HPLC法同时测定毛发样本中地西泮、去甲西泮和奥沙西泮

Simultaneous determination of diazepam,nordiazepam and oxazepam in hair by HPLC

目的:建立测定毛发样本中地西泮、去甲西泮和奥沙西泮的HPLC分析方法。方法:采用超声法结合液液萃取法作为前处理方法。采用Athena C18-WP(250 mm×4.6 mm,5μm)色谱柱;以甲醇-0.02 mol·L^(-1)磷酸二氢钠溶液(磷酸调pH 3.4)-乙腈(30∶43∶27)为流动相;柱温50℃;流速0.7 mL·min^(-1);检测波长254 nm。内标为氯硝西泮。结果:本方法在0.1~25 ng·mg^(-1)范围内具良好的线性关系,定量限为0.1 ng·mg^(-1),检测限为0.05 ng·mg^(-1),批内准确度(n=6)在88.2%~103.9%,批间准确度(n=24)在88.5%~106.2%,精密度小于或等于9.19%,提取回收率稳定,符合方法学要求。将该方法应用于实际病人的毛发样本,测得地西泮及其代谢物去甲西泮、奥沙西泮浓度分别是(0.307±0.016)ng·mg^(-1)、(0.244±0.012)ng·mg^(-1)和(0.478±0.053)ng·mg^(-1),其中C去甲西泮/C地西泮为0.795,与文献中报道相符。结论:优化后的方法操作快捷简便,重现性好,具适用性和可靠性。

Objective:To establish an HPLC analysis method for the determination of diazepam,nordiazepam and oxazepam in hair samples.Methods:The determination of drug content and related substances was performed by high performance liquid chromatography(HPLC).Ultrasonic method combined with liquid-liquid extraction method was used as the pretreatment method.The chromatographic column was Athena C_(18)-WP column(250 mm×4.6 mm,5μm);the mobile phase was the mixture of methanol:0.02 mol·L^(-1)sodium dihydrogen phosphate(adjusted to pH 3.4 with phosphoric acid):acetonitrile(30∶43∶27,v/v/v)in an isometric elution;the column temperature was 50℃;the flow rate was 0.7 mL·min^(-1);the running time was 30 min;the injection volume was 20μL.The detection wavelength was 254 nm.The internal standard was clonazepam.Results:The linearity of all the analytes were good in the concentration range of 0.1 to 25 ng·mg^(-1)(r>0.995).The limit of quantitative was 0.1 ng·mg^(-1)and the limit of detection was 0.05 ng·mg^(-1).The intra-batch accuracies were 88.2%-103.9%(n=6)and the inter-batch accuracies were 88.5%-106.2%with imprecisions≤9.19%.The recoveries were stable for three anlytes,which met the requirement of methodology.The present method was applied to the authetic hair samples,in which the concentrations of diazepam,norazepam and oxazepam were(0.307±0.016)ng·mg^(-1),(0.244±0.012)ng·mg^(-1)and(0.478±0.053)ng·mg^(-1)respectively.Besides,C_(norazepam)/C_(diazepam)was 0.795,which was in consistent with the literature reported.Conclusion:The established HPLC method is fast and easy to operate,with good reproducibility,applicability,and reliability.