Development and application of a simple, high-throughput magnetic bead-assisted liquid chromatography-tandem mass spectrometry method for the quantification of vitamin K1 and vitamin K2 (menaquinone-4 and menaquinone-7) in human serum.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the preferred method for vitamin K quantification, yet conventional workflows are labor-intensive and scale poorly. We developed and validated a simple, high-throughput automated magnetic bead-assisted LC-MS/MS method for simultaneous quantification of vitamin K1 (VK1), menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human serum according to Clinical and Laboratory Standards Institute (CLSI) C62 (2nd ed.) guidelines. This method used MSi050/PLS magnetic beads (5 mg/mL) for extraction via methanol loading and acetonitrile elution, requiring only 100 μL of serum to complete sample pretreatment within 17 min. All three calibration curves were linear from 0.20 to 10.00 ng/mL (r > 0.99), with a lower limit of quantification of 0.10 ng/mL. Total coefficients of variation for VK1, MK-4, and MK-7 ranged from 1.98% to 3.54%, 2.53% to 3.73%, and 2.77% to 4.59%, respectively. The recoveries of VK1, MK-4, and MK-7 were 94.8% to 112%, 98.0% to 113%, and 90.3% to 106%, respectively. Both matrix effects and carryover met CLSI acceptance criteria. Because vitamin K2 is photosensitive, all samples were processed under light-protected conditions. Unextracted samples were stable for 14 days at -20 °C, and extracted samples were stable for 48 h at 4 °C. Additionally, our method was applied to 245 healthy volunteers to characterize serum VK distribution profiles. By integrating automated magnetic bead extraction with rapid LC-MS/MS detection, this robust strategy markedly improves analytical throughput and reproducibility while maintaining high accuracy, offering a practical solution for large-scale clinical vitamin K assessment.