液相色谱串联质谱法测定血清中25-羟基维生素D2及25-羟基维生素D3含量

Determinations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum by liquid chromatography tandem-mass spectrometry

目的建立液相色谱串联质谱法（LC-MS／MS）测定血清25-羟基维生素D2[25（OH）D2]及25-羟基维生素D3[25（OH）D3]含量的方法。方法采用乙醇沉淀蛋白，正己烷液液萃取目标组分，乙腈复溶。采用LC-MS／MS[正离子电喷雾离子化（ESI＋）的多反应监测模式（MRM）]氘代同位素内标法检测血清25（OH）D2及25（OH）D3含量并进行相关方法学验证。结果LC-MS／MS检测血清25（OH）D2及25（OH）D3的批内精密度为0．88％-7．69％，批间精密度为1．56％～9．90％；25（OH）D2在0．5～10．0ng/mL、25（OH）D3在5～100ng／mL范围内线性良好，线性相关系数分别为0．9991、0．9999，校准品测试结果正确度为95．5％-101．2％。281名志愿者男、女之间25（OH）D2含量差异无统计学意义（P〉0．05），25（OH）D3含量差异有统计学意义（P〈0．05）。结论LC-MS／MS检测血清25（OH）D2及25（OH）D3的敏感性高，结果准确、稳定，可应用于临床分析。

Objective To establish a method for the determinations of 25-hydroxyvitamin D2 [ 25 （OH） D2 ] and 25- hydroxyvitamin D3 [25（OH） D3 ] contents in serum by liquid chromatography tandem-mass spectrometry （LC-MS/ MS）. Methods After proteins in serum were precipitated by ethanol, liquid-liquid extraction by n-Hexane, then the residuals were re-dissolved in acetonitrile and analyzed by LC-MS/MS in the positive electrospray ionization （ESI ＋ ） mode and multiple reaction monitor（MRM） mode. The quantitative analysis for 25 （OH）D2 and 25 （OH）D3 contents was carried out by deuterium isotope as internal standard, and the methodology validation was performed. Results The within-run precision of 25 （OH） D2 and 25 （OH） D3 was 0. 88% -7.69% , and between-run precision was 1.56% - 9.90%. The good correlation coefficients were 0. 999 1 and 0.999 9 at the concentration of 0.5-10.0 ng/mL 25 （OH） D2 and 5-100 ng/mL 25 （OH） D3, respectively. The accuracy of quality control materials of 25 （OH） D2 and 25 （OH） D3 was 95.5 % -101.2%. 25 （OH） D2 content did not have statistical significance （ P 〉 0.05 ）, and 25 （OH） D3 content had statistical significance （P 〈 0.05 ） between males and females of 281 volunteers. Conclusions This method is sensitive, accurate and stable, satisfying the quantification requirements of 25 （OH）D2 and 25 （OH）D3 in serum, and it can be used in clinical analysis.