This article reports the results obtained from the investigation of the influence of miconazole administration on the physiological fluctuation of the markers of the steroid profile included in the "steroidal module" of the Athlete Biological Passport. Urines collected from male Caucasian subjects before, during, and after either systemic (i.e., oral and buccal) or topical (i.e., dermal) treatment with miconazole were analyzed according to validated procedures based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) (to determine the markers of the steroid profile) or liquid chromatography coupled to MS/MS (LC-MS/MS) (to determine miconazole urinary levels). The results indicate that only after systemic administration, the markers of the steroid profile were significantly altered. After oral and buccal administration, we have registered (i) a significant increase of the 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol ratio and (ii) a significant decrease of the concentration of androsterone, etiocholanolone, 5β-androstane-3α,17β-diol, and 5α-androstane-3α,17β-diol and of the androsterone/etiocholanolone, androsterone/testosterone, and 5α-androstane-3α,17β-diol/epitestosterone ratios. Limited effects were instead measured after dermal intake. Indeed, the levels of miconazole after systemic administration were in the range of 0.1-12.5 μg/ml, whereas after dermal administration were below the limit of quantification (50 ng/ml). Significant alteration started to be registered at concentrations of miconazole higher than 0.5 μg/ml. These findings were primarily explained by the ability of miconazole in altering the kinetic/efficacy of deglucuronidation of the endogenous steroids by the enzyme β-glucuronidase during the sample preparation process. The increase of both incubation time and amount of β-glucuronidase was demonstrated to be effective countermeasures in the presence of miconazole to reduce the risk of uncorrected interpretation of the results.

Keywords: antidoping analysis; athletes biological passport; confounding factors; miconazole; steroidal module.

The Paternò-Büchi reaction is a common organic reaction in which a carbonyl radical formed by exposure to UV radiation reacts with an alkene to form an oxetane ring. Recent analytical applications of this reaction have included the determination of C=C bond position in lipid fatty acyl tails using tandem mass spectrometry. Our group has recently investigated methods for structurally modifying steroid isomers to improve their identification and resolution using ion mobility spectrometry. Herein we report the first application of the Paternò-Büchi reaction to form steroid oxetanes using a simple, low-cost, and high efficiency method with a low pressure mercury lamp. This methodology is performed on several endogenous steroid isomers, resulting in unique ion mobility spectra that provide a unique fingerprint for each. These fingerprint spectra can add confidence in identification of those com-pounds, especially in complex biological matrices. Testosterone and epitestosterone, an epimer pair commonly interrogated in a number of applications such as for their use as performance enhancing drugs, displayed one and three unique ion mobility peaks, respectively. These spectra and their measured collision cross sections (CCS) allow for unambiguous differentiation of these and several other steroid isomer groups analyzed in this work. Finally, multiple anabolic androgenic steroids (AAS) prohibited by the World Anti-Doping Agency (WADA) were tested with this method and resulted in unique CCS for their PB reaction products This approach can offer improved confidence in their identification, as well as for many other banned substances.

The knowledge of the biotransformation of compounds prohibited by the World Anti-Doping Agency is of high concern as doping analyses are mostly based on the detection of metabolites instead of the parent compounds abused by athletes. While the self-administration of doping-relevant compounds is from an ethical point of view a rather problematic method to investigate metabolism, the usage of cell culture systems allows for studies on biotransformation in vitro. Five cell culture models with different tissue origin (liver, ovary, skin, kidney, testis) were comparatively incubated with testosterone and epitestosterone as well as with the synthetic testosterone derivatives 17α-methyltestosterone and 4-chlorotestosterone to investigate the impact of synthetic modifications on phase I metabolic pathways. Cell culture supernatants were analysed by high-performance liquid chromatography-tandem mass spectrometry. All cell lines possessed the default steroid phase I biotransformation reactions. The highest conversion rate was observed in ovarian (BG-1) and liver cells (HepG2). For BG-1 and skin cells (HaCaT), the 5α-reductase products 5α-dihydrotestosterone (for both) and 5α-androstane-3α/β,17β-diol (for BG-1 solely) were found to be prevailing after testosterone incubation. In kidney (COS-1) and HepG2 cells, the 17β-hydroxysteroid dehydrogenase activity was predominant as supported by the observation that the 17α-OH (epitestosterone) and the methyl group (17α-methyltestosterone) impeded the conversion rate in these cell lines. In conclusion, future work should extend the characterization of the BG-1 and HepG2 cells on phase II metabolic pathways to examine whether they are suitable models for the generation of metabolite reference collections comparable to those obtained by human excretion studies.

Keywords: LC-MS/MS; cell culture; doping; metabolism; testosterone.

At the Swedish national forensic toxicology laboratory, a measured testosterone/epitestosterone (T/E) ratio ≥12 together with testosterone/luteinizing hormone (T/LH) in urine >400 nmol/IU is considered as a proof of exogenous testosterone administration. However, according to the rules of the World Anti-Doping Agency (WADA), samples with T/E ratio >4 are considered suspicious and shall be further analyzed by gas chromatography-combustion-isotope-ratio mass spectrometry (GC-C-IRMS) to confirm the origin of testosterone and its metabolites. The aim of this study was to investigate the possibility of false negative results and to estimate the frequency of negative results using the current criteria for detection of abuse of testosterone in forensic investigations. Urine and serum samples were collected by the police at suspected infringement of the doping law in Sweden. Fifty-eight male subjects were included in the study. Urinary testosterone was determined by GC-MS, serum testosterone and LH - by immunoassay. The origin of testosterone and its metabolites was confirmed by means of GC-C-IRMS. Twenty-six of the 57 analyzed subjects tested positive for exogenous testosterone using the criteria T/E ≥12 combined with T/LH >400 nmol/IU. The IRMS analyses confirmed 47 positives, thus 21 were considered false negatives. Negative predictive value was 32% (95% CI: 16-50%) and sensitivity 55%. No false positive subjects were found. The number of false negative cases using the current criteria for the detection of testosterone abuse and hence the low sensitivity indicates a need to discuss introduction of new strategies in forensic doping investigations.

Keywords: Doping; GC-C-IRMS; LH; T/E; Testosterone.

Epitestosterone (17 alpha-hydroxy-4-androsten-3-one) inhibited competitively 17 alpha-hydroxylation of pregnenolone and subsequent C17,20-side chain cleavage of resulting 17 alpha-hydroxypregnenolone using microsomal preparations from the human testis. The inhibition constants for 17 alpha-hydroxylase and C17,20-lyase with 5-ene-precursors of C21-steroids were 96 and 12.4 mumol/l, respectively.

From Day 268 of gestation till two days after parturition blood samples were collected from the jugular vein of 5 cows with normal and 4 cows with flumethasone induced parturition and centrifuged immediately after sampling. In another experiment blood samples were taken from the jugular and uterine vein of 3 cows during the last week before normal parturition. The dehydroepiandrosterone (DHA) and epitestosterone (ET) concentrations in plasma were measured using radioimmunoassay. In the control group the DHA concentration was in the range of 2 to 5 nmol/l, ET levels varied from 4 to 8 nmol/l before term. In both groups, there was an increase in DHA concentrations during the last periparturient period whereas ET levels increased only after flumethasone induced parturition. The concentrations of both androgens declined after the expulsion of the placenta and they were higher in the uterine vein than in the jugular vein. It is concluded, that both androgens are secreted by the bovine placenta and that the delta 5-pathway of steroidogenesis is active in vivo.

Careful epoxidation of the delta 16-olefins 3 and 4 yielded 16 alpha,17 alpha-epoxides 5 and 6 which were reduced by lithium aluminium hydride, oxidized, and dehydrated to 17 alpha-hydroxycholest-4-en-3-one 20, i.e., an epitestosterone homolog containing a well tolerated alkyl group at position 17. Under catalysis of acids, epoxide 5 was rearranged to delta 13-16 alpha-alcohol 10. Less careful epoxidation of delta 16-olefin 4 with excess of peroxy acid led to products of double epoxidation (i.e., epoxidation, rearrangement, and another oxidation) 7 and 12. Structures of products of rearrangement were studied mainly by NMR spectroscopy.

4-chloro-androst-4-ene-3,17-dione (CLAD) and 4-chlorotestosterone (clostebol, beta-CLT or CLT) were made immunogenic by coupling to protein carriers via the 3 and 17 positions, respectively. These immunogens were used to elicit polyclonal and monoclonal antibodies to CLAD and to clostebol. The antibodies were characterized in an enzyme immunoassay for sensitivity and specificity. Polyclonal antisera generated through position 17 reacted preferentially with 4-chlorotestosterone-17-acetate (clostebol acetate, CLTA), 4-chloro-epitestosterone (epi-clostebol, 17alpha-clostebol, 17alpha-CLT), and clostebol, whereas polyclonal antisera generated through the 3 position almost did not react with these derivatives. Interestingly, the monoclonal antibody generated through the 3 position recognized (35%) epi-clostebol. These results suggest that polyclonal antisera generated through the 17 position have a broad specificity profile and can be used to analyze by immunoassay methods urinary metabolites of clostebol acetate and thereby detect the illegal use of clostebol acetate in livestock farming.

A recommended confirmatory procedure for detecting 5 alpha-dihydrotestosterone (DHT) doping in male athletes proposed the use of the urinary concentration ratio of DHT to epitestosterone (EpiT) as the primary marker and those of 5 alpha-androstane-3 alpha,17 beta-diol (5 alpha-Adiol) to EpiT, luteinizing hormone (LH), and 5 beta-androstane-3 alpha,17 beta-diol (5 beta-Adiol) as secondary markers. Here we investigate the effects on these markers of intramuscular administration of DHT heptanoate (250 mg) to six healthy men. Within 24 h of administration all four markers greatly exceeded the published discrimination limits, remaining above these limits for 10-14 days. All ratios returned to basal values by day 28. In contrast to results after percutaneous administration, 5 beta-Adiol excretion decreased, probably as a consequence of greater suppression of testicular steroidogenesis. Results were largely in agreement with those obtained after percutaneous administration, although probably augmented by the larger dose and the different route of delivery.

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.

With regard to previous finding of an inhibitory activity of furosemide on 11 beta-hydroxysteroid dehydrogenase, 16 other commonly used diuretics have been tested as to their ability to inhibit rat renal, and in four instances also testicular 11 beta-hydroxysteroid dehydrogenase, using glycerrhetinic acid as a standard. In addition, epitestosterone has been tested as well, with respect to its recently demonstrated inhibitory activity on several other enzymes of androgen biosynthesis. Besides corticosterone, 11 beta-hydroxy-4-androstene-3,17-dione has been used as a substrate. Of all drugs studied, quinapril, dihydralazin, trandolapril, metipamid, methyldopa, betaxolol only appeared to be weak inhibitors of 11 beta-hydroxysteroid dehydrogenase, with an inhibitory activity 10-28% of that of glycyrrhetinic acid. Using corticosterone as a substrate, epitestosterone displayed a weak inhibitory activity with Ki 850, 1200 nmol/l and Vmax 2420, 3900 nmol/l.min for renal and testicular enzyme, respectively. In contrast to kidneys, the testicular 11 beta-hydroxysteroid dehydrogenase accepted also 11 beta-hydroxy-4-androstene-3,17-dione as a substrate, which could be inhibited by epitestosterone (Ki 1490 nmol/l, Vmax 1150 nmol/l.min). The results represent further evidence for different substrate specificity of renal and testicular 11 beta-hydroxysteroid dehydrogenase.

The ability of androstane and androstene neurosteroids with modifications at C-17, C-5, and C-3 (compounds <b>1</b>-<b>9</b>) to influence the functional activity of inhibitory glycine and γ-aminobutyric acid (GABA) receptors was estimated. The glycine- and GABA-induced chloride current (<i>I</i>_Gly and <i>I</i>_GABA) were measured in isolated pyramidal neurons of the rat hippocampus and isolated rat cerebellar Purkinje cells, correspondingly, using the patch-clamp technique. Our results demonstrate that all the nine neurosteroids display similar biological activity, namely, they strongly inhibited <i>I</i>_Gly and weakly inhibited <i>I</i>_GABA. The threshold concentration of neurosteroids inducing effects on <i>I</i>_Gly was 0.1 μM, and for effects on <i>I</i>_GABA was 10-50 μM. Moreover, our compounds accelerated desensitization of the <i>I</i>_Gly with the IC₅₀ values varying from 0.12 to 0.49 μM and decreased the peak amplitude with IC₅₀ values varying from 16 to 22 μM. Interestingly, our study revealed that only compounds <b>4</b> (epiandrosterone) and <b>8</b> (dehydroepiandrosterone) were able to cause a significant change in <i>I</i>_GABA in 10 μM concentration. Moreover, compounds <b>3</b> (testosterone), <b>5</b> (<em>epitestosterone</em>), <b>6</b> (dihydroandrostenedione), and <b>9</b> (etiocholanedione) did not modulate <i>I</i>_GABA up to the concentration of 50 μM. Thus, we conclude that compounds <b>3</b>, <b>5</b>, <b>6</b>, and <b>9</b> may be identified as selective modulators of <i>I</i>_Gly. Our results offer new avenues of investigation in the field of drug-like selective modulators of <i>I</i>_Gly.

Blood samples collected from eight Braunvieh cows between the sixth and eighth month of gestation were allowed to stand with and without anticoagulant at 20 degrees C and 0 degrees C for different time periods. In these samples the degree of in vitro conversion of gestagens, androgens and estrogens was investigated. The concentrations were measured by radioimmunoassay. After 24 h at 20 degrees C, the levels of pregnenolone, progesterone, 17alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone and estrone decreased to 62, 29, 25, 10, 34 and 44%, respectively, of the initial value and those of 17alpha, 20beta-dihydroxyprogesterone, epitestosterone and estradiol-17alpha increased to 385, 800 and 852%, respectively. The conversion was slower in clotted blood. The concentrations of testosterone and estradiol-17beta were consistent over the 24 h period. There was no marked decrease of the steroid concentration after 24 h of incubation of whole blood at 0 degrees C and of plasma at 20 degrees C. After the addition of (3)H-steroids, conversion could be demonstrated by thin-layer chromatography and autoradiography. These results demonstrate that all investigated hormones except testosterone and estradiol-17beta were metabolized by bovine blood cells.

Cannabis, or marijuana, the most commonly used illicit drug in the world, has been shown to be responsible for suppressing the production and secretion of androgens, particularly testosterone. However, despite such findings in animals, the chronic effects of marijuana use on human endocrine systems have proved to be inconsistent. Here, we investigated the reference ranges of urinary levels of testosterone (T) and epitestosterone (E) as well as their metabolic ratio of T/E in a Korean male population (n=337), which would enable an evaluation of abnormal changes in steroid metabolism induced by habitually administered cannabis. The T/E ratio was significantly decreased in the marijuana group (n=18), while the urinary testosterone concentrations were also tended to decrease. This study is the first to provide data for the reference values of two urinary androgens and T/E values among control Korean males, and, furthermore, suggests that the T/E ratio, though not testosterone levels, might be used to understand the suppression of human male gonadal function affected by smoking marijuana.

The action of the endogenous steroid epitestosterone administered to castrated male mice substituted with testosterone propionate is manifested by reduced weight increments and a reduced relative weight of their seminal vesicles and kidneys. Epitestosterone in vitro displaces androgens from their bond with receptors in cytosol from rat prostates and markedly inhibits the testosterone transformation to the more effective androgen dihydrotestosterone. Epitestosterone can be thus defined as a true endogenous antiandrogen; to its action at the receptor level a potent inhibitory effect on 5 alpha-reductase must be added.

To detect doping with pseudo-endogenous anabolic steroids in sports, a urinary steroid profile with glucuronidated plus unconjugated androgens is used. In addition to analyze androgen glucuronide metabolites it can be of interest to also include sulfate metabolites in the urinary steroid profile. The combined ratios of epitestosterone sulfate/epitestosterone glucuronide to the ratios of testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) has previously been investigated as a complementary biomarker for testosterone doping. In this restudy the aim was to evaluate this biomarker in a larger study sample population. A single dose of 500 mg testosterone enanthate was administered to 54 healthy male volunteers. Urine was collected prior to (day 0) administration and throughout fifteen days and analyzed for the sulfate and glucuronide conjugates of testosterone and epitestosterone. The results show that the combined ratio increased to a larger extent than the traditional T/E ratio in all subjects. This increase was independent on UGT2B17 gene polymorphism. Moreover, a delayed peak of the combined ratio was observed in ~ 60 % of the participants. The results confirm that complementary analyses of the sulfate metabolites may be useful approach to detect testosterone doping in men.

Keywords: steroid glucuronides; steroid sulfates; testosterone; urinary steroid profile.

An isocratic HPLC method for the determination with screening purposes of anabolic androgenic steroids (AASs: fluoxymesterone, boldenone, nortestosterone, metandrostenolone, norethindrone, methyltestosterone and bolasterone), used as growth promoting agents, in finishing pig feed samples has been developed and validated. The separation was achieved by using a reversed-phase Chromolith RP-18e column at controlled temperature, UV-detection at 245nm and epitestosterone as internal standard. The method development involved optimization of different aqueous-organic mobile phases using methanol or acetonitrile as organic modifiers, flow-rate and temperature. The optimum separation for these compounds was achieved at 40 degrees C using ultrapure water:acetonitrile (71:29, v/v) as mobile phase and 3mLmin(-1) flow-rate, allowing the separation of AASs with baseline resolution in about 15min. The optimized method was applied to the analysis of AASs in finishing pig feed samples. Prior to HPLC, sample preparation procedure was used by leaching using acetonitrile, saponification in a basic medium and solid-phase extraction using polymeric Abselut Nexus cartridges. Method validation has been carried out according to the European Commission Decision 2002/657/EC. The extraction efficiencies, decision limits (CCalpha) and detection capabilities (CCbeta) for these compounds were in the range 83-96%, 27-37 and 32-47microgkg(-1) range, respectively. The within-laboratory reproducibility at 1, 1.5 and 2 CCbeta concentration levels were smaller than 13, 10 and 8%, respectively. Finally, the proposed method was successfully applied to nine different kinds of animal feed.

BACKGROUND

Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment.

METHODS

Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification.

RESULTS

The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R²>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases.

CONCLUSIONS

Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

The solubility of testosterone, boldenone, androstenone, etiocholanolone, and epitestosterone are measured in pure supercritical CO2. Testosterone exhibited the highest solubility in supercritical CO2. The solubility of all steroids except epitestosterone increased by one order of magnitude with increasing pressure from 100 to 400 atm. Epitestosterone had the lowest solubility in supercritical CO2 and its solubility was not affected by pressure. The extraction efficiency of steroids from an aqueous saline environment exceeded 95%. Because of the partial solubility of water in supercritical CO2, the addition of a moisture trap after the aqueous vessel is necessary to prevent the plugging and deterioration of the gas chromatographic (GC) column. It is demonstrated that on-line supercritical fluid extraction-GC-mass spectrometry is feasible for the quantitative extraction and analysis of steroids from both saline and urine solutions. However, it is determined that the adsorbent vessel filled with Hydromatrix is not sufficient to trap all the moisture, and after 3 to 4 extractions, the GC column efficiency lowered.

The main purpose of this study was to investigate if the specificity of the binding of testosterone to plasma proteins could be defined as a preferential binding of this steroid over epitestosterone. The amount of testosterone that is specifically bound was calculated using the formula: (see article) concentration, where "Ri" is the ratio (14C) testosterone: (3H) epitestosterone in plasma prior to centrifugation, "Ru" is the isotope ratio in the protein-free supernatant obtained after ultracentrifugation (149,000 x g, at 0 C, for 18 h) and "T concentration" is the testosterone concentration in plasma resulting from addition of (14C) testosterone, the endogenous steroids having been removed by preliminary charcoal extraction. The theoretical separation of the binding sites for testosterone into two populations, one non-specific with no preference for testosterone over epitestosterone, and another with absolute specificity for testosterone over its physiologically inactive stereoisomer, proved to be useful. Ovalbumin was found to be an example for non-specific, non-preferential binding. Determination of the ratio (14C) testosterone: (3H) epitestosterone in the successive fractions of various ultracentrifuged preparations showed a small but significant preference for (14C) testosterone by human and bovine serum albumin, while alpha1-acid glycoprotein had a preference for (3H) epitestosterone. Saturation curves showed at least two components: the first one, presumably corresponding to TeBG, had a higher affinity and lower capacity. This binding capacity can be accurately determined by extrapolation to the ordinate or the second component, a straight line corresponding to a binding of somewhat lower affinity and much larger capacity.

A 37-year-old woman presented with a history of secondary amenorrhoea and hirsutism for 4 years. She had elevated serum levels of testosterone and dihydrotestosterone, and decreased serum levels of sex hormone binding globulin and oestradiol. Almost daily use of a testosterone-containing ointment in the vulvar region for 6 years was disclosed as the cause of the hyperandrogenism. Serum testosterone, testosterone excretion rate in urine and testosterone/epitestosterone ratio in urine were determined at fixed intervals 24 h before and 48 h after application of the testosterone-containing ointment. There was a rapid increase in serum testosterone, with a peak level after 4-6 h. The testosterone excretion rate and the testosterone/epitestosterone ratio in urine peaked after 2-4 h. After 48 h the serum testosterone level was still about twice the basal value. The testosterone/epitestosterone level was over the 'doping limit' of 6 for 28 h. We conclude that determination of the testosterone/epitestosterone ratio in urine would have disclosed exogenous testosterone administration in this patient. We recommend this test for patients in whom exogenous testosterone administration is suspected.

Urinary testosterone and epitestosterone were determined in 90 normal healthy women and in 90 women with idiopathic hirsutism, both groups aged between 16 and 46 years. Testosterone and epitestosterone excretion values were above the normal range in 27 of the 90 hirsute women (30%), and these 27 women had much more prominent hair growth than the others. When these results were statistically analysed according to the age groups or for all ages as a whole, they were found to be highly significant (P less than 0.0005). Therefore, it is concluded that the estimation of urinary testosterone and epitestosterone could be meaningfully applied to study the androgen status of hirsute women.