Jennifer Orwa

Isolation and structural characterization of polymyxin B components.

Polymyxin B is a peptide antibiotic complex present as sulphate. The components were separated preparatively on a poly(styrene-divinylbenzene) (PLRP-S), 1000 A, 8 microm, 250 x 12.5 mm I.D. stationary phase maintained at 60 degrees C and using 215 nm detection. Elution was carried out with acetonitrile-sodium sulphate solution (0.7%, m/v; pH adjusted to 2.5 with trifluoroacetic acid)-water (18:50:32, v/v) at a flow-rate of 4.0 ml/min. Seven polymyxin B components were isolated and characterized using 1H and 13C NMR. The molecular masses were confirmed by mass spectrometry. The structures of two components were determined for the first time. Polymyxins B5 and B6 were identified as having the same composition as polymyxin B1 except that the fatty acid moiety was nonanoic acid and 3-hydroxy-6-methyloctanoic acid, respectively.

Liquid chromatography of polymyxin B sulphate

A reversed-phase liquid chromatography method for analysis of polymyxin B sulphate is described. The method uses a YMC-Pack Pro, C18, 5 microm, 250x4.6 mm I.D. column maintained at 30 degrees C. The mobile phase comprises acetonitrile-sodium sulphate (0.7%, m/v)-phosphoric acid (6.8%, v/v dilution of 85%, m/m phosphoric acid)-water (22.25:50:5:22.75) at a flow-rate of 1.0 ml/min. Detection was by UV at 215 nm. The method is able to resolve polymyxin B1, the major component, from more than thirty other components present in the complex. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.

Study of the stability of polymyxins B1, E1 and E2 in aqueous solution using liquid chromatography and mass spectrometry

Polymyxins B(1), E(1) (colistin A) and E(2) (colistin B) were subjected to degradation in aqueous solutions of different pH values (1.4, 3.4, 5.4 and 7.4) and at different temperatures (37, 50 and 60 degrees C) in order to investigate the characteristics of decomposition. The progress of decomposition was followed by reversed-phase liquid chromatography on YMC-Pack Pro, C-18 stationary phase. The degradation curves showed (pseudo) first order kinetics. The pH-rate profiles indicate that colistin is more susceptible to degradation in solutions of pH above 5 and is more stable in acidic media. The degradation of polymyxin B(1) was most rapid at pH 7.4. Qualitative analysis of the degradation products by LC/MS reveals that racemization is the major mechanism of degradation in both acidic and neutral media.

Analysis of polymyxin B sulfate by capillary zone electrophoresis with cyclodextrin as additive. Method development and validation.

A capillary zone electrophoresis method for analysis of polymyxin B sulfate is described. In this method, triethanolamine (TEA)-phosphate buffer at pH 2.5 was employed to reduce the adsorption of analyte onto the capillary wall. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found to be necessary for selectivity enhancement. In order to optimize the method and to control its robustness, a central composite design was performed with four parameters, i.e. concentration of M-beta-CD, TEA, IPA and buffer pH. The optimal separation conditions were as follows: capillary, 55 cm (50 microm I.D., 47 cm effective length); 130 mM TEA-phosphate buffer (pH 2.5) containing 5 mM M-beta-CD and 5% IPA; 24 kV (51 microA) applied voltage; column temperature, 20 degrees C. Further, linearity and limits of detection quantification were examined. Three commercial samples were analyzed quantitatively.

Liquid chromatography method for separation of clindamycin from related substances

A reversed-phase liquid chromatography method has been developed for the separation of clindamycin from 7-epiclindamycin, clindamycin B, lincomycin, lincomycin B, 7-epilincomycin and other impurities of unknown identity. The method uses a Hypersil ODS, 5 microm, 250 x 4.6 mm i.d. column maintained at 45 degrees C. The mobile phase comprises acetonitrile phosphate buffer (1.35% v/v phosphoric acid, adjusted to pH 6.0 with ammonium hydroxide)-water (35:40:25, v/v) at a flow rate of 1.0 ml/min. UV detection is performed at 210 nm. The method was tested on several C-18 columns and showed good robustness. Robustness was further evaluated by performing a full-fraction factorial design experiment. The method showed good selectivity, linearity, and repeatability. It is also suitable for analysis of clindamycin formulations.

Liquid chromatography-ion trap tandem mass spectrometry for the characterization of polypeptide antibiotics of the colistin series in commercial samples.

A selective reversed-phase liquid chromatography-tandem mass spectrometry method is described for the characterization of related substances in the colistin complex. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ionization probe operated in the positive ion mode. The main advantage of this technique is the characterization of novel related substances without time-consuming isolation and purification procedures. Using this method seven new related substances were partially identified in colistin bulk sample and tablets. Four components were assigned as isomers of the main components of colistin.

Liquid chromatographic method for separation of lincomycin from its related substances

Abstract A reversed-phase ion-pair liquid chromatographic method with UV detection at 210 nm is described for the separation of lincomycin from 7-epilincomycin and lincomycin B. The method utilizes a base-deactivated Supelcosil LC-ABZ, C12–18, 5 μm, 250×4.6 mm I.D. column maintained at 45°C. The mobile phase consists of 2.25% (v/v) acetonitrile, 5% (v/v) phosphate buffer (2.72%, m/v KH2PO4 adjusted to pH 5.0 with 3.48%, m/v K2HPO4), 0.067% (v/v) methanesulfonic acid and water to 100%. The flow-rate is 1.0 ml/min. The separation of lincomycin from 7-epilincomycin is only possible on base-deactivated (BDS) columns. Other octadecylsilyl columns examined showed insufficient selectivity. The method was also tested on other BDS columns (Spherisorb S5-ODS-B, 5 μm, Hypersil BDS, C18, 5 μm and Supelcosil LC-ABZ, C18, 5 μm, all 250×4.6 mm I.D.) and showed good robustness. Robustness was further evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity. It is also suitable for analysis of lincomycin formulations.

Analysis of colistin sulfate by capillary zone electrophoresis with cyclodextrins as additive

A method for the quantitative analysis of colistin sulfate by capillary zone electrophoresis is described. Since colistin components have five free amino groups, they tend to adsorb onto the capillary wall and cause peak tailing. It was found that triethanolamine (TEA)‐phosphate buffer at pH 2.5 was useful to reduce such adsorption. Methyl‐β‐cyclodextrin (M‐β‐CD) and 2‐propanol (IPA) were found necessary for selectivity enhancement. In order to optimize the separation parameters and predict the method robustness, a central composite design was performed including three variables, namely concentration of M‐β‐CD, TEA, and IPA. The effects of capillary length and applied voltage on separation were also investigated. The optimal conditions established were: 140 mM TEA‐phosphate buffer containing 5 mM M‐β‐CD and 6% v/v IPA, a capillary with 55 cm total length (50 μm inner diameter, 47 cm from inlet to detection window) and 24 kV applied voltage. The method was found to be robust when the variables were changed in the following range: 4—6 mM M‐β‐CD, 5—7% v/v IPA, and 130—150 mM TEA. Further, the linearity, limit of detection (LOD), and limit of quantitation (LOQ), as well as repeatability for both colistin A and B were examined and three commercial samples were quantitatively analyzed.

Liquid Chromatography of Gramicidin

SummaryA simple, selective method is described for separation of more than 10 gramicidin components on Spherisorb ODS B, 5 μm,250×4.6 mm I.D. column, maintained at 50°C. The mobile phase comprised methanol-water (71:29) at a flow rate of 1.0 mL min−1. Detection was by UV at 282 nm. Valine gramicidins A, B and C were very well separated from the isoleucine gramicidins A, B and C. Four new gramicidin components were also resolved and their structures determined by liquid chromatography/ mass spectrometry. The names 10-methionine valine gramicidin C, 4-methionine valine gramicidin A, valine gramicidin A hydroxypropyl and isoleucine gramicidin A hydroxypropyl were proposed. Robustness of the liquid chromatography method was evaluated by performing a full factorial design experiment. The method also showed good repeatability, linearity and sensitivity.

Structure elucidation of four related substances in gramicidin with liquid chromatography/mass spectrometry

A selective reversed phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of related substances in commercial gramicidin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and the negative ion mode. The LCQ is ideally suited for identification of related substances because it provides on-line LC/MS(n) capability. Compared with UV detection the main advantage of this hyphenated LC/MS(n) technique is the efficient identification of novel related substances without time-consuming isolation and purification procedures. Using this method four novel related substances were separated and identified in a commercial sample.