高效液相色谱法测定血清中25-羟基维生素D_3

Determination of serum 25(hydroxyl)vitamin D3 concentration by high performance liquid chromatography

目的建立测定血清25-羟基维生素D3[25（OH）VitD3]的高效液相色谱（HPLC）法，并评价其应用。方法血清经甲醇沉淀蛋白质，以二氯甲烷-正己烷溶液提取后进行HPLC分析。以25（OH）VitD3标准品外标法建立标准曲线，并检测47名正常健康儿童的血清25（OH）VitD3浓度。结果该方法在10～200μg／L浓度范围内线性关系良好（r=0．9971），日内变异系数（CV）为3．5％～4．4％，日间CV为3．9％～4.7％。方法回收率为93．4％～106．2％，提取回收率为88．8％～94．1％。正常健康儿童血清25（OH）VitD3夏季参考范围为14．8～34．5μg／L。结论本法具有快速、灵敏、准确和重复性好等优点，可用于临床实验室测定血清25（OH）VitD3浓度。

Objective To establish a high performance liquid chromatography（HPl,C）assay for the determination of serum 25 （ hydroxyl ） vitamin D3 [ 25 （ OH ） VitD3 ] and evaluate the application value. Methods The serum after deproteinization with absolute methanol was extracted with dichloromethane/n-hexane and determined by HPLC. The standard curve was established with outer standard assay with standardization of 25 （OH） VitD3. The levels of 25 （OH） VitD3 in 47 healthy children were determined. Results The linear correlation coefficient of the method was satisfactory （r =0. 997 1 ） in 10-200 μg/L. The intra-day coefficient of variation （CV）was 3.5%-4.4% and inter-day CV was 3.9% -4.7%. The method recovery rate was 93.4% -106.2% and the extracted recovery rate was 88.8% -94.1%. The reference range of healthy children was 14.8-34.5μg/L in summer. Conclusions The method is quick,sensitive,accurate and reproducible. It is suitable for the determination of serum 25 （OH） VitD3 concentration.