Marie-Dominique Blanchin

Influence of polyelectrolyte capillary coating conditions on protein analysis in CE

CE of biomolecules is limited by analyte adsorption on the capillary wall. To prevent this, monolayer or successive multiple ionic‐polymer layers (SMILs) of highly charged polyelectrolytes can be physically adsorbed on the inner capillary surface. Although these coatings have become commonly used in CE, no systematic investigation of their performance under different coating conditions has been carried out so far. In a previous study (Nehmé, R., Perrin, C., Cottet, H., Blanchin, M. D., Fabre, H., Electrophoresis 2008, 29, 3013–3023), we investigated the influence of different experimental parameters on coating stability, repeatability and peptide peak efficiency. Optimal coating conditions for monolayer and multilayer (SMILs) poly(diallyldimethylammonium) chloride/ poly(sodium 4‐styrenesulfonate) coated capillaries were determined. In this study, the influence of polyelectrolyte concentration and ionic strength of the coating solutions, and the number of coating layers on coating stability and performance in limiting protein adsorption was carried out. EOF magnitude and repeatability were used to monitor coating stability. Coating ability to limit protein adsorption was investigated by monitoring variations of migration times, time‐corrected peak areas and separation efficiency of test proteins. The separation performance of polyelectrolyte coatings were compared with those obtained with bare silica capillaries.

Determination of aminoglycosides in pharmaceutical formulations — II. High-performance liquid chromatography

A post-column derivatization procedure using OPA and fluorescence detection has been used for the determination of seven aminoglycosides (dibekacin, framycetin, kanamycin, netilmicin, sisomicin, tobramycin and gentamicin) in commercial pharmaceutical formulations. The linearity, precision and detection limits were satisfactory. Recoveries from eye drops, ointments, injections and capsules were comparable (P = 0.05) to those obtained with TLC or microbiological assays. A ruggedness test showed that the method was not sensitive to minor variations in the mobile phase composition, post-column derivatization system or detection wavelength.

Stability of capillaries coated with highly charged polyelectrolyte monolayers and multilayers under various analytical conditions—Application to protein analysis

The stability of capillaries coated with highly charged polyelectrolytes under various analytical conditions was studied, as well as their performance for the analysis of proteins by Capillary Electrophoreis (CE) over a wide range of pH (2.5-9.3). In this study, fused silica capillaries were modified either with a poly(diallyldimethylammonium) chloride (PDADMAC) monolayer or PDADMAC/poly(sodium 4-styrenesulfonate) (PSS) multilayer coatings, using optimal coating conditions previously determined. Results show that the coated capillaries are remarkably stable and efficient to limit protein adsorption under a variety of extreme electrophoretic conditions even in the absence of the coating agent in the background electrolyte which is exceptional for non-covalent coatings. Monolayer coated capillaries were demonstrated for the first time to be stable to acidic rinses and to organic solvents which proves that the stability of the capillaries is highly dependent on the coating procedure used. In addition, PDADMAC/PSS multilayer coatings were found to be stable to alkaline treatments. PDADMAC/PSS coated capillaries gave excellent performances for the analysis of proteins covering a large range of pI (4-11) and of molecular weight (14-65 kDa) over a wide pH range (i.e. 2.5-9.3). Even at high pH 9.3, protein analysis was possible with very good repeatabilities (RSD(tm)<1% and RSD(CPA)<2.6% (n ≥ 8)) and high peak efficiencies in the order of 700,000.

Determination of cephalosporins utilising thin-layer chromatography with fluorescamine detection.

A thin-layer chromatographic procedure utilising fluorescamine detection is described for the quantitation of cephradine, cefaclor, cephalexin and cefadroxil. The repeatability, sensitivity and detectability of the method are compared with those for o-phthalaldehyde fluorescence emission and UV absorption measurements on the plate. The applicability of the fluorescamine method to the determination of these cephalosporins in serum, urine and pharmaceutical formulations is discussed.

Liquid chromatography with amperometric detection for the determination of cephalosporins in biological fluids

The amperometric detection of cephalosporins with an aminothiazole substituent in the side-chain after HPLC separation has been investigated. The electrochemical characteristics of the cephalosporins were determined at three different pH values. A limiting current is reached at a potential of + 1.00–1.10 V vs. Ag-AgCl under the conditions used. The linearity and precision are comparable to those obtained with UV detection. The detection limits are about five times lower than those obtained with UV detection. The applicability of the method to the determination of cephalosporins in serum and urine is shown for cefmenoxine. The method is also of interest for stability studies in vitro.

Capillary electrophoresis methods for the analysis of antimalarials. Part I. Chiral separation methods

This paper presents an overview on the current status of enantiomeric and diastereomeric separations of chiral antimalarials and derivatives by capillary electrophoresis (CE). The wide variety of chiral selectors which have been employed to resolve successfully antimalarial enantiomers: oligosaccharides (cyclodextrins, oligomaltodextrins), neutral (amylose, dextrin and dextran) and charged (chondroitin sulfate, heparin, dextran sulfate) polysaccharides and proteins are reviewed. Cyclodextrins were the most employed. Chiral additives added to the background electrolyte often facilitated separations of quinine/quinidine and cinchonine/cinchonidine diastereomers. However, in a few cases, using micellar electrokinetic capillary chromatography or non aqueous CE, resolution of diastereomers could be achieved without additives. Quantitative applications of CE to the quality control of antimalarial drugs and their analysis in biological and food matrices are presented.

Validation of a capillary electrophoresis procedure for the determination of calcium in calcium acamprosate

The quantitative aspects of a capillary electrophoresis (CE) technique for the determination of calcium in calcium acamprosate are reported. Separation was carried out on a 57-cm (50 cm to the detector)×75-μm I.D. fused-silica capillary at a potential of 10 kV and 25°C, using as electrolyte 10 mM imidazole containing 1 mM tetrabutylammonium sulfate adjusted to pH 4.5 with sulfuric acid. Standard solutions of calcium carbonate and test solutions of calcium acamprosate containing 10 ppm of Ca2+ and Mg2+ (internal standard) were injected hydrodynamically for 3 s. Indirect UV detection was carried out at 214 nm. A satisfactory agreement was found between CE, complexometry and theoretical content for determination in calcium acamprosate. Precision of CE on different capillaries using an internal standard complies with the requirements of quality control for the drug substance.

Determination of cefotaxime, desacetylcefotaxime, cefmenoxime and ceftizoxime in biological samples by fluorescence detection after separation by thin-layer chromatography

Cephalosporins bearing an aminothiazole group conjugated with a methoxyimino group give a strong fluorescence on a silica gel plate with a fluorescence indicator after development with an acidic mobile phase. A sensitive thin-layer chromatographic method based on this property has been developed for the determination of cefotaxime, desacetylcefotaxime, cefmenoxime and ceftizoxime. The detection limits and reliability of the proposed procedure allow the method to be used for the determination of these cephalosporins in therapeutic monitoring.

Capillary electrophoresis for the assay of fixed-dose combination tablets of artesunate and amodiaquine

BackgroundQuality control of drugs in formulations is still a major challenge in developing countries. For the quality control of artesunate and amodiaquine tablets in fixed-dose combination, only liquid chromatographic methods have been proposed in the literature. There are no capillary electrophoretic methods reported for the determination of these active substances, although this technique presents several advantages over liquid chromatography (long lifetime, low price of the capillary, low volumes of electrolyte consumption) in addition to simplicity. In this paper, a reliable capillary electrophoresis method has been developed and validated for the quality control of these drugs in commercial fixed-dose combination tablets.MethodsArtesunate and amodiaquine hydrochloride in bilayer tablets were determined by micellar electrokinetic capillary chromatography (MEKC). Analytes were extracted from tablets by sonication with a solvent mixture phosphate buffer pH 7.0-acetonitrile containing benzoic acid as internal standard. Separation was carried out on Beckman capillary electrophoresis system equipped with fused silica capillary, 30 cm long (20 cm to detector) × 50 μm internal diameter, using a 25 mM borate buffer pH 9.2 containing 30 mM sodium dodecyl sulfate as background electrolyte, a 500 V cm−1 electric field and a detection wavelength of 214 nm.ResultsArtesunate, amodiaquine and benzoic acid were separated in 6 min. The method was found to be reliable with respect to specificity,linearity of the calibration line (r2 > 0.995), recovery from synthetic tablets (in the range 98–102%), repeatability (RSD 2–3%, n = 7 analytical procedures). Application to four batches of commercial formulations with different dosages gave content in good agreement with the declared content.ConclusionThe MEKC method proposed is reliable for the determination of artesunate and amodiaquine hydrochloride in fixed-dose combination tablets. The method is well-suited for drug quality control and detection of counterfeit or substandard medicines.

Stability-indicating assay for oxyphenbutazone. Part I. Thin-layer chromatographic determination of oxyphenbutazone and its degradation products

A high-performance thin-layer chromatographic procedure for the separation and determination of oxyphenbutazone and its six main potential degradation products in situ is reported. The method avoids degradation of oxyphenbutazone in situ by chelating iron in the silica plate and allows the simultaneous assay of oxyphenbutazone and its decomposition products using a chromatographic spectrophotometer. The method has been validated as a stability-indicating assay of oxyphenbutazone in tablets and capsules. It allows the determination of 0.5% of decomposition products (with respect to oxyphenbutazone). In the formulations analysed, only trace amounts of two oxidation products were found.