色谱2025，Vol.43Issue(3)：228-236,9.DOI:10.3724/SP.J.1123.2024.02003
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
摘要
Abstract
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∶10 000 and stabilized after 1 h.Compared with Silica and C18 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 ho-mogenized samples,which were saponified with alkali solution,extracted and concentrated,purified using a SPE column,derivatized,and separated on a Waters Acquity UPLC BEH C18 column(100 mm×2.1 mm,1.7 μm)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 posi-tive 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 0.2-50 μg/L,with correlation coefficients of 0.999 5-0.999 9.The limits of detection(LODs)and limits of quantification(LOQs)were in the range of 0.018-0.066 and 0.06-0.22 μg/kg,re-spectively.Recoveries were 92.6%-99.4% at three spiked levels(0.20,1.0,and 5.0 μg/kg),and the relative standard deviations(RSDs)were 3.6%-6.2% .This highly sensitive method yields reproducible and accurate results for the quantitative determination of vitamin D and 25-hydroxyvitamin D in animal-derived foods.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Key words
ultra performance liquid chromatography(UPLC)/tandem mass spectrometry(MS/MS)/vitamin D/25-hydroxyvitamin D/derivatization/4-phenyl-1,2,4-triazoline-3,5-dione(PTAD)分类
化学引用本文复制引用
刘宇,谢继辉,章萍萍,周迪,赵维克,张居舟..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[J].色谱,2025,43(3):228-236,9.基金项目
安徽省市场监督管理局科技计划项目(2022MK031) （2022MK031）
市场监管总局科技计划项目(2023MK064).Science and Technology Planning Project of Anhui Administration for Market Regulation(No.2022MK031) （2023MK064）
Science and Technology Planning Project of State Administration for Market Regulation(No.2023MK064). （No.2023MK064）
