Long‐term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

Abstract

The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called "steroid profiling" method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses.The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.

Biomarker ratios

“…Esquivel et al investigated 14 ratios of steroid sulphates to show ES and dehydroandrosterone sulphate (DHAS) are effective ERCs in biomarker values that improved the retrospectivity for detection of oral TU administration compared with T/E values alone 31 . This coincided with a population reference study of steroid sulphate ratios in male ( n = 780) and female ( n = 373) routine antidoping urine samples reported by Saad et al 32 Forsdahl et al returned to the combination of glucuronide and sulphate forms of T and E to propose ((ES/EG)/(TS/TG)) as a complementary biomarker to improve the detection of intramuscular testosterone administration, 33 which was supported by Schulze et al 34 Further, the work of De Wilde et al has provided a number of insights into the analysis of intact steroid conjugates 35 . First, no degradation effects were observed over long periods (e.g., >10 years) in samples stored at −20°C.…”

“…Interestingly, the MRR is subsequently used in a ratio with the CV from preadministration samples to provide an initial measure of biomarker quality. In addition, the TPR is itself a ratio for diagnostic sensitivity derived from true positive samples relative to the total administration samples at a specificity level of 99% 35 . The BBQ scores were used to assess the utility of ratios from profiling three free steroids and 14 intact steroid conjugates following single‐dose administrations of oral TU, intramuscular TU, topical T gel, topical dihydrotestosterone (DHT) gel and oral DHEA 35 …”

“…has provided a number of insights into the analysis of intact steroid conjugates. 35 First, no degradation effects were observed over long periods (e.g., >10 years) in samples stored at À20 C. Second, four parameters; maximum relative response (MRR), coefficient of variance (CV), true positive rate (TPR) and receiver operator characteristic (ROC), were combined to generate a best biomarker quality (BBQ) score for improved biomarker ratio selection. MRR is calculated as the maximal postadministrative increase relative to baseline, which is averaged for all volunteers.…”

“…In addition, the TPR is itself a ratio for diagnostic sensitivity derived from true positive samples relative to the total administration samples at a specificity level of 99%. 35 The BBQ scores were used to assess the utility of ratios from profiling three free steroids and 14 intact steroid conjugates following single-dose administrations of oral TU, intramuscular TU, topical T gel, topical dihydrotestosterone (DHT) gel and oral DHEA. 35 It is instructive from investigations of endogenous steroids and their intact conjugates that ratios can improve biomarker interpretations by removing some of the analytical imprecision associated with quantification at low concentrations.…”

Rapidity and Precision of Steroid Hormone Measurement

“…Due to the above reason, samples containing the steroids should be stored at −20 • C and pH 5-7. The temperature stability tests of steroids have also been evaluated by LC-MS [83]. Stability experiments of dehydroepiandrosterone sulfate (DHEAS), epiandrosterone sulfate (EpiAS), androsterone sulfate (AS), testosterone glucuronide, and testosterone sulfate showed no degradation effects in the steroid profile of samples stored for long periods (>10 years) at −20 • C.…”