Detecting chlorophylls and carotenoids utilizing a novel chromatographic method designed for HPLC-PDA-MS analyses and application to environmental and cultured algae and cyanobacteria

Abstract Chlorophylls and carotenoid analyses are important in aquaculture and freshwater and marine research as they are used to quantify algal biomass, food and supplement analyses, and to determine ecological diversity. The methods used for these analyses have often been limited to photometric detection, with some methods incorporating chromatographic separation. Mass spectrometric detection offers improved sensitivity, selectivity, and structural information compared to spectroscopy, but requires a full redesign of the separation system as previous chromatographic separations used solvent additives incompatible with mass spectrometry. The separation developed here used a YMC C30 “Carotenoid Column” with a novel mobile phase and elution scheme to separate a suite of algal pigments. Detection can be performed using a photodiode array alone, or in conjunction with mass spectrometry (MS). We report that the carotenoid echinenone (CAS 432-68-8) is actually an isomeric pair, which may have important implications for cyanobacterial photosynthesis and the orange carotenoid protein complex. New chlorophyll analogs, that could be hydroxylated or dehydroxylated derivatives, were detected in Caulerpa and Planktothricoides sp. BLCC-F182. This cyanobacterium produced novel chlorophylls, and it uniquely contained no echinenone or 3-OH echinenone. There was marked diversity in the types of pigments produced in the multiple strains analyzed, and the analyses demonstrated that there is likely to be more to discover in the pigments produced by algae and cyanobacteria. The inclusion of MS added significant value for the analysis of carotenoids and chlorophylls, and novel methodologies such as this may lead the field into important discoveries and advancements.