用HPLC-MS/MS法同时测定人血浆中9种镇静催眠类药物浓度及其临床应用

Clinical application and determination of nine sedative-hypnotics in human plasma by HPLC-MS/MS

目的 建立高效液相色谱-串联质谱(HPLC-MS/MS)法并验证同时检测人血浆中9种镇静催眠类药物浓度的方法,并进行临床应用的初步探索。方法 人血浆样品经乙腈沉淀蛋白后,用高效液相色谱-串联质谱仪测定。经Agilent Poroshell 120 EC-C18 (2.1 mm×50.0 mm, 2.7μm)色谱柱分离,以乙腈-水(0.1%甲酸)为流动相等度洗脱。流速:0.3 mL·min^(-1),柱温:20℃,进样量:5μL,采用电喷雾离子源,正离子模式,多重离子监测法进行测定。考察该方法的专属性、标准曲线与最低定量限、精密度与回收率、基质效应、稳定性和稀释效应。结果 空白人血浆中内源性物质对待测物及内标的测定不产生干扰,专属性良好。艾司唑仑、阿普唑仑、奥沙西泮、氯硝西泮、劳拉西泮、三唑仑、咪达唑仑、地西泮和唑吡坦质量浓度分别在18~1 800、4.5~450、25~2 500、3.5~350、25~2 500、1.5~150、5.5~550、35~3 500和4~400 ng·mL^(-1)线性关系良好,相关系数r2均大于0.997 7;最低定量限分别为18、4.5、25、3.5、25、1.5、5.5、35和4 ng·mL^(-1);最低定量限质量浓度样品的日间、日内精密度相对标准偏差均小于20%,低、中、高质量浓度质控样品的日间、日内精密度相对标准偏差均小于15%,准确度为86.21%~112.38%,回收率为93.07%~110.50%;内标归一化的基质因子为86.61%~108.41%,相对标准偏差均小于15%;在多种储存条件下,血浆样品稳定性良好;血浆样品进行稀释后分析精密度与准确度良好。结论 该方法操作简单、检测速度快、灵敏度高、特异性强,可用于人血浆中上述9种镇静催眠类药物的同时检测。

Objective To establish and validate a method for simultaneous determination of 9 sedative-hypnotics in human plasma,and to explore the preliminary clinical application.Methods Plasma samples were precipitated with acetonitrile and determined by high performance liquid chromatography tandem mass spectrometry.The column was Agilent Poroshell 120 EC-C18(2.1 mm×50.0 mm,2.7μm)and eluted with acetonitrile water containing 0.1%formic acid in an equal degree program at a flow rate of 0.3 mL·min^(-1).The column temperature was 20℃and injection volume was 5μL.The deprotonated ions of analytes were ionized by positive ion,electron spray ionization and multiple reaction monitoring mode.The specificity,standard curve and lower limit of quantification,precision and recovery,matrix effect,stability and dilution effect of the method were investigated.Results Excellent linear relationship with correlation coefficient of r~2≥0.9977 was obtained.The linear of esazolam,alprazolam,oxazepam,clonazepam,lorazepam,triazolam,midazolam,diazepam and zolpidem were 18-1800,4.5-450,25-2500,3.5-350,25-2500,1.5-150,5.5-550,35-3500,4-400 ng·mL^(-1),respectively.The lower limit of quantification were 18,4.5,25,3.5,25,1.5,5.5,35,4 ng·mL^(-1).The method was accurate and precise with acceptable intra-day and inter-day precisions(relative standard deviations were less than 20%for a lower limit of quantification and less than15%for other quality control samples)and an accuracy of 86.21%-112.38%.The extraction recovery rate were93.07%-110.50%.The matrix factors normalized by internal standard were 86.61%-108.41%,relative standard deviations were less than 15%.Plasma samples remained stable under various storage conditions.The precision and accuracy of plasma samples were acceptable after dilution.Conclusion The method is simple,rapid,sensitive and specific,and it can be used for simultaneous detection of the 9 sedative-hypnotics in human plasma.