基于QuEChERS提取的HPLC-MS/MS法检测人血浆中6种常用利尿剂

Detection of 6 common diuretics in human plasma by HPLC-MS/MS based on QuEChERS extraction

目的:建立液相色谱-串联质谱(HPLC-MS/MS)同时检测人血浆中6种常用利尿剂氢氯噻嗪、呋塞米、托拉塞米、螺内酯、吲达帕胺和氨苯蝶啶的检测方法。方法:血浆样品酶解后,用提取溶剂[乙酸乙酯-乙醚-异丙醇(6∶3∶1)]溶液提取,经QuEChERS(分散固相萃取)方法净化后,以乙腈与水为流动相梯度洗脱,Eclipse Plus C18(3.0 mm×100 mm,1.8μm)色谱柱分离,电喷雾正负离子同时扫描模式,多级反应监测(MRM)模式进行检测。结果:6个化合物质量浓度在1.0~250.0μg·L-1范围内呈良好线性,相关系数均大于0.996。其中,氢氯噻嗪、托拉塞米、吲达帕胺和氨苯蝶啶的检测下限(S/N≥3)为0.30μg·L-1,呋塞米和螺内酯为0.5μg·L-1;氢氯噻嗪、托拉塞米、吲达帕胺和氨苯蝶啶的定量下限(S/N≥10)为1.0μg·L-1,呋塞米和螺内酯为2.0μg·L-1,样品在其定量下限1倍、2倍、10倍3个加标水平下回收率在75.2%~90.6%之间,RSD在3.2%~8.1%之间。10名健康成年男性志愿者口服吲达帕胺2.5 mg后1 h的血药浓度最高(平均为65.3μg·L-1),随之急速下降,口服24 h后下降到19.4μg·L-1,96 h时平均浓度为3.2μg·L-1,120 h后所有志愿者血浆检测均显示阴性。结论:该方法灵敏、准确,操作性强,可满足于血浆样品中6种常用利尿剂的同时定量检测分析。

Objective:To establish a method for simultaneous detection of 6 common diuretics,such as hydrochlorothiazide,furosemide,torasemide,spironolactone,indapamide and triamterene residues in human plasma by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Methods:The plasma samples were digested by enzyme and purified with Qu ECh ERS method after extraction by extracting solution[ethyl acetate-diethyl ether-isopropyl alcohol(6∶3∶1)],then separated on an Eclipse Plus C18 column(3.0 mm×100 mm,1.8μm)using gradient elution with mobile phases of acetonitrile and water.The electrospray was operated in both positive and negative mode at the same time and was detected using multiple reaction monitoring(MRM).Results:The calibration curves showed good linearity in the range of 1.0-250.0μg·L-1 for 6 compounds with correlation coefficient(r)greater than 0.996.The limits of detection(LODs,S/N≥3)for 4 diuretics(hydrochlorothiazide,torasemide,indapamide and triamterene)were 0.30μg·L-1 and the limits of quantitation(LOQs,S/N≥10)were 1.0μg·L-1.LODs of furosemide and spironolactone were 0.30μg·L-1 and LOQs were 2.0μg·L-1.On the basis of LOQs,the average recoveries for 6 compounds in human plasma samples at three spiked levels(1 times,2 times,10 times)ranged from 75.2%to 90.6%with relative standard deviations(RSDs)of 3.2%-8.1%.10 volunteers of healthy adult male was selected for oral 2.5 mg indapamide,the highest blood concentration(average 65.3μg·L-1)was observed after oral indapamide 1 hours,followed by a rapid decline,then drop to 19.4μg·L-1 after 24 hours.The average concentration was 3.2μg·L-1 at 96 hours and all volunteers showed negative after 120 hours.Conclusion:The method is sensitive,accurate and easy to operate,and can be used for simultaneous quantitative analysis of 6 compounds used diuretics in plasma samples.