液相串联质谱法检测糖尿病患者血浆25-羟基维生素D3

Quantitative measurement of vitamin D in diabetic plasma by liquid chromatography tandem mass spectrometry

目的建立液相串联质谱(LC-MS/MS)检测人血浆中维生素D(VitD)的方法,并评价糖尿病患者血浆VitD与健康人之间的差异。方法采用2倍量的甲醇/乙腈(80/20,v/v)去除血浆中蛋白质,上清液采用甲基叔丁基醚萃取VitD,萃取物经N2吹干后直接或采用PTAD柱前衍生法处理后行LC-MS/MS分析。结果 PTAD柱前衍生法测得25(OH)-D2、25(OH)-D3的定量限分别0.05和0.07 ng,直接检测法定量限0.2和0.3 ng;25(OH)-D3在15例II型糖尿病患者和18例健康人血浆中水平分别为(12.1±3.9)和(20.8±4.8)ng/ml。结论 PTAD柱前衍生法较直接检测法可提高25(OH)-D2和25(OH)-D3的定量限大约3倍;Ⅱ型糖尿病患者血浆25(OH)-D3水平显著低于健康人水平(P<0.05)。

Objective： To establish a quantitative method for determining human plasma vitamin D level by liquid chromatography tandem mass spectrometry,and investigate vitamin D difference between diabetic and healthy participant. Methods： Proteins in plasma were precipitated by adding 2 folds of methanol / acetonitrile（ 80 /20,v / v）,the supernatant after centrifugation was extracted with tert-butyl methyl ether,the upper phase after centrifugation was dried by N2,and the dryness was dealt with pre-column derivatization with PTAD or re-dissolved in mobile phase directly for LC-MS / MS analysis. Results： The limitation of detection（ LOD） of 25（ OH）-D2 and 25（ OH）-D3 before and after derivatiation were 0. 2 and 0. 3 ng,0. 05 and 0. 07 ng,respectively; 25（ OH）-D3 levels in 15 type II diabetic and 18 healthy participant were 12. 1 ± 1. 9 and 20. 8 ± 4. 8 ng / mL,respectively. Conclusion： Pre-column derivatization with PTAD could improve the LOD of 25（ OH）-D2 and 25（ OH）-D3 about 3 fold,respectively; 25（ OH）-D3 level is significantly lower in diabetic than that in healthy participant（ P 0. 05）.