Fluorimetric analysis of five amino acids in chocolate: Development and validation

Abstract

Amino acids present ergogenic action, helping to increase, protect, and restore the mus-cular system of young athletes. Moreover, the encapsulation of five relevant amino acids in chocolate pellet form will appeal to them, facilitating their daily consumption. A reliable HPLC fluorimet-ric method was developed to detect and quantitatively determine L-Leucine, L-Isoleucine, L-Histi-dine, L-Valine, and β-Alanine in chocolate using aniline as an internal standard. Experimental design methodology was used to investigate and optimize the clean-up procedure of the samples. Therefore, three extraction techniques (solid-phase extraction (by two different SPE cartridges) and liquid–solid extraction (LSE)) were compared and evaluated. The LOQ values in chocolate varied from 24 to 118 ng/g (recovery 89.7–95.6%, %RSD < 2.5). Amino acids were pre-column derivatized with o-phthalaldehyde (OPA), while derivatization parameters were thoroughly investigated by experimental design methodology. The analysis was performed by HPLC-fluorescence (emission: λ = 455 nm, excitation: λ = 340 nm) method using a C18 column and a mixture of phosphate buffer (pH = 2.8; 20 mM)-methanol as a mobile phase in gradient elution. The method was validated (r2 >0.999, %RSD < 2, LOD: 10 ng mL−1 for histidine and leucine, 2 ng mL−1 for alanine and valine, and 4 ng mL−1 for Isoleucine) according to the International Conference on Harmonization guidelines.