An UFLC-DAD Method for the Quantification of Menaquinone-4 in Spiked Rabbit Plasma

Objective: The present study is aimed to develop and validate an Ultra-Fast Liquid Chromatography-Diode Array Detector (UFLC-DAD) method for the quantification of vitamin K2 as Menaquinone-4 (MK-4) in rabbit plasma. Method: Standard solutions and spiked plasma samples of MK-4 and Internal Standard (IS) were prepared from primary stock solutions of 1mg/ml concentration in ethanol each. Protein precipitation was carried out for the MK-4 and IS extraction from plasma spiked samples. Chromatographic separation was employed using Isopropyl Alcohol and Acetonitrile (50:50 v/v) as mobile phase and a C-18 column with 1ml/min flow rate and a run time of 10 mins. Detection was carried out in the range 190-600 nm with 269 nm set as a reference wavelength. Result The retention times of MK-4 and IS were at 5.5 +/- 0.5 mins and 8 +/- 0.5 mins respectively. Calibration curve for MK-4 was found to be linear in the range of 0.374 to 6 mu g/ml with an R-2 value of 0.9934. The % RSD for accuracy was <15%, inter and intraday precisions were <10%. The samples were found to be stable throughout the study. Conclusion: This method can be applied to the estimation of MK-4 in rabbit plasma using UFLC-DAD.