A Fluorescent-Based HPLC Assay Using 4-Chloro-7-nitrobenzo-2-oxa-1, 3-diazole as Derivatization Agent for the Determination of Iron Bioavailability to Red Tide Phytoplankton

Abstract

A new fluorescent-based high performance liquid chromatography (HPLC) assay using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) was employed to determine iron (Fe) bioavailability to red tide phytoplankton in seawater. After growing four red tide species (Prymnesium parvum, Heterosigma akashiwo, Eutreptiella gymnastica, and Oltmannsiellopsis viridis) in f/2 artificial seawater under different Fe conditions, soluble extracts of the phytoplankton were derivatized using different fluorescent reagents (NBD-C1, 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole; NBD-F, fluorescamine, and ortho-phthalaldehyde; OPA) followed by HPLC assay. Among the four fluorescent reagents, NBD-C1 was most effective for derivatizing the phytoplankton extracts which would consist of proteins and peptides. HPLC chromatograms of the NBDderivatized extracts showed gradual changes (decrease/increase) of six peaks for different Fe conditions. Four of the peaks decreased, while two peaks increased with the increase of Fe concentrations in the culture medium. Considering the consistency and sensitivity of chromatogram peaks E and A to different Fe, phosphate and nitrate conditions for all phytoplankton studied, the ratio of these two peaks (I E/A ) has been proposed as the indicator of Fe bioavailability to red tide phytoplankton.