Determination of artemether and lumefantrine in anti-malarial fixed-dose combination tablets by microemulsion electrokinetic chromatography with short-end injection procedure.

BACKGROUND: Artemether-lumefantrine (AL) combination therapy is now the most used anti-malarial treatment in the world. Quality control of AL formulations is still a major challenge in developing countries. Until now, only liquid chromatographic methods have been reported in the literature for their analysis. Capillary electrophoretic methods, which present various advantages (low price of capillary, low volumes of electrolyte consumption), may be an alternative to liquid chromatography methods. In this paper, a reliable method was developed and validated for the determination of AL in commercial fixed-dose combination tablets commercialized in Côte d'Ivoire. METHODS: Artemether and lumefantrine were determined by microemulsion electrokinetic chromatography using short-end injection procedure. The two analytes were extracted from tablets by acidified methanol. Pyrimethamine was used as internal standard. Separation was carried out in an uncoated fused silica capillary, 30 cm long × 50 µm internal diameter, using an effective length of 10 cm and a microemulsion composed of octane, butanol, sodium dodecyl sulfate and borate buffer as background electrolyte, a - 500 V x cm(-1) electric field and a detection wavelength of 214 nm. RESULTS: Artemether, lumefantrine and pyrimethamine were separated in 6 min. The method was reliable with respect to selectivity towards formulation excipients, linearity of the response function (r2 > 0.998), recovery studies from synthetic tablets (in the range 99-101%), repeatability (relative standard deviation 1-3%, n = 7 analytical procedures). Application to four commercial formulations containing 20/120 mg of AL per tablet gave a content in good agreement with the declared content. However, the electropherogram of one tablet formulation showed the presence of an ingredient which was not declared. CONCLUSION: The developed MEEKC method can be proposed as an alternative method to liquid chromatography for the determination of artemether and lumefantrine in fixed-dose combination tablet formulations.

Capillary electrophoresis methods for the analysis of antimalarials. Part II. Achiral separative methods.

An exhaustive review of applications of achiral capillary electrophoresis methods to the analysis of antimalarials is presented. It covers quality control of formulations, analysis in biological fluids and food, determination of physico-chemical properties (pKa, partition coefficient and interaction studies). Miscellaneous applications are also considered.

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.

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

BACKGROUND: Quality 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. METHODS: Artesunate 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. RESULTS: Artesunate, 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 (r(2) > 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. CONCLUSION: The 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.

Capillary zone electrophoresis as a potential technique for the simultaneous determination of sulfadoxine and pyrimethamine in tablet formulations.

A novel, simple and rapid capillary zone electrophoresis method with UV detection has been developed for the simultaneous determination of pyrimethamine and sulfadoxine in tablet formulations. The compounds are separated in 6 min in a fused silica capillary, 30 cm long (20 cm to detector)× 50 µm using a 100 mM phosphate buffer pH 7.2 as background electrolyte, a 330 V cm(-1) electric field and a detection wavelength of 214 nm. Analysis of different tablet formulations has shown a good agreement with the liquid chromatography method described in the United States Pharmacopoeia. Main advantages of the CZE method are the rapid set-up of instrumentation and capillary equilibration, short analysis time and low running cost.

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.

Use of coated capillaries for the electrophoretic separation of stereoisomers of a growth hormone secretagogue.

The diastereoisomeric separation of peptidomimetics of hexarelin, a strong growth hormone secretagogue, in CE has been studied. Highly sulfated-gamma-CD was found to be an appropriate selector for the separation of the stereoisomers. However, non-repeatable analyses were obtained on bare fused silica capillary due to the progressive adsorption of the analytes on the capillary wall. Two types of polyelectrolyte coating agents were tested to prevent this phenomenon. Coating with neutral polyethylene oxide was found to be efficient but resulted in a very long analysis time (about 40 min). Coating with cationic poly(diallyldimethylammonium) chloride was found both to prevent analyte adsorption, reduce analysis time and alter separation selectivity. EOF measurement revealed that the highly sulfated-gamma-CDs were strongly adsorbed on the poly(diallyldimethylammonium) chloride coating surface yielding a stable strong cathodic EOF, which considerably reduced analysis time (about 12 min). Very good repeatability of analysis was obtained (RSD(migration time)<1%).

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.

Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis.

Polyelectrolytes are widely used in capillary electrophoresis as coating agents of silica capillaries to prevent adsorption phenomena and improve the repeatability of peptide and protein analysis. A systematic study of the coating experimental conditions has been carried out to optimize coating stability and performance. The main experimental parameters studied were the type and concentration of polyelectrolytes used in several monolayer and multilayer coatings, the ionic strength of coating and stabilizing solutions, and the procedures used for coating and capillary storage. Electroosmotic flow magnitude, direction and repeatability were used to monitor coating stability. Coating ability to limit adsorption was investigated by monitoring variations of migration times, time-corrected peak areas and separation efficiency of test peptides. Capillary-to-capillary and batch-to-batch reproducibility was also studied. In addition, the separation performance of polyelectrolyte coatings were compared to those obtained with bare silica capillaries.

LIF detection of peptides and proteins in CE.

CE- and microchip-based separations coupled with LIF are powerful tools for the separation, detection and determination of biomolecules. CE with certain configurations has the potential to detect a small number of molecules or even a single molecule, thanks to the high spatial coherence of the laser source which permits the excitation of very small sample volumes with high efficiency. This review article discusses the use of LIF detection for the analysis of peptides and proteins in CE. The most common laser sources, basic instrumentation, derivatization modes and set-ups are briefly presented and special attention is paid to the different fluorogenic agents used for pre-, on- and postcapillary derivatization of the functional groups of these compounds. A table summarizing major applications of these derivatization reactions to the analysis of peptides and proteins in CE-LIF and a bibliography with 184 references are provided which covers papers published to the end of 2005.

Precision study on capillary electrophoresis methods for metacycline.

A CE method for metacycline (MTC) determination was investigated in an inter-laboratory experiment. Many problems were encountered in this study, most of which were related to the transfer of the method to different CE equipment. The reported problems could be classified into different categories: problems related to the precision, to the parameters in the protocol, and to the MTC peak shape. As the peak shape problem was partially responsible for the poor precision, a new CE method was developed in order to obtain a good MTC peak shape on all equipment. The precision of this new method for MTC determination was examined in an intermediate precision study, where the influence of the factors "time" and "equipment" was investigated. Although the new method could be transferred to different instruments, the precision remained poor mainly due to the contributions of the between-replicate and the between-injection variances.

Separation of 3'-azido-2',3'-dideoxythymidine pronucleotide diastereoisomers in biological samples by CZE with cyclodextrin addition.

The possibility of using capillary electrophoresis as an alternative technique to HPLC for the separation of pronucleotide diastereoisomers of AZT was investigated. In the pH range 6.2-7.2 where the analytes are stable, a chiral additive, carboxymethyl-beta-CD, was found appropriate to enable the separation of the uncharged diastereoisomers. An experimental design strategy was used to study the influence of several parameters (CD and phosphate buffer concentration, methanol content of the electrolyte, injected volume, capillary length, electric field and separation temperature) on the separation and find suitable analytical conditions for monitoring the prodrugs in cell extracts. The diastereoisomers of the three tBuSATE phenylphosphotriester derivatives of AZT studied could be fully resolved within short analysis time (less than 10 min). Method validation results showed satisfactory results for linearity, accuracy and repeatability.

Robustness testing of chiral separations by capillary electrophoresis using highly-sulfated cyclodextrins.

The robustness of a generic method for chiral separation in capillary electrophoresis using highly-sulfated cyclodextrins in a low pH phosphate buffer and the "short-end injection technique" was studied. In this study, we focused on the robustness of the separations and not of the quantitative analysis of the enantiomers. The robustness was evaluated for the enantiomeric separation of a basic (propranolol), a neutral (praziquantel) and an acidic (warfarin) compound. The influence of eight factors which were believed to affect significantly the separations was studied using a 11-factor, 12-experiment Plackett-design. Statistical interpretation of the factor effects on different analytical responses (selectivity and resolution) was performed. The separations of the three compounds could be considered as rather robust as the factor effects were generally not significant (alpha = 0.05) and small.

Influence of peak measurement parameters on the quality of chiral electrophoretic separations.

The effect of nine peak measurement/analysis parameters on chiral capillary electrophoresis (CE), followed by UV detection, was studied. The parameters pertained UV-detection (detection wavelength, reference wavelength, and wavelength bandwidths), signal processing (data acquisition rate, type and amount of filtering) and peak detection (detection threshold and peak width). The influence of these factors on the chiral separation of dimethindene enantiomers was studied at two different concentrations (i.e., at high and low signal-to-noise (S/N) ratio) by the means of experimental designs. The electropherogram characteristics considered were the resolution between the two enantiomers, the peak areas, and the S/N ratio. A D-optimal design was first used as screening design to identify the most critical parameters. Afterwards, a modelling of the different responses as a function of these critical parameters was performed based on the results of a face-centered central composite design. The results showed that the signal-processing parameters should be carefully selected when developing a CE separation since very important variations in the separation, the S/N ratio and the peak area of the substances can occur by setting these parameters at different levels. The detection wavelength should also be carefully chosen for optimal peak area measurement. The role of these parameters becomes more important with decreasing concentration of the analytes (i.e., low S/N ratio). This study showed that the peak measurement/analysis parameters should be optimized as the chemical and physical parameters of a method. They also should always be well specified in order to allow a good transfer of a method from one instrument to another.