High-performance liquid chromatography-electrospray mass spectrometry of retinoids.

Abstract

High-performance liquid chromatography (HPLC)-electrospray mass spectrometry (LC-MS) was used to analyze vitamin A-active retinoids including retinoic acid, retinol, retinal, and retinyl acetate. Unlike previous LC-MS methods such as negative ion electron capture chemical ionization, no derivatization of retinoic acid was required. HPLC separations were carried out on a C30 reversed phase column with gradient elution using mobile phases containing water, methanol, and methyl-tert-butyl ether. Ammonium acetate (5 mM) was added to the mobile phase to facilitate 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 protonation during LC-MS analysis of nonacidic retinoids. During negative ion electrospray, retinoic acid formed abundant deprotonated molecules, [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 retinal and base peaks of m/z 269 corresponding to elimination of water or acetic acid from the protonated molecules of retinol or retinyl acetate, respectively. No ions from retinoic acid were detected during positive ion electrospray. Limits of detection for retinoic acid, retinal, retinol, and retinyl acetate were 23 pg, 1.0 ng, 0.5 ng, and 10 ng, respectively.