High resolution orbitrap mass spectrometry in comparison with tandem mass spectrometry for confirmation of anabolic steroids in meat

A prominent trend which has been observed in recent years in the analysis of veterinary drugs and growth-promoting agents is the shift from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform (FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS for confirmatory analysis of growth-promoting agents in meat was compared to that of a QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most compounds >0.99) but somewhat inferior sensitivity. Indeed, CC_αs reached from 0.04–0.88 μg kg⁻¹ for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS detection a range of 0.07–2.50 μg kg⁻¹ was observed. Using QqQ-MS adequate precision was obtained since relative standard deviations, associated with the repeatability and intra-laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds (i.e. some estrogens) this threshold was exceeded and thus poor precision was observed, which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity for some of the compounds.     

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.     

Development and validation of a method for the confirmation of nicarbazin in chicken liver and eggs using LC-electrospray MS-MS according to the revised EU criteria for veterinary drug residue analysis.

A method is described for the quantitative confirmation of 4,4'-dinitrocarbanilide (DNC), the marker residue for nicarbazin in chicken liver and eggs. The method is based on LC coupled to negative ion electrospray MS-MS of tissue extracts prepared by liquid-liquid extraction. The [M-H]- ion at m/z 301 is monitored along with two transition ions at m/z 137 and 107 for DNC and the [M-H]- ion at m/z 309 for the internal standard, d8-DNC. The method has been validated according to the new EU criteria for the analysis of veterinary drug residues at 100, 200 and 300 microg kg(-1) in liver and at 10, 30 and 100 microg kg(-1) in eggs. Difficulties concerning the application of the new analytical limits, namely the decision limit (CCalpha) and the detection capability (CCbeta) to the determination of DNC in both liver and eggs are discussed.     

Standards and GC-MS analysis: An answer to the requirement of compound confirmation

Summary The attribution of an unknown GC-MS chromatographic peak to a specific substance should be confirmed whenever possible by comparison with a standard compound analysed under the same experimental conditions. The range of chemicals supplied by various companies is very wide but can never be completely comprehensive. This paper proposes two simple ways by which a reference compound may be obtained. Firstly, it may occur as an impurity in a commercial sample as a synthesis by-product. Alternatively, the compound may be synthesized by a simple procedure, preferably a single step one, by putting small amounts of reagents in a sealed glass vial placed in a programmable oven. Examples of these two approaches are described.     

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.     

Development and validation of an improved method for confirmation of the carbadox metabolite, quinoxaline-2-carboxylic acid, in porcine liver using LC-electrospray MS-MS according to revised EU criteria for veterinary drug residue analysis

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA), the marker residue for the veterinary drug carbadox, in swine liver. Tissue is subjected to alkaline hydrolysis followed by liquid-liquid extraction. QCA residues are cleaned up using automated solid phase extraction (SPE), before a final liquid-liquid extraction step. Analysis is based on LC coupled to positive ion electrospray MS-MS, monitoring product ions at m/z 129, 102 and 75 for QCA and at m/z 106 for the internal standard (d4-QCA). The method has been validated according to draft revised EU criteria for analysis of veterinary drug residues, and is suitable for monitoring tissues taken under national surveillance schemes. The method has been validated at 3, 10, 30, 100 and 300 microg kg(-1). The method performance characteristics, CCalpha (decision limit) and CCbeta (detection limit) were determined to be 0.16 and 0.27 microg kg(-1), respectively. The described method, which is relatively rapid and applicable to large sample numbers, correlates well (r2 = 0.9799) with a widely used GC-MS assay for QCA.     

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 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.     

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.     

头发中内源性类固醇激素的气相色谱-串联质谱分析

建立了建康人头发中内源性类固醇兴奋剂睾酮、表睾酮、雄酮、苯胆烷醇酮和脱氢表雄酮的气相色谱-串联质谱(GC-MS/MS)分析方法。头发经碱水解后,以乙醚提取,经衍生化后采用GC-MS/MS的多反应监测模式(MRM)分析。方法的线性关系良好,检出限达0.1～0.2 pg/mg;提取回收率为74.6%～104.5%;日内测定的准确度为90.1%～113.7%,日内及日间测定的精密度均小于17.5%。应用所建立的方法测定了80例中国健康人头发中睾酮、表睾酮、雄酮、苯胆烷醇酮和脱氢表雄酮的生理水平,为内源性类固醇兴奋剂滥用的判断提供了方法和基础数据。     

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。     

Determination of quinolone residues in shrimp using liquid chromatography with fluorescence detection and residue confirmation by mass spectrometry

The quinolones, oxolinic acid (OXO), flumequine (FLU), and nalidixic acid (NAL), are antibacterial drugs effective against Gram-negative bacteria. Quinolones are used in both human and veterinary medicine, but are currently not approved by the U.S. Food and Drug Administration for use in food fish. A liquid chromatography-fluorescence (LC-FL) method was developed to determine OXO, FLU, and NAL residues in shrimp. An additional liquid chromatography-mass spectrometry (LC-MSⁿ) method was created to confirm these residues using the same sample extract. Samples were prepared with a simple ethyl acetate extraction followed by solvent exchange into 0.2% formic acid and cleaned-up with hexane. Reverse phase chromatography was used to separate the three compounds in both procedures. For the LC-FL determinative method, fluorescence emission was monitored at 369 nm with excitation at 327 nm. With electrospray ionization, the three most abundant ions from the MS³ product ion spectrum were used to identify OXO, FLU, and NAL in the confirmation procedure. Shrimp samples fortified at levels ranging from 7.5 to 100 ng g⁻¹ were used to validate both methods.     

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.     

Metabolites in feces can be important markers for the abuse of anabolic steroids in cattle.

In Belgium, to control the abuse of anabolic steroids in cattle, urine samples have been gradually replaced by feces samples, because the latter can be obtained more easily from living animals. Urine and feces samples were collected from heifers after administration of boldenone, norethandrolone or ethylestrenol. Metabolites present in feces or urine were determined by GC-MS. Large qualitative and quantitative differences in the metabolic profiles were observed. In feces, in contrast to urine, the parent compounds or their major metabolites were detectable only shortly after administration. On the other hand, metabolites resulting from the reduction of the 3-oxo group and the unsaturated carbon-carbon bonds, present on the A-ring, allow for long-term detection in feces. A-ring reduced metabolites have been identified in samples found positive for norgestrel, boldenone, methylboldenone and methyltestosterone, respectively. These results are in agreement with concomitant in vivo experiments.     

Application of fast gas chromatography/mass spectrometry for the rapid screening of synthetic anabolic steroids and other drugs in anti-doping analysis

This paper describes a fast gas chromatographic/mass spectrometric (GC/MS) screening method for the detection, in urine, of 36 xenobiotics (30 synthetic anabolic steroids, four narcotics, one diuretic and one stimulant) excreted free or as glucuro-conjugates in urine and detectable as trimethylsilyl (TMS) derivatives. These drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase and then assayed as TMS derivatives by GC/MS using electron ionisation (EI) and single ion monitoring (SIM) acquisition mode. The total time needed for the GC run is less than 8 min. Good reproducibility of the retention times (CV% <1) and the relative abundances of the diagnostic fragment ions (CV% <10) was observed for all target analytes. The sensitivity of the method is sufficient to match the requirements of the World Anti-Doping Agency (WADA) for the accredited laboratories, with limits of detection (LODs) that are lower than the corresponding WADA minimum required performance limits (MRPLs) for all target compounds.     

Method for the analysis of thyreostats in meat tissue using gas chromatography with nitrogen phosphorus detection and tandem mass spectrometric confirmation

An analytical method not requiring a mercury column cleanup step is described for the isolation and detection of four thyreostatic agents in meat tissue. The use of these growth promotants in livestock has been banned by regulatory agencies. The meat tissue is homogenized with acetonitrile-water, centrifuged, and the supernatant is partitioned with petroleum ether. The acetonitrile-water is concentrated and then passed through a silica-gel column. The solvent is then removed and the residue derivatized with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. The total amount of organic solvent used for the analysis is merely 35 mL. The derivatized thyreostats are detected and quantitated by gas chromatography (GC) equipped with a nitrogen-phosphorus detector. Percent recoveries from fortified meat tissue (n = 6) at the 0.1-microg/g (parts per million) level are 93.5 +/- 2.9 for 2-thiouracil, 90.3 +/- 3.0 for tapazole, 87.5 +/- 2.9 for 6-methyl-2-thiouracil, and 85.1 +/- 5.8 for 6-n-propyl-2-thiouracil. For the confirmation of analyte identities, GC-tandem mass spectrometry with an ion-trap instrument is used. The estimated minimum level for a reliable measurement is 0.050 microg/g in meat tissue.     

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

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

衍生固相萃取GC-MS检测牛可食组织中克伦特罗的残留量

盐酸克伦特罗(clenbuterol,CLB),俗名"瘦肉精",化学名为"2-[(叔丁氨基)甲基]-4-氨基-3,5-二氯苯甲醇盐酸盐"(相对分子质量313.5),是一种β-兴奋剂。近年来动物性产品中CLB残留对人类健康造成的危害和对生态食物链所造成的破坏已经引起高度重视。1986年开始,欧美等发达国家已严禁畜牧生产中应用CLB。中国农业部在1997年3月以[农牧发(1997)3号文]严令禁止β-肾上腺素能激动剂在动物生产中应用。但目前国内外非法使用情况仍存在。国际上发展了一系列畜禽     

Multiresidue determination of acidic pesticides in water by HPLC-DAD with confirmation by GC-MS using conversion to the methyl ester with trimethylsilyldiazomethane

A multiresidue pesticide methodology is studied and results for acidics are reported here, with base/neutral to follow. This work studies a literature procedure as a possible general approach to many pesticides and potentially other analytes that are considered to be liquid chromatographic (LC) candidates, rather than gas chromatographic (GC) ones. The analysis of the sewage effluent of a major southwestern U.S. city serves as an example of the application of the methodology to a real sample. Recovery studies were also conducted to validate the proposed extraction step. A gradient elution program was followed for the high-performance LC (HPLC) leading to a general approach for acidics. Confirmation of identity was by electron ionization-GC-mass spectrometry (MS) after conversion of the acids to the methyl ester (or other appropriate methylation) by means of trimethylsilyldiazomethane. The 3,4-dichlorophenoxyacetic acid is used as an internal standard to monitor the reaction, and polychlorobiphenyl #19 is used for the quantitation internal standard. Although others have reported similar analyses of acids, conversion to the methyl ester is by means of diazomethane itself, rather than by the more convenient and safe trimethylsilyldiazomethane. Thus, the present paper supports the use of trimethylsilyldiazomethane with all of these acids (trimethylsilyldiazomethane has been used in environmental work with some phenoxyacetic acid herbicides) and further supports the usefulness of this reagent as a potential replacement for diazomethane. The HPLC approach here could also serve as the separation basis for an LC-MS solution for confirmation of identity, as well as quantitation.     

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

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