Synthesis of recently reported phase II metabolites for their inclusion in routine doping control analysis

Code: 12A13OP 

Availability of reference materials is crucial in doping control since they are needed to confirm adverse analytical findings and to develop quantitative methods.  Most of the analytes included in doping control routine methods are phase I metabolites released to urine after enzymatic hydrolysis. However, the direct detection of phase II metabolites by LCMS/ MS is a common trend in doping control analysis. Therefore, the synthesis of phase II metabolites will become more important for the field in the next years. 
In previous WADA funded projects, our research group has identified several phase II metabolites which are useful for the detection of the misuse of several anabolic steroids. Among them, preliminary results indicate that 6β-hydroxyandrosterone glucuronide and 6βhydroxyetiocholanolone glucuronide can improve the detection of testosterone misuse. Several sulphate metabolites like epiboldenone sulphate or 17β-methyl-5β-androstane-3α,17α-diol 3αsulphate can help in the detection of boldenone and methyltestosterone misuse. The structure of these metabolites has been confirmed by their synthesis at small scale. This synthesis at small scale was necessary because none of the detected metabolites is commercially available. 
Larger amounts of the metabolites are needed for their complete characterization by NMR techniques. Besides, the availability of these larger amounts would also allow for the development of analytical approaches able to detect and confirm their presence in urine samples. 
Therefore, the goals of this project are (i) to synthesize at a larger scale several phase II metabolites previously identified as useful for the doping control field (6β-hydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide, epiboldenone sulphate, 17α-methyl-5βandrostane-3α,17β-diol 3α-sulphate, 17β-methyl-5β-androstane-3α,17α-diol 3α-sulphate and 17βmethyl-5α-androstane-3α,17α-diol 3α-sulphate), (ii) to fully characterize them by well established techniques (NMR spectroscopy, mass spectrometry), (iii) to study their stability and (iv) to develop analytical approaches to quantify conjugated metabolites of testosterone (6βhydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide). The synthetized standards will be distributed among WADA accredited laboratories. 

Main Findings: 

Availability of reference materials is crucial in doping control since they are needed to confirm adverse analytical findings and to develop quantitative methods. Most of the analytes included in doping control routine methods are phase I metabolites released to urine after enzymatic hydrolysis. However, the direct detection of phase II metabolites by LC-MS/MS is a common trend in doping control analysis. Therefore, the synthesis of phase II metabolites will become more important for the field in the next years.In previous WADA funded projects, our research group has identified several phase II metabolites which are useful for the detection of the misuse of several anabolic steroids. Among them, preliminary results indicate that 6β-hydroxyandrosterone glucuronide and 6β-hydroxyetiocholanolone glucuronide can improve the detection of testosterone misuse. Several sulfate metabolites like epiboldenone sulfate or 17β-methyl-5β-androstane- 3α,17α-diol 3α-sulfate can help in the detection of boldenone and methyltestosterone misuse. The structure of these metabolites has been confirmed by their synthesis at small scale since none of the detected metabolites is commercially available.

This project aimed to (i) synthesize selected phase II metabolites at large scale, (ii) characterize these metabolites (iii) develop analytical methodology for the quantitation of conjugated metabolites of testosterone by LC-MS/MS and (iv) distribute of the synthesized material among WADA accredited laboratories.

The main goals of this project have been reached. Firstly, we have synthesized at a larger scale 6β-hydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide, epiboldenone sulfate, 17α-methyl-5βandrostane-3α,17β diol 3α-sulfate, 17β-methyl-5α-androstane-3α,17α-diol 3α-sulfate and 17βα-methyl-5α-androstane-3α,17α-diol 3α-sulfate. Additionally, we have fully characterized these metabolites by well established techniques like NMR spectroscopy and mass spectrometry. We have developed and validated an analytical method based on LC-MS/MS for the quantification of glucuronide metabolites of testosterone. Finally, we have distributed the synthesized sulfates among WADA accredited laboratories.