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

目的 建立血清25- 羟维生素D325-hydroxyvitamin D3, 25-(OH)D3] 及3-epi-25- 羟维生素D33-epi-25-hydroxyvitaminD3, 3-epi-25-(OH)D3] 超高效液相色谱- 串联质谱检测新方法（ultra-performance liquid chromatography-tandem massspectrometry，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（pentafluorophenylpropyl，PFPP）色谱柱分离25-(OH)D3 与3-epi-25-(OH)D3；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)D3 及3-epi-25-(OH)D3 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0.992 2，0.992 8；25-(OH)D3 与3-epi-25-(OH)D3 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)D3 平均浓度22.35±13.28nmol/L，3-epi-25-(OH)D3 平均浓度5.43±4.35 nmol/L；3-epi-25-(OH)D3 占总25-(OH)D3 的平均比例为19.96%±12.08%，范围0.00% ～ 60.62%。以25-(OH)D3 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)D3 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（χ2=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)D3 浓度，在早产儿Vit D 评估方面具有较好的应用价值。

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

Objective To establish a novel ultra-performance liquid chromatography-tandent mass spectrometry （UPLC-MS/ MS） method for quantification of serum 25-hydroxyvitamin D3 25-(OH)D3] and 3-epi-25-hydroxyvitamin D3 3-epi-25(OH) D3], 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)D3 and 3-epi- 25(OH)D3. Then used the established method for a clinical application in 134 premature infants after performance validation. Results For 25(OH)D3 and 3-epi-25(OH)D3, 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 0.992 2, 0.992 8. The relative standard deviations (RSD) of intra-day and inter-day for 25(OH)D3 and 3-epi-25(OH)D3 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)D3 and 3-epi-25-(OH)D3 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)D3 to total 25-(OH)D3 was 19.96%±12.08%，ranging from 0.00% to 60.62%. Based on 25-(OH)D3, 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)D3. The P values of Vit D deficiency and Vit D sufficiency were both less than 0.05（χ2=5.351，1.719，4.823，all P ＜ 0.05）. Conclusion The novel UPLC-MS/MS method successfully detects the 25-(OH)D3 and 3-epi-25-(OH)D3 in premature infants, which has a great value in clinical applications for Vit D assessment of premature infants.