UPLC-MS/MS法检测人尿液中8种苯二氮?药物及其临床应用

Determination of 8 kinds of benzodiazepines in human urine by UPLC-MS/MS and its clinical application

目的建立一种定量检测人尿液中8种常见苯二氮■类药物的方法,并将其应用于临床中毒检测。方法尿液样本经液液萃取后,用液相-串联质谱联用仪测定,色谱柱为BEH C18(2.1 mm×50.0 mm,1.7μm),以含0.01%甲酸乙腈溶液-含0.01%甲酸水溶液为流动相进行梯度洗脱,流速为0.4 mL·min^-1,电喷雾正离子模式,多反应监测。考察该方法的专属性、标准曲线与定量下限、精密度与提取回收率、基质效应和稳定性。结果硝西泮、地西泮、艾司唑仑、阿普唑仑、氯硝西泮、劳拉西泮、咪达唑仑和三唑仑浓度分别在0.5~1000.0(r=0.992 5)、0.5~1000.0(r=0.991 0)、0.5~1000.0(r=0.991 3)、0.5~1000.0(r=0.998 4)、1.0~1000.0(r=0.996 0)、1.0~1000.0(r=0.997 9)、0.5~1000.0(r=0.990 9)和1.0~1000.0(r=0.992 1)ng·mL^-1内线性关系良好;批内相对标准偏差(RSD)和相对误差(RE)分别为5.07%~19.66%和-9.75%~19.00%,批间RSD和RE分别为5.75%~17.86%和-13.83%~19.00%;提取回收率和基质效应均满足检测要求;在多种储存条件下,处理前后的尿液样本稳定性好。结论该方法简便,专属性高,准确度高,重复性好,可有效检测中毒浓度范围的8种BZDs。本方法成功用于68例临床中毒样本的检测。

Objective To establish and validate a method for quantitative detection of 8 common benzodiazepines in human urine, which can be applied to clinical poisoning detection. Methods The urine samples were measured by liquid phase-tandem mass spectrometry after liquid-liquid extraction. The chromatographic column was BEH C18 (2.1 mm×50.0 mm,1.7 μm). Acetonitrile solution and water solution both with 0.01% formic acid were used as mobile phase to perform gradient elution. The flow rate was 0.4 mL·min^-1. Multiple reaction monitoring and electrospray ionization positive ion mode were used for monitoring. The specificity, linearity, limit of quantitation, precision, recovery rate, matrix effect and stability were investigated. Results The linear ranges of nitrazepam, diazepam, estazolam, alprazolam, clonazepam, lorazepam, midazolam and triazolam were 0.5-1000.0(r=0.992 5), 0.5-1000.0(r=0.991 0),0.5-1000.0(r=0.991 3), 0.5-1000.0(r=0.998 4), 1.0-1000.0(r=0.996 0),1.0-1000.0(r=0.997 9), 0.5-1000.0(r=0.990 9),1. 0-1000. 0( r = 0. 992 1) ng·mL^-1,respectively. The intra-day relative standard deviation( RSD) and relative error( RE) were 5. 07%-19. 66% and -9. 75%-19. 00%. The inter-day RSD and RE were 5. 75%-17. 86% and -13. 83%-19. 00%. The extraction recovery and matrix effect met the test requirements. Urine samples were stable before and after treatment under various storage conditions. Conclusion The method is simple,specific,accurate and repeatable,which can be used for detection of 8 drugs in different concentration. The method was successfully used to detect 68 cases of clinical poisoning samples.