Evaluation of a Hydrophilic Interaction Liquid Chromatography Design Space for Amino Acids

A column ranking system was developed for the separation of all 20 proteinogenic amino acids using hydrophilic interaction liquid chromatography mode across 18 polar stationary phases with acetonitrile/aqueous mobile phases. Each amino acid was individually evaluated on all columns using gradient elution, and the retention time data were compiled into a statistical ranking algorithm that enables selection of any analyte subset and identifies the column providing the greatest separation between successively eluting analytes. Since most of these analytes lack strong chromophores, evaporative light scattering detection was employed for quantitative detection. Mobile phase pH (3.5 to 6.0) and ammonium formate concentration (10 to 100 mM) were then optimized on the three highest-ranked columns (Accucore-150-Amide HILIC, TSKgel Amide-80, and Poroshell 120 HILIC-OH5). Retention of most amino acids was insensitive to pH, whereas arginine and lysine showed inverse dependence on buffer concentration compared with all other analytes, and 50 mM ammonium formate without pH adjustment gave the best compromise for the complete analyte set. Re-querying the ranking algorithm for smaller analyte subsets returned a different optimal column in each case, confirming that no single stationary phase is universally preferable. Knowledge gained from evaluating the design space facilitated the rapid development of a quantitative method for the determination of the amino acid composition of a capsule formulation. The isocratic assay was linear for leucine, isoleucine, and valine over approximately 0.1 to 1.0 mg/mL (R² ≥ 0.997), recovered 102%-104% of label claim across six independent preparations, and gave injection precision of ≤ 0.45% relative standard deviation with quantitation limits of ≤ 114 µg/mL.