Quantitative analysis of the pyridinium crosslinks of collagen in urine using ion-paired reversed-phase high-performance liquid chromatography.
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.
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Biochemistry Division, Rowett Research Institute, Bucksburn, Aberdeen, Scotland.
Duncan
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Robins
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D000328:Adult; D000596:Amino Acids / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003094:Collagen / Q000652:urine*; D006801:Humans; D006868:Hydrolysis; D011726:Pyridinium Compounds / Q000652:urine*
D000596:Amino Acids; D011726:Pyridinium Compounds; C015484:pyridinoline; C036020:deoxypyridinoline; D003094:Collagen