This study aimed to develop a simultaneous analysis method for cis/trans MK-7 and MK-6 to manage specifications when listing vitamin K₂ (trans MK-7) as a nutritional ingredient for health functional food. Dimethyl sulfoxide was chosen as the extraction solvent due to its compatibility with various dosage forms, including powders, capsules, and microcapsules. To improve the cis/trans selectivity of MK-7, we used the YMC Carotenoid C₃₀ column and optimized the wavelength range (268 nm). The method was validated in terms of specificity, linearity, limits of detection and quantification (LOD/LOQ), accuracy, and precision. High specificity and linearity were demonstrated, with correlation coefficients of 0.999 for MK-6 and 0.998 for trans MK-7 within the linear quantification ranges. The LOD/LOQ values were 0.5/1.5 for MK-6 and 0.7/2.2 for trans MK-7. The recovery rate of MK-6 and trans MK-7 were 96.8∼106.0%, 95.6∼101.5%, respectively. The relative standard deviations (RSDs) of repeatability were 3.046% for MK-6, and 2.254% for trans MK-7, respectively. Reproducibility was assessed using an inter laboratory trial which showed high reproducibility across three laboratories with RSD below 3%. To confirm the applicability, the labeled amounts of the distributed products were monitored. The recovery of trans MK-7 in all the products was more than 80% of the labeled amounts, whereas the ratio of cis MK-7 and MK-6 was within 2% and 3%, respectively. Thus, the new method is expected to increase consumer confidence by appropriately managing the specification of health functional foods containing vitamin K₂.

Keywords: health functional food, vitamin K₂, MK-6, trans MK-7