Methanol

Phospholipid extraction was performed with modifications to a previously published protocol^{47 (link)}. All extractions were performed under dim red light. Retina homogenates from 8-week-old strain 129/Sv mice or bovine OS were extracted by the addition of 3 ml of 1:2 (vol/vol) mixture of chloroform (Sigma) and acidified methanol (Fisher) (one L methanol + 8 µl trifluroacetic acid (Sigma)) followed by brief vortexing and incubation on ice for 10 min. Next, 1.3 ml of 0.3 M NaCl were added and the samples extracted twice into one ml chloroform with centrifugation at 1750 × g for 10 min at 10 °C to separate phases. The pooled chloroform layers were transferred to 16 × 100 mm borosilicate glass test tubes and evaporated to dryness under a stream of nitrogen. Samples were dissolved in 100 µl of acidified methanol and analyzed by reverse-phase LC^{48 (link)} on an Agilent 1100 series chromatograph equipped with a photodiode-array detector using an Phenomenex Primeshere C18-HC 110 A column (250 × 4.6 mm) and a 15–0% water gradient in acidified methanol (8 µl TFA/L methanol) at a flow rate of 1.0 (gradient) to 2.4 (isocratic) ml per min. Spectra (190–550 nm) were acquired for all eluted peaks. The identity of each eluted peak was established by comparing the spectra and elution times with those of authentic retinoid and N-ret-PE standards. Sample peaks were quantitated by peak area.