超高效液相色谱-串联质谱法测定血清中25-羟基维生素D和维生素K1

Simultaneous determination of 25-hydroxyvitamin D and vitamin K1 in serum by ultra-performance liquid chromatography-tandem mass spectrometry

目的建立超高效液相色谱-串联质谱(ultra-performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)同时测定血清中25-羟基维生素D2[25-hydroxyvitamin D2,25(OH)D2]、25-羟基维生素D3[25-hydroxyvitamin D3,25(OH)D3]和维生素K1(vitamin K1,VK1)的方法,并用此方法诊断维生素缺乏症,评估人群营养状况。方法血清标本采用乙腈沉淀血清蛋白,正己烷提取。采用BEH C18色谱柱(100 mm×2.1 mm,1.7μm)进行分离,以甲醇-0.1%甲酸水溶液梯度洗脱。质谱采用电喷雾正离子模式和多反应监测检测。以稳定同位素标记的d6-25(OH)D3、d7-维生素K1作为内标定量。结果25(OH)D2、25(OH)D3和维生素K1分别在浓度为5.0~75.0 ng/mL、2.0~81.5 ng/mL和0.3~12.0 ng/mL的范围内呈良好线性关系,相关系数r>0.995;检出限分别为1、0.25和0.1 ng/mL,定量限分别为3、0.75和0.3 ng/mL;血清基质中3种维生素高中低3个浓度水平的精密度均<5.0%、加标回收率为98.5%~104.3%(n=6)。结论本研究建立的UPLC-MS/MS方法检测血清中25(OH)D2、25(OH)D3和维生素K1灵敏度高、准确性好,适用于人群维生素D及维生素K1营养状况的监测。

OBJECTIVE To establish a method for analysis of 25-hydroxylvitamin D2(25(OH)D2),25-hydroxylvitamin D3(25(OH)D3)and vitamin K1 in serum by ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS),which can be applied in diagnosis of vitamin deficiency and estimation on the nutritional status of people.METHODS Serum samples mixed with d6-25(OH)D3,d7-vitamin K1(internal standard)were precipitated with acetonitrile and extracted with n-hexane.The sample solution was separated using BEH C18 column(2.1 mm×100 mm,1.7μm)with gradient elution using methanol-water containing 0.1%formic acid as mobile phase.The target molecule was detected in positive electrospray ionization(ESI+)and multiple reaction monitoring. RESULTS The concentration of 25( OH) D2,25( OH) D3 and vitamin K1 showed good linearity in the range 5. 0-75. 0 ng /mL,2. 0-81. 5 ng /mL and 0. 3-12. 0 ng /mL,respectively,with the correlation coefficients greater than 0. 995. The limits of detection( LOD) of 25( OH) D2,25( OH) D3 and vitamin K1 were 1,0. 25 and 0. 1 ng /mL,and the limits of quantification ( LOQ) for 25 ( OH) D2,25 ( OH) D3 and vitamin K1were 3,0. 75 and 0. 3 ng /mL,respectively. The recoveries of three levels in the matrix were 98. 5% - 104. 3%,the relative standard deviation( RSD) were all less than 5. 0%( n = 6) .CONCLUSION An UPLC-MS /MS method for analysis of 25( OH) D2,25 ( OH) D3 and vitamin K1 in serum is sensitive,rapid,accurate and suitable for the nutritional surveillance of vitamin D and K1 in the population.