Improved HPLC-method for estimation and correction of amino acid losses during hydrolysis of unknown samples

Abstract

Amino acid analysis, commonly done by acid hydrolysis of proteins and HPLC analysis, faces one major problem: incomplete hydrolysis of stable amino acids and degradation of unstable amino acids are causing amino acid losses. As a result, amino acid recovery of unknown samples cannot be estimated. Some methods have been reported for correction of these factors in the past. This paper shows an improved and integrated method to overcome this problem by using stillage as an exemplary unknown sample material. Amino acid recovery from an unknown sample can be estimated by standard addition of a known protein. If the sample does not cause matrix effects during amino acid hydrolysis, recoveries of the standard protein are transferable to the sample. If the sample does cause matrix effects correction of amino acid losses can instead be done by determination of hydrolysis kinetics. Therefore, first order kinetics were used for amino acids that undergo degradation during hydrolysis. For all stable amino acids higher order kinetics were used, a novel approach to determine hydrolysis kinetics. The presented method can be a helpful tool for scientists who want to optimize amino acid analysis of a particular biomass substrate.

Keywords: Amino acid analysis; HPLC; Hydrolysis kinetics; Matrix effects; Non-linear least-squares regression.