Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay.

Hypoxia-inducible factor (HIF) stabilizers are listed in the World Anti-Doping Agency's prohibited list as they can increase aerobic exercise capacity. The rapid pace of emergence of highly structurally diverse HIF stabilizers could pose a risk to conventional structure-based methods in doping control to detect new investigational drugs. Therefore, we developed a strategy that is capable of detecting the presence of any HIF stabilizer, irrespective of its structure, by detecting biological activity. Previously developed cell-based HIF1/2 assays were optimized to a stable format and evaluated for their screening potential toward HIF stabilizers. Improved pharmacological characterization was established by the stable cell-based formats, and broad specificity was demonstrated by pharmacologically characterizing a diverse set of HIF stabilizers (including enarodustat, IOX2, IOX4, MK-8617, JNJ-42041935). The methodological (in solvent) limit of detection of the optimal HIF1 stable bioassay toward detecting the reference compound roxadustat was 100 nM, increasing to 50-100 ng/mL (corresponding to 617-1233 nM in-well) in matching urine samples, owing to strong matrix effects. In a practical context, a urinary limit of detection of 1.15 µg/mL (95% detection rate) was determined, confirming the matrix-dependent detectability of roxadustat in urine. Pending optimization of a universal sample preparation strategy and/or a methodology to correct for the matrix effects, this untargeted approach may serve as a complementing method in antidoping control, as theoretically, it would be capable of detecting any unknown substance with HIF stabilizing activity.

Investigating the detection of the novel doping­relevant peptide kisspeptin­10 in urine using liquid chromatography high­resolution mass spectrometry.

Kisspeptin-10 is a peptide hormone capable of increasing circulating follicle-stimulating hormone, luteinizing hormone and testosterone levels in humans. Clinically, these effects suggest its use as a treatment for infertility. However, its testosterone-increasing effect indicates potential misuse in sports. As such, it is included in the 2024 World Anti-Doping Agency Prohibited List. This work describes the successful validation of an initial testing procedure (screening) and a confirmation procedure for kisspeptin-10 in urine using liquid chromatography-mass spectrometry. Additionally, kisspeptin-10 was incubated in human serum to mimic endogenous metabolism to improve method sensitivity, as previous research had demonstrated a rapid elimination time of only 30 min after injection (in rats). Four metabolites, corresponding to peptide fragments y9, y8, y7 and y5, were found and added to the ITP in full scan mode. A degradation product discovered during early experimentation was found to probably be caused by oxidation of the tryptophan residue into a kynurenine residue. Further research should elucidate the kinetic parameters of the reaction to improve product stability. Using the validated confirmation procedure, a black-market vial of kisspeptin-10 was analysed. The product contained no unexpected impurities, although it appeared to have undergone more degradation than the purchased reference standard.

The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction.

BACKGROUND: Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. RESULTS: Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. CONCLUSION: Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.

Automated sample preparation for the detection and confirmation of hypoxia-inducible factor stabilizers in urine.

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation.

Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5.

The tendency of peptides to adsorb to surfaces can raise a concern in variety of analytical fields where the qualitative/quantitative measurement of low concentration analytes (ng/mL-pg/mL) is required. To demonstrate the importance of using the optimal glassware/plasticware, four doping relevant model peptides (GHRP 5, TB-500, Insulin Lispro, Synachten) were chosen and their recovery from various surfaces were evaluated. Our experiments showed that choosing expensive consumables with low-bind characteristics is not beneficial in all cases. A careful selection of the consumables based on the evaluation of the physico/chemical features of the peptide is recommended.

Gas chromatography/chemical ionization triple quadrupole mass spectrometry analysis of anabolic steroids: ionization and collision-induced dissociation behavior.

RATIONALE: The detection of new anabolic steroid metabolites and new designer steroids is a challenging task in doping analysis. Switching from electron ionization gas chromatography triple quadrupole mass spectrometry (GC/EI-MS/MS) to chemical ionization (CI) has proven to be an efficient way to increase the sensitivity of GC/MS/MS analyses and facilitate the detection of anabolic steroids. CI also extends the possibilities of GC/MS/MS analyses as the molecular ion is retained in its protonated form due to the softer ionization. In EI it can be difficult to find previously unknown but expected metabolites due to the low abundance or absence of the molecular ion and the extensive (and to a large extent unpredictable) fragmentation. The main aim of this work was to study the CI and collision-induced dissociation (CID) behavior of a large number of anabolic androgenic steroids (AAS) as their trimethylsilyl derivatives in order to determine correlations between structures and CID fragmentation. Clarification of these correlations is needed for the elucidation of structures of unknown steroids and new metabolites. METHODS: The ionization and CID behavior of 65 AAS have been studied using GC/CI-MS/MS with ammonia as the reagent gas. Glucuronidated AAS reference standards were first hydrolyzed to obtain their free forms. Afterwards, all the standards were derivatized to their trimethylsilyl forms. Full scan and product ion scan analyses were used to examine the ionization and CID behavior. RESULTS: Full scan and product ion scan analyses revealed clear correlations between AAS structure and the obtained mass spectra. These correlations were confirmed by analysis of multiple hydroxylated, methylated, chlorinated and deuterated analogs. CONCLUSIONS: AAS have been divided into three groups according to their ionization behavior and into seven groups according to their CID behavior. Correlations between fragmentation and structure were revealed and fragmentation pathways were postulated.

In vitro and in vivo metabolism studies of dimethazine.

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti-doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA(+/+) -SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called 'Xtreme DMZ'. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC-High Resolution Mass Spectrometry (HRMS) and GC-MS(/MS) analysis methasterone and six other dimethazine metabolites (M1-M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro-conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley & Sons, Ltd.

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.

In vitro models for metabolic studies of small peptide hormones in sport drug testing.

Peptide hormones represent an emerging class of potential doping agents. Detection of their misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore, investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic studies with human volunteers are often not allowed because of ethical constraints, and therefore alternative models are needed. This study was performed in order to evaluate in vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of peptidic doping agents and to compare them with the established models. The peptides that were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and leuprolide. Several metabolites were detected for each peptide after incubation with human liver microsomes, S9 fraction, and serum, which all showed endopeptidase and exopeptidase activity. In vitro models from different organs (liver vs. kidney) were compared, but no significant differences were recorded. Deamidation was not observed in any of the models and was therefore evaluated by incubation with α-chymotrypsin. In conclusion, in vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic markers in biological fluids.

GC-C-IRMS in routine doping control practice: 3 years of drug testing data, quality control and evolution of the method.

In order to detect the misuse of endogenous anabolic steroids, doping control laboratories require methods that allow differentiation between endogenous steroids and their synthetic copies. Gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) is capable of measuring the carbon isotope ratio of urinary steroids and this allows differentiation between both. GC-C-IRMS and its application to doping control has evolved a lot during the last decade and so have the World Anti-Doping Agency (WADA) technical documents that describe how GC-C-IRMS should be applied. Especially the WADA technical document of 2014 introduced a number of obligatory quality controls and a fixed methodology that should be used by all the doping control laboratories. This document imposed more uniform methods between the laboratories in order to decrease the interlaboratory standard deviation and acquire similar results for the analysis of the same urine samples. In this paper, 3 years of drug testing data of our GC-C-IRMS method in routine doping control practice is described, with an emphasis on the new WADA technical document and its implementation. Useful data for other doping control laboratories is presented focussing on general method setup, quality control and data collected from routine samples. The paper concentrates on how IRMS results shift or remain similar by switching to the 2014 WADA technical document and gives insight in a straightforward approach to calculate the measurement uncertainty.

Detection of the selective androgen receptor modulator S-23 and its metabolites in equine urine and plasma following oral administration.

S-23 is an arylpropionamide selective androgen receptor modulator that has been investigated in animal models for use as a male hormonal contraceptive but is not yet available therapeutically. S-23 is available alongside other selective androgen receptor modulators (SARMs) to purchase online via uncontrolled sites, sold as supplement products. It has been detected in several human doping cases, highlighting the importance of identifying the best analytical targets for equine doping control. The purpose of this study was to investigate the detection of S-23 and its phase I metabolites in equine urine and plasma following a multiple dose oral administration to two Thoroughbred racehorses. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification, and liquid chromatography-tandem mass spectrometry was used for full sample analysis and generation of urine and plasma profiles. S-23 and seven phase I metabolites were observed in urine following enzyme hydrolysis and solvolysis. The most abundant analyte detected was the hydroxylated 4-amino-2-(trifluoromethyl)benzonitrile metabolite, which also allowed the longest duration of detection in urine from both horses, for up to 360 h following administration. The data suggest that this metabolite was likely to be highly conjugated with both sulphate and glucuronide moieties. In plasma, S-23 and two phase I metabolites were observed. S-23 was the most abundant analyte detected for both horses, allowing detection for up to 143 h post-administration. To the best of the authors' knowledge, this is the first report of S-23 and metabolites in equine urine and plasma samples.

Prevalence of nandrolone preparations with endogenous carbon isotope ratios in Australia.

Nandrolone and its prohormones, including 19-norandrost-4-ene-3,17-dione and 19-norandrost-4-ene-3ß,17ß-diol, are anabolic steroids forbidden at all times in sports according to the World Anti-Doping Code Prohibited List and its metabolite 19-norandrosterone (19NA) is the preferred urinary target compound to identify their abuse. In recent years, an increasing number of 19NA isotope ratio mass spectrometry (IRMS) cases have arisen that, based on the initial testing procedure, were likely to result in an adverse analytical finding but were concluded negative after IRMS analysis. The current study was therefore set up to gain a better insight on the prevalence of nandrolone preparations with endogenous carbon isotope ratio values in Australia. Suitable workplace (non-athlete) urine samples that had previously been reported positive for 19NA were identified and analysed on IRMS. A total of 82% of the samples that were analysed were reported with enriched carbon isotope ratios of 19NA (i.e., 19NA greater than -26‰). This indicates that there is a high prevalence of nandrolone-containing anabolic androgenic steroid preparations in Australia that have 'endogenous' carbon isotope ratios which reduces the ability to identify exogenous nandrolone.

In vivo and in vitro metabolism of the synthetic cannabinoid JWH-200.

RATIONALE: The synthetic cannabinoid JWH-200 (1-[2-(4-morpholinyl)ethyl]-3-(1-naphthoyl)-indole) appeared on the market around 2009. In order to identify markers for misuse of this compound and allow for the development of adequate routine methods, the metabolism of this compound was investigated using two models. METHODS: In vitro and in vivo (both with and without enzymatic hydrolysis) samples were purified by solid-phase extraction and analyzed using liquid chromatography. Electrospray ionization high-resolution Orbitrap mass spectrometry was used for the identification of the metabolites. To confirm the results in vivo, triple-quadrupole mass spectrometry was employed RESULTS: In the in vitro model, using human liver microsomes, 22 metabolites were detected which could be divided into 11 metabolite classes. By using the chimeric mouse model with humanized liver, most of these metabolites were confirmed in vivo. It was found that all metabolites are excreted in urine as conjugates, mostly as glucuronides with varying conjugation rates. CONCLUSIONS: The metabolite formed by consecutive morpholine cleavage and oxidation of the remaining side chain to a carboxylic group was detected in the highest amounts with the longest detection time. Therefore, it is the best candidate metabolite to detect JWH-200 abuse in urine.

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.

Development of a sensitive GC-C-IRMS method for the analysis of androgens.

The administration of anabolic steroids is one of the most important issues in doping control and is detectable through a change in the carbon isotopic composition of testosterone and/or its metabolites. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), however, remains a very laborious and expensive technique and substantial amounts of urine are needed to meet the sensitivity requirements of the IRMS. This can be problematic because only a limited amount of urine is available for anti-doping analysis on a broad spectrum of substances. In this work we introduce a new type of injection that increases the sensitivity of GC-C-IRMS by a factor of 13 and reduces the limit of detection, simply by using solvent vent injections instead of splitless injection. This drastically reduces the amount of urine required. On top of that, by only changing the injection technique, the detection parameters of the IRMS are not affected and there is no loss in linearity.

Evaluation of alternative gas chromatographic and mass spectrometric behaviour of trimethylsilyl-derivatives of non-hydrolysed sulfated anabolic steroids.

Sulfated metabolites have shown to have potential as long-term markers (LTMs) of anabolic-androgenic steroid (AAS) abuse. The compatibility of gas chromatography-mass spectrometry (GC-MS) with trimethylsilyl (TMS)-derivatives of non-hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M-H2 SO4 ]•+ . Sulfated reference standards are in limited commercial availability, and therefore, the current knowledge of the GC-MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in-house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC-MS in their TMS-derivatised non-hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono-hydroxy-diones, 17-methyl-3,17-diols and 17-keto-3,16-diols as these are commonly encountered AAS conformations. The ability to predict the GC-MS behaviour of non-hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential, for example, for understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure.

Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

Characterization and identification of a C-terminal amidated mechano growth factor (MGF) analogue in black market products.

RATIONALE: Mechano growth factor (MGF) is a splice variant of insulin-like growth factor that possesses anabolic properties and has not yet been approved for therapeutic use. Nevertheless, it is readily available on the black market. Although the World Anti-Doping Agency (WADA) has banned the use of MGF in sports, no routinely performed methods have been reported for its detection. In this work, two preparations from the black market containing an unknown MGF analogue were characterized. METHODS: Mass spectrometry characterizations of unknown preparations and a reference human MGF were performed on an Orbitrap and a triple quadrupole mass spectrometers after separation by liquid chromatography. High accuracy measurements allowed protein identification from full scan MS data, and low-resolution full scan MS/MS provided further information on fragmentation. RESULTS: HCD scans of the analytes showed the presence of common b series product ions in the black market preparations and the human MGF reference standard, but all the y series ions starting from (y(1))(+) exhibited a difference of 1 m/z unit in nominal mass. This difference was demonstrated to be due to a C-terminal amidation of MGF. High-resolution data demonstrated that the black market products were both C-terminal amidated analogues of human MGF. In addition, low-resolution MS/MS characterization revealed a potentially diagnostic transition (m/z 717.8 → 431.1) for the discrimination of C-amidated MGF from the endogenous form. CONCLUSIONS: Qualitative identification of a MGF C-terminal amidated analogue in two black market products was successfully achieved. This report demonstrates that illegal MGF preparations are commercially available for use as doping agent in sports.

Qualitative detection of desmopressin in plasma by liquid chromatography-tandem mass spectrometry.

This work describes a liquid chromatography-electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (n = 10), the ion suppression as 5% (n = 10), and the limit of detection was 50 pg/ml (signal-to-noise ratio greater than 3). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.

An improved gas chromatography screening method for doping substances using triple quadrupole mass spectrometry, with an emphasis on quality assurance.

A GC-QqQ-MS method was developed for the detection of over 150 compounds from different classes (steroids, narcotics, stimulants, ß-blockers, ß-2-agonists and hormone antagonists) in a qualitative way. In the quantitative part, the traditional steroid profile with the most important endogenous steroids is expanded with six minor metabolites, which further improves the detection and identification of endogenous steroid abuse. In addition to these, norandrosterone, salbutamol and the major metabolite of cannabis are also quantified. Methods developed for anti-doping purposes should be subjected to the highest level of quality. Here, the addition of a combination of (deuterated) internal standards allows for an accurate quality control of every single step of the methodology: hydrolysis efficiency, derivatization efficiency and microbiological degradation are monitored in every single sample. Additionally, special attention is paid to the relationships between parameters indicating degradation by micro-organisms and the reliability of the steroid profile. The impact of the degradation is studied by evaluation of the quantities and percentages of 5α-androstane-3,17-dione and 5ß-androstane-3,17-dione. The concept of measurement uncertainty was introduced for the evaluation of relative abundances of mass-to-charge ratios and the obtained ranges were compared with the World Anti-Doping Agency regulations on tolerance windows for relative ion intensities. The results indicate that the approaches are similar.