RATIONALEDue to their health benefits, there is growing interest in the production and use of carotenoids from natural sources, e.g. microalgae. To date, only Haematococcus pluvialis and Dunaliella, that accumulate, respectively, astaxanthin and β‐carotene in large quantities, are grown commercially. However, interest is also being focused on other xanthophylls, such as (all‐E)‐fucoxanthin characterized by anti‐obesity and anti‐carcinogenic effects. In this regard, rigorous chemical and analytical techniques following preparative isolation of components are needed to unequivocally identify individual carotenoids in microalgae.METHODSThe carotenoid profile of Isochrysis sp. biomass, produced in closed photobioreactors, was determined by reversed‐phase C30 (RP‐30) high‐performance liquid chromatography coupled with diode‐array detector mass spectrometry using positive electrospray ionization (HPLC/DAD‐MS/ESI+) analysis. Additionally, multistage mass spectrometry (MSn) analyses, together with fine structures of the UV–vis spectra, were used to differentiate structural and geometrical isomers.RESULTSThis technique allowed the simultaneous determination of geometrical, isomers of fucoxanthin (all‐E‐fucoxanthin, 13Z‐, 13'Z‐ and 9'Z‐fucoxanthin), diatoxanthin and 5,8‐epoxydiadinoxanthin diasteroisomers (R/S). The analyzed extracts contained fucoxanthin isomers as the major carotenoids and, in particular, (all‐E)‐fucoxanthin was the main geometrical isomer (~̴ 85%) found at a concentration of 17 mg/g of the lyophilized biomass.CONCLUSIONSConsidering the high content of fucoxanthin in Isochrysis sp. biomass, the microalga could be proposed as a source of this compound for nutraceutical and pharmaceutical applications. Copyright © 2013 John Wiley & Sons, Ltd.