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.     

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.     

Mass spectrometric analysis of pharmaceutical adulterants in products labeled as botanical dietary supplements or herbal remedies: a review

The increased availability and use of botanical dietary supplements and herbal remedies among consumers has been accompanied by an increased frequency of adulteration of these products with synthetic pharmaceuticals. Unscrupulous producers may add drugs and analogues of various classes, such as phosphodiesterase type 5 (PDE-5) inhibitors, weight loss, hypoglycemic, antihypertensive and anti-inflammatory agents, or anabolic steroids, to develop or intensify biological effects of dietary supplements or herbal remedies. The presence of such adulterated products in the marketplace is a worldwide problem and their consumption poses health risks to consumers. Analytical methods that allow rapid and reliable testing of dietary supplements for the presence of synthetic drugs are needed to address such fraudulent practices. Mass spectrometry (MS) and hyphenated techniques such as liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) have become primary tools in this endeavor. The present review critically assesses the role and summarizes the applications of MS in the analysis of pharmaceutical adulterants in botanical dietary supplements and herbal remedies. The uses of MS techniques in detection, confirmation, and quantification of known pharmaceutical adulterants as well as in screening for and structure elucidation of unexpected adulterants and novel designer drugs are discussed.     

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC-MS3)

Within the scope of the European Community member states' residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC-MS(n) analysis was no longer required. Analytical results could be obtained within 1 day and only 5 mL urine was needed to carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved. A higher GC-MS technique's specificity was achieved by detecting the steroids using GC-MS3. Nevertheless, it was decided to screen routinely sampled urine with GC-MS2 whereas GC-MS3 was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.     

Comparison between triple quadrupole, time of flight and hybrid quadrupole time of flight analysers coupled to liquid chromatography for the detection of anabolic steroids in doping control analysis

Triple quadrupole (QqQ), time of flight (TOF) and quadrupole-time of flight (QTOF) analysers have been compared for the detection of anabolic steroids in human urine. Ten anabolic steroids were selected as model compounds based on their ionization and the presence of endogenous interferences. Both qualitative and quantitative analyses were evaluated. QqQ allowed for the detection of all analytes at the minimum required performance limit (MRPL) established by the World Anti-Doping Agency (between 2 and 10 ng mL(-1) in urine). TOF and QTOF approaches were not sensitive enough to detect some of the analytes (3'-hydroxy-stanozolol or the metabolites of boldenone and formebolone) at the established MRPL. Although a suitable accuracy was obtained, the precision was unsatisfactory (RSD typically higher than 20%) for quantitative purposes irrespective of the analyser used. The methods were applied to 30 real samples declared positives either for the misuse of boldenone, stanozolol and/or methandienone. Most of the compounds were detected by every technique, however QqQ was necessary for the detection of some metabolites in a few samples. Finally, the possibility to detect non-target steroids has been explored by the use of TOF and QTOF. The use of this approach revealed that the presence of boldenone and its metabolite in one sample was due to the intake of androsta-1,4,6-triene-3,17-dione. Additionally, the intake of methandienone was confirmed by the post-target detection of a long-term metabolite.     

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.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements

A sensitive and selective method for the screening of 28 different compounds including testosterone and prohormones, nandrolone and prohormones, stanozolol and metandienone in solid nutritional supplements is described and validated. The different substances are extracted from the solid nutritional supplements by liquid-liquid extraction with a mixture of pentane and freshly distilled diethylether (9/1) after dissolving the supplement in NaOH (1 N). The anabolizing agents are derivatized with a mixture of MSTFA/NH(4)I/ethanethiol (320/1/2), routinely used for the derivatization of anabolic steroids extracted from urine. The TMS-derivatives are analysed by GC-MS in the SIM mode. The limits of detection were in the range from 2 to 40 ng/g. One supplement was analysed with this method and was found to contain several forbidden substances according to IOC doping regulations. All detected compounds, except dihydrotestosterone, could be confirmed with GC-MS(2), proving that the proposed method is suitable for the screening of anabolizing agents in solid nutritional supplements.     

Case Study: Doping Substances in Equestrian Food Supplements

In the course of investigations on equestrian supplemental products for the presence of doping substances, two products were found to contain forbidden substances. As reported earlier a plant extract (Mexican cactus extract) named “Energy 5” contained the anabolic androgenic steroids (AAS) stanozolol, 17β-hydroxy-17α-methyl-5α-androstane-3β-ol (3β,5α-THMT) as well as mestanolone not declared on the label. In the present study, a product called “Super Kalm Paste” was tested. Analysis by gas chromatography - mass spectrometry (GC-MS) revealed that the preparation contained the class I anti-arrhythmics quinine (trade names Kinidin^TM, Durules) and cinchonine. The samples were prepared according to a sample preparation procedure established for anabolic steroids in nutritional supplements for humans. The sample treatment comprised the extraction and purification of the analytes as well as the chemical conversion with N-methyl-N-trimethylsilyl-trifluoracetamide (MSTFA) to yield the trimethylsilyl (TMS)-derivatives. To verify whether the administration of such products could lead to positive doping tests, a pilot excretion study on “Energy 5” was conducted with two geldings, and urine samples were collected. Gas chromatography - high resolution mass spectrometry (GC-HRMS) after solid phase extraction and mixed derivatisation has demonstrated the presence of the stanozolol metabolite 16β-hydroxy-stanozolol in urine samples after “Energy 5” application.     

Simultaneous determination of synthetic phosphodiesterase-5 inhibitors found in a dietary supplement and pre-mixed bulk powders for dietary supplements using high-performance liquid chromatography with diode array detection and liquid chromatography-electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography-diode array detection (HPLC-DAD) method and a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method were developed to screen for the presence of synthetic phosphodiesterase-5 (PDE-5) inhibitors and their analogues, namely sildenafil, vardenafil, tadalafil, homosildenafil, acetildenafil and hydroxyhomosildenafil. The methods were applied to pre-market samples submitted to the Health Sciences Authority of Singapore (HSA) for testing. One sample was in the form of capsules while six other samples were pre-mixed bulk powder samples for dietary supplements to be repackaged or formulated into the final dosage forms (usually capsules). Identification of PDE-5 inhibitors and their analogues was achieved by comparing individual peak retention times, UV spectra and mass spectra with those of reference standards. The seven samples were found to contain at least one of the following compounds: sildenafil, vardenafil, hydroxyhomosildenafil, homosildenafil and acetildenafil. The five compounds were simultaneously determined by LC-ESI-MS/MS in multiple reactions monitoring (MRM) scan mode. The method has been validated for accuracy, precision, linearity and sensitivity.     

Simultaneous quantitation of six major quassinoids in Tongkat Ali dietary supplements by liquid chromatography with tandem mass spectrometry

Tongkat Ali (Eurycoma longifolia) is one of the most popular traditional herbs in Southeast Asia and generally consumed as forms of dietary supplements, tea, or drink additives for coffee or energy beverages. In this study, the liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of six major quassinoids of Tongkat Ali (eurycomanone, 13,21‐dihydroeurycomanone, 13α(21)‐epoxyeurycomanone, 14,15β‐dihydroxyklaineanone, eurycomalactone, and longilactone) was developed and validated. Using the developed method, the content of the six quassinoids was measured in Tongkat Ali containing dietary supplement tablets or capsules, and the resulting data were used to confirm the presence of Tongkat Ali in those products. Among the six quassinoids, eurycomanone was the most abundant quassinoid in all samples tested. The developed method would be useful for the quality assessment of Tongkat Ali containing dietary supplements.     

The Usefulness of X-ray Diffraction and Thermal Analysis to Study Dietary Supplements Containing Iron

X-ray powder diffraction (XRPD) and thermal analysis (differential scanning calorimetry/derivative of thermogravimetry (DSC/DTG)) are solid-state techniques that can be successfully used to identify and quantify various chemical compounds in polycrystalline mixtures, such as dietary supplements or drugs. In this work, 31 dietary supplements available on the Polish market that contain iron compounds, namely iron gluconate, fumarate, bisglycinate, citrate and pyrophosphate, were evaluated. The aim of the work was to identify iron compounds declared by the manufacturer as food supplements and to try to verify compliance with the manufacturer’s claims. Studies performed by X-ray and thermal analysis confirmed that crystalline iron compounds (iron (II) gluconate, iron (II) fumarate), declared by the manufacturers, were present in the investigated dietary supplements. Iron (II) bisglycinate proved to be semi-crystalline. However, depending on the composition of the formulation, it was possible to identify this compound in the tested supplements. For amorphous iron compounds (iron (III) citrate and iron (III) pyrophosphate), the diffraction pattern does not have characteristic diffraction lines. Food supplements containing crystalline iron compounds have a melting point close to the melting point of pure iron compounds. The presence of excipients was found to affect the shapes and positions of the endothermic peaks significantly. Widening of endothermic peaks and changes in their position were observed, as well as exothermic peaks indicating crystallization of amorphous compounds. Weight loss was determined for all dietary supplements tested. Analysis of the DTG curves showed that the thermal decomposition of most food supplements takes place in several steps. The results obtained by a combination of both simple, relatively fast and reliable XRPD and DSC/DTG methods are helpful in determining phase composition, pharmaceutical abnormalities or by detecting the presence of the correct polymorphic form.     

Screening for unknown synthetic steroids in human urine by liquid chromatography-tandem mass spectrometry

Chemically modified steroids (designer steroids), including tetrahydrogestrinone and norbolethone, pose a threat to the integrity of the sport community. These compounds have recently been detected in urine specimens from athletes, resulting in temporary or permanent suspension from amateur and/or professional competition. Triple quadrupole mass spectrometers enable doping control laboratories to screen for unknown, anabolic, androgenic steroids utilizing precursor ion scans. On the basis of common dissociation patterns of steroids with common structural features, characteristic product ions were selected to serve as diagnostic markers for previously unidentified drugs or drug metabolites in human urine samples. An assay was established to complement standard screening procedures. Urine specimens were enzymically hydrolyzed, partitioned into ether, concentrated, and analyzed by precursor ion scanning. Spectra from samples fortified with eight standard compounds (methyltestosterone, ethyltestosterone, 1-testosterone, gestrinone, dihydrogestrinone, tetrahydrogestrinone, norbolethone, and propyltrenbolone) and one deuterium-labeled analog (d(4)-tetrahydrogestrinone) at 50 ng/ml of urine, had precursor ion peaks other than those from common endogenous steroids. Subsequent product ion scan experiments on precursor ions of peaks of unknown origin provided structural identification of the unknown compounds.     

Orbitrap高分辨质谱用于保健食品中15种非法添加减肥类药物的筛查鉴定

建立了筛查保健食品中非法添加的15种减肥类化合物的液相色谱-Orbitrap高分辨质谱联用法和TraceFinder筛查数据库。样品以甲醇为提取溶剂，上清液过滤后直接进行液相色谱-质谱联用分析。质谱采用Full MS/dd-MS²扫描模式，正负离子同时检测。将采集的数据文件导入TraceFinder筛查软件，利用软件构建了化合物数据库及筛查方法进行快速、自动、高精度筛查，确定样品中是否违法添加了减肥类药物，并对阳性样品进行定量分析。方法学验证结果表明，所有化合物均显示出优异的线性关系，标准曲线回归系数（r）均大于0.998，回收率范围为79.7%~95.4%，精密度在3.3%~8.7%之间。应用该方法对29批保健食品进行了测定，在6批阳性样品中检出了4种化合物。该方法可实现自动、高精度筛查鉴定，为打击非法添加提供了新的手段。     

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.     

Simultaneous determination of a broad spectrum of nonconjugated xenobiotics by high-performance liquid chromatography-tandem mass spectrometry

A procedure was developed for screening 23 corticosteroids, 12 anabolic steroids, 23 diuretics, and 49 central nervous system stimulants and narcotics. The sample preparation includes extraction, the evaporation of the organic extract in a nitrogen flow, and the dissolution of the dry residue. The extract was analyzed by high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure electrospray ionization under different recording conditions. Negatively charged ions were recorded to determine corticosteroids and diuretics, while positively charged ions, to determine anabolic steroids, diuretics, stimulants, and narcotics. Mass spectra were obtained for all test compounds; the characteristic ions, retention times, detection limits, degree of ionization suppression by the matrix, and recovery were determined for all analytes.     

Rapid screening test for adulteration in raw materials of dietary supplements

Fourier transform infrared (FTIR) spectroscopy with attenuated total reflectance (ATR) sampling was evaluated for use in screening for adulteration in raw materials used in the formulation and manufacture of dietary supplements. ATR requires minimal-to-no sample preparation and the method runs in less than ten minutes, providing a robust, rapid screening test for a variety of possible adulterants in the raw materials of dietary supplements. Spectral comparison methods targeting structural similarities of known adulterants were developed. In this study, FTIR-ATR was used to detect the presence of known adulterants intentionally spiked into dietary ingredients, including erectile dysfunction drugs, steroids, weight loss drugs and Melamine.