HPLC-MS/MS法测定人血浆中儿茶酚胺浓度的方法学建立

Establishment of a LC/MS/MS method to determine content of catecholamines in human plasma

目的:建立LC/MS/MS法测定人血浆中儿茶酚胺(肾上腺素、去甲肾上腺素、多巴胺)的含量。方法:选用PFP column(150 mm×2.1 mm,3μm),流动相A:甲醇(0.01%甲酸):B水(0.01%甲酸),流速:0.3 m L·min-1,柱温:40℃,进样量:20μL,正离子方式检测,多反应监测(MRM)扫描,以异丙肾上腺素为内标,采用弱阳离子固相萃取(WCX-SPE)血浆中儿茶酚胺后进行LC/MS/MS法分析。结果:去甲肾上腺素(NE)在100~2 000 pg·m L-1(n=6,r=0.994 2)、肾上腺素(E)在100~2 000 pg·m L-1(n=6,r=0.992 2)、多巴胺(DA)在100~2 000 pg·m L-1(n=6,r=0.996 2)浓度范围内线性关系良好,最低检测限均为100 pg·m L-1(S/N≥10),批内、批间准确度、精密度、精密度RSD均<15%,儿茶酚胺样品4℃放置12 h,以及预处理后16 h均稳定。结论:该方法灵敏度高、选择性高、操作简便、结果准确可靠,能满足检测血浆中儿茶酚胺的浓度,适合于临床诊疗需要。

Objective:To establish a LC-MS-MS method to measure concentration of catecholamines(epinephrine,noradrenaline,dopamine)in Human plasma.Methods:The assay was conducted with a PFP column(150 mm×2.1 mm,3μm),mobile phase was methanol(0.01%formic acid)-water(0.01%formic acid)at a flow rate of 0.3 mL·min-1,the temperature was kept at 40℃,and the injection volume was 20μL.Catecholamines in human plasma were pretreated by weak cation solid phase extraction(WCX-SPE),with isoproterenol as internal standard,and were operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive mode for LC/MS/MS method analysis.Results:The linearity of norepinephrine(NE),adrenaline(E),and dopamine(Da)were good in the ranges of 100~2 000 pg·mL-1(n=6,r=0.994 2)and100~2 000 pg·mL-1(n=6,r=0.992 2),and 100~2 000 pg·mL-1(n=6,r=0.996 2),respectively.Limits of detection for NE,E and DA were 100 pg·mL-1(S/N≥10).The accuracy and precision RSD of intra and inter batch were less than 15%.The catecholamine samples were stable after placing in 4℃for 12 h and pretreatment for 16 h.Conclusion:The established method for determination of catecholamine in human plasma is suitable for clinical application,with high sensitivity,simple operation,high accuracy,and reliability.