Liu Yu, Xie Ji-Hui, Zhang Ping-Ping, Zhou Di, Zhao Wei-Ke, Zhang Ju-Zhou
Anhui Institute for Food and Drug Control, China National Center for Quality Supervision and Test of Agricultural-Avocation Processed Food, Hefei 230051, China.
Se Pu. 2025 Mar;43(3):228-236. doi: 10.3724/SP.J.1123.2024.02003.
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 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 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.
动物源性食品是人类饮食中维生素D和25-羟基维生素D的重要来源。目前，中国相关监管方法仅限于使用非衍生化方法测定维生素D含量。本研究以4-苯基-1,2,4-三唑啉-3,5-二酮（PTAD）作为衍生试剂，通过狄尔斯-阿尔德反应引入易于电离的基团，提高了维生素D和25-羟基维生素D的电离效率。该方法采用衍生化结合超高效液相色谱-串联质谱（UPLC-MS/MS）同时测定动物源性食品中的维生素D和25-羟基维生素D。对衍生化、样品预处理、色谱分离和质谱检测条件进行了优化。结果表明，衍生化反应在乙腈溶剂中，目标化合物与PTAD质量比为1∶10000时有效，1 h后反应稳定。与硅胶和C固相萃取（SPE）柱相比，亲水亲脂平衡（HLB）SPE柱对目标化合物的回收率更高，同时降低了基质效应。在水-甲醇流动相体系中加入5 mmol/L甲胺可显著提高检测灵敏度。向匀浆样品中加入同位素内标，用碱溶液皂化，提取浓缩，经SPE柱净化，衍生化后在Waters Acquity UPLC BEH C柱（100 mm×2.1 mm，1.7 μm）上分离，以0.1%甲酸-5 mmol/L甲胺水溶液和0.1%甲酸-5 mmol/L甲胺甲醇溶液为流动相进行梯度洗脱。采用正电喷雾电离模式（ESI）下的多反应监测（MRM）对分析物进行测定，并以内标法进行定量。维生素D和25-羟基维生素D的结果在0.2 - 50 μg/L范围内均呈现良好的线性，相关系数为0.9995 - 0.9999。检测限（LOD）和定量限（LOQ）分别在0.018 - 0.066和0.06 - 0.22 μg/kg范围内。在三个加标水平（0.20、1.0和5.0 μg/kg）下回收率为92.6% - 99.4%，相对标准偏差（RSD）为3.6% - 6.2%。这种高灵敏度方法对动物源性食品中维生素D和25-羟基维生素D的定量测定具有可重复性和准确性。
