Simultaneous Determination of Menaquinone-4 and Its Metabolite in Human Osteoblasts by High-Performance Liquid Chromatography/Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry

A simple and sensitive method is described for the determination of menaquinone-4 and its metabolite menaquinone-4 epoxide in human periosteal osteoblasts. The method is based on high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry, using deuterated menaquinone-4 as an internal standard. Selected reaction monitoring (SRM) is employed for selectivity and sensitivity. This enables quantification in a short period of time (within 5 min) over the extended ranges 1 - 1000 ng/ml for menaquinone-4 and 2.5 - 5000 ng/ml for menaquinone-4 epoxide with acceptable precision and accuracy. The method was used to determine both compounds in human osteoblasts, following incubation with menaquinone-4. It is suggested that menaquinone-4 is metabolized to menaquinone-4 epoxide in bone as well as in liver, indicating the drug has the same role in γ-carboxylation of vitamin K dependent proteins in both tissues. It is expected that this method will contribute to elucidating the pharmacological mechanism of action of menaquinone-4 on bone metabolism.