Sallustio B C, Kassapidis C, Morris R G
Department of Clinical Pharmacology, Queen Elizabeth Hospital, Woodville, South Australia.
Ther Drug Monit. 1994 Apr;16(2):174-8. doi: 10.1097/00007691-199404000-00011.
The use of high-performance liquid chromatography for therapeutic drug monitoring of clonazepam has previously been limited by low sensitivity and labor-intensive liquid-liquid extractions. The present method was developed employing a rapid solid-phase extraction, thus minimising sample workup and providing analytical sensitivity down to 2 micrograms/L using 1 ml of plasma. Plasma samples were loaded onto C18 solid-phase extraction columns, and clonazepam and its internal standard (methyl-clonazepam) were eluted with methanol, dried, and reconstituted in 130 microliters of mobile phase. Chromatographic separation was achieved using a 3-microns RP18 column at 40 degrees C and a mobile phase of 32% acetonitrile and 0.5% glacial acetic acid in distilled water at 0.5 ml/min. Detection was carried out using ultraviolet absorbance at 306 nm. Retention times for clonazepam and methyl-clonazepam were approximately 7 and 12 min respectively. Standard curves were linear over a range of 5-200 micrograms/L with intraassay coefficients of variation of 1.2 and 4.8% at 200 and 5 micrograms/L, respectively. Plasma concentrations measured in patient samples were not statistically different from those obtained using an established gas chromatographic method, and quality control specimens from the Heathcontrol EQA Scheme were consistently within +/- 1.2 SD of the group means. There was no chromatographic interference from other benzodiazepines or other drugs used for the treatment of epilepsy.
高效液相色谱法用于氯硝西泮的治疗药物监测，此前一直受限于灵敏度低以及液 - 液萃取操作繁琐。目前所开发的方法采用了快速固相萃取，从而将样品预处理降至最低限度，并使用1毫升血浆实现了低至2微克/升的分析灵敏度。血浆样品加载到C18固相萃取柱上，氯硝西泮及其内标（甲基氯硝西泮）用甲醇洗脱，干燥后，再用130微升流动相复溶。使用3微米的RP18柱在40℃下进行色谱分离，流动相为32%乙腈和0.5%冰醋酸的蒸馏水混合液，流速为0.5毫升/分钟。采用306纳米的紫外吸光度进行检测。氯硝西泮和甲基氯硝西泮的保留时间分别约为7分钟和12分钟。标准曲线在5 - 200微克/升范围内呈线性，在200微克/升和5微克/升时的批内变异系数分别为1.2%和4.8%。患者样品中测得的血浆浓度与使用既定气相色谱法获得的浓度无统计学差异，并且来自Heathcontrol EQA计划的质量控制样本始终在组均值的±1.2标准差范围内。未观察到来自其他苯二氮䓬类药物或用于治疗癫痫的其他药物的色谱干扰。
