Rapid method for the isolation of hydroxylysylpyridinoline and lysylpyridinoline from concentrated bone hydrolysates by liquid chromatographic techniques.

A rapid method for the isolation of hydroxylysylpyridinoline and lysylyridinoline from bone by liquid chromatographic methods is described. Decalcified bone is hydrolysed in 7 M hydrochloric acid. After evaporation of the acid, the high molecular mass and dark coloured degradation products are removed by adsorption on non-polar adsorbents. The pyridinolines are separated from the majority of the amino acids by adsorption on cellulose. Separation of HP and LP is performed either by cation-exchange chromatography or by reversed-phase ion-pair chromatography. The pyridinoline containing fractions are desalted by size-exclusion chromatography. The progress of the hydrolytic cleavage of collagen and the optimal parameters for purification and separation were examined. As a result the existing method allows the isolation of high amounts of pyridinolines with low amounts of adsorbents and chemicals within a short time.