Validation of an HPLC method for the determination of amino acids in feed

Abstract

The subject of this study was the validation of a high-performance liquid chromatography method for the analysis of amino acids in fodder. The contents of amino acids were determined in maize, soybean, soybean meal, as well as in their mixtures enriched with different amounts of methionine, threonine and lysine. The method involved the acid hydrolysis of the sample (6 h at 150 °C), automated derivatisation of the amino acids with the aid of o-phthaldialdehyde and 9-fluorenylmethyl chloroformate reagents, separation on a ZORBAX Eclipse-AAA column and detection using a diode-array detector. The method is characterized by high specificity (the difference between the retention times of the fodder samples and standard mixtures were below 1.7 %), wide linear range (from 10 to 1000 nmol cm-3, r2 = 0.9999), high accuracy (recovery 93.3-109.4 %), and the precision of the results (RSD below 4.14 % in the case of repeatability and below 4.57 % in the case of intermediate precision). The limit of detection and the limit of quantification were in the ranges 0.004-1.258 μg cm-3 and 0.011-5.272 μg cm-3, respectively. The results demonstrated that the procedure could be used as a method for the determination of the composition of primary amino acids of fodder proteins.