Determination of Five Retinol Isomers in Animal Livers Using Ultra-High Performance Supercritical Fluid Chromatography

Abstract

A novel and efficient ultra-high performance supercritical fluid chromatography method was developed for the quantitative analysis of five retinol isomers in animal livers. The separation of the five retinol isomers was carried out using an Acquity UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 µm) with acetonitrile as a co-solvent. By optimizing the columns, gradient program, co-solvent, column temperature and backpressure, the five retinol isomers and the internal standard 11-cis-13,14-dihydroretinol were successfully separated within 20 min. Samples were saponified and extracted by solid-supported liquid–liquid extraction using a diatomaceous earth cartridge. Comparing with the traditional liquid–liquid extraction, the extraction enables the reduction of time-consuming and laborious procedures. This method used 11-cis-13,14-dihydroretinol as an internal standard to improve the precision and accuracy of quantitative analysis. The correlation coefficients (r2) of the calibration curves were all above 0.999, the limits of detection for the five retinol isomers were in the range of 0.10–0.20 µg mL− 1, and the limits of quantification were in the range of 0.33–0.66 µg mL− 1. The mean recoveries were from 92.5 to 102.5%. The interday and intraday relative standard deviations were within 10%. This method was successfully applied to the determination of retinol isomers in ten raw animal livers and animal liver products (chicken, duck, pig, cattle, and sheep).