Recently, new physiological roles of vitamin K homologues have been established in the treatment of
rheumatoid arthritis, osteoporosis, hepatocellular carcinoma and leukemia. However, relatively high
plasma protein binding, low plasma concentrations and occurrences of interfering lipids make accurate
determination of vitamin K homologues a challenging task. Therefore, a sensitive and reliable salting-out
assisted liquid/liquid extraction (SALLE) method coupled with HPLC-Fluorescence detection was de-
signed for efficient extraction and quantification of trace levels of vitamin K homologues in human
plasma. The investigated vitamin K homologues were phylloquinone (PK, vitamin K1), menaquinone-4
(MK-4) and menaquinone-7 (MK-7). The method employed a new efficient fluorescence derivatization
reaction using ethanolic solution of stannous chloride in acidic solution to generate highly fluorescent
naphthohydroquinone derivatives. Correlation coefficients were more than 0.998 in the concentration
ranges of 0.3–100 ng mLÀ1 with detection limits of 0.1–0.17 ng mLÀ1 in human plasma. The developed
HPLC-FL system was successfully applied for sensitive determination of vitamin K homologues in plasma
of healthy volunteers. The developed method may provide a valuable tool in the pharmacoinformatic
studies concerning the roles of vitamin K homologues.