An improved protocol for determining ratios of retinol-d4 to retinol isolated from human plasma

An improved protocol for assessing vitamin A status in humans using stable isotope dilution is described. Human subjects were given an oral dose of retinyl-d4 acetate, and blood specimens were collected after a period of pseudoequilibration. After the precipitation of proteins, the plasma was extracted with hexane and partitioned against acetonitrile. Retinol was isolated from the acetonitrile fraction using HPLC, and the retinol was converted to its tert-butyldimethylsilyl ether and analyzed by gas chromatography/mass spectrometry using selected-ion monitoring. The new method allows for more rapid isolation of retinol from plasma with fewer interferences than were seen in our earlier work. Significant improvements in the reliability, sensitivity, and precision of the method have been obtained in the present study, and these improvements should allow for the use of smaller doses of isotopically labeled vitamin A for evaluation of the vitamin A status of human populations.