液相色谱-串联质谱(LC-MS/MS)检测血浆肾素活性(PRA)的方法学建立和临床应用评估

 目的    建立高血压患者血浆肾素活性(plasma renin activity,PRA)液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)的检测方法,用于早期筛查高血压患者中的原发性醛固酮增多症(primary aldosteronism,PA)。方法    arginine 13C 15N作为内标,采用Phenomenex Kinetex C18柱(2.6 μm,100 mm×3.0 mm)进行分离,柱温55 ℃。流动相为0.2%甲醇水溶液和0.2%甲酸甲醇溶液,流速为0.5 mL/min,梯度洗脱。考察该方法的特异性、基质效应、携带污染、重复性、定量下限(lower limit of measuring interval,LLMI)、线性、不精密度、回收率、稀释一致性和血浆标本稳定性。采用LC-MS/MS方法测定296名表面健康人群(男性153名,女性143名,平均年龄48岁)及360名高血压患者(平均年龄52岁,确诊为PA 27例)的血浆样本PRA,并与放射免疫法(radioimmunoassay,RIA)进行比较。结果    该方法通过了特异性、基质效应、携带污染、重复性的性能评价。PRA的检测线性范围为0.084 4～30.000 0 ng/mL;PRA的LLMI为0.084 4 ngmL-1h-1;日间和批内的不精密度变异系数(coefficient of variation,CV)均＜15%;准确度偏差均<15%,回收率为92.28%～102.62%;建立的生物参考区间为0.25～5.12 ngmL-1h-1 (立位);与RIA比较,结果相关性较好(r2=0.81,P＜0.05),但存在-18.5%的偏倚;LC-MS/MS所得PRA的醛固酮/肾素活性比值(aldosterone to renin ratio,ARR)对PA的诊断特异性与灵敏度分别为96.3%与91.0%,诊断性能更佳(AUC:0.983 vs.0.894)。结论    我们建立了准确检测PRA的LC-MS/MS方法,适合于临床早期快速筛查PA。

Method development and clinical application assessment of plasma renin activity (PRA) by liquid chromatography-tandemmass spectrometry (LC-MS/MS)

Objective    A method for the detection of plasma renin activity (PRA)was established by LC-MS/MS for the early screening of primary aldosteronism (PA) in hypertensive patients.Methods    Arginine 13C and15N were used as internal standards,and we selected Phenomenex Kinetex C18 (2.6 μm,100 mm×3.0 mm) for separation with column temperature of 55 ℃,mobile phase of 0.2%  aqueous methanol solution and 0.2% formic acid methanol solution,flow rate of 0.5 mL/min and gradient elution.The method was investigated for specificity,matrix effect,carryover contamination,repeatability,lower limit of measuring interval (LLMI),linearity,imprecision,recovery,dilution consistency and plasma specimen stability.LC-MS/MS method was used to determine the plasma renin activity of 296 healthy individuals (153 males and 143 females with mean age of 48 years) and 360 hypertensive patients (mean age of 52 years,27 patients diagnosed as PA),and compared with radioimmunoassay (RIA) methods.Results    The method passes the performance evaluation of specificity,matrix effect,carryover contamination,and repeatability.The linear range of PRA was 0.084 4-30.000 0 ng/mL.The LLMI of PRA was 0.084 4 ngmL-1h-1.The imprecision coefficient of variations (CV) were less than 15% between days and batch.The accuracy deviation was less than 15%,and the recoveries was 92.28% to 102.62%.The biological reference interval ranged from 0.25 to 5.12 ng.mL-1.h-1 (standing position).Compared with RIA,the correlation was acceptable (r2=0.81,P＜0.05),but there was a bias of -18.5%.The specificity and sensitivity of aldosterone to renin ration ARR (LC-MS/MS method) for diagnosis of PA were 96.3% and 91.0%,and diagnostic performance was better (AUC:0.983 vs.0.894).Conclusions    Our study established an accurate LC-MS/MS method for the determination of PRA,which is suitable for rapid screening of PA early in the clinical.