Objective To establish a liquid chromatography-tandem mass spectrometry ( LC-MS/MS) for the quantitation determination of serum 25-hydroxyvitamin D [25(OH) D] and perform the general method performance verification.Methods The LC-MS/MS was used for the quantitation determination of 25(OH)D2 and 25(OH)D3 in serum by Waters® XevoTM TQ MS ACQUITY UPLC® mass spectrometry instrument.According to US Food and Drug Administration ( FDA)′s guideline ( Guidance for Industry Bioanalytical Method Validation), the general method performance verification was performed for linearity, determination limit, precision and accuracy.Results The linear ranges of 25(OH)D2 and 25(OH)D3 were 6.25-500.00 nmol/L.The LC-MS/MS had quantitation determination limits of 2.50 nmol/L for 25(OH) D2 and 1.25 nmol/L for 25(OH) D3.The within-run and between-run coefficients of variation (CV) of 25(OH)D2 and 25(OH)D3 were <4% and <6%, respectively.The recovery rates were 93.26%-112.16%.The result of the Vitamin D External Quality Assessment Scheme ( DEQAS) interlaboratory quality assessment had a bias <10%.Conclusions The basic performance of LC-MS/MS meets the evaluation standards, and LC-MS/MS is sensitive and accurate for detecting the concentrations of 25(OH)D2 and 25(OH)D3 in serum.%目的：建立定量检测血清25－羟基维生素 D[25（OH）D]的液相色谱－串联质谱（LC－MS／MS）方法，并对此方法进行基本分析性能验证。方法使用 Waters® XevoTM TQ MS ACQUITY UPLC®液质联用仪（LC－MS／MS）同时定量检测血清中的25（OH）D2和25（OH）D3含量。参照美国食品和药品管理局（FDA）的生物分析方法验证导则标准对所建方法进行线性、检出限、精密度和准确性等基本性能的验证。结果LC－MS／MS检测25（OH）D2和25（OH）D3的线性范围为6．25～500．00 nmol／L；25（OH）D2和25（OH）D3的定量检出限分别为2．50和1．25 nmol／L；批内、批间变异系数（CV）分别为＜4％、＜6％；回收率为93．26％～112．16％；维生素D 国际室间质量评价计划（DEQAS）室间质评结果偏倚＜10％。结论建立的 LC－MS／MS 基本性能符合评价标准，能够灵敏且准确的检测出血清中25（OH）D2和25（OH）D3的浓度。
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