High Performance Liquid Chromatographic Determination of Free Amino Acids in Shrimp

A reverse phase high performance liquid chromatographic method for the analysis of total free amino acids in shrimp, utilizing pre column fluorescence derivatization is described. The primary amino acids were treated with o-phthalaldehyde (OPA). The reaction products were separated on a Microsorb Short-ones 3-μm reversed-phase column with gradient elution development. 15-OPA amino acids were separated in 21 min. Secondary amino acids were reacted with 4-chloro-7-nitrobenzofurazan (NBD). The separation was carried out on a Lichrosorb RP-C18, 5μm column. Wild and cultured Mexican shrimp species (Penaeus vannamei) were analyzed. Free amino acid contents of glycine, alanine and proline were higher than those of other amino acids. Total Free amino acid content was significantly higher in cultured than wild shrimp. The OPA-retention time could be measured within ±0.1 relative standard deviation and the relative peak areas, based on the internal standard calculation methodology, were within ±3% or less. The coefficient of variation of 18 kinds of amino acid samples were about 10%, the detection limit was 100 fmol.