"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.

Measurement of tobramycin by reversed-phase high-performance liquid chromatography with mass spectrometry detection.

Analysis of tobramycin faces challenges owing to its significant basicity, hydrophilicity and lack of a UV absorbing chromophore. Chromatographic methods, coupled with derivatization to introduce chromophores for tobramycin analysis, were extensively studied. A direct reversed-phase HPLC method for tobramycin analysis has not been reported. Here, we would like to report a simple LC/MS method for quantitative analysis of tobramycin in pharmaceutical formulations. Reversed-phase HPLC analysis of tobramycin was achieved using a pH stable C18 column with basic (pH 11) aqueous mobile phase (ammonium hydroxide buffer), while direct detection was carried out employing a single quadruple mass detector in negative mode via electrospray ionization. This unique separation-detection combination provided simple and specific determination of tobramycin. This method was found to be linear at a tobramycin concentration range of 0.2-0.8 mg/mL with a correlation coefficient value of 0.999. The quantitation limit and detection limit were calculated as 0.210 and 0.063 microg/mL, respectively, with 99.994% confidence. This method was successfully applied to measure tobramycin content in matrices containing tobramycin and other pharmaceutical formulation ingredients. Recoveries of 101.8, 97.8 and 106.7% were obtained for tobramycin spiked in the pharmaceutical formulation at concentrations of 1.68, 1.0 and 0.35 mg/mL, respectively. The relative standard deviations for six injections of spiked samples ranged from 0.2 to 3.2%, indicating good method repeatability.