Stability Studies of Testosterone and Epitestosterone Glucuronides in Urine

Home , Epitestosterone

RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 858–864 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2387 Stability studies of testosterone and epitestosterone glucuronides in urine

C. Jime´nez1,2, R. de la Torre1,2, J. Segura1,2 and R. Ventura1,2* 1Unitat de Farmacologia, Institut Municipal d’Investigacio´ Me`dica, Barcelona, Spain 2CEXS, Universitat Pompeu Fabra, Barcelona, Spain Received 7 November 2005; Revised 9 January 2006; Accepted 9 January 2006

The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with b-glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was obtained from an excretion study after the administration of T to healthy volunteers. The homogeneity of the sample was veriﬁed before starting the stability study. The stability of TG and EG was evaluated at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 48C and S208C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 378C for 3 and 7 days. The effect of repeated freezing (at S208C) and thawing (at room temperature) was studied for up to three cycles. Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized urine samples stored at 4 and S208C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 378C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio was not affected. These results show the feasibility of preparing reference materials containing TG and EG to be used for quality control purposes. Copyright # 2006 John Wiley & Sons, Ltd.

Knowledge of the stability of drugs in biological fluids is transport and to establish recommended storage conditions critical for the proper interpretation of analytical results. for a given reference material. Since the analysis of biological samples for drug testing is not Most data available in the literature in the area of analytical usually performed immediately after sample collection, it is toxicology refer to the stability of drugs of abuse in biological very important to use optimal storage conditions for which specimens,6–10 mainly because their presence in these the drug has been demonstrated to be stable during the matrices may involve legal consequences for individuals. storage time. For this reason, stability assessment is The misuse of drugs as ergogenic aids by athletes constitutes considered a fundamental parameter for the validation of an offence in regulated sport practice and conceptually the bioanalytical methods.1 The stability of drugs also has to be control of these substances in biological fluids, known as evaluated in order to prepare samples for inter-comparison antidoping control, is similar to drugs of abuse testing. exercises or reference materials to ensure that differences in However, few stability studies of doping agents have been results between laboratories are not related to drug reported.11–13 instability or the lack of sample homogeneity. Participation In human doping control, testosterone (T) misuse is in inter-laboratory comparisons and the regular use of forbidden in sports by the International Olympic Committee reference materials are two of the requirements of testing (IOC) since 1983 and the World Anti-Doping Agency laboratories to enforce international quality standards such (WADA). The concentration ratio of T to E (T/E ratio) in as ISO/IEC 17025.2 In both cases, homogeneity and drug urine extracts obtained after hydrolysis of their glucuronic stability should be verified from production until end-use.3–5 acid conjugates is used to raise suspicion of T abuse by Additionally, stability data may also be useful in planning athletes. Samples with a T/E ratio higher than 4 have to be subjected to further investigation in order to determine *Correspondence to: R. Ventura, Unitat de Recerca en Farmaco- whether this finding is related to a physiological or logia, Institut Municipal d’Investigacio´ Me`dica (IMIM), Doctor Aiguader 80, 08003 Barcelona, Spain. pathological condition, or to a T misuse. At present, and E-mail: [email protected] due to the potential misuse of T metabolic precursors (i.e. Contract/grant sponsor: European Commission grant ALADIN androstenedione, dehydroepiandrosterone) other than T, a 2002: Analytical Laboratories for AntiDoping Control: Interna- tional Network for External Quality Assessment; contract/grant sample with a T/E ratio higher than 4 is subjected to follow- number: G7RT-CT-2000-05022. up studies and to isotope ratio (13C/12C) mass spectrometric

Copyright # 2006 John Wiley & Sons, Ltd. Stability studies of T and E glucuronides in urine 859 analysis. On the other hand, since the T/E ratio can be arti- phosphate solution and the pH was adjusted to 7 by adding ficially modified by the administration of epitestosterone (E), dihydrogen phosphate solution dropwise. a urinary concentration of E higher than 200 ng/mL has been Filters for clarification of urine and for sterilizing filtration established as indicative of its misuse as a masking agent.14 were supplied by Millipore. For clarification of the urine one Detection of doping with exogenous T has been a matter of cellulose reinforced disc membrane filter (90 mm diameter, concern for years.15–18 Estimating the T/E ratio is a critical 100 mm minimum thickness and 75% pore size) and two glass aspect of controlling the misuse of T. This has led to fiber filters (90% pore size, one of 90 mm diameter and discussions on the optimal method for estimating the T/E 380 mm minimum thickness and another of 75 mm diameter ratio, and means for ensuring that results are consistent and 1200 mm minimum thickness) were used. For the between laboratories. Previous work has provided evidence sterilizing filtration of the urine a membrane filter of of the variability in the estimation of the T/E ratios by GC/ modified polyvinylidene fluoride (0.22 mm pore size) was MS between laboratories.19 Results showed the need for used. antidoping laboratories to standardize methods for estimat- Cryotubes (4.5 mL) for storage at 808C were supplied by ing the urinary T/E ratio. For this purpose, the availability of Labclinics (Barcelona, Spain). Polypropylene tubes (5 mL) for reference materials containing T and E glucuronides with a storage at 208C and 48C were supplied by Vidra Foc T/E ratio higher than 4 has to be considered. (Barcelona, Spain). The effect of different factors, e.g. urine contamination by microorganisms, on the results of the T/E ratio has already Standard solutions been investigated.20 However, the stability of testosterone Separate stock standard solutions (1 mg/mL) of the steroids glucuronide (TG), epitestosterone glucuronide (EG) and the (T, E, T-d , and E-d ) were prepared using methanol as a T/E ratio in sterile samples (to be used as reference 3 3 solvent. The working standard solution of 100 mg/mL was materials) have never been studied. prepared by a 1:10 dilution of the stock standard solution The aim of this work has been the systematic study of the with methanol. A working standard solution of T and E with stability of TG, EG and the T/E ratio in sterilized urine a T/E ratio of 6 and a concentration of E of 1 ng/mL was used samples in order to evaluate the feasibility of preparing to prepare the calibration samples for quantification. All reference materials to be used for internal quality control or solutions were stored at 208C. to be distributed in inter-comparison exercises among antidoping laboratories. Analytical method m To 2.5 mL of urine samples, 20 L of the IS solution (T-d3, 9 mg/mL, and E-d , 1.5 mg/mL) were added. Urine samples EXPERIMENTAL 3 were applied to XAD-2 columns previously washed with Chemical and reagents methanol (2 mL) and water (2 mL). The column was washed Testosterone (17b-hydroxyandrost-4-en-3-one), epitesto- with water (2 mL) and analytes were eluted with 2 mL of a sterone (17 -hydroxyandrost-4-en-3-one), testosterone-d3 methanol. The solvent was evaporated and reconstituted 2 b ([16,16,17- H3]-17 -hydroxyandrost-4-en-3-one) and epites- with 1 mL of 0.2 M sodium phosphate buffer (pH 7). The 2 a tosterone-d3 ([16,16,17- H3]-17 -hydroxyandrost-4-en-3-one) mixture was extracted with 5 mL of tert-butyl methyl ether by used as internal standards (IS) were supplied by NARL rocking the mixture at 40 movements/min for 20 min. After (Sidney, Australia). centrifugation (3500 rpm), the urines were kept at 208C b-Glucuronidase from Escherichia coli (K12) was provided until the aqueous fraction was completely frozen. The by Roche Diagnostics GmbH (Mannheim, Germany). organic fraction (free fraction) was separated and evaporated Reagents for derivatization were N-methyl-N-trimethylsilyl- to dryness under a stream of nitrogen in a water bath at 408C. trifluoroacetamide (MSTFA) of GC grade, purchased from The residue was kept in a desiccator (maintained at 608Cina Macherey-Nagel (Du¨ ren, Germany), and ammonium iodide vacuum oven, using phosphorous pentoxide as desiccant) and 2-mercaptoethanol from Sigma Chemical Co. (St. Louis, for at least 30 min before derivatization (see below). MO, USA). Ultra-pure water was obtained using a Milli-Q After separation of the organic fraction (free fraction), the purification system (Millipore Ibe´rica, Barcelona, Spain). urine sample was allowed to reach room temperature before DetectabuseTM solid-phase extraction columns (XAD-2) were being submitted to hydrolysis of the glucuronide conjugates. purchased from Biochemical Diagnostics Inc. (New York, Enzymatic hydrolysis was performed by adding about 5000 NY, USA). Fishman units of b-glucuronidase (50 mL) to the aqueous Methanol was of high-performance liquid chromato- fraction and incubating the mixture in a water bath for 1 h 8 m graphic (HPLC) grade. Other reagents were of analytical- at 55 C. After incubation, 20 L of the IS solution (T-d3, m m reagent grade and purchased from Merck (Darmstadt, 9 g/mL, and E-d3, 1.5 g/mL) were added. The pH of the Germany). Potassium carbonate (5%) was prepared by aqueous solution was adjusted to around 10 with 250 mLofa dissolving 25 g of potassium carbonate in 500 mL of Milli-Q 5% potassium carbonate solution. The mixture was extracted water. A 0.2 M phosphate buffer, pH 7, was prepared by with 5 mL of n-pentane by rocking the mixture at 40 dissolving 14.2 g of sodium hydrogen phosphate in 500 mL of movements/min for 20 min. The organic fraction was Milli-Q water and 6.9 g of sodium dihydrogen phosphate in separated and evaporated to dryness under a stream of 250 mL of Milli-Q water. The sodium hydrogen phosphate nitrogen in a water bath at 408C. The residue was kept in a solution was mixed with 200 mL of the sodium dihydrogen desiccator for at least 30 min before derivatization.

Trimethylsilyl (TMS)-enol-TMS ether derivatives were compounds obtained after the analysis of spiked samples formed by redissolving the dry extracts in 50 mL of a mixture with the mean value of those obtained when the standards of MSTFA, ammonium iodide and 2-mercaptoethanol were added to extracted blank urine samples (representing (1000:2:6) and incubating them at 608C for 20 min. 100% of extraction recovery). Three concentrations were A HP 6890 series GC system equipped with a quadrupole studied: 24, 120 and 240 ng/mL for T, and 4, 20 and 40 ng/mL MS (HP 5973 mass selective detector) and an autosampler for E (n ¼ 4). (HP 7683 series injector) was used. Separation was per- Intra-assay stability, defined as the influence of time (i.e. formed using a methylsilicone fused-silica capillary column time expended in the plate of an autosampler before analysis) (17 m 0.2 mm i.d., 0.11 mm film thickness) (HP, Ultra-1). on the response (peak areas) of the analytes and the IS, was Helium was used as carrier gas at a flow rate of 0.8 mL/min studied with an analysis of the variance (ANOVA) test (measured at 1808C). The oven was set at an initial (a ¼ 5%). temperature of 1808C, and then the following rates were Intra-assay precision and accuracy were determined by the programmed: from 1808C to 2308Cat38C/min; then to 3108C analysis of three replicates of control urine samples at three at 408C/min, and held for 3 min, with a total run time of different concentrations (48, 128 and 176 ng/mL for T, and 6, 21.33 min. Samples were injected then in splitless mode. The 16 and 22 ng/mL for E), on the same day. Inter-assay injector and the interface temperatures were set at 2808C. The precision and accuracy were calculated by the analysis of the mass spectrometer was operated in electron ionization (EI) control samples on three different days. Precision was mode (70 eV) and in selected ion monitoring (SIM) acquisi- expressed as the relative standard deviation (RSD) of the tion mode. Three ions (m/z 432, 417 and 327) were monitored control sample concentrations calculated using the calibra- for T and E TMS derivatives and used as qualifying ions for tion curve, and accuracy was expressed as the relative error their identification (deviations in ion abundance ratios (ERR) of these concentrations. higher than 20% were not accepted). The ions used for quantification were m/z 432 for T and E and m/z 435 for T-d 3 Experimental design for stability testing and E-d3 TMS derivatives. Preparation of samples for stability testing Assay validation Urine containing TG and EG was obtained after adminis- The following parameters were evaluated during the tration by the intramuscular route of single doses (n ¼ 2) of validation of the analytical method: homoscedasticity/ 100 mg of testosterone (Testoviron Depot-100, Schering, heteroscedasticity, linearity, limits of detection and quanti- Madrid, Spain) to two healthy, male volunteers according fication, recovery, stability, and intra- and inter-assay preci- to the clinical protocol approved by the local ethical sion and accuracy. committee (CEIC-IMAS). Urine samples were collected at For the study of linearity, T and E calibration curves were 0–12 and 12–24 h intervals on days from 1 to 6. Overnight prepared by direct derivatization of the standard solution urine samples were collected on days from 7 to 10 and stored with a T/E ratio of 6 (1 mg/mL of E). For T, calibration at 208C until analysis. Urines with a pH value higher or samples were prepared to final concentrations of 24, 60, 120, equal to 6.5 were adjusted to pH 5.5 with 0.1 M hydrochloric 180 and 240 ng/mL and for E, at 4, 10, 20, 30 and 40 ng/mL. acid. After analysis of each fraction of urine collected, For the first assay of validation, the calibration samples were different urines were pooled to obtain the required TG and prepared and analyzed in quadruplicate. The peak area EG concentrations. A urine sample with a final concentration ratios between the analyte and the corresponding IS were of 171 ng/mL of TG and 19 ng/mL of EG (T/E ratio 9:1) was used for calculations. Dixon’s test (a ¼ 5%) was applied to prepared. T and E concentrations were chosen to obtain a detect outliers in the replicates at each concentration level. sample with a T/E ratio above the current criterion for The behavior of the variance over the calibration range reporting positive results established by the IOC (T/E ratio (homoscedasticity/heteroscedasticity) was evaluated by of 6) at the beginning of the stability study (2003). applying Levene’s test (a ¼ 5%) to the results obtained after The final pool of urine was preserved by the addition of analysis of the calibration curve in quadruplicate. The sodium azide (0.1% w/v) and clarified by filtration using analytical procedure is homoscedastic when the measure- three different filters (one cellulose-reinforced disc mem- ment variance is constant over the calibration range, i.e. it brane filter and two glass fiber filters of different pore size). does not change with concentration. Otherwise, it is The urine was filtered through a sterilizing membrane filter considered heteroscedastic. For the remaining validation of modified polyvinylidene fluoride (0.22 mm pore size) and assays, the calibration curve was prepared in duplicate. distributed in aliquots under sterile conditions in a laminar The goodness of fit of the linear model (i.e. comparison of the flow cabinet. Sterilized aliquots were placed in appropriate variance assigned to the lack of fit with that related to the containers for storage at the different temperatures intended random error) was demonstrated by an F test (a ¼ 5%). for stability testing. Some of the aliquots were subjected to The limits of detection (LODs) and quantification (LOQs) freeze-drying.13 were calculated to be 3.3 and 10 times the noise level, The potential adsorption of TG and EG onto the sterilizing respectively, for the analytical system employed. The noise filter was investigated by comparing the concentrations of level was set to be the equivalent of the standard deviation the analytes obtained before and after the filtration process. calculated for the lowest calibration concentration. About 500 mL of urine sample were filtered, and aliquots of Extraction recoveries of the analytes and the IS were 50 mL collected. One aliquot taken before the sterilizing calculated by comparison of the peak areas of the filtration (aliquot 0) and four aliquots taken at regular

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 858–864 Stability studies of T and E glucuronides in urine 861 intervals after the sterilizing filtration process (aliquots 2, 4, 7 of change) were determined for each storage condition. To and 10) were analyzed. further investigate any deviation from the reference value Five aliquots of the sample were taken at random at the suggesting sample degradation, a linear trend analysis of end of the production step and analyzed for homogeneity concentrations and ratios of concentrations to the reference testing. value (CX/C808C) at different storage times was also determined for each storage condition. Storage conditions The following stability conditions were studied according to RESULTS AND DISCUSSION a protocol previously described:21 Validation results – Long-term stability was evaluated in urine samples stored The analytical method used in this study to quantify TG and 8 8 at 4 C and 20 C for 1, 2, 4, 6, 9, 12, and 22 months. An EG in urine has been demonstrated to comply with the 8 additional stock of samples was stored at 80 C as refer- criteria for the validation of quantitative methods established ence condition for comparison purposes. according to the requirements of different international – Short-term stability was evaluated in urine samples stored organizations and regulatory authorities.1,22–27 8 at 37 C for 3 and 7 days. A stock of samples was stored at The heteroscedasticity of the analytical procedure was 8 20 C for comparison purposes. detected by Levene’s test, so a proportional weighted (1/ – The stability of the analytes after going through three concentration) least-squares regression analysis was selected 8 freeze ( 20 C)/thaw (room temperature) cycles was also as the calibration model. Determination coefficients (r2)upto evaluated. 0.990 in all calibrations were observed. The F test for comparison of variances was not significant, indicating adequate adjustment of the data to the proposed linear model Sample analysis over the calibration range. Five aliquots of the sample (five replicates) from each storage Limits of quantification of T and E are listed in Table 1. condition tested were analyzed at different time intervals Extraction recoveries for T and E were 68.2 6.4% and using the corresponding quantitative method described 73.8 6.3%, respectively. Recoveries of 65.6 5.8% and above. Calibration samples were prepared in duplicate. A 67.0 9.9% were also found for T-d and E-d , respectively. control sample was analyzed in triplicate in each analytical 3 3 No influence of injection time on the responses of the batch for internal quality control purposes. The replicates of analytes and the internal standards was found. each aliquot of sample were analyzed at random in the Precision and accuracy were determined by the analysis of analytical batch. three replicates of control urine samples at three different concentration levels. Results obtained for intra-assay preci- Calculations sion and accuracy, and inter-assay precision and accuracy, a ¼ Dixon’s test ( 5%) was applied to detect outliers in the are presented in Table 1. For T, precision and accuracy ¼ replicates (n 5) of each aliquot of sample. Homogeneity, obtained in intra- and inter-assay studies did not exceed 7% adsorption of the analytes on the sterilizing filter and stability and 14%, respectively and, for E, precision and accuracy did a ¼ were evaluated by applying an ANOVA test ( 5%) to the not exceed 6% and 18%, respectively. aliquots, once outliers of replicates (if any) had been excluded (SPSS for Windows, version 11.0). For stability testing, the ANOVA test was used to compare concentrations Stability data obtained at each storage condition (CX) with the reference The adsorption of TG and EG in the sterilizing filter was value (concentration of aliquots of sample stored at the evaluated. Data obtained for different drugs of abuse (e.g. reference condition). THC-COOH) in the context of external quality control In addition, to evaluate long-term stability, ratios of the activities in drugs of abuse testing28,29 indicate that some mean values of concentrations obtained at each test condition analytes are adsorbed on the filter used for the sterilizing

(CX) to the mean value of concentrations obtained at the ﬁltration of the sample. This phenomenon may alter the reference condition (C808C) were monitored over time. homogeneity of the different aliquots of a ﬁltrated sample Differences with respect to the reference value (percentages and, therefore, it has to be investigated for each analyte. The

Table 1. Results of the validation of the analytical method for the quantiﬁcation of T and E

Intra-assay Inter-assay

QC sample (ng/mL) Precision % Accuracy% Precision % Accuracy% LOQ (ng/mL)

Testosterone 48 2.3–7.2 3.6–6.9 7.2 5.8 5.3 128 2.7–3.8 5.7–14.1 4.5 9.0 176 1.2–2.8 1.0–8.3 4.3 4.8 Epitesosterone 6 2.3–5.6 1.5–10.7 5.7 7.0 1.1 16 1.5–6.1 1.1–7.8 5.8 3.9 22 3.6–5.4 7.9–18.2 6.7 12.0

Figure 1. Long-term stability of TG, EG and T/E ratio. Ratios between concentration

mean values (n ¼ 5) determined under the different storage conditions studied (Cx) and

the mean value of concentrations of sample aliquots stored at 808C(C808C). *, 208C; &,48C. verification procedure consisted of assessing the minimum term stability testing usually include the evaluation of volume of sample that had to be wasted at the beginning of lyophilized aliquots of the samples stored at 48C. Results the sterilizing filtration process in order to minimize obtained for the lyophilized samples containing TG and EG adsorption. Differences between T and E concentrations have recently been published.13 obtained before and after the filtration process were not The use of mass spectrometry for estimating the T/E ratio statistically significant (p < 0.05). These results indicated that in urine samples is mandatory in doping control.15,17,19,31 To TG and EG were not retained on the filtration system. Thus, a reduce the imprecision of the GC/MS assay, a sample stored waste of 100 mL of sample (dead volume of the filtration under reference conditions (808C) analyzed at the same equipment) was considered to be appropriate. time as the test conditions samples has been chosen to obtain The homogeneity of the sample prepared for stability the best information on actual analyte (in)stability.21 testing was also verified. Differences between concentration Much research has been done to investigate the factors that values of the five aliquots analyzed for homogeneity testing could influence the outcome of a T/E ratio analysis.30,32,33 In were not statistically significant (p < 0.05), indicating the this context, the effect of urine contamination by micro- homogeneity of the sample batch. organisms has been extensively investigated. Both T and E The protocol for stability testing was mainly focused on the are essentially present in urine as their glucuronides and evaluation of the suitability of urine samples after being there is evidence that some bacterial degradation in urine exposed to those temperature conditions most commonly could be responsible for enzymatic transformation of some encountered for their intended use as test samples in external steroids giving rise to the non-controlled hydrolysis of their quality assurance schemes, or as reference materials.21 As glucuronides.20,33 Sources of microorganisms may be well as the storage conditions studied in this work (liquid physiological or pathological conditions (e.g. urinary tract urine samples stored at 4 and 208C), the protocols for long- infection), contamination by use of non-sterilized material

Table 2. Results of T,E and the T/E ratio in the conjugated fraction and after spontaneous hydrolysis (free fraction). Mean values of concentration (n ¼ 5) and differences (percentages of change) to the reference condition (aliquots of sample stored at 208C)

208C378C–Day 3 378C–Day 7

Fraction Mean S.D. Mean SD %change Mean SD %change

Testosterone Conjugated 175.8 5.7 167.3 9.4 4.8 160.8 9.8 8.5 Epitestosterone 17.6 0.5 17.5 0.1 0.8 16.4 0.8 7.0 T/E ratio 9.9 0.03 9.8 0.3 0.3 9.8 0.2 0.2 Testosterone Free 6.8 0.1 7.5 0.8 9.2 9.1 0.5 33.3 Epitestosterone 1.1 0.2 1.0 0.04 10.5 1.4 0.2 19.3 T/E ratio 7.0 1.0 7.3 0.7 4.3 6.8 1.1 2.9

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 858–864 Stability studies of T and E glucuronides in urine 863

Figure 2. Freeze/thaw stability of TG, EG and T/E ratio. Mean values of concentrations (n ¼ 5) obtained for the aliquots of sample not subjected (F/T 0) and subjected to freeze/ thaw cycles (F/T 1, F/T 2 and F/T 3). during sample collection, or by manipulation or storage at accordance with those obtained by de la Torre et al.,20 who the laboratory. For this reason, urine for stability testing was reported deconjugation rates of TG and EG lower than 10% in sterilized by filtration and stored in sterile tubes. In addition, sterilized urine samples after incubation at 378C for 2 weeks. sodium azide was added as a preservative to prevent In spite of changes in T and E concentrations observed after 1 bacterial degradation of steroids and hydrolysis of steroid week of storage at 378C, the differences in T/E ratios were conjugates.15,34 The sterility of the urine sample was not statistically significant. monitored by using previously described methods.20,28,29 Evaluation of freeze/thaw stability was performed by The long-term stability data of T and E in the glucuro- using the concentration of the aliquot not subjected to freeze/ nide fraction and the T/E ratio showed non-statistically thaw cycles as a reference (F/T 0) (Fig. 2). The stability of TG, significant changes (p < 0.05) in concentration under any of EG and the T/E ratio was also demonstrated in urine the storage conditions tested (208C and 48C for up to samples subjected to three freeze/thaw cycles. Overall 22 months; Fig. 1). These changes did not exceed the intra- differences between cycles were found to be not statistically assay precision of the method for any of the analytes significant (p < 0.05) in all cases. investigated, demonstrating good stability over the entire In summary, data obtained in the different stability studies period of study. To avoid any misinterpretation of the T/E carried out in this work demonstrate the stability of T and E ratio, the concentration of free T and E in urine was also glucuronides and the T/E ratio in preserved sterilized urine determined. A significant amount of free steroids present in samples stored at 48C and 208C for 22 months, and after the urine (>5% of the total) could be interpreted as an going through three freeze/thaw cycles. T and E glucuronide indication of bacterial degradation of glucuronides. The conjugates are spontaneously hydrolyzed to a low extent presence of T and E in the free fraction was almost negligible. after 7 days of storage at 378C; however, the T/E ratio Although the stability of steroid conjugates in urine has not remained constant. been studied extensively, other small studies have also These results show the feasibility of preparing reference shown the stability of TG in non-preserved urine samples materials containing TG and EG to be used for quality control stored at 208C or lower temperatures for up to 1 year.15,16 purposes. Urine samples with a certain T/E ratio may be The short-term stability was evaluated by comparing the of particular interest in antidoping control for their use as concentrations obtained at 378C with the concentration of Certified Reference Materials (CRM) to test the accuracy of aliquots of the sample stored at 208C. In Table 2, T and E their methods in an analogous way to those provided by the concentrations, and the T/E ratio in the glucuronide and free National Institute of Standards and Technology (NIST) for fractions, are presented under different storage conditions. drugs of abuse testing. Statistically significant (p < 0.05) decreases in the conjugated steroid concentrations were obtained after 7 days of storage Acknowledgements due to the hydrolysis of the glucuronide conjugates during The present work was supported by European Commission storage at 378C. This was corroborated by a relevant increase grant ALADIN 2002: Analytical Laboratories for AntiDoping in concentrations of free T and E. Our results are in Control: International Network for External Quality Assessment

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