UHPLC-based C-phycocyanin quantification method to support the validation of remote sensing models for harmful cyanobacterial blooms

The deployment of hyperspectral satellite missions has opened new opportunities for integrated approaches to address the escalating issue of cyanobacterial algal blooms in marine and inland waters. Despite these advancements, the validation of satellite data concerning C-phycocyanin (C-PC) content requires robust analytical methods. Currently, the available techniques, predominantly spectrophotometric or fluorometric, exhibit low reproducibility due to the interference of chlorophyll a and perform optimally with water samples. The samples collected during ship-based campaigns and commonly used for satellite data validation are concentrated samples on filters, which limiting the application of these methods. In this study, we aim to establish an Ultra High Pressure Liquid Chromatography (UHPLC) -based method for C-PC quantification in water samples concentrated on filters to validate satellite derived algorithm for monitoring cyanobacteria blooms. This involves the extraction of C-PC from pure algal cultures (Synechococcus spp. and Anabaena spp.) and natural samples fixed on filters and the quantification employing UHPLC analysis-a recognized gold standard for phytoplankton pigment analysis. Based on a Design of Experiment (DoE) full factorial design approach, we compared different extraction techniques. Subsequently, we compared different methods and developed and validated a rapid, simple, and sensitive reverse phase UHPLC method. The optimized chromatographic parameters for C5 phase, were acetonitrile with trifluoroacetic acid (0.1 % v/v) in a 5' linear gradient (from 20 % to 100 %), flowrate 0.8 mL min^-1, with an accuracy of 10 %, a reproducibility of 9.8 % and a limit detection of 0.025 mg L^-1.