Fast and Efficient Separation of Eleven Mycosporine-like Amino Acids by UHPLC-DAD and Their Quantification in Diverse Red Algae.

Abstract

Due to their hostile habitats, characterized by a high exposure to UV-A and UV-B radiation, red algae are known to synthesize unique secondary metabolites: mycosporine-like amino acids (MAAs). These small molecules possess an extremely high UV absorption capacity and therefore mainly act as photoprotective agents. In this study, the first ultrahigh-performance liquid chromatography (UHPLC) method with diode-array detection (DAD) was developed for the determination of eleven MAAs in various algal species. All of the analytes could be separated in under 8 min on a Phenomenex Luna Omega C18 1.6 µm column. The mobile phase comprised water with 0.25% formic acid and 20 mM ammonium formate (A) and acetonitrile (B). Elution was carried out in gradient mode. Method validation following ICH guidelines confirmed excellent linearity (R2 ≥ 0.9998), selectivity, precision and accuracy (from 97.41 to 103.38%) for all analytes. The assay’s LOD was always 0.01 µg/mL; its LOQ was not higher than 0.04 µg/mL. Practical applicability was assured by analyzing several algae (e.g., Gracilaria chilensis, Pyropia plicata) using the developed method, and results indicated a high variation in MAA profiles as well as content. Whilst some MAAs were only found in specific samples, shinorine, which was always present, occurred in concentrations from 0.05 to 4.14 mg/g of dried biomass. As UHPLC-MS was also feasible, this method showed high flexibility concerning the detection mode, surpassing established procedures for MAA analysis not only concerning separation efficiency and analysis time.