Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration

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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 selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A‐S/T‐S) which prolonged the DT 1.2–2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3–1.5 times in two Asian volunteers. Other ratios between A‐S or etiocholanolone sulfate and E‐S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T‐S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended.

Section: Resultsmentioning

confidence: 71%

“…Comparison of the DTs obtained for six Caucasian and six Asian volunteers of the fifteen important ratios and of T/E (obtained from data of Kotronoulas et al). *last collected sample.…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of sulfate metabolites as markers of intramuscular testosterone administration in Caucasian and Asian populations

Esquivel

Alechaga

Monfort

et al. 2019

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“…Consequently, an important research activity was developed in the recent years to look for additional markers, [5][6][7][8][9][10][11][12][13][14] including other endogenous metabolites excreted in the glucuronide fraction, 5,6 metabolites excreted as conjugates with cysteine, 7,8 or glucuronide metabolites resistant to enzymatic hydrolysis. Consequently, an important research activity was developed in the recent years to look for additional markers, [5][6][7][8][9][10][11][12][13][14] including other endogenous metabolites excreted in the glucuronide fraction, 5,6 metabolites excreted as conjugates with cysteine, 7,8 or glucuronide metabolites resistant to enzymatic hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

Esquivel

Alechaga²,

Monfort³

et al. 2018

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A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantitation of endogenous steroid sulfates has been developed to be able to evaluate these metabolites as biomarkers to detect the misuse of endogenous androgenic anabolic steroids in sports. For sample preparation, a mixed-mode solid-phase extraction was optimized to eliminate the glucuronide fraction in the washing step thus obtaining only the sulfate fraction. Chromatographic separation was optimized to achieve adequate resolution between isomers. The electrospray ionization and the product ion mass spectra of the sulfates were studied in order to obtain the most specific and selective transitions. The method was validated for quantitative purposes for 11 steroid sulfates obtaining satisfactory values for linearity, accuracy, and intra- and inter-day precision (relative standard deviation better than 16.2%). Limits of quantitation ranged between 0.5 and 2 ng/mL. Extraction recoveries for sulfate metabolites were between 90 and 94%. Matrix effect ranged from 90 to 110% showing the absence of significant ion suppression/enhancement. Samples were found to be stable after 2 freeze/thaw cycles. The applicability of the method was checked by the analysis of 75 urine samples from healthy volunteers (54 males, 37 Caucasian and 17 Asian, and 21 Caucasian females) to evaluate the concentration levels of endogenous sulfate metabolites in basal conditions.

“…In this first part, the potential usefulness of the studied metabolites for the screening of multiple transdermal administration of T is evaluated. Their utility for the detection of intramuscular administration of testosterone undecanoate is discussed in a second part of the study . In order to reach the goal of this first part, urine samples were collected at different times before and after the administration of 3 doses of T gel to 12 volunteers (6 L‐T/E and 6 M‐T/E) in 3 consecutive days.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration

Kotronoulas

Gómez-Gómez

Fabregat

et al. 2017

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Although the introduction by the World Anti-Doping Agency (WADA) of the steroid module of the athlete biological passport (ABP) marked an important step forward in the screening of testosterone (T) misuse, it still remains one of the most difficult challenges in doping control analysis. The urinary determination of alternative markers has been recently reported as a promising tool for improving the screening of T oral administration. However, their evaluation for other, commonly used, administration routes is still required. The main goal of this study is the evaluation of the potential of 2 groups of metabolites (cysteinyl conjugated and glucuronoconjugated) after transdermal and intramuscular administration of T. Their suitability was evaluated in individuals with both low basal (L-T/E) and medium basal (M-T/E) values of T/E. In this Part I, we evaluated the urinary excretion profile of these 2 groups of T metabolites after the administration of 3 doses of T gel to 12 volunteers (6 L-T/E and 6 M-T/E) for 3 consecutive days. For this purpose, 9 different concentration ratios (5 cysteinyl conjugated and 4 glucuronoconjugated markers) were studied. Both, the intra-individual variability and the detection windows (DW) obtained by each ratio were evaluated. Cysteinyl conjugates showed a general low intra-individual variability and DWs that were shorter than any other tested marker. Despite the relatively large intra-individual variability, the DWs reached by glucuronoconjugates (2-3 days) were similar to those obtained by markers currently included in the ABP. Overall; this evaluation advises for the introduction of additional glucuronoconjugated markers in the screening of transdermal T administration.