Artifactual formylation of the secondary amine of duloxetine hydrochloride by acetonitrile in the presence of titanium dioxide: implications for HPLC method development.

Duloxetine hydrochloride, a secondary amine containing pharmaceutical, currently marketed as Cymbalta, is shown to undergo N-formylation as an artifact of sample preparation prior to HPLC analysis for impurities. The reaction was discovered as a result of an investigation into variability in the levels of N-formyl duloxetine observed upon HPLC analysis. The reaction is catalyzed by sonication and/or light in the presence of titanium dioxide and is proposed to occur via a radical-initiated mechanism. The mechanism is supported by controlled sample preparation studies with deuterium-labeled acetonitrile and LC/MS studies that showed incorporation of one deuterium into N-formyl duloxetine. This discovery is broadly relevant because sonication is commonly used to aid dissolution of pharmaceuticals in acetonitrile for HPLC analysis, titanium dioxide is a commonly used excipient, the amount of light found in modern analytical laboratories is sufficient to cause the reaction to occur, and secondary amines are present in the structures of many pharmaceuticals. The artifactual reaction was effectively eliminated by changing the sample solvent to methanol and replacing sonication with shaking to aid sample dissolution.

"Orthogonal" separations for reversed-phase liquid chromatography.

A general procedure is proposed for the rapid development of a reversed-phase liquid chromatographic (RP-LC) separation that is "orthogonal" to a pre-existing ("primary") method for the RP-LC separation of a given sample. The procedure involves a change of the mobile-phase organic solvent (B-solvent), the replacement of the primary column by one of very different selectivity, and (only if necessary) a change in mobile phase pH or the use of a third column. Following the selection of the "orthogonal" B-solvent, column and mobile phase pH, further optimization of peak spacing and resolution can be achieved by varying separation temperature and either isocratic %B or gradient time. The relative "orthogonality" of the primary and "orthogonal" RP-LC methods is then evaluated from plots of retention for one method versus the other. The present procedure was used to develop "orthogonal" methods for nine routine RP-LC methods from six pharmaceutical analysis laboratories. The relative success of this approach can be judged from the results reported here.

Aqueous acidic degradation of the carbacephalosporin loracarbef.

The aqueous degradation of the carbacephalosporin loracarbef under moderately acidic conditions (pH range, 2.7-4.3) is described. Structures of a total of 10 compounds isolated by preparative reversed-phase HPLC have been proposed. Five of these 10 degradation compounds arose from hydrolysis of the beta-lactam ring followed by structural changes in the six-membered heterocyclic ring. Four compounds form from intermolecular reactions of loracarbef to form dimeric structures with peptide linkages. The remaining compound resulted from oxidation of the primary amine to a hydroxylamine. Pathways for the formation of these compounds from the parent loracarbef are proposed.