摘要
目的建立液相色谱-串联质谱(LC-MS/MS)方法同时检测血清中维生素A、维生素D(25-OH-D_(2)、25-OH-D_(3))、维生素E和维生素K_(1)水平,并通过血清样品验证。方法以同位素标记的维生素A-d4、25-OH-D_(2)-d6、25-OH-D_(3)-d6、维生素E-d6和维生素K_(1)-d7作为内标,在血清样品中利用甲醇/乙腈混合溶液沉淀蛋白质,然后用正己烷萃取脂溶性维生素,采用Phenomenex Kinetex C18(50.0 mm×3.0 mm,2.6μm)色谱柱,柱温设置为40℃,流动相分别为0.1%甲酸水溶液和0.1%甲酸甲醇溶液,以0.6 mL/min的流速进行梯度洗脱,质谱采用大气压化学电离正离子MRM模式。结果维生素A、25-OH-D_(2)、25-OH-D_(3)、维生素E和维生素K_(1)在相应浓度范围内线性关系良好,线性相关系数均大于0.998,其日内精密度和日间精密度在1.87%~9.81%,加标回收率在91.47%~107.30%,携带污染率在-6.03%~2.14%,均在可接受范围之内。结论成功建立了LC-MS/MS方法同时测定人血清中维生素A、25-OH-D_(2)、25-OH-D_(3)、维生素E和维生素K_(1)的水平,该方法前处理简便,分析速度快,灵敏度高,专属性强且各被检测物质的线性关系良好,整个检测过程简单、规范,适合临床血清样品的检测。
Objective To establish the LC-MS/MS method for simultaneously determining serum vitamin A,vitamin D(25-OH-D_(2),25-OH-D_(3)),vitamin E and vitamin K_(1) levels,and to pass the serum sample validation.Methods Isotopically labeled vitamin A-d4,25-OH-D_(2)-d6,25-OH-D_(3)-d6,vitamin E-d6 and vitamin K_1-d7 served as the internal standards.The methanol/acetonitrile mixture in serum samples was used to precipitate the protein,then normal hexane was used to extract the fat-soluble vitamins.A Phenomenex Kinetex C18(50.0 mm×3.0 mm,2.6 μm) column was used with the column temperature set at 40 ℃ and the mobile phases were 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution,and the gradient elution was performed at a flow rate of 0.6 mL/min.The mass spectrometry adopted the atmospheric pressure chemical ionization positive ion MRM mode.Results Vitamin A,25-OH-D_(2),25-OH-D_(3),vitamin E and vitamin K_(1) showed good linearity in the corresponding concentration ranges with linear correlation coefficients greater than 0.998,and their intra-day precision and inter-day precision were 1.87%-9.81%.The spiked recoveries ranged 91.47%-107.30%,and the carryover contamination rates ranged-6.03%-2.14%,which all were within the acceptable range.Conclusion The LC-MS/MS method is successfully established for the simultaneous determination of vitamin A,25-OH-D_(2),25-OH-D_(3),vitamin E and vitamin K_(1) in human serum.The method is simple in pretreatment and fast in analysis with high sensitive and strong specificity,moreover the linear relationship among various detected materials is good,and the whole detection procedure is simple and standardized,which is suitable for the determination of clinical serum samples.
作者
于鹏
周传贵
楚士颖
李艳
迟伟
田晗
程文播
YU Peng;ZHOU Chuangui;CHU Shiying;LI Yan;CHI Wei;TIAN Han;CHENG Wenbo(Tianjin Guoke Medical Technology Development Co.,Ltd.,Tianjin 300399,China;Suzhou Institute of Biomedical Engineering Technology,Chinese Academy of Sciences,Suzhou,Jiangsu 215163,China;Shenyang Municipal Maternal and Child Health Care Hospital,Shenyang,Liaoning 110014,China)
出处
《检验医学与临床》
2024年第6期721-725,732,共6页
Laboratory Medicine and Clinic
基金
国家重点研发计划资助项目(2021YFC2401100)。
作者简介
于鹏,男,工程师,主要从事医用质谱的应用研究;通信作者:程文播,E-mail:chengwb@sibet.ac.cn。
