Elsevier

Analytica Chimica Acta

Volume 683, Issue 2, 10 January 2011, Pages 221-226
Analytica Chimica Acta

Urine stability and steroid profile: Towards a screening index of urine sample degradation for anti-doping purpose

https://doi.org/10.1016/j.aca.2010.10.003Get rights and content

Abstract

The presence of microorganisms in urine samples, under favourable conditions of storage and transportation, may alter the concentration of steroid hormones, thus altering the correct evaluation of the urinary steroid profile in doping control analysis. According to the rules of the World Anti-Doping Agency (WADA technical document TD2004 EAAS), a testosterone deconjugation higher than 5% and the presence of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the deconjugated fraction, are reliable indicators of urine degradation. The determination of these markers would require an additional quantitative analysis since the steroids screening analysis, in anti-doping laboratories, is performed in the total (free + conjugated) fraction. The aim of this work is therefore to establish reliable threshold values for some representative compounds (namely 5α-androstane-3,17-dione and 5β-androstane-3,17-dione) in the total fraction in order to predict directly at the screening stage the potential microbial degradation of the urine samples. Preliminary evidence on the most suitable degradation indexes has been obtained by measuring the urinary concentration of testosterone, epitestosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione by gas chromatography–mass spectrometric every day for 15 days in the deconjugated, glucuronide and total fraction of 10 pools of urines from 60 healthy subjects, stored under different pH and temperature conditions, and isolating the samples with one or more markers of degradation according to the WADA technical document TD2004EAAS. The threshold values for 5α-androstane-3,17-dione and 5β-androstane-3,17-dione were therefore obtained correlating the testosterone deconjugation rate with the urinary concentrations of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the total fraction. The threshold values suggested as indexes of urine degradation in the total fraction were: 10 ng mL−1 for 5α-androstane-3,17-dione and 20 ng mL−1 for 5β-androstane-3,17-dione. The validity of this approach was confirmed by the analysis of routine samples for more than five months (i.e. on a total of more than 4000 urine samples): samples with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione higher than the threshold values showed a percentage of free testosterone higher than 5 of its total amount; whereas free testosterone in a percentage higher than 5 of its total amount was not detected in urines with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione lower than the threshold values.

Introduction

The transformation of steroids by microorganisms has been widely studied and it was proved in 1937 when Mamoli and Vercellone used an anaerobic yeast culture to convert androst-4-en-3-one to testosterone [1]. Afterwards other studies demonstrated microbial formation of testosterone under both aerobic or anaerobic conditions in the presence of a wide variety of microorganisms, commonly found in human urine, using different steroids as substrates [2], [3], [4]. Moreover, it has generally been observed that microbial contamination induces hydrolysis of glucuronide and sulphate conjugates, followed by modifications of the steroid structure by oxidoreductive reactions [5], [6], [7]. The above degradation processes may cause changes to the urinary endogenous steroid profiles of the samples leading to misinterpretation of analytical results in the anti-doping field [8], [9], [10], [11], [12]. This is because the interpretation of results for endogenous androgenic steroids is based completely on alterations of the natural androgenic steroid baseline profile.

Up to the present time, much work has been done: (i) to identify urine degradation markers [8], [9], [10], [11], (ii) to identify microorganisms (whose presence may be caused by either urinary tract infection and/or by use of non sterilized material during sample collection or by manipulation, transport to and/or storage at the laboratory) potentially involved in alterations of the urinary endogenous steroid profile under controlled conditions [3], [4], and (iii) to find suitable stabilization methods to prevent degradation of urinary steroids during transportation [12]. Taking into account these results in order to avoid the risk of obtaining results of reduced utility by the analysis of the steroid profile, the World Anti-Doping Agency (WADA), established several criteria to identify microbial metabolic activity in urine sample, such as the presence of 5α-androstane-3,17-dione, 5β-androstane-3,17-dione and the presence of free testosterone in a percentage higher than 5 of the total testosterone [13]. Nevertheless, being the steroids screening analysis performed in the total fraction, none of the criteria established until now, allow to predict directly at the screening stage the degradation state of the urine samples.

In our laboratory after a preliminary, retrospective evaluation of the samples received in the summer months, we have confirmed the empirical evidence [5], [6], [7] that the degradation of the samples was often associated to an increase of testosterone (T)/epitestosterone (E) ratio value. The impossibility with the present rules to evaluate the sample degradation at the screening stage has led our laboratory to carry out an additional workload in the summer months, due to the T/E confirmation analysis for the degraded samples. The aim of this work was, therefore, to evaluate the possibility to establish one or more indexes to understand directly from the steroid screening analysis when a sample has to be considered degraded. These indexes were then validated according to ISO 17025.

Section snippets

Samples

Urine samples from 60 healthy subjects (30 females and 30 males) were collected for two days into non-sterile plastic containers. Ten pools (5 from female urines and 5 from male urines) of urines were then obtained mixing different urines: six for each pool. The 5 male pools and the 5 female pools were then divided in 2 aliquots: one for the experiments at different incubation pHs (5, 7 and 9, fixed temperature 37 °C; the pH was checked every day and corrected, see Fig. 1) and one for the

Effect of temperature

Under the experimental conditions of this study, steroids were deconjugated in those samples incubated at 37 and 25 °C. More specifically in Fig. 3C and D was reported the T and E profile during the 15 days of incubation at 37 °C in one of the 5 male pools. We can notice that the T and E deconjugation rate was very rapid: in less than 7 days more than 70% of compounds were present in the free fraction. The deconjugation kinetics was slower but still pronounced at 25 °C, while no difference was

Discussion and conclusions

According to the WADA technical document TD2004 EAAS, a deconjugation rate higher than 5% of the total amount of T and the presence in the free fraction of 5a-AND and for 5b-AND are reliable indicators of sample degradation [13]. Taking into account the WADA criteria and the results obtained previously by other investigators, the results obtained in the present study allow to suggest a reliable threshold value for the concentration, in the steroid total fraction, of the two most reliable

Acknowledgement

This work has been supported in part by a Research Grant of the Italian Department of Health (“Ministero della Salute, Commissione per la vigilanza sul doping e sulla tutela sanitaria delle attività sportive”).

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1

Present address: Cycling Anti-Doping Foundation, International Cycling Union (UCI), Ch. de la Mêlée 12, 1860 Aigle, Switzerland.

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