Eugene Roets

The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors

A quantitative nonisotopic solution assay for gelatinases and inhibitors was developed using biotinylated gelatin as enzyme substrate. In this assay, residual biotinylated substrate is sandwiched between avidin-coated plates and streptavidin-peroxidase and is quantified by the peroxidase reaction. This assay was useful for measuring gelatinase activities and defining the activities of gelatinase inhibitors. When 23 tetracycline analogues were compared, significant differences in gelatinase B inhibition were found between various compounds. 4-epioxytetracycline base, 4-epichlortetracycline, meclocyclinesulfosalicylate, and unmodified metacycline and minocycline proved to be the most potent gelatinase B (EC 3.4.24.35) inhibitors. The gelatinase B inhibitory activity of tetracyclines was clearly dissociated from their antimicrobial activity. The effect of high-molecular-weight inhibitors, such as monoclonal antibodies, was also demonstrable in the microtiter plate assay. In view of the pathophysiological function of gelatinases, the definition of gelatinase inhibitors with known efficacy, safety, and side effects is crucial for the treatment of diseases such as rheumatoid arthritis and multiple sclerosis. Particular tetracyclines fulfil these criteria and the described assay is useful for defining other gelatinase-inhibiting lead compounds.

Characterisation of reversed-phase liquid chromatographic columns by chromatographic tests. Evaluation of 36 test parameters: repeatability, reproducibility and correlation.

The European Pharmacopoeia (Ph. Eur.) or other official compendia give only a general description of the stationary phase in the description of a liquid chromatographic method. Therefore the selection of a column giving suitable selectivity presents difficulties. Earlier, a test procedure was proposed that allows measurement of a number of parameters which are reported to be representative for stationary phase characteristics. This paper describes how the test procedure was applied on 69 RP-LC C18 columns. Chromatographic parameters obtained as test results were evaluated, and their repeatability, reproducibility and correlation were examined.

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.

Characterisation of reversed-phase liquid chromatographic columns by chromatographic tests. Rational column classification by a minimal number of column test parameters.

The European Pharmacopoeia (Ph. Eur.) and other official compendia give only a general description of the stationary phase in the description of a liquid chromatographic method. Therefore the selection of a column giving suitable selectivity presents difficulties. Earlier, a test procedure was proposed that allows to measure 36 chromatographic parameters which have been described for characterising stationary phases. This procedure was carried out on 69 reversed-phase liquid chromatography (RP-LC) columns. This paper focuses on the classification of RP-LC stationary phases based on chromatographic parameters. A chemometric study was conducted using 24 parameters that could be measured in a repeatable and reproducible way. Principal component analysis was used to classify the columns and to estimate the minimal number of parameters necessary for a rational classification. It is shown that after reducing the number of parameters from 24 to four or three, similar classifications were obtained. The column classifications were compared to the European Pharmacopoeia stationary phase description and to the column properties obtained from the manufacturers.

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.

Determination of neomycin sulfate by liquid chromatography with pulsed electrochemical detection

The determination of neomycin sulfate by liquid chromatography using a column packed with a poly(styrenedivinylbenzene) co-polymer and pulsed electrochemical detection on a gold electrode is described. The mobile phase consisted of an aqueous solution containing 70 g/l of sodium sulfate, 1.4 g/l of sodium 1-octanesulfonate and 50 ml/l of a 0.2 M phosphate buffer (pH 3.0). Sodium hydroxide was added post-column. The influence of the different chromatographic parameters on the separation was investigated. The method shows good linearity and repeatability and is stability indicating. A number of commercial samples was analyzed using this method and the results were compared with results obtained with the European Pharmacopoeia method and a previously described thin-layer chromatographic method.

Development and validation of a simple capillary zone electrophoresis method for the analysis of kanamycin sulfate with UV detection after pre-capillary derivatization

Capillary zone electrophoresis was successfully applied to separate eight related substances of kanamycin and several minor unknowns from the main component. Strategies to enhance derivatization and selectivity and to optimize separation parameters involved the application of experimental designs. This chemometrical approach considers main effects as well as interactions of the influential parameters, thus conducting a more thorough investigation of the method than the common step-by-step approach. Central composite face centered designs established optimal separation conditions: 30 mM borax buffer, pH 10.0 containing 16.0% (v/v) methanol and optimal composition of derivatization reagent: 27 mg/ml 1,2-phthalic dicarboxaldehyde and 25 microl/ml mercaptoacetic acid in borate buffer, pH 10.4. The standard curves were linear over the concentration range of 0.007-1.01 mg/ml for the main component and 0.003-0.1 mg/ml for the related substances. The limit of quantitation was 0.14% (m/m) for the related substances and impurities (S/N= 10). The assay method was used to determine the composition of several commercial samples. Quantitative analysis indicates potential usefulness of capillary electrophoresis as an alternative to the assay method prescribed in the European Pharmacopoeia and the United States Pharmacopeia.

Liquid chromatography of erythromycin A and related substances on poly(styrene-divinylbenzene)

SummaryAn isocratic liquid chromatography method for assay and purity control of erythromycin is presented. Erythromycin A is separated from all its potential impurities, except erythromycin D. The selectivity depends on the pore size of the poly(styrene-divinylbenzene) stationary phase. Wide pore PLRP-S 8 μm 1000 Å shows the best selectivity. The column is heated at 70°C. The mobile phase is acetonitrile-2-methyl-2-propanol-0.2 M potassium phosphate buffer pH 9.0-water (3:16.5:5:75.5). The flow rate is 2 ml/min. UV detection is performed at 215 nm. The total analysis time is about 30 min. The method was used to compare official standards.

Quantitative analysis of oxytetracycline and related substances by high-performance liquid chromatography

Isocratic high-performance liquid chromatography on PLRP-S 8-microns poly(styrene-divinylbenzene) copolymer allows complete separation of oxytetracycline, 4-epioxytetracycline, tetracycline, anhydrooxytetracycline, alpha- and beta-apooxytetracycline. The mobile phase was tert.-butanol-0.2 M phosphate buffer pH 8.0-0.02 M tetrabutylammonium sulphate pH 8-0.0001 M sodium ethylenediaminetetraacetate pH 8.0-water (5.9:10:5:10:78.1, m/v/v/v/v). With this isocratic method, 2-acetyl-2-decarboxamidooxytetracycline is only partly resolved from oxytetracycline. The separation and the detection limits can be improved by the use of gradient elution. Gradient elution was used for the comparison of official standards and for the analysis of a number of commercial samples, and to monitor the stability of oxytetracycline hydrochloride during storage in the solid state for about 6 years at various temperatures.

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.