Preconcentration of Phenylalanine in Cheese and Yoghurt Using Deep Eutectic Solvent and Determination by HPLC Technique

Background: In the current research, a green, fast and cheap extraction method based on deep eutectic solvent was developed for the preconcentration of phenylalanine. Then, high performance liquid chromatography (HPLC) as a sensitive and accurate technique was used to determine amounts of preconcentrated phenylalanine. Objective: The combination of the HPLC technique and extraction procedure using deep eutectic solvent makes it possible to find a good procedure for the preconcentration of phenylalanine in different media with acceptable precision and accuracy. Methods: Decanoic acid and tetrabuthylammonium bromide were used as the components of the green solvent. Then, the synthesized green solvent was used for the preconcentration of phenylalanine. HPLC equipped with a UV-VIS detector, a C18 column, a mixture of sodium dihydrogen phosphate 2-hydrate (20 mM, pH 6.60)-ACN (70-30%) as the mobile phase, and the wavelength of 245 nm were selected as the best separation. Results: The chief characteristic of deep eutectic solvent was identified using Fourier transform infrared spectroscopy. Central composite design to evaluate the effects of preconcentration parameters showed that the pH value of 6.31, salt concentration of 0.272 M, DES volume of 226 μL, and stirrer time of 5.60 min were suggested as the best conditions of separation. Total analysis time was 5.50 min. Conclusion: Validation of designed analysis exposed good linearity (0.015-1.50 μg/mL), suitable sensitivity, excellent preconcentration (32.50) and enrichment (21.15) factors, acceptable relative standard deviation (3.07%), and low amounts of detection limit (0.015 μg/mL). Finally, the designed method was effectively used for the determination of phenylalanine in cheese and yoghurt with relative recoveries of 113.33 to 125.00% and 94.00 to 100.00%, respectively.