Capillary zone electrophoretic (CZE) separations and mass spectrometric analysis of salmon calcitonin and related analogues were performed to generate electrophoresis and mass fingerprints for quality control of the recombinant polypeptide pharmaceutical salmon calcitonin. The calcitonins and their corresponding tryptic digests were successfully separated by CZE at low pH in fused silica capillaries dynamically modified with poly-cationic polymers. The poly-cationic modified inner surface of the fused silica capillaries generated a strong anionic electroosmotic flow (EOF). Analytes of negative, neutral, and positive charge were all swept through the capillary toward the positive electrode. Compared to Polybrene-coated capillaries, capillaries coated with PEI showed a markedly slower but much more stable electroosmotic flow. The migration order of the analytes was predicted by comparing approximate values of the charge to (molecular mass)2/3 ratios. The predicted migration order was confirmed by off-line analysis of CZE fractions with matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

No abstract

Three microdialysis methods, the "tritium" method, the "point-of-no-net-flux" method, and a method using the low perfusion rate of 0.1 microliter/min, were compared with respect to their ability to generate estimates of unbound steady-state concentrations (Cu(ss)) of the antiasthmatic drug theophylline in blood and brain tissue in anesthetized rats. Concomitantly, the influence of the perfusion flow rate on the estimated extracellular Cu(ss) obtained with the point-of-no-net-flux method was investigated. Theophylline was administered as a rapid intravenous bolus dose followed by constant intravenous infusion. Changes in perfusion flow rate from 2.0 to 0.75 microliter/min and, finally, to 0.25 microliter/min, using the point-of-no-net-flux method, had no significant effect on the estimated Cu(ss) of theophylline in blood and striatum. This observation, particularly in the case of brain tissue, is not consistent with the theory that the process of dialysis drains a significant amount of substance from the immediate vicinity of the dialysis probe. Similar estimates of Cu(ss) in blood as well as in brain tissue were obtained with all three methods. Their accuracy in estimating Cu(ss) in blood was further strengthened by observations of unbound fractions similar to those reported in the literature. Furthermore, all three methods gave striatum/blood ratios at steady state of approximately 0.5, indicating that there is active transport of theophylline from brain tissue. It is concluded that the tritium method, when validated, can be used to study the time course of unbound drug concentrations in blood and tissues.

No abstract