血清中维生素D活性代谢物的LC-MS/MS定量方法研究  

A quantitation method for the determination of Vitamin D metabolite in human serum using LC-MS/MS

作　　者：阮亮亮 李金蓉 RUAN Liangliang;LI Jinrong(Department of Bioanalysis,Shanghai Fenglin Clinical Laboratory Co.,Ltd,Shanghai 200231,China;Shanghai Clinical Research Center,Shanghai 200032,China;Shanghai Clinical Research Center Co.Ltd,Shanghai 200032,China;Shanghai Engineering Research Center of Biobank,Shanghai 200231,China)

机构地区：[1]上海枫林医药医学检验有限公司生物分析部,上海200231 [2]上海医药临床研究中心,上海200032 [3]上海医药临床研究中心公司,上海200032 [4]上海生物样本库工程技术研究中心,上海200231

出　　处：《国际检验医学杂志》2024年第S01期80-84,共5页International Journal of Laboratory Medicine

摘　　要：目的1α,25-二羟基维生素D_(3)是维生素D的活性代谢产物。但该物质在人血清中的含量非常低,常规的检测方法无法准确定量。建立并验证一种准确、选择性高、快速定量1α,25-二羟基维生素D3的液质联用方法,对满足临床研究、临床诊断的需求非常有帮助。方法因该分析物为内源性物质,方法中采用自配并经过验证的替代基质配制标准曲线和质控品。人血清样品经过液液萃取纯化,用衍生化试剂消除内源性干扰,再进行衍生化反应以提高灵敏度。采用反相柱分离,质谱电喷雾电离源正离子模式采集,离子对为574.3→314.3和577.4→317.2的多反应监测(MRM)模式,监测1α,25-二羟基维生素D3和内标。在15~1000 pg/mL范围内线性良好,最低定量限(LLOQ)为15 pg/mL。结果经过方法学验证,分析物的精密度和准确度(日内及日间)偏差均满足±20%以内的要求。血清样品稳定性实验结果良好,满足评判标准。结论该方法准确、稳定,具有耐用性及灵敏度高等优点,适用于临床样品的分析。Objective 1α,25-dihydroxyvitamin D3 is an active metabolite of vitamin D.However,it is difficult to analyze accurately for its very low concentration in human serum.To develop and validate an accurate,highly selective and rapid LC-MS/MS method for the quantification of 1α,25-dihydroxyvitamin D3 was helpful to clinical research and diagnosis.Methods Since the analyte was an endogenous substance,standard curves and quality control samples were prepared using self-prepared and validated surrogate matrix.Human serum samples were purified by liquid-liquid extraction,and eliminated endogenous interference with one of the derivatization reagent,then derivatized to improve the sensitivity.The analyte was separated with reverse phase column,electrospray ionization(ESI)source was operated in positive ion mode.Transition ions m/z 574.3→314.3 and m/z 577.4→317.2 were used to quantify 1α,25-dihydroxyvitamin D3 and internal standard(I.S.),respectively.The linearity was obtained over the concentration range of 15.0-1000 pg/mL,and the LLOQ was up to 15.0 pg/mL.Results The precision and accuracy(intra-day and inter-day)deviations of the analyte was within±20%.The serum samples were stable and all met the acceptance criteria.Conclusion This method is accurate,reliable,and sensitive for the analysis of clinical samples.

关 键 词：维生素D代谢物 1α 25-二羟基维生素D_(3) 化学衍生法 

分 类 号：R446.11[医药卫生—诊断学]