液相色谱-串联质谱法测定尿液中的内源性类固醇激素

建立了液相色谱-串联质谱(LC-MS/MS)测定尿液中的内源性类固醇激素的方法。尿样经葡萄糖醛酸甙酶酶解后进行液-液提取,以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68:32)为流动相,采用Cosmosil C18色谱柱分离,并以三重四极杆串联质谱多反应监测扫描方式对尿样中的脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素进行检测。方法的最低检出限为0.01～10 ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可替代气相色谱-质谱法用于体液中内源性类固醇激素兴奋剂的常规分析。     

液相色谱-串联质谱法检测血清中15种甾体激素的动态变化

建立了用液相色谱-串联质谱检测血清中15种甾体激素的方法。在血清样品中加入含内标的乙腈沉淀蛋白质后,用100 mmol/L盐酸羟胺衍生,经Agilent-C18柱(50 mm×3.0 mm,2.7μm)分离,内标法定量。质谱分析采用电喷雾电离(ESI)正离子扫描,多反应监测(MRM)模式检测。数据显示,13种激素在0.05~20 ng/mL范围内线性关系良好(相关系数(R2)不小于0.995 6),检出限不大于1 ng/mL;皮质醇及脱氢表雄酮硫酸酯在50~2 000ng/mL范围内线性关系良好(R2不小于0.997 9),检出限不大于0.5 ng/mL。将该方法应用于女性月经周期激素变化规律的研究,结果表明,15种甾体激素在一个完整的月经周期内有不同特征的动态变化,其中雄激素在滤泡期的后期达到最高值,孕(雌)激素在黄体期达到最高值,而皮质激素则波动不明显。该方法灵敏度高、重复性好,为临床诊疗提供了参考。     

Characterization of chemically modified steroids for doping control purposes by electrospray ionization tandem mass spectrometry

The discovery of the designer steroid tetrahydrogestrinone (THG) in elite athletes' doping control samples in 2003 demonstrated the availability of steroid derivatives prepared solely for doping purposes. Modern mass spectrometers utilizing electrospray ionization and collisionally activated dissociation (CAD) of analytes allow the structural characterization of steroids and their derivatization sites by the elucidation of fragmentation behaviors. A total of 21 steroids comprising either a 4,9,11-triene, a 3-keto-4-ene or a 3-keto-1-ene nucleus were investigated regarding their dissociation pathways, deuterated analogues were synthesized and fragmentation routes were postulated, permitting the identification of steroidal structures and modifications. Compounds based on a 4,9,11-triene steroid with an ethyl residue at C-13 (gestrinone analogues) generate abundant fragment ions at m/z 241 and 199, whereas the substitution of the C-13 ethyl group by a methyl residue (trenbolone analogues) results in a shift of m/z 241 to 227. Substances related to testosterone with a 3-keto-4-ene structure give rise to abundant fragment ions at m/z 109 and 97 whereas steroids with a 3-keto-1-ene nucleus eliminate the A-ring including the carbons C-1-C-4, in addition to C-19 that is proposed to migrate from C-10 to C-1 under CAD conditions.     

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.     

Development and validation of a qualitative screening method for the detection of exogenous anabolic steroids in urine by liquid chromatography-tandem mass spectrometry

A screening method for the urinary detection of 34 exogenous anabolic steroids has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography-tandem mass spectrometry. The use of some adducts such as [M+NH₄]⁺, [M+CH₃COO]⁻ and [M+H+MeOH]⁺ was necessary in order to detect some analytes at the required level (lower than 10 ng/ml). Two transitions were selected for each analyte. Different concentration factors have been studied in order to increase the sensitivity. A concentration factor of 50 was selected for the screening method although the high ion suppression observed under these conditions can hamper its application as a quantitative method. The method was validated and limits of detection were obtained by spiking ten different blank urine samples at five different concentration levels. Up to 29 analytes were detected in all spiked urines at the required level. Limits of detection between 1 and 10 ng/ml were obtained for most analytes which fulfil current requirements. The applicability of the method was shown by analysing positive samples.     

Simultaneous analysis of fourteen tertiary amine stimulants in human urine for doping control purposes by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

A method for the simultaneous screening and confirmation of the presence of fourteen tertiary amine stimulants in human urine by gas chromatography-mass spectrometry (GC-MS) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Solid phase extraction (SPE) and liquid-liquid extraction (LLE) approaches were utilized for the pre-treatment of the urine samples. The study indicated that the capillary temperature played a significant role in the signal abundances of the protonated molecules of cropropamide and crotethamide under positive ion electrospray ionization (ESI) conditions. In addition, comparison studies of two different pre-treatment approaches as well as the two ionization modes were conducted. The LODs of the developed method for all the analytes were lower than the minimum required performance limit (MRPL) as set forth in the World Anti-Doping Agency (WADA) technical document for laboratories. The human urine sample obtained after oral administration of prolintane·HCl was successfully analyzed by the developed method, which demonstrated the applicability and reliability of the method for routine doping control analysis.     

Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times.     

Rapid screening of polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch in human urine by liquid chromatography-tandem mass spectrometry

The increasing number of samples and target substances in doping control requires continuously improved screening methods, combining high-throughput analysis, simplified sample preparation, robustness and reliability. Hence, a rapid screening procedure based on liquid chromatography-electrospray ionization-tandem mass spectrometry with in-source collision-induced dissociation was developed. The detection of the polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch (HES) in human urine was established without further sample preparation. The in-source fragmentation strategy of the approach represented a valuable tool in the analysis of the polysaccharide-based compounds, allowing the use of tandem mass spectrometry. After direct injection of urine specimens, analytes were chromatographically separated on a monolithic reverse-phase column and detected via multiple reaction monitoring of diagnostic ions at detection limits of 10 microg/mL for HES and 30 microg/mL for dextran. Validation was performed regarding the parameters specificity, linearity, precision (8-18%) and accuracy (77-105%) and the method was applied to the investigation of approximately 400 doping control samples and seven dextran and two hydroxyethyl starch post-administration samples. The approach demonstrated its capability as a rapid screening tool for the detection of dextran and hydroxyethyl starch and represents an alternative to existing screening procedures since time consuming hydrolysis or derivatization steps were omitted.     

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。     

反相高效液相色谱-串联质谱法测定尿液中儿茶酚胺和肾上腺素(英文)

The measurement of urine catecholamine and metanephrine concentrations is important for biochemical screening and diagnosis of pheochromocytoma.The goal of this work was to develop a simple liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for determining catecholamines and metanephrines in urine to replace an existing liquid chromatographic method using electrochemical detection.Urine samples were prepared using Oasis weak-cation-exchange cartridges.The eluate was analyzed on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column in 3 min.Adrenaline,noradrenaline,dopamine,metanephrine,normetanephrine,and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring(MRM).No evidence of ion suppression was observed.The assay was linear up to 5μmol/L for adrenaline,5μmol/L for noradrenaline,6.1μmol/L for dopamine,5.6μmol/L for metanephrine,and 34.6μmol/L for normetanephrine,with lower limits of quantification of 5,5,12,6 and 7nmol/L,respectively.The intra-day and inter-day precisions for all analytes ranged from 0.59%to 4.64%and1.98%to 4.80%,respectively.External quality assurance samples were assayed and showed excellent agreement with the target values.This simple method provides an improved assay for determining urine catecholamines and metanephrines.     

基于液相色谱-串联质谱的结肠癌血清氧固醇标志物筛选

结肠癌(CC)是全球常见恶性肿瘤之一,发病率呈逐年上升趋势,目前没有有效的标志物用于疾病早期诊断和干预跟踪。胆固醇及其氧化衍生物氧固醇在众多恶性肿瘤发生发展中发挥关键作用。该研究采用液相色谱-串联质谱(LC-MS/MS)技术,对CC临床血清样本中胆固醇及相关10种氧固醇代谢物进行了定性定量分析,并采用偏最小二乘判别分析(PLS-DA)和正交偏最小二乘判别分析(OPLS-DA)进行多元统计分析,发现上述目标代谢物能够较好地区分CC组与健康对照组。为防止数据过拟合,该研究在PLS-DA模型各代谢物变量投影重要性(VIP)基础上,结合最优组分数及K-均值聚类结果,筛选得到3种代谢标志物。通过受试者操作特征曲线(ROC)的曲线下面积(AUC)分析,发现筛选得到的3种潜在标志物联合预测CC达到0.998,说明模型性能优良。GO(基因本体论)富集分析显示3种潜在标志物主要分布在内质网和包被囊泡上,参与胆固醇代谢、运输、低密度脂蛋白重塑等生物进程,发挥胆固醇运输活性和低密度脂蛋白颗粒受体结合的分子功能。KEGG(京都基因与基因组百科全书)通路分析显示3种潜在标志物富集于类固醇生物合成、PPAR(过氧化物酶体增殖物激活受体)信号通路及ABC(ATP结合盒)转运等通路上。该研究为寻找CC标志物及进一步阐明胆固醇及氧固醇在CC发病过程中的作用奠定了一定的基础。     

高效液相色谱-串联质谱法同时测定人尿液中7种邻苯二甲酸酯代谢物

建立了同时检测人尿液中7种邻苯二甲酸酯代谢物的高效液相色谱-串联三重四极杆质谱法。尿液经酶水解后,采用萃取柱净化,以2%(v/v)甲酸甲醇溶液为洗脱剂,经苯基柱分离,以0.1%(v/v)乙酸水溶液和0.1%(v/v)乙酸乙腈溶液为流动相进行梯度洗脱,采用电喷雾离子源负离子模式和多反应监测模式采集信号,用同位素内标法进行定量分析。尿液中7种邻苯二甲酸酯代谢物在0.2~200.0 μg/L范围内定量离子的相对峰面积比值与质量浓度均呈良好线性关系(r≥0.99976);检出限(LOD)为13.43~80.21 ng/L,定量限为44.77~267.37 ng/L; 3个水平的加标回收率为88.8%~108.9%,日内和日间精密度均不大于17.05%。该方法可同时准确、灵敏、简便地测定人尿液中7种邻苯二甲酸酯代谢物的暴露水平。     

Quantification of testosterone undecanoate in human hair by liquid chromatography–tandem mass spectrometry

Testosterone undecanoate (T‐C11) can be used by athletes in order to improve performance. After oral intake, T‐C11 is rapidly metabolized, hampering discrimination between exogenous and endogenous testosterone. A possible alternative is to detect the intact ester in hair. A method based on liquid chromatography–tandem mass spectrometry was developed for the determination of T‐C11 in hair. The sample procedure consisted of digestion of 200 mg of pulverized hair with tris(2‐carboxyethyl)phosphine hydrochloride and liquid–liquid extraction with n‐pentane. Several parameters such as the mobile phase, the ionization source and the washing step were optimized. The method was validated at different spiked levels obtaining satisfactory values for accuracy (between 92 and 102%) with relative standard deviations lower than 7% and a limit of detection of 0.2 ng/g. The applicability of the method was checked by the analysis of three samples from patients using T‐C11. A peak for the analyte was detected in all samples with concentrations between 0.4 and 8.4 ng/g. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Analysis of chloramphenicol in honey by on-line pretreatment liquid chromatography-tandem mass spectrometry

Chloramphenicol is an antibiotic and one of the potential contaminants in honey.Solid-phase extraction is the key pretreatment procedure for analysis of chloramphenicol in honey.In this work,an on-line pretreatment liquid chromatography-tandem mass spectrometer system for sensitive,reliable and higher throughput analysis was developed.With the methylcellulose-immobilized reversed-phase column,sugars in a honey sample were efficiently removed in 1 min.As a result,the limit of quantitation of chloramphenicol was 20 pg/mL(0.2 μg/kg honey).     

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.     

Ionization of anabolic steroids by adduct formation in liquid chromatography electrospray mass spectrometry

The ionization of 46 anabolic steroids has been studied. The absence of basic or acidic moieties in most of these analytes makes their direct ionization as [M + H]+ by atmospheric pressure interfaces difficult. The formation of adducts with different components of the mobile phase has been found to be an efficient way to ionize anabolic steroids by electrospray. Different mobile phases using methanol (MeOH) or acetonitrile as organic solvent and HCOOH, Na+ or NH4+ as additives have been tested to favor the adduct formation. A direct correlation between the chemical structure of the anabolic steroid and the possibility to ionize it in a particular chromatographic condition has been found. According to their ionization, anabolic steroids can be divided into seven different groups depending on both the nature and the relative position of their functional groups. The formation of different adducts such as [M + Na + MeOH]+ or [M + H + CH3 CN - H2O]+ is required in order to ionize some of these groups and the optimal mobile phase composition for each group of anabolic steroids is proposed. Despite the ionization limitations due to their chemical structure, most of tested anabolic steroids could be ionized using the adduct formation approach.     

Relationships between structure,ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I–IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H]⁺ or [M + H–nH₂O]⁺ in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05–20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05–1, 2–5 and 10–20 ng/mL, respectively. Steroids including the conjugated keto‐functional group at C3 showed good proton affinity and stability, and generated the [M + H]⁺ ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H]⁺ ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H ? H₂O]⁺ or [M + H ? 2H₂O]⁺ ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC‐MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I–V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of aflatoxins in olive oil by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric with electrospray ionization (LC/ESI-MS/MS) method for determining the four naturally occurring aflatoxins (AFs) B1, B2, G1, and G2 in olive oil is proposed. AFs were extracted from oil sample by means of matrix solid phase dispersion (MSPDE), utilizing C18 as dispersing material. No further purification step, such as lipid removal, was performed. Aflatoxin M1, the hepatic metabolite of AFB1, was employed as internal standard. Olive oil extract was analyzed by LC/ESI-MS/MS in positive ionization mode, with multireaction monitoring acquisition. Due to a signal suppression ranging between 4 and 23%, quantitation was performed by matrix-matched calibration curves. The regression line coefficients of determination were above 0.9991. Sample recoveries ranged from 92 to 107%, with relative standard deviations below 13% for spiking levels between 0.5 and 5 ng g⁻¹; method quantification limits ranged between 0.04 and 0.12 ng g⁻¹. The developed LC/ESI-MS/MS method, although not as sensitive as LC coupled to fluorescence detection, is rapid, selective, accurate and precise, thus it can be used as confirmatory assay. The MSPDE appears suitable for application to other oleaginous matrices and for multiresidue investigation.     

基于非靶标筛查的超高效液相色谱-三重四极杆质谱法检测尿液样品中的酰基肉碱

建立了一种基于母离子扫描模式的超高效液相色谱-三重四极杆质谱检测尿液中酰基肉碱的分析方法。对酰基肉碱类化合物所共有的m/z为60、85和144的碎片离子进行选择性检测,结合化合物母离子扫描的结果及其对应的保留时间,选取一致性较好的化合物进行筛选,再利用高分辨质谱确认,最终检测到37种酰基肉碱化合物,其中有14种尚未被HMDB和LIPID MAPS数据库收录。该方法可应用于其他生物样本(如血液、组织)中酰基肉碱的定性、定量分析,可作为检测酰基肉碱化合物的新选择。