Doran T C
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53792.
Ther Drug Monit. 1988;10(4):474-9. doi: 10.1097/00007691-198804000-00018.
An improved reversed-phase liquid chromatographic assay for the anticonvulsant clonazepam in human serum is presented. The drug and two internal standards are extracted from serum with n-butyl chloride at pH 9.2, back-extracted into 2 N HCl, and then extracted at pH 11 into a solvent mixture of chloroform/isopropanol (9:1 by volume). The final extract is evaporated to dryness, reconstituted, and injected into a cyanopropylsilane liquid chromatography column with a mobile phase of 0.5 N acetic acid, acetonitrile, and n-butylamine (59:41:0.005 by volume, respectively). Linearity to 400 micrograms/L was demonstrated, and the detection limit was found to be approximately 5 micrograms/L. Relative recovery from serum as compared to water was approximately 100% for clonazepam and the two internal standards, flunitrazepam and desmethyldiazepam. Within-run precision at 40 micrograms/L was demonstrated by a coefficient of variation (CV) of 2.60% (flunitrazepam internal standard) and 2.53% (desmethyldiazepam internal standard). Between-day multiple analyst precision was demonstrated by a CV of 8.98% (mean 10.09 micrograms/L, n = 77) and 6.51% (mean 28.14 micrograms/L, n = 81) using either internal standard. Capacity factor diagrams reveal at least a dual mechanism separation. Chromatograms of the analysis of clonazepam and other benzodiazepines are presented. The extraction has been optimized for selectivity, thereby minimizing interference from drugs and other extraneous compounds. The chromatographic system has been optimized for speed, efficiency, resolution, and stability and avoids the problem of carbamazepine interference that is commonly encountered with other reversed-phase procedures using octylsilane or octadecylsilane columns.
本文介绍了一种改进的反相液相色谱法，用于测定人血清中的抗惊厥药物氯硝西泮。该药物和两种内标物在pH 9.2条件下用正丁基氯从血清中萃取，反萃取至2N盐酸中，然后在pH 11条件下萃取至氯仿/异丙醇（体积比9:1）的溶剂混合物中。最终萃取液蒸发至干，复溶后注入氰丙基硅烷液相色谱柱，流动相为0.5N乙酸、乙腈和正丁胺（体积比分别为59:41:0.005）。结果表明，线性范围可达400μg/L，检测限约为5μg/L。与水相比，氯硝西泮以及两种内标物氟硝西泮和去甲基地西泮在血清中的相对回收率约为100%。在40μg/L浓度下，批内精密度通过变异系数（CV）表示，氟硝西泮内标为2.60%，去甲基地西泮内标为2.53%。使用任一内标物，多分析人员日间精密度的CV分别为8.98%（平均值10.09μg/L，n = 77）和6.51%（平均值28.14μg/L，n = 81）。容量因子图显示至少存在双重机制分离。文中给出了氯硝西泮和其他苯二氮䓬类药物的分析色谱图。萃取过程已针对选择性进行了优化，从而最大限度地减少了药物和其他外来化合物的干扰。色谱系统已针对速度、效率、分离度和稳定性进行了优化，避免了使用辛基硅烷或十八烷基硅烷柱的其他反相方法中常见的卡马西平干扰问题。
