HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ELECTROSPRAY MASS-SPECTROMETRYOF RETINOIDS

High-performance liquid chromatography (HPLC)-electrospray mass spectr ometry (LC-MS) was used to analyze vitamin A-active retinoids includin g retinoic acid, retinol, retinal, and retinyl acetate. Unlike previou s LC-MS methods such as negative ion electron capture chemical ionizat ion, no derivatization of retinoic acid was required, HPLC separations were carried out on a C-30 reversed phase column with gradient elutio n using mobile phases containing water, methanol, and methyl-tert-buty l ether, Ammonium acetate (5 mM) was added to the mobile phase to faci litate ion pair formation during reversed phase HPLC of retinoic acid, and acetic acid (0.5% v/v) was added to the mobile phase to enhance p rotonation during LC-RIS analysis of nonacidic retinoids, During negat ive ion electrospray, retinoic acid formed abundant deprotonated molec ules, [M-H](-), of m/z 299 without significant fragmentation, Although retinol, retinal, and retinyl acetate did not ionize during negative ion electrospray, the positive ion electrospray mass spectra of these retinoids showed an abundant protonated molecule of m/z 285 for retina l and base peaks of m/z 269 corresponding to elimination of water or a cetic acid from the protonated molecules of retinol or retinyl acetate , respectively. No ions from retinoic acid were detected during positi ve ion electrospray. Limits of detection for retinoic acid, retinal, r etinol, and retinyl acetate were 23 pg, 1.0 ng, 0.5 ng, and 10 ng, res pectively.