LC-MS/MS法测定人血浆霉酚酸浓度及与EMIT法的相关性分析  

Determination of mycophenolic acid in human plasma by LC-MS/MS and its correlation with EMIT

作　　者：刘焕君[1] 张蕊[1] 李荣[1] 孔祥麟[1] 袁桂艳[1] LIU Huan-jun;ZHANG Rui;LI Rong;KONG Xiang-lin;YUAN Gui-yan(Department of Pharmacy,Qilu Hospital of Shandong University,Jinan 250012,Shandong Province,China)

机构地区：[1]山东大学齐鲁医院药学部,山东济南250012

出　　处：《中国临床药理学杂志》2020年第22期3802-3806,共5页The Chinese Journal of Clinical Pharmacology

基　　金：山东省自然科学基金面上基金资助项目(ZR2017MH064)。

摘　　要：目的建立LC-MS/MS法测定人血浆中霉酚酸浓度,并与传统的EMIT方法检测结果进行相关性分析,考察两种方法的准确性和可靠性。方法用乙腈沉淀蛋白处理血浆样本,以格列齐特为内标,以5 mmol·L-1醋酸铵-乙腈(50∶50,v/v)为流动相,用Diamonsil C18(2)(150 mm×4.6 mm, 5μm)为色谱柱,柱温25℃,流速0.5 mL·min-1。电喷雾离子源(ESI),负离子模式,毛细管电压4000 V,干燥气(N2)温度350℃,干燥气流速9 L·min-1,雾化器压力275 kPa,霉酚酸和格列奇特内标的碎片电压分别为140和135 V,碰撞能分别为25和12 eV,多级反应监测(MRM)质荷比为m/z 319.3→m/z 191.4(霉酚酸)和m/z 322.5→m/z 170.4(格列奇特)的碎片离子峰。收集临床上服用霉酚酸制剂的患者血浆样本249份,用建立的LC-MS/MS法测定血浆中霉酚酸浓度。用Passing-Bablok回归分析和Bland-Altman分析进行LC-MS/MS和EMIT的相关性分析,考察方法的准确性和可靠性。结果 LC-MS/MS法检测霉酚酸的标准曲线范围是0.05~10μg·mL-1。批内和批间精密度小于11.60%,提取回收率在108.87~111.42%,基质效应为90.91%~92.62%,霉酚酸在设定的各种存储条件下均稳定。用本方法检测了249份服用霉酚酸患者的血浆样本,血浓度范围为0.057~24.90μg·mL-1,平均值为3.26μg·mL-1。EMIT法检测的霉酚酸浓度范围为0.16~24.87μg·mL-1,平均值为4.72μg·mL-1,略高于LC-MS/MS法,但相关性分析显示两种方法具有良好相关性,提示两种方法的测定结果均准确可靠。结论建立的LC-MS/MS法操作简单、快速、灵敏,检测结果略低于传统的EMIT法,但二者相关性良好,提示两种方法均可用于临床上霉酚酸的血浓度监测。Objective To develop a LC-MS/MS method for determination of mycophenolic acid in human plasma, analyze the correlation of established LC-MS/MS and traditional EMIT method, and to evaluate the accuracy and reliability of the two methods. Methods Mycophenolic acid and gliclazide(internal standard) in plasma were precipitated with acetonitrile and separated on a Diamonsil C18(2)(150 mm×4.6 mm, 5 μm) column using 5 mmol·L-1 ammonium acetate-acetonitrile(50∶50, v/v) as mobile phase at a flow rate of 0.5 mL·min-1. The mass spectrometry parameters were as follows: Electrospray ion source(ESI), negative ion mode, capillary voltage 4000 V,dry gas(N2) temperature 350 ℃,dry gas flow rate 9 L·min-1,nebulizer pressure 275 kPa. The fragment voltage for mycophenolic acid and gliclazide were 140 and 135 V,respectively,and collision energies were 25 and 12 eV,respectively. Mycophenolic acid and gliclazide were determined using multilevel reaction monitoring(MRM) mode at the transition of m/z 319. 3 → m/z 191. 4 and m/z 322. 5 →m/z 170. 4. 249 plasma samples from patients using mycophenolic acid preparations were collected and analyzed by the established LC-MS/MS method. The correlation of LC-MS/MS and EMIT for determination of mycophenolic acid was analyzed using Passing-Bablok and Bland-Altman to evaluate the accuracy and reliability of the results. Results The calibration curve for mycophenolic acid was linear within the range of 0. 05-10 μg·mL-1. Either inter-or intra-day precision was less than 11. 60%. The extraction recoveries were ranged from 108. 87% to 111. 42% for mycophenolic acid. Mycophenolic acid in plasma was stable at various storage conditions. The concentration of mycophenolic acid ranged from 0. 057 to 24. 90 μg·mL-1 with an average of 3. 26 μg·mL-1. The concentration of mycophenolic acid determined by EMIT ranged from 0. 16 to24. 87 μg·mL-1 with an average of 4. 72 μg·mL-1. Conclusion The established LC-MS/MS method was simple,rapid and sensitive. The results of LC-MS/MS were lower than

关 键 词：霉酚酸 液相色谱串联二级质谱 酶放大免疫分析技术 治疗药物监测 相关性 

分 类 号：R97[医药卫生—药品]