超高效液相色谱串联质谱联用法快速筛选3-羟基-3-甲基戊二酰辅酶A还原酶抑制剂

Rapid Screening of 3-Hydroxy-3-methylglutaryl CoA Reductase Inhibitors by Ultra Performance Liquid ChromatographyTandem Mass Spectrometry

建立了超高效液相色谱串联质谱仪(UPLC-MS/MS)检测酶促反应体系中还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)浓度变化,快速筛选3-羟基-3-甲基戊二酰辅酶A还原酶(HMGCR)抑制剂的方法。优化了HMGCR酶促反应体系和检测NADPH的色谱-质谱条件,探讨了NADPH的离子化试剂的作用和质谱碎裂机理。采用ACQUITY UPLC BEH Amide色谱柱(100 mm×2.1 mm,1.7μm),以三乙胺/六氟异丙醇缓冲液(pH=8.0)为流动相,流速0.3 mL/min,柱温30℃,电喷雾离子源-多反应监测模式,对酶促反应体系中的NADPH进行分析。NADPH的峰面积与其浓度在0.05～50μmol/L范围内呈良好的线性关系(r>0.9996),定量限(S/N=10)为0.05μmol/L。

A rapid and accurate method has been developed for screening 3-hydroxy-3-methylglutaryl CoA reductase （HMGCR） inhibitors by measuring the concentration of reduced nicotinamide adenine dinucleotide phosphate （NADPH） in the enzymatic reaction system using ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. Several parameters, including the enzymatic reaction system, liquid chromatography and mass spectrometric conditions were optimized for improving the chromatographic performance and MS signal. The effect of ionization reagent and the fragmentation mechanism of NADPH were also discussed. The separation was performed on an ACQUITY UPLC BEH Amide column （100 mm × 2.1 mm, 1.7 μm） with a mobile phase consisting of triethylamine/hexafluoroisopropanol buffer at a flow of 0.3 mL/min, column temperature of 30℃. Identification and quantification were achieved by UPLC-MS/MS in an electrospray ionization-negative mode and multiple reactions monitoring （MRM）. A good linearity was obtained for determination of NADPH at the concentration of 0.05 -50 μmol/L with the linear correlation coefficient more than 0. 9996. The limit of quantification for NADPH was 0.05 μmoL/L （S/N= 10）.