3-Hydroxy-beta,epsilon-carotene-3'-one (3'-oxolutein) is the major oxidative metabolite of dietary carotenoids in the retina of the human eye. Elucidating the biochemical mechanism of its formation may provide helpful insight into the pathogenesis of age-related macular degeneration; however, it is found in relatively low quantities that require highly sensitive methods for quantitation from individual retinas. Normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry allowed us to do quantitative analysis of 3'-oxolutein from central and peripheral retinas obtained from individual human donors. The limit of quantification for 3'-oxolutein in human retina at a signal-to-noise ratio of 10 was 6 pg. The precision of the assay yielded a coefficient of variation ranging from 4.7 to 7.4% and accuracies of 106-108%. A statistically significant (R = 0.99, p < or = 0.001) linear working range was achieved between 5 and 7200 pg. The 3'-oxolutein contents from 8-mm punches of the central macula and peripheral retina were found to be 375+/-192 and 191+/-95 pg/tissue, respectively.