Selective analysis of secondary amino acids in gelatin using pulsed electrochemical detection

A method was developed for selective analysis of the secondary amino acids proline and 4-hydroxyproline from gelatin hydrolysates using anion-exchange high-performance liquid chromatography followed by integrated pulsed amperometric detection (HPLC-IPAD). An extraction scheme was implemented prior to HPLC-IPAD analysis to isolate the secondary amino acids by the removal of primary amino acids through derivatization with o-phthalaldehyde followed by solid-phase extraction with C18 packed columns. The use of the IPAD technique eliminated the need for a second derivatization step to detect secondary amino acids. The removal of interfering primary amino acids prior to chromatographic analysis allowed the use of isocratic mobile-phase conditions to achieve effective and efficient separation of the amino acids. This led to a more precise and accurate quantitation of their content in gelatin hydrolysates. Detection limits approach 10 parts per billion ( approximately 2 pmol/injection) with a chromatographic analysis time under 8 min. The ratios of secondary amino acids, in addition to their abundances, were used to distinguish gelatin manufactured from bovine, porcine, and fish raw material sources.