Microwave Digestion and On-line Pre-Column Derivatization UHPLC Method for Analysis of Total Amino Acids in Feed (AD-0216)

Introduction

Analysis of total amino acid (AA) composition in feed is routinely performed in feed manufacture. This is because dietary amino acids in feed are crucial for lactation, growth, and health of pet and livestock. Animal diet needs amino acids as supplement, especially the essential AAs (histidine etc.) that cannot be synthesized within the animal body. Conventional HPLC analysis for determination of the total AA composition involves a tedious procedure. A feed sample is first pre-treated by acid hydrolysis (6N, HCl) to liberate AAs from proteins. Due to oxidation, tryptophan is destroyed during acid hydrolysis. Hence, alkaline hydrolysis is needed to assess tryptophan. In this study, a microwave digestion procedure was employed to carry out both acid and alkaline hydrolysis in less than 30 minutes, which is much faster in comparison to the conventional hydrolysis at 110oC for up to 24 hours. On-line pre-column derivatization of the amino acids obtained was adopted to further speed up and simplify the analysis using the automatic pre-treatment function of the autosampler. This on-line derivatization uses ophthalaldehyde (OPA) for primary amino acids and 9- fluorenylmethyl chloroformate (FMOC) for secondary amino acids to produce fluorescent substances. Fluorescence (RF) detection was used for nmol/L level of high sensitivity analysis of AA quantification whereas UV detection allowed µmol/L level of AAs quantification.

May 22, 2020 GMT