steroids 73 (2008) available at journal homepage:

steroids 73 (2008) 751 759 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/steroids Gas chromatography mass spectrometry method for the analysis of 19-nor-4-androstenediol and metabolites in human plasma: Application to pharmacokinetic studies after oral administration of a prohormone supplement Susana Torrado a,b, Jordi Segura a,c,magí Farré a,d, Rosa Ventura a,c, a Pharmacology Research Unit, Institut Municipal d Investigació Mèdica, IMIM, Dr. Aiguader 88, 08003 Barcelona, Spain b Facultat de Química, Universitat de Barcelona, UB, Martí i Franquès 1-11, 08028 Barcelona, Spain c Department of Experimental and Health Sciences CEXS, Universitat Pompeu Fabra, UPF, Dr. Aiguader 88, 08003 Barcelona, Spain d Universitat Autònoma de Barcelona, UAB, E08193 Barcelona, Spain article info abstract Article history: Received 5 July 2007 Received in revised form 21 February 2008 Accepted 24 February 2008 Published on line 8 March 2008 Keywords: Norandrostenediol Plasma GC/MS Prohormones 19-Nor-4-androstenediol is a prohormone of nandrolone. Both substances are included in the WADA list of prohibited classes of substances. The aim of this study is to determine the plasma levels of 19-nor-4-androstenediol and its metabolites after oral administration of a nutritional supplement containing the drug. Two capsules of Norandrodiol Select 0 were orally administered to six healthy male volunteers. Plasma samples were collected up to 24 h. Samples were extracted to obtain free and glucuronoconjugated metabolic fractions. Trimethylsilyl derivatives of both fractions were analyzed by gas chromatography coupled to mass spectrometry (GC MS). The method was validated to determine linearity, extraction recovery, limit of detection and quantification, intra- and inter-day precision and accuracy. After administration of 19-nor-4-androstenediol, the main metabolites detected were norandrosterone and noretiocholanolone, mainly in the glucuronide fraction. Nandrolone, norandrostenedione and 19-nor-4-androstenediol were also detected at lower concentrations. 2008 Elsevier Inc. All rights reserved. 1. Introduction Nandrolone (ND) is an anabolic androgenic steroid that promotes muscle growth similar to that of the endogenous male hormone, testosterone. For this reason, it is considered a performance-enhancing drug and it was included in the list of banned substances in sport [1]. ND is one of the most detected drugs in doping control in sports [2]. ND is metabolised to two major metabolites, 19-norandrosterone (NA) and 19- noretiocholanolone (NE), which are then excreted in the urine mainly as conjugates with glucuronic acid. Illegal use of ND is detected by the presence of NA in urine at a concentration greater than 2 ng ml 1 [3]. The limit was set because it has been shown that NA can be present in urine of males and females as an endogenous substance [4 11]. In addition, formation of NA from endogenous androsterone in stored urines Corresponding author at: Unitat de Recerca en Farmacologia, Institut Municipal d InvestigacióMèdica, Dr. Aiguader 88, 08003 Barcelona, Spain. Tel.: +34 93 310471; fax: +34 93 310499. E-mail address: rventura@imim.es (R. Ventura). 0039-128X/$ see front matter 2008 Elsevier Inc. All rights reserved. doi:10.101/j.steroids.2008.02.012

752 steroids 73 (2008) 751 759 with bacterial contamination has recently been demonstrated [12]. Besides, consumption of boar meat has been reported as another unintentional source for the presence of NA in urine after [13,14]; also, several studies have shown that the consumption of nutritional supplements contaminated with prohormones can result in a positive doping case [15 19]. Prohormones of ND, such as 19-norandrostenedione (NONE), 19-nor-5-androstenediol and 19-nor-4-androstenediol (NOL), were synthesised as precursors of ND and can be administered via oral, transdermal or sublingual routes. They were introduced on the US market as a result of the Dietary Supplement and Health Education Act of 1994 [20]. They were considered nutritional supplements and sold over-thecounter. However, they were included in the list of anabolic steroids forbidden in sports in 1999 by the International Olympic Committee (IOC), and they remain on the current WADA list. Their illegal use is demonstrated by the detection of NA in urine. Legislative classification has recently changed in the US and currently they are among the controlled substances (Anabolic Steroid Control Act of 2004 [21]). Introduction of prohormones on the market of nutritional supplements and their availability without restriction during one decade may be another reason for the high number of adverse analytical findings of NA in doping control. As mentioned, oral intake of contaminated nutritional supplements has been shown to result in positive doping tests. These findings led to investigate the composition of a wide variety of nutritional supplements, including prohormones, for the presence of steroids not mentioned in the label, and this was confirmed in some of the supplements tested [22 27]. Many studies have been published dealing with the topic of prohormones of ND from a doping point of view. Most of these studies have focused attention on the risk of failing a doping test after consumption of nutritional supplements contaminated with prohormones. Pharmacokinetic studies are necessary to know the behaviour of prohormones in the body in order to assess their capacity to enhance performance in sports or to be harmful for the health, where their actual conversion to the active hormone plays a crucial role. Some authors have focused their attention in the physiological effects [28 31] or metabolites in urine [32 3] after administration of prohormones itself or as a nutritional supplement [37,38]. A study has been recently published dealing with the conversion of these hormone precursors to their active form ND in the body [39]. Only ND was measured in free and total fraction, whereas the rest of the metabolites were detected in the total fraction. Due to the doses administered, very low concentrations of free ND were detected after oral administration of NOL, while higher concentrations were detected after sublingual administration. The aim of the present study was to determine plasma concentrations of NOL and its metabolites in the free and glucuronide metabolic fractions after oral administration of NOL at the doses recommended by the manufacturer of the nutritional supplement. The analytical method to quantify free and glucuronide metabolic fractions has been optimized and validated. 2. Experimental 2.1. Chemical and reagents 19-Norandrosterone (3 -hydroxy-5 -estran-17-one, NA), 19-noretiocholanolone (3 -hydroxy-5 -estran-17-one, NE), methyltestosterone (17 -methyl-4-androsten-17 -ol-3-one) and 19-norandrosterone-d 4 ([2,2,4,4-2 H 3 ]-3 -hydroxy-5 estran-17-one) were supplied by NMI, Reference Materials (Pymble, Australia). 17 -Nandrolone (4-estren-17 -ol-3-one, ND) was supplied by Sigma (St. Louis, Mo, USA), 19-nor-4- androstenediol (4-estren-3,17 -diol, NOL) was purchased from Steraloids Inc. (Newport Rhode Island, USA) and 19- norandrostenedione (4-estren-3,17-one, NONE) was supplied by Research Plus Inc. (Manasquan, NJ, USA). Methanol (HPLC grade), di-sodium hydrogen phosphate, sodium hydrogen phosphate, tert-butylmethylether, ammonium iodide, 2-mercaptoethanol, potassium carbonate and acetone (all analytical grade) were purchased from Merck (Darmstadt, Germany). -Glucuronidase from Escherichia coli K12 was provided by Roche Diagnostics GmbH (Mannheim, Germany). N-Methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) was purchased from Macherey-Nagel (Düren, Germany). Ultra-pure water was obtained using a Milli-Q purification system (Millipore Ibérica, Barcelona, Spain). Detectabuse TM solid-phase extraction columns (XAD-2) were purchased from Biochemical Diagnostics Inc. (New York, NY, USA). Blank human plasma was kindly provided by the Emergency Service Laboratory of Hospital del Mar (Barcelona, Spain). 2.2. Standard solutions Separate stock standard solutions (1 mg ml 1, in free base form) of each analyte were prepared using methanol as solvent. Successive 1:10 dilutions with methanol of the 1mgmL 1 stock solutions were made to prepare working solutions of 100, 10, 1 and 0.1 gml 1. All solutions were stored at 20 C. 2.3. Sample preparation 2.3.1. Free fraction extraction To 1 ml plasma samples, the solutions of internal standards (20 and 25 L of the 1 gml 1 19-norandrosterone-d 4 and methyltestosterone solutions, respectively) were added and, then, samples were adjusted to ph 7 with 1.5 ml of sodium phosphate buffer (0.2 M, ph 7). The mixture was extracted with 5 ml of tert-butylmethylether by rocking it at 40 movements/min for 20 min. After centrifugation (2000 g, 5 min), organic layers were separated and evaporated to dryness under a stream of nitrogen in a water bath at 40 C. The extracts were kept in desiccators containing P 2 O 5 and maintained under vacuum for at least min before derivatization. 2.3.2. Glucuronide fraction extraction Plasma samples (1 ml) were extracted for the free fraction as described before. The organic layers were discarded and the small volume of organic solvent still present on top of the

steroids 73 (2008) 751 759 753 Table 1 Diagnostic ions used for identification and quantification, and absolute and relative retention times (RT and RRT) for the bis-o-tms derivatives of the analytes under study, and limits of quantification (LOQ) and extraction recoveries obtained in the free and conjugated fractions Diagnostic ions RT RRT Free fraction Conjugated fraction LOQ (ng/ml) % recovery (mean; S.D.) LOQ (ng/ml) % recovery (mean; S.D.) a Norandrosterone-d 4 409, 424, 319 8.9 0.59 NA 405, 420, 315 8.9 0.0 0.9 100.; 9.8 1.0 52.8; 11.0 NE 405, 420, 315 9.9 0. 1.4 94.7; 4.2 0.5 51.9; 11.3 NOL 420, 405, 3 11.1 0.74 0.4 95.4; 1.2 0.4 78.5; 15.8 NONE 41, 401, 311 11.9 0.80 0.1 92.2; 5.8 0.4 40.4; 13.5 ND 418, 403, 194 12.4 0.82 0.5 84.9; 4.7 0.4 4.7; 17.1 Methyltestosterone a 44, 1 14.9 1.00 a Internal standards. aqueous phase was evaporated under a nitrogen stream. To the aqueous phase, the solutions of internal standards (20 and 25 L of the 1 gml 1 19-norandrosterone-d 4 and methyltestosterone solutions, respectively) were added and, then, the samples were applied to XAD-2 cartridges previously conditioned with methanol (2 ml) and water (2 ml). The cartridges were washed with water (2 ml) and analytes were eluted with 2 ml of methanol. The organic extracts were evaporated to dryness under nitrogen stream in a bath at 50 C and reconstituted with 1 ml of sodium phosphate buffer (0.2 M, ph 7). Enzymatic hydrolysis was performed by adding L of glucuronidase from E. coli and incubating the mixture at 55 C for 1 h. Then, samples were allowed to reach room temperature and 250 L of5%k 2 CO 3 solution was added. The mixture was extracted with 5 ml of tert-butylmethylether by rocking it at 40 movements/min for 20 min. After centrifugation (2000 g, 5 min), organic layers were separated and evaporated to dryness under a stream of nitrogen in a water bath at 40 C. The extracts were kept in desiccators containing P 2 O 5 and maintained under vacuum for at least min before derivatization. 2.3.3. Derivatization The dry extracts were derivatized by adding 50 L ofamixture of MSTFA:NH 4 I:2-mercaptoethanol (1000:2:, v/w/v) and incubating at 0 C for 20 min. After incubation, the derivatized extracts were transferred to injection vials and analyzed by GC/MS. 2.4. Chromatographic conditions An Agilent (Palo Alto, CA, USA) 890N GC system equipped with an autosampler (Agilent 783 series injector) and connected to a quadrupole MS (Agilent 5973 mass selective detector) was used. Separation was performed using a 100% methylsilicone fused silica capillary column [HP Ultra-1; 17 m 0.2 mm i.d.; film thickness, 0.11 m]. Helium was used as carrier gas with a flow rate of 0.8 ml min 1 (measured at 180 C). The oven was set at an initial temperature of 180 C, and then the following rates were programmed: at 3 Cmin 1 from 180 to 2 C, then at 40 Cmin 1 to 310 C and held for 3 min. 2 L of samples was injected with split mode (10:1). The injector and the detector were set at 280 C. The mass spectrometer was operated in electron ionization (EI) mode (70 ev) and in selected ion monitoring acquisition mode. Three diagnostic ions were monitored for each compound of interest (Table 1). 2.5. Assay validation The following parameters were evaluated during the validation of the analytical method for the free and glucuronide fraction: selectivity, homoscedasticity/heteroscedasticity, linearity, limits of detection (LOD) and quantification (LOQ), extraction recovery, and intra- and inter-assay precision and accuracy. The validation protocol included four different assays. In the first assay of validation, the calibration curves were prepared with five concentration levels in quadruplicate. For the remaining validation assays, calibration curves with five concentration levels in duplicate and quality control (QC) samples containing two different concentrations of each analyte in triplicate were prepared. Calibration samples were prepared daily by adding appropriate volumes of working solutions to blank human plasma. QC samples were prepared in batches by adding appropriate volumes of working solutions to blank human plasma; each batch was distributed into aliquots of 1 ml and they were kept at 20 C until processing. Peak area ratios between the selected ion of the analyte and the selected ion of the ISTD 19-norandrosterone-d 4 (after area correction due to the contribution of isotopic ion of NA) were used for calculations. The Dixon s test ( = 5%) was applied to detect outliers in the replicates at each concentration level in the first assay and then the behaviour of the variance over the calibration range (homoscedasticity/heteroscedasticity of the procedure) was evaluated by applying the Levene test ( = 5%). In the rest of validation assays, to demonstrate the goodness of fit using the linear model, and an F test ( = 5%) was applied to compare the variance attributable to lack of fit with that due to random error. Selectivity and specificity were studied by the analysis of five different human plasma samples. The presence of any interfering substance at the retention time of the compounds of interest and the internal standards was verified. The LOD and LOQ were calculated to be 3.3 and 10 times the noise level, respectively. The noise level was set to be the equivalent of the

754 steroids 73 (2008) 751 759 standard deviation calculated for the lowest calibration level in the first assay. Extraction recoveries of each analyte were calculated by comparison of peak areas of the compounds obtained after the analysis of samples spiked with 20 ng ml 1 of each compound (n = 4) with the mean value of those obtained when the standards were added to extracted blank human plasma and subjected to the derivatization process (representing 100% of extraction recovery). Precision and accuracy were determined by comparing the results of QC samples analyzed the same day and in three different days. Precision was expressed as the relative standard deviation (R.S.D.) of the control samples concentration calculated using the calibration curve and accuracy was expressed as the relative error (%) of the estimated concentration. 2.. Nutritional supplement analysis The content of NOL in the nutritional supplement Norandrodiol Select 0 (Ergopharm, Illinois, USA) was verified. A portion of approximately 35 mg (n = ) of the dietary supplement was weighed and dissolved in 10 ml of methanol. The mixture was shaken for 5 min in an ultrasonic bath and excipients not dissolved were allowed to settle. Then, a 1:10 dilution was made with 1 ml of the supernatant. 50 L of this solution was transferred to a glass tube and was evaporated to dryness under nitrogen stream. The dry extracts were reconstituted in 0 L of mobile phase (n-hexane:isopropanol, 9:4, v/v), and they were injected into the HPLC system. Calibrations standards with 2, 5 and 10 g of NOL in 0 L of mobile phase were prepared in duplicates. The calibration curve was constructed by plotting the peak areas of the standards versus the amounts. Analysis was performed using a Series II 1090L liquid chromatograph equipped with a diode array detector (Hewlett-Packard, Palo Alto, CA, USA). The instrument was linked to an HP ChemStation (Hewlett-Packard). The column was a Dimethylaminopropyl EC Nucleosil 100-5 N (CH 3 ) 2 (4.0 mm 250 mm 5 m) protected by a pre-column CC 8/4 Nucleosil 100-5 N (CH 3 ) 2. The mobile phase was a mixture of n-hexane (A) and isopropanol (B), and the following gradient elution was used: 4% B for 15 min, to 80% B in 4 min, 80% B for 3 min, to 4% B in 2 min, and 4% B for min. The flow rate was 1 ml min 1 and the system was operated at ambient temperature. The UV detector was set at 210 nm. The injection volume was 20 L. 2.7. Clinical protocol and sample collection Administration studies of NOL to six healthy male volunteers were performed. The mean age of the participants was 27.5 years (range, 21 37 years), the mean weight was 74.3 kg (range, 2.3 81.8 kg), and the mean height was 177.9 cm (range, 10 187 cm). All participants gave written informed consent before inclusion in the study. The study was conducted in accordance with the Declaration of Helsinki, and following a protocol approved by the local Ethical Committee (CEIC- IMAS 2002/1519), and authorized by the Agencia Española del Medicamento y Productos Sanitarios (reference AEMPS 04-0012) of the Spanish Ministry of Health. Two capsules of Norandrodiol Select 0 were orally administered. 15 ml of blood samples were collected in tubes with EDTA immediately before administration and at 0.5, 1, 2, 4, 8 and 24 h after the supplement administration. Samples were immediately centrifuged and the plasma obtained was transferred to polyethylene tubes and stored at 20 C until analysis. 2.8. Pharmacokinetic and statistics evaluation Pharmacokinetic parameters and statistic evaluation were calculated using WinNonlin software (WinNonlin Professional, Version 4.1, Pharsight). The parameters t max (time of peak plasma concentration), C max (peak plasma metabolite concentration), and AUC 0 24 (area under plasma concentration time curve from 0 to 24 h) were calculated for each metabolite. AUC 0 24 was determined by the trapezoidal rule method. 3. Results 3.1. Validation of the analytical method The analytical method was optimized for the quantification of NOL and its metabolites in human plasma in two metabolic fractions, free and glucuronide fraction. Two solvents (n-pentane and tert-butylmethylether) were compared for the liquid liquid extractions. The extraction recoveries were higher with tert-butylmethylether for all the compounds in both fractions, as shown in Fig. 1. The hydrolysis efficiency with -glucuronidase from E. coli was studied. Hydrolysis efficiency for NA and NE glucuronides were 1.7 ± 2.7% and 82.9 ± 4.2%, respectively (n = 4). Fig. 1 Comparison of extraction recoveries with n-pentane (light grey) and tert-butylmethylether (dark grey) in the free and conjugated fraction.

steroids 73 (2008) 751 759 755 The method was validated to determine linearity, extraction recovery, LOD and LOQ, intra- and inter-day precisions and accuracies. The heteroscedasticity of the procedure was detected by Levene s test, so a proportional weighted (1/concentration) least-squares regression analysis was selected as the calibration model. Determination coefficients (r 2 ) higher than 0.99 in all calibrations were obtained. The F test for comparison of variances was not significant, indicating adequate adjustment of the data to the proposed linear model over the calibration range. Limits of quantification and extraction recoveries of both fractions are listed in Table 1. No interfering compounds were observed after analysis of five blank human plasmas obtained from different donors. Results obtained for intra- and inter-assay precision and accuracy in the free fraction are presented in Table 2. Ascan be observed, values did not exceed 10% in most cases and they were always lower than 20%. Results obtained for intra- and inter-assay precision and accuracy in the glucuronide fraction for the different analytes are presented in Table 3. Values lower than 15% were obtained for all compounds at the studied concentrations. 3.2. Supplement analysis The content of NOL per capsule was experimentally quantified. The amount found was 90.7 ± 11.2 mg, representing a 0.5% of the theoretical content of 150 mg per capsule announced in the label. The nutritional supplement was also analyzed by GC/MS in the conditions used for plasma samples with SIM and scan acquisition and the presence of other prohormones, such as NONE, was not detected. 3.3. Pharmacokinetic study Individual concentrations of NOL and metabolites detected in the glucuronide and free fractions are presented in Figs. 2 and 3, respectively. The median value for t max, and the mean values for C max and AUC 0 24 calculated for each metabolite are presented in Table 4. As can be seen, NOL and its metabolites were mainly detected in the glucuronide fraction. The main metabolites detected in the glucuronide fraction were NA and NE. Both metabolites were still present 24 h after administration. ND was detected in glucuronide form in five volunteers for dif- Table 2 Intra- and inter-assay precisions and accuracies in the free fraction QC (ng/ml) Intra-assay Inter-assay Assay n Precision (R.S.D., %) Accuracy (% relative error) n Precision (R.S.D., %) Accuracy (% relative error) NA 1 3 0.8 2.7 9 5.0 7.5 2 3 4.2 12.3 3 3 1.7 7.5 1 3 1.2 1.3 9 4. 10.2 2 3 1.4 9.3 3 3 1.4 5.1 NE 1 3 8.3 8.7 9 4.9 9.0 2 3 4.1 10.5 3 3 1. 7.8 1 3 4.9. 9 3.5.9 2 3 1.8 9.8 3 3 1.1 4.3 NONE 1 3 20.5 14.2 9 17.1 12.3 2 3 10.4 13.2 3 3 10. 9.5 1 3 3.4 17.5 9 8.7 11.7 2 3 3. 13.3 3 3.1 4.3 ND 3 15 1 3 19.1 15.5 9 15.7 18.2 2 3 8.8 22.3 3 3 10.7 1.7 1 3. 5.7 9 8.5.7 2 3 3.0 5.7 3 3 5.7 8.7 NOL 1 3 20. 14.2 9 12.4 7. 2 3 5.5 3. 3 3 4.2 4.8 1 3 4.2 1.9 9 3.1 15.3 2 3 2.4 1.5 3 3 1.3 12.

75 steroids 73 (2008) 751 759 Table 3 Intra- and inter-assay precisions and accuracies in the conjugated fraction QC (ng/ml) Intra-assay Inter-assay Assay n Precision (R.S.D., %) Accuracy (% relative error) n Precision (R.S.D., %) Accuracy (% relative error) NA 0 0 1 3 0.5 2.7 9 3.3 4.2 2 3 0. 8.0 3 3 3.1 1.9 1 3 0.7 4.3 9 5.9 5.0 2 3 1.1 2.8 3 3 9.7 7.9 NE 0 0 1 3 3.7 5.3 9 4.8 5.7 2 3 1.9 8.8 3 3 3.9 2.9 1 3 1.2 1.4 9 5.2 5. 2 3 0. 7.7 3 3 8.9 7.8 ND 1 3 7.0 7.5 9.4 8.8 2 3 4. 14.7 3 3 3.4 4.1 1 3 3.0 8.4 9.4 8.1 2 3 4.5 8.0 3 3 10.7 7.9 NOL 1 3 4.1 8.8 9 4. 7.1 2 3.0.0 3 3 4.2.4 1 3 5.2 11.2 9 9.5 8.5 2 3 4.2 4.4 3 3 10.7 9.9 ferent times, and for two of them, it was still present 24 h after administration at concentrations near the LOQ. Very low concentrations of NOL could be detected in the glucuronide fraction of all volunteers for different times as can be seen in Fig. 2. In the free fraction, ND, NA and NONE were detected at low concentrations (Fig. 3). NOL was not detected. NONE was the main metabolite detected in the free fraction. It was detected in five volunteers, in three of them up to 8 h and in two of them it was still present after 24 h. Free ND could be quantified in three volunteers. Free NA was detected in samples of all volunteers, however free NE could only be found in one volunteer in samples collected 1 and 2 h after administration at very low concentrations (1.5 and 2.8 ng ml 1, respectively). Fig. 2 Plasma concentrations of norandrostenediol and metabolites in the glucuronide fraction for each volunteer.

steroids 73 (2008) 751 759 757 Table 4 t max (median values and range) and C max and AUC 0 24 (mean values and 95% confidence interval) for each metabolite t max (h) C max (ng/ml) AUC 0 24 (ng h/ml) n a Median Range n Mean CI 95% n Mean CI 95% Glucuronide fraction NOL 5 2 0.5 4 0.9 0.2 1.5 5.7 1.3 10.0 ND 2 0.5 4 17.1 0.8 33.4 7.4 2.1 132. NA 2 1 2 3.0 19.2 43.8 2003.0 1312.9 293.0 NE 2 0.5 24 107.8 145.5 504.8 1139.2 5.9 112.5 Free fraction ND 3 2 1 2 0.4 0 0.8 1.0 0 2. NA 2 0.5 4 7.7 2.5 12.9 44.9 20.1 9.8 NONE 5 1 0.5 2 13.2 0.0 58. 0 117.7 a Number of volunteers where the parameter was calculated. Fig. 3 Plasma concentrations of norandrostenedione, nandrolone and norandrosterone in the free fraction for each volunteer. 4. Discussion The validated method allows the quantification of NOL and its metabolites in human plasma in two metabolic fractions, free and glucuronide fractions. Two different solvents (npentane and tert-butylmethylether) were compared for the liquid liquid extractions. In agreement with results obtained with other steroid metabolites [40,41], the extraction with n- pentane was found to be more selective but provided lower extraction yields than tert-butylmethylether for all the compounds, especially for the most polar compounds like NOL. As NOL is the parent compound in the present study and it is expected to be detected at low concentrations, tertbutylmethylether was selected as extraction solvent in both fractions. Other attempts have been described using two liquid liquid extraction steps with different solvents, but the LOQ obtained were higher than those obtained in the present study [39]. The hydrolysis efficiency was evaluated for some of the compounds and results confirmed the adequacy of the conditions used. The analytical method used in this study to quantify the free and conjugated fraction of NOL and its metabolites in plasma has demonstrated to comply with the criteria for the validation of quantitative methods established according to the requirements of different international organizations and regulatory authorities [42 44]. The content of NOL in the nutritional supplement was verified and results revealed the presence of the drug at a concentration representing the 0.5% of the theoretical content. No other compounds were detected. The discrepancy between the actual and the theoretical content of drugs in nutritional supplements has been widely described [22 27]. The posology recommended by the manufacturer was used for the clinical study, and two capsules of NOL were administered to the healthy volunteers. The parent compound could only be found in the conjugated fraction of some volunteers at very low concentrations. The main metabolites detected were NA, NE and ND, present mainly in the glucuronide fraction. Oxidation to ND with further transformation to NA and NE and conjugation with glucuronic acid has an important role in the metabolic clearance of NOL. Other metabolic routes, such as conjugation with sulphates, have not been studied in the present study. Besides, low concentrations of NONE were detected in the free fraction. NONE was not present in the nutritional supplement, thus it could only be explained as a result of a metabolic transformation from NOL. The median values of t max for the different metabolites ranged from 1 to 2 h post-administration, although individual

758 steroids 73 (2008) 751 759 values showed great variability, indicating high interindividual variability in the formation and elimination of the metabolites. Shorter t max have been found in our study compared with data presented in a previous study where total plasma concentrations of NOL metabolites were evaluated [39], although direct evaluation of the data is not possible because mean values of t max are presented in the former study. On the other hand, the higher values for C max and AUC 0 24 obtained in the present study compared with data previously reported [39], are related to the higher doses of the drug administered in the present study. The pharmacokinetic profile of NE glucuronide has different behaviour than that of NA glucuronide (Fig. 2). For NA glucuronide, after an initial peak in concentrations between 1 and 4 h, plasma concentrations show a slow decrease until 24 h. In contrast, plasma concentrations of NE glucuronide increase from 8 to 24 h post-administration after an initial decline. This observation might suggest the existence of two different metabolic pathways with different rates of NE formation, that could be related with two different isoforms of 5 -reductase enzyme. The increase in plasma concentrations of NE glucuronide from 8 to 24 h is accompanied with an increase in urinary concentrations of NE glucuronide with respect to NA glucuronide from 8 to 9 h post-administration as described by the authors [45]. These results have important implication in antidoping control in order to define the best marker for the detection of the administration of ND precursors. Quantification of plasma levels of ND in the free fraction is relevant to evaluate the pharmacological effect of the steroid. It has been reported that the minimum effective concentration of the drug that will result in a pharmacological effect is in the range of 0.3 1.2 ng ml 1 [39]. After ingestion of two capsules of norandrodiol (equivalent to a dose of 180 mg of NOL, approximately), concentrations in the range of 0.5 1.1 ng ml 1 were detected only in three volunteers for up to 2 4 h. Thus, by increasing the dose, higher concentrations of free ND are obtained for some of the volunteers compared to previous data [39]. However, the concentrations of free ND obtained, even at high doses of oral NOL, are much lower than those reported using sublingual route of administration [39]. Hence, it can be concluded that after oral administration of NOL it is difficult to reach effective levels of free ND. Acknowledgements Helpful comments on pharmacokinetic parameters by Dr. Marcel.lí Carbó are gratefully appreciated. 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