Magnetic bead-assisted extraction combined with ID-LC-MS/MS for simultaneous quantification of fat-soluble vitamins A, D, E, and K in human serum.

This study developed and validated a high-throughput method based on automated magnetic solid-phase extraction (MSPE) with isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the simultaneous and accurate quantification of five fat-soluble vitamins (VA, 25(OH)D3, 25(OH)D2, VE, and VK1) in human serum. For the MSPE, a functionalized silica-based magnetic core-shell material was synthesized, featuring a poly (polystyrene-co-divinylbenzene-co-N-vinylpyrrolidone) coating and a covalently immobilized 2-vinylbenzofuran affinity ligand linked via a hydrophilic polyethylene glycol spacer. This design facilitated efficient enrichment and purification of the target analytes. The validated method demonstrated excellent specificity, with complete chromatographic separation, without significant matrix effects or interferences. It exhibited superior sensitivity and linearity, with a satisfactory linear response (R2 > 0.995 for all analytes) and a low limit of quantification (0.15 ng/mL-0.49 μg/mL). Recoveries were between 90.48 % and 101.95 %, while precision was satisfactory with intra-day and inter-day coefficients of variation (CV) below 7.14 % and 4.68 %, respectively. The accuracy of the method was further demonstrated by analyzing NIST standard reference material SRM 968f and SRM 972a, with a mean bias within ±7 %. Compared to traditional liquid-liquid extraction (LLE) process, the automated MSPE process efficiently extracted the target analytes from serum within 11 min through systematic optimization. Furthermore, method comparison showed a good agreement between the clinical routine LLE method and the MSPE method established. In addition, the method performed well in external quality assessment schemes, also confirming high accuracy and reliability. This robust methodology provides a powerful analytical tool for the precise monitoring of fat-soluble vitamins and nutritional status assessment in clinical practice and has the potential to promote the development of laboratory automation.