衍生化-超高效液相色谱-串联质谱法测定动物源性食品中维生素D和25-羟基维生素D  

Determination of vitamin D and 25-hydroxyvitamin D in animal-derived foods by derivatization-ultra performance liquid chromatography-tandem mass spectrometry

作　　者：刘宇 谢继辉 章萍萍 周迪 赵维克 张居舟 LIU Yu;XIE Jihui;ZHANG Pingping;ZHOU Di;ZHAO Weike;ZHANG Juzhou(Anhui Institute for Food and Drug Control,China National Center for Quality Supervision and Test of Agricultural-Avocation Processed Food,Hefei 230051,China)

机构地区：[1]安徽省食品药品检验研究院,国家农副加工食品质量检验检测中心,安徽合肥230051

出　　处：《色谱》2025年第3期228-236,共9页Chinese Journal of Chromatography

基　　金：安徽省市场监督管理局科技计划项目(2022MK031);市场监管总局科技计划项目(2023MK064)。

摘　　要：动物源性食品是人体摄入维生素D和25-羟基维生素D的重要食物来源。目前我国相关标准限于维生素D的含量测定,使用非衍生化方法进行检测。本研究以4-苯基-1,2,4-三唑啉-3,5-二酮(PTAD)作为衍生试剂,通过Diels-Alder反应,在维生素D和25-羟基维生素D上引入易电离基团,提高离子化效率,建立了衍生化-超高效液相色谱-串联质谱法(UPLC-MS/MS)同时测定动物源性食品中维生素D和25-羟基维生素D的检测方法。本研究对衍生化条件、样品前处理条件、色谱分离条件和质谱检测条件进行了优化。结果表明,在乙腈溶剂中,目标化合物与衍生试剂PTAD质量比为1∶10000的条件下,衍生反应充分;衍生反应1 h后达到稳定;相较于Silica和C 18固相萃取(SPE)柱,HLB SPE柱对目标化合物的回收率更高,同时可有效降低基质效应的影响;在水-甲醇流动相体系中添加5 mmol/L甲胺,可显著提升目标化合物的检测灵敏度。样品经过均质,加入同位素内标,碱液皂化,萃取浓缩,固相萃取柱净化和化学衍生,在Waters Acquity UPLC BEH C 18色谱柱(100 mm×2.1 mm,1.7μm)上,以0.1%甲酸-5 mmol/L甲胺水溶液和0.1%甲酸-5 mmol/L甲胺甲醇溶液作为流动相梯度洗脱。分析物在电喷雾正离子模式(ESI+)下进行多反应监测(MRM)测定,内标法定量。结果表明,维生素D和25-羟基维生素D在0.2~50μg/L内线性良好,相关系数为0.9995~0.9999,方法检出限为0.018~0.066μg/kg,方法定量限为0.06~0.22μg/kg,在0.20、1.0、5.0μg/kg 3个加标水平下,加标回收率为92.6%~99.4%,相对标准偏差为3.6%~6.2%。该方法灵敏度高,重复性好,适用于动物源性食品中维生素D和25-羟基维生素D灵敏、准确的定量检测。Animal-derived foods are essential sources of vitamin D and 25-hydroxyvitamin D in the human diet.Currently,the relevant regulatory methods in China are limited to using non-derivatization methods to determine vitamin D content.In this study,4-phenyl-1,2,4-triazoline-3-5-dione(PTAD)was used as a derivative reagent,and the ionization efficiencies of vitamin D and 25-hydroxyvitamin D were improved by introducing readily ionizable groups via the Diels-Alder reaction.This method allowed for the simultaneous determination of vitamin D and 25-hydroxyvitamin D in animal-derived foods using derivatization and ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The conditions for derivatization,sample pretreatment,chromatographic separation,and MS detection were optimized.The results showed that the derivatization reaction was effective in acetonitrile solvent at a target compound to PTAD mass ratio of 1∶10000 and stabilized after 1 h.Compared with Silica and C 18 solid-phase extraction(SPE)columns,hydrophilic lipophilic balanced(HLB)SPE columns yielded higher recoveries of the target compounds,while simultaneously reducing the matrix effect.The detection sensitivity was significantly improved by adding 5 mmol/L methylamine to the water-methanol mobile phase system.An isotopic internal standard was added to the homogenized samples,which were saponified with alkali solution,extracted and concentrated,purified using a SPE column,derivatized,and separated on a Waters Acquity UPLC BEH C 18 column(100 mm×2.1 mm,1.7μm)with a gradient elution using 0.1%formic acid-5 mmol/L methylamine aqueous solution and 0.1%formic acid-5 mmol/L methylamine of methanol as the mobile phases.The analytes were determined by multiple reaction monitoring(MRM)in positive electrospray ionization mode(ESI+)and quantified using the internal standard method.The results for both vitamin D and 25-hydroxyvitamin D showed good linearity in the range of 0.2-50μg/L,with correlation coefficients of 0.9995-0.9999.The limits of detec

关 键 词：超高效液相色谱 串联质谱 维生素D 25-羟基维生素D 衍生化反应 4-苯基-1 2 4-三唑啉-3 5-二酮 

分 类 号：O658[理学—分析化学]