A RAPID PROCEDURE FOR PURIFYING LARGE AMOUNTS OF PYRIDINOLINE CROSS-LINKS OF BONE

HPLC assessment of urinary Pyridinoline (Pyr) and Deoxypyridinoline (Dpyr) requires the use of large amounts of purified Pyr and Dpyr as external standards. We have developped a procedure for large-scale pyridinoline (Pyr and Dpyr) purification from sheep bone combining successively gel filtration partition chromatography and semi-preparative HPLC. After bone powder (500 g) hydrolysis in 6N HCl (5 liters), the concentrated hydrolysate (600 ml) was separated by gel filtration on a Biogel P2 column (2.4 Liters), allowing the elimination of 95% of impurities and the reduction of the pyridinolines solution to 150 ml. Then partition chromatography was carried out on CF1 cellulose where non-polar contaminants were suppressed. Finally, drawing on analytical HPLC knowledge, an isocratic semi-preparative HPLC was developed using a reversed phase C-18 column (250 mm x 10 mm) with HFBA as the ion pairing agent. The last impurities were thus eliminated, and the Pyr was separated from the Dpyr. By this sequence of processes, 15 mg of pyridinoline and 1.8 mg of deoxypyridinoline were purified. This optimized procedure allows the large-scale production of Pyr and Dpyr from large amounts of bone or other tissue in a relatively short time, and requires only conventional biochemical reagents.