Andreu Fabregat

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.

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.

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.

Alternative markers for the long-term detection of oral testosterone misuse

The screening of testosterone misuse in the doping control field is normally performed by the measurement of the ratio between the concentrations of testosterone and epitestosterone excreted as glucuronides (T/E). Despite the satisfactory results obtained with this approach, the measurement of T/E presents some limitations like the long-term detection of oral testosterone administration. Recently, several testosterone metabolites released after basic treatment of the urine have been reported (androsta-1,4-dien-3,17-dione, androsta-4,6-dien-3,17-dione, 17β-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione). In the present work, the usefulness of these metabolites for the detection of oral testosterone misuse has been evaluated and compared with the conventional T/E measurement. For this purpose, 173 urine samples collected from healthy volunteers were analysed in order to obtain reference concentrations for the four metabolites released after alkaline treatment. On the other hand, urine samples collected from five volunteers before and after testosterone undecanoate administration were also analysed. Concentrations of androsta-4,6-dien-3,17-dione and 17β-hydroxy-androsta-4,6-dien-3-one showed a similar behaviour as the T/E, allowing the detection of the misuse for several hours after administration. More promising results were obtained by quantifying androsta-1,4-dien-3,17-dione and 15-androsten-3,17-dione. The time in which the concentrations of these analytes could be differentiated from the basal level was between 3 and 6 times longer than the obtained with T/E, as a result, an improvement in the detection of testosterone abuse can be achieved. Moreover, several ratios between these compounds were evaluated. Some of them improved the detection of testosterone misuse when comparing with T/E. The best results were obtained with those ratios involving androsta-1,4-dien-3,17-dione.

Detection of dihydrotestosterone gel, oral dehydroepiandrosterone, and testosterone gel misuse through the quantification of testosterone metabolites released after alkaline treatment.

The natural occurrence of endogenous anabolic steroids together with their availability in different administration forms makes the detection of their misuse a great challenge for doping control laboratories. Nowadays, the detection of endogenous steroids abuse is performed by the analysis of the steroid profile. Recently, androst-1,4-dien-3,17-dione (1,4-AD), androst-4,6-dien-3,17-dione (4,6-AD), 17β-hydroxy-androst-4,6-dien-3-one (6-T), and androst-15-en-3,17-dione (15-AD) have been described as testosterone (T) metabolites released after basic treatment of the urine. In the present work, the usefulness of these metabolites has been evaluated detecting the use of three different forms of endogenous steroids in a single dose: dihydrotestosterone gel (DHT), oral dehydroepiandrosterone (DHEA), and T gel. After the independent administration of these endogenous steroids, a rise in the value of several of the ratios calculated between the tested metabolites was noticed. For DHT, a small increase was observed for the ratios 1,4-AD/15-AD, 6-T/15-AD and 4,6-AD/15-AD although only for one volunteer. Better results were obtained for oral DHEA and T gel where an increase was observed in all volunteers for several of the tested ratios. The detection time in which the misuse can be detected (DT) has been evaluated using two different approaches: (1) comparison with population based reference limits, and (2) comparison with individual threshold levels. The obtained DTs were compared with the results of previously published markers for the misuse of such substances. When using basic released metabolites, shorter DTs were obtained for DHT, similar DTs for DHEA, and the detectability was substantially improved for T gel.

Untargeted metabolomics in doping control: detection of new markers of testosterone misuse by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry.

The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated.

Detection and characterization of urinary metabolites of boldione by LC-MS/MS. Part I: Phase I metabolites excreted free, as glucuronide and sulfate conjugates, and released after alkaline treatment of the urine.

Boldione (1,4-androstadien-3,17-dione) is included in the list of prohibited substances, issued by the World Anti-Doping Agency (WADA). Endogenous production of low concentrations of boldione has also been reported. The objective of this study was to assess boldione metabolism in humans. Detection of boldione metabolites was accomplished by analysis by liquid chromatography coupled to tandem mass spectrometry of urine samples obtained after administration of the drug and subjected to different sample preparation procedures to analyze the different metabolic fractions (free, glucuronides, sulpfates and released in basic media). In addition to boldione, eight metabolites were detected in the free fraction. Four of them were identified by comparison with standards: 6β-hydroxy-boldenone (M3), androsta-1,4,6-triene-3,17-dione (M5), (5α)-1-androstenedione (M6) and (5α)-1-testosterone (M8). Metabolite M7 was identified as the 5β-isomer of 1-androstenedione, and metabolites M1, M2 and M4 were hydroxylated metabolites and tentative structures were proposed based on mass spectrometric data. After β-glucuronidase hydrolysis, five additional metabolites excreted only as conjugates with glucuronic acid were detected: boldenone, (5β)-1-testosterone (M9), and three metabolites resulting from reduction of the 3-keto group. Boldenone, epiboldenone, and hydroxylated metabolites of boldione, boldenone and 1-testosterone were detected as conjugates with sulfate. In addition, boldione and seven metabolites (boldenone, M2, M3, M4, M5, M7 and M9) increased their concentration in urine after treatment of the urine in alkaline conditions. In summary, 15 boldione metabolites were detected in all fractions. The longer detection time was observed for metabolite M4 after alkaline treatment of the urine, which was detected up to 5 days after boldione administration.

Quantification of testosterone and metabolites released after alkaline treatment in human urine.

A rapid, accurate, and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of the testosterone (T) fraction released under basic conditions and its metabolites 1,4-androstadien-3,17-dione (1,4-AD), 4,6-androstadien-3,17-dione (4,6-AD) and 4,6-androstadien-17β-ol-3-one (6-T) obtained in the same way has been developed and validated. Urine samples (5 ml) were alkalinized and the released analytes were extracted by liquid-liquid extraction. The chromatographic separation was performed in 8 min. MS/MS determination was performed under selected reaction monitoring mode using electrospray ionization in positive mode. The method was shown to be linear from 0.05 to 50 ng/ml for 1,4-AD, 6-T and T while for 4,6-AD the linearity from 0.5 to 500 ng/ml was demonstrated (r > 0.99). Limits of detection below 0.05 ng/ml were obtained for all analytes. Intra-assay precision and accuracies, evaluated at three concentrations levels (0.1, 1 and 20 ng/ml) were below 20%. The applicability of the analytical method was confirmed by analysis of several samples collected from healthy volunteers and samples collected after oral testosterone undecanoate administration. The analytical method was found to be able to quantify the metabolites either at the low concentrations levels present in the urine from untreated volunteers or at the high concentrations found after T administration. Short analysis time, simple sample preparation, and satisfactory quantitative parameters make this method potentially useful for anti-doping control purposes.

Current status and bioanalytical challenges in the detection of unknown anabolic androgenic steroids in doping control analysis

Androgenic anabolic steroids (AAS) are prohibited in sports due to their anabolic effects. Doping control laboratories usually face the screening of AAS misuse by target methods based on MS detection. Although these methods allow for the sensitive and specific detection of targeted compounds and metabolites, the rest remain undetectable. This fact opens a door for cheaters, since different AAS can be synthesized in order to evade doping control tests. This situation was evidenced in 2003 with the discovery of the designer steroid tetrahydrogestrinone. One decade after this discovery, the detection of unknown AAS still remains one of the main analytical challenges in the doping control field. In this manuscript, the current situation in the detection of unknown AAS is reviewed. Although important steps have been made in order to minimize this analytical problem and different analytical strategies have been proposed, there are still some drawbacks related to each approach.

Adrenal hormonal imbalance in acute intermittent porphyria patients: results of a case control study.

BackgroundAcute Intermittent Porphyria (AIP) is a rare disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers are at risk of presenting acute life-threatening neurovisceral attacks. The disease induces overproduction of heme precursors in the liver and long-lasting deregulation of metabolic networks. The clinical history of AIP suggests a strong endocrine influence, being neurovisceral attacks more common in women than in men and very rare before puberty. To asses the hypothesis that steroidogenesis may be modified in AIP patients with biochemically active disease, we undertook a comprehensive analysis of the urinary steroid metabolome.MethodsA case–control study was performed by collecting spot morning urine from 24 AIP patients and 24 healthy controls. Steroids in urine were quantified by liquid chromatography-tandem mass spectrometry. Parent steroids (17-hydroxyprogesterone; deoxycorticosterone; corticoesterone; 11-dehydrocorticosterone; cortisol and cortisone) and a large number of metabolites (N = 55) were investigated. Correlations between the different steroids analyzed and biomarkers of porphyria biochemical status (urinary heme precursors) were also evaluated. The Mann–Whitney U test and Spearman’s correlation with a two tailed test were used for statistical analyses.ResultsForty-one steroids were found to be decreased in the urine of AIP patients (P < 0.05), the decrease being more significant for steroids with a high degree of hydroxylation. Remarkably, 13 cortisol metabolites presented lower concentrations among AIP patients (P < 0.01) whereas no significant differences were found in the main metabolites of cortisol precursors. Nine cortisol metabolites showed a significant negative correlation with heme precursors (p < 0.05). Ratios between the main metabolites of 17-hydroxyprogesterone and cortisol showed positive correlations with heme-precursors (correlation coefficient > 0.51, P < 0.01).ConclusionsComprehensive study of the urinary steroid metabolome showed that AIP patients present an imbalance in adrenal steroidogenesis, affecting the biosynthesis of cortisol and resulting in decreased out-put of cortisol and metabolites. This may result from alterations of central origin and/or may originate in specific decreased enzymatic activity in the adrenal gland. An imbalance in steroidogenesis may be related to the maintenance of an active disease state among AIP patients.