Sequential Extraction and Purification of 50 Distinct Carotenoids from Aquatic Organisms

Abstract

A system of sequential extraction and purification, coupled with ultra-performance liquid chromatography coupled with quadrupole electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS), was established and subsequently applied to the analysis of common carotenoids with very different physicochemical properties from aquatic organisms. In the first stage of extraction, free carotenoids were extracted with methanol (0.1% butylated hydroxytoluene (BHT)) and purified using hydrophile-lipophile balance column (HLB). In the second stage of extraction, extremely hydrophobic carotenoid esters were mainly extracted with methanol-dichloromethane (0.1% BHT) and purified using Alumina-N. In total, nearly 50 distinct carotenoid species were identified from common aquatic organisms by combining first-stage and second-stage solutions with recoveries of no less than 86.9%, which contained 25 free carotenoids and 24 carotenoid esters, respectively. Additionally, astacin diester 22:6/16:0, oxidized-astaxanthin diester 22:6/16:0, and adonixanthin diester 22:6/20:5 were identified for the first time from Penaeus monodon. Our results show that this methodology was a significant improvement over other alternatives for analyzing carotenoids because of its compatibility with free carotenoids and carotenoid esters and provides a technical guarantee not only for enriching carotenoid species but also for the tracing of metabolic pathways in aquatic organisms.