Yao T, Zeng S, Ding H
Department of Pharmaceutical Analysis, Zhejiang Medical University, Hangzhou, 310031.
Se Pu. 1997 Jul;15(4):316-8.
A chiral RP-HPLC method was developed to assay the 5-(p-hydroxyphenyl)-5-Phenylhydantoin (p-HPPH) enantiomers, the major metabolite of antiepileptic drug phenytoin, in rat hepatic microsomes. A 50 mm FLC-C8 column was used as the analytical column, beta-cyclodextrin (beta-CD) as chiral mobile phase additive and phenobarbital as the internal standard. The detection wavelength was 250 nm. The linear range of p-HPPH enantiomers was 0.5-110 mg/L. The detection limit was 5 ng (S/N = 3). The recoveries of S- and R-p-HPPH were 93.6% +/- 2.8% and 94.7% +/- 1.8% respectively. The RSD within day and between days were less than 2%. The concentration of beta-CD played an important role in separating chiral enantiomers. When the concentration of beta-CD was between 8.8 and 13.2 mmol/L the resolution of p-HPPH enantiomers had the largest value Rs = -1.1. In this work, 8.8 mmol/L beta-CD solution (4 g beta-CD, 6 g urea, and 1.5 g ammonium acetate in 400 mL water) was selected in considering some factors such as column efficiency, solubility of beta-CD etc. Urea can increase the solubility of beta-CD. When urea: beta-CD = 1:1-1.5:1 (g/g), the solubilization of beta-CD was significant. Methanol concentration in mobile phase affected retention time, resolution of p-HPPH enantiomers and solubility of beta-CD.
建立了一种手性反相高效液相色谱法，用于测定大鼠肝微粒体中抗癫痫药物苯妥英的主要代谢产物5-(对羟基苯基)-5-苯基乙内酰脲(p-HPPH)对映体。采用50 mm FLC-C8柱作为分析柱，β-环糊精(β-CD)作为手性流动相添加剂，苯巴比妥作为内标。检测波长为250 nm。p-HPPH对映体的线性范围为0.5-110 mg/L。检测限为5 ng(S/N = 3)。S-和R-p-HPPH的回收率分别为93.6%±2.8%和94.7%±1.8%。日内和日间相对标准偏差均小于2%。β-CD的浓度在分离手性对映体中起重要作用。当β-CD的浓度在8.8至13.2 mmol/L之间时，p-HPPH对映体的分离度Rs值最大，为-1.1。在本研究中，综合考虑柱效、β-CD的溶解度等因素，选择了8.8 mmol/L的β-CD溶液(4 g β-CD、6 g尿素和1.5 g醋酸铵溶于400 mL水中)。尿素可以增加β-CD的溶解度。当尿素:β-CD = 1:1-1.5:1(g/g)时，β-CD的增溶效果显著。流动相中甲醇浓度影响p-HPPH对映体的保留时间、分离度及β-CD的溶解度。
