DEVELOPMENT OF A METHOD FOR QUANTITATION OF RETINOL AND RETINYL PALMITATE IN HUMAN SERUM USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ATMOSPHERIC-PRESSURE CHEMICAL-IONIZATION MASS-SPECTROMETRY

A method for the quantitative analysis of the vitamin A compounds all- trans-retinol and all-trans-retinyl palmitate was developed using high -performance liquid chromatography-atmospheric pressure chemical ioniz ation-mass spectrometry (APCI-LC-MS). Unlike previous quantitative mas s spectrometric methods for vitamin A, HPLC separations were carried o ut using a C-30,, reversed-phase column instead of GC separation. Beca use no sample hydrolysis or derivatization was necessary, retinyl palm itate was preserved for analysis instead of being hydrolyzed to retino l. Human serum was analyzed following simple hexane extraction without saponification or any additional purification. A comparison of APCI a nd electrospray ionization showed that only APCI produced a linear res ponse over all four orders of magnitude of retinol and three orders of magnitude of retinyl palmitate concentrations. Selected ion monitorin g of the fragment ion of m/z 269 was used for APCI quantitation of bot h retinol an; retinyl palmitate, since it was the base peak and the on ly abundant ion in the mass spectra of both compounds and the internal standard, retinyl acetate. The ion of mit 269 corresponded to loss of water, loss of palmitic acid, or elimination of acetic acid from the protonated molecules of retinol, retinyl palmitate and retinyl acetate , respectively. The limit of detection of APCI-LC-MS for all-trans-ret inol and all-trans-retinyl palmitate was determined to be approximatel y 34 fmol/mu l and 36 fmol/mu l (0.670 pmol all-trans-retinol and 0.72 0 pmol all-trans-retinyl palmitate injected in 20 mu l on-column), res pectively. The limit of quantitation was approximately 500 fmol/mu l a nd 250 fmol/mu l (10 pmol and 5 pmol injected in 20 mu l on-column) fo r retinol and retinyl palmitate, respectively; (C) 1998 Published by E lsevier Science B.V.