Briefly, isolated RPE-choroid complexes were processed in 80% cold methanol via homogenization and metabolites were analyzed using Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex, Framingham, MA, USA). Chromatographic separation was achieved using ACQUITY UPLC BEH Amide analytic column (2.1×50 mm, 1.7 μm, Waters) with mobile phase A (water with 10 mM ammonium acetate with pH 8.9) and mobile phase B (acetonitrile/water (95/5) with 10 mM ammonium acetate with pH 8.2) (All solvents were LC-MS Optima grade from Fisher Scientific, Pittsburgh, PA, USA). Chromatographic separation was completed in a total run time of 11min with a gradient as previously described 20 (link), 21 (link). Multiple reaction monitoring was performed for 97 transitions listed in Table S3. MultiQuant 3.0.2 software (AB Sciex, Framingham, MA, USA) was used to integrate the extracted peaks and volcano plots were used to analyze the data. Different metabolites and enriched metabolic pathways were identified by MetaboAnalyst software.