ZirChrom Separations (United States)

BackgroundThe activity and haemolytic toxicity associated with primaquine has been linked to its reactive metabolites. The reactive metabolites are thought to be primarily formed through the action of cytochrome P450-mediated pathways. Human erythrocytes generally are not considered a significant contributor to drug biotransformation. As erythrocytes are the target of primaquine toxicity, the ability of erythrocytes to mediate the formation of reactive oxidative primaquine metabolites in the absence of hepatic enzymes, was evaluated.MethodsPrimaquine and its enantiomers were incubated separately with human red blood cells and haemoglobin. Post-incubation analysis was performed with UPLC–MS/MS to identify products of biotransformation.ResultsThe major metabolite detected was identified as primaquine-5,6-orthoquinone, reflecting the pathway yielding putative active and haematotoxic metabolites of primaquine, which was formed by oxidative demethylation of 5-hydroxyprimaquine. Incubation of primaquine with haemoglobin in a cell-free system yielded similar results. It appears that the observed biotransformation is due to non-enzymatic processes, perhaps due to reactive oxygen species (ROS) present in erythrocytes or in the haemoglobin incubates.ConclusionThis study presents new evidence that primaquine-5,6-orthoquinone, the metabolite of primaquine reflecting the oxidative biotransformation pathway, is generated in erythrocytes, probably by non-enzymatic means, and may not require transport from the liver or other tissues.

An increased interest in non-caloric natural sweeteners has generated significant attention on glycosides from Stevia. The GRAS acceptance for Rebaudioside A and Stevioside has led to their utilization in foods, beverages, etc. However these major glycosides have an undesirable lingering aftertaste which has raised interest for characterization of new and/or minor glycosides from Stevia. The most commonly accepted analytical HPLC method, established by Joint FAO/WHO Expert Committee on Food Additives (JECFA), is appropriate for the major glycosides but does not provide good resolution for the minor glycosides that co-elute with the major glycosides present. The JECFA analytical method also does not resolve the polar region of the Stevia extract. The major interest has been in glycosides found in the polar region because of their potential taste profiles. We've established three alternative HPLC methods to the JECFA method which better resolve different regions of the Stevia extract. The complexity of the Stevia extract is such that no single method is suitable to resolve all compounds found in the extract.

Abstract Colloidal zirconia was spray dried to yield zirconia particles, which were further modified with N, N, N′, N′‐ Ethylenediamine tetra methylenephosphonic acid (EDTPA) to yield a support for use in bioseparations. EDTPA modified zirconia particles will be further referred to as, r_PEZ. Cell culture supernatants rich in monoclonal antibody (Mab) subtypes IgG1, IgG2a, IgG2b, and IgG3 were chromatographed on a r_PEZ column, and on a protein A‐hyper D column that was purchased commercially. All Mab subtypes bound to r_PEZ and process yields in the range of 88 to 99% were obtained. The purity of the Mab products were ascertained by gel electrophoretic analysis and were estimated to be greater than 95%. The purified Mab products obtained from r_PEZ and protein A columns were compared to the reference Mab standard in biological and enzymatic assays. The value of the dissociation constant (Kd) was found to be comparable and was in the range to that obtained with reference Mab standard (0.231±0.03 M). In ad...