液相色谱-串联质谱法同时测定对乙酰氨基酚及其相关代谢产物

Simultaneous Determination of Acetaminophen and its Related Metabolites by LC-MS/MS

目的:建立一种检测小鼠血浆中对乙酰氨基酚(APAP)及其5个代谢产物的液相色谱-串联质谱(LC-MS/MS)方法,将之应用于对乙酰氨基酚的代谢研究。方法:血浆样品经甲醇沉淀蛋白后,以对氨基苯甲酸(PABA)为内标,采用LC-MS/MS法,C18色谱柱,流动相为甲醇-5 mmol·L^(-1)乙酸铵水溶液(含0.1%甲酸)(55∶45),流速为0.5 ml·min^(-1),柱温为25℃,采用电喷雾离子化(ESI),正离子多反应监测(MRM)检测方式进行定量分析:APAP,[M-H]^-,m/z 152.0→110.0;APAP-cys,[MH]^-,m/z 271.2→140.1;APAP-glut,[M-H]^-,m/z 457.0→328.0;APAP-NAC,[M-H]-,m/z 313.4→208.0;APAP-sulf,[MH]^-,m/z 232.4→152.1;APAP-gluc,[M-H]^-,m/z 328.2→152.1;IS,[M-H]^-,m/z 138.2→120.0。结果:在0.2~10μg·ml^(-1)(APAP)、1.0~20μg·ml^(-1)(APAP-gluc)、1.0~20μg·ml^(-1)(APAP-sulf)、1.0~20μg·ml^(-1)(APAP-glut)、0.4~15μg·ml^(-1)(APAP-NAC)和0.2~10μg·ml^(-1)(APAP-cys)范围内线性良好(r≥0.990 0)。各测定物的日内精确度均在85%~115%间,精密度均小于15%,加样回收率均在85%~115%之间,RSD均低于15%。结论:该方法快速、灵敏、准确,适用于小鼠血浆中对乙酰氨基酚及其5个代谢产物的定量测定。

Objective ： To establish an LC-MS/MS method for the simuhaneous determination of acetaminophen and its five metabolites in mice plasma, and inveatigate the metabolism of acetaminophen by using the method. Methods： Para aminobenzoic acid was used as the internal standard. The plasma samples were precipitated by methanol, and then separated on a C18 column with the mobile phase of methanol and 5 mmol·ml^-1 ammonium acetate buffer solution containing 0.1% formic acid （ 55 ： 45 ）. The flow rate was 0.5 ml· min^-1, and the column temperature was 25℃. An electrospray ionization source was applied and operated in a positive ion mode using MRM ： APAP, [ M-H ]^-, m/z 152.0→110.0; APAP-cys, [ M-H ]^ -, m/z 271.2 → 140.1 ; APAP-glut, [ M-H ]^ -, m/z 457.0→ 328.0 ; APAP-NAC, [ M-H ]^- , m/z 313.4 →208.0 ; APAP-sulf, [ M-H ]^ -, m/z 232.4→152.1 ; APAP-gluc, [ M-H ] ^-, m/z 328.2→152.1 ;IS, [ M-H]^ - m/z 138.2→120.0. Results： The method exhibited good linearity over the concentration range of 0.2-10μg·ml^-1 for APAP, 1.0-20μg·ml^-1 for APAP-glue, 1.0-20 μg·ml^-1 for APAP-sulf, 1.0-20μg·ml^-1 for APAP-glut, 0.4-15μg·ml^-1 for APAP-NAC and 0.2-10μg·ml^-1 for APAP-eys （r ≥0. 990 0）. The inter-day accuracy and precision of acetaminophen and its five metabolites were all below 15%. The average recovery was between 85% and 115% , and RSDs were all below 15%. Conclusion： The LC-MS/MS method is proved to be quick, sensitive and accurate, and suitable for the determination of acetaminophen and its five metabolites in mice plasma.