Electrospray Tandem Mass Spectrometric Analysis of Macular Pigment Oxidation Products in Human Eyes

Abstract

Abstract: : Purpose: Carotenoids have been implicated in protection of the eye from light mediated photo–toxicity caused by free radicals. Under conditions of normal oxidative stress the carotenoids serve as protective antioxidants, however, when the oxidative stress exceeds the antioxidant capacity, carotenoids can be oxidized into numerous cleavage products. The determination and identification of oxidized carotenoids in biological samples remains a major challenge due to the small sample size and low stability of these compounds. We investigated the reaction of various zeaxanthin cleavage products with O–ethyl hydroxylamine to evaluate their levels in a biological sample. Methods: For this, a sensitive and specific electrospray tandem mass spectrometry (ESI–MS/MS) was developed, avoiding the classical lower sensitive and specific HPLC–UV and fluorescence absorption methods. Protonated molecular ions [M+H]⁺ of carotenoids upon collision induced dissociation produced a number of structurally characteristic product ions. Results: A series of complicated clusters of product ions differing in 14 (CH₂) and 26 (C₂H₂) Da was characteristic of the polyene chain of intact carotenoids. All carotenoid ethyl oximes of zeaxanthin cleavage products were characterized by the losses of 60 and 61 Da in their MS/MS spectra. Through the application of the LC/MS/MS method, we identified two oxime derivatives of 3–hydroxy–ß–ionone and 3–hydroxy–14’–apocarotenal with protonated molecular ions at m/z 252 and m/z 370 in human eye samples, and increased levels were found in human Age–related Macular Degeneration (AMD) eyes.Conclusions: Oxidation products of macular pigment are present in the human macula and may play a role in the patho–biochemistry of AMD.

Keywords: macular pigment • age-related macular degeneration • oxidation/oxidative or free radical damage