Quantification of Amino Acids in Fermentation Media by Isocratic HPLC Analysis of Their alpha-Hydroxy Acid Derivatives

In this paper we describe a novel method for quantification of amino acids. First, a-hydroxy acid derivatives of amino acids were formed after reaction with dinitrogen trioxide by the van Slyke reaction. Second, the a-hydroxy acid derivatives were separated on an Aminex HPX-87H column (Bio-Rad) eluted isocratically with 5 mM H2SO4 and quantified by refractive index detection. We were able to measure the reaction products of 13 of the 20 classical amino acids: glycine, L-alanine, L-valine, L-isoleucine, L-methionine, L-serine, L-threonine, L-asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, and L-proline. We obtained linear relationships between the product peak areas and initial amino acid concentration, whereby the concentrations of these amino acids could be quantified on the basis of the quantification of their products. The method can be used to analyze amino acids in parallel with other small molecules, such as sugars or short chain fatty acids, and was used for parallel quantification of glycine, L-alanine, or L-glutamic acid, and glucose uptake in cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii. The method can also be used to quantify other amines, as demonstrated by detection of Tris (2-amino-2-(hydroxymethyl)-propane-1,3-diol).