Targeting prohibited substances in doping control blood samples by means of chromatographic–mass spectrometric methods

Urine samples have been the predominant matrix for doping controls for several decades. However, owing to the complementary information provided by blood (as well as serum or plasma and dried blood spots (DBS)), the benefits of its analysis have resulted in continuously increasing appreciation by anti-doping authorities. On the one hand, blood samples allow for the detection of various different methods of blood doping and the abuse of erythropoiesis-stimulating agents (ESAs) via the Athlete Biological Passport; on the other hand, targeted and non-targeted drug detection by means of chromatographic–mass spectrometric methods represents an important tool to increase doping control frequencies out-of-competition and to determine drug concentrations particularly in in-competition scenarios. Moreover, blood analysis seldom requires in-depth knowledge of drug metabolism, and the intact substance rather than potentially unknown or assumed metabolic products can be targeted. In this review, the recent developments in human sports drug testing concerning mass spectrometry-based techniques for qualitative and quantitative analyses of therapeutics and emerging drug candidates are summarized and reviewed. The analytical methods include both low and high molecular mass compounds (e.g., anabolic agents, stimulants, metabolic modulators, peptide hormones, and small interfering RNA (siRNA)) determined from serum, plasma, and DBS using state-of-the-art instrumentation such as liquid chromatography (LC)–high resolution/high accuracy (tandem) mass spectrometry (LC-HRMS), LC–low resolution tandem mass spectrometry (LC-MS/MS), and gas chromatography–mass spectrometry (GC-MS).     

An overview of the doping control analysis during the Olympic Games of 2004 in Athens, Greece

This study summarizes the results obtained from the doping control analysis during the period of the XXVIII summer Olympic Games (30 July-29 August 2004). The analysis of all doping control samples was performed at the Doping Control Laboratory (DCL)—the World Anti-Doping Agency (WADA) Accredited Laboratory of Athens. Three thousand six hundred and seventeen tests were conducted in total throughout the games. In 23 specimens the presence of a prohibited substance was confirmed. Sixteen of those were related to anabolic agents. The screened results were confirmed with various mass spectrometry analytical techniques, such as gas chromatography/high resolution mass spectrometry (GC/HRMS), gas chromatography/mass spectrometry (GC/MS), gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography/mass spectrometry (ion trap) (LC/MS). The results of the first time applied screening and confirmatory procedures for the detection of recombinant human growth hormone in serum were also presented. Besides, 107 therapeutic use exemptions (TUE) were verified for glucocorticosteroid and beta2-agonist use.     

Preventive doping control screening analysis of prohibited substances in human urine using rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometry

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, β₂‐agonists, β‐blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single‐step liquid‐liquid extraction of hydrolyzed urine and the use of a rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4‐methyl‐2‐hexanamine, which resulted in re‐reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives. Copyright © 2010 John Wiley & Sons, Ltd.     

Doping und Dopinganalytik: Wirkstoffe und Methoden

Since 1997, alterations regarding the anti‐doping rules of sport federations have led to the prohibition of new classes of substances such as plasma volume expanders, anti‐estrogens, aromatase inhibitors and artificial oxygen carriers (e.g. perfluorocarbons, cross‐linked hemoglobins) besides the classical doping agents including stimulants, narcotics, anabolic agents, diuretics and peptide hormones. The determination of doping substances, which has been based mainly on GC‐MS procedures, is more and more performed employing LC‐MS and LC‐MS/MS instruments. For instance, the classes of betablockers, diuretics, corticosteroids and newly proteins such as the cross‐linked hemoglobin 'Hemopure' are effectively detected utilizing LC‐MS(/MS) systems. The urinary identification of erythropoietin (EPO)‐doping is accomplished by means of an assay composed by isoelectric focusing with subsequent visualisation of characteristic EPO bands with monoclonal EPO antibodies (double blotting).     

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.     

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.     

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.     

Determination of nandrolone metabolites in human urine: comparison between liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry

Nandrolone (19‐nortestosterone) is an androgenic anabolic steroid illegally used as a growth‐promoting agent in animal breeding and as a performance enhancer in athletics. Therefore, its use was officially banned in 1974 by the Medical Commission of the International Olympic Committee (IOC). Following nandrolone administration, the main metabolites in humans are 19‐norandrosterone, 19‐norethiocolanolone and 19‐norepiandrosterone, and their presence in urine is the basis of detecting its abuse. The present work was undertaken to determine, in human urine, nandrolone metabolites (phase I and phase II) by developing and comparing multiresidue liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) methods. A double extraction by solid‐phase extraction (SPE) was necessary for the complete elimination of the interfering compounds. The proposed methods were also tested on a real positive sample, and they allow us to determine the conjugated/free fractions ratio reducing the risk of false positive or misleading results and they should allow laboratories involved in doping control analysis to monitor the illegal use of steroids. The advantages of LC/MS/MS over GC/MS (which is the technique mainly used) include the elimination of the hydrolysis and derivatization steps: it is known that during enzymatic hydrolysis several steroids can be converted into related compounds and deconjugation is not always 100% effective. The validation parameters for the two methods were similar (limit of quantification (LOQ) <1 ng/mL and percentage coefficient of variance (CV%) <16.4), and both were able to confirm unambiguously all the analytes, thus confirming the validity of both techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

Targeted phase II metabolites profiling as new screening strategy to investigate natural steroid abuse in animal breeding

The use of steroid hormones as growth promoters in cattle is banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. While efficient targeted confirmatory methods have been implemented in control laboratories for many years, fast and reliable screening methods are still required, especially in the case of natural hormones abuse, but more globally for new "fishing" strategies allowing to reveal the use of even unknown anabolic agents. The development of focused profiling or untargeted metabolomic approaches is thus emerging in this context. The present study was a focused profiling study using steroids phase II metabolites, with the aim to get a better understanding of the steroid metabolism disruptions after exogenous administration of androstenedione and finally reveal potential biomarkers signing its administration. A sample preparation procedure was first developed, based on a separation of 31 glucuronide and sulphate conjugate compounds using an anion exchange SPE system. Each fraction was then analysed by UPLC-MS/MS in MRM mode showing a rapid (between 4h and 4 days after treatment) and huge excretion of several direct metabolites of androstenedione such as etiocholanolone-glucuronide or epiandrosterone-sulphate.     

Generic sample preparation combined with high-resolution liquid chromatography–time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories

A unification of doping-control screening procedures of prohibited small molecule substances—including stimulants, narcotics, steroids, β2-agonists and diuretics—is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography–time-of-flight mass spectrometry method and one liquid chromatography–time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, β2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples.     

Introduction to the application of capillary gas chromatography of performance-enhancing drugs in doping control.

Performance-enhancing drugs banned by antidoping rules are detected in doping control preferably by hyphenated chromatographic techniques, capillary gas chromatography in particular. Based on the prohibited classes of substances and on the general aspects of sample collection and preparation, a survey is given about the usual procedures of screening, identification and confirmation of the most important doping agents: stimulants, narcotics, anabolics, diuretics, beta-blockers. In addition to gas chromatography itself, the application of various MS techniques doping is outlined.     

High resolution separation methods for the determination of intact human erythropoiesis stimulating agents. A review

Human erythropoietin (hEPO), a hormone involved in the formation of red blood cells, is a 30 kDa glycoprotein with a high carbohydrate content. The production of recombinant hEPO has made possible its widespread therapeutic use and its banned use in competition sports. Methods to analyze EPO and other erythropoiesis stimulating agents (ESAs) are necessary for the characterization and quality control of these biopharmaceuticals and also for doping control. In this paper, high resolution separation methods, namely high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), with special attention to CE-coupled mass spectrometry, are reviewed. The usefulness of these techniques when applied in different modes to separate the glycoprotein isoforms, aggregates or excipients are detailed. In addition, sample preparation methods that have been applied to ESA samples for subsequent determination by HPLC or CE, as well as the potential compatibility of other preparation methods, are discussed. Applications of the HPLC and CE methods regarding regulatory considerations for biopharmaceuticals analysis, with emphasis on biosimilars, and doping control are also included. Finally, limitations of the present methods and their possible solutions are considered.     

Identification of the aromatase inhibitor aminoglutethimide in urine by gas chromatography/mass spectrometry

Aminoglutethimide is used therapeutically as an aromatase inhibitor in the treatment of metastatic breast cancer in post-menopausal women. For doping purposes, aminoglutethimide may be used for treatment of adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase the testosterone concentration and stimulation of testosterone biosynthesis. The use of aromatase inhibitors has been prohibited for male athletes since September 1, 2001. The purpose of this study was to develop methods for the identification of the parent compound or its main metabolite and the inclusion of this information into established screening procedures in doping analysis. An excretion study was conducted using oral application of one single therapeutic dose (500 mg) of Orimeten. The analysis was performed by gas chromatography/mass spectrometry (GC/MS). Aminoglutethimide is excreted almost totally as unconjugated parent compound and is detectable by different screening procedures for up to 165 h. Most suitable for the detection of aminoglutethimide is the screening procedure for heavy volatile nitrogen-containing drugs ('Screening 2'). However, since only competition samples are analysed in that screening procedure, the additional inclusion of aminoglutethimide in the screening procedure for anabolic androgenic agents ('Screening 4') is recommended. Full mass spectra and diagnostic ions for the analysis of aminoglutethimide are presented.     

Fast screening of anabolic steroids and other banned doping substances in human urine by gas chromatography/tandem mass spectrometry

A fast and sensitive method for the comprehensive screening of anabolic agents and other banned doping substances using gas chromatography/tandem mass spectrometry (GC/MS/MS) with an external ionization ion trap mass spectrometer is presented. The method takes advantage of the resolving power of MS/MS to eliminate background interferences, thus speeding up the chromatographic analysis. For each compound, different fragmentation reactions were studied and their collision energies optimized to obtain the best sensitivity in terms of their signal-to-noise ratio (S/N). A dramatic reduction in overall analysis time was achieved compared with other common approaches. More than 50 substances could finally be monitored in less than 7.4 min with detection limits (S/N >3) lower than 0.5 ng ml(-1) for most of the compounds with special sensitivity requirements according to the International Olympic Committee (IOC). A validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the analytes. Good intermediate precision, below 25% for most of the compounds, and robustness were observed. The optimized method was successfully applied to analyse more than 100 real human urine samples with optimum sensitivity and specificity rates.     

Anabolic, doping, and lifestyle drugs, and selected metabolites in wastewater—detection, quantification, and behaviour monitored by high-resolution MS and MS n before and after sewage treatment

Municipal wastewater has been examined for steroids, β₂-agonists, stimulants, diuretics, and phosphodiesterase type V inhibitors (PDE type V inhibitors), which are “dual-use-drugs” applied either as anabolic, doping, and lifestyle drugs or for treatment of diverse diseases. To identify their origin, fitness centre discharges under suspicion of being point sources and sewage-treatment plant feed and effluents were sampled and concentrations determined. Sensitive and selective methods for determination and quantification based on solid-phase extraction (SPE) followed by high-performance liquid chromatography–high resolution mass and tandem mass spectrometry (HPLC–(HR)MS and HPLC–MS–MS) were developed and established for analysis of these compounds in wastewater and to assess their effect on the environment. The methods developed enabled quantification at trace concentrations (limit of quantification (LOQ): 5 ng L⁻¹). Of the steroids and stimulants under investigation, testosterone, methyltestosterone, and boldenone or ephedrine, amphetamine, and MDMA (3,4-methylendioxy-N-methylamphetamine) were observed at up to 5 μg L⁻¹ (ephedrine). Of the β₂-agonists salbutamol only, and of the diuretics furosemide and hydrochlorothiazide were confirmed in the extracts. Quite high concentrations of the PDE type V inhibitors sildenafil, tadalafil, and vardenafil and their metabolites were confirmed in fitness centre discharges (sildenafil: 1,945 ng L⁻¹) whereas their concentrations in municipal wastewater did not exceed 35 ng L⁻¹. This study identified anabolic and doping drugs in wastewater for the first time. Results obtained from wastewater treatment plant effluents proved that these “dual-use-drugs”, with the exception of hydrochlorothiazide, were mostly eliminated.     

A fast, comprehensive screening method for doping agents in urine by gas chromatography-triple quadrupole mass spectrometry

The use of performance enhancing drugs in sports is prohibited. For the detection of misuse of such substances gas chromatography or liquid chromatography coupled to mass spectrometry are the most frequently used detection techniques. In this work the development and validation of a fast gas chromatography tandem mass spectrometric method for the detection of a wide range of doping agents is described. The method can determine 13 endogenous steroids (the steroid profile), 19-norandrosterone, salbutamol and 11-nor-Δ9-tetrahydrocannabinol.9carboxylic acid in the applicable ranges and to detect qualitatively over 140 substances in accordance with the minimum required performance levels of the World Anti-Doping Agency in 1ml of urine. The classes of substances included in the method are anabolic steroids, β2-agonists, stimulants, narcotics, hormone antagonists and modulators and beta-blockers. Moreover, using a short capillary column and hydrogen as a carrier gas the run time of the method is less than 8min.     

A high-throughput multicomponent screening method for diuretics, masking agents, central nervous system (CNS) stimulants and opiates in human urine by UPLC-MS/MS

A simple and rapid multicomponent screening method of 130 substances for direct injections of urine samples has been developed. The fully automated method based on ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) is used for three different classes of doping agents: diuretics, central nervous system stimulants (CNS stimulants) and opiates. The samples are diluted with buffer containing internal standards (IS) by a pipetting robot system into 96-well plates. Samples are injected on a reversed phase sub 2-microm particle column connected to a fast polarity switching and rapid scanning tandem mass spectrometer with an electrospray interface. The software used to evaluate the results produced reports containing a small-sized window for each component and a data table list with flags to indicate any adverse analytical findings in the sample. The report can also be processed automatically using an application software, which interpret the data and indicate if there is a suspicious sample. One 96-well plate can be analyzed within 16 h.     

‘Wrong‐way‐round ionization’ and screening for doping substances in human urine by high‐performance liquid chromatography/orbitrap mass spectrometry

To free analytical resources for new classes of doping substances, such as banned proteins, maximization of the number of compounds that can be determined with high sensitivity in a single run is highly urgent. This study demonstrates an application of ‘wrong‐way‐round ionization’ for the simultaneous detection of multiple classes of doping substances without the need to switch the polarity. A screening method for the detection of 137 compounds from various classes of prohibited substances (stimulants, diuretics, β₂‐agonists, β‐blockers, antiestrogens, glucocorticosteroids and anabolic agents) has been developed. The method involves an enzymatic hydrolysis, liquid–liquid extraction and detection by liquid chromatography/orbitrap mass spectrometry with wrong‐way‐round ionization. Up to 64% of compounds had a 10‐fold lower limit of detection (LOD) than the minimum required performance limit. To compare the efficiency of conventional ionization relative to wrong‐way‐round ionization of doping substances in + ESI, a fortified blank urine sample at the minimum required performance limit was analyzed using two ESI approaches. All compounds were detected with markedly better S/N in a high‐pH mobile phase, with the exception of acetazolamide (minimal change in S/N, < 20%).The method was validated by spiking 10 different blank urine samples at five different concentrations. Validation parameters included the LOD, selectivity, ion suppression, extraction recovery and repeatability. Copyright © 2012 John Wiley & Sons, Ltd.     

Analytical challenges in doping control: Comprehensive two-dimensional gas chromatography with time of flight mass spectrometry,a promising option

Doping control screening based on the enhanced resolution of comprehensive two-dimensional (2D) gas chromatography hyphenated to time of flight mass spectrometer was investigated. The identification of anabolic agents (clenbuterol, norandrosterone, epimetendiol, two methyltestosterone metabolites and 3′-hydroxystanozolol) contained in a spiked urine sample (2 ng/ml) was demonstrated. Special emphasis was given to 3′-hydroxystanozolol, mainly considering the difficulty in its detection. In contrast to conventional GC–MS approaches that must use single-ion monitoring, the GC × GC–TOFMS method enabled the identification of that metabolite through the deconvolution of the full mass spectrum and also resolved the co-eluted peaks of 3′-hydroxystanozolol and an endogenous component.     

High- and low-resolution mass spectrometry coupled to liquid chromatography as confirmatory methods of anabolic residues in crude meat and infant foods

Within the European Union the use of anabolic steroids for promoting growth and improving meat-to-fat ratio in food-producing animals has been banned since 1988. For the unequivocal identification of hormone residues in a complex matrix such as meat we have developed a rapid, specific and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, in combination with a simple extraction procedure based on the matrix solid-phase dispersion (MSPD). The performances of a triple quadrupole (QqQ) and a quadrupole/time-of-flight (QqTOF) were compared: the QqQ mass spectrometer was found to be more sensitive for almost all studied analytes, but the selectivity was superior using the QqTOF system; the full-scan spectra (acquired without losing sensitivity), mass accuracy and resolution of the hybrid instrument enabled a more probatory analyte identification than that obtained selecting two multiple-reaction monitoring (MRM) transitions with a QqQ. Average recoveries ranged from 80 to 100%, and the detection capabilities (CCbetas) were less than 1.06 ppb with the QqQ instrument and less than 5.20 ppb with the QqTOF instrument for the bovine meat, which proved to be the most complex matrix.