HPLC-UV法测定人血浆中青霉胺浓度

摘要

Aim To develop a HPLC-UV method for the determination of penicillamine in human plasma and apply it to a phannacokinetic study of penicillamine in healthy Chinese volunteers.Method Plasma samples were added with derivatization reagent 5 , 5'-dithiobis ( 2-nitrobenzoic acid ), and then vortexed and incubated at room temperature for 5 min before acidification with 10% perchloric acid to deposit proteins.The supernatant liquid was injected into the HPLC system.Shimadzu VP-ODS C18 column ( 150 mm×4.6 mm, 5 μm ) was used.The chrom-atographic conditions were as follows : The mobile phase was methanol -0.05 mol·L-1 sodium acetate solution (12: 88 ), the flow rate was 1 ml·min-1 , the column temperature was at 25℃ and the UV detection wavelength was set at 320 nm, the injection volume was 20 ΜL.Result Calibration curve was linear over the range of 0.1 ~ 10.0 mg·L-1 .The lower limit of quantification was 0.1 mg·L-1.The extraction recoveries were higher than 60%.The intra-and inter-day precision (RSD %) were lower than 6%.Conclusion A simple, rapid, accurate and precise HPLC method for determining penicillamine in human plasma is successfully developed and validated and successfully applied to a phannacokinetic study of penicillamine in healthy Chinese volunteers.%目的 建立人血浆中青霉胺浓度的HPLC测定方法,并用于人体药动学试验中青霉胺血药浓度测定.方法取血浆样品加入衍生化试剂DTNB,室温反应5 min,10%的高氯酸溶液沉淀蛋白,上清液进样分析.色谱柱:岛津 VP-ODS C18色谱柱(150 mm×4.6 mm,5 μm),流动相为甲醇-0.05 mol·L-1醋酸钠缓冲溶液(12:88),流速1.0 ml·min-1,检测波长320 nm,柱温25 ℃,进样量20 μl.结果 血浆中青霉胺在0.1~10.0 mg·L-1浓度范围内线性关系良好,定量下限为0.1 mg·L-1 (S/N>10,n=5;RSD=6.4%,准确度为94.2%),在低、中、高浓度血浆样品日内、日间RSD均小于6%,准确度在92%～96%范围内,提取回收率>60%,且稳定 (RSD<10%),符合生物样品分析要求.结论所建立的HPLC法样品前处理简便、操作简便、能满足青霉胺在人体内的药代动力学研究.