Carotenoids and apocarotenoids determination in intact human blood samples by online supercritical fluid extraction-supercritical fluid chromatography-tandem mass spectrometry.

A direct on-line method based on the coupling of supercritical fluid extraction and supercritical fluid chromatography with triple quadrupole mass spectrometry detection (SFE-SFC-QqQ/MS) for selected carotenoids determination and apocarotenoids detection in intact human blood was developed for the first time. Carotenoids and apocarotenoids were identified by using the available standard together with full scan, selected ion monitoring (SIM), and multiple reaction monitoring (MRM) experiments. Moreover, beta-Cryptoxanthin, Zeaxanthin, beta-Carotene and Capsanthin were directly quantified by the developed methodology, using a multiple reaction monitoring (MRM) approach; the determined average content of beta-carotene was 123.8 nmol L-1 (range 18.7-485.1 nmol L-1), of beta-cryptoxanthin was 385.3 nmol L-1 (range 72.5-1920.3 nmol L-1), of zeaxanthin was 396.9 nmol L-1 (range < LoD - 1795.8 nmol L-1) and of capsanthin was 38.9 nmol L-1 (range < LoD - 188.4 nmol L-1). Analyses were carried out on 10 mu L aliquots of intact blood samples without any preliminary treatment; the online extraction and chromatographic separation time was just over 20 min. The method was validated in terms of linearity, precision, limits of detection and quantification, and accuracy. Interestingly, beta-apo-120-carotenal, apo100- zeaxanthinal, apo-120-zeaxanthinal, apo-140-zeaxanthinal, epsilon-apo-8-luteinal, epsilon-apo-12-luteinal and epsilon-apo-14-luteinal were detected in human blood, together with two zeaxanthin fatty acid esters, for the first time.