Fluorimetric Analysis of Five Amino Acids in Chocolate: Development and Validation

Amino acids present ergogenic action, helping to increase, protect, and restore the muscular 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 fluorimetric method was developed to detect and quantitatively determine L-Leucine, L-Isoleucine, L-Histidine, 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.