Nobilis Milan, Jira Thomas, Lísa Miroslav, Holcapek Michal, Szotáková Barbora, Lamka Jirí, Skálová Lenka
Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS Praha a.s. and Academy of Sciences of the Czech Republic, Heyrovského, Hradec Králové, Czech Republic.
J Chromatogr A. 2007 May 11;1149(1):112-20. doi: 10.1016/j.chroma.2007.01.013. Epub 2007 Jan 10.
Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.
氟苯达唑，即[5-(4-氟苯甲酰基)-1H-苯并咪唑-2-基]氨基甲酸甲酯，属于苯并咪唑类驱虫药，广泛应用于兽医学和人类医学。氟苯达唑的I相生物转化包括氨基甲酰甲基部分的水解并伴随脱羧反应（水解氟苯达唑）以及氟苯达唑的羰基还原反应（还原氟苯达唑）。氟苯达唑是一种前手性药物，因此在羰基的非立体选择性还原过程中会形成外消旋混合物。开发并验证了两种生物分析HPLC方法，用于测定猪和雉鸡肝微粒体及胞质组分中氟苯达唑及其代谢产物。分析物从生物基质中提取到叔丁基甲基醚中。第一种非手性方法使用一根250 mm×4 mm的辛基硅烷硅胶柱（5μm），等度流动相为乙腈-0.025 M KH₂PO₄缓冲液（pH 3，28:72，v/v）。阿苯达唑用作内标。整个分析在流速为1 ml/min的条件下持续27分钟。第二种手性HPLC方法在Chiralcel OD-R 250 mm×4.6 mm柱上进行，流动相为乙腈-1 M NaClO₄（4:6，v/v）。该方法能够分离还原氟苯达唑的两种对映体。评估了对映体过量情况。使用光电二极管阵列检测器（在λ=246 nm处扫描或单波长检测）监测柱流出物。所研究的每种分析物都有特征性的紫外光谱，此外，它们的化学结构通过高效液相色谱-质谱联用（HPLC-MS）实验得到确认。观察到氟苯达唑酶促羰基还原反应中的立体特异性。虽然合成的还原氟苯达唑外消旋混合物被分离为等摩尔量的两种对映体，但在所有孵育物的提取物中实际仅检测到一种对映体。
