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)