高效液相色谱-串联质谱法定量测定大鼠血浆中LMV-12(HE003)及其代谢产物M4

Determination of LMV-12(HE003)and its metabolite M4 in SD rats plasma by high performance liquid chromatography tandem mass spectrometry

目的建立一种高效液相色谱-串联质谱法(HPLC-MS/MS)同时测定SD大鼠血浆中LMV-12(HE003)(以下简称LMV-12)及其活性代谢产物M4的方法,并开展完整的生物分析方法学验证。方法采用蛋白沉淀提取血浆中LMV-12及M4,用HPLC-MS/MS方法进行定量分析。采用ACQUITY HPLC^(■)CSH C18色谱柱,以0.3%甲酸/40 mmol/L甲酸铵水溶液-乙腈为流动相进行梯度洗脱实现快速分离。质谱采用电喷雾离子化电离源(ESI)、正离子检测模式和选择反应监测(SRM),用于定量分析的离子反应为m/z 674.300→169.200(LMV-12),m/z 661.200→156.000(M4)和m/z 688.200→183.000(内标CH3-LMV-12)。结果完整的生物分析方法学验证结果显示,在本方法中LMV-12血浆浓度在20~8000 ng/ml范围内、M4浓度在5~2000 ng/ml范围内线性关系良好,方法的选择性、残留、准确度、精密度、基质效应、稳定性均满足生物样品定量分析要求。结论本方法检测快速、灵敏度高、操作简便、重现性好,能同时检测出LMV-12原型药及其代谢产物M4,适用于LMV-12的大鼠药代动力学和毒代动力学研究。

Objective We aim to establish a method for simultaneous determination of LMV-12(HE003)(called LMV-12 for short)and its metabolite M4 in SD rat plasma by ultra-high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Methods Protein precipitation was used for extraction.HPLC-MS/MS was used for quantitative analysis with CH3-LMV-12 as the internal standard.An ACQUITY HPLC^(■)CSH C18 column was used for chromatographic separation,and 0.3%formic acid/40 mmol/L ammonium formate solution-acetonitrile was used as mobile phase for gradient elution to achieve rapid separation.The electrospray ionization source(ESI),positive ion detection mode and selective reaction monitoring(SRM)were used.The ionic reactions for quantitative analysis were m/z 674.300→169.200(LMV-12),m/z 661.200→156.000(M4)and m/z 688.200→183.000(CH3-LMV-12)respectively.Result Results showed that the quantification linear relationship was qualified for LMV-12 in range of 20-8000 ng/ml and for M4 in range of 5-2000 ng/ml.The selectivity,residue,accuracy,precision and matrix effect of the method can well meet the requirements.The stability was proved for LMV-12 and M4 frozen at–70℃for 135 days,after 3 freeze-thaw cycles or stored at room temperature for 6 h.The post-extraction plasma sample was stable for 96 h in automatic sampler.Conclusion The method is rapid,sensitive,simple and reproducible for simultaneous detection of the prototype and metabolites of LMV-12.