人血浆中吡柔比星HPLC测定方法及其在药动学研究中的应用

Development of a High Performance Liquid Chromatographic Method of Pirarubicin in Human Plasma and Its Application in Clinical Pharmacokinetic Study

目的建立人血浆中吡柔比星的HPLC测定方法,并用于吡柔比星化疗病人的药动学研究。方法取血浆300μL,以柔红霉素为内标,采用甲醇-氯仿(1∶3)作为提取溶剂提取,有机相氮气吹干,残渣溶于60μL流动相进样分析。色谱柱:Shim-packCLC-ODS柱(6mm×15cm,5μm);流动相:乙腈-磷酸盐-冰醋酸(35∶65∶0.5);流速1mL.min-1;荧光检测,激发波长480nm,发射波长560nm。乳腺癌化疗病人15min内静注吡柔比星60mg.m-2,在注射开始0,5,10min、注射结束时、结束后5,10,30min,1,2,4,8,12,24,48h取血测定浓度,使用WinNonlin软件拟合,计算药动学参数。结果本方法在5～500μg.L-1内线性良好,相关系数r=0.9994(n=6)。高、中、低3个质量浓度质控样品批内误差的RSD为3.01%～4.87%,批间RSD为1.49%～4.39%,最低定量限为5μg.L-1;药物代谢为三室模型,半衰期分别为(0.034±0.01),(0.858±0.416)和(18.81±4.47)h。结论本方法采样量小,操作简便,为药动学和生物等效性研究提供了方法学基础。

OBJECTIVE To develop a HPLC method for the determination of pirarubicin in human plasma,and to investigate the pharmacokinetic profiles of pirarubicin in patients. METHODS With daunorubicin （DM） as the internal standard,300 μL plasma was extracted by methanol/chloroform （1：3）. The organic layer was evaporated to dryness under nitrogen stream,and the residue was reconstituted with mobile phase. A Shim-pack CLC-ODS（6 mm×15 cm,5 μm） column was selected and the mobile phase consisted of acetonitrile-hydrogen phosphate bufferacetic acid （35：65：0. 5） at a/low rate of 1 mL· min^-1. The excitation wavelength was at 480 nm and the emission wavelength was at 560 nm for the/luorescence detector. The patients were treated with a single dose of 60 mg·m^-2for 15 min i. v. infusion. The blood samples were obtained at the following times： before infusion,5,10 min after the beginning of the infusion, and 0,5,10,30 min, 1,2,4,8,12,24,48 h after the end of the injection, then the program of WinNonlin was used to calcu- late the pharmacokinetic parameters. RESULTS A linearity was obtained over the range of 5～500 μgμ·L^-1 of pirarubicin in plasma with a good correlation coefficient （r=0.999 4,n =6）. The RSDs of intraand interrun validation were less than 5%. The mean recoveries were 101.64% ,102.17% and 107.59% for the high,middle and low concentrations of check samples,respectively. The de- tection limit was 5 μg·L^-1. The profiles of pirarubicin were simulated with a three-compartment open model with α ,β and γ half-lives of （0.034±0.010） , （0.858±0.416） and （18.81±4.47 ）h. CONCLUSION The method is sensitive, specific and simple. It is suitable for clinical pharmacokinetic study.