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

Abstract

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.

Overview of chromatographic and electrophoretic methods for the determination of branched‐chain amino acids

“…In conclusion, a pre‐column derivatization is a crucial tool for analyzing BCAAs due to its ability to enhance detection sensitivity and its versatility in accommodating various sample types and detectors. Table 1 shows a compilation of 19 commonly used derivatization reagents, along with a summary of their respective advantages and disadvantages [33–55]. The different reactions with AAs are described in Figure 2.…”

In Vitro Permeability Study of Homotaurine Using a High-Performance Liquid Chromatography with Fluorescence Detection Pre-Column Derivatization Method

“…Under these conditions, one of the most important factor to be studied was pH (Table 2) in the range of 8.5-11.0. A value of 9 was chosen as Regarding the selection of the appropriate buffer, the use of a borate solution is recommended since, due to its high buffering capacity (pH 8.0-10.0), it prevents the ionization of secondary amino groups [21]. Under these conditions, one of the most important factor to be studied was pH (Table 2) in the range of 8.5-11.0.…”

“…A value of 9 was chosen as optimal since derivatization was assured and the chromatogram was improved (lower tailing factor). Then, the time required for the reaction was also critical [21] and was investigated between 0 and 30 min (Table 3). Based on the results, it was observed that the derivatization is completed immediately, and up to about 30 min, the HOM derivative is relatively stable.…”

“…Ortho-phthalaldehyde (OPA) is a widely used reagent for the derivatization of substances containing primary amino groups in their molecule, as it reacts rapidly and selectively (under alkaline conditions) to form strongly fluorescent derivatives. The reaction is conducted in the presence of a thiol-reducing reagent, such as 2-mercaptoethanol, urea and sodium sulfite [20], N-acetyl-L-cysteine [21] and 3-mercaptopropanoic acid [16]. In such cases, preliminary research is deemed necessary to select a stable and effective OPA derivative.…”