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In this work, a GC-MS/MS method was developed for the determination of anabolic-agent residues in bovine urine. The optimized sample preparation was as follows: enzymatic hydrolysis by β-glucuronidase-sulfatase enzyme from Helix pomatia for 16 h at 37.5 °C, liquid-liquid extraction with diethyl ether, solid-phase extraction with HLB and aminopropylsilane cartridges, and microwave-assisted derivatization using 25 μL of MSTFA/NH 4 I/ethanethiol and full microwave power for 2 min. The method was validated according to Decision 657/2002/EC, Codex Alimentarius, and Manual da Garantia da Qualidade Analítica guidelines. The acceptability criteria for quantitative analysis were met for α-ethinylestradiol, α-nandrolone, β-estradiol, β-zearalanol, β-zearalenol, drostanolone, ethisterone, dienestrol, diethylstilbestrol, hexestrol, megestrol, methyltestosterone, and zearalenone. The analytes α-zearalenol, α-zearalanol, and norethandrolone were validated for qualitative analysis.

In livestock production, illegal use of natural steroids is hard to prove because metabolites are either unknown or not significantly above highly fluctuating endogenous levels. In this work we outlined for the first time a metabolomics based strategy for anabolic steroid urine profiling. Urine profiles of controls and bovines treated with the prohormones dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-TOFMS). The obtained full scan urinary profiles were compared using sophisticated preprocessing and alignment software (MetAlign) and multivariate statistics, revealing hundreds of mass signals which were differential between untreated control and prohormone-treated animals. Moreover, statistical testing of the individual accurate mass signals showed that several mass peak loadings could be used as biomarkers for DHEA and pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and verification by standards or Orbitrap mass spectrometry demonstrated that the observed differential masses are most likely steroid phase I and glucuronide metabolites excreted as a direct result from the DHEA and pregnenolone administration, thus underlining the relevance of the findings from this untargeted metabolomics approach. It is envisaged that this approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and possibly in sports doping as well.

The detection of new anabolic steroid metabolites and new designer steroids in urine is a challenge in doping analysis. An approach based on precursor ion scanning for the detection of unknown anabolic steroids and metabolites is proposed. The study of the MS/MS spectra of selected anabolic steroids revealed different fragmentation pathways at low and medium collision energy depending on the steroid structure. However, after analysis at high collision energy three common ions at m/z 105, m/z 91, and m/z 77 were found for all studied anabolic steroids. These ions can be explained by the fragmentation of the steroid structure and corresponded to the methyl tropylium, tropylium, and phenyl ions, respectively. Because of the theoretical low specificity of these ions, the simultaneous presence of all of them was used as a starting point to consider a substance as a possible anabolic steroid. Hence, the developed approach is based on the simultaneous acquisition of the precursor ion scan of m/z 105, 91, and 77. The specificity of this approach has been checked by the injection of several doping agents including beta-agonists, corticosteroids, beta-blockers, and diuretics. In general, only compounds with a steroidal structure showed a signal at all three selected m/z values although some exceptions have been found. The applicability of the method was tested for three different scenarios: the detection of steroid metabolites, the detection of unknown steroids, and the analysis of prohormones. In metabolic studies, several recently reported fluoxymesterone metabolites were also found using this method. For detection of unknown steroids, some negative urine samples were spiked with the designer steroid THG and 33 other anabolic steroids and treated as blind samples. Finally, the applicability of the developed approach for the analysis of dietary supplements was checked by the analysis of a prohormone where several impurities and/or degradation products were found.

Fast screening for anabolic steroids in whole urine is achieved by combining reactive desorption electrospray ionization (reactive DESI) and tandem mass spectrometry. Spray solutions containing hydroxylamine allow heterogeneous reactions of hydroxylamine with the carbonyl group of the steroids during the ionization process. Seven steroids, including a glycosteroid, were examined. The ion/molecule reaction adduct and the oxime formed via its dehydration were observed using reactive DESI; the protonated and sodiated forms of the ionized steroid were also observed both in reactive DESI and in DESI performed without the added hydroxylamine reagent. Paper, glass, and polytetrafluoroethylene were tested as sample substrates, but the glycosteroid was ionized intact without hydrolysis only from polytetrafluoroethylene. Limits of detection for the pure compounds were less than 1 ng, dynamic ranges were typically 2 orders of magnitude, and analysis times were just a few seconds. Concentration levels of ketosteroids in raw urine relevant to screening for sports doping (approximately 20 ng/mL) can be reached using a simple solid-phase microextraction (SPME) preconcentration step. Reactive DESI provided significant improvements in ionization efficiency of these steroids in raw undiluted urine as compared to conventional DESI; suppression effects due to the sample matrix were minimal and the urine matrix had no deleterious effect on steroid detection limits. Tandem mass spectrometry provided confirmation of analyte identification in this rapid screening process.

Twenty-six veal calves were split into two groups and fed two milk replacers with a different content of phytosterols for 26 days; then, 14 calves (7 animals from each diet) were kept as controls and 12 calves (6 per diet) received daily, per os, a combination of 17beta-boldenone (17beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) for 38 days. The urinary elimination of 17 alpha-/17beta-boldenone conjugates (17 alpha/beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) was followed by liquid chromatography-tandem mass spectrometry from all of the animals until slaughtering. In urine from treated animals, 17 alpha-Bol concentrations, despite a great variability, were greater than 17beta-Bol, both detected always as conjugates. At days 1, 2, and 3, the mean urine concentration of 17 alpha-Bol was higher than 12 ng/mL. A remarkable decrease was observed during the following days, but the 17 alpha-Bol concentration was still higher than the attention level of 2 ng/mL in 58% of the samples; the concentration of 17beta-Bol was around the action level of 1 ng/mL; two days after treatment withdrawal, no 17beta-Bol was detected in the urine. In urine from control animals, the 17 alpha-Bol concentration was strictly related to the phytosterol content of the diet, while, in urine from treated animals, the much higher 17 alpha-Bol levels were not modified by the production from diet precursors. The results confirmed that a 17 alpha-Bol level higher than 2 ng/mL should be considered as evidence of suspected illegal treatment and that the urinary excretion of 17beta-Bol is due to exogenous administration of 17beta-Bol. The discontinuous rate of elimination of both 17 alpha- and 17beta-Bol, despite the daily administration of 17beta-Bol plus ADD, indicates the necessity for further research to detect other urinary boldenone metabolites to strength surveillance strategy.

Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

A conjugated hapten microarray based on miniature immunoassay for fast and multiplex detection of anabolic steroids is reported for the first time. This preliminary study investigated the possibility of using a microarray technology as a multisteroid detection assay. The microarray system used eight monoclonal antibodies raised against three steroid conjugates, 4-androsten-4-chloro-17beta-ol-3-one, 1,5alpha-androsten-1beta-methyl-17beta-ol-3-one, and 5beta-androsten-1-en-17beta-ol-3-one, which were conjugated to BSA by the active ester method. In addition to 4 commercial conjugated haptens, 18 steroid-BSA conjugates were synthesized and from all these a conjugated hapten microarray was fabricated. The analyzed substances included 42 types of anabolic steroid reference materials and 28 positive urine samples. Of these, 24 anabolic steroids and 12 positive urines were successfully detected.

Pyrolysis coupled to metastable atom bombardment (MAB) and time-of-flight mass spectrometry (TOFMS) is used for generating mass spectra from bovine urine samples obtained from cattle treated with anabolic steroids. These spectra constitute fingerprints, which can be discriminated by multivariate statistical analysis. Four main conclusions can be drawn from this work: (i) The use of different metastable gases, such as Xe*, Kr*, or N2*, as an energy-tunable ionization beamline allows control of the internal energy and the dissociation processes of the produced odd electron molecular ions, thus giving rise to complementary mass spectra fingerprints. (ii) A variable transformation depending on the biofluid matrix suitably contracts the frequency distribution of the generated data for low m/z ratios holding information related to endogenous metabolites encountered in urine. (iii) Coupling variable selection to statistical pattern recognition methods results in low error rates (< 1%) for predicting MAB mass fingerprints, especially using lineardiscriminant analysis (LDA). (iv) LDA discriminates controls from treated animals and also correlates to quantitative physiological responses induced by anabolic steroids. This work shows that Py-MAB-TOFMS could be a suitable method for complementary monitoring anabolic use in sports, medicine, and cattle breeding, as well as monitoring many other long-lasting although weak physiological disruptions.