Ion Exchange HPLC Determination of Pyridinium Crosslinks in Urine as Markers of Bone Resorption

Abstract

The aim of this study was to improve the published method¹ for rapid and sensitive determination of the urinary pyridinoline as well as deoxypyridinoline, i.e. the crosslinking elements of bone and cartilage collagen. This method is based on the HPLC analysis of previously prepared urinary hydrolysates. Uríne hydrolysate (6 N HCl, 110°C, 16 hours) was purified by selective fractionation using washing with a mixture of water -acetic acid-butanol (1-1-4 by volume) on small disposable columns filled with microgranular cellulose. Liquid chromatography was based on ion-exchange counteractions and was properly performed with the use of a strong cation exchanger as a stationary phase and with a buffered solution at pH=3.35 as the mobile phase, by keeping the oven temperature at 48°C and with the injection of a volume of 10 μl. The time desired for HPLC determination alone didn't exceed 15 min and by using a fluorescence detector (Shimadzu RF 535) set at 297 and 400 nm for excitation and emmision wavelengths, respectively, it reached a sensitivity of about 200 femtomoles for both the crosslinks. The method developed was experimentally tried in testing excretion levels of both markers of collagen tissue breakdown in different groups of subjects. The results obtained are in high correlation with the assumed conditions in the investigated groups and, therefore, this procedure seems to be very useful and effective as a sensitive indication of bone mass resorption processes.