基于UPLC-MS/MS建立早产儿血清25-(OH)D3和3-epi-25-(OH)D3检测新方法及临床初步应用

A Novel UPLC-MS/MS Method for Simultaneous Quantification of Serum 25-(OH)D3 and 3-epi-25(OH)D3 Followed by the Preliminary Application in Clinics

目的建立血清25-羟维生素D_(3)[25-hydroxyvitamin D_(3),25-(OH)D_(3)]及3-epi-25-羟维生素D_(3)[3-epi-25-hydroxyvitamin D_(3),3-epi-25-(OH)D_(3)]超高效液相色谱-串联质谱检测新方法(ultra-performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS),并在早产儿中开展初步应用。方法采用液-液萃取法提取化合物,五氟苯基丙基(pentafluorophenyl propyl,PFPP)色谱柱分离25-(OH)D_(3)与3-epi-25-(OH)D_(3);从最低定量限、线性关系、精密度与准确度等四个方面对新建方法进行验证,并对134例早产儿血清样本进行检测分析。结果25-(OH)D_(3)及3-epi-25-(OH)D_(3)的最低定量限分别为6.05±0.78 nmol/L,1.48±0.20 nmol/L;线性范围3.78~480.00 nmol/L,1.00~128.00 nmol/L;相关系数r2为0.9922,0.9928;25-(OH)D_(3)与3-epi-25-(OH)D_(3)低、中、高浓度质控品的日内精密度和日间精密度均小于15.00%;回收率109.92%,102.25%,98.76%;97.75%,95.25%,99.80%。134例早产儿血清25-(OH)D_(3)平均浓度22.35±13.28nmol/L,3-epi-25-(OH)D_(3)平均浓度5.43±4.35 nmol/L;3-epi-25-(OH)D_(3)占总25-(OH)D_(3)的平均比例为19.96%±12.08%,范围0.00%~60.62%。以25-(OH)D_(3)计算,维生素D(Vitamin D,Vit D)缺乏率、不足率、充足率分别为78.36%,19.40%,2.24%;以总25-(OH)D_(3)计算,Vit D缺乏率、不足率、充足率分别为65.67%,26.12%,8.21%;二者Vit D缺乏率及充足率的比较,差异均有统计学意义(χ^(2)=5.351,1.719,4.823,均P<0.05)。结论本研究建立的UPLC-MS/MS检测法性能良好且能精准检测早产儿血清25-(OH)D_(3)浓度,在早产儿Vit D评估方面具有较好的应用价值。

Objective To establish a novel ultra-performance liquid chromatography-tandent mass spectrometry(UPLC-MS/MS)method for quantification of serum 25-hydroxyvitamin D_(3)[25-(OH)D_(3)]and 3-epi-25-hydroxyvitamin D_(3)[3-epi-25(OH)D_(3)],and to assess the Vitamin D(Vit D)levels in premature infants.Methods The liquid-liquid extraction was used to extract compounds after protein precipitation,and pentafluorophenyl propyl(PFPP)column was used to resolve 25-(OH)D_(3) and 3-epi-25(OH)D_(3).Then used the established method for a clinical application in 134 premature infants after performance validation.Results For 25(OH)D_(3) and 3-epi-25(OH)D_(3),the low limit of quantification(LLOQ)were 6.05±0.78nmol/L,1.48±0.20 nmol/L.The linear ranges were 3.78~480.00nmol/L,1.00~128.00nmol/L.The correlation coefficients(r2)were 0.9922,0.9928.The relative standard deviations(RSD)of intra-day and inter-day for 25(OH)D_(3) and 3-epi-25(OH)D_(3) with low,middle,high concentrations were all less than 15.00%.The recovery rates were 109.92%,102.25%,98.76%and 97.75%,95.25%,99.80%.respectively.The average serum concentrations of 25-(OH)D_(3) and 3-epi-25-(OH)D_(3) in 134 premature infants were 22.35±13.28nmol/L and 5.43±4.35 nmol/L,respectively.The percentage of 3-epi-25-(OH)D_(3) to total 25-(OH)D_(3) was 19.96%±12.08%,ranging from 0.00%to 60.62%.Based on 25-(OH)D_(3),Vit D deficiency,insufficiency and sufficiency were 78.36%,19.40%and 2.24%.While these values were 65.67%,26.12%and 8.21%,respecitively based on total 25-(OH)D_(3).The P values of Vit D deficiency and Vit D sufficiency were both less than 0.05(χ^(2)=5.351,1.719,4.823,all P<0.05).Conclusion The novel UPLC-MS/MS method successfully detects the 25-(OH)D_(3) and 3-epi-25-(OH)D_(3) in premature infants,which has a great value in clinical applications for Vit D assessment of premature infants.