高效液相色谱-质谱法测定大鼠不同脑区的硫酸酯型神经甾体

应用高效液相色谱-质谱联用技术,以雌酮硫酸酯为内标,建立了大鼠不同脑区硫酸酯型神经甾体的测定方法。甾体分两步萃取,第一步用氯仿-仲丁醇(体积比为1∶1)提取甾体硫酸酯,然后经固相萃取纯化。溶剂解使甾体硫酸酯形成游离型甾体,然后用衍生化试剂进行衍生化,再用液相色谱-质谱分离测定。初步研究发现,雄性SD大鼠不同脑区神经甾体孕烯醇酮硫酸酯和脱氢表雄酮硫酸酯的含量分别为(4.14±1.33) ng/g和(2.26±0.76) ng/g(垂体),(1.98±1.13) ng/g和(1.80±0.93) ng/g(下     

超声提取辅助顶空式固相微萃取/气相色谱-质谱法测定塑胶跑道面层中7种挥发性硫化物

建立了一种检测塑胶跑道面层中二甲基硫（DMS）、二硫化碳（CS2）、甲基乙基硫（MES）、二乙基硫（DES）、二甲基二硫（DMDS）、二乙基二硫（DEDS）、二甲基三硫（DMTS）7种挥发性硫化物（VSCs）的超声提取辅助顶空式固相微萃取/气相色谱-质谱（HS-SPME/GC-MS）分析方法。实验优化了顶空固相微萃取的萃取条件、超声提取条件及气相色谱-质谱参数。在优化条件下,DES、DMDS的线性范围为5～500 ng/g,其余5种VSCs的线性范围为10～500 ng/g,相关系数（r）为0.984 7～0.998 9,方法的检出限（S/N=3）为0.4～1.4 ng/g,定量下限（S/N=10）为1.3～4.7 ng/g。在低、中、高3个加标水平下的平均回收率为89.6%～104%,相对标准偏差（RSD,n=6）为3.5%～12%。该方法可实现分离、富集和进样一体化,适用于塑胶跑道面层中VSCs含量的日常检测。     

亚临界水萃取及气相色谱-串联质谱法检测红茶中多种农药残留

建立了亚临界水萃取及气相色谱-串联质谱(GC-MS/MS)检测红茶中21种有机氯和拟除虫菊酯农药残留的方法。在萃取压力为5 MPa条件下,样品经150 ℃的亚临界水提取15 min后,将目标物转移至丙酮-正己烷(1:1, v/v)中,经ENVI-Carb固相萃取净化小柱净化,DB-5毛细管气相色谱柱分离,在多反应监测(MRM)模式下进行MS/MS检测,基质匹配溶液内标法定量。各目标物在5.0～320.0 μg/L范围内线性关系良好,相关系数均大于0.99,其定量限(信噪比(S/N)＞10)为50 ng/g,检出限(S/N＞3)为10 ng/g。茶叶基质中添加50、100和200 ng/g的标准品时,21种农药的回收率为70.18%～119.98%,相对标准偏差(RSD)为5.01%～11.76%。该方法的灵敏度、准确度和精密度均符合农药残留测定的技术要求,适用于红茶中有机氯和拟除虫菊酯农药残留的检测。     

固相萃取-气相色谱/负化学电离-质谱法测定酸水解植物蛋白及酱油中的3-氯-1,2-丙二醇

建立了酸水解植物蛋白及酱油中3-氯-1,2-丙二醇(3-MCPD)的固相萃取-气相色谱/质谱测定方法。样品经Aoisa-HBL固相萃取柱萃取,正己烷-乙酸乙酯净化提取,七氟丁酰咪唑衍生,衍生物经气相色谱/负化学电离-质谱（GC/NCI-MS）选择离子模式(SIM)检测,外标法定量。3-MCPD的定量检测限为0.5 μg/kg,平均回收率为92.2%～97.4%,相对标准偏差为3.6%～10.9%。该方法检测灵敏度高,定性定量准确。     

微波辅助萃取/气相色谱-质谱联用分析蔬菜中的有机磷农药

建立了微波辅助萃取（MAE）/气相色谱-质谱联用法（GC-MS）测定蔬菜样品中二嗪磷、水胺硫磷的分析方法，研究了不同溶剂的萃取效率。选择二氯甲烷为萃取溶剂，采用二因素三水平的正交设计实验优化了萃取溶剂体积和萃取时间。方法的线性范围分别为二嗪磷和对硫磷4ng/g-400ng/g，水胺硫磷20ng/g-400ng/g，检出限分别为二嗪磷和对硫磷4ng/g-400ng/g、水胺硫磷20ng/g-400ng/g，检出限分别为二嗪磷0.29ng/g、对硫磷1.70ng/g、水胺硫磷2.30ng/g。测定200.0ng/g和50.0ng/g加标蔬菜样品，回收率为72.2%-102.0%,RSD为1.5%-11.0%。与传统的机械振荡萃取法相比，不仅萃取效率相当，而且还具有省时省溶剂的优点。     

顶空固相微萃取-气相色谱-串联质谱法测定香精香料中黄樟素

提出了顶空固相微萃取-气相色谱-串联质谱法测定香精香料中黄樟素含量的方法。为使固相微萃取达到更高的效率,选用65μm聚二甲基硅氧烷/二乙烯苯作为微萃取的涂层,萃取温度及时间为（25±5）℃和30 min。用DB-5MS毛细管色谱柱分离,电子轰击离子源串联质谱模式检测。选定黄樟素的母离子和子离子分别为m/z 162和m/z 131,内标丙酸苯乙酯的定量离子为m/z 104。黄樟素的线性范围为20～1 000 ng·g^-1,方法的检出限（3S/N）为2.2 ng·g^-1。在3个浓度水平上做回收试验,加标回收率在71.1%～114.0%之间,相对标准偏差（n=5）在3.4%～16%之间。     

微波辅助萃取-气相色谱质谱法检测生物体内的多溴联苯醚

建立了微波辅助萃取-气相色谱质谱法测定生物样品中的多溴联苯醚(PBDEs)的方法,优化了萃取剂的种类、萃取剂用量、萃取时间等微波萃取条件和GC-MS仪器分析条件。正己烷-丙酮混合溶剂提取后,经实验室自制的多层硅胶柱分离纯化,用气相色谱-质谱进行测定,该方法基质加标回收率在60%～77%之间,相对标准偏差在11%～18%之间,方法的检测限为0.03～0.20ng/g,适用于生物样品中PBDEs的测定。     

体液中-叶萩碱检测方法的研究

本文报道了用气相色谱(GC)及气相色谱-质谱联用(GC/MS)检出人体液中一叶萩碱的方法.尿样经碱化后用乙醚直接萃取,回收率为72.1％;血浆样品用Sep-pak小柱提取,回收率为 43.9％,GC/MS的检测限为 20ng.方法快速、简便、准确.     

固相萃取–气相色谱–串联质谱法检测土壤中15种邻苯二甲酸酯

建立了固相萃取结合气相色谱串联质谱联用仪检测土壤中15种邻苯二甲酸酯残留的方法。样品采用丙酮和石油醚超声提取,取上清液浓缩,上弗罗里硅土固相萃取柱净化,收集洗脱液,定容,以气相色谱串联质谱法分析。该方法在20~2 000 ng/g范围内线性关系良好(r~20.999 0),检出限(S/N=3)为0.12~0.61 ng/g。20,50,200 ng/g 3个添加浓度的15种邻苯二甲酸酯的加标回收率在78.9%~101.8%之间,测定结果的相对标准偏差为0.19%~8.34%(n=5)。该方法准确、灵敏,符合痕量分析的要求,适用于土壤中邻苯二甲酸酯类残留的分析。     

顶空固相微萃取-气相色谱质谱法测定蜂蜜中的苯酚残留量

建立了一种顶空固相微萃取-气相色谱质谱法(SPME-GC/MS)测定蜂蜜中苯酚的分析方法。对SPME纤维头、萃取温度、萃取时间及解吸时间等萃取条件进行了优化。结果表明:用85μm的聚丙烯酸酯(PA)萃取涂层对蜂蜜中的苯酚萃取效果很好,苯酚在0.5~1000 ng/g的浓度范围内,方法的检出限为0.1ng/g,相对标准偏差(n=9)为3.3%,平均回收率为85.79%~99.35%。     

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

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

Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography–Tandem Mass Spectrometry for Automated Analysis of Four Sulfated Steroid Metabolites in Saliva Samples

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC–MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01–2 ng mL⁻¹ for PREGS, DHEAS, and CRTS and of 0.05–10 ng mL⁻¹ for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL⁻¹, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6–112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.     

Studies on neurosteroids XXII. Liquid chromatography-tandem mass spectrometric method for profiling rat brain 3-oxo-4-ene-neuroactive steroids

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous determination of five 3-oxo-4-ene-neuroactive steroids, i.e. androstenedione, testosterone (T), progesterone (PROG), 20alpha-dihydroprogesterone and 20beta-dihydroprogesterone, in rat brain has been developed and validated. The brain steroids were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges and subjected to LC-ESI-MS/MS. The method does not require derivatization. Deuterium-labeled T and PROG were used as the internal standards, and quantification was based on the selected reaction monitoring mode. This method allowed the reproducible and accurate quantification of the brain neuroactive steroids using 100 mg of tissue; the intra- and inter-assay relative standard deviations were below 4.7 and 4.3%, respectively, and the accuracy values were 97.6-103.2% for all the steroids. The limits of quantitation were 0.1 ng/g tissue for all the steroids. The application of this developed method for the analysis of changes in the brain neuroactive steroid levels by immobilization stress is also presented.     

Routine method for the simultaneous quantification of 17alpha-hydroxyprogesterone, testosterone, dehydroepiandrosterone, androstenedione, cortisol, and pregnenolone in human serum of neonates using gas chromatography-mass spectrometry

Steroid determination by immunoassays results in significant interferences and inaccurate results. This study describes the development and validation of a new gas chromatographic-mass spectrometric method for the simultaneous quantification of 17alpha-hydroxyprogesterone (17alphaOHP), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (Delta4-A), cortisol (F) and pregnenolone (Preg) in serum of neonates. Steroids were extracted and purified from 0.5 mL serum using diethyl ether and Extrelut mini NT1 column. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA)/trimethylsilyl iodide (TMSI)/dithioerythritol (DTE) and the resulting trimethylsilyl derivatives were quantified by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The detection limit for all steroids was lower than 0.1 ng/mL. The limit of quantification was 0.1 ng/mL for all steroids except cortisol which was at 0.25 ng/mL. d3-Testosterone and methyltestosterone served as internal standards. Precision for all compounds at the concentrations of 0.5, 1, 5 and 10 ng/mL (n = 10) in fortified steroid-free serum samples ranged from 0.8% to 16.6%. Accuracy was calculated at the concentrations of 0.5, 1, 5 and 10 ng/mL and ranged from -9.2% to 10.6% (n = 10). Linear calibration equations were obtained for all five steroids (0.125-31.25 ng/mL) and for cortisol (0.125-200 ng/mL). Relative recoveries at concentrations 1.0 and 12.5 ng/mL ranged from 70.5% to 97.5%. Absolute recoveries at the same concentrations ranged from 73.2% to 96.6%. Reference intervals were estimated for infants aged from 9 to 40 days. The proposed steroid profile is suitable for routine analysis and provides meaningful data for samples within normal range as well as those with elevated levels.     

Derivatization in Liquid Chromatography/Mass Spectrometric Analysis of Neurosteroids

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Development and application of a UHPLC‐MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17‐OH‐pregnenolone, progesterone, 17‐OH‐progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11‐deoxycortisol, 11‐deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra‐high‐performance liquid chromatography–tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra‐high‐performance liquid chromatography–tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922–0.9986, and the limits of detection and limits of quantification were in the range of 0.002–2 and 0.0055–5.5 ng ml⁻¹, respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml⁻¹) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml⁻¹ free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25–51.26 ng ml⁻¹) and cortisol (range 32.57–52.24 ng ml⁻¹), followed by 17‐OH‐pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.     

Mass Spectrometry Combinations for Structural Characterization of Sulfated-Steroid Metabolites

Steroid conjugates, which often occur as metabolites, are challenging to characterize. One application is female-mouse urine, where steroid conjugates serve as important ligands for the pheromone-sensing neurons. Although the two with the highest abundance in mouse urine were previously characterized with mass spectrometry (MS) and NMR to be sulfated steroids, many more exist but remain structurally unresolved. Given that their physical and chemical properties are similar, they are likely to have a sulfated steroid ring structure. Because these compounds occur in trace amounts in mouse urine and elsewhere, their characterization by NMR will be difficult. Thus, MS methods become the primary approach for determining structure. Here, we show that a combination of MS tools is effective for determining the structures of sulfated steroids. Using 4-pregnene analogs, we explored high-resolving power MS (HR-MS) to determine chemical formulae; HD exchange MS (HDX-MS) to determine number of active, exchangeable hydrogens (e.g., OH groups); methoxyamine hydrochloride (MOX) derivatization MS, or reactive desorption electrospray ionization with hydroxylamine to determine the number of carbonyl groups; and tandem MS (MSⁿ), high-resolution tandem MS (HRMS/MS), and GC-MS to obtain structural details of the steroid ring. From the fragmentation studies, we deduced three major fragmentation rules for this class of sulfated steroids. We also show that a combined MS approach is effective for determining structure of steroid metabolites, with important implications for targeted metabolomics in general and for the study of mouse social communication in particular.

Studies on neurosteroids X. Determination of pregnenolone and dehydroepiandrosterone in rat brains using gas chromatography-mass spectrometry-mass spectrometry

An assay method for pregnenolone and dehydroepiandrosterone in rat brains is developed using gas chromatography (GC)-electron ionization-mass spectrometry (MS)-MS. The extract of the rat brain homogenate containing deuterated internal standard with organic solvent is purified by silica gel minicolumn chromatography. The obtained fraction is derivatized into methyloxime, treated with dimethylisopropylsilylimidazole, and then subjected to GC-MS-MS. The method is applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which are divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increases more than three times after stress, dehydroepiandrosterone in both regions is not so clearly influenced by stress.     

Derivatization in Liquid Chromatograhy/Mass Spectrometric Analysis of Neurosteroids

 Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances However, some compounds do not show enough sensitivity in LC/MS and soft ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information This report presents an overview