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

HPLC assessment of urinary Pyridinoline (Pyr) and Deoxypyridinoline (D pyr) requires the use of large amounts of purified Pyr and Dpyr as ext ernal standards. We have developped a procedure for large-scale pyridi noline (Pyr and Dpyr) purification from sheep bone combining successiv ely gel filtration partition chromatography and semi-preparative HPLC. After bone powder (500 g) hydrolysis in 6N HCl (5 liters), the concen trated hydrolysate (600 ml) was separated by gel filtration on a Bioge l P2 column (2.4 Liters), allowing the elimination of 95% of impuritie s and the reduction of the pyridinolines solution to 150 ml. Then part ition chromatography was carried out on CF1 cellulose where non-polar contaminants were suppressed. Finally, drawing on analytical HPLC know ledge, an isocratic semi-preparative HPLC was developed using a revers ed phase C-18 column (250 mm x 10 mm) with HFBA as the ion pairing age nt. The last impurities were thus eliminated, and the Pyr was separate d from the Dpyr. By this sequence of processes, 15 mg of pyridinoline and 1.8 mg of deoxypyridinoline were purified. This optimized procedur e 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.