Development of amino acids identifying method by UPLC-PDA in mixture solution for MMR vaccine production

Abstract

Objective: Development of an analytical UPLC method to identify each one of 15 amino acids in a mixture used for MMR vaccine production, for implementation in Bio-Manguinhos physicochemical quality control. Methodology: Amino acids mixture was derivatized with AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) for detection by Photodiode Array (PDA). Analysis was performed by UPLC reverse-phase, in a gradient with mobile phases A as 140mM sodium acetate and 17 mM triethylamine (TEA) and B as 60% (volume) acetonitrile in water with column at 37 o C. The influence of pH were evaluated at range of 3,4 – 6,8 in mobile phase A and the identification of amino acids was made by comparison to reference standards. A full factorial design with 3 factors (pH, mass of sodium acetate and volume of TEA) and 2 levels with center point, was conducted to evaluate robustness. Selectivity was evaluated against other amino acids absent in the mixture. Results: All 15 amino acids were efficiently separated with a total run time of 19 min. The use of lower pH values allowed initial peaks with better symmetry and sharper. Isoleucine/Leucine were separated with Resolution > 1,5 in all experiments. Pareto’s graphic showed that pH was the only significant factor. From experimental data an optimization was made and was achieved a critical pair resolution of R > 1,6. The optimized conditions was: pH = 3,444; 140,7 mM sodium acetate; and 16,8 mM TEA. The method was selective in relation to Asparagine, Aspartic Acid, Glutamic Acid and Cysteine, and could be evaluated as internal standards for quantification. AQC derivatization products shown to be stable for more than 3 weeks for a qualitative analysis. Conclusion: The method developed shown to be ideal for quality control analysis considering its short derivatization step with a long product stability, speed and robustness for qualitative analysis, and the potential for quantify pure and mixed amino acid. This method is now ready to be validated and implemented at Bio-Manguinhos physicochemical quality control routine.