Genangeli Michele, Caprioli Giovanni, Cortese Manuela, Laus Fulvio, Matteucci Mara, Petrelli Riccardo, Ricciutelli Massimo, Sagratini Gianni, Sartori Stefano, Vittori Sauro
School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
School of Biosciences and Veterinary Medicine, University of Camerino, Via Circonvallazione 93/95, 62024, Matelica, Italy.
J Mass Spectrom. 2017 Jan;52(1):22-29. doi: 10.1002/jms.3896.
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 , respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml ) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml 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 ) and cortisol (range 32.57-52.24 ng ml ), followed by 17-OH-pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.
已采用超高效液相色谱 - 串联质谱技术，开发出一种全新、快速且简便的分析方法，该方法能够同时鉴定和定量马血清中的17种类固醇激素及其代谢物（孕烯醇酮、17 - 羟基孕烯醇酮、孕酮、17 - 羟基孕酮、雄酮、雄烯二酮、脱氢表雄酮、硫酸脱氢表雄酮、睾酮、皮质醇、皮质酮、醛固酮、11 - 脱氧皮质醇、11 - 脱氧皮质酮、双氢睾酮、雌酮和雌二醇）。总共400μl样品用1000μl乙腈进行脱蛋白处理，蒸发后，用50μl含25%甲醇的溶液复溶，并注入超高效液相色谱 - 串联质谱三重四极杆中。通过在两种不同浓度下向基质中加入类固醇激素标准混合物获得的回收率在所有情况下均高于85.60%，变异系数百分比低于8.37%。所分析化合物校准曲线的相关系数范围为0.9922 - 0.9986，检测限和定量限分别在0.002 - 2和0.0055 - 5.5 ng/ml范围内。睾酮的检测定量限（即50 pg/ml）相较于去势公马血浆中允许的阈值（100 pg/ml游离睾酮）低两倍。该方法的高灵敏度和定量特性使得能够检测马血清中的大多数类固醇。经验证后，该方法用于定量母马、种马和去势公马血清样品中的17种类固醇激素。检测到的主要类固醇为皮质酮（范围37.25 - 51.26 ng/ml）和皮质醇（范围32.57 - 52.24 ng/ml），其次是17 - 羟基孕烯醇酮、双氢睾酮和孕烯醇酮。版权所有© 2016 John Wiley & Sons, Ltd.
