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 me naquinone-4 and its metabolite menaquinone-4 epoxide in human perioste al osteoblasts. The method is based on high-performance liquid chromat ography/atmospheric pressure chemical ionization mass spectrometry, us ing deuterated menaquinone-4 as an internal standard. Selected reactio n monitoring (SRM) is employed for selectivity and sensitivity. This e nables quantification in a short period of time (within 5 min) over th e extended ranges 1-1000 ng/ml for menaquinone-4 and 2.5-5000 ng/ml fo r menaquinone-4, epoxide with acceptable precision and accuracy. The m ethod was used to determine both compounds in human osteoblasts, follo wing 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 gamma-carboxylation of vitam in K dependent proteins in both tissues. It is expected that this meth od will contribute to elucidating the pharmacological mechanism of act ion of menaquinone-4 on bone metabolism.