Solvent partition of 14C-unconjugated bilirubin to remove labeled polar contaminants

Polar derivatives contaminating unconjugated bilirubin (UCB) are customarily extracted with weak alkali. As UCB degrades rapidly at alkaline pH, regeneration of polar derivatives may limit purification. This problem is especially important when trying to eliminate radiolabeled contaminants from C-14-UCB. As polar derivatives of UCB should have a much greater aqueous to CHCl3 partition ratio (PR) than UCB even at neutral pH, where degradation of UCB is minimal, 14C-UCB in CHCl3 was serially extracted with an aqueous buffer at pH 7.0 to determine whether labeled derivatives could be preferentially removed. A single extraction of customarily purified C-14-UCB removed 0.18 +/- 0.06% of the radioactivity as labeled derivatives. Subsequent serial extractions yielded a stable, 67% lower C-14-PR with only 0.03% of radioactivity as labeled derivatives. Reverse-phase high-performance liquid chromatography (HPLC) of phases from later extractions revealed, however, that up to 1.1% of the disintegration per minute (dpm) in CHCl3 phases and up to 50% in aqueous phases were polar impurities. HPLC of partition phases spiked with purified 14C-UCB revealed that these impurities derived from incomplete extraction of the least polar impurities and their regeneration during HPLC. In the dark under argon, C-14-UCB in CHCl3 or dimethyl sulfoxide (DMSO) solution degraded very slowly to polar derivatives. Extraction of impurities from a solution of C-14-UCB in CHCl3 is best done using pH 7.0 buffer, with removal of over 80% of the labeled contaminants remaining after customary purification by alkaline extraction.