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Danielle B. Gutierrez, Dan Higbee, Kevin L. Schey, Rosalie K. Crouch, Zsolt Ablonczy; Sensitive and Specific Mass Spectrometric Identification and Quantitation of A2E. Invest. Ophthalmol. Vis. Sci. 2011;52(14):902. doi: https://doi.org/.
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A2E is a major component of liposfuscin, an autofluorescent, complex mixture that increases with age in the retinal pigment epithelium (RPE). Currently, the standard method of identifying and quantitating A2E is HPLC separation followed by UV absorption detection; however, the specificity of this method for A2E is limited to absorption maximum and chromatographic retention time. The aim of this work was to develop and apply a mass spectrometric method for specific and sensitive identification and quantitation of A2E from tissue extracts.
A2E was chlorofrom-methanol extracted from the eyecups of ABCR-/-, RPE65-/-, and C57bl6 (wt) mice of various ages. Samples were analyzed via liquid chromatography tandem mass spectrometry (LC-MS/MS), using selected ion monitoring of A2E and its oxides during the MS scan. The amount of A2E present in a tissue extract was determined by comparing the area under the curve of the most intense fragment ion of A2E to a standard curve produced using various amounts (5-100 fmol) of synthesized A2E.
The mass spectrometric approach developed here for the analysis of A2E and its oxides provided unambiguous identification based on retention time, mass-to-charge ratio (m/z), and fragmentation pattern. Furthermore this method enabled identification and quantitation of low femtomole amounts of A2E. The levels of A2E detected from tissue extracts varied with each mouse model, with the highest levels found in the ABCR-/- model. As expected, no A2E was detected in the RPE65-/- model. Oxidized A2E was also detected in these experiments.
An approach has been developed that offers specific and sensitive identification and quantitation of A2E and its oxides. This approach has applications in determining changes in the levels and distributions of A2E in aging normal and diseased eyes.
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