Rapid and efficient high-performance liquid chromatography-ultraviolet determination of total amino acids in protein isolates by ultrasound-assisted acid hydrolysis

Abstract

This study presents the optimization and validation of an ultrasound-assisted acid method for the HPLC-UV determination of amino acids in plant-based proteins. The research focuses on enhancing hydrolysis efficiency and reducing environmental impact. Ultrasound treatment significantly accelerated hydrolysis by creating cavitation, which increases local pressure and temperature, leading to faster reaction rates. The optimal condition was a 30-minute treatment at 90 °C with 6 M hydrochloric acid. The 9-fluorenylmethyloxycarbonyl chloride derivatization was best performed at pH 9.0 using borate buffer, ethanol as the organic solvent, and a 5-minute derivatization time with a 5 mM concentration. The method’s analytical performance, validated according to FDA guidelines, showed excellent selectivity, specificity, linearity (r² > 0.999), accuracy (recovery between 80–118 %), and precision (RSD<10.9). The analysis of 15 plant-based proteins revealed distinct amino acid profiles. Compared to traditional acid hydrolysis methods, the ultrasound-assisted approach demonstrated no significant difference in results (p-value > 0.05), confirming its reliability. The optimized ultrasound-assisted method is a reliable and efficient alternative for amino acid analysis, offering significant cost and time savings while maintaining high analytical performance. These findings are crucial for nutritional planning and developing functional foods to improve health outcomes.