超高效液相色谱串联质谱检测α-羟基丁酸方法的建立

Ultra-high performance liquid chromatography tandem mass spectrometry method for detection of α-hydroxybutyrate

目的建立超高效液相色谱串联质谱(UHPLC-MS/MS)检测血清α-羟基丁酸(α-HB)的方法。方法方法学建立及评价。使用电喷雾电离源负离子及多反应监测模式检测α-HB,对方法的线性范围、定量下限、精密度、回收率、干扰等进行评估,并检测上海市东方医院收集的表观健康人群血清标本130份(其中来自男62例,女68例)初步建立该方法的参考区间,用Dixon法判断离群值,采用K-S检验进行数据正态性分析,标准曲线采用一元线性回归分析。结果所建立的方法分析时间为4 min,在0.5~40.0 mg/L浓度范围内线性良好(r=0.999 4);定量下限为0.5 mg/L;批内和批间精密度分别小于4.1%和6.3%;回收率在95.8%~103.8%;溶血样本(血红蛋白浓度约为5 g/L)、脂血样本(三酰甘油浓度约为12 mmol/L)、黄疸样本(总胆红素浓度约为150 μmol/L)对检测无明显干扰,表观健康人群参考区间为1.46~6.48 mg/L。结论本研究建立了UHPLC-MS/MS检测血清α-HB的方法,方法性能满足临床检测,且简便快速,可用于临床样本检测。

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for detecting α-hydroxybutyrate (α-HB) in serum.Methods Electrospray ionization negative ion and multiple reaction monitoring mode were used to detect serum α-HB. The linearity, low limits of quantification, precision, recovery and interference of UHPLC-MS/MS were evaluated. The reference interval of this method was established in 130 serum samples (62 males and 68 females) from Shanghai East Hospital. Dixon method was used to judge the outliers and K-S test was used to analyze the data normality. The standard curve was scored by linear regression analysis.Results The total run time was 4 min of UHPLC-MS/MS method for the determination of α-HB. It has a good linear relationship in the range of 0.5-40.0 mg/L(r=0.999 4);the low limit of quantification was 0.5 mg/L;the in-batch and inter-batch coefficient of variation precision were less than 4.1% and 6.3%, respectively;the recovery ranged between 95.8% and 103.8%. Hemolytic samples (about 5 g/L hemoglobin), lipemic samples (about 12 mmol/L triglyceride), icteric samples (about 150 μmol/L total bilirubin) had no significant interference to the detection. The reference range of the apparent healthy population was 1.46-6.48 mg/L.Conclusions A method for the determination of serum α-HB by UHPLC-MS/MS was established. The method was simple, rapid, and could be used for the detection of clinical samples.