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A novel method for determining the testosterone/epitestosterone concentration ratio in human urine was established by capillary electrophoresis with diode-array detector. The urine samples were firstly purified by the solid extraction. The optimal experimental conditions were: running buffer pH = 4.74, 15.0 mmol L -1 HAc-NaAc, separation voltage 25 kV, temperature 25 °C, sample injection pressure 3.43 × 10 3  Pa, and duration 10 s. The testosterone and epitestosterone linear range were determined as 8.0-960.0 ng mL -1 , respectively. The testosterone and epitestosterone detection limits were determined as 4.6 and 4.5 ng mL -1 , respectively. The relative standard deviation was less than 0.36%., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Testosterone is one of the most abused pseudo-endogenous anabolic steroids in sport doping. The current method adopted to detect the abuse of testosterone and other pseudo-endogenous steroids (endogenous steroids when administered exogenously) is first based on the longitudinal monitoring of several urinary biomarkers, which constitute the so called "steroidal module" of the Athlete Biological Passport (ABP): atypical samples undergo a confirmation analysis based on the measurement of the 13 C/ 12 C isotopic ratio of selected target compounds, to distinguish their endogenous or exogenous origin. At the same time, testosterone administration can be allowed in athletes diagnosed with hypogonadism, provided they are granted a therapeutic use exemption by the relevant medical authority. In this pilot study we have investigated whether the approach based on the preliminary determination of the urinary steroid profile, in the format considered in the steroidal module of the ABP, also integrated with the inclusion of the sulfo-conjugates and of additional target steroids, can retain its validity also in the case of hypogonadal athletes. We have studied the effects of a single low dose (40 mg) of testosterone gel (T-gel) on the urinary concentration of the markers of steroidal module of the ABP, as well as on some additional steroid markers. The study was based on the analysis of urinary samples from 19 non-hospitalized hypogonadal men, 10 of them with late-onset hypogonadism (LOH), collected before, after 4 h and after 24 h the transdermal self-administration of 40 mg of T-gel. None of the patient had any co-morbidities possibly affecting the urinary excretion of the steroidal markers. The steroidal markers were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) after the enzymatic hydrolysis of the respective glucuro-conjugates and the chemical hydrolysis of the respective sulfo-conjugates. Targeted GC-MS/MS analysis was carried out operating in electron impact (EI) ionization mode, with acquisition in multiple reaction monitoring (MRM) mode. Our preliminary results show that, as expected, the treatment with T-gel leads, in all hypogonadal men, to an increase of the urinary concentration of the glucuro-conjugate metabolites of testosterone and its main metabolites, with special relevance to those with 5α-reduction. Furthermore, samples collected from non-LOH hypogonadal men showed an increase also in the levels of epitestosterone glucuronide, testosterone sulfate and epitestosterone sulfate. Apart from their biochemical and pharmacological relevance, these outcomes could be leveraged to refine the analytical strategy currently followed in the antidoping field for the analysis of the urinary steroidal markers, with potential implications also in other forensic and/or clinical investigations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This article studies the genetic influence of polymorphism of the UGT2B17 gen on the urinary steroid profile and its implications for the anti-doping field. The study presents the results of a triple-blind randomized placebo-controlled crossover trial with healthy athletes submitted to a single dose of 250 mg of testosterone cypionate. Forty urine samples were collected from each participant. Mass spectrometry-based techniques commonly used in Anti-Doping laboratories, were employed to measure the urinary concentration and the Δδ 13 C values of a selection of target compounds for testosterone (T) administration together with LH. Twelve volunteers were included in the study; the polymorphism was evenly distributed among them. After T administration, the most meaningful change affected the Testosterone/Epitestosterone ratio (T/E) and the urinary concentration of LH. In relation with T/E, the wild type homozygous (ins/ins) group there was a mean relative increase of 30 (CI 95%: 25.2 to 36.7); in the heterozygous mutant (del/ins) group it was 19.8 (CI 95%:15.9 to 24.7); and in the homozygous mutant (del/del) group it was 19.7 (CI 95% 14.9 to 26.2). In the case of LH, it́s observed how LH values decrease significantly after the administration of Testex homogeneously among the three groups. The main outcome was related to the (del/del) group (homozygous mutant), where due to the depressed basal level of the steroid profile, if the longitudinal steroid profile of the athlete was not available, the analysis by GC/MS would not produce an "atypical" result according to the WADA TD2016EAAS despite the T administration. However, the genotyping of the UGT2B17 polymorphism, the follow up of LH and the use of GC-C-IRMS makes it possible to identify most of these samples as Adverse., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chronic and heavy alcohol consumption disrupts lipid metabolism and hormonal balance including testosterone levels. However, studies doubt the relationship between moderate alcohol intake and sex hormone levels. Therefore, the aim of the present investigation was to establish the direct impact of chronic and moderate alcohol intake on cholesterol homeostasis and steroid hormone synthesis. Asymptomatic chronic and moderate alcoholics (n=12) without chronic liver disease and healthy volunteers (n=14) were selected for the study. Furthermore, effects of standardized water extract of Tinospora cordifolia (Willd) Mier. (Menispermaceae) (TCJ), a well reported anti-alcoholic herbal drug, on urinary steroids was studied. This study included four groups, i.e. a) healthy; b) healthy+TCJ; c) alcoholic; d) alcoholic+TCJ. The blood and urine samples from each group were collected on day 0 and 14 of the post-treatment with TCJ and analyzed. Alcoholic blood samples showed the significantly higher values of traditional biomarkers γ-GT and MCV along with cholesterol, LDL, TGL and urinary methylglucuronide compared to healthy. Qualitative analysis of steroids showed that moderate alcohol intake in a chronic manner increased the cholesterol synthesis and directed its flow toward C-21 steroids; shown by increased levels of corticosterone (2.456 fold) and cortisol (3.7 fold). Moreover, alcohol intake also increased the synthesis of estradiol and clearance rate of other steroids through the formation of glucuronides. Therefore, it decreased the synthesis and increased the clearance rate of testosterone (T) and androstenedione (A). Quantitative analysis confirmed decreased T/A ratio from 2.31 to 1.59 in plasma and 2.47 to 1.51 in urine samples of alcoholics. TCJ intervention normalized the levels of steroids and significantly improved the T:A ratio to 2.0 and 2.12 in plasma and urine. The study revealed that TCJ modulated lipid metabolism by inhibiting cholesterol and glucuronides synthesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The present study was performed to investigate the influence of the intake of selective oestrogen receptor modulators on the urinary endogenous steroids profile. For this purpose the circadian variability of luteinizing hormone, follicle-stimulating hormone, testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, androsterone and etiocholanolone were measured on eight subjects (four males and four females) by gas chromatography-mass spectrometry and chemiluminescent immunometric assay techniques before and after oral administration of multiple doses of either tamoxifen (80 mg for 2 days) or toremifene (120 mg for 2 days) or clomiphene (100 mg for 2 days). The individual baseline variability of the steroids studied was set up by collecting the urine samples every 3 h, for 3 days prior to the treatment; whereas the evaluation of the effects of the oral administration of multiple doses of selective oestrogen receptor modulators on the steroid urinary profile was assessed by collecting urine samples every three hours for at least five days from the first administration. The results of our measurements showed that, only in male subjects, the relative urinary concentrations of testosterone, epitestosterone and 4-androstenedione were significantly altered generally after the second day of drug administration. While no significant effects were recorded in both sexes on the luteinizing hormone, follicle-stimulating hormone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol and 5β-androstan-3α,17β-diol urinary levels and on testosterone/epitestosterone, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol and androsterone/etiocholanolone ratios., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3β-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3β-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17β-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17β-diol, and 5α-androst-1-ene-3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Oestrogens, such as oestrone (E(1)), 17β-oestradiol (E(2)), oestriol (E(3)) and their biologically active metabolites 2-methoxyoestrone (2-MeOE(1)), 2-hydroxyoestradiol (2-OHE(2)) 16-ketooestradiol (16-OE(2)), 16-epioestriol (16-epiE(3)), as well as testosterone (T) play an important role in physiological and pathological developmental processes during human development. We therefore aimed at developing an isotope dilution/bench top gas chromatography-mass spectrometry (ID/GC-MS) method, based on benchtop GC-MS, for the simultaneous determination ('profiling') of the above analytes in children. The method consisted of equilibration of urine (5 ml) with a cocktail containing stable isotope-labelled analogues of the analytes as internal standards ([2,4-(2)H(2)]E(1), [2,4,16,16-(2)H(4)]E(2), [2,4,17-(2)H(3)]E(3), [16,16,17-(2)H(3)]T, [1,4,16,16-(2)H(4)]2-MeOE(1), [1,4,16,16,17-(2)H(5)]2-OHE(2), [2,4,15,15,17-(2)H(5)]16-OE(2) and [2,4-(2)H(2)]16-epiE(3)). Then, solid-phase extraction (C(18) cartridges), enzymatic hydrolysis (sulphatase from Helix pomatia (type H-1)), re-extraction, purification by anion exchange chromatography and derivatisation to trimethylsilyl ethers followed. The samples were analysed by GC-MS (Agilent GC 6890N/5975MSD; fused silica capillary column 25 m × 0.2 mm i.d., film 0.10 μm). Calibration plots were linear and showed excellent reproducibility with coefficients of determination (r(2)) between 0.999 and 1.000. Intra- and inter-assay coefficients of variation (CV) were <2.21% for all quantified metabolites. Sensitivity was highest for 2-OHE(2) (0.25 pg per absolute injection: signal-to-noise ratio (S/N)=3) and lowest for 16-epiE(3) (2 pg per absolute injection: S/N=2.6), translating into corresponding urine sample analyte concentrations of 0.025 ng ml(-1) and 0.2 ng ml(-1), respectively. Accuracy - determined in a two-level spike experiment - showed relative errors ranging between 0.15% for 16-OE(2) and 11.63% for 2-OHE(2). Chromatography showed clear peak shapes for the components analysed. In summary, we describe a practical, sensitive and specific ID/GC-MS assay capable of profiling the above-mentioned steroids in human urine from childhood onwards., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Steroid profiling provides valuable information to detect doping with endogenous steroids. Apart from the traditionally monitored steroids, minor metabolites can play an important role to increase the specificity and efficiency of current detection methods. The applicability of several minor steroid metabolites was tested on administration studies with low doses of oral testosterone (T), T gel, dihydrotestosterone (DHT) gel and oral dehydroepiandrosterone (DHEA). The collected data for all monitored parameters were evaluated with the respective population based reference ranges. Besides the traditional markers T/E, T and DHT, minor metabolites 4-OH-Adion and 6α-OH-Adion were found as most sensitive metabolites to detect oral T administration. The most sensitive metabolites for the detection of DHEA were identified as 16α-OH-DHEA and 7β-OH-DHEA but longest detection up to three days (after oral administration of 50 mg) was obtained with non-specific 5β-steroids and its ratios. Steroids applied as a gel had longer effects on the metabolism but were generally not detectable with universal decision criteria. It can be concluded that population based reference ranges show limited overall performance in detecting misuse of small doses of natural androgens. Although some minor metabolites provide additional information for the oral testosterone and DHEA formulations, the topical administered steroids could not be detected for all volunteers using universal reference limits. Application of other population based threshold limits did not lead to longer detection times., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17beta-boldenone-sulphate. The analytical procedure included direct extraction-purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap). Decision limit (CCalpha) and detection capability (CCbeta) were respectively 0.2 microg L(-1) and 0.4 microg L(-1) on triple quadrupole and 0.1 microg L(-1) and 0.2 microg L(-1) on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17beta-Boldenone-sulphate was measurable up to 36h after oral administration of boldione, and 30 days after 17beta-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.

Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. A deletion polymorphism in the gene coding for UGT2B17 is strongly associated with reduced testosterone glucuronide (TG) levels in urine. Many of the individuals devoid of the gene would not reach a T/E ratio of 4.0 after testosterone intake. Future test programs will most likely shift from population based- to individual-based T/E cut-off ratios using Bayesian inference. A longitudinal analysis is dependent on an individual's true negative baseline T/E ratio. The aim was to investigate whether it is possible to increase the sensitivity and specificity of the T/E test by addition of UGT2B17 genotype information in a Bayesian framework. A single intramuscular dose of 500mg testosterone enanthate was given to 55 healthy male volunteers with either two, one or no allele (ins/ins, ins/del or del/del) of the UGT2B17 gene. Urinary excretion of TG and the T/E ratio was measured during 15 days. The Bayesian analysis was conducted to calculate the individual T/E cut-off ratio. When adding the genotype information, the program returned lower individual cut-off ratios in all del/del subjects increasing the sensitivity of the test considerably. It will be difficult, if not impossible, to discriminate between a true negative baseline T/E value and a false negative one without knowledge of the UGT2B17 genotype. UGT2B17 genotype information is crucial, both to decide which initial cut-off ratio to use for an individual, and for increasing the sensitivity of the Bayesian analysis.

The classical analytical method for detection of anabolic steroid abuse is gas chromatography followed by mass spectrometry (GC/MS). However, even molecules with a chemical structure typical for this class of substances, are sometimes not identified in routine screening by GC/MS when their precise chemical structure is still unknown. A supplementary approach to identify anabolic steroid abuse could be a structure-independent identification of anabolic steroids based on their biological activity. To test the suitability of such a system, we have analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer demonstrated that the yeast reporter gene system is able to detect a variety of different anabolic steroids and their metabolites with high specificity, including the so-called 'designer steroid' tetrahydrogestrinone. In contrast, other non-androgenic steroids, like glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to stimulate transactivation. To test whether the system would also allow the detection of androgens in urine, experiments with spiked urine samples were performed. The androgen reporter gene in yeast responds very sensitive to 5alpha-dihydrotestosterone (DHT), even at high urine concentrations. To examine whether the test system would also be able to detect anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples previously characterized by GCMS were analyzed with the reporter gene assay. Even when the concentration of the anabolic metabolites was comparatively low in some positive samples it was possible to identify the majority of positive samples by their biological activity. In conclusion, our results demonstrate that the yeast reporter gene system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid abusing athletes. Therefore we believe that this system can be developed towards a powerful (pre) screening tool for the established doping tests. The system is easy to handle, robust, cost-efficient and needs no high-tech equipment. But most importantly, a biological test system does not require knowledge of the chemical structure of androgenic substances and therefore suitable to detect previously unidentified substances, especially those of the class of so-called designer steroids.

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography-mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3beta-hydroxy-5alpha-androstane-4,17-dione (2) and 3alpha-hydroxy-5beta-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3xi,4xi-dihydroxy-5xi-androstan-17-one (4, 6-11) except 3alpha,4alpha-dihydroxy-5beta-androstan-17-one (5). Out of the 17beta-hydroxylated analogs 4-hydroxytestosterone (18), 3beta,17beta-dihydroxy-5alpha-androstan-4-one (19), 3alpha,17beta-dihydroxy-5beta-androstan-4-one (20), 5alpha-androstane-3beta,4beta,17beta-triol (21), 5alpha-androstane-3alpha,4beta,17beta-triol (26) and 5alpha-androstane-3alpha,4alpha,17beta-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation.

Boldenone is an androgenic steroid that improves the growth and food conversion in food producing animals. In most countries worldwide, this anabolic steroid is forbidden for meat production. Until recently, the control of its illegal use was based either on 17beta-boldenone or 17alpha-boldenone (its main metabolite in cattle) identification in edible tissues, hair, faeces or urine. Recent observations and data tend to demonstrate the natural occurrence (but not ubiquitous) in cattle of these steroids, making the analytical strategy of the control more complicated. We investigated the metabolism of boldenone in cattle after intramuscular and oral treatment of boldenone, boldenone esters and boldione. The central objective was to elucidate the structures of the main metabolites (phase I and phase II) in urine, with main objective to be further in position to compare boldenone urinary profiles of treated and non-treated animals. Nine metabolites have been identified, only four were present whatever the treatment and the administered boldenone source. Nevertheless, all of them have been detected at least once in non-treated animals which did not permit us to use them as biomarkers of an illegal treatment. At last, but not at least, all metabolites were found mainly glucuro-conjugated, and rarely sulfo-conjugated, with the only exception of 17beta-boldenone. Current investigations are showing the absence of 17beta-boldenone sulfoconjugate in non-treated animals; that would permit to distinguish non-treated from treated animals with boldione, boldenone and boldenone esters.

Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone-bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG-horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3',5,5'-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9+/-4.3 pg ml(-1) for boldenone and 120.2+/-8.2 pg ml(-1) for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC(50)=1.0+/-0.3 ng ml(-1)(n=3, N=3), R(2)=0.969, R.S.D.=9.6% for boldenone and 1.5+/-0.3 ng ml(-1), 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of alpha-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.

The metabolic effect of multiple oral testosterone undecanoate (TU) doses over 4 weeks was assessed in seven voluntary men. The protocol was designed to detect accumulation of the substance by choosing the appropriate spot urines collections time and to study the urinary clearance of the substance after weeks of treatment. Urines were analysed by a new GC/C/isotope ratio mass spectrometry (IRMS) method to establish the delta(13)C-values of testosterone metabolites (androsterone and etiocholanolone) together with an endogenous reference compound (16(5alpha)-androsten-3alpha-ol). The significant differences in inter-individual metabolism following TU intake was illustrated by large variations in delta(13)C-values of both T metabolites (maximum Deltadelta(13)C-values = 5.5 per thousand), as well as by very stable longitudinal T/E profiles and carbon isotopic ratios in the first hours following administration. According to T/E ratios and delta(13)C-values, the washout period after 80 mg TU intake was less than 48 h for all subjects and no accumulation phenomenon was observed upon chronic oral administration.

Pregnenolone (PREG) can potentially be abused by athletes to maintain an equilibration of the steroidal environment after sex steroids administrations. Five men volunteers orally ingested 50 mg PREG to determine optimal urinary markers for detection of this steroid. Our findings show that ingestion of PREG has no significant effects on the testosterone/epitestosterone (T/E) and testosterone/luteinizing hormone (T/LH) ratios, whereas variable changes on the carbon isotopic values of three T metabolites: androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (pregnanediol) have been observed. The difference between the carbon isotopic values (delta13C-values) of androstenol and pregnanediol is potentially the most reliable marker of exogenous PREG administration in males. For all subjects, the differences differ by 3.0 per thousand or more over a period of about 10 h and for both of them the detection window for positivity is extended over 40 h.

Excretory samples from adult female mice were collected non-invasively during development, estrous cycling, pregnancy, and postpartum. In initial studies, urinary measures were statistically more dynamic over days than were fecal measures; thus subsequent studies focused on urine. Higher 17beta-estradiol levels were present in isolated females than in those exposed to males. In cycling females, urinary 17beta-estradiol was more variable than were measures of testosterone or progesterone, showing peaks with an approximate 5-day periodicity. When urinary estradiol and progesterone were monitored in conjunction with vaginal smear cell counts, patterns were idiosyncratic; most females showed distinct peaks in urinary steroids, not in clear synchrony with vaginal cell cornification. Levels of progesterone rose markedly during the first 10 days of pregnancy, then declined before birth. Estradiol showed a substantial peak on days 7-8 of gestation in all females measured. Urinary testosterone was not dynamic during pregnancy, but rose in immediate prenatal and postpartum measures. During post-weaning, pre-pubertal development, urinary levels of progesterone remained constant but levels of estradiol rose substantially over time.

Androstenedione is a steroid hormone sold over-the-counter to individuals who expect that it will enhance strength and athletic performance. Endogenous androstenedione is the immediate precursor of testosterone. To evaluate the metabolism of oral androstenedione, we randomly assigned 37 healthy men to receive 0 (group 1), 100 mg (group 2), or 300 mg (group 3) of androstenedione in a single daily dose for 7 days. Eight-hour urines were collected 1 day before the start of androstenedione, and on days 1 and 7. Using gas chromatography-mass spectrometry, we measured excretion rates of glucuronide-conjugated epitestosterone, its putative precursor (E-precursor), and metabolites (EM-1 and EM-2), and we evaluated possible markers of androstenedione administration. Day 1 and 7 rates were not different: the means were averaged. The means (microg/h) for groups 1, 2, and 3, respectively were, for epitestosterone 2.27, 7.74, and 18.0; for E-precursor, 2.9, 2.0, and 1.5; for EM-1/E-precursor 0.31, 1.25, and 2.88; for EM-2/E-precursor 0.14, 0.15, and 1.15; for testosterone/epitestosterone (T/E) 1.1, 3.5, and 3.2. Epitestosterone, EM-1, and EM-2 excretion was greater in groups 2 and 3 versus group 1 (0.0001 < P < 0.03), as were EM-1/E-precursor, EM-2/E-precursor, and T/E. E-precursor excretion was lower in groups 2 (P = 0.08) and 3 (P = 0.047) versus group 1. Androstenedione increases excretion of epitestosterone and its two metabolites, while decreasing that of its precursor. Elevated ratios of EM-1- and EM-2/E-precursor, and the presence of 6alpha-hydroxyandrostenedione are androstenedione administration markers.

Orally administered testosterone undecanoate (TU), an anabolic, androgenic steroid, can potentially be abused by athletes. Indirect evidence for detecting oral TU intake could be deduced from the changes in steroid profile post-administration. Direct evidence could be obtained by detection of unchanged TU in plasma. To this end, both urinary and plasma steroid profiles of six healthy male subjects given a single oral dose of 120 mg of TU were studied by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The increased concentration of glucuronidated testosterone in plasma appears to be the most characteristic sign of oral TU intake. The testosterone glucuronide (TG)/nonconjugated testosterone (T) ratio, TG/17-hydroxyprogesterone (17OHP) ratio, and TG/luteinizing hormone (LH) ratio were observed to be significantly elevated above their basal levels for 10 h, 10 h, and 6 h, respectively. Urinary ratios of TG/epitestosterone glucuronide (EG) were found to be higher than the cut-off value of 6 for the period 4 approximately 8 h post-administration, but only in three subjects. One subject failed to respond with respect to all of the above-mentioned indirect markers, as TG was not significantly increased in either plasma or urine. Unchanged TU was directly detected in plasma of all six subjects from 1 approximately 1.5 h to 4 approximately 6 h after oral TU intake by GC/MS/MS, providing unequivocal proof of exogenous testosterone intake. Distinct and complementary markers for detecting oral TU intake could be obtained from plasma and urine, respectively.

Dehydroepiandrosterone (DHEA) replacement therapy as compensation for high age-related decline of DHEA and DHEA sulfate production is a matter of intense investigation, since many beneficial effects have been proven, or are suggested and expected. Therefore, DHEA abuse by athletes has been considered by the International Olympic Committee, which banned the substance recently. As DHEA for oral supplementation is easily available, we decided to investigate the effect on the urinary androgen profile of administration along this route of a single substitution dose of 50 mg. Quantitative analysis by gas chromatography-mass spectrometry with selected ion monitoring demonstrated that the drug was readily absorbed with 50 to 75% recovery of dosing after 24 h, and with glucuro- and sulfoconjugates of DHEA, androsterone, and etiocholanolone as the most abundant metabolites. In agreement with reported data found in blood, conversion of exogenous DHEA to the principal biologically active androgen, testosterone, was low but proven to be real by the administration of deuterium-labeled DHEA and the subsequent identification and quantification of deuterium-labeled testosterone. A concentration threshold of 300 micrograms/L of DHEA glucuronide is proposed for the screening of DHEA abuse in sport, but a single replacement dose can only be detected during 8 h. Such a short detection period is the consequence of considerable first-pass hepatic metabolism and also of the high interindividual variability of circulating and urinary DHEA and DHEA sulfate concentrations.

A mixture of 3H-testosteron (T) and 14C-4-androstene-3, 17-dione (A) was injected intravenously into 2 (I and II) rhesus monkeys (Macaca mulatta). A third monkey (III) was injected with 3H-T only. Urine and bile samples were collected at intervals for 6 hours following the injection. The excretion, conjugation and aglycone metabolites of the steroids injected were studied using these samples. Of the injected dose, animal I (male) excreted 32% 3H and 23% 14C in the bile and 30% 3H and 21% 14C in the urine in 6 hours. Animal II (female), however, had a comparatively higher biliary excretion (66% 3H, 40% 14 C), but a urinary excretion (18% 3H, 13% 14C) comparable to that of animals I and III. The averages in the bile of the 3 animals were: unconjugated compounds 3%, glucosiduronates 78%, sulfates 9%, sulfoglucosiduronates 5% and disulfates 3%; and in urine, 5% unconjugated, 92% glucosiduronates and 3% sulfates. The aglycones obtained following hydrolysis were separated gy chromatography on Lipidex 5000, further purified by thin layer and paper chromatography and identified by co-crystallization. The major matabolites from 3H-T were androsterone and 5beta-androstane-3alpha,17beta-diol, whereas that from 14C-A was androsterone. Other metabolites identified were: etiocholanolone (3beta-hydroxy-5-beta-androstan-17-one); T, epitestosterone (epi-T), (17alpha-hydroxy-4-androsten-3-one); epiandrosterone (3-beta-hydroxy-5alpha-androstan-17-one) and 5alpha-androstane-3alpha, 17beta-diol. The results indicate that while androgen metabolism in the rhesus monkey is similar to that of the baboon and human in conjugate and metabolite formation, the rate of excretion was significantly different, resembline more closely that of the baboon than the human.

Intravenous infusions of 14C-testosterone (Te) either alone or in combination with 3H-estradiol (E2) were given to five normal male subjects, twelve male patients on haemodialysis (HD) treatment and to one patient with a very restricted renal function. The elimination of radioactivity was measured in urine, HD fluid and faeces. Urinary excretion diminished with renal function. It was negligible at a creatinine clearance of less than one ml per minute. A quarter of both isotopes was eliminated by six HD treatments within three weeks. No difference was found in this respect between nephrectomized patients and those who were still in possession of their kidneys. The main excretion occurred in the stools. E2 metabolites, and to a lesser extent Te metabolites, appeared in the faeces within 24 hours, which might be explained by biliary excretion only. More 3H (E2 metabolites) than 14C (Te metabolites) was found in the faeces; more 14C than 3H found in HD fluid.

A method for the measurement of unconjugated testosterone and androstenedione in human urine is described. The method uses chromatographic separation followed by radioimmunoassay and has been examined for reliability. The mean 24-hour excretion of androstenedione by adult male subjects was 2.5 mug and of testosterone was 0.8 mug. For women, the mean excretion was 2.9 mug of androstenedione and 0.25 mug of testosterone. In pregnancy, androstenedione excretion was occasionally elevated above the normal range, but testosterone excretion was quite commonly increased. Some hirsute subjects exhibited an increase in androstenedione excretion, which was decreased by administration of dexamethasone. The results suggest that the amount of unconjugated testosterone in urine is not a direct reflection of the plasma free testosterone, but urinary androstenedione may be a useful reflection of plasma androstenedione levels.

Testosterone-1,2-3H was injected intravenously into a male dog with a bile fistula and bile and urine collected. The radioactivity was excreted preponderantly in bile (52% of the injected dose) in 6 hours; only 12% appeared in the urine. Methods to study the biliary metabolites of testosterone in this and other animals were developed. Satisfactory conjugate patterns were obtained by fractionation on DEAE-Sephadex A-25 columns using two different elution systems. In addition to an unchanged fraction, six different monoglucuronide fractions were separated. No other conjugates were isolated. Lipidex 5000 column chromatography, TLC and paper chromatography were used for the isolation and purification of aglycone metabolites, which were further identified by co-crystallization methods. The biliary metabolites of testosterone were epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one), etiocholanlone (3alpha-hydroxy-5beta-androstan-17-one), 5alpha-androstan-3beta, 17beta-diol, 5beta-androstan-3alpha, 17beta-diol and 5beta-androstan-3beta,17beta-diol.

A simple, reliable and rapid radioimmunoassay (RIA) for the determination of testosterone glucosiduronate (TG) in crude urine is described. Two protein-TG complexes were investigated in raising antibodies: a) Bovine serum albumin (BSA)-TG and b) human plasma Cohn's fraction IV-4 (CF)-TG. In rabbits, high titers of antibodies were obtained after the injection of CF-TG. The specificity of the antiserum was sufficiently high (cross reaction with free testosterone 27%, with 5alpha-dihydrotestosterone-glucosiduronate 20%). TG was estimated in small aliquots of male and female urine after evaporation overnight at 50 degrees C in order to eliminate interfering material. The intraassay coefficient of variation (CV) was found to be 6% and the interassay CV 11%. TB has been determined in 40 samples of urine simultaneously by "direct" RIA and by a "classical" RIA following hydrolysis with beta-glucuronidase. The coefficient of correlation was found to be 0.89. The mean excretion of TG in the urine of 26 healthy men amounted to 164+/-51 mug/24 hours with a range from 97 to 346 mug/24 hours. In a group of 16 women a mean urinary excretion of TG of 24+/-10 mug/24 hours was determined. The method allows a technician to assay 40 samples per day.