Capillary electrophoresis analysis of gentamicin sulphate with UV detection after pre-capillary derivatization with 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid

A selective, sensitive, and rapid pre-capillary derivatization method for determination of the multicomponent aminoglycoside antibiotic gentamicin is described. The derivatization reagents 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid were used and the thioisoindole derivative was UV detected at 330 nm. A central composite experimental design was performed to optimize selectivity and derivatization conditions. Baseline separation of gentamicin C1, C1a, C2, C2a, C2b, sisomicin and several minor components was achieved with a background electrolyte containing 30 mM sodium tetraborate, 7.5 mM beta-cyclodextrin and 12.5% (v/v) methanol at pH 10. Quantitative analysis was performed and illustrated the potential use of capillary electrophoresis for the identification and quantitation of gentamicin as an alternative to methods prescribed in the United States Pharmacopeia and European Pharmacopoeia.     

Development of a capillary electrophoretic method for the separation of diastereoisomers of a new human immunodeficiency virus protease inhibitor

A capillary electrophoretic (CE) method was developed for the separation of diastereoisomers of a new human immunodeficiency virus (HIV) protease inhibitor TMC114. In total 16 isomers of this drug have been synthesized (eight pairs of enantiomers). We succeeded in the separation of the eight diastereoisomers, but no enantiomers could be separated. Because of the high similarity and water-insolubility of these isomers, the separation is a real challenge. Different CE modes were tried out: capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), micellar electrokinetic capillary chromatography (MEKC), and microemulsion electrokinetic capillary chromatography (MEEKC). Only MEEKC offered resolution of these compounds.     

Minimal number of chromatographic test parameters for the characterisation of reversed-phase liquid chromatographic stationary phases

This paper focuses on the classification or differentiation of RP-HPLC columns based on measured chromatographic properties. A chemometric study has been conducted on a published data set consisting of 85 RP-HPLC columns and on a data set consisting of 47 self-tested columns. Principal component analysis enables determination of the number of parameters necessary for a rational differentiation. The results show that reducing the number of parameters for such differentiation still allows classification of the columns just as a higher number did. It is shown that three test parameters produce a classification similar to that obtained with five parameters.     

Mass spectrometric study to characterize thioisoindole derivatives of aminoglycoside antibiotics

Aminoglycoside antibiotics are widely used to treat serious Gram-negative and Gram-positive bacterial infections. The lack of a UV chromophore presents a problem in the analysis of aminoglycosides. Derivatization with 1,2-phthalic dicarboxaldehyde (OPA) in the presence of a thiol made it possible to introduce a UV chromophoric thioisoindole moiety. A qualitative mass spectrometry study was carried out to confirm the molecular identity of the products formed. The conditions described earlier to derivatize gentamicin and kanamycin yielded products in which all primary amino groups are fully derivatized. On the other hand, with tobramycin and amikacin, there was also formation of incompletely derivatized products that contained one thioisoindole group less than the fully derivatized product. This study has therefore brought an additional insight into the nature of the OPA-aminoglycoside derivatives studied.     

Liquid chromatography of josamycin on poly(styrene-divinylbenzene)

An isocratic liquid chromatographic method for the assay and purity control of josamycin using wide-pore poly(styrene-divinylbenzene) as the stationary phase is presented. Josamycin has been well separated from all the related leucomycins, present as impurities in commercial samples. Wide-pore PLRP-S 8 m 1000 Å has shown the best selectivity. The separation has been carried out at 65°C with a mobile phase of 2-methyl-2-propanol/0.2 mol/l potassium phosphate buffer pH 10.5/water (24.5: 5: 70.5, V/V) and a flow rate of 1.0 ml/min. UV detection has been performed at 232 nm. The total analysis time has been about 40 min. The method has been used to analyse commercial bulk samples and tablets containing josamycin base.Dedicated to Professor Dr. Dr. h.c. mult. J.F.K. Huber on the occasion of his 70th birthday     

Investigation of unknown related substances in commercial erythromycin samples with liquid chromatography/mass spectrometry

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

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-beta-cyclodextrin (M-beta-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-beta-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-beta-CD and 6% v/v IPA, a capillary with 55 cm total length (50 microm 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-beta-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.     

Kinetic study of angiotensin converting enzyme activity by capillary electrophoresis after in-line reaction at the capillary inlet

The in-capillary reaction of angiotensin converting enzyme (ACE) with the tripeptide substrate hippuryl-L-histidyl-L-leucine was studied. ACE activity was determined by the quantitation of the product, hippuric acid, at 230 nm. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the compounds. When the set-up was reversed, i.e. reaction at the opposite side after short-end injection of enzyme and substrate, separation was achieved in less than 5 min. Using the Lineweaver-Burk equation, an average Michaelis constant for ACE from rabbit lung was calculated to be 1.16 +/- 0.12 mM, a value consistent with previously reported data.     

Analysis of unknown compounds in azithromycin bulk samples with liquid chromatography coupled to ion trap mass spectrometry

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of azithromycin impurities and related substances in commercial azithromycin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization interface operated in positive ion mode. The LCQ provides on-line LC/MS(n) capability, making it ideally suited for identification purposes. In comparison with UV detection, this hyphenated technique provides as its main advantage efficient identification of novel substances without time-consuming isolation and purification procedures. Using this technique, six novel related substances detected in commercial azithromycin samples have been studied.