Mouse eyecups (2–4 eyes/sample as indicated) were homogenized, extracted in chloroform/methanol (2:1), and analyzed for bisretinoids (A2E, iso-A2E, A2-DHP-PE, atRAL di-PE) by reversed-phase HPLC using an Alliance System (Waters, Milford, MA) and Atlantis dC18 column or Waters Acquity UPLC-MS system and Acuity BEH phenyl column (A2GPE) (Waters, Milford, MA) as described (22 (link)). Molar quantities per eye were calculated by comparison with synthesized standards. The pyridinium bisretinoid A2E and its cis isomer, iso-A2E, were measured separately and summed (A2E/iso-A2E).
For retinoid quantification, mouse eyecups (1 eye/sample) were homogenized and derivatized using O-ethylhydroxylamine (63 (link)). Retinal O-ethyloxime was extracted with hexane and resuspended in acetonitrile. The Waters Acquity UPLC-MS system was used with a CSH C18 column (1.7 μm, 2.1 × 100 mm; Waters, Milford, MA) and gradients of water (A) and acetonitrile (B) with 0.1% of formic acid as follows: beginning at 60% B, holding for 5 min, followed by a linear increase to 70% B over 55 min, followed by a linear increase to 100% B over 10 min and holding for 20 min (flow rate of 0.3 ml/min) (64 (link)).
For retinoid quantification, mouse eyecups (1 eye/sample) were homogenized and derivatized using O-ethylhydroxylamine (63 (link)). Retinal O-ethyloxime was extracted with hexane and resuspended in acetonitrile. The Waters Acquity UPLC-MS system was used with a CSH C18 column (1.7 μm, 2.1 × 100 mm; Waters, Milford, MA) and gradients of water (A) and acetonitrile (B) with 0.1% of formic acid as follows: beginning at 60% B, holding for 5 min, followed by a linear increase to 70% B over 55 min, followed by a linear increase to 100% B over 10 min and holding for 20 min (flow rate of 0.3 ml/min) (64 (link)).