A VALIDATED HIGH-THROUGHPUT CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS DETERMINATION OF VITAMIN K HOMOLOGUES

Abstract

Vitamin K homologues are highly lipophilic compounds that require long separation times on chromatographic analysis which does not meet the demand of higher sample throughputs in quality control laboratories. Therefore, this study aimed to develop a new validated high-throughput high-performance thin-layer chromatographic (HPTLC) method to quantify vitamin K homologues including phylloquinone (PK, vitamin K₁), menaquinone-4 (MK-4, vitamin K₂), and menaquinone-7 (MK-7, vitamin K₂). The densitometric analysis was carried out using HPTLC silica gel G 60 F254 plates as the stationary phase. The plates were developed with methanol-ethanol-isopropanol-water (75:5:5:15, v/v/v/v) in the absorbance mode at 254 nm. The retention factors of MK-4, PK, and MK-7 were 0.56, 0.43, and 0.23, respectively. Linearity was found to be in the range of 1–200 ng band⁻¹ for PK and MK-4 and 2–200 ng band⁻¹ for MK-7 with correlation coefficient of 0.9990 or more. The limits of detection and quantitation were 0.19–0.85 and 0.76–2.5 ng band⁻¹, respectively. The method was validated in accordance ICH guidelines. The method was applied for determination of vitamin K homologues in pharmaceutical formulations and food samples after extraction without prior clean-up procedures. The developed HPTLC method provides a useful tool for rapid and efficient high-throughput analysis of vitamin K homologues.

Keywords:

• food analysis
• high-performance thin-layer chromatography
• high-throughput
• pharmaceutical analysis
• phylloquinone
• vitamin K homologues

Notes

^a Peak area ratio of vitamin K and internal standard versus concentration (ng band⁻¹).

^b Data presented as mean (±SD) of three experiments.

^a Values are mean of five determinations.

^b USP 2007.^{[ 34 ]}

^c JP XV.^{[ 35 ]}

^d Theoritical values at 95% confidence limit; t = 2.306, F = 6.388.

^a Average of five determinations ± SD.

^a Average of four determinations ± SD.

^b Reference.^{[ 30 ]}

^a Average of five determinations ± SD after subtracting the existed amount.