Spherisorb s3w column

Cardiac RDHs activity assay was performed as described by others^{41 (link)}, and the details of the experiment were as follows.
100 μg of cardiac microsomal fractions protein was incubated with 3 μM all-trans-retinol (17772, Sigma-Aldrich, St. Louis, MO) solubilized with bovine serum albumin and 1 mM NAD + (NAD98-RO, Sigma-Aldrich, St. Louis, MO) in 0.5 ml of the reaction buffer for 20 min at 37 °C. Reactions were stopped by the addition of an equal volume of ice-cold methanol, and Retinaldehyde were extracted twice with 2 ml of hexane. Hexane layers were dried, and the dry residue was reconstituted in 0.2 ml of acetonitrile. Retinaldehyde were separated by normal-phase HPLC using Spherisorb S3W column (4.6 mm × 100 mm; Waters) and isocratic mobile phase consisting of acetonitrile at 1 ml/min and analyzed.

Samples of subcellular fractions (100 μg of protein for mitochondrial and microsomal fractions, and 30 or 40 μg for LD) were incubated with 3 μM all-trans-retinol or all-trans-retinaldehyde (Toronto Research Chemicals, Toronto, Canada) solubilized with bovine serum albumin as described (63 (link)) and 1 mM NAD⁺ or NADPH (Sigma-Aldrich) in 0.5 ml of the reaction buffer for 15 min (MT and MS) or 20 min (LD) at 37 °C. Reactions were stopped by the addition of equal volume of ice-cold methanol, and retinoids were extracted twice with 2 ml of hexane. Hexane layers were dried, and the dry residue was reconstituted in 0.2 ml of hexane:ethyl acetate (90:10). Retinoids were separated by normal-phase HPLC using Spherisorb S3W column (4.6 mm × 100 mm; Waters) and isocratic mobile phase consisting of hexane:ethyl acetate (90:10) at 1 ml/min and analyzed as described (63 (link)).

Fifty µg of microsomal skin fractions was incubated with 3 µM all-trans-retinol (Toronto Research Chemicals, Toronto, Canada) solubilized with bovine serum albumin and 1 mM NAD⁺ (Sigma-Aldrich, St. Louis, MO) in 0.5 ml of reaction buffer for 15 min at 37 °C^{65 (link)}. Reactions were stopped by the addition of an equal volume of ice-cold methanol and retinoids were extracted with 2 ml of hexane. Hexane layers were dried, and dry residue was reconstituted in 0.1 ml of hexane:ethyl acetate (90:10). Retinoids were separated by normal-phase HPLC using a Spherisorb S3W column (4.6 mm × 100 mm; Waters, Milford, MA) and an isocratic mobile phase consisting of hexane:ethyl acetate (90:10) at 1 ml/min^{65 (link)}.