HPLC-UV法测定人血浆中青霉胺浓度

HPLC-UV determination of penicillamine in human plasma

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,5μm),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20μl。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。

Aim To develop a HPLC-UV method for the determination of penicillamine in human plasma and apply it to a pharmacokinetic 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 pharmacokinetic study of penicillamine in healthy Chinese volunteers.