Quantitative analysis of the pyridinium crosslinks of collagen in urine using ion-paired reversed-phase high-performance liquid chromatography

✍ Scribed by Darcey Black; Alexander Duncan; Simon P. Robins

• Publisher: Elsevier Science
• Year: 1988
• Tongue: English
• Weight: 472 KB
• Volume: 169
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(88)90274-6

✦ Synopsis

A method has been developed for the simultaneous and rapid analysis of the 3-hydroxypyridinium crosslinks of mature collagen, pyridinoline and deoxypyridinoline, in samples of urine and tissue. After hydrolysis in 6 M HCl, samples were prefractionated by partition chromatography using columns packed with cellulose CF1. The appropriate fractions were freeze-dried and then subjected to high-performance liquid chromatography using a gradient system with 20 mM NH4Cl, pH 3.5, and acetonitrile, incorporating 1-octanesulfonic acid as an ion-pairing agent; the pyridinium crosslinks were detected fluorometrically. The limit of detection of both crosslinks was 1 pmol. The mean recoveries of added standard to samples ranged between 94.2 and 97.2%, and the reproducibility of the complete procedure was between 12 and 16%. An application of the method in the study of degenerative disorders of bone and connective tissue is illustrated.