Determination of eleven total amino acids including cyst(e)ine by HPLC-DAD/FLD in complete dry and wet pet foods and their feed materials

Abstract

A single-step analytical method for the simultaneous determination of eleven amino acids (Arginine, Histidine, Threonine, Tyrosine, Cystine, Valine, Methionine, Phenylalanine, Isoleucine, Leucine, Lysine) materials was developed and validated in complete pet food and feed. Samples were hydrolysed, purified using a polytetrafluorethylene (PTFE) syringe filter, and then analysed by high-performance liquid chromatography (HPLC), equipped with photodiode array (DAD) and fluorometric (FLD) detectors, after an online pre-column derivatisation with o-phthalaldehyde. The use of two detection systems allows the simultaneous identification and quantification of different amino acids by a single chromatographic with high sensitivity and specificity. The chromatographic separations were carried out using an Advance Bio AAA column under a gradient mode with a mobile phase of acetonitrile, methanol, water (45:45:10), and a phosphate buffer solution. Linearity, precision, recovery, specificity, limit of detection (LOD) and limit of quantification (LOQ) were assessed. Linearity was evaluated over the range of 4–250 µM with diode array detector and 4–100 µM with the fluorometric detector. Method validation was carried out using certified reference materials; each analyte recovery ranged from 71% to 111% with a precision (CV%) less than 5%. Ninety one samples (ie, n = 30 dry and wet complete pet foods and n = 61 feed materials) were analysed using the validated method to define the nutritional quality of feed materials and to verify the completeness of complete pet foods for dogs and cats. All complete pet food samples complied with the minimum recommended levels of amino acids.