微量血清中25-羟维生素D的柱前衍生-高效液相色谱-串联质谱法的建立及其临床应用

目的 建立微量血清中25-羟维生素D2（25-(OH)D2）和25-羟维生素D3（25-(OH)D3）的高效液相色谱-串联质谱法（LC-MS/MS），并用于临床常规检验。方法 10微升血清样本中加入同位素内标，用无水乙醇沉淀蛋白再用正己烷萃取，氮气吹干后衍生，复溶后经液相色谱分离、质谱定性定量。结果 25-(OH)D2和25-(OH)D3的线性相关系数（r）分别为0.9958和0.9937；批内精密度小于13.2%，批间精密度小于11.9%，平均加样回收率均在93.6%~109.7%之间；25-(OH)D2和25-(OH)D3的LOD分别为0.1ng/ml和0.025ng/ml，LOQ分别为1.5ng/ml和5ng/ml。在所检测的213份血样中约有6%的儿童的25-羟维生素D低于15ng/ml。结论 本法满足方法学要求，可用于微量血清中25-羟维生素D2和25-羟维生素D3的测定，对儿科临床诊断治疗有重要提示意义。

Establishment of pre-column derivatization high-performance liquid chromatography-tandem mass spectrometry for the determination of 25-hydroxyvitamin D in trace serum and its clinic application

Objective To establish a method for determination of 25-hydroxyvitamin D2 (25-(OH)D2) and 25-hydroxyvitamin D3(25-(OH)D3) by pre-column derivatization liquid chromatography-tandem mass spectrometry（LC-MS/MS）in trace serum, so as to use for clinical routine examination. Methods Isotopic internal standard was added to 10 microliter serum samples. The protein in samples was precipitated with anhydrous ethanol and the target chemicals were extracted with n-hexane. After extraction, the supernatants were evaporated to dryness under a stream of nitrogen. The residue was experienced the derivation process and the derivatizing products were redissolved and chromatographically separated and quantified by mass spectrometry. Results The linear correlation coefficients of 25-(OH) D2 and 25-(OH) D3 were 0.9958 and 0.9937, respectively. The RSDs within batch was less than 13.2%, and the RSDs between batches were less than 11.9%. The average recoveries were 93.6% - 109.7%. The LOD of 25-(OH) D2 and 25-(OH) D3 were 0.1ng/ml and 0.025ng/ml, respectively, and the LOQs were 1.5ng/ml and 5ng/ml, respectively. 25-hydroxyvitamin D in about 6% of the 213 blood samples tested were less than 15ng/ml. Conclusion This method meets the methodological requirements and can be applied to the determination of 25-(OH) D2 and 25-(OH) D3 in trace serum. This method has important significance for the pediatric clinical diagnosis and treatment.