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

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ACQUITYUPLCBEHAmide色谱柱（100mmx2．1mm，1．7μm），以三乙胺／六氟异丙醇缓冲液（pH=8．0）为流动相，流速0．3mL／min，柱温30℃，电喷雾离子源一多反应监测模式，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—50tx...

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Bibliographic Details
Published in分析化学 Vol. 41; no. 7; pp. 1013 - 1018
Main Author 吴琼 王世聪 马俊锋 李婉南 邢述 付学奇
Format Journal Article
LanguageChinese
Published 吉林大学生命科学学院,分子酶学工程教育部重点实验室,长春130012 2013
Subjects
3-羟基-3-甲基戊二酰辅酶A还原酶
抑制剂筛选
超高效液相色谱串联质谱
还原型炯酰胺腺嘌呤二核苷酸磷酸
Inhibitor screening
抑制剂筛选
还原型烟酰胺腺嘌呤二核苷酸磷酸
Ultra performance liquid chromatography-tandem mass spectrometry
3-Hydroxy-3-methylglutaryl CoA reductase
超高效液相色谱串联质谱
3-羟基-3-甲基戊二酰辅酶A还原酶
Reduced nicotinamide adenine dinucleotide phosphate
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ISSN0253-3820
DOI10.3724/SP.J.1096.2013.20961

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Summary: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ACQUITYUPLCBEHAmide色谱柱（100mmx2．1mm，1．7μm），以三乙胺／六氟异丙醇缓冲液（pH=8．0）为流动相，流速0．3mL／min，柱温30℃，电喷雾离子源一多反应监测模式，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—50txmol／L范围内呈良好的线性关系（r〉0．9996），定量限（S／N=10、为0．05LLmol／L。
Bibliography:WU Qiong, WANG Shi-Cong, MA Jun-Feng, LI Wan-Nan,XING Shu , FU Xue-Qi （Key Laboratory Molecular Enzymology and Engineering of the Ministry of Education, College of Life Sciences, Jilin University, Changchun 130012, China）
22-1125/O6
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
ISSN:0253-3820
DOI:10.3724/SP.J.1096.2013.20961