Petters I, Peng D R, Rane A
J Chromatogr. 1984 Mar 9;306:241-8. doi: 10.1016/s0378-4347(00)80886-5.
A high-performance liquid chromatographic method is reported for the determination of clonazepam and its metabolites 7-amino- and 7-acetamidoclonazepam. Extraction from buffered plasma is carried out at pH 9.5 with hexane-ethyl acetate (7:3) for clonazepam and with chloroform for the metabolites. Flunitrazepam, 7-aminodemethylflunitrazepam and 7-acetamidoflunitrazepam are used as the internal standards for clonazepam and its 7-amino and 7-acetamido metabolites, respectively. To prevent decomposition of 7-aminoclonazepam a high concentration of 7-aminomethylclonazepam is added to the plasma. Chromatography is carried out on a reversed-phase column with detection at 254 nm for clonazepam and 240 nm for the metabolites. Using the method it was possible to determine 5 ng/ml clonazepam, 7-aminoclonazepam and 7-acetamidoclonazepam in plasma with coefficients of variation of 9.5%, 5.9% and 8.9%, respectively. This method can be used to measure clonazepam in plasma from patients treated with other antiepileptics. It may also be utilized for in vitro studies on the metabolism of clonazepam in subcellular fractions from the liver.
本文报道了一种高效液相色谱法，用于测定氯硝西泮及其代谢产物7-氨基氯硝西泮和7-乙酰氨基氯硝西泮。氯硝西泮在pH 9.5的缓冲血浆中用己烷 - 乙酸乙酯（7:3）萃取，代谢产物用氯仿萃取。氟硝西泮、7-氨基去甲基氟硝西泮和7-乙酰氨基氟硝西泮分别用作氯硝西泮及其7-氨基和7-乙酰氨基代谢产物的内标。为防止7-氨基氯硝西泮分解，向血浆中加入高浓度的7-氨基甲基氯硝西泮。色谱分析在反相柱上进行，氯硝西泮在254 nm处检测，代谢产物在240 nm处检测。使用该方法可以测定血浆中5 ng/ml的氯硝西泮、7-氨基氯硝西泮和7-乙酰氨基氯硝西泮，变异系数分别为9.5%、5.9%和8.9%。该方法可用于测定接受其他抗癫痫药物治疗患者血浆中的氯硝西泮。它也可用于氯硝西泮在肝脏亚细胞组分中代谢的体外研究。
