超高效液相色谱-串联质谱法测定动物肌肉组织和鸡蛋中残留的11种甾体激素类药物

建立了采用超高效液相色谱-串联质谱(UPLC-MS/MS)同时测定猪、牛、羊和鸡肌肉组织及鸡蛋中睾酮、甲基睾酮、黄体酮、群勃龙、勃地龙、诺龙、美雄酮、司坦唑醇、丙酸诺龙、丙酸睾酮及苯丙酸诺龙等11种甾体激素多残留的分析方法。试样在碱性条件下用叔丁基甲醚提取,冷冻离心脱脂净化,以乙腈和甲酸水溶液为流动相,梯度洗脱,反相液相色谱分离。采用电喷雾离子化、多反应监测方式(MRM),对11种甾体激素同时进行定性定量测定。动物肌肉和鲜蛋中睾酮、甲基睾酮、勃地龙、美雄酮及司坦唑醇的检出限为0.3 μg/kg,群勃龙、诺龙、黄体酮、丙酸诺龙、丙酸睾酮及苯丙酸诺龙的检出限为0.4 μg/kg。在动物组织及鸡蛋中添加1,2及10 μg/kg 水平的药物回收试验中,睾酮、甲基睾酮、勃地龙、美雄酮及司坦唑醇的回收率均在62.3%～105%之间,相对标准偏差为0.5%～15%;群勃龙、诺龙、黄体酮、丙酸诺龙、丙酸睾酮及苯丙酸诺龙的回收率大于50.0%,相对标准偏差小于16%。11种甾体激素在1～100 μg/L范围内,线性关系良好,相关系数都大于0.99。该方法的样品前处理简单、快速,测定灵敏、准确,选择性好,可满足动物源食品中甾体激素类药物多残留的同时测定。     

高效液相色谱-串联质谱法同时检测鸡肉和鸡蛋中合成类固醇类激素和糖皮质激素

建立了鸡肉和鸡蛋中合成类固醇类激素(睾酮、甲基睾酮、群勃龙、勃地龙、诺龙、美雄酮、司坦唑醇、丙酸诺龙、丙酸睾酮及苯丙酸诺龙)和糖皮质类激素(泼尼松、泼尼松龙、地塞米松、氟氢可的松、甲基泼尼松、倍氯米松及氢化可的松)多残留的液相色谱-串联质谱(LC-MS/MS)检测方法.样品经乙腈超声提取,正己烷脱脂净化,以甲醇-甲酸水溶液为流动相,经C18柱分离后进行LC-MS/MS选择反应监测模式下的定性及定量分析.合成类固醇类激素采用正离子模式检测,糖皮质激素则采用负离子模式检测,正、负离子化模式一次进样同时检测.类固醇类和糖皮质类激素的定量检出限为0.5 μg/kg.在0.5、 1.0和5.0 μg/kg 3种浓度添加水平,上述激素的平均回收率为73.4%～108.9%;相对标准偏差为3.4%～13.4%.可实现样本灵敏、准确地定性定量分析.     

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。     

Analysis of anabolic steroids in hair by GC/MS/MS

A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.     

Detection of testosterone,nandrolone and precursors in horse hair

Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid–liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.     

Statistical discrimination of steroid profiles in doping control with support vector machines

Due to their performance enhancing properties, use of anabolic steroids (e.g. testosterone, nandrolone, etc.) is banned in elite sports. Therefore, doping control laboratories accredited by the World Anti-Doping Agency (WADA) screen among others for these prohibited substances in urine. It is particularly challenging to detect misuse with naturally occurring anabolic steroids such as testosterone (T), which is a popular ergogenic agent in sports and society.     

Investigations into the feasibility of routine ultra high performance liquid chromatography–tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids,anabolic steroid esters and related compounds

The detection of the abuse of anabolic steroids in equine sport is complicated by the endogenous nature of some of the abused steroids, such as testosterone and nandrolone. These steroids are commonly administered as intramuscular injections of esterified forms of the steroid, which prolongs their effects and improves bioavailability over oral dosing. The successful detection of an intact anabolic steroid ester therefore provides unequivocal proof of an illegal administration, as esterified forms are not found endogenously. Detection of intact anabolic steroid esters is possible in plasma samples but not, to date, in the traditional doping control matrix of urine. The analysis of equine mane hair for the detection of anabolic steroid esters has the potential to greatly extend the time period over which detection of abuse can be monitored.     

Studies related to the origin of C18 neutral steroids isolated from extracts of urine from the male horse: the identification of urinary 19-oic acids and their decarboxylation to produce estr-4-en-17beta-ol-3-one (19-nortestosterone) and estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) during sample processing

For almost two decades we have known that enzymatic hydrolysis of "normal" urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts. Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of "normal" urine from male horses may not be derived from the 17beta-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17beta-sulphate conjugate. Thus, if the 19-nortestosterone-17beta-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses. These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in "normal" urine from the entire male horse. Purified fractions have then been studied by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to identify the precursors.     

Multivariate optimization of a derivatisation procedure for the simultaneous determination of nine anabolic steroids by gas chromatography coupled with mass spectrometry

The medical commission of the International Olympic Committee forbids the use of anabolic androgenic steroids to improve sporting performances. Nine anabolic steroids (androsterone (A), nandrolone, estradiol, testosterone propionate, nandrolone-17 propionate, dydrogesterone, testosterone, epitestosterone, boldenone) and alpha-cholestane as internal standard were studied by gas chromatography coupled with mass spectrometry (GC/MS). The derivatisation reagent employed for the derivatisation of anabolic steroids was a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and 2-mercaptoethanol (1000:2:6, v/w/v). Trimethylsilyl (TMS) derivatives were obtained. Anabolic steroids can be derivatised into one or two forms, mainly for androsterone into A-monoTMS and A-diTMS. The aim of this study was to research the optimization conditions of the derivatisation process (maximum yield of silylation reaction) of each anabolic steroid into only one form. A two-level factorial Doelhert design was used to determine the influence of different parameters and their interactions on each compound, thanks to response surface methodology. The parameters to be optimized were the reaction time and the temperature. The interaction "temperature-reaction time" is significant and has a positive effect on the improvement of the effectiveness of the derivatisation. Considering the large amount of information, often not convergent, a global desirability function was applied for multi-responses optimization. Thus, the optimized temperature and the reaction time of silylation were 85 degrees C and 24 min, respectively. Several GC/MS analytical parameters were also studied: linearity (regression coefficient upper than 0.99 for each compound, sensibility (range of concentration 0.05-0.30 microg/ml). Confirmatory experiments were applied to check the predicted values and to validate the model. The confirmatory assay responses are relatively close to the responses predicted. We observed satisfactory resolutions by GC/MS and a run lower than 12 min.     

Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionization (DESI) mass spectrometry (MS). In this work the feasibility of DESI application for the rapid screening of intact esters of anabolic steroids in bovine hair has been studied. Using a linear ion trap both full scan and data-dependent collision induced dissociation MS(n) spectra were acquired in minutes for testosterone cypionate, testosterone decanoate and estradiol benzoate standard solutions deposited on a glass or PTFE surface. However direct analysis of incurred hair failed due to inefficient desorption ionization and the minute quantities of steroid esters present. Therefore a simplified ultrasonic liquid extraction procedure was developed, allowing rapid DESI analysis of a few microliters of the concentrate and a total analysis time of 2-4h per batch instead of 3 days. The potential of this DESI approach is clearly demonstrated by MS(3) data from hair samples incurred with high levels (300-800 μg kg(-1)) of steroid esters, levels which do occur in samples from controlled- and illegally treated animals. For much lower levels state-of-the-art ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) screening methods remain the method of choice and might benefit from the proposed simplified extraction as well.     

Measurement of 19-nortestosterone and its esters in equine plasma by high-performance liquid chromatography with tandem mass spectrometry

A high-performance liquid chromatographic-tandem mass spectrometric (HPLC/MS/MS) method for the determination of 19-nortestosterone and its esters (cyclopentanepropionate, phenylpropionate, and decanoate) in equine plasma is achieved using an atmospheric pressure chemical ionization (APCI) interface in selected reaction monitoring (SRM) mode. The two internal standards used were 16,16, 17-(2)H(3)-19-nortestosterone for 19-nortestosterone and methenolone acetate for its esters. The steroids studied were extracted from plasma samples with a mixture of diethyl ether/n-hexane (9:1, v/v). The quantification limits for 19-nortestosterone, 19-nortestosterone cyclopentanepropionate, 19-nortestosterone phenylpropionate, and 19-nortestosterone decanoate were 0.16, 5.0, 0.1, and 2.0 ng/mL, respectively, when 2 mL of plasma were used. The recoveries of most of the steroids were 71.6-101.0% except for the decanoate, which could be recovered to about 39.8%. The responses were linear, with correlation coefficients varying from 0.9897 to 0.9999 in the concentration range of 0.1 to 50.0 ng/mL for the steroids studied. When applied to equine (mare) plasma samples, the present method allowed detection of 19-nortestosterone up to 23 days after an intra-muscular injection of 400 mg as the decanoate.     

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.     

Properties of the Adsorption Equilibrium Isotherms Used and Measured in RPLC

Monitoring anabolic steroids in meat-producing animals is a challenging task. It implies very specific and sensitive analytical methods able to detect and identify sub-μg kg^?1 residue levels in complex biological matrices such as meat, urine, or hair. Gas and liquid chromatography coupled to mass spectrometry are the most efficient means of achieving these objectives. In this paper we review how developments in mass spectrometry have been rapidly applied to this problem, how efficient analysis of anabolic steroids in urine, edible tissue, and hair has been achieved, and, later, how measurement of conjugate steroids and determination of the origin of natural steroid hormones has been achieved. The performance characteristics of different mass spectrometers (quadrupole, ion-trap, electromagnetic, isotope-ratio, tandem, and hybrid instruments), the efficiency of different acquisition techniques (LR-SIM, HR-SIM, MRM), and, finally, sample introduction (gas chromatography and liquid chromatography, with discussion of alternative interfaces) are discussed, with numerous applications.     

多重机制杂质吸附萃取净化-快速液相色谱-串联质谱法测定鱼组织中11种同化激素

建立了准确、灵敏的鱼组织中11种同化激素(勃地酮、雄烯二酮、诺龙、美雄酮、甲睾酮、睾酮、醋酸睾酮、群勃龙、丙酸睾酮、康力龙、氟甲睾酮)的多重机制杂质吸附萃取净化-快速液相色谱-串联质谱的分析方法。鱼组织均质样品经甲醇提取后,在上清液中加入一定量的C18固体吸附剂、中性氧化铝吸附剂和氨基功能化纳米吸附剂实现快速净化。采用Shim-Pack XR-ODSII色谱柱(100 mm×2.0 mm, 2.2 μm)分离,以乙腈(含0.1%甲酸)和水(含0.1%甲酸)为流动相进行梯度洗脱,电喷雾正离子多反应监测(MRM)模式下检测,外标法定量。结果表明,11种目标化合物在线性范围内具有良好的线性关系,相关系数大于0.999,其在鱼组织中的检出限(S/N＞3)为0.03～0.4 μg/kg,定量限(S/N＞10)为0.1～1.5 μg/kg,平均回收率为80.9%～98.1%,相对标准偏差(RSD)为5.2%～11.5%。该方法简便、快速、准确,可用于鱼组织中同化激素的定性、定量监测。     

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.     

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).     

Surface-enhanced Raman spectroscopy for classification of testosterone propionate and nandrolone residues in chicken

There is a critical need for a rapid and simple screening method of androgens in chicken. In this study, we evaluated surface-enhanced Raman spectroscopy (SERS) coupled with multivariate techniques for the classification of two androgens (i.e., testosterone propionate and nandrolone) in chicken from 294 samples. Raw Raman spectra were pretreated by using the methods of baseline correction, normalization and second derivative. Support vector machines (SVM) model using the score values of the first four principal components as the inputs was developed to classify all the chicken samples into the four groups (i.e., control, nandrolone, testosterone propionate, and testosterone propionate combined with nandrolone groups) with accuracy of 96.9%. Furthermore, the particle swarm optimization (PSO) was adopted to automatically optimize the penalty parameter C and the kernel parameter g of SVM model for improving the classification accuracy. The experimental results demonstrated that SERS, in combination with multivariate methods, could be utilized as a rapid and simple classification assay of androgens in chicken and exhibited great potential in practical applications as a screening tool to better serve customers.     

Determination of ethyl glucuronide and fatty acid ethyl esters in hair samples

Hair testing for alcohol biomarkers is an important tool for monitoring alcohol consumption. We propose two methods for assessing alcohol exposure through combined analysis of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) species (ethyl myristate, palmitate, stearate and oleate) in hair (30 mg). EtG was analysed by liquid chromatography–tandem mass spectrometry, while FAEEs were analysed by gas chromatography–tandem mass spectrometry using electron impact ionization. Both methods were validated according to internationally accepted guidelines. Linearity was proven between 3 and 500 pg/mg for EtG and 30–5000 pg/mg for FAEEs, and the limits of quantification were 3 pg/mg for EtG and 30 pg/mg for each of the four FAEEs. Precision and accuracy were considered adequate, processed EtG samples were found to be stable for up to 96 h left in the injector and processed FAEEs samples for up to 24 h. Matrix effects were not significant. Both methods were applied to the analysis of 15 authentic samples, using the cut‐off values proposed by the Society of Hair Testing for interpretation. The results agreed well with the self‐reported alcohol consumption in most cases, and demonstrated the suitability of the methods to be applied in routine analysis of alcohol biomarkers, allowing monitoring consumption using low sample amounts.     

Effect of extended use of single anabolic steroids on urinary steroid excretion and metabolism

Long-term use of single anabolic steroids by weightlifters and body builders at dosages greater than or equal to 25 mg per 24 h resulted in reduced excretion of urinary androgen metabolites, androsterone and etiocholanolone, compared to values prior to anabolic use. The excretion of major urinary metabolites of glucocorticoids was not affected by anabolic use. Urinary excretion of anabolic steroids or anabolic metabolites averaged 20-25% of total anabolic steroid administered. The major excreted metabolites of methandrostenolone, nandrolone, oxandrolone and oxymetholone were identified by gas chromatography-mass spectrometry based on the major mass spectral ion peaks.     

Gas chromatography/mass spectrometry based hair steroid profiling may reveal pathogenesis in hair follicles of the scalp

A method of steroid profiling, including androgens, progestins, corticoids and sterols, was developed to evaluate the concentrations of steroids as well as the activities of the enzymes responsible for steroidogenesis in hair by gas chromatography/mass spectrometry. The extraction efficiencies of steroids from the hair matrix were improved by ultrasonication for 1 h at 50 °C. The overall recoveries ranged from 71 to 132%, with a limit of quantification for all analytes ranging from 1 to 50 ng/g. The devised method was used to identify the metabolic changes for both male‐pattern baldness (MPB) and the drug efficiency of dutasteride, which inhibits 5α‐reductase. Increased dihydrotestosterone levels and the dihydrotestosterone/testosterone (DHT/T) ratio, which is responsible for the 5α‐reductase activity, were observed in the MPB patients. A dutasteride treatment resulted in decreases in the DHT and 5α‐androstanedione concentrations and DHT/T ratio in the hair samples. Hair steroid profiling reflects the sebaceous status in the scalp and may be useful for monitoring the metabolic responses to both the disease and drug actions. Copyright © 2011 John Wiley & Sons, Ltd.