Varfaj Ina, Mancinelli Alice Cartoni, Migni Anna, Mercolini Laura, Castellini Cesare, Galli Francesco, Bartolini Desirée, Sardella Roccaldo
Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.
Department of Agricultural, Food and Environmental Sciences, University of Perugia, Perugia, Italy.
J Sep Sci. 2025 Jan;48(1):e70087. doi: 10.1002/jssc.70087.
The objective of this study is to develop an HPLC-UV method for the cost-effective and quantitative determination of vitamin D3 in food, even in the presence of vitamin D2, with a specific focus on egg yolk. During method development, the performance of three stationary phases in resolving the peak of vitamin D2 from that of vitamin D3 was investigated. The physicochemical properties of these phases differed particularly in the extent of hydrophobicity and silanophilic activity, including a GraceSmart RP C18 column without silanol endcapping, a Robusta RP C18 column with silanol endcapping, and a Waters Xbridge RP C18 column with ethylene-bridged hybrid (BEH) particle technology. The Xbridge C18 stationary phase exhibited the most favorable performance, leading to an R of 1.6 under the following nonaqueous reversed-phase (NARP) experimental conditions: mobile phase, acetonitrile, methanol, and trifluoroacetic acid in a (99/1/0.1, v/v/v) ratio; column temperature, 15°C. The developed chromatographic method does not require preanalytical purification steps and is also compatible with mass spectrometry. The identity of the vitamin D3 peak observed in the HPLC analysis was verified via GC-MS. The NARP-HPLC-UV method was partially validated, demonstrating satisfactory linearity, precision, accuracy, limit of quantification, and robustness. The HPLC method was then successfully applied to the analysis of real egg yolk samples, revealing average concentrations of vitamin D3 of 4-5 µg/g of wet weight sample.
本研究的目的是开发一种高效液相色谱 - 紫外检测法，用于经济高效且定量地测定食品中的维生素D3，即使存在维生素D2时也能测定，特别关注蛋黄中的情况。在方法开发过程中，研究了三种固定相在分离维生素D2和维生素D3峰方面的性能。这些固定相的物理化学性质在疏水性和硅醇活性程度上存在差异，包括未进行硅醇封端的GraceSmart RP C18柱、进行了硅醇封端的Robusta RP C18柱以及采用乙烯桥杂化（BEH）颗粒技术的Waters Xbridge RP C18柱。在以下非水反相（NARP）实验条件下，Xbridge C18固定相表现出最有利的性能，分离度R为1.6：流动相为乙腈、甲醇和三氟乙酸，比例为（99/1/0.1，v/v/v）；柱温为15°C。所开发的色谱方法不需要分析前的纯化步骤，并且也与质谱兼容。通过气相色谱 - 质谱联用（GC - MS）验证了高效液相色谱（HPLC）分析中观察到的维生素D3峰的同一性。对NARP - HPLC - UV方法进行了部分验证，结果表明该方法具有令人满意的线性、精密度、准确度、定量限和稳健性。然后，该HPLC方法成功应用于实际蛋黄样品的分析，结果显示湿重样品中维生素D3的平均浓度为4 - 5 μg/g。
