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Background: Most current state-of-the-art strategies to generate individual adaptive reference ranges are designed to monitor one clinical parameter at a time. An innovative methodology is proposed for the simultaneous longitudinal monitoring of multiple biomarkers. The estimation of individual thresholds is performed by applying a Bayesian modeling strategy to a multivariate score integrating several biomarkers (compound concentration and/or ratio). This multimodal monitoring was applied to data from a clinical study involving 14 female volunteers with normal menstrual cycles receiving testosterone via transdermal route, as to test its ability to detect testosterone administration. The study samples consisted of urine and blood collected during 4 weeks of a control phase and 4 weeks with a daily testosterone gel application., Results: Integrating multiple biomarkers improved the detection of testosterone gel administration with substantially higher sensitivity compared with the distinct follow-up of each biomarker, when applied to selected urine and serum steroid biomarkers, as well as the combination of both. Among the 175 known positive samples, 38% were identified by the multimodal approach using urine biomarkers, 79% using serum biomarkers and 83% by combining biomarkers from both biological matrices, whereas 10%, 67% and 64% were respectively detected using standard unimodal monitoring., Significance and Novelty: The detection of abnormal patterns can be improved using multimodal approaches. The combination of urine and serum biomarkers reduced the overall number of false-negatives, thus evidencing promising complementarity between urine and blood sampling for doping control, as highlighted in the case of the use of transdermal testosterone preparations. The generation in a multimodal setting of adaptive and personalized reference ranges opens up new opportunities in clinical and anti-doping profiling. The integration of multiple parameters in a longitudinal monitoring is expected to provide a more complete evaluation of individual profiles generating actionable intelligence to further guide sample collection, analysis protocols and decision-making in clinics and anti-doping., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The Technical Document TD2014EAAS was drafted by the World Anti-Doping Agency (WADA) in order to fight the spread of endogenous anabolic androgenic steroids (EAAS) misuse in several sport disciplines. In particular, adoption of the so-called Athlete Biological Passport (ABP) - Steroidal Module allowed control laboratories to identify anomalous EAAS concentrations within the athletes' physiological urinary steroidal profile. Gas chromatography (GC) combined with mass spectrometry (MS), indicated by WADA as an appropriate technique to detect urinary EAAS, was utilized in the present study to develop and fully-validate an analytical method for the determination of all EAAS markers specified in TD2014EAAS, plus two further markers hypothetically useful to reveal microbial degradation of the sample. In particular, testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, dehydroepiandrosterone, 5α-dihydrotestosterone, were included in the analytical method. Afterwards, the multi-parametric feature of ABP profile was exploited to develop a robust approach for the detection of EAAS misuse, based on multivariate statistical analysis. In particular, Principal Component Analysis (PCA) was combined with Hotelling T(2) tests to explore the EAAS data obtained from 60 sequential urine samples collected from six volunteers, in comparison with a reference population of single urine samples collected from 96 volunteers. The new approach proved capable of identifying anomalous results, including (i) the recognition of samples extraneous to each of the individual urine series and (ii) the discrimination of the urine samples collected from individuals to whom "endogenous" steroids had been administrated with respect to the rest of the samples population. The proof-of-concept results presented in this study will need further extension and validation on a population of sport professionals., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ(13)C value). However, (13)C labeled standards can be used to control the δ(13)C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the (13)C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ(13)C values between Andro and ANAD (Δδ(13)CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different (13)C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ(13)CAndro-ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ(13)CAndro-ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-(13)C labeled standards., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The confirmation by GC/C/IRMS of the exogenous origin of pseudo-endogenous steroids from human urine samples requires extracts of adequate purity. A strategy based on HPLC sample purification prior to the GC/C/IRMS analysis of human urinary endogenous androgens (i.e. testosterone, androsterone and/or androstenediols), is presented. A method without any additional derivatization step is proposed, allowing to simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis, without the need of correcting the measured delta values for the contribution of the derivatizing agent. The HPLC extracts were adequately combined to both reduce the number of GC/C/IRMS runs and to have appropriate endogenous reference compounds (ERC; i.e. pregnanediol, 11-keto-etiocholanolone) on each GC-IRMS run. The purity of the extracts was assessed by their parallel analysis by gas chromatography coupled to mass spectrometry, with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision below 0.3‰(13)C delta units and between assay precision below 0.6‰(13)C delta units for most of the compounds investigated) fulfilling the World Anti-Doping Agency requirements., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this research, a novel stacking capillary electrophoresis method, repetitive large volume sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze the presence of three androgenic steroids considered as sport doping drugs, testosterone (T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides better sensitivity enhancement than the traditional large volume sample stacking-sweeping strategies due to sensitivity enhancement by repetitive injections. This multiple sampling method enhances sensitivity of monitoring of urine samples by UV detection (254 nm). Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the samples were injected into the capillary at 10 psi for 20s, and then stacked at -10 kV for 1 min using phosphate buffer containing SDS. The above injecting and stacking steps were repeated five times. Finally, separation was performed at -20 kV, using phosphate buffer containing methanol, SDS and (2-hydroxypropyl)-β-cyclodextrin. Method validation showed that calibration plots were linear (r≥0.997) over a range of 5-200 ng mL(-1) for T, 20-200 ng mL(-1) for E and 0.5-500 ng mL(-1) for EG. The limits of detection were 1.0 ng mL(-1) for T, 5.0 ng mL(-1) for E and 200.0 pg mL(-1) for EG. When evaluating precision and accuracy, values of RSD and RE in intra-day (n=3) and inter-day (n=5) analysis were found to be less than 10.0%. Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method further improves sensitivity up to 25 folds (~2500 folds with MEKC without preconcentration). This method was applied to monitor 10 athletes' urine, and did not detect any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The presence of microorganisms in urine samples, under favourable conditions of storage and transportation, may alter the concentration of steroid hormones, thus altering the correct evaluation of the urinary steroid profile in doping control analysis. According to the rules of the World Anti-Doping Agency (WADA technical document TD2004 EAAS), a testosterone deconjugation higher than 5% and the presence of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the deconjugated fraction, are reliable indicators of urine degradation. The determination of these markers would require an additional quantitative analysis since the steroids screening analysis, in anti-doping laboratories, is performed in the total (free+conjugated) fraction. The aim of this work is therefore to establish reliable threshold values for some representative compounds (namely 5α-androstane-3,17-dione and 5β-androstane-3,17-dione) in the total fraction in order to predict directly at the screening stage the potential microbial degradation of the urine samples. Preliminary evidence on the most suitable degradation indexes has been obtained by measuring the urinary concentration of testosterone, epitestosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione by gas chromatography-mass spectrometric every day for 15 days in the deconjugated, glucuronide and total fraction of 10 pools of urines from 60 healthy subjects, stored under different pH and temperature conditions, and isolating the samples with one or more markers of degradation according to the WADA technical document TD2004EAAS. The threshold values for 5α-androstane-3,17-dione and 5β-androstane-3,17-dione were therefore obtained correlating the testosterone deconjugation rate with the urinary concentrations of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the total fraction. The threshold values suggested as indexes of urine degradation in the total fraction were: 10 ng mL(-1) for 5α-androstane-3,17-dione and 20 ng mL(-1) for 5β-androstane-3,17-dione. The validity of this approach was confirmed by the analysis of routine samples for more than five months (i.e. on a total of more than 4000 urine samples): samples with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione higher than the threshold values showed a percentage of free testosterone higher than 5 of its total amount; whereas free testosterone in a percentage higher than 5 of its total amount was not detected in urines with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione lower than the threshold values., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Voltammetric investigation of two corticoid isomers--testosterone and epitestosterone has been carried out at bare and single-wall carbon nanotubes (SWNT)-modified edge plane pyrolytic graphite electrode (EPPGE). Square wave voltammetry (OSWV) has been used for the simultaneous determination of isomeric steroids. The reduction of the two isomers occurred in a pH dependent, 2e, 2H+ process and well-defined voltammetric peaks were observed. Under the optimum experimental conditions, linear calibration curves are obtained within the concentration range 5-1000 nM for both the steroids with the limit of detection 2.8 x 10(-9) and 4.1 x 10(-9) M for testosterone and epitestosterone respectively. The developed protocol is successfully implemented for the analysis of both the compounds in the urine samples of normal subjects as well as in patients undergoing treatment with testosterone. The results obtained from the proposed voltammetric method were also compared with HPLC analysis and found to be similar.

This paper describes the effects of oral administration of non-steroidal anti-inflammatory drugs on the endogenous and synthetic anabolic androgenic steroids urinary excretion as assessed by gas-chromatography mass-spectrometry. Experiments were carried out on 5 male subjects, with pathologies and/or diseases, treated with non-steroidal anti-inflammatory drugs. To set up the individual baseline variability of testosterone and its main metabolites, urine samples were collected for 3 days, every 2 h prior to the administration of the drug(s); whereas the study of the effects of a single dose of each drug, here considered, on the endogenous androgen steroid urinary concentrations, was assessed by collecting urine samples for 2 days, every 2 h. Data obtained after drugs administration were then evaluated taking into account the individual baseline variability. The results showed that, only in the case of propyphenazone administration, the relative urinary concentrations of some testosterone metabolites were significantly altered. More specifically, the urinary levels of dehydroepiandrosterone, 11keto-etiocholanolone, 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, androsterone, etiocholanolone and some metabolite ratios decrease significantly, generally between 2 and 10 h after administration of the drug, whereas no effects were observed on urinary calculated concentrations of testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and testosterone/epitestosterone ratio. The observed effects do not depend on alterations on pharmacokinetics (excretion/metabolism), but on steroid sample preparation steps (hydrolysis and derivatization) inhibition. More specifically the significant decrease of dehydroepiandrosterone and testosterone metabolites urinary levels was due to a reduced yield of the steroid derivatization step for the presence in urine of the main metabolites of propyphenazone, namely hydroxyl-propyphenazone metabolites.

The residue profiles of boldenone (17beta-Bol), its epimer (17alpha-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17alpha-Bol decreased rapidly after end of treatment; detectable amounts of 17alpha-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17beta-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.

Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incubated with fresh horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation. The results showed that the major biotransformation of turinabol was hydroxylation at the C6, C16 and C20 sites to give metabolites 6beta-hydroxyturinabol (M1), 20-hydroxyturinabol (M2), two stereoisomers of 6beta,16-dihydroxyturinabol (M3a, M3b) and 6beta,20-dihydroxyturinabol (M4). The metabolite 6beta-hydroxyturinabol was confirmed using an authentic reference standard. The structures of all other turinabol metabolites were tentatively identified by mass spectral interpretation. For the in vivo studies, two horses were administered orally with turinabol. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that turinabol was extensively metabolised and the parent drug was not detected in urine. Two metabolites detected in the in vitro studies, namely 20-hydroxyturinabol and 6beta,20-dihydroxyturinabol, these were also detected in post-administration urine samples. In addition, 17-epi-turinabol (M5) and six other metabolites (M6a-M6c and M7a-M7c), derived from D-ring hydroxylation and A-ring reduction, were also detected. Except for 17-epi-turinabol, none of these metabolites has ever been reported in any species. All in vivo metabolites were detected within 48 h after administration.

The residue profiles of 17alpha-/17beta-boldenone conjugated (17alpha/beta-Bol) and ADD were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male veal calves fed two commercial milk replacers, with different content of cholesterol and phytosterols. The urine samples were collected within 4 h after feeding and further from all the animals. Detectable amounts of 17alpha-Bol conjugated were measured in urine collected from all calves, but the concentrations of 17alpha-Bol were higher in urine from calves receiving the milk replacer with the greater amount of phytosterols. During the whole experiment, 17beta-Bol and ADD were never detected in urine samples collected.

Natural occurrence or illegal treatment of boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17alpha-boldenone (alpha-BOLD) and 17beta-boldenone (beta-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid-liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC-MS-MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17beta-boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCalpha) smaller than 0.5 ng mL(-1) were obtained for each analyte.

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) substrate system, at +100 mV. The EC50 values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL(-1) and 960 pg mL(-1), respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL(-1) to 40 ng mL(-1); while that in urine ranged from 0.03 ng mL(-1) to 1.6 ng mL(-1). The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL(-1) and 1.8 pg mL(-1). Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV<13%, n=3) and repeatability (CV<9%, n=3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 degrees C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.