ANALYSIS of Acid-Soluble Hydroxy-Proline, Free Proline and Collagen-Bound Hydroxyproline in Rat Liver by High Performance Liquid Chromatography With Pre-Column Derivatization

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Abstract
A High Performance Liquid Chromatographic analysis of acid-soluble hydroxyproline, free proline and collagen-bound hydroxyproline from rat liver is described. A precolumn derivatisation with the fluorogenic reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was adopted. The chromatographic assay was performed by using a Spherisorb ODS2 reversed phase column, with fluorometric detection. Elution was carried out isocratically with acetonitrile 0.1 M sodium phosphate buffer, pH 7.2 (9:91, v/v). The derivatives of standard imino acids and of internal standard (3,4-dehydro-L-proline) can be separated in less than 15 min and quantitated with high sensitivity (1 injected pmole of hydroxyproline and 5 injected pmoles of proline). Key steps in the approach with the biological sample include initial extraction of acid-soluble hydroxyproline, free proline and collagen; acid hydrolysis of collagen and of hydroxyproline-containing peptides; selective derivatisation of imino acids with the fluorogenic reagent, after a previous reaction of the sample with o-phthalaldehyde; finally, chromatographic analysis of the derivatives. The assay of acid-soluble hydroxyproline requires a clean-up step on a Sep-Pak C18 cartridge prior to the analytical chromatography. Owing to its high sensitivity and reliability, the presented procedure can be used in studies on collagen metabolism and it should be preferred over the time-consuming and less sensitive colorimetric assays previously described.