用液相色谱-串联质谱法测定人血清中25-羟基维生素D的浓度及临床应用

Determination of 25-hydroxyvitamin D in serum by liquid chromatography-tandem mass spectrometry and its clinical application

目的建立一种血清中25-羟基维生素D_(2)(25-OH VD_(2))和25-OH VD_(3)的定量分析方法,进行方法的全验证并应用于临床血清分析。方法血清样本经乙腈沉淀蛋白,以d3-25-OH VD_(2)和d6-25-OH VD_(3)内标。色谱柱:Kinetex C_(18)(3.0 mm×50.0 mm,2.6μm);流动相:0.1%甲酸水溶液-0.1%甲酸甲醇溶液;梯度洗脱;流速:0.5 mL·min^(-1);柱温:30℃,进样量:10μL。电喷雾离子化源,正离子模式,多重反应监测。考察该方法的专属性、标准曲线与定量下限、精密度与准确度、稳定性等。结果25-OH VD_(2)和25-OH VD_(3)的保留时间分别为2.0和1.9 min,在0.5~50.0 ng·mL^(-1)和1.0~100.0 ng·mL^(-1)内具有良好的线性关系。3个质量浓度的质控样本的加标回收率在94.92%~111.39%内,批内和批间精密度≤7.99%,该方法具备较高的准确度和精密度。室温放置6 h、4~8℃冷藏5 d、-20℃冰冷放置30 d存放条件不影响对分析物浓度进行准确测定。结论本研究方法前处理简便,分析测试快速,结果准确可靠,可实现血清中25-OH VD_(2)和25-OH VD_(3)的准确测定。

Objective To establish a quantitative analysis method of 25-hydroxyvitamin D_(2)(25-OH VD_(2))and 25-OH VD_(3) in serum was established,and apply it to clinical serum analysis after full validation.Methods Serum samples were precipitated with acetonitrile,and internal standards were d3-25-OH VD_(2) and d6-25-OH VD_(3).Chromatographic column was Kinetex C_(18)(3.0 mm×50.0 mm,2.6μm);mobile phase was 0.1%formic acid aqueous solution-0.1%formic acid methanol solution,flow rate was 0.5 mL·min^(-1),ith gradient elution;column temperature was 30℃C,sample injection was 10μL.Electrospray ionization source,positive ion mode,multiple reaction monitoring.The specificity,standard curve and lower limit of quantification,precision and accuracy,and stability of the method were investigated.Results The retention time of 25-OH VD_(2) and 25-OH VD_(3) was 2.0 and 1.9 min respectively.They had a good linear relationships in the range of 0.5-50.0 ng·mL^(-1)and 1.0-100.0 ng·mL^(-1).The spiked recoveries of quality control samples at three concentrations were in the range of 94.92%^(-1)11.39%,and the intra-batch and inter-batch precision were≤7.99%.This method had high accuracy and precision.The storage conditions of 6 h at room temperature,5 d at 4-8℃and 30 d at-20℃didn't affect the accurate determination of analytes.Conclusion This method is simple in pretreatment,rapid in analysis and test,accurate and reliable in results,which can realize the accurate determination of 25-OH VD_(2)and 25-OH VD_(3) in serum.