Simultaneous determination of all-trans, 9-cis, 13-cis retinoic acid and retinol in rat prostate using liquid chromatography-mass spectrometry

Since retinoic acid (RA) and RA receptors are key developmental regulators during organogenesis, they might participate in the abnormal development of the prostate caused by early estrogen exposure. In order to test this assu mption, a sensitive analytical method that can differentiate 9-cis, 13-cis, and all-trans RA in small tissue samples (similar to8 mg) is required. Sin ce retinol is the metabolic precursor to RA, simultaneous quantification of retinol would also provide valuable information. Here, we report a liquid chromatography-mass spectrometry method for simultaneous determination of r etinol and 9-cis, 13-cis, and all-trans RA in rat prostate. Mass spectromet ric signal responses for RA were compared using positive ion atmospheric-pr essure chemical ionization (APCI) and electrospray, as well as positive ion and negative ion APCI. Positive ion APCI was selected for all subsequent a nalysis for its better sensitivity, and to provide simultaneous determinati on of retinol and RA. Ventral prostate tissue samples were homogenized and extracted following simple protein precipitation without derivatization. Ba seline separation of 9-cis, 13-cis, and all-trans RA standards was obtained by using a non-porous silica C-18 column. Selected ion monitoring of the i ons m/z 301 and m/z 269 was carried out for mass spectrometric quantitative analysis. The ion of m/z 301 corresponded to the protonated molecule of RA , whereas the ion of m/z 269 corresponded to loss of water or acetic acid f rom the protonated molecule of retinol or the internal standard retinyl ace tate respectively. The method has a linear response over a concentration ra nge of at least three orders of magnitude. The limit of quantitation was de termined to be 702 fmol all-trans RA injected on-column. The method showed excellent intra- and inter-assay reproducibility and good recovery, and is suitable for analyzing RA and retinol in small tissue samples (similar to8 mg). Copyright (C) 2001 John Wiley & Sons, Ltd.