基于高效液相色谱-串联质谱的新生儿脂溶性维生素定量检测新方法的建立与应用研究

Nutritional level and metabolism of fat soluble vitamins (FSVs) is closely related to children’s development and health, its deficiency and excess can be a serious impact on child development. research on variety of fat-soluble vitamins affecting development of fetals and infants growth has been limited to detection method and failed to go in depth. This investigation plans to collect, transport and store trace amount of peripheral blood or venous blood with dried blood filter paper and develop a new, simple, rapid, sensitive, stable and low sample demand quantitative detection method using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) to measure the concentration of FSVs and standardize quality control of this method. Reference measurement procedure is developed to measure amount of FSVs of newborns and young children, measurements of FSVs and the metabolites for reference measurements are also established.Screening the metabolic markers of FSVs in the blood of children with developmental delay or malnutrition through correlation analysis, and extending this method to the central laboratory which takes part in child care and epidemiological investigation. This would help to understand the relationship between fat-soluble vitamins metabolism and child development in depth. It would also provide a powerful technical support in improving the children’s malnutrition and the population quality, preventing vitamin deficiency.

脂溶性维生素的营养和代谢水平与儿童的生长发育和健康状况密切相关，其缺乏和过量均会对儿童发育产生较为严重的影响。而从营养代谢角度同时研究多种脂溶性维生素水平对胚胎和婴幼儿生长发育的影响一直受到检测方法的限制未能深入的进行。本研究拟以干血点法采集和储存运输微量静脉血或末梢血，利用液质联用技术（HPLC-MS/MS）开发一种简单、快速、灵敏、稳定且样品需求量低的血中脂溶性维生素及代谢物同时定量检测方法。对此方法进行标准化质控，运用该方法测定新生儿和低龄儿童血液中脂溶性维生素及代谢物含量，制定参考测量值；通过相关性分析筛选出发育迟缓或营养不良儿童血中脂溶性维生素代谢标志物；并将此方法推广到儿童保健和流行病调查的中心实验室。对儿童脂溶性维生素的检测将有助于从代谢的角度深入理解脂溶性维生素代谢与儿童发育的关系，为改善儿童营养不良，预防维生素缺乏，有效促进儿童早期发育，提高人口素质提供有力的技术支持。

本研究成功建立了液相色谱-串联质谱（LC-MS/MS）同时定量测定儿童血浆中维生素A（VA）、25-羟基维生素D3（25-OH VD3）和α-生育酚（VE）的简单、灵敏、快速稳定且样品需求量低的方法。样品预处理选择固相萃取。取血浆100μL与含4μL同位素内标的300μL乙醇混合。经过一系列操作后离心，将上清液应用于固相萃取板（HLBμ洗脱板）。收集洗脱液，氮吹后用100μL甲醇复溶。然后，将6μL复溶样品注入LC-MS/MS中，用正向电喷雾离子源（ESi+）进行多反应监测（MRM）定量分析。Acquity UPLC反相苯基己基分析柱(CSH，2.1×50 mm，1.7μm)对VA、25-OH VD3和VE进行色谱分离。流速0.4mL/min，流动相为0.1%甲酸溶液，0.1%甲酸、5mM甲酸铵的乙腈为流动相梯度洗脱。总分析时间为10 min。该方法对甲醇中VA、25-OH VD3和VE的定量限分别为10.03、1.20和0.04 ng/mL，线性范围分别为0.14~14.32μg/mL、1.80~180.29 ng/mL和6.03~602.99 ng/mL，相关系数均大于0.998。所有分析物的回收率在80%~120%之间，日间和日内精密度(相对标准偏差，RSD%)小于10.0%。对反复呼吸道感染患儿血浆和贫血儿指尖血中VA、25-OH、VD3和VE进行定量分析，并用社会科学统计软件包（SPSS20.0）对数据进行统计分析。应用该方法可同时定量检测多种脂溶性维生素，固相萃取方法被用于简化样品预处理，方法中采用μElution 96-孔板可减少样品体积，仅用100μL样品即可同时检测出VA，25-OH VD3和VE，同时96-孔板适合于大批量样品操作，此外，该方法进样量小，仅需6μL。这些都使该方法适用于儿科临床中进行大批量样本检测，尤其是体内循环血量有限，静脉血采集非常困难的婴儿和新生儿样本的检测。这是首次针对儿童样品的特点开发的能够全面检测儿童（特别是婴幼儿）血液中脂溶性维生素的方法，并应用于临床儿童样品检测中，具有重要的应用前景。