GC-MS method development and validation for anabolic steroids in feed samples

A GC-MS method for the determination of AAS used as growth promoting agents using SIM in piglet feed samples has been developed and validated, using testosterone as internal standard. The formation of volatile steroid derivatives was carried out by derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The optimum separation was achieved using a Zebron ZB-5 column under a gradient temperature elution, allowing the separation of steroids in 18 min. The required sample treatment process was discussed. A leaching using ACN, saponification using a binary NaOH/MgCl2 solution, and LLE using ethyl acetate were finally selected. Method validation has been carried out according to the Commission Decision 2002/657/EC criteria established for quantitative confirmatory methods. The extraction efficiencies, CCalpha and CCbeta for these compounds were in the ranges 78-98%, 10-21 and 18-35 mug/kg, respectively. The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta concentration levels were smaller than 8.2, 7.5, and 5.8% and 12.2, 9.5, and 7.5%, respectively. Accuracy was in the 99-103% range. The robustness was evaluated using the Youden robustness test. The proposed method was applied to the analysis of steroids spiked in different kinds of animal feed samples with satisfactory results.     

Multi-residue screening of a minimum package of anabolic steroids in urine with GC-MS

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography-mass spectrometry (GC-MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCalpha and CCbeta values are based on the detection of the most abundant ion. Results of validation experiments are presented. The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.     

Application of the revised EU criteria for the confirmation of anabolic steroids in meat using GC-MS

The EU criteria for the confirmation of the presence of illegal compounds in biological matrices were recently revised. The old and the revised criteria were applied to relative ion intensities obtained for five anabolic steroids (methylboldenone, methyltestosterone, ethynylestradiol, -boldenone and -nortestosterone) in meat (cow, pig, turkey) and fish at concentrations ranging from 0.5 to 5.0 g/kg. Confirmatory analysis was done by GC-MS; therefore four diagnostic ions had to be monitored and three ion ratios had to be calculated and tested against the criteria. Application of the old and revised criteria, with either standards or fortified samples as reference, showed mutually rather divergent results. Confirmation according to the revised EU criteria and using fortified samples as a reference gave the best results; in other words the highest percentage of diagnostic ion ratios within the tolerance intervals. A correlation was found between the percentage of these ion ratios and the signal/noise (S/N) ratio of the least intense ion of interest in the recorded MS spectrum. Although there were distinct differences in the results obtained for different analytes and sample types, it is safe to conclude that at S/N=3 the percentage of ratios within the tolerance intervals generally will be at or below 50%, while for S/N10, the percentage increases to over 90%. In the present study, fully satisfactory results were obtained down to about 2 g/kg, but not for lower analyte concentrations.     

Direct determination of anabolic steroids in pig urine by a new SPME-GC-MS method

A new solid phase microextraction (SPME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of four anabolic steroids such as 3α-hydroxy-5α-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 °C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8-640 pg/mL for HA and DHT and 16-510 pg/mL for AD and MT, respectively. The detection limits (S/N = 3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.     

Validation of multi-residue methods for the detection of anabolic steroids by GC-MS in muscle tissues and urine samples from cattle.

The use of anabolic steroids is prohibited in the European Community by Directive 96/22/EC and the control of compliance is regulated by Directive 96/23/EC. Multi-residue methods are necessary for screening for the use of forbidden substances. Because accreditation is gaining more and more importance, validation of the methods used and of the results obtained has become indispensable. The developed GC-MS methods for the detection of anabolic steroids in urine and muscle tissue were validated with regard to the following parameters: specificity, recovery at the 2 micrograms kg-1 level and limit of detection. For urine the recoveries ranged from 17 to 81% and for muscle tissue from 26 to 65%. The limit of detection ranged from 0.1 to 2.6 micrograms kg-1 for urine and from 0.3 to 4.6 micrograms kg-1 for muscle tissue. Specificity was guaranteed in both matrices by the selection of four specific ions. Blank samples were evaluated for interferences and it could be concluded that in no case did the four selected ions appear simultaneously at the correct retention time. The practicability of the criteria for low resolution mass spectrometry set in Decision 93/256 in the low micrograms kg-1 range is also briefly discussed.     

Determination of anabolic steroids in creatine nutritional supplements after supercritical fluid extraction

The effects of various parameters, i.e. extraction pressure, temperature, time, and modifier on the efficiency of extraction were investigated using an analytical-scale supercritical fluid extraction system. An optimal set of conditions for the extraction and determination by gas chromatography-mass spectrometry of trimethylsilyl derivatives of 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione, nandrolone, and testosterone in nutritional supplements was developed. The optimum amount of creatine supplement was 1 g, while the optimum pressure and temperature were determined to be 35 MPa and 80 °C, respectively. The optimum dynamic extraction time was 45 min. The limit of detection (LOD) of the investigated compounds ranged from 5 to 25 ng · g⁻¹ of supplement, while recoveries ranged from 76.1 to 86.6%. Correspondence: Petra Mikulcikova, Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, nám. Cs. Legií 565, CZ 532 10 Pardubice, Czech Republic     

Liquid chromatographic purification and detection of anabolic compounds.

The role of liquid chromatography within methods of analysis for steroids, related compounds and beta-agonists in biological samples is discussed. Special attention is given to the application of liquid chromatography in sample preparation and extract clean-up. Different forms of liquid chromatography, including immunoaffinity chromatography, are compared and evaluated. Methods for confirmation based on gas chromatography-mass spectrometry and cryotrapping Fourier transform infrared spectrometry are discussed.     

General and selective isolation procedure for high-performance liquid chromatographic determination of anabolic steroids in tissues

A multi-residue method has been developed for the determination of anabolic steroids in animal tissue. The analytes are extracted from tissue with methanol and the extract is subjected to two solid-phase extractions, one using a non-specific adsorbing material, such as graphitized carbon black (Carbopack B), and the other Amberlite CG-400 I in the OH form. This procedure allowed the neutral anabolics (testosterone, trenbolone and progesterone) to be isolated and separated from the acidic type (phenolic group), such as diethylstilbestrol, oestradiol, zeranol/zearalenone and their respective metabolites. The determination was effected using high-performance liquid chromatography with different detectors (ultraviolet, fluorimetric and electrochemical). Several analytical parameters were studied: chromatographic conditions, recoveries, evaporation step, solvent flow-rate, cartridges reusability, interference of plastic cartridges. For all the anabolics investigated the recoveries were greater than 83.6%.     

GC-MS determination of steroids related to androgen biosynthesis in human hair with pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation

An efficient method for the simultaneous determination of eight steroids, androstenedione, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone, androsterone, etiocholanolone, progesterone and pregnenolone, in human hair by gas chromatography-mass spectrometry (GC-MS) using d3-testosterone as internal standard is described. The method involves alkaline digestion, liquid-liquid extraction and subsequent conversion to mixed pentafluorophenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected ion monitoring (SIM) mode. This method showed good overall repeatability and reproducibility of 4.88-11.24 and 3.19-9.58%, respectively. For the first time, the quantification of DHT, DHEA and pregnenolone in human hair has been achieved by GC-MS, testosterone was also quantified. The detection of four steroids in hair samples was possible in the concentration range 0.12-8.45 ng g-1. The other four steroids, androstenedione, androsterone, etiocholanolone and progesterone, were not detected. The detection limits for SIM of the steroids varied in the range 0.02-0.5 ng g-1, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.996 for most of the steroids studied. The concentrations of the four steroids detected were different in male and female hair samples.     

液相色谱-电喷雾电离质谱法检测尿液中的3种合成类固醇药物

研究建立了合成类固醇药物群勃龙、四氢孕三烯炔酮和孕三烯酮的液相色谱-电喷雾质谱的检测方法。尿样经过β-葡萄糖醛酸酶酶解和叔丁基甲醚提取后,采用Zorbax SB-C18分析柱(150 mm×2.1 mm,5 μm),在流动相为pH 3.5的甲酸铵缓冲液-乙腈的条件下进行梯度洗脱,在正离子模式下检测。考察了不同的质谱条件对这类化合物检测结果的影响。建立了人尿液中合成类固醇药物的液相色谱-电喷雾电离质谱的初筛和确证方法。     

Separation of anabolic steroids by micellar electrokinetic capillary chromatography

Summary The separation of seven analogous anabolic steroids was studied by micellar electrokinetic capillary chromatography (MECC). The retention order was found to be dependent on polarity. All of these steroids were well separated by the addition of organic modifiers to the separation buffer. Of the organic modifiers tested, 1-propanol gave the best separation, better than methanol or acetonitrile.     

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。     

Studies on anabolic steroids. I. Integrated methodological approach to the gas chromatographic-mass spectrometric analysis of anabolic steroid metabolites in urine

The analytical and methodological imperatives for large-scale and routine gas chromatographic-mass spectrometric screening of anabolic steroid urinary metabolites are described. Several aspects of their isolation, enzymatic hydrolysis, derivatization and metabolism in humans are discussed. Gas chromatographic-mass spectrometric data illustrating artifacts arising from enzymatic hydrolysis of 3 beta-ol-5-en steroids, and describing new metabolites of boldenone, methanedienone and stanozolol, as well as the conversion of norethisterone into 19-nortestosterone metabolites through de-ethylation at C-17, are presented. The analytical approach developed for gas chromatographic-mass spectrometric screening of anabolic steroids is based on the sequential selection-ion monitoring of specific and discrete ion groups characteristic to the steroids of interest under high-resolution chromatographic conditions. The major analytical and methodological requirements necessary to provide irrefutable evidence, in the case where the presence of a synthetic anabolic steroid or a testosterone to epitestosterone ratio higher than 6:1 is suspected in a given urine specimen, are also 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.     

Simultaneous determination of nandrolone, testosterone, and methyltestosterone by multi-immunoaffinity column and capillary electrophoresis

A multitarget antibody immunoaffinity column was proposed for the purification and enrichment of nandrolone, testosterone, and methyltestosterone from urine. Nandrolone-3-site substituted antigen was designed and synthesized and the polyclonal antibody was prepared with immunizing rabbits. The stationary phase of the immunoaffinity column was synthesized by covalently bonding the antibodies specific to nandrolone, testosterone, and methyltestosterone onto CNBr-actived Sepharose 4B. The analytes of interest were extracted with a methanol/water mixture in one step. The immunoaffinity column showed high affinity and high selectivity to a class of structurally related compounds. The elution was then transferred to a micellar electrokinetic CE system with a running buffer of sodium borate and sodium cholate for separation and determination. Recoveries of the three steroids from complex matrix were 88-94% with RSD values less than 5.2%. Optimization of the immunoaffinity column purification was achieved and the feasibility of the technique for the analysis of steroid hormone was discussed. The results indicated that the combination of multi-immunoaffinity column and CE was an effective technique, which was rapid, simple, and sensitive for the assay of steroids.     

Multiresidue analysis of anabolic agents in muscle tissues and urines of cattle by GC-MS.

The illegal use of anabolic steroids in livestock breeding has taken enormous proportions the last few decades. To protect the consumer against possible harmful effects due to the consumption of contaminated meat or meat products, a multiresidue analysis of anabolic steroids has been developed for muscle tissues and urine. The pretreatment of the meat and urine samples consists of an enzymatic digestion, liquid or solid-phase extraction, and finally high-performance liquid chromatography (HPLC) fractionation. Five fractions or windows are collected, each containing a number of analytes. The residues are derivatized prior to the detection by gas chromatography-mass spectrometry (GC-MS). Both gas chromatographic retention data and mass spectral data are used for identification of nortestosterone, testosterone, estradiol, ethynylestradiol, trenbolone, zeranol, diethylstilbestrol, boldenone, methandienone, methyltestosterone, megestrol acetate, chlormadinone acetate, medroxyprogesterone acetate, chlorotestosterone, progesterone, and chlorotestosterone acetate. The limit of detection varies from matrix to matrix and from analyte to analyte but is, in the most favorable case, on the order of 0.3 ppb (micrograms/kg).     

Multiplexed immunoassay to detect anabolic androgenic steroids in human serum

A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The multiplexed method was developed according to a planar strategy in which the analytes are identified by their location in the microtiter plate. In the immunochemical procedure established here, human serum samples are mixed with a cocktail of antibodies and added to the distinct sections of a microplate biofunctionalized with different haptenized biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole immunochemical analytical procedure takes around 2 h including sample preparation, and many samples can be processed simultaneously to screen for the presence of the three AAS in a single run. Using this ELISA, ST, B, and THG can be detected and quantified individually. When used as a screening method, due to the cross-reactivity profiles of the immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The detectabilities achieved by this method in human serum are below the MRPLs (minimum required performance limits) proposed by WADA (World Anti-Doping Agency) and reference laboratories of the European Community.