色谱

色谱 ›› 2025, Vol. 43 ›› Issue (3): 228-236.DOI: 10.3724/SP.J.1123.2024.02003

• 研究论文 • 上一篇    下一篇

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

刘宇, 谢继辉, 章萍萍, 周迪, 赵维克, 张居舟()   

  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
  • 收稿日期:2024-02-05 出版日期:2025-03-08 发布日期:2025-03-03
  • 通讯作者: 张居舟
  • 基金资助:
    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)

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

LIU Yu, XIE Jihui, ZHANG Pingping, ZHOU Di, ZHAO Weike, ZHANG Juzhou()   

  1. Anhui Institute for Food and Drug Control, China National Center for Quality Supervision and Test of Agricultural-Avocation Processed Food, Hefei 230051, China
  • Received:2024-02-05 Online:2025-03-08 Published:2025-03-03
  • Contact: ZHANG Juzhou
  • Supported by:
    Science and Technology Planning Project of Anhui Administration for Market Regulation(2022MK031);Science and Technology Planning Project of State Administration for Market Regulation(2023MK064)

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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C18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 C18色谱柱(100 mm×2.1 mm, 1.7 μm)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0.2~50 μg/L内线性良好,相关系数为0.9995~0.9999,方法检出限为0.018~0.066 μg/kg,方法定量限为0.06~0.22 μg/kg,在0.20、1.0、5.0 μg/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。

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

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∶10000 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 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 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 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 0.2-50 μg/L, with correlation coefficients of 0.9995-0.9999. 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, respectively. 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.

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)

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