高效液相色谱法测定指甲真菌病患者指甲中特比萘芬含量的方法研究

HPLC Determination of Concentration of Terbinafine in Onychomycosis Nails

目的：建立测定指甲真菌病患者指甲中特比萘芬含量的高效液相色谱法。方法：将患者指甲标本20 mg加2mol/L氢氧化钠溶液0.5 ml,置60℃水浴2 h碱水解,调节pH值至9.0后,于37℃下水浴至指甲标本完全溶解。用2 ml正己烷及100μl甲醇提取,取有机层,加入0.5 mol/L硫酸-异丙醇（85∶15）混合物0.2 ml进行酸化,取水相20μl进样测定。色谱柱为Kromasil C18柱（250 mm×4.6 mm,5μm）,流动相为乙腈-0.01 mol/L磷酸盐缓冲液-水-三乙胺（66∶10∶24∶0.1）,流速为1.0 ml/min,紫外检测波长224 nm。结果：指甲中内源性物质对样品测定无干扰。本方法在0.003 7~0.89 mg/L浓度范围内线性关系良好（r=0.998 9）,方法回收率为94.15%~100.85%（RSD〈10%）,日内RSD〈2%。结论：本法简便、灵敏,准确,适用于特比萘芬药动学的研究。

Objective：To establish an HPLC method for the determination of terbinafine in fingernails and toenails.Methods：About 20 mg of patient＇s nail was weighed and placed into a glass tube.0.5 ml of 2 mol/L NaOH was added to hydrolyze the nail at 60 ℃ in water bath for 2 h.The pH of the content in the tube was then adjusted to 9.0 with 2 mol/L HCl to dissolve the neil completely at 37 ℃ in water bath.Terbinafine was extracted from the watery solution by 2 ml of hexane combined with 100 μl of methol.Then the organic phase was acidified with a solution of 0.5 mol/L sulfuric acid and isopropanol（85∶15） and 20 μl of aqueous part was taken to inject into the HPLC apparatus.The components in the mixture were separated on a Kromasil C18 column（250 mm×4.6 mm,5 μm）.The mobile phase was composed of acetonltrile-0.01 mol/L phosphate buffer-water-triethylamine（66∶10∶24∶0.1）.The flow rate of mobile phase was 1.0 ml/min.The detection wavelength was at 224 nm.Results：All substances except terbinafine did not interfere with the determination of terbinafine.The linear range was from 0.003 7 to 0.89 mg/L（r=0.998 9）.The recovery was 94.15%～100.85%（RSD10%）.Within-day RSD was less than 2%.Conclusion：The method is simple,sensitive and accurate,being suitable for the pharmacokinetic study of terbinafine.