DETERMINATION OF VITAMINS E, D3, AND K1 IN PLASMA BY LIQUID CHROMATOGRAPHY-ATMOSPHERIC PRESSURE CHEMICAL IONIZATION-MASS SPECTROMETRY UTILIZING A MONOLITHIC COLUMN

This paper reports a rapid, simple, and sensitive method for determination of vitamin D3, vitamin E acetate, and vitamin K1 in plasma using atmospheric pressure chemical ionization -high performance liquid chromatography-mass spectrometry. Plasma samples were prepared using solid phase extraction. The separation of compounds was achieved using a C-18 monolithic column and a mobile phase composed of methanol and 0.1% formic acid in gradient elution mode at a flow rate of 1.0mLmin(-1). Analytes were ionized using atmospheric chemical ionization in positive mode. Mass spectra were recorded at m/z=385.23, 473.47, and 451.41 for vitamin D3, vitamin E, and vitamin K1, respectively. Vitamin D2 was used as an internal standard and its mass spectra was recorded at 397.28 m/z. The method was validated using ICH guidelines. The system suitability responses were calculated for retention time, number of theoretical plates, capacity factor, resolution, and the selectivity factor. System validation was evaluated for precision, specificity, and linearity of all compounds. The limits of detection for vitamin D3, vitamin E, and vitamin K1 were determined to be 0.1, 1.36, and 0.052ngmL(-1), respectively. The accuracy, evaluated as % of recovery, was in the range of 96.4 to 102.4% and precision determined as the coefficient of variation was between 1.24 and 3.6%. The validated method was applied to real plasma samples.