17. Detection/ Analysis of Anabolic Steroids

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17. Detection/ Analysis of Anabolic Steroids

 

 

 

CATEGORY: Anabolic Steroids 100 Courses

COURSE NUMBER: 17

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Syllabus

ABSTRACT 3

ACKNOWLEDGEMENTS 5

CONTENTS 6

LIST OF ORIGINAL PUBLICATIONS 8

ABBREVIATIONS 10

1 REVIEW OF THE LITERATURE 12

1.1 Anabolic-androgenic steroids (AAS) 12

1.1.1 Origin and effects 12

1.1.2 Clinical use and abuse in sports 14

1.1.3 Structure and nomenclature 14

1.1.4 Human metabolism 15

1.1.5 Anabolic steroid glucuronides 17

1.2 Analysis of AAS 19

1.2.1 Requirements 19

1.2.2 Sample matrixes 20

1.2.3 Sample preparation 20

1.2.4 Analytical methods 22

1.2.4.1 GCMS 22

1.2.4.2 LCMS 25

2 AIMS OF THE STUDY 28

3 MATERIALS AND METHODS 29

3.1 Reagents and solvents 29

3.2 Steroids and steroid glucuronides 29

3.3 Production and characterization of steroid glucuronides 33

3.4 Urine samples and sample preparation 34

3.5 LCMS analysis of conjugated steroid glucuronides 34

3.5.1 Validation and inter-laboratory comparison 35

3.6 GCMS analysis of anabolic steroids 36

4 RESULTS AND DISCUSSION 38

4.1 Production of AAS glucuronides 38

4.1.1 Enzyme assisted synthesis 38

4.1.2 Characterization 40

4.2 Sample preparation 41

4.3 Development of LCESI-MS/MS method for AAS glucuronides 41

4.3.1 LC separation of AAS glucuronides 41

Detection/ Analysis of Anabolic Steroids


A method is described for analysis of multi-component mixtures of steroid metabolites in biological fluids by means of capillary gas chromatography with glass and fused-silica columns and simultaneous detection of methoxylamine-trimethylsilyl derivatives with universal flame-ionization and selective nitrogen alkali flameionization detectors. A data system was applied to the on-line treatment of the results. Computer programs were designed for precise calculation of Kováts retention indices from the known values for selected natural urinary steroids. The programs allow the selection of nitrogen-containing components, normalized chromatogram plotting for both detection channels and qualitative and quantitative analysis. Results are presented on the detection of metabolites of methandrostenolone, 17 alpha-methyltestosterone, 19-nortestosterone and fluoxymesterone.


Anabolic steroids are banned substances in equine sports. Gas chromatography‐mass spectrometry (GC‐MS) has been the traditional technique for doping control analysis of anabolic steroids in biological samples. Although liquid chromatography‐mass spectrometry (LC/MS) has become an important technique in doping control, the detection of saturated hydroxysteroids by LC‐MS remains a problem due to their low ionization efficiency under electrospray. The recent development in fast‐scanning gas‐chromatography‐triple‐quadrupole mass spectrometry (GC‐MS/MS) has provided a better alternative with a significant reduction in chemical noise by means of selective reaction monitoring. Herein, we present a sensitive and selective method for the screening of over 50 anabolic steroids in equine urine using gas chromatography‐tandem mass spectrometry.


Anabolic steroids are prohibited substances in both equestrian and human sports events as specified in Article 6 of the International Agreement on Breeding, Racing, and Wagering published by the International Federation of Horseracing Authorities (IFHA) and the World Anti‐Doping Agency (WADA) Prohibited List, respectively. The effective screening of anabolic steroids in doping control is critical, as according to the recently published Anti‐Doping Testing Figures Report by WADA, anabolic agents are still the most reported misused prohibited substances amongst all substance groups. The conventional method for the detection of anabolic steroids in biological samples is by the use of gas chromatography‐mass spectrometry (GC‐MS). Liquid chromatography‐mass spectrometry (LC‐MS) has become a compatible technique for doping control testing in sports in recent years as LC‐MS has the ability to handle heat labile and large biomolecules, with faster instrument turnaround time and better sensitivity. Although LC‐MS has been applied successfully to the doping control testing of many anabolic steroids in biological matrices, the detection of saturated hydroxysteroids or oxosteroids remains a problem as they exhibit low ionization efficiency under electrospray ionization (ESI). Derivatization by different classes of reagents such as the introduction of a permanently charged moiety, 2‐hydrazino‐1‐methylpyridine into some oxosteroids, or the use of dansyl chloride to incorporate one or more dansyl moieties (with high proton‐affinity) into some hydroxysteroids, have been reported to improve the ESI efficiency of some saturated hydroxy and oxosteroids. The authors have reported an LC‐MS screening method incorporating a derivatization step with the use of a pyridine‐carboxylate analogue, picolinic acid, for the detection of over 30 anabolic steroids in equine urine. The recent development in fast‐scanning gas‐chromatography‐triple‐quadrupole mass spectrometry (GC‐MS/MS) has provided a better alternative with a significant reduction in chemical noise by means by selective reaction monitoring (SRM). The use of GC‐MS/MS fills in the gap for doping analysis of anabolic steroids which exhibits low sensitivity by GC‐MS or low ionization efficiency under ESI by LC‐MS. This paper describes the first reported GC‐MS/MS screening method for the detection of over 50 anabolic steroids in equine urine as well as the determination of testicular steroid markers in intact male horse within a single run. Validation results showed that this method has sufficient sensitivity and robustness to be used as a regular qualitative screening method.


Methasterone is a designer anabolic steroid that is prohibited for athletes and is monitored by anti-doping laboratories. In this work, our objective is to discover new human phase I metabolites, define their excretion kinetics for 30 days and analyze their phase II metabolism (sulfate, cysteine and N-acetylcysteine conjugates). Urine samples from four volunteers were analyzed by chromatographic techniques. Through gas chromatography coupled to mass spectrometry analysis it was possible to detect methasterone and its nine phase I metabolites in the urine samples after glucuronide enzymatic hydrolysis, from which one were previously unreported. These nine compounds were not excreted in free form. The new proposed metabolite is 17β-hydroxy-2α,17α-dimethyl-5β-androstan-3-one, obtained from the epimerization at C5. The 3α-hydroxy metabolite, currently monitored by anti-doping laboratories, was the most abundant and was detected for the longest time. Furthermore, four other long-term metabolites were identified. By ultra-performance liquid chromatography coupled to tandem mass spectrometry, only the drug and a known metabolite were detected after glucuronide hydrolysis, and phase II metabolites were not found. Thus, our results contribute to elucidating methasterone metabolism, including long-term metabolites besides of the 3α-hydroxy in routine doping analysis, which is very important due to variation in human metabolism.
Recently developed radioimmunoassays (RIA) for the analysis of anabolic steroids and their metabolites in biological fluids were tested for cross-reactivity with other types of steroids. Results show that the degree of desirable cross-reactivity within the two classes or orally active anabolic steroids vary widely and that the antiserum for 19-Nortestosterone (the active principle of intramuscular preparations) has a very high degree of undesirable cross-reactivity with components of oral contraceptives. Single and multiple dose studies in human volunteers demonstrate that the detection level and degree of retrospectivity are likewise variable but that the test easily detects most anabolic steroids during treatment. At the present time, the combination of the three antisera for the assay of a sample appears to be a relatively rapid and economic method for screening large numbers of samples in situations where doping control of anabolic steroids is required. The importance of utilizing physico-chemical means for identification of RIA potential positives is emphasized.
In the early 1950s, attention began to focus on the potential use of hormones in sport with the allegation that Soviet weightlifters were using testosterone to increase their strength (Todd, 1987). During the 1960s, many pharmaceutically licensed chemical analogues of testosterone were released on to the market, these analogues being designed to enhance the anabolic effect for clinical purposes. However, competitors also desired these drugs, wanting the muscle building effects of testosterone but without the androgenic effects. By 1968, anabolic steroid use was rife; for example, it was estimated at that time that a third of the US track and field team had used steroids. In 1967, the International Olympic Committee (IOC) re-established a medical commission, which banned the practice of doping in sport and introduced a drug testing programme in 1968.

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