Measurement of cis and trans isomers of vitamin K1 in rat tissues by liquid chromatography with a C30 column

The purpose of this study was to validate a method for measuring vitamin K isomers in rat tissues by liquid chromatography (LC) with fluorescence detection after simple solvent extraction. This method uses separation on a C30 column, followed by zinc reduction and fluorescence measurement (243 nm, excitation; 430 nm, emission) to detect and quantitate vitamin K isomers. We were able to separate cis- and trans-vitamin K1 in methylene chloride extracts of homogenized rat livers and in hexane extracts of rat plasma. Tissue extracts were evaporated and rediluted with tetrahydrofuran-methanol (1 + 1) or methanol before being injected under isocratic conditions onto the LC column. Liver tissue of Fischer 344 rats fed a vitamin K1-containing diet ad libitum contained approximately 20 and 60 ng/g cis- and trans-vitamin K1, respectively. Mean recoveries of vitamin K1 isomers from spiked liver were 92 +/- 11% for cis-vitamin K1 and 106 +/- 5% for trans-vitamin K1. We recovered 96 +/- 8% of trans-vitamin K1 added at 1, 3, and 6 ng/mL to plasma (containing an endogenous level of 4 ng/g) from the same rats; we recovered 112 +/- 5% when trans-vitamin K1 was added to human serum (National Institute of Standards and Technology Standard Reference Material 968C). This direct method shows significant potential for the selective measurement of vitamin K1 isomers in tissues.