Stability studies of testosterone and epitestosterone glucuronides in urine.
ABSTRACT 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 beta-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 4 degrees C and -20 degrees C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing (at -20 degrees C) 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 -20 degrees C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37 degrees C 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.
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ABSTRACT: Testosterone doping in sport is detected by measurement of an increased testosterone/epitestosterone (T/E) ratio in urine. The critical limit is 6. The present study concerns calibration curves for the T/E ratio measured by gas chromatography/mass spectrometry (electron impact) according to the guidelines of the International Olympic Committee. Testosterone (T) and epitestosterone (E) are measured as trimethylsilyl (TMS)-enol-TMS ethers in selected ion monitoring mode using m/z 432 with methyltestosterone (MT) (m/z 446) as internal standard. Calibration curves corresponding to T/E = 1, 6 and 12 prepared directly, i.e. without extraction of T and E, were non-linear. The non-linearity was caused by an increase of the relative molar response of T with respect to the internal standard MT with increasing concentration level. A mean increase of 82% was observed from T/E = 1 to T/E = 12 (E fixed). Adding T/E corresponding to 1/1, 6/1 and 12/1 to urine without endogeneous hormone content resulted in an almost linear calibration curve along the diagonal, with only a slight increase of the relative molar response of testosterone (16% from T/E = 1 to 12). Apparently, the biological matrix stabilizes the relative molar response over a wide concentration range. At a molar ratio of about 1/1 for T/MT, the relative molar response for direct measurement of T is identical to that observed in the presence of urine matrix, which is explained on the basis of a simple mathematical model. The practical conclusion of this study is that, contrary to the present-day practice, calibration curves for the T/E ratio should be based on T/E added to blank urine taken through the extraction procedure. Otherwise, the T/E ratio of urine sample is systematically easily underestimated by 30% or more.Biological Mass Spectrometry 08/1993; 22(7):412-8.
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ABSTRACT: Stability is an important consideration in the use of specimens for accurate determination of analyte concentrations. To determine the long-term stability for analytes routinely analyzed by mass spectrometry in this laboratory, quality-control (QC) results were plotted versus time. The time required for the initial concentration to reach a specified level of deviation (i.e., 15%) was then determined from the slopes. QCs were prepared at 1-3 concentrations in drug-free matrix and stored at approximately -20 degrees C; urines were fortified with 1% sodium fluoride; plasmas (except for cocaines) were prepared with heparin. For cocaine and metabolites, the plasma was either fortified with 2% sodium fluoride or with 2% sodium fluoride and 1 mg% physostigmine after adjustment of the plasma pH to 6.0. In urine, amphetamine, methamphetamine, codeine, morphine, benzoylecgonine (BZE), and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) slopes did not exceed a 15% deviation before 852 days. Cocaine, however, reached a 15% reduction at 165 days. When cocaine and BZE were prepared in plasma with just 2% sodium fluoride, negative slopes reached 15% deviation within 154 and 111 days, respectively. Further fortification with physostigmide and adjustment of the pH extended this time frame significantly. Delta9-Tetrahydrocannabinol (THC) and THCA in plasma had negative slopes that deviated by 15% just prior to one year. l-Alpha-acetylmethadol (LAAM), methadone, and their N-demethylated metabolites in urine did not have any negative slopes exceeding 15% before 686 days. Several of the compounds had positive slopes. Those for 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine reached the 15% mark within 98 days. Those for LAAM, norLAAM, and dinorLAAM were concentration dependent. The 25-ng/mL controls reached 15% at 158-216 days. The 700-ng/mL controls reached 15% at 784-1340 days. In plasma, only naltrexone and buprenorphine displayed negative slopes at all three concentrations, reaching the 15% mark as early as 576 and 272 days, respectively. LAAM, norLAAM, dinorLAAM, ibogaine, 6-beta-naltrexone, risperidone, and 9-OH-risperidone did not exceed a 15% deviation before 416 days. To attempt to validate this method, two sets of clinical plasma samples that had been analyzed for buprenorphine were reanalyzed 644 and 869 days after the initial analyses. Those reanalyzed after 644 days were not statistically different from initial analyses, whereas those stored for 869 days were statistically different (p < 0.05). As the average time to reach 15% deviation for the three concentrations of buprenorphine QCs was 782 days, this suggests that extrapolation of QC results gathered over time may provide a reliable method to estimate long-term stability limits for drugs stored under the same conditions as the QC samples.Journal of analytical toxicology 11/1999; 23(6):535-40. · 2.11 Impact Factor
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ABSTRACT: The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.Journal of analytical toxicology 04/2000; 24(2):102-15. · 2.11 Impact Factor