José Diana

Classification of reversed-phase columns based on their selectivity towards vancomycin compounds.

The selection of a reversed-phase liquid chromatographic (RP-LC) column with suitable selectivity for a particular separation is difficult if the brand name of the column is not known. The monographs of the European Pharmacopoeia and other official compendia for drug analysis only give a general description of the stationary phase to be used in the operating procedure of a liquid chromatographic method. A project to develop a chromatographic test procedure to characterise RP-LC C(18) columns was started earlier and resulted in a fast, simple, repeatable and reproducible test procedure. Four column parameters, determined on 69 RP-LC C(18) columns, allowed the characterisation and ranking of these columns. In this paper, an overview of this column classification system is given with an application on the separation of vancomycin and some of its impurities. It is shown that the column ranking system is a helpful tool in the selection of a suitable column.

Development and validation of an improved method for the analysis of vancomycin by liquid chromatography selectivity of reversed-phase columns towards vancomycin components.

The current method prescribed in official monographs for the purity control of vancomycin is inappropriate in that several components are not separated from each other and other components are coeluted with the main component vancomycin B. The method uses an ODS column at pH 3.2. In this study, several changes were introduced in order to improve the separation. The optimization of the separation method at low pH indicated that pH 1.7 was optimum and that the use of dioxane as organic modifier drastically improved the separation. These conditions were used to test a set of more than 40 reversed-phase columns for their selectivity towards vancomycin components. The selection of the most suitable columns was performed by means of principal component analysis. Most of these columns did not allow the separation of didechlorovancomycin from monodechlorovancomycin 1. It was found that neutral to slightly alkaline mobile phases allowed better separation. Further optimization of the separation method and a robustness study were performed by means of experimental design. This optimization indicated that pH 7.7 was optimum and gradient elution was also used to effect complete analysis. The final method uses a Kromasil column and the mobile phase comprises dioxane, water and ammonium formate solution pH 7.7. The separation of monodechlorovancomycin 2 and of some unknown impurities from the main component vancomycin B is described for the first time. The method shows good repeatability, linearity and sensitivity.

Separation of tetracycline and its potential impurities by liquid chromatography on XTerra RP-18

A linear gradient elution method is described for the separation of tetracycline, its fermentation impurities, and its degradation products. This method uses XTerra RP-18, 5 μm (25 cm×4.6 mm ID), a silica-based stationary phase with reduced silanol activity. Gradient elution with mobile phases containing acetonitrile, 0.3 M tetrabutylammonium hydrogen sulfate pH 7.5, 0.3 M ethylenediaminetetraacetic acid pH 7.5, water was used at a flow rate of 1.0 mL/min. The column temperature was set at 40°C. The UV detection was performed at 280 nm. A central composite experimental design was performed to optimize the separation. The method permits a good separation of TC and its impurities. Some of these were not reported before and have now been identified for the first time. Some unidentified impurities were also separated.

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

SummaryAn improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min−1, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.

Development and validation of an improved liquid chromatographic method for the analysis of dirithromycin.

The official method for the determination of dirithromycin and related substances in the European Pharmacopoeia (Ph. Eur.) and in the United States Pharmacopeia (USP) is an isocratic liquid chromatographic (LC) method using an ODS column. With this method, the separation of the main component dirithromycin from its epimer is not complete. Moreover, this method suffers sometimes from drift of the baseline and from subsequent quantitation problems. The required resolution is not easy to obtain. Using an adapted method derived from the one prescribed in the pharmacopoeias, the selectivity of a set of more than 40 reversed-phase columns towards dirithromycin components was investigated. The selection of the most suitable column was achieved by the chromatographic response function (CRF) approach. Several changes were introduced to the method in order to improve the separation and to overcome the baseline drift problem. The resulting method uses a Zorbax Extend column maintained at 30 degrees C and a mobile phase containing acetonitrile, methanol, 2-propanol, water and a phosphate buffer at pH 7.5. The method allows a good separation of dirithromycin components, which is much better than that obtained with the existing methods. Several impurities of unknown identity are also separated. The method shows good repeatability, linearity and sensitivity, and it is robust. In addition, it proved to be applicable to a wide number of C18 reversed-phase columns.