用液相色谱串联质谱法检测脐带血血清25-羟基维生素 D2和25-羟基维生素 D3浓度

摘要

Objective To establish a method for determining the concentrations of 25-hydroxyvitamin D2[25(OH) D2] and 25-hydroxyvitamin D3[25(OH)D3] in cord blood.Methods The serum samples from cord blood were pretreated by acetonitrile and ZnSO4 .High performance liquid chromatography tandem-mass spectrometry ( LC-MS/MS) was used for the determinations of 25(OH)D2 and 25(OH)D3 concentrations.The analysis was carried on a Poroshell®120 EC-C18 column.Electrospray ionization ( ESI), multiple reaction monitor ( MRM), nebulizer 35.0 psi, gas temperature 180℃, sheath gas temperature 350 ℃ and sheath gas flow 11.0 L/min were performed.The column temperature was 50℃ with linear gradient elution using methanol ( 80%) and water ( 20%) , and the flow rate was 0.5 mL/min.The quantitative analysis was performed by selective reaction monitoring mode of heater ESI.Results A good linearity was detected in the linear range of 3.125-100μg/L for 25(OH) D2 and 25(OH) D3 (R2 =0.998 7 and 0.999 1).Blood samples had frozen and thawed for 3 times.Both intra-day and inter-day relative standard deviations (RSD) were <15%.The quality control materials of serum 25(OH)D2 and 25(OH)D3 were analyzed, and the results met the requirements of quality control.Conclusions LC-MS/MS is sensitive, rapid and suitable for the determinations of serum 25(OH)D2 and 25(OH)D3 concentrations in cord blood.%目的：建立一种测定脐带血25-羟基维生素D2[25（OH）D2]和25-羟基维生素D3[25（OH）D3]浓度的方法。方法脐带血血清样本前处理采用饱和硫酸锌和乙腈的蛋白沉淀法。采用液相色谱串联质谱法（LC-MS／MS）测定25（OH）D2和25（OH）D3浓度。固定相为 Poroshell®120 EC-C18正向柱；电喷雾离子源（ESI）；多反应监测模式（MRM）；喷雾电压35．0 psi；喷雾温度180℃；鞘气温度350℃；鞘气流速11．0 L／min。以甲醇（80％）、超纯水（20％）为流动相进行梯度洗脱，流速为0．5 mL／min，柱温为50℃；质谱采用高温ESI，以选择反应监测模式进行定量分析。结果25（OH）D2和25（OH）D3在3．125～100μg／L范围内呈良好的线性关系（R2＝0．9987、0．9991）。血样冻融3次进行检测，日内、日间相对标准偏差（RSD）均＜15％。对血清25-（ OH） D2和25（ OH） D3质控品进行检测，结果均符合要求。结论 LC-MS／MS敏感性高，分析时间短，适用于脐带血25（OH）D2和25（OH）D3浓度检测。