This paper describes a method for the determination in urine of PDE5Is and their metabolites by gas chromatography/mass spectrometry (GC/MS) after liquid/liquid extraction of the analytes from urine and derivatisation to obtain trimethylsilyl derivatives. The metabolic profile was studied on real samples collected from subjects taking PDE5Is (Viagra, Levitra or Cialis); the main urinary metabolites were identified and their MS fragmentation characterized. The sample pre-treatment and GC/MS conditions for the detection of the metabolites have been optimised. A method for their preliminary screening and subsequent confirmation is described that takes into account the general requirements of a routine doping analysis to be used for the screening of large numbers of samples. The main metabolites identified can be included in a general purpose screening method and all the metabolites in a more specific confirmation method. The method developed has been applied for the screening of PDE5Is in 5000 urine samples. Based on the obtained results, the proposed method appears to be of practical use in analytical and forensic toxicology, including doping analysis., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

A fast method has been developed for the simultaneous determination of 52 stimulants and narcotics excreted unconjugated in urine by gas chromatography/mass spectrometry (GC/MS). The procedure involves the liquid/liquid extraction of the analytes from urine at strong alkaline pH and the injection of the extract into a GC/MS instrument with a fast GC column (10 m x 0.18 mm i.d.); the short column allows the complete separation of the 52 analytes in a chromatographic run of 8 min. The method has been fully validated giving lower limits of detection (LLODs) satisfactory for its application to antidoping analysis as well as to forensic toxicology. The repeatability of the concentrations and the retention times are good both for intra- and for inter-day experiments (%CV of concentrations always lower than 15 and %CV of retention times lower than 0.6). In addition, the analytical bias is satisfactory (A% always >15%). The method proposed here would be particularly useful whenever there are time constraints and the analyses have to be completed in the shortest possible time., (Copyright 2010 John Wiley & Sons, Ltd.)

Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were considered for the characterization of tamoxifene metabolites in human urine for anti-doping purpose. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 166, 152 and 129) and neutral loss scan (neutral loss of 72 Da and 58 Da) in positive ion mode were assessed to recognize common ions or common losses of tamoxifene metabolites. The applicability of these methods was checked first by infusion and then by the injection of solution of a mixture of reference standards of four tamoxifene metabolites available in our laboratory. The data obtained by the analyses of the mixture of the reference standards showed that the five methods used exhibited satisfactory results for all tamoxifene metabolites considered at a concentration level of 100 ng/mL, whereas the analysis of blank urine samples spiked with the same tamoxifene metabolites at the same concentration showed that the neutral loss scan of 58 Da lacked sufficient specificity and sensitivity. The limit of detection in urine of the compounds studied was in the concentration range 10-100 ng/mL, depending on the compound structure and on the selected product ion. The suitability of these approaches was checked by the analysis of urine samples collected after the administration of a single dose of 20 mg of tamoxifene. Six metabolites were detected: 4-hydroxytamoxifene, 3,4-dihydroxytamoxifene, 3-hydroxy-4-methoxytamoxifene, N-demethyl-4-hydroxytamoxifene, tamoxifene-N-oxide and N-demethyl-3-hydroxy-4-methoxytamoxifene, which is in conformity to our previous work using a time-of-flight (TOF) mass spectrometer in full scan acquisition mode., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

A gas chromatographic/mass spectrometric (GC/MS) study aimed at identifying the metabolites of sibutramine (1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl)cyclobutanemethanamine) in urine is described. Urinary excretion of sibutramine metabolites following the oral administration of a single dose of sibutramine was followed by GC/MS analysis. After identification of the chromatographic signals corresponding to the six main urinary metabolites, the fragmentation pattern was studied in electron ionization (EI) mode after derivatization to the corresponding methyl and trimethylsilyl derivatives. Urine samples were pretreated according to a reference procedure (liquid/liquid separation, enzymatic hydrolysis, pre-concentration under a stream of nitrogen and derivatization, either under thermal incubation and by microwave irradiation). All sibutramine metabolites were excreted as glucuroconjugates, and retain the chiral carbon present in the sibutramine skeleton. The metabolites identified included mono-desmethylsibutramine (nor-sibutramine), bi-desmethylsibutramine (nor-nor-sibutramine), and the corresponding hydroxylated compounds, the hydroxylation taking place either on the cyclobutane or on the isopropyl chain. The excretion profiles of the different metabolites were also evaluated. From an analytical point of view, the method can be applied to different fields of forensic analytical toxicology, including anti-doping analysis. Although the lack of certified reference materials does not allow a precise determination of the limits of detection (LODs) of all the sibutramine metabolites, an estimation taking into account the response factor of similar compounds ensures that all metabolites are still clearly detectable in a range of concentrations between 10 and 50 ng/mL, thus satisfying the minimum required performance limits (MRPLs) of the World Anti-Doping Agency (WADA)., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

This paper describes a fast gas chromatographic/mass spectrometric (GC/MS) screening method for the detection, in urine, of 36 xenobiotics (30 synthetic anabolic steroids, four narcotics, one diuretic and one stimulant) excreted free or as glucuro-conjugates in urine and detectable as trimethylsilyl (TMS) derivatives. These drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase and then assayed as TMS derivatives by GC/MS using electron ionisation (EI) and single ion monitoring (SIM) acquisition mode. The total time needed for the GC run is less than 8 min. Good reproducibility of the retention times (CV% <1) and the relative abundances of the diagnostic fragment ions (CV% <10) was observed for all target analytes. The sensitivity of the method is sufficient to match the requirements of the World Anti-Doping Agency (WADA) for the accredited laboratories, with limits of detection (LODs) that are lower than the corresponding WADA minimum required performance limits (MRPLs) for all target compounds., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

A simple and efficient digestion method for rapid sample preparation and quantification of 25 chemical elements in lichens by sector field inductively coupled plasma mass spectrometry is described. A microwave (MW)-assisted acid digestion was carried out at atmospheric pressure simultaneously handling up to 80 samples in screw-capped disposable polystyrene tubes. This digestion procedure was compared with the established MW digestion in closed vessels in order to examine its potential applicability in routine analysis for environmental monitoring. Three certified reference materials, i.e. BCR 482 (lichens), BCR 62 (olive leaves) and BCR 100 (beech leaves), as well as a small set of real samples were analyzed. Limits of quantification, accuracy and precision of the method were assessed. The majority of the elements were totally recovered from the lichens and from the other vegetable matrices. Low contamination risk, simplicity, time-saving, and applicability in routine analyses make this method very suitable for use in extensive screening campaigns., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

A fast liquid chromatographic/mass spectrometric (LC/MS/MS) screening method for the detection, in urine, of synthetic glucocorticoids, stimulants (formoterol, modafinil and mesocarb), anti-oestrogens (finasteride, exemestane, anastrozole, letrozole and formestane) and synthetic anabolic steroids (stanozolol, gestrinone and tetrahydrogestrinone) is described. All these drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid extraction step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase, and assayed in 7 min by LC/MS/MS using electrospray ionization in positive ion mode and multiple reaction monitoring as the acquisition mode. All compounds show good reproducibility of both the retention times (CV% <2%) and the relative abundances (CV% <10%). The limits of detection for the anti-oestrogens, glucocorticoids and steroids are in the range of 1-30 ng/mL, and for the stimulants are in the range of 100-200 ng/mL, thus satisfying the minimum required performance limits of the World Anti-Doping Agency., (Copyright 2006 John Wiley & Sons, Ltd.)

This paper describes a rapid gas chromatographic/mass spectrometric (GC/MS) screening method for the detection of drugs of abuse and/or their metabolites in urine. Synthetic stimulants, opiates, cocaine metabolites, cannabinoids--and specifically the acid metabolite of tetrahydrocannabinol (THC-COOH)--can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, and assayed as trimethylsilyl derivatives by GC/MS in SIM (selected ion monitoring) mode. All the analytes show a good linearity (R2 > 0.99 for most of the considered substances) in the range 25-1000 ng/mL, with a good reproducibility of both the retention times (CV% <0.7) and the relative abundances of the characteristic diagnostic ions (CV% <13). The limit of detection (LOD) of the method is 25 ng/mL of target compound in human urine for most of the substances investigated, 3 ng/mL for THC-COOH, and 10 ng/mL for norbuprenorphine. Validation of the method allows its application to different fields of forensic analytical toxicology, including antidoping analysis.