Isolation and partial characterization of pyridoxal 5'-phosphate hemoglobins by high-performance liquid chromatography as a quality-control method for hemoglobin-based blood substitutes.

Pyridoxal 5'-phosphate hemoglobin (PLP-Hb), prepared from hemoglobin and a four-fold excess of pyridoxal 5'-phosphate by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205], has been chromatographically resolved into six components via a quaternary ammonium monobead support. On an analytical scale, the separations have been found to be rapid (ca. 50 min) and highly reproducible. The results also indicate that the preparation of PLP-Hb yields a reproducible product ratio. The potential of the analytical method for the routine quality control of blood substitutes derived from PLP-Hb is discussed. All five of the PLP derivatives (components II-VI), isolated and purified via a combination of conventional and preparative monobead anion-exchange chromatography, gave single peaks when analyzed by high-performance liquid chromatography. Total phosphate analyses indicated that components II and III each contain two PLPs per Hb, IV and V four and VI six.