Comparison of sulfo-conjugated and gluco-conjugated urinary metabolites for detection of methenolone misuse in doping control by LC-HRMS, GC-MS and GC-HRMS.

Methenolone (17ß-hydroxy-1-methyl-5α-androst-1-en-3-one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1-methylene-5α-androstan-3α-ol-17-one) excreted conjugated with glucuronic acid using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for the parent molecule, after hydrolysis with ß-glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC-high resolution (HR)MS and the estimation of the long-term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC-HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti-doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC-HRMS using electrospray ionization in negative mode searching for [M-H](-) ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1-methylene-5α-androstan-3α-ol-17-one, 3z-hydroxy-1ß-methyl-5α-androstan-17-one and 16ß-hydroxy-1-methyl-5α-androst-1-ene-3,17-dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z-hydroxy-1ß-methyl-5α-androstan-17-one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC-HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC-MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction.

Estimating measurement uncertainty in quantitative methods not based on chromatography for doping control purposes.

Estimation of measurement uncertainty (MU) for quantitative results is a requirement of ISO/IEC17025. This concept is well established for chromatographic methods in doping control and forensic analysis. For non-chromatographic methods, however, very few practical methodologies have been published. In this paper, the applicability of a top-down model, established for estimating uncertainty in chromatography, was evaluated for two other methodologies with different sets of raw data as a starting point. The first case study involves the estimation of MU for the determination of haematological parameters. In this case, a large data set of quality control material and proficiency testing results was available to establish MU. The second case study involves the estimation of MU for the recently approved method for the determination of human growth hormone misuse. In this case the amount of data available to establish MU was limited to results from method validation and a basic set of analysis data. In both cases a methodology based upon long-term bias, long-term imprecision and--eventually--a correction for standard impurity is proposed. The proposed methodology can be regarded as a dynamic procedure, which allows re-evaluation of MU on a regular basis. Finally, a concept for the verification and evaluation of MU estimations using proficiency testing results is proposed.

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin.

BACKGROUND: The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. METHODS: The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. RESULTS: The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 degrees C or storage at -20 degrees C. CONCLUSIONS: The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.

Stabilization of human urine doping control samples: II. microbial degradation of steroids.

The transportation of urine samples, collected for doping control analysis, does not always meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency (WADA) accredited laboratories. Because sample collection is not conducted under sterile conditions, microbial activity may cause changes to the endogenous steroid profiles of samples. In the current work, funded by WADA, a chemical mixture consisting of antibiotics, antimycotic substances and protease inhibitors was applied in urine aliquots fortified with conjugated and deuterated steroids and inoculated with nine representative microorganisms. Aliquots with and without the chemical mixture were incubated at 37 degrees C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at -20 degrees C as reference. Microbial growth was assessed immediately after inoculation and at the end of the incubation period. Variations in pH and specific gravity values were recorded. Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical stabilization mixture to urine samples inhibited microorganism growth and prevented steroid degradation at 37 degrees C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 degrees C.

Two-step silylation procedure for the unified analysis of 190 doping control substances in human urine samples by GC-MS.

BACKGROUND: While a number of different derivatization procedures for screening GC-MS analysis of prohibited substances are followed by doping control laboratories, a unified derivatization procedure for the GC-MS analysis of 190 different doping agents was developed. RESULTS: Following preliminary experiments, a two-step derivatization procedure was selected. The evaluation of various silylation parameters, such as reagent composition, reaction time, reaction temperature, catalysts and microwave oven reaction time, for this procedure was carried out. CONCLUSION: The suitability of the developed procedure was demonstrated through application on urine samples at concentration levels of the minimum required performance limit for all tested substances. This new derivatization procedure, which significantly decreases time and cost, is suitable for a routine basis application.

ISO/IEC 17025 Sysmex R-500 hematology reticulocyte analyzer validation.

The Sysmex R-500 (R-500) Hematology Analyzer is a bench-top system appropriate for the analysis of limited batches of blood samples. The R-500 provides percentage proportional (RET%), absolute reticulocyte (RET#), and absolute red blood cell (RBC#) counts. The system was validated at the Doping Control Laboratory of Athens, according to the International Committee for Standardization in Hematology, International Standards Organization (ISO/IEC) 17025, and World Antidoping Agency (WADA) specifications. The instrument calibration was performed according to the manufacturer and validation parameters comprised linearity, precision, uncertainty (intermediate and long-term precision), comparability, effect of drift, carryover, stability, and accuracy. The linearity and the comparability studies for RET#, RET%, and RBC# were expressed in regression factors (R2) and coefficients of correlation [r(x, y)], respectively. For the precision studies, the coefficients of variation for RET#, RET%, and RBC# were 9.49%, 9.83%, and <1.5%, respectively. For the intermediate precision studies, the coefficients of variation for RET#, RET%, and RBC# were 3.1%, 3.6%, and 0.6%, respectively. Carryover was found to be negligible. Sample stability was demonstrated at both room temperature and at 4 degrees C over a 24-hour period. Comparability studies for the R-500 were performed using a Sysmex SE-9500. The total evaluation led to the conclusion that the R-500 is an accurate and precise analyzer and because of to its relatively limited size, it can be considered a portable instrument, capable to be used in sports competition and training sites, where doping control and health tests are conducted. The analytical methodology of RET% measurement by the R-500 has been incorporated into the Doping Control Laboratory of Athens' Scope of Accreditation according to the ISO/IEC 17025 and WADA specifications.

Determination of xylazine and its metabolites by GC-MS in equine urine for doping analysis.

Xylazine and its main metabolites were detected in equine urine after a single-dose intravenous administration of 0.98 and 1.01 mg/kg body weight xylazine, respectively, in two horses, in order to be used for equine doping control routine analysis. The urine levels of the parent drug and its metabolites were determined using gas chromatography-mass spectrometry (GC-MS). Xylazine is metabolised rapidly, down to a concentration level of about 1.0 microg/ml after 1-3h administration. Seven metabolites were identified in urine. 4-Hydroxy-xylazine, the major metabolite, could be traced for 25 h and it is regarded as the long-term metabolite of xylazine in horse. 2,6-Dimethylaniline was, for the first time, reported as metabolite in equine.

Study of excretion of ecdysterone in human urine.

A study of excretion in human urine of ecdysterone, which is the active component of several over-the-counter supplements such as "Ecdysten", reportedly used by athletes, is presented. The study was performed after oral administration of 20 mg of ecdysterone. The collected urine samples were prepared using the standard screening extraction procedure for the free and conjugated fraction of anabolic steroids, and analyzed by gas chromatography (GC) coupled with quadrupole mass spectrometry (MS) and also with high-resolution mass spectrometry (HRMS). Two ecdysterone metabolites were identified and detected along with unchanged ecdysterone. Accurate mass measurements were made for diagnostic ions, including the molecular ion of the main metabolite of ecdysterone, deoxyecdysone, which, to our knowledge, has not previously been reported in the literature. These accurate mass measurements support the proposed fragmentation scheme.

Determination of ephedrines in urine by gas chromatography-mass spectrometry.

A selective gas-liquid chromatographic method with mass spectrometry (GC-MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines--one derivative was formed for each ephedrine--were prepared and analyzed by GC-MS, after alkaline extraction of urine and evaporation of the organic phase, using d3-ephedrine as internal standard. Calibration curves, with r2>0.98, ranged from 3.0 to 50 microg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.

Elimination profiles of flurbiprofen and its metabolites in equine urine for doping analysis.

Flurbiprofen and its main acidic metabolites were detected in equine urine after a single-dose administration of 500 mg flurbiprofen to two 2.5-3.5-years-old mares, in order to be used in equine doping control routine analysis. The urine levels of the parent drug were determined using GC/MS. Five acidic metabolites were found in the urine. The structure of the proposed metabolites was confirmed by HRMS accurate mass measurements. The highest flurbiprofen concentration was 204 mug ml(-1) at 1-3 h post administration. Flurbiprofen could be detected for 24-37 h in urine using the standard screening procedure. All metabolites were present 25 h post administration, while 4'-hydroxyflurbiprofen could be traced for more than 48 h and it is regarded as the long-term metabolite of flurbiprofen in horse.

Excretion study of the beta2-agonist reproterol in human urine.

An excretion study of the beta2-agonist 7-[3-[(beta-3,5-trihydroxyphenethyl)amino]-propyl]theophylline (reproterol) in human urine, which is reportedly misused by athletes and horses as a doping agent, is presented. The study was performed after an oral administration of 20 mg of reproterol hydrochloride. The collected urine samples were prepared using the standard anabolic steroid extraction procedure and analyzed by gas chromatography coupled with quadrupole mass spectrometry and, also, with high-resolution mass spectrometry (HRMS). The main reproterol metabolite was found, whereas unchanged reproterol was not detected. The structure of the main metabolite was confirmed by an accurate HRMS measurement of diagnostic ions. Finally, an excretion urine profile of the main metabolite is presented. The mass spectrum of another possible unidentified reproterol metabolite is also reported.

Quantitative structure-retention relationships in doping control.

Regression equation modelling was used for the correlation of gas chromatographic relative retention times tRR of anabolic steroids, stimulants and narcotics with their molecular characteristics in order to create a model for the prediction of tRR values of unanalyzed molecules. Predicting chromatographic retention parameters is one of the main goals of the quantitative structure-retention relationships (QSRR) methodology. To be performed, QSRR studies require two tools; a methodology for the extraction of the structural characteristics and a statistical program for the correlation of these characteristics with the chromatographic data.

Prediction of gas chromatographic relative retention times of stimulants and narcotics.

The ADAPT software system was used to create models for the prediction of gas chromatographic relative retention times (RRTs) of stimulants and narcotics that are analyzed in doping control of athletes. The two main methods that were followed for building the models were the quantitative structure-retention relationship (QSRR) and multiple linear regression analysis. The main proposed model for the entire data set had a multiple correlation coefficient R = 0.991 and standard error s = 0.046 or approximately 4.5%. Because of the relatively high standard error of the main model, a second model was built on a subset of compounds with R = 0.982 and s = 0.027 or approximately 2.5%.

Prediction of gas chromatographic relative retention times of anabolic steroids.

The prediction of gas chromatographic relative retention times (RRTs) of anabolic steroids, used in the doping control of athletes, was performed by a quantitative structure-retention relationship (QSRR) and multiple linear regression analysis study. A nine-variable model was generated with a multiple correlation coefficient R = 0.991 and relative standard error of less than 3%. Preliminary results indicated that the application of the model, especially in the prediction of RRTs of metabolites of the anabolic steroids, will be helpful.