Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols

doi:10.1016/S0378-4347(99)00066-3

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 727, Issues 1–2, 30 April 1999, Pages 95-105

https://doi.org/10.1016/S0378-4347(99)00066-3 Get rights and content

Abstract

We propose a new screening method for testosterone (T) doping in sport. The current method for detecting T administration is based on finding a T to epitestosterone ratio (T/E) in urine that exceeds six. The difficulties with T/E are that T administration does not always result in a T/E>6 and that a rare individual will have T/E>6 in the absence of T administration. Our previous studies reveal that carbon isotope ratio helps to determine the origin of the urinary T because the values for T and its metabolites decrease after the administration of exogenous T. In this study, we present a rapid and efficient screening sample preparation method based on three successive liquid–solid extractions, deconjugation with E. coli β-glucuronidase after the first extraction, acetylation after the second extraction, and a final extraction of the acetates. The ¹³C/¹²C of two T metabolites (5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol) and one pregnanediol as endogenous reference (5β-pregnane-3α,20α-diol) was measured by gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) on 10 ml of urine collected from 10 healthy men before and after T administration. Following T administration, the ¹³C/¹²C of 5β-androstane-3α,17β-diol diacetate and 5α-androstane-3α,17β-diol diacetate declined significantly from −26.2‰ to −30.8‰ and from −25.2‰ to −29.9‰, respectively and the ¹³C/¹²C of 5β-pregnane-3α,20α-diol diacetate was unchanged. In addition, the ratio of androstanediols to pregnanediol increased in the post-T urines.

Introduction

After 1976, when the use of anabolic androgenic steroids (AAS) was prohibited by the International Olympic Committee (IOC), several techniques were developed to detect these compounds. At present, AAS are detected in urine samples by gas chromatography–mass spectrometry (GC–MS). However, pharmaceutical or exogenous testosterone presents special challenges as it is not possible to distinguish it from natural endogenous testosterone by GC–MS.

New possibilities for detecting exogenous T emerged following reports that the carbon isotope ratio (¹³C/¹²C) of exogenous T was different from that of endogenous T [1]. The ¹³C/¹²C of natural human testosterone is a function of the aggregate sum of the ¹³C/¹²C of the plants and animals we consume, and any additional effects of human biological processing. Synthetic testosterone is prepared from one plant source with an unusually low ¹³C/¹²C. These slight differences can be detected through isotope ratio mass spectrometry [2], [3], [4], [5], [6]. In a T administration study that collected urines from subjects before and after T administration, we reported that the ¹³C/¹²C of urinary T and metabolites of T were significantly lower after T, and that the ¹³C/¹²C of metabolic precursors of T were unchanged [2]. Shackleton et al. [5] confirmed that T administration results in lower ¹³C/¹²C values for T metabolites, showed that the values remain low for 8 days following a dose of 250 mg of T enanthate, and demonstrated the potential applicability of the method for detecting administration of dehydroepiandrosterone (DHEA) and other steroids. These studies and others [7] are consistent with the view that carbon isotope ratio methods could be very useful in discovering the origin of T and other steroids in an athlete’s urine.

The present method for detecting T administration is finding a ratio of T to epitestosterone (T/E) in urine by GC–MS that exceeds six [8]. While T/E>6 is an excellent indicator of T administration, it is not a definitive test because a few individuals who have not used T have a T/E greater than six [9], [10], and, other factors may alter T/E [11]. Further, since the median T/E is ∼1 [12], individuals with low or normal T/E may take T and not exceed the reporting threshold of six, thus a negative report (T/E<6) does not exclude T administration. Lowering the T/E ratio threshold would result in detecting more cases of naturally elevated T/E, but would still allow some use of synthetic T. Other indirect means of detecting T administration have been discussed [13], but unlike ¹³C/¹²C, none have the potential to serve as a direct indicator of T administration.

If a ¹³C/¹²C method is to be useful for increasing the sensitivity of detecting T administration, it is necessary to develop a screening method that is rapid, efficient, reliable, and requires a small volume of urine. In our first reports [2], [3], [4], the method required ∼30 ml and it was relatively slow due to the high-performance liquid chromatography (HPLC) step. Furthermore, in the interest of understanding the method, we determined the ¹³C/¹²C of several compounds: cholesterol, DHEA, androst-5-ene-3β,17β-diol (T precursors), T itself, and T metabolites 5α-androstane-3α,17β-diol (5α-adiol) and 5β-androstane-3α,17β-diol (5β-adiol).

In this paper, we present a new sample preparation method that only requires 10 ml of urine, does not require HPLC fractionation, and provides chromatograms with excellent separation of the compounds of interest and virtually flat baselines.

Section snippets

Urine samples

Urines were obtained from two groups of healthy male subjects. Subjects A–E were 20–59 years of age and were participating in a 25-week study concerned with the effects of T on behavior. The protocol was approved by the Harvard Medical School institutional review board. The subjects received intramuscular T enanthate (300 mg/week) or placebo, and urines were collected weekly for 26 weeks. Subjects F–J were participating in an 8-day study to discover the effects of T infusions on the

Results and discussion

A principal objective of the new sample preparation scheme presented here was to develop a rapid method for screening urine samples by GC–C–IRMS. To accomplish this, it was necessary to eliminate the HPLC purification step from our previous methods [2], [3], [4]. Secondly, we aimed to reduce the sample volume requirements from ∼30 to 10 ml or less. Thirdly, a screening method must be rapid and efficient, therefore we wished to adapt the method to batch processing in order to improve sample

Conclusions

We propose a new screening method for the detection of exogenous testosterone administration using GC–C–IRMS. The method meets the requirements for use in routine screening. Firstly, only 10 ml of male urine was required to obtain satisfactory estimates of δ¹³C‰. Secondly, by replacing the HPLC step in our previous procedure with three successive solid-phase extractions, greater speed and efficiency was achieved by processing samples in batches. Thirdly, the choice of deconjugating with

Acknowledgements

We are grateful for financial support from the National Collegiate Athletic Association, the National Football League, and the United States Olympic Committee. K. Schramm skillfully assisted with the analyses, and T. Callahan and J. Wilson provided outstanding editorial assistance.

References (20)

R. Aguilera et al.
J. Chromatogr. B
(1996)
C.H.L. Shackleton et al.
Steroids
(1997)
C.H.L. Shackleton et al.
Steroids
(1997)
E. Raynaud et al.
Lancet
(1992)
M. Garle et al.
J. Chromatogr. B
(1996)
P. Deines
R. Massé et al.
J. Chromatogr.
(1989)
L. Dehennin et al.
Steroids
(1998)
G. Southan, A. Mallet, J. Jumeau, S. Craig, N. Poojara, D. Mitchell, M. Wheeler, R.V. Brooks, Programme and Abstracts...
M. Becchi et al.
Rapid Commun. Mass Spectrom.
(1994)

There are more references available in the full text version of this article.

Cited by (95)

Application of comprehensive 2D chromatography in the anti-doping field: Sample identification and quantification
2021, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Anti-doping analysis requires an exceptional level of accuracy and precision given the stakes that are at play. Current methods rely on the application of chromatographic techniques linked with mass spectrometry to provide this. However, despite the effectiveness of these techniques in achieving good selectivity and specificity, some issues still exist. In order to reach the minimum required performance level as set by WADA, labs commonly use selective monitoring by quadrupole mass spectrometry. This can be potentially fooled through the use of masking agents or by moving the peaks, as often only a small portion of the spectrum is used for analysis. Further issues exist in the inability to detect new or modified compounds, or to reanalyse samples/spectra. One technique that could overcome these problems is that of comprehensive 2D chromatography. Here a second separation column is employed to generate greater separative power. Compared to conventional separation, GCxGC allows for a greater peak capacity (i.e., number of peaks that can be resolved within a given time) and greater separation of coeluting compounds, which makes the technique promising for the complex task required in anti-doping. When combined with Time of Flight Mass Spectrometry this technique demonstrates vast potential allowing for full mass range datasets to be obtained for retroactive analysis. Similarly, LCxLC provides improvements in resolving power compared to its 1D counterpart and can be used both online as part of the analysis or offline solely as a purification step. In this review we summarise the work in this field so far, how comprehensive chromatography has been applied to anti-doping studies, and discuss the future application for this technique.
Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration
2017, Journal of Steroid Biochemistry and Molecular Biology
Testosterone (T) has traditionally been the most commonly reported doping agent by doping control laboratories. The screening of T misuse is performed by the quantification of six endogenous androgenic steroids and the ratio T/E included in the Athlete Biological Passport (ABP). The inclusion of additional metabolites can improve the screening capabilities of ABP. In this study, the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-Andros3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio3G) as markers of T oral administration was evaluated. These glucuronides have been shown to be resistant to enzymatic hydrolysis and their quantification by means of liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was reported as the only way to obtain feasible results. Urine samples were collected from five volunteers before and after the oral administration of 40 mg of T undecanoate and were analyzed by a LC–MS/MS method recently developed. Concentration of 6OH-Andros3G and 6OH-Etio3G compounds and those of the glucuronides of T (TG), epitestosterone (EG), androsterone and etiocholanolone were established and different concentration ratios were calculated. The detection windows (DWs) for the T administration obtained by each selected ratio were compared to the one of TG/EG. The results showed that four out of the nine tested markers presented DWs much larger for all volunteers than those obtained by the World Anti-Doping Agency established T/E marker or other alternative markers. The 6OH-Andros3G/EG, 6OH-Etio3G/EG, 6OH-Andros3G/TG and 6OH-Etio3G/TG markers were able to identify the T abuse up to 96 h after the administration, extending our detection capability for the misuse up to 84 h more than the classic marker. The importance of these markers was also highlighted by their prolonged capacity to detect the T misuse in the case of one volunteer whose TG/EG barely exceeded his individual threshold. As a consequence, the four markers presented in this study seem to have an exceptional potential as biomarkers of T oral administration.
The use of gas chromatography-mass spectrometry/combustion/isotope ratio mass spectrometry to demonstrate progesterone treatment in bovines
2016, Journal of Chromatography A
Currently, no analytical method is available to demonstrate progesterone administration in biological samples collected in rearing animals, and therefore, tracking the abuse of this popular growth promoter is arduous. In this study, a method is presented to reveal progesterone (PG) treatment on the basis of carbon isotope measurement of 5β-pregnane-3α, 20α-diol (BAA-PD), a major PG metabolite excreted in bovine urine, by gas chromatography–mass spectrometry/combustion/isotope ratio mass spectrometry (GC–MS/C/IRMS). 5-Androstene-3β,17α-diol (AEdiol) is used as endogenous reference compound. Intermediate precisions (n = 11) of 0.56‰ and 0.68‰ have been determined for AEdiol and BAA-PD, respectively. The analytical method was used for the very first time to successfully differentiate urine samples collected in treated and untreated animals.
Analytical approach for the determination of steroid profile of humans by gas chromatography isotope ratio mass spectrometry aimed at distinguishing between endogenous and exogenous steroids
2015, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
Thus, the optimization of the conditions used during the sample preparation requires careful selection of solid phase for SPE, buffer used for the stabilization of pH, enzyme used for hydrolysis, organic solvent used for LLE as well as internal standards for MS detection. According to the literature, the SPE was performed on octadecyl sorbent (C18), followed by the use of methanol [6–12], isopropanol [13], ethyl acetate or acetonitryl [14–17] for elution. Enzymatic hydrolysis was performed with β-glucuronidase—Escherichia coli [6–8,12,13] or Helix pomatia [16].
The contamination of commonly used supplements by unknown steroids as well as their metabolites (parent compounds) become a challenge for the analytical laboratories. Although the determination of steroids profile is not trivial because of the complex matrix and low concentration of single compound, one of the most difficult current problem is to distinguish, during analytical procedure, endogenous androgens such as testosterone, dehydrotestosterone or dehydroepiandrosterone from their synthetic equivalents.
The aim of this work was to develop and validate an analytical procedure for determination of the steroid profile in human urine by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) toward distinguishing between endogenous and exogenous steroids. Beside the optimization of the experimental parameters for gas chromatography separation and mass spectrometry, attention was focused on urine sample preparation. Using an optimized sample preparation protocol it was possible to achieve better chromatographic resolutions and better sensitivity enabling the determination of 5 steroids, androsterone, etiocholanolone, testosterone, 5-androstandiol, 11-hydroxyandrdostane, pregnandiol, with the expanded uncertainty (k = 2) below 1‰. This enable to evaluate the significant shift of the δ¹³C/¹²C [‰] values for each of examined steroids (excluding ERC). The analytical protocol described in this work was successfully used for the confirmation of positive founding urine by evaluation T/E ratio after GC/C/IRMS analysis.
Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. A review
2013, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
A T/EpiT ratio greater than 6 strongly suggests T doping. However, subsequent observations indicated that urinary T/EpiT ratio varies a lot among individuals, suggesting an influence of genetic factors [51–54], hypothesis confirmed by comparing the excretion of T, EpiT and other androgens in a large sample of Swedish and Korean people [3]. Studies performed to evaluate how DHEA administration can affect T/EpiT ratio showed a great variability of response [55].
The use of doping agents, once restricted to professional athletes, has nowadays become a problem of public health, since it also concerns young people and non-competing amateurs in different sports. The use is also diffused in social life for improving physical appearance and enhancing performance and even dietary supplements assumed to improve performance often contain anabolic steroids. While decades ago the so-called “classical doping agents” (like stimulants and narcotics) were used, to-day anabolic steroids are more widely diffused. Anabolic steroids are synthetic substances prepared by introducing modifications in the molecular structure of testosterone, the main natural androgenic anabolic steroid that forms in testes interstitial cells. The first report concerning the use of anabolic steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports was banned by the International Olympic Committee and control tests were implemented in 1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a limited number of exogenous steroids. Over the years, always new doping substances are synthesized and, as a consequence, the list of prohibited compounds is continuously updated and new suitable analytical methods for their detection and determination in biological matrices are continuously required. In doping control analysis the knowledge of steroid metabolism pathway in human body is of primary importance and the analytical methods must permit the simultaneous detection and determination not only of the forbidden precursor agents but also of their metabolites. In addition, the potential presence and amount in the biological samples of species that can interfere in the analysis should be evaluated. Also the several anabolic steroids, specifically designed to circumvent doping control, put on the market have been incorporated in the list of the prohibited substances of the World Anti-Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly reminded that assumption of doping agents not only leads to athletes the possible failing of doping tests but causes important health risk and WADA prohibited list establishes criteria to highlight the alteration of the natural steroid profile caused by exogenous administration. Doping control analyses are generally performed in urine, a matrix that provides a prolonged detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To identify the chemical structures of anabolic steroids the use of mass spectrometry detection is very advantageous. Gas chromatography–mass spectrometry (GC–MS) techniques allowed for the development of comprehensive screening methods. GC–MS methods are sensitive and robust but present the disadvantages of time-consuming sample pretreatment, that is often based on hydrolysis and derivatisation reactions. Liquid chromatography–mass spectrometry (LC–MS) methods have been successfully used to identify and determinate steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has become the technique of choice for steroid analysis. Due to its generally higher speed, sensitivity, reproducibility and specificity with respect to HPLC, it can be used to simultaneously separate and determinate multi component steroid mixtures. The technique is of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency enhancement due to the small particle (sub-2 μm) column packing, which provides high peak capacity within analysis times even 5–10 fold shorter than conventional HPLC methods. Modern multiplex instruments can analyze thousands of samples per month so that, notwithstanding the generally high instrumental costs, the cost of the individual assay is affordable. In addition, the improved specificity and resolution offered by time-of-flight or quadrupole time-of-flight mass spectrometry allow their application in doping control analysis or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of the present review is to consider, compare and discuss the applications of the UHPLC/MS methods present in literature for the identification and determination of forbidden steroids and their metabolites in human biological matrices.
Use of isotope ratio mass spectrometry to differentiate between endogenous steroids and synthetic homologues in cattle: A review
2013, Analytica Chimica Acta
Although substantial technical advances have been achieved during the past decades to extend and facilitate the analysis of growth promoters in cattle, the detection of abuse of synthetic analogs of naturally occurring hormones has remained a challenging issue. When it became clear that the exogenous origin of steroid hormones could be traced based on the ¹³C/¹²C isotope ratio of the substances, GC/C/IRMS has been successfully implemented to this aim since the end of the past century. However, due to the costly character of the instrumental setup, the susceptibility of the equipment to errors and the complex and time consuming sample preparation, this method is up until now only applied by a limited number of laboratories. In this review, the general principles as well as the practical application of GC/C/IRMS to differentiate between endogenous steroids and exogenously synthesized homologous compounds in cattle will be discussed in detail, and will be placed next to other existing and to be developed methods based on isotope ratio mass spectrometry. Finally, the link will be made with the field of sports doping, where GC/C/IRMS has been established within the World Anti-Doping Agency (WADA) approved methods as the official technique to differentiate between exogenous and endogenous steroids over the past few years.

View all citing articles on Scopus

View full text

Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols

Abstract

Introduction

Section snippets

Urine samples

Results and discussion

Conclusions

Acknowledgements

J. Chromatogr. B

Steroids

Steroids

Lancet

J. Chromatogr. B

J. Chromatogr.

Steroids

Rapid Commun. Mass Spectrom.