Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized amino acid mixtures

Recent improvements in HPLC-based methods for analyzing amino acids have made it feasible to analyze accurately a wide variety of sample types. The compatibility of amino acid derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the presence of salts and lipids makes the reagent an ideal choice for the analysis of complex samples. Method development was accomplished through the use of a quaternary HPLC eluent system with optimized pH and ionic strength. One chromatographic system was developed to achieve high resolution for a collagen sample containing hydroxyproline (Hyp) and hydroxylysine (Hyl) in addition to the hydrolyzate amino acids. A second procedure optimized resolution for a sample mixture of 24 amino acids. The quaternary HPLC streamlined routine operation and improved reproducibility as the multi-solvent eluent blends allowed precise, rapid changes in pH during the gradient separation. The resulting optimized methods blend the buffered eluents to achieve the accurate pH control required. Run times were reduced to less than 1 h by employing an increased flow-rate during the final third of the run. For the 24-amino acid mixture, high resolution was achieved for a number of important amino acids including Asn, Gin, Trp, gamma-aminobutyric acid, ornithine, Hyp, and Hyl, as well as the 16 hydrolyzate amino acids in a single analysis. Examples of collagen hydrolyzate analysis and cell culture media samples are given to illustrate the utility of the methods.