Gas chromatography–combustion–isotope ratio mass spectrometry analysis of 19-norsteroids: application to the detection of a nandrolone metabolite in urine

Abstract

Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54–60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the ¹³C/¹²C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.

Introduction

The International Olympic Committee (IOC) first prohibited anabolic androgenic steroids for the Montreal Olympic games in 1976. Nandrolone or 19-nortestosterone (19-NT) belongs to this class of compounds and is one of the most widely used anabolic steroids. Administration of Nandrolone (or other 19-nonsteroid precursors such as norandrostendione or norandrostendiol) can be detected in urine from the presence of their two major glucuronic metabolites, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) [1], [2] (Fig. 1). Other metabolites have been described but are presented in urine in smaller amounts [3]. By the IOC anti-doping code, an adverse report will be issued and may lead to sanction if a concentration of at least one metabolite in excess of 2 ng/ml or 5 ng/ml of urine is found in a male or female competitor, respectively. This threshold has been established in males in order to exclude low endogenous production of 19-NA which is well known in stallions [4] and boars [5], [6]. Different authors have recently reported such natural production in human beings [7], [8], [9], [10], [11]. No evidence of endogenous production of 19-NA producing a level of >2 ng/ml was found in males in these previous studies. The problem is more complex for females as nandrolone has been found in human ovarian follicular fluid [12] and in the placenta during pregnancy [13]. Mareck-Engelke et al. [14] reported that the concentration of 19-NA may reach 20 ng/ml in urine during pregnancy. These cases are, however, unusual in the context of anti-doping testing and are easily detected by a test for HCG. It has also been reported that some certain norsteroid (e.g. norethisterone) contained in contraceptive pills can be metabolised to 19-NA and 19-NE. In these cases, however, other relevant metabolites (tetrahydro-metabolites of norethisterone) are also present in urine.

Various sources of contamination have also been put forward in order to explain the presence of 19-NA in the urine of sports players who have claimed to be innocent. Food contaminated by endogenous production of 19-NA [15] or by exogenous administration of nandrolone used as a growth promoter animals [16] has been investigated, as have permitted nutritional supplements which contain ‘unexpected’ traces of anabolic steroid. In this context, differentiation between 19-NA in urine of endogenous and exogenous origin represents an important challenge for anti-doping laboratories. Since the introduction of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) into the field of anti-doping testing, a similar problem which arises with testosterone abuse is in the process of being resolved. It has been shown that exogenous administration of testosterone can be differentiated from naturally high production by measuring the ¹³C/¹²C ratio [17], [18], [19], [20], [21], [22], [23]. Discrimination is made from the lower level of the ¹³C stable isotope in the source material used in the chemical synthesis of testosterone compared to endogenous testosterone production. Such differentiation between synthetic and endogenous origins has also been reported for other hormones such as DHEA [24] and corticosteroids [25], [26]. This report presents the first results of ¹³C/¹²C ratio measurement in 19-norsteroids. We describe a purification procedure for 19-NA in urine for use in GC–C–IRMS analysis and discuss the reliability of carbon isotope results obtained by GC–C–IRMS.