Chromatography

Biography

Biography: Rafea M Naffa

Abstract

The natural crosslinks between tropocollagen molecules in skin contribute to its physical properties of strength and flexibility. Despite advances in analytical techniques such as mass spectrometry, the methods used for their analysis and quantitation date back to the 70s through to the 90s, are challenging and time consuming. As a result, it is difficult to obtain standards to verify analyses of these compounds. We have developed methods to isolate highly pure, natural crosslinks from skin, and have verified their structures using mass spectrometry and NMR. Fragmentation studies of these crosslinks will enable the development of a method for label-free quantitation in skin hydrolysates. We have also developed a novel chromatographic analytical method for the simultaneous determination of natural crosslinks. Seven crosslinks were separated on Cogent Diamond Hydride HPLC column using isocratic and gradient conditions then detected by mass spectrometry without derivatization. Total run time of less than 10 minutes was achieved under isocratic conditions using water and acetonitrile. To the best of our knowledge, this is the first method in which histidinolysinonorleucine (HHL) and histidinohydroxymerodesmosine (HHMD) were separated and identified by the mass spectrometry. This technique was applied on skin, elastin and cartilage in which strong evidence suggested the presence of undocumented crosslinks. The developed method will be widely used for quantitative and qualitative analysis of natural crosslinks in biological samples as well as characterization of new crosslinks.