A Novel Fast HPLC-UV Method for Menaquinone-7 Determination in Fermentation Broth

Menaquinone-7 (MK-7) plays a key role in bone and cardiovascular health, and it is often included in multivitamin supplements and functional foods. The aim of this study is the development and validation, according to ICH guidelines, of a fast, highly sensitive, and reliable reverse-phase HPLC-UV quantification of vitamin K₂ as MK-7 in a common biological matrix sample, such as fermentation broth medium from Bacillus subtilis, with the advantage of single-step extraction and short analysis time. MK-7 was successfully isolated from the fermentation broth and its interfering residues by thermo-acidic extraction using 5% H₂SO₄ and ethanol, without derivatization, and was retained using a C8 column. The isocratic elution and UV detection at 268 nm within a 3-min run time allowed quantification over a wide accuracy range of 0.10–18.00 μg/mL, with an LOQ value of 0.10 μg/mL and an LOD value of 0.03 μg/mL. The method was found to be precise, accurate, and reliable, with RSD% lower than 5% and recoveries varying between 96.0% and 108.9%. The proposed method unlocks the opportunity to be suitable for routine analysis in the screening of bacterial strains producing MK-7, as well as to be successfully employed in manufacturing processes in the nutraceutical and food industries where complex matrices are involved.