A high-resolution reverse-phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms

Abstract

Mycosporine-like amino acids (MAAs) are a group of about 20 structurally related water-soluble compounds, widely distributed among freshwater and marine organisms. To provide a better assessment of the diversity and concentration of MAAs in aquatic environments a high-performance liquid chromatography (HPLC) method of analysis based on reverse-phase C18 column and trifluoroacetic acid and an ammonium-containing mobile phase was developed. The improvements with respect to previous methods and the extraction and clean-up procedures are described here. With this method the clean-up recovery of MAAs of high polarity (shinorine), medium polarity (palythinol), and low polarity (palythene) is greater than 99% (±1%). The method is selective enough to resolve in a single run most of the characterized MAAs found in marine organisms, including the critical and highly polar compounds shinorine, mycosporine-2-glycine, and palythine-serine, the medium polarity pair palythenic acid and shinorine methyl ester (M-333), and the low polarity isomeric pair usujirene and palythene. A chromatogram of a mixture of over 20 MAAs such as might be found in complex samples of marine organisms is given. Good precision was obtained in the separations. The relative standard deviation for retention times was below 1% and the mean relative standard deviation for integrated area estimations was below 2%. A mean column recovery of standards was 99% (±1%) whereas limits of detection (signal-to-noise, S/N=2) for different MAAs varied between 0.08 and 0.47 pmol injected. The applicability of the method was tested using extracts of three microalgae cultures, three natural phytoplankton populations, two scleractinian corals, and one species of sea anemone. Results reveal the occurrence of several unknown MAAs not previously reported in the literature. The selectivity of the method toward some recently discovered MAAs makes it especially suitable not only for studying new field samples, but also for re-examining the MAA composition of previously studied organisms.