基于QuEChERS提取的HPLC-MS/MS法检测人血浆中6种常用利尿剂

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，1.8 μm）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1.0~250.0 μg·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0.30 μg·L^-1，呋塞米和螺内酯为0.5 μg·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1.0 μg·L^-1，呋塞米和螺内酯为2.0 μg·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65.3 μg·L^-1），随之急速下降，口服24 h后下降到19.4 μg·L^-1，96 h时平均浓度为3.2 μg·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 QuEChERS 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,1.8 μm)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 1.0-250.0 μg·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 0.30 μg·L^-1 and the limits of quantitation(LOQs,S/N ≥ 10)were 1.0 μg·L^-1. LODs of furosemide and spironolactone were 0.30 μg·L^-1 and LOQs were 2.0 μg·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 65.3 μg·L^-1)was observed after oral indapamide 1 hours,followed by a rapid decline,then drop to 19.4 μg·L^-1 after 24 hours. The average concentration was 3.2 μg·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. 

[1] BI HG,COOPER SF,COTE MG.Determination and identification of amiloride in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry[J].J Chromatogr,1992,582(7):93
[2] LEGORBURU MJ,ALONSO RM,JIMENEZ RM,et al.Quantitative determination of indapamide in pharmaceuticals and urine by high-performance liquid chromatography with amperometric detection[J].J Chromatogr Sci,1999,37(6):283
[3] HANG TJ,ZHAO W,LIU J,et al.A selective HPLC method for the determination of indapamide in human whole blood:application to a bioequivalence study in Chinese volunteers[J].J Pharm Biomed Anal,2006,40(l):202
[4] 王媛,张文韬,李斯特,等.固相萃取-高效液相色谱法同时减肥功能食品中五种违禁药[J].四川大学学报(医学版),2010,41(5):873 WANG Y,ZHANG WT,LI ST,et al.Simultaneous determination of five illegal drugs in weight control foods with solid phase extraction-high performance liquid chromatography[J].J Sichuan Univ(Med Sci Ed),2010,41(5):873
[5] 吴迪,郭伟英.HPLC法同时测定缬沙坦氢氯噻嗪胶囊中缬沙坦和氢氯噻嗪的含量[J].中国药房,2012,23(9):846 WU D,GUO WY.Content determination of valsartan and hydrochlorothiazide in valsartan and hydrochlorothiazide capsules by HPLC[J].China Pharm,2012,23(9):846
[6] MARGALHO C,BOER D,GALLARDO E,et al.Determination of furosemide in whole blood using SPE and GC-EI-MS[J].J Anal Toxicol,2005,29(5):309
[7] DING L,YANG LH,LIU F,et al.A sensitive LC-ESI-MS method for the determination of indapamide in human plasma:method and clinical applications[J].J Pharm Biomed Anal,2006,42(4):213
[8] 佟玲,齐丹丹,毕开顺,等.高效液相色谱-串联质谱法测定人血浆中叫哒帕胺及其生物等效性研究[J].中国新药与临床杂志,2006,25(11):851 TONG L,QI DD,BI KS,et al.Determination of indapamide in human plasma with high performance liquid chromatography-mass spectrometry method and bioequivalence study[J].Chin J New Drugs Clin Rem,2006,25(11):851
[9] 秦旸,徐友宣,吴侔天.液相色谱-质谱法快速筛选人尿中26个利尿剂[J].质谱学报,2007,28(4):229 QIN Y,XU YX,WU MT.Screening for 26 diuretics in human urine by rapid HPLC-MS[J].J Chin Mass Spectroc Soc,2007,28(4):229
[10] 朱永林,贡玉清,邵德佳,等.高效液相色谱-串联质谱法测定动物尿液中8种利尿剂残留量[J].中国兽药杂志,2008,42(9):6 ZHU YL,GONG YQ,SHAO DJ,et al.Determination of 8 diuretic residues in animal urine by HPLC-MS/MS[J].Chin J Vet Drug,2008,42(9):6
[11] 曾祥林,何颂华,黄晓东,等.HPLC-MS/MS检测中药制剂及保健食品中添加6个减肥药物的研究[J].中国药学杂志,2009,44(17):1339 ZENG XL,HE SH,HUANG XD,et al.Identification of six anti-obesity chemical drugs added into traditional Chinese medicines and health foods by liquid chromatography-ion trap mass spectrometry method[J].Chin Pharm J,2009,44(17):1339
[12] 梁宇光,曲恒燕,李媛媛,等.HPLC-MS/MS法测定人体血浆中吲哒帕胺的浓度[J].中国临床药理学与治疗学,2012,17(2):206 LIANG YG,QU HY,LI YY,et al.Quantitative determination of indapamide in human plasma by high performance liquid chromatography-tandem mass spectrometry[J].Chin J Clin Pharmacol Ther,2012,17(2):206
[13] 丁宝月,屠婕红,薛磊冰,等.UPLC-MS/MS法快速测定降压类中成药及保健食品中非法添加34种化学药的研究[J].中草药,2015,46(5):688 DING BY,TU JH,XUE LB,et al.Rapid determination of 34 chemicals illegally added into Chinese patent medicines and health foods with blood pressure lowering function by UPLC-MS/MS[J].Chin Tradit Herb Drugs,2015,46(5):688
[14] ZHANG L,WANG R L,TIAN Y,et al.Determination of torasemide in human plasma and its bioequivalence study by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry[J].J Pharm Anal,2016,6(2):95
[15] 宫旭,芦丽,冯有龙,等.UPLC-MS/MS法测定减肥降脂通便三类保健食品中添加的37种药物[J].药物分析杂志,2016,36(5):918 GONG X,LU L,FENG YL,et al.Determination of 37 drugs illegally added in weight-losing,lipid-lowering and laxative health foods by UPLC-MS/MS[J].Chin J Pharm Anal,2016,36(5):918
[16] 马艳华,闫宽,董颖,等.液相色谱-动态串联质谱技术同时检测人尿中120种刺激剂和利尿剂等禁用物质[J].中国运动医学杂志,2016,35(5):461 MA YH,YAN K,DONG Y,et al.Mass screening of 120 stimulants,diuretics and other prohibited substances in human urine by LC-MS/MS with dynamic MRM[J].Chin J Sports Med,2016,35(5):461
[17] SHAH JV,SHAH PA,SANYAL M,et al.Simultaneous quantification of amiloride and hydrochlorothiazide in human plasma by liquid chromatography-tandem mass spectrometry[J].J Pharm Anal,2017,7(5):288
[18] 郭礼强,宫小明,丁葵英,等.基于QuEChERS提取的液相色谱-串联质谱法测定干腌火腿中15种真菌毒素[J].分析测试学报,2015,34(2):141 GUO LQ,GONG XM,DING KY,et al.Determination of 15 mycotoxins in dry-cured hams with QuEChES-based extraction and liquid chromatography-tandem mass spectrometry[J].J Instrum Anal,2015,34(2):141