Determination of free and deconjugated testosterone and epitestosterone in urine using SPME and LC–MS/MS (original) (raw)
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Talanta, 2011
A sensitive and rapid liquid chromatographic (LC) method for the simultaneous determination of testosterone (T) and epitestosterone (E) in human urine samples has been developed and elaborated. The ratio of the both steroids (T/E) in human urine is a widely used as doping control indicator. A sample pretreatment by solid-phase extraction (SPE) after hydrolysis using 36% hydrochloric acid for determination of total level of T has been applied. Unconjugated (free) form of the both androgens were determined without hydrolysis steps, what makes novelty of the method, because simplifies the proposed procedure. In turn, the measurements of urinary free T and E provided the diagnostic information for excess adrenal production of steroids. The proposed LC assay was evaluated by analyzing a series of urine samples containing T, E and methyltestosterone (MT) as internal standard at the range of concentration 2-300 ng −1 mL of both analyzed hormones. The proposed method was fully validated for specificity, linearity, limits of detection and quantitation, precision and trueness according to the current requirements concerning analytical methods. Interestingly, the developed LC method allows to obtain a sensitive enhancement with respect to UV detection with the quantitation limit for T and E equaled 2 ng mL −1 . The method was selective and reliable for identity and enable to detect changes of endogenous levels of T and E in urine independently of fluctuations characteristic for both analyzed endogenous hormone level in plasma. .pl (L. Konieczna). androgen deficiency in clinical conditions . The normal amounts of total endogenous T and epitestosterone (E) practically measured in healthy male in urine are in the range 30-60 ng mL −1 [11]. T and E and their ratio T/E is stable in males, what was well established . Since 1983, T was forbidden in sports by the International Olympic Committee (IOC). The detection of illicit use of T is currently carried out measuring the ratio between the concentration of T and its isomer E. A ratio of their concentrations (T/E ratio) higher than 4 is considered as potentially indicative of T administration. On the other hand, because the T/E ratio can be artificially modified by the administration of E, a urinary concentration of epitestosterone above 200 ng mL −1 has been established as indicative of its misuse as a masking agent . The World Anti-Doping Agency (WADA) indicated that if the T/E ratio was equal or above 4, or concentration of E higher than 200 ng mL −1 , a confirmation procedure to prove doping would be necessary .
Steroids, 2020
For an effective detection of doping with pseudo-endogenous anabolic steroids, the urinary steroid profile is of high value. In this work, the aim was to investigate steroid metabolism disruption after exogenous intramuscular administration of different testosterone esters. The investigation focused on both sulfo-and glucoro conjugated androgens. A single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido®) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanone®), was given to six healthy volunteers. Urine was collected throughout a testing period of 60 days. A LC-MS method was developed and validated for the analysis of eight conjugated steroids in their intact form. The results show that urinary changes in both sulfo-and glucuro conjugated steroid levels are prominent after the injection of testosterone esters. A promising potential marker for the intake of exogenous testosterone is the combined ratio of epitestosterone sulfate/epitestosterone glucuronide to testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) as a complementary biomarker for testosterone abuse. This represents a new piece of evidence to detect testosterone doping, representing a new approach and being independent from the metabolic connections of the markers in the steroid passport.
Quantification of testosterone and epitestosterone in human urine by capillary liquid chromatography
Journal of Microcolumn Separations, 2000
A capillary-liquid chromatography LC method was developed for the quantification of the endogenous steroids testosterone and epitestosterone in human urine. One milliliter of urine was used for the overall method. Free testosterone was first separated by liquid᎐liquid extraction with n-pentane at pH 7. Glucuronides of testosterone and epitestosterone were enzymatically hydrolyzed and the free compounds were extracted with n-pentane at pH 11. A capillary Ž . column switching system with a low back pressure precolumn PC was used for Ž . fast loading of large sample volumes 20 L . Chromatographic separation was Ž . carried out on a 15 cm = 300 m inner diameter i.d. column, packed with 3 m Hypersil BDS-C at a flow rate of 4 Lrmin with isocratic elution and UV 18 Ž . absorbance detection 240 nm . Limit of detection for free testosterone was established at 0.5 ngrmL. Limits of detection were established at 1.5 and 3.2 ngrmL for testosterone and epitestosterone, respectively, after being hydrolysed from their glucuronides. Good reproducibility and robustness were observed Ž . through the entire calibration range up to 250 ngrmL . ᮊ
Androgen Sulfation in Healthy UDP-Glucuronosyl Transferase 2B17 Enzyme-Deficient Men
The Journal of Clinical Endocrinology & Metabolism, 2011
The conspicuous interindividual differences in metabolism and urinary excretion of testosterone and its metabolites make it challenging to reveal testosterone doping. The variation in testosterone glucuronide excretion is strongly associated with a deletion polymorphism in the uridine diphosphate-glucuronosyltranferase (UGT) 2B17 gene.
Rapid Communications in Mass Spectrometry, 2000
A gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method is described and validated for measurement of d 13 C values of the acetate derivatives of urinary etiocholanolone and androsterone. The analysis was performed with only 2 mL of urine. The sample preparation consisted of deconjugation with b-glucuronidase, solid phase extraction, and derivatization with acetic anhydride and pyridine. The within-assay precision of two quality control (QC) urine samples ranged from 0.5 to 2.1 CV%. The between-assay precision in the same QC urines ranged from 1.7 to 3.4 CV%. Administration of testosterone enanthate to a subject resulted in a 6% decrease in d 13 C values from À25% (baseline) to À31%. Two weeks after testosterone administration was discontinued, the d 13 C values remained abnormally low while the urine testosterone/epitestosterone (T/E) ratio returned to less than 6. This relatively simple method is useful for rapidly screening a large number of urine samples, including those with T/E`6.
Drug Testing and Analysis
In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted.
Journal of Analytical Toxicology, 2004
The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ralio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5o~-androstanediol, and 5[~-androstanediol) together with an endogenous reference (5[3-pregnanediol) were extracted from the urines and the ~t3C/12C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum 613C-value variations (parts per thousand difference of 813C/12C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5u.-androstanediol 813C-values returned to the baseline 15 h after the second T administration, a decrease of the 5~-androstanediol ~-values could be detected for over 40 h. This suggests that measurements of 5~-androstanediol 8-values allow lhe detection of a testosterone ingestion over a longer post-administration period than other T metabolites 813C-values or than the usual T/E ratio approach.
Steroids, 1997
A gas chromatographic combustion isotope ratio mass spectrometric (GC/C/IRMS) method was used for studying the incorporation of exogenous testosterone enanthate into excreted urinary 5or-and 515-androstane-3c~,176diols. A multistep but straigh(forward work-up procedure produced a simple GC chromatogram of urina~ steroid acetates composed principally of two androstanediols and pregnanedioL It is anticipated that such a method may form the basis of a doping control test for testosterone that could be used as a primary method during major sporting events or alternatively as a verification technique. Urine samples from five individuals were collected before and after administration of testosterone enanthate (250 rag). The ~/3C °/00 value of androstanediols was around -26 to -28 during the baseline period and decreased to about -29 to -30 in the days following synthetic testosterone administration. One of the other major steroids in the chromatogram, pregnanediol, was utilized as the "internal standard," because its ~13C°/00 values did not markedly change following testosterone administration, remaining at -25 to -27. In all subjects studied, the ~13C°/00 values for androstanediols were reduced sufficiently over 8 days to confirm administration of synthetic testosterone. Although steroids isolated from urine of normal individuals from 12 different countries gave values between -24 and -28, this seemed not to be related to nationality or region. The most likely variable is the proportion of plants with low and high carbon 13 content in the diet. This variable is likely to be more affected by individual food preferences than broad ethnic food divisions. In this paper, we propose a ratio of ~13 C°/OO for androstanediols to pregnanediol as a useful discriminant of testosterone misuse, a value above 1.1:1.0 being indicative of such misuse. The work-up procedure was designed for batch analysis and to use only simple techniques, rather than employ further instrumentation, such as high-performance liquid chromatography (HPLC), in purifying steroids for GC/C/IRMS. (Steroids 62:379-387, 1997) © 1997 by Elsevier Science Inc.
The Journal of Steroid Biochemistry and Molecular Biology, 2013
The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse.
Drug Testing and Analysis, 2019
The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed‐mode solid‐phase extraction and analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Urine samples after a single IM injection (100 mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre‐ and post‐administration samples. For Caucasian volunteers, a separation between pre‐ and post‐administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selec...