Retinoïds were extracted using the method described by Kim and Quadro (2010) (link) and quantified by reverse-phase HPLC from isolated chylomicrons. Briefly, 100 μL of an 1 μg.μL−1 retinyl acetate internal standard was added to 500 μL of each sample of isolated chylomicrons. The samples were then extracted with 4 ml hexane. Hexane fraction was recovered by centrifugation and extracted with 500 μL deionized water. After recovery, the hexane fraction was dried under vacuum at room temperature, reconstituted with 200 μL HPLC mobile phase and injected into the HPLC system.
The HPLC system was equipped with a Waters Spherisorb 5 μm ODS2 4.6 × 250 mm analytical column (Waters, Millford, MA, United States) and a Gilson UV/Vis-155 detector set at 325 nm (Gilson, Middleton, WI, United States). The mobile phase consisted in acetonitrile, methanol and methlyenechloride (70 : 15 : 15 v/v) at a flow rate of 1.8 ml.min−1. Based on the internal standard quantification, recovery was >98%. Retinol and retinyl palmitate were quantified from calibration curves that exhibited the same linear correlation factors of R2 = 0.997. Their respective detection limits were determined to be 1.95 and 3.91 ng per injection.
The HPLC system was equipped with a Waters Spherisorb 5 μm ODS2 4.6 × 250 mm analytical column (Waters, Millford, MA, United States) and a Gilson UV/Vis-155 detector set at 325 nm (Gilson, Middleton, WI, United States). The mobile phase consisted in acetonitrile, methanol and methlyenechloride (70 : 15 : 15 v/v) at a flow rate of 1.8 ml.min−1. Based on the internal standard quantification, recovery was >98%. Retinol and retinyl palmitate were quantified from calibration curves that exhibited the same linear correlation factors of R2 = 0.997. Their respective detection limits were determined to be 1.95 and 3.91 ng per injection.
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