Chromatography-Based Determination of Anabolic Steroids in Biological Fluids: Future Prospects Using Electrochemistry and Miniaturized Microchip Device

Anabolic steroids form one of the important classes of doping agents and their consumption has been found to give benefits to the athletes. Inspite of the fact that they also produce adverse effects and damage several organs and systems, their consumption is continuously increasing in competitive games. The World Anti-Doping Agency and International Olympic Committee have banned the use of anabolic steroids and several other compounds. This article focuses an over view of various chromatographic techniques commonly used for detecting anabolic steroids and their metabolites during last 3 years in human biological fluids to establish the cases of doping. The possibility of electrochemistry- and microchip-based techniques have been considered as possible techniques for future to achieve simple and fast analysis at the site of competitive games as the first tool to detect the cases of doping.

     

Nutritional supplements cross-contaminated and faked with doping substances

Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods (GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances. These investigations showed that nutritional supplements contained prohibited stimulants as ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were purchased in 13 different countries showed that about 15% of the nonhormonal nutritional supplements were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products intentionally faked with high amounts of 'classic' anabolic steroids such as metandienone, stanozolol, boldenone, dehydrochloromethyl-testosterone, oxandrolone etc. have been detected on the nutritional supplement market. These anabolic steroids were not declared on the labels either. The sources of these anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-contaminations of stanozolol and metandienone. Since 2002 new 'designer' steroids such as prostanozol, methasterone, androstatrienedione etc. have been offered on the nutritional supplement market. In the near future also cross-contaminations with these steroids are expected. Recently a nutritional supplement for weight loss was found to contain the beta2-agonist clenbuterol. The application of such nutritional supplements is connected with a high risk of inadvertent doping cases and a health risk. For the detection of new 'designer' steroids in nutritional supplements, mass spectrometric strategies (GC/MS, LC/MS/MS) are presented.     

Simultaneous Doping Analysis of Main Urinary Metabolites of Anabolic Steroids in Horse by Ion-trap Gas Chromatography-Tandem Mass Spectrometry

The use of anabolic steroids in racehorses is strictly regulated. We have developed a method for the simultaneous analysis of 11 anabolic steroids: fluoxymesterone, 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, oxymetholone, boldenone, furazabol, methenolone, nandrolone, and stanozolol, for possible application to a doping test in racehorses. We selected 15 kinds of target substances for a doping test from the main metabolites of these anabolic steroids, and established a method for simultaneous analysis. Urine was hydrolyzed and subjected to solid-phase extraction. Then, the residue from the extracts was derivatized by trimethylsilylation. The derivatized samples were subjected to ion-trap gas chromatography-tandem mass spectrometry, and their mass chromatograms and product ion spectra were obtained. The limit of detection of the target substances was 5 - 50 ng/mL, and the mean recovery and coefficient of variation were 71.3 - 104.8% and 1.1 - 9.5%, respectively.     

Investigations into the feasibility of routine ultra high performance liquid chromatography–tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids,anabolic steroid esters and related compounds

The detection of the abuse of anabolic steroids in equine sport is complicated by the endogenous nature of some of the abused steroids, such as testosterone and nandrolone. These steroids are commonly administered as intramuscular injections of esterified forms of the steroid, which prolongs their effects and improves bioavailability over oral dosing. The successful detection of an intact anabolic steroid ester therefore provides unequivocal proof of an illegal administration, as esterified forms are not found endogenously. Detection of intact anabolic steroid esters is possible in plasma samples but not, to date, in the traditional doping control matrix of urine. The analysis of equine mane hair for the detection of anabolic steroid esters has the potential to greatly extend the time period over which detection of abuse can be monitored.     

Statistical discrimination of steroid profiles in doping control with support vector machines

Due to their performance enhancing properties, use of anabolic steroids (e.g. testosterone, nandrolone, etc.) is banned in elite sports. Therefore, doping control laboratories accredited by the World Anti-Doping Agency (WADA) screen among others for these prohibited substances in urine. It is particularly challenging to detect misuse with naturally occurring anabolic steroids such as testosterone (T), which is a popular ergogenic agent in sports and society.     

Ionization of anabolic steroids by adduct formation in liquid chromatography electrospray mass spectrometry

The ionization of 46 anabolic steroids has been studied. The absence of basic or acidic moieties in most of these analytes makes their direct ionization as [M + H]+ by atmospheric pressure interfaces difficult. The formation of adducts with different components of the mobile phase has been found to be an efficient way to ionize anabolic steroids by electrospray. Different mobile phases using methanol (MeOH) or acetonitrile as organic solvent and HCOOH, Na+ or NH4+ as additives have been tested to favor the adduct formation. A direct correlation between the chemical structure of the anabolic steroid and the possibility to ionize it in a particular chromatographic condition has been found. According to their ionization, anabolic steroids can be divided into seven different groups depending on both the nature and the relative position of their functional groups. The formation of different adducts such as [M + Na + MeOH]+ or [M + H + CH3 CN - H2O]+ is required in order to ionize some of these groups and the optimal mobile phase composition for each group of anabolic steroids is proposed. Despite the ionization limitations due to their chemical structure, most of tested anabolic steroids could be ionized using the adduct formation approach.     

Metabolism of some anabolic agents: toxicological and analytical aspects

The metabolism of the animal growth promotants diethylstilbestrol, zeranol and 17 beta-trenbolone and of a few anabolizing steroids used in humans is briefly reviewed. The possible role of reactive metabolic intermediates in the toxicity of some anabolic agents is discussed. Analytical implications of the metabolism of anabolizing agents are described and examples of the analysis of metabolites by means of recently developed techniques are given. It is proposed to utilize the covalent binding of reactive metabolites of anabolic compounds to blood proteins such as haemoglobin and serum albumin for retrospective doping analysis.     

High amounts of 17-methylated anabolic-androgenic steroids in effervescent tablets on the dietary supplement market

In numerous studies it has been demonstrated that several nutritional supplements contain prohormones not declared on the label. In the current study two products (effervescent tablets) containing high amounts of the 17-methylated anabolic androgenic steroids metandienone (product 1: 16.8 mg/tablet) and stanozolol (product 2: 14.5 mg/tablet) were identified. Additionally in both products norandrostenedione was detected, in product 2 with minor amounts of several other steroids. The substances identified can cause enormous health risks. In addition, the use of the analyzed tablets can lead to positive doping results for metabolites of the respective steroids in sports. This study again shows the insufficient surveillance of the production and trade of dietary supplements. Consumers should be aware of the enormous health and doping risks connected with the use of such products. For GC-MS identification of the analytes the trimethylsilyl derivatives of the steroids and the mixed N-t-butyldimethylsilyl,O-trimethylsilyl derivatives were used. The quantitation of metandienone, norandrostenedione, and stanozolol was performed using HPLC-DAD.     

Analytical methods for anti-doping control in sport: anabolic steroids with 4,9,11-triene structure in urine

Doping control in sport is mandatory to detect and to control the use of prohibited substances. Due to the growing number of targets, the analysis of doping compounds and their metabolites is carried out using established screening methods. However, detection of anabolic steroids with 4,9,11-triene structure in urine is problematic, so it is necessary to improve the methods.We review the state of the art in doping-control analysis of 4,9,11-trien-3-one steroids, providing an overview of the screening and confirmatory methods developed for these analytes in human urine. First, we review chromatographic techniques. We discuss difficulties in the derivatization of those compounds prior to gas chromatography analyses. In recent years, liquid chromatography has been the preferred technique in drug testing in sport, due to the reduced sample pre-treatment, improved limits of detection and comprehensiveness. We also report on advances and limitations of immunochemical techniques for the analysis of this group of substances.     

Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography–tandem mass spectrometry

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography–tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1′‐carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H]⁺ signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M‐CO₂+H]⁺ and [M‐ΙmCO₂+H]⁺ as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti‐Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples.     

类固醇兴奋剂分析方法的研究进展

蛋白同化雄性类固醇是一类滥用最为普遍的兴奋剂物质,对其进行有效的控制和检测关系到运动员的身心健康和体育比赛的公平公正。对类固醇兴奋剂分析方法的改进和发展是目前兴奋剂检测的重要任务。本文主要是对自2002年以来类固醇兴奋剂样品的预处理和检测手段的研究进展做一概述,包括气相色谱-质谱法、液相色谱-质谱法、免疫法、电化学方法以及质谱法等。     

Emerging drugs: mechanism of action,mass spectrometry and doping control analysis

The number of compounds and doping methods in sports is in a state of constant flux. In addition to ‘traditional’ doping agents, such as anabolic androgenic steroids or erythropoietin, new therapeutics and emerging drugs have considerable potential for misuse in elite sport. Such compounds are commonly based on new chemical structures, and the mechanisms underlying their modes of action represent new therapeutic approaches arising from recent advances in medical research; therefore, sports drug testing procedures need to be continuously modified and complementary methods developed, preferably based on mass spectrometry, to enable comprehensive doping controls. This tutorial not only discusses emerging drugs that can be categorized as anabolic agents (selective androgen receptor modulators, SARMs), gene doping [hypoxia‐inducible factor stabilizers, peroxisome‐proliferator‐activated receptor (PPAR)δ‐agonists] and erythropoietin‐mimetics (Hematide) but also compounds with potentially performance‐enhancing properties that are not classified in the current list of the World Anti‐Doping Agency. Compounds such as ryanodine‐calstabin‐complex modulators (benzothiazepines) are included, their mass spectrometric properties discussed, and current approaches in sports drug testing outlined. Copyright © 2009 John Wiley & Sons, Ltd.     

Preliminary study for simultaneous detection and quantification of androgenic anabolic steroids using ELISA and pattern recognition techniques

A first step towards the multidetection, identification and quantification of anabolic androgenic steroids by enzyme-linked immunosorbent assay (ELISA) has been performed in this study. This proposal combines multiple competitive ELISA assays with different cross-reactivity profiles and multivariate data analysis techniques. Data have been analyzed by principal component analysis in conjunction with a novel K-nearest line classifier. This proposal allows simultaneous detection of up to four different steroids in the range of concentration from 0.1 to 316.2 nM with a total rate of 90.6% of correct detection, even in the presence of cross-reactivities. A methodology for concentration prediction is also presented with satisfactory results.     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

An LC-MS screening method with library identification for the detection of steroids in dietary supplements

For many years anabolic-androgenic steroids (AAS) are by far the most frequently detected pharmacological substances in doping control. In order to improve their performances, professional sportsmen are often tempted to take dietary supplements. However, due to the frequent and widespread occurrence of contaminated supplements, the use of such products is not without risk for the athletes involved. In order to minimize the chances of an unattended positive doping test or serious health problems, fast and reliable screening methods for the detection of anabolic steroids in dietary supplements are needed. A general screening procedure requires the fast and unambiguous detection of a large range of steroids. Gas chromatography-mass spectrometry (GC-MS) has been used intensively in the detection of doping substances for the past 40 years. Over time, many laboratories have delivered spectra to be included in standard reference databases, one of which is maintained by the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). In recent years, however, liquid chromatography coupled to mass spectrometry (LC-MS) has gained popularity. Unfortunately, existing GC-MS libraries are not applicable to LC-MS analysis. In the present study, a new mass spectral library of 88 steroids was developed, along with a fast UPLC-MS method. For the construction of this mass spectral library, three different mass spectra were measured for each steroid, with a sample cone voltage of 30, 60 and 100 V, respectively. This method was then successfully tested on contaminated dietary supplements which had previously been tested by means of a targeted LC-MS/MS method. Overall, the library search was shown to identify the same compounds as the MRM method.     

Direct analysis of anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption-dielectric barrier discharge ionization mass spectrometry

Rapid detection of trace level anabolic steroids in urine is highly desirable to monitor the consumption of performance enhancing anabolic steroids by athletes. The present article describes a novel strategy for identifying the trace anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption (LPTD) coupled to dielectric barrier discharge (DBD) ionization mass spectrometry. Using this method the steroid molecules are enriched within a liquid droplet during the thermal desorption process and desorbed all-together at the last moment of droplet evaporation in a short time domain. The desorbed molecules were ionized using a dielectric barrier discharge ion-source in front of the mass spectrometer inlet at open atmosphere. This process facilitates the sensitivity enhancement with several orders of magnitude compared to the thermal desorption at a lower temperature. The limits of detection (LODs) of various steroid molecules were found to be in the range of 0.05–0.1 ng mL⁻¹ for standard solutions and around two orders of magnitude higher for synthetic urine samples. The detection limits of urinary anabolic steroids could be lowered by using a simple and rapid dichloromethane extraction technique. The analytical figures of merit of this technique were evaluated at open atmosphere using suitable internal standards. The technique is simple and rapid for high sensitivity and high throughput screening of anabolic steroids in urine.     

Liquid chromatographic and spectral analysis of the 17-hydroxy anabolic steroids

The liquid chromatographic properties of various 17-hydroxy anabolic steroids are examined under reversed-phase conditions. These anabolic steroids are now listed as controlled drugs in many states due to their abuse potential in athletics, body building, and other areas. These nonesterified steroids are separated on a C18 stationary phase with a 70% methanol in water mobile phase. In a few cases, two compounds display very similar retention properties. However, dual-wavelength detection at 254 and 280 nm allows for their differentiation. Reversed-phase retention parallels steroid lipophilicity based on hydroxyl and methyl group substituents. Also, those steroids containing a dienone substructure are more polar than steroids containing an enone moiety.     

Feasibility of gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry in analysis of anabolic steroids

Mass spectrometers equipped with atmospheric pressure ion sources (API-MS) have been designed to be interfaced with liquid chromatographs (LC) and have rarely been connected to gas chromatographs (GC). Recently, we introduced a heated nebulizer microchip and showed its potential to interface liquid microseparation techniques and GC with API-MS. This study demonstrates the feasibility of GC-microchip atmospheric pressure photoionization-tandem mass spectrometry (GC-μAPPI-MS/MS) in the analysis of underivatized anabolic steroids in urine. The APPI microchip provides high ionization efficiency and produces abundant protonated molecules or molecular ions with minimal fragmentation. The feasibility of GC-μAPPI-MS/MS in the analysis of six selected anabolic steroids in urine samples was studied with respect to intra-batch repeatability, linearity, linear range, and limit of detection (LOD). The method showed good sensitivity (LODs 0.2-1 ng/mL), repeatability (relative standard deviation<10%), and linearity (regression coefficient≥0.9995) and, therefore, high potential for the analysis of anabolic steroids. Quantitative performance of the method was tested with two authentic urine samples, and the results were in good agreement with those obtained with conventional GC-electron ionization-MS after derivatization.