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。     

Screening for anabolic steroids in doping analysis by liquid chromatography/electrospray ion trap mass spectrometry

A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human urine has been developed and validated. Liquid-liquid extraction with diethyl ether was applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds. The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10 ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-hydroxystanozolol, were assessed using the described method.     

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.     

Isotopic fractionation of endogenous anabolic androgenic steroids and its relationship to doping control in sports

The use of gas chromatography (GC)-combustion (C)-isotope ratio mass spectrometry (IRMS) demonstrates that a single oral administration of dehydroepiandrosterone (DHEA, 100 mg) to a male subject significantly lowers the 13C content of etiocholanolone (Et) and androsterone (A) in the subject's urine. The difference in carbon isotope ratio (d13C per thousand) values between Et and A increases from 1.6 per thousand at the time of administration to 5.1 per thousand at 26 h post-administration, indicating preferential metabolism of administered DHEA to form Et in relation to A. Multiple oral administrations of DHEA to a male subject reveals lower d13C values during the excretion period of Et (-31.7 per thousand to -34.6 per thousand) and A (-31.4 per thousand to -33.0 per thousand) to that of the d13C value of the administered DHEA (-31.3 per thousand). Reference distributions of d13C Et and d13C A constructed from normal athlete populations within Australia and New Zealand show a small natural discrimination against 13C in the formation of Et relative to A (mean=0.3 per thousand, n=167, p=0.007). Amplified differences between d13C Et and d13C A, and in vivo 13C depletion measured by GC-C-IRMS are shown to be potentially useful for doping control.     

External calibration in gas chromatography-combustion-isotope ratio mass spectrometry measurements of endogenous androgenic anabolic steroids in sports doping control

An alternative calibration procedure for the Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry (GC–C–IRMS) measurements of the World Antidoping Agency (WADA) Accredited Laboratories is presented. To alleviate the need for externally calibrated CO₂ gas for GC–C–IRMS analysis of urinary steroid metabolites, calibration using an external standard mixture solution of steroids with certified isotopic composition was investigated. The reference steroids of the calibration mixture and routine samples underwent identical instrumental processes. The calibration standards bracketed the entire range of the relevant δ¹³C values for the endogenous and exogenous steroids as well as their chromatographic retention times. The certified δ¹³C values of the reference calibrators were plotted in relation to measured m/z¹³CO₂/¹²CO₂ (i.e. R(45/44)) mass spectrometric signals of each calibrator. δ¹³C values of the sample steroids were calculated from the least squares fit through the calibration curve. The effect of the external calibration on δ¹³C values, using the same calibration standards and set of urine samples but different brands of GC–C–IRMS instruments, was assessed by an interlaboratory study in the WADA Accredited Laboratories of Sydney, Australia and Athens, Greece. Relative correspondence between the laboratories for determination of androsterone, etiocholanolone, 5β-androstane-3α,17β-diacetate, and pregnanediacetate means were SD(δ¹³C) = 0.12‰, 0.58‰, −0.34‰, and −0.40‰, respectively. These data demonstrate that accurate intralaboratory external calibration with certified steroids provided by United States Antidoping Agency (USADA) and without external CO₂ calibration is feasible and directly applicable to the WADA Accredited Laboratories for the harmonization of the GC–C–IRMS measurements.     

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.     

Analysis of meat samples for anabolic steroids residues by liquid chromatography/tandem mass spectrometry

A rapid, specific and highly sensitive multi-residue method for the determination of anabolic steroid residues in bovine, pork and poultry muscle tissues was developed. The sample preparation involves enzymatic digestion followed by extraction with methanol. The crude extract was cleaned up by solid-phase extraction (SPE) combining C18 and NH2 columns. The detection was carried out by a highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using both positive and negative ionization modes. Natural and synthetic steroids covering different polarities could be extracted, concentrated and purified using one single method. Mobile phase composition and additives were optimized to achieve the highest sensitivity. The linearity was not good enough for quantitative analysis but the method was well-suited for qualitative confirmation. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.5 ng g(-1) for all the compounds in the three types of meat studied. The developed method is suitable for routine analysis in our laboratories.     

Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine

The applicability of in-vial two-phase liquid-phase microextraction (LPME) in porous hollow polypropylene fiber was studied for the sample preparation of unconjugated anabolic steroids in urine. Four different anabolic steroids - metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol - were used as test compounds and methyltestosterone as an internal standard. A standard two-phase LPME method for use with liquid chromatography/mass spectrometry (LC/MS) was set up and the influence of different parameters, including the nature of organic solvent, extraction time, salting-out and temperature, on the LPME process was investigated. Taking advantage of the preliminary studies, a novel two-phase LPME method utilizing simultaneous in-fiber silylation was developed and validated for gas chromatographic/mass spectrometric (GC/MS) analysis of a danazol metabolite in urine. In all, LPME allowed a very straightforward, simple and selective way to prepare urine samples for steroid analysis, being most suitable for hydrophobic steroids. The LPME method with in-fiber derivatization for GC/MS analysis exhibited high sensitivity, repeatability and linearity and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine matrix without any other steps in sample pretreatment.     

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.     

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.     

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.     

高效液相色谱法同时测定血浆中的10种蛋白同化激素

建立了一种用于10种蛋白同化激素的同时分离检测的高效液相色谱法。根据被分析物的性质,以C18反相色谱柱为分离柱,以乙腈和水为流动相,采用梯度洗脱方式,并在194～290 nm的范围内快速调节检测波长,使各物质均在最大吸收波长处被检出。在优化的条件下,10种被测组分在10 min内实现了快速的基线分离,检出限在0.01～0.10 μg/mL范围内。在兔血浆中进行加标回收率测定,10种被测组分的加标回收率为70.3%～120%。选取美雄醇为代表进行实际动物实验,成功检测到耳脉注射美雄醇后兔血浆内的美雄醇成分。实验结果表明该方法可行,快速简便,准确可靠。     

Feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization mass spectrometry in analyzing anabolic steroids in urine samples

We examined the feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization tandem mass spectrometry (capLC/µAPPI‐MS/MS) for the analysis of anabolic steroids in human urine. The urine samples were pretreated by enzymatic hydrolysis (with β‐glucuronidase from Helix pomatia), and the compounds were liquid‐liquid extracted with diethyl ether. After separation the compounds were vaporized by microchip APPI, photoionized by a 10 eV krypton discharge lamp, and detected by selected reaction monitoring. The capLC/µAPPI‐MS/MS method showed good sensitivity with detection limits at the level of 1.0 ng mL^?1, good linearity with correlation coefficients between 0.9954 and 0.9990, and good repeatability with relative standard deviations below 10%. These results demonstrate that microchip APPI combined with capLC/MS/MS provides a new potential method for analyzing non‐polar and neutral compounds in biological samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Fast analysis of 19 anabolic steroids in bovine tissues by high performance liquid chromatography with tandem mass spectrometry

For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120‐EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid‐phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 µg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10–0.48 and 0.17–0.95 µg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients  > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.     

Comprehensive screening of anabolic steroids, corticosteroids, and acidic drugs in horse urine by solid-phase extraction and liquid chromatography-mass spectrometry

This paper reports two highly efficient liquid chromatography-mass spectrometry (LC-MS) methods for the screening of anabolic steroids, corticosteroids, and acidic drugs for the purpose of doping control in equine sports. Sample extraction was performed using a mixed-mode C8-SCX solid-phase extraction (SPE) cartridge. The first eluted fraction (acidic/neutral fraction) was base-washed and the resulting organic extract was used for the screening of anabolic steroids and corticosteroids by LC-MS using multiple reaction monitoring (MRM) in the positive electrospray ionisation (ESI) mode. The remaining aqueous extract was re-adjusted to pH 6 and acidic drugs were recovered by liquid/liquid extraction. Detection was again achieved using LC-MRM but in the negative ESI mode. A total of 40 anabolic steroids and corticosteroids, and over 50 acidic drugs, including some cyclooxygenase-2 (COX-2) inhibitors, oxicams, anti-diabetics, sedatives, diuretics and Delta(9)-tetrahydro-11-norcannabinol-9-carboxylic acid, could be covered by the two LC-MS methods. Both methods utilized a high efficiency reversed-phase column (3.3 cm L x 2.1 mm I.D. with 3 microm particles) coupled with a fast-scanning triple-quadrupole mass spectrometer to achieve fast turnaround times. The overall turnaround times for both methods were 10 min, inclusive of post-run and equilibration times.     

Liquid chromatographic method development for anabolic androgenic steroids using a monolithic column Application to animal feed samples

An isocratic HPLC method for the determination with screening purposes of anabolic androgenic steroids (AASs: fluoxymesterone, boldenone, nortestosterone, metandrostenolone, norethindrone, methyltestosterone and bolasterone), used as growth promoting agents, in finishing pig feed samples has been developed and validated. The separation was achieved by using a reversed-phase Chromolith RP-18e column at controlled temperature, UV-detection at 245 nm and epitestosterone as internal standard. The method development involved optimization of different aqueous-organic mobile phases using methanol or acetonitrile as organic modifiers, flow-rate and temperature. The optimum separation for these compounds was achieved at 40 °C using ultrapure water:acetonitrile (71:29, v/v) as mobile phase and 3 mL min⁻¹ flow-rate, allowing the separation of AASs with baseline resolution in about 15 min. The optimized method was applied to the analysis of AASs in finishing pig feed samples. Prior to HPLC, sample preparation procedure was used by leaching using acetonitrile, saponification in a basic medium and solid-phase extraction using polymeric Abselut Nexus cartridges. Method validation has been carried out according to the European Commission Decision 2002/657/EC. The extraction efficiencies, decision limits (CCα) and detection capabilities (CCβ) for these compounds were in the range 83-96%, 27-37 and 32-47 μg kg⁻¹ range, respectively. The within-laboratory reproducibility at 1, 1.5 and 2 CCβ concentration levels were smaller than 13, 10 and 8%, respectively. Finally, the proposed method was successfully applied to nine different kinds of animal feed.     

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.     

The importance of cooling of urine samples for doping analysis

Storing and transporting of urine samples for doping analysis, as performed by the anti-doping organizations associated with the World Anti-Doping Agency, does not include a specific protocol for cooled transport from the place of urine sampling to the doping laboratory, although low cost cooling facilities can easily be made available. As a result, microbial and thermal degradation of the chemical substances in the urine may occur, which may lead to false negative or false positive results in the subsequent doping analysis. This scientifically and morally unacceptable practice is still maintained in spite of publications demonstrating that immediate cooling is an absolute requirement. Given the enormous societal consequences of positive tests, the lack of a controllable chain of custody during transport should be outlawed. This paper proposes a simple method, based on immediate cooling and cooled transport, which can easily be implemented in developed countries at low cost.     

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.