高效液相色谱串联质谱法检测末梢全血中25-羟维生素D含量的方法及其临床应用  

Determination of 25-hydroxyvitamin D in Peripheral Whole Blood by High Performance Liquid Chromatography-tandem Mass Spectrometry and Clinical Applications

作　　者：周丽君 陈博一 刘纪松 张远 赵芳艳 王红英 ZHOU Li-jun;CHEN Bo-yi;LIU Ji-song;ZHANG Yuan;ZHAO Fang-yan;WANG Hong-ying(Wujiang District Children's Hospital,Suzhou 215200,China;Hangzhou Biozon Medical Co.,Hangzhou 310030,China)

机构地区：[1]苏州市吴江区儿童医院,江苏苏州215200 [2]杭州佰辰医学检验所有限公司,浙江杭州310030

出　　处：《解放军药学学报》2023年第1期60-66,共7页Pharmaceutical Journal of Chinese People's Liberation Army

基　　金：苏州民生科技项目,No.SS202091;吴江科教兴卫项目,No.WWK202122。

摘　　要：目的建立快速、灵敏的微量末梢全血样本中25-羟维生素D_(2)和25-羟维生素D_(3)的高效液相色谱串联质谱检测方法,并分析苏州地区0~14岁儿童的25-羟维生素D营养状况。方法采用甲醇蛋白沉淀和正己烷液液萃取进行样本前处理,上清液氮气吹干,80%乙腈复溶后,使用高效液相色谱串联质谱(ESI+)多反应监测模式、同位素内标法检测25-羟维生素D_(2)和25-羟维生素D_(3)的含量,对方法进行验证,并分析苏州地区2858例0~14岁体检儿童的25-羟维生素D营养状况。结果25-羟维生素D_(2)和25-羟维生素D_(3)在2.50~125.00 ng·ml^(-1)浓度范围内线性良好,线性相关系数分别为0.9997、0.9998。25-羟维生素D_(2)低浓度质控样本和高浓度质控样本的批内和批间精密度均<6.5%,准确度为97.3%~105.5%;25-羟维生素D_(3)低浓度质控样本和高浓度质控样本的批内和批间精密度均<8.6%,准确度为88.7%~102.2%。经考察无明显基质效应,样本可在常温(25℃)稳定保存3 d,可在4℃稳定保存7 d。2858例儿童25-羟维生素D检测结果显示秋季水平较高,为35.96(27.81~44.04)ng·ml^(-1),4个季节间的数据差异有统计学显著差异(P<0.01);随着年龄的增长,25-羟维生素D水平呈下降趋势,0~12个月儿童的水平最高,为36.90(26.56~45.80)ng·ml^(-1),8~14岁组水平最低,为16.91(13.41~23.46)ng·ml^(-1),4个年龄组间的数据差异有统计学显著差异(P<0.01)。结论本研究建立了高效液相色谱串联质谱法测定微量末梢全血中25-羟维生素D_(2)和25-羟维生素D_(3)的方法,简单快捷、灵敏度高、准确度好,可应用于临床。Objective To establish a quick and sensitive method for determination of 25-hydroxyvitamin D_(2)〔25-(OH)D_(2)〕and 25-hydroxyvitamin D_(3)〔25-(OH)D_(3)〕in fingertip blood using high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS),and study the levels of 25-(OH)D in children aged 0 to 14 in Suzhou.Methods Samples were prepared via protein precipitation with methanol and via liquid-liquid extraction with n-hexane.The supernatant was dried with nitrogen and resolved in 80%acetonitrile.Method validation was performed before the concentrations of 25-(OH)D_(2)and 25-(OH)D_(3)were determined using the isotope internal standard dilution technique in the LC-MS/MS(ESI+)MRM mode.The level of 25-(OH)D in 2858 children aged 0 to 14 in Suzhou was also studied.Results 25-(OH)D_(2)and 25-(OH)D_(3)showed good linearity in the concentration range of 2.50 to 125.00 ng·ml^(-1),with linear correlation coefficients of 0.9997 and 0.9998,respectively.In both low-concentration and high-concentration quality control samples,the intra-day and inter-day precision of 25-(OH)D_(2)was less than 6.5%,and the accuracy ranged from 97.3%to 105.5%,the level of 25-(OH)D_(3)was less than 8.6%,the accuracy was between 88.7%and 102.2%.No obvious matrix effect was observed,the samples could be stably stored at room temperature(25℃)for 3 days and at 4℃for 7 days.The results of detection of 25-(OH)D in 2858 children showed that the level was highest in autumn,which was 35.96(27.81-44.04)ng·ml^(-1).The data was significantly different between the four seasons(P<0.01).The level of 25-(OH)D tended to increase with age,which was the highest among 0-to 12-month-olds〔36.9(26.56-45.80)ng·ml^(-1)〕,and was the lowest in the group aged 8 to 14〔16.91(13.41-23.46)ng·ml^(-1)〕.The data was significantly different between the four age groups(P<0.01).Conclusion In this study,an LC-MS/MS method for the determination of 25-(OH)D_(2) and 25-(OH)D_(3) in fingertip blood is established,which is simple,quick,sensitive,accurate,and ap

关 键 词：末梢全血 25-羟维生素D 高效液相色谱串联质谱法 

分 类 号：R937.1[医药卫生—生药学]