Josep Marcos

Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50-300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed.

New potential markers for the detection of boldenone misuse

Boldenone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Boldenone misuse is commonly detected by the identification of the active drug and its main metabolite, 5β-androst-1-en-17β-ol-3-one (BM1), by gas chromatography-mass spectrometry (GC-MS), after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. However, some cases of endogenous boldenone and BM1 have been reported. Nowadays, when these compounds are detected in urine at low concentrations, isotope ratio mass spectrometry (IRMS) analysis is needed to confirm their exogenous origin. The aim of the present study was to identify boldenone metabolites conjugated with sulphate and to evaluate their potential to improve the detection of boldenone misuse in sports. Boldenone was administered to a healthy volunteer and urine samples were collected up to 56h after administration. After a liquid-liquid extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using electrospray ionisation in negative mode by monitoring the transition of m/z 365-350, specific for boldenone sulphate. Boldenone sulphate was identified in the excretion study urine samples and, moreover, another peak with the same transition was observed. Based on the MS/MS behaviour the metabolite was identified as epiboldenone sulphate. The identity was confirmed by isolation of the LC peak, solvolysis and comparison of the retention time and MS/MS spectra with an epiboldenone standard. These sulphated metabolites have not been previously reported in humans and although they account for less than 1% of the administered dose, they were still present in urine when the concentrations of the major metabolites, boldenone and BM1, were at the level of endogenous origin. The sulphated metabolites were also detected in 10 urine samples tested positive to boldenone and BM1 by GC-MS. In order to verify the usefulness of these new metabolites to discriminate between endogenous and exogenous origin of boldenone, four samples containing endogenous boldenone and BM1, confirmed by IRMS, were analysed. In 3 of the 4 samples, neither boldenone sulphate nor epiboldenone sulphate were detected, confirming that these metabolites were mainly detected after exogenous administration of boldenone. In contrast, boldenone sulphate and, in some cases, epiboldenone sulphate were present in samples with low concentrations of exogenous boldenone and BM1. Thus, boldenone and epiboldenone sulphates are additional markers for the exogenous origin of boldenone and they can be used to reduce the number of samples to be analysed by IRMS. In samples with boldenone and BM1 at the concentrations suspicion for endogenous origin, only if boldenone and epiboldenone sulphates are present, further analysis by IRMS will be needed to confirm exogenous origin.

Alternative long-term markers for the detection of methyltestosterone misuse.

Methyltestosterone (MT) is one of the most frequently detected anabolic androgenic steroids in doping control analysis. MT misuse is commonly detected by the identification of its two main metabolites excreted as glucuronide conjugates, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol. The detection of these metabolites is normally performed by gas chromatography-mass spectrometry, after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. The aim of the present work was to study the sulphate fraction of MT and to evaluate their potential to improve the detection of the misuse of the drug in sports. MT was administered to healthy volunteers and urine samples were collected up to 30days after administration. After an extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry using electrospray ionisation in negative mode by monitoring the transition m/z 385 to m/z 97. Three diol sulphate metabolites (S1, S2 and S3) were detected. Potential structures for these metabolites were proposed after solvolysis and mass spectrometric experiments: S1, 17α-methyl-5β-androstan-3α,17β-diol 3α-sulphate; S2, 17β-methyl-5α-androstan-3α,17α-diol 3α-sulphate; and S3, 17β-methyl-5β-androstan-3α,17α-diol 3α-sulphate. Synthesis of reference compounds will be required in order to confirm the structures. The retrospectivity of these sulphate metabolites in the detection of MT misuse was compared with the obtained with previously described metabolites. Metabolite S2 was detected up to 21days after MT administration, improving between 2 and 3 times the retrospectivity of the detection compared to the last long-term metabolite of MT previously described, 17α-hydroxy-17β-methylandrostan-4,6-dien-3-one.

Testosterone metabolism revisited: discovery of new metabolites

AbstractThe metabolism of testosterone is revisited. Four previously unreported metabolites were detected in urine after hydrolysis with KOH using a liquid chromatography–tandem mass spectrometry method and precursor ion scan mode. The metabolites were characterized by a product ion scan obtained with accurate mass measurements. Androsta-4,6-dien-3,17-dione, androsta-1,4-dien-3,17-dione, 17-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione were proposed as feasible structures for these metabolites on the basis of the mass spectrometry data. The proposed structures were confirmed by analysis of synthetic reference compounds. Only 15-androsten-3,17-dione could not be confirmed, owing to the lack of a commercially available standard. That all four compounds are testosterone metabolites was confirmed by the qualitative analysis of several urine samples collected before and after administration of testosterone undecanoate. The metabolite androsta-1,4-dien-3,17-dione has a structure analogous to that of the exogenous anabolic steroid boldenone. Specific transitions for boldenone and its metabolite 17β-hydroxy-5β-androst-1-en-3-one were also monitored. Both compounds were also detected after KOH treatment, suggesting that this metabolic pathway is involved in the endogenous detection of boldenone previously reported by several authors. FigureFour previously unreported testosterone metabolites released after basic treatment of the urine have been characterized using mass spectrometric techniques. The structure of three of them has been confirmed by comparison with reference material.

Maternal separation induces neuroinflammation and long-lasting emotional alterations in mice.

Early life experiences play a key role in brain function and behaviour. Adverse events during childhood are therefore a risk factor for psychiatric disease during adulthood, such as mood disorders. Maternal separation is a validated mouse model for maternal neglect, producing negative early life experiences that result in subsequent emotional alteration. Mood disorders have been found to be associated with neurochemical changes and neurotransmitter deficits such as reduced availability of monoamines in discrete brain areas. Emotional alterations like depression result in reduced serotonin availability and enhanced kynurenine metabolism through the action of indoleamine 2, 3-dioxygenase in response to neuroinflammatory factors. This mechanism involves regulation of the neurotransmitter system by neuroinflammatory agents, linking mood regulation to neuroinmunological reactions. In this context, the aim of this study was to investigate the effects of maternal separation with early weaning on emotional behaviour in mice. We investigated neuroinflammatory responses and the state of the tryptophan-kynurenine metabolic pathway in discrete brain areas following maternal separation. We show that adverse events during early life increase risk of long-lasting emotional alterations during adolescence and adulthood. These emotional alterations are particularly severe in females. Behavioural impairments were associated with microglia activation and disturbed tryptophan-kynurenine metabolism in brain areas related to emotional control. This finding supports the preeminent role of neuroinflammation in emotional disorders.

Circadian Variation of Melatonin, Light Exposure, and Diurnal Preference in Day and Night Shift Workers of Both Sexes

Background: Light-at-night has been shown in experimental studies to disrupt melatonin production but this has only partly been confirmed in studies of night shift workers. In this cross-sectional study, we examined the circadian variation of melatonin in relation to shift status, individual levels of light-at-night exposure, and diurnal preference, an attribute reflecting personal preference for activity in the morning or evening. Methods: One hundred and seventeen workers (75 night and 42 day) of both sexes, ages 22 to 64 years, were recruited from four companies. Participants collected urine samples from all voids over 24 hours and wore a data logger continuously recording their light exposure. Sociodemographic, occupational, lifestyle, and diurnal preference information were collected by interview. Concentrations of urinary 6-sulfatoxymelatonin (aMT6s), the main melatonin metabolite, were measured. Results: Mean aMT6s levels were lower in night [10.9 ng/mg creatinine/hour; 95% confidence interval (CI), 9.5–12.6] compared with day workers (15.4; 95% CI, 12.3–19.3). The lowest aMT6s levels were observed in night workers with morning preference (6.4; 95% CI, 3.0–13.6). Peak time of aMT6s production occurred 3 hours later in night (08:42 hour, 95% CI, 07:48–09:42) compared with day workers (05:36 hour, 95% CI, 05:06–06:12). Phase delay was stronger among subjects with higher light-at-night exposure and number of nights worked. Conclusions: Night shift workers had lower levels and a delay in peak time of aMT6s production over a 24-hour period. Differences were modified by diurnal preference and intensity of light-at-night exposure. Impact: Night shift work affects levels and timing of melatonin production and both parameters may relate to future cancer risk. Cancer Epidemiol Biomarkers Prev; 23(7); 1176–86. ©2014 AACR.

Investigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometry.

The accurate and precise measurement of endogenous corticosteroids in urine is a powerful tool to understand the biochemical state in several diseases. In this study, a rapid, accurate, and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 67 endogenous gluco- and mineralo-corticosteroids and progestins has been developed and validated. Sample preparation, chromatographic separation, and mass spectrometric detection were optimized. Urine samples (0.5 mL) were hydrolyzed with β-glucuronidase and the released analytes were extracted by liquid-liquid extraction. The chromatographic separation was performed in 20 min after redisolution of the extract. MS behavior of endogenous corticosteroids was evaluated in order to select the most specific precursor ion ([M+H](+), [M+NH4](+), or [M+H-nH2O](+)) for the detection. MS/MS determination was performed under selected reaction monitoring mode using electrospray ionization in positive mode. The method was shown to be linear (r>0.99) in the range of endogenous concentrations for all studied metabolites. Limits of detection (LOD) below 1 ng mL(-1) were typically obtained for analytes with a 3-oxo-4-ene structure whereas LODs below 15 ng mL(-1) were common for the rest of analytes. Recoveries were higher than 80% and intra-assay precisions below 20%, evaluated at three concentration levels, were found for most steroids. No significant or moderate matrix effect, ranging from 54 to 155%, was observed for most of the analytes. The applicability of the method was confirmed by analyzing 24h urine samples collected from twenty healthy volunteers and comparing the results with previously established normal ranges. The wide coverage of corticosteroid metabolism, together with short analysis time, low sample volume, simple sample preparation, and satisfactory quantitative results make this method useful for clinical purposes.

Derivatization of steroids in biological samples for GC–MS and LC–MS analyses

The determination of steroids in biological samples is essential in different areas of knowledge. MS combined with either GC or LC is considered the best analytical technique for specific and sensitive determinations. However, due to the physicochemical properties of some steroids, and the low concentrations found in biological samples, the formation of a derivative prior to their analysis is required. In GC-MS determinations, derivatization is needed for generating volatile and thermally stable compounds. The improvement in terms of stability and chromatographic retention are the main reasons for selecting the derivatization agent. On the other hand, derivatization is not compulsory in LC-MS analyses and the derivatization is typically used for improving the ionization and therefore the overall sensitivity achieved.

Detection, synthesis and characterization of metabolites of steroid hormones conjugated with cysteine

The occurrence of several polyunsaturated testosterone related compounds (including 4,6-androstadien-3,17-dione and 4,6-androstadien-17β-ol-3-one) in urine after alkaline treatment of the sample has been recently reported. Although several experiments seem to indicate that they are testosterone metabolites, their origin is still unknown. In this study, it is demonstrated that these metabolites are produced from the degradation of cysteine conjugates. Several testosterone metabolites conjugated with cysteine have been synthesized and characterized by NMR techniques. Their detection in human urine has been performed by LC-MS/MS. The acquisition of several transitions in the SRM mode and the comparison between ion ratios and retention times allowed for the unequivocal confirmation of the presence of cysteine conjugates in urine. The analysis of urine samples collected after testosterone administration confirmed that synthesized cysteine conjugates are testosterone metabolites. The fact that these conjugates result in polyunsaturated compounds in urine after alkaline treatment was demonstrated by fraction collection and alkaline treatment of each fraction. Besides, the presence of these metabolites was also confirmed in human plasma. The formation of these metabolites implies an unreported metabolic biotransformation: 6,7-dehydrogenation as phase I metabolism followed by conjugation with glutathione and subsequent transformation to cysteine conjugates. Finally, the existence of similar metabolites for cortisol and progesterone was also confirmed by LC-MS/MS indicating that the presented metabolic pathway is not exclusively active in androgens, but common to progestagens and glucocorticoids.

Sensitive and robust method for anabolic agents in human urine by gas chromatography–triple quadrupole mass spectrometry

A rapid, sensitive and robust gas chromatography-triple quadrupole mass spectrometry method was developed for the determination of seven anabolic agents in human urine. The selection of analytes includes the main metabolites of all anabolics with higher sensitivity requirements. After optimizing the fragmentation conditions for each compound, a validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the compound. Adequate intermediate precision, below 14%, was observed for all of the compounds at the lower concentration tested. The concentrations assayed were in accordance with the performance limits required by the World Anti-Doping Agency (WADA). Unlike a previously published GC/QqQ method, detection of 17α-methyl-5β-androstane-3α,17β-diol (the main metabolites of methyltestosterone) at 2 ng/mL was accomplished under routine conditions. The qualitative method was applied to the analysis of 1367 samples in the span of 2 weeks, as part of the doping control of the XVI Pan American Games which took place in Mexico (14th-30th October, 2011). The high sensitivity was maintained during the analysis of all analytical batches, proving for the first time the excellent ruggedness of GC/QqQ methods.