液相色谱-串联质谱结合谱库检索法同时测定猪组织中12种类固醇激素

Determination of 12 steroid hormone residues in pig tissues by liquid chromatography-tandem mass spectrometry combining with library search

建立了猪组织中12种类固醇类激素(炔诺酮、雄诺龙、醛固酮、去氢睾酮、达那唑、去氢甲睾酮、甲基睾丸酮、诺龙、孕酮、康力龙、睾酮和丙酸睾酮)多残留的液相色谱-四极杆/线性离子阱串联质谱(QTrap LC-MS/MS)的检测方法。组织样品经β-葡萄糖醛苷酶/芳基硫酸酯酶酶解提取,混合型强阳离子交换固相萃取小柱(MCX柱)净化后,以含0.1%(体积分数)甲酸的乙腈和水为流动相,经Venusil MP C18色谱柱(100mm×2.1mm,3μm)分离,按照多级反应监测(MRM)、信息相关采集(IDA)、增强子离子扫描(EPI),结合建立的12种类固醇类激素的标准谱图库检索的多步模式进行分析。结果表明,12种类固醇激素的线性范围为0.5～100.0μg/L,线性相关性良好(r>0.99);样品在5.0μg/kg添加水平下的平均回收率为72.0%～98.1%,相对标准偏差为3.1%～12.5%;方法检出限(S/N=3)为0.2～0.5μg/kg。实际样品检测结果表明,应用本方法可以实现对猪组织样本中类固醇类激素残留的定性定量分析,且具有灵敏、准确的特点。

A method was developed for the simultaneous determination and identification of 12 steroid hormone residues in pig tissues,including stanolone,aldosterone,boldenone,danazol,metandienone,methyltestosterone,nadrolone,norethindrone,progesterone,stanozolol,testosterone and testosterone propionate,using liquid chromatography-tandem triple-quadrupole linear ion trap mass spectrometry（LC-MS/MS）.Homogenized pig tissue samples were purified with a Waters MCX solid phase extraction column after enzymatic hydrolysis by β-glucuronidase,then separated on a Venusil MP C18 column（100 mm×2.1 mm,3 μm） using gradient elution with the mobile phases of acetonitrile and water with 0.1%（v/v） formic acid.A multiple reaction monitoring（MRM） as survey scan and an enhanced product ion（EPI） scan as dependent scan were performed in an information dependent acquisition（IDA） experiment.The compound identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode.The results showed that the limits of detection（LODs,S/N=3） were in the range of 0.2-0.5 μg/kg for the steroid hormones,and with a good linearity（r〉0.99） ranged from 0.5 to 100.0 μg/L.The average recoveries（n=6） of the 12 steroid hormones spiked in pig tissue samples at 5.0 μg/kg ranged from 72.0% to 98.1% with the relative standard deviations（RSDs） between 3.1% and 12.5%.The method was applied for the qualitative and quantitative determination of steroid hormone residues in pig tissues with sensitive and accurate characteristics.