Evaluation of a performic acid oxidation method for quantifying amino acids in freshwater species

Abstract

Thiol amino acids in proteins store metals like mercury, but established methods for their quantitation in freshwater species have had limited application and evaluation. As such, literature on the amino acid composition of aquatic species often lacks the thiols cysteine and methionine. Here, we evaluated a performic acid (PFA) oxidation method to determine its suitability to measure cysteine and methionine, as well as 15 other amino acids, in novel matrices (zooplankton, benthic invertebrates, fishes, and algae). Protein-bound amino acids were oxidized with PFA, hydrolyzed in hydrochloric acid, derivatized with AccQ·Tagultra (Waters), and separated by ultra-high performance liquid chromatography with fluorescence detection. PFA oxidation was successful in determining precise results for 15 amino acids, including the sulfur amino acids, with the complete loss of tyrosine (TYR) and poor precision of phenylalanine (PHE). The overall variability of the method was 11% (excluding TYR and PHE), or 6% when reported as relative percentages, comparable to methods without PFA oxidation in other matrices. Except for TYR and thiols, PFA oxidation did not affect the amino acid composition of these biota. Overall, the findings were reproducible and comparable to other approaches and suggest this is a rigorous method for measuring sulfur amino acids and the overall amino acid composition for aquatic biota, both of which are rare in the literature.