HPLC-MS/MS法同时测定大鼠血浆中多柔比星及其代谢产物的浓度

HPLC-MS/MS method to simultaneously determine doxorubicin and its five metabolites in rat plasma

目的建立同时测定大鼠血浆中多柔比星和5种代谢产物(M1-M5)的HPLC-MS/MS方法。方法以柔红霉素作为内标,血浆采用固相萃取方法进行处理。色谱柱为ZORBAX Extend-C18(150 mm×4.6 mm,5μm),流动相为含0.1%甲酸水和乙腈,进行梯度洗脱分离。用API4000质谱仪,电喷雾离子源(ESI),正离子扫描,多反应监测模式(MRM)测定。用于定量分析的离子对分别为m/z 544.3→m/z 397.1(多柔比星)、m/z 545.9→m/z 399.1(M1)、m/z 397.0→m/z 361.0(M2)、m/z 399.2→m/z 363.0(M3)、m/z 399.2→m/z 381.1(M4)、m/z 401.2→m/z 297.0(M5)和m/z 528.3→m/z 321.1(柔红霉素)。结果多柔比星血药浓度在5~5000 ng·mL^-1,5种代谢产物血药浓度分别在5~1500 ng·mL^-1线性良好。待测物的日内和日间精密度良好,提取回收率和基质效应均符合生物样品测定要求。结论本方法前处理简单,分析快速、灵敏、准确,为盐酸多柔比星相关制剂的药代动力学研究奠定基础。

Objective To establish a HPLC-MS/MS method to si-multaneous determination free doxorubicin,and five metabolites(M1-M5).Methods Daunorubicin was selected as internal standard.After plasma samples were treated with solid phase extraction,chromatographic separation was achieved on a ZORBAX Extend-C18(150 mm×4.6 mm,5μm)column through gradient elution by 0.1%formic acid with acetonitrile.The analytes were detected by API 4000 mass spectrometer with the electrospray ionization(ESI)in multiple reaction monitoring(MRM)mode.Doxorubicin and five metabolites were detected using positive ion mode.The precursor/product ion transitions of m/z 544.3→m/z 397.1(doxorubicin),m/z 545.9→m/z 399.1(M1),m/z 397.0→m/z 361.0(M2),m/z 399.2→m/z 363.0(M3),m/z 399.2→m/z 381.1(M4),m/z 401.2→m/z 297.0(M5)and m/z 528.3→m/z 321.1(daunorubicin).Results The quantitative linear ranges were 5-5000 ng·mL^-1 for doxorubicin and 5-1500 ng·mL^-1 for its five metabolites in rat plasma.Intra-day and inter-day precision for quality control samples of doxorubicin and five metabolites were good.Extraction recovery and matrix effects were in line with requirements.Conclusion This method is simple,rapid,sensitive and suitable for the pharmacokinetic studies of doxorubicin hydrochloride preparation.