Sodium Dodecyl Sulfate Buffer Research Articles

Delicate Analysis of Interacting Proteins and Their Assemblies by Flow Field-Flow Fractionation Techniques.

We study the efficiency of several asymmetrical flow field-flow fractionation (AF4) techniques to investigate self-associating wheat gluten proteins. We compare the use of a denaturing buffer including sodium dodecyl sulfate (SDS) and a mild chaotropic solvent, water/ethanol, as the eluent, on a model gluten sample. Through a thorough analysis of the data obtained from coupled light scattering detectors and with the identification of molecular composition of the eluted protein, we evidence coelution events in several conditions. We show that the focus step used in conventional AF4 with the SDS buffer leads to the formation of aggregates that coelute with monomeric proteins. By contrast, a frit-inlet device enables the fractionation of individual wheat proteins in the SDS buffer. Interestingly conventional AF4, using water/ethanol as eluent, is an effective method for fractionating gluten proteins and their complex dynamic assemblies, which involve weak forces and are composed of both monomeric and polymeric proteins.

Cationic and anionic detergent buffers in sequence yield high-quality genomic DNA from diverse plant species

Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of elite germplasms. High-quality DNA, free of RNA, proteins, polysaccharides, secondary metabolites, and shearing, is mandatory for downstream molecular biology applications, especially for next-generation genome sequencing and pangenome analysis aiming to capture the complete genetic diversity within a species. The study aimed to accomplish an efficient protocol for the extraction of high-quality DNA suitable for diverse plant species/tissues. We describe a reliable, and consistent protocol suitable for the extraction of DNA from 42 difficult-to-extract plant species belonging to 33 angiosperm (monocot and dicot) families, including tissues such as seeds, roots, endosperm, and flower/fruit tissues. The protocol was first optimized for the outbreeding recalcitrant trees viz., Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera, which are rich in proteins, polysaccharides, and secondary metabolites, and the quality of the extracted DNA was confirmed by downstream applications. Nine procedures were attempted to extract high-quality, impurities-free DNA from these three plant species. Extraction of the ethanol-precipitated DNA from cetyltrimethylammonium bromide (CTAB) protocol using sodium dodecyl sulfate (SDS) buffer, i.e., the extraction using a cationic (CTAB) detergent followed by an anionic (SDS) detergent was the key for high yield and high purity (1.75–1.85 against A260/280 and an A260/230 ratio of >2) DNA. A vice versa extraction procedure, i.e., SDS buffer followed by CTAB buffer, and also CTAB buffer followed by CTAB, did not yield good-quality DNA. PCR (using different primers) and restriction endonuclease digestion of the DNA extracted from these three plants validated the protocol. The accomplishment of the genome of P. cineraria using the DNA extracted using the modified protocol confirmed its applicability to genomic studies. The optimized protocol successful in extracting high-quality DNA from diverse plant species/tissues extends its applicability and is useful for accomplishing genome sequences of elite germplasm of recalcitrant plant species with quality reads.

Eco-friendly micellar HPLC approach for assessment of two anti-asthmatic breezhalers; Application of central composite design

Short-term breathing problems are brought on by asthma, a chronic lung disease. Mometasone furoate (MOF) and Indacaterol (IDT), are co-formulated alone or with Glycopyrronium (GLY) in the form of inhalation powder to control the symptoms of asthma. A novel green hybrid micellar liquid chromatographic method was developed to analyze the three mentioned medications. Contrary to routine analysis, Quality by Design methodology was applied to maximize the resolution with the fewest number of trials. Fractional factorial design was used to study the impact of three significant variables: sodium dodecyl sulphate (SDS) concentration, methanol %, and the mobile phase pH. Separation of the analytes was optimized by central composite design using a mobile phase consists of (191.49 mM) SDS and (10.00 mM) potassium dihydrogen phosphate buffer: methanol (77.50: 22.50 v/v), pH 3.00 on a reversed phase C18 column, within eight minutes. The flow rate was 1.2 mL/min, with 20 µL injection volume, and UV detection at 210 nm. Green Analytical Procedure Index, and Analytical Greenness Calculator were used for the method’s greenness evaluation, and it was deemed to be an effective green methodology. The developed approach was effectively applied for quantitative determination of the three drugs in bulk powder and two pharmaceutical dosage forms. International Conference of Harmonization requirements were adhered to when validating the approach. Under optimized conditions, calibration curves of the studied drugs were linear over the concentration range of (10.00 – 100.00 µg/mL) with high accuracy results and correlation coefficients of 0.9997 for MOF, 0.9996 for IDT and 0.9996 for GLY. Additionally, a precise approach has been demonstrated by the intra- and inter-day coefficients of variation ≤ 2%. The LOD and LOQ were (2.39 μg/mL) and (7.25 μg/mL) for MOF, (2.67 μg/mL) and (8.08 μg/mL) for IDT, and (2.80 μg/mL) and (8.48 μg/mL) for GLY, respectively.

Comparison of protein characterization using In solution and S-Trap digestion methods for proteomics

Protein extraction and digestion are important analytical steps in the study of proteomics. The use of sodium dodecyl sulfate (SDS) buffer makes it possible to effectively analyze various proteins. Its use was evaluated using the S-Trap digestion method and compared to the traditional In solution digestion method. Differences in protein composition were examined for each protein preparation method. S-Trap digestion followed by SDS buffer extraction clearly increased the number of identified proteins, including more mitochondrial and membrane-related proteins. The S-Trap digestion method with 5% SDS buffer was applied to the pellet remaining from the removal of RIPA buffer-soluble proteins, which identified more extracellular space proteins than the conventional S-Trap digestion method. S-Trap digestion of the pellet was particularly advantageous for identifying proteins located inside multilayer membranes.

Optimizing Decellularization Strategies for the Efficient Production of Whole Rat Kidney Scaffolds.

Renal dysfunction remains a global issue, with chronic kidney disease being the 18th most leading cause of death, worldwide. The increased demands in kidney transplants, led the scientific society to seek alternative strategies, utilizing mostly the tissue engineering approaches. Unlike to perfusion decellularization of kidneys, we proposed alternative decellularization strategies to obtain acellular kidney scaffolds. The aim of this study was the evaluation of two different decellularization approaches for producing kidney bioscaffolds. Rat kidneys from Wistar rats, were submitted to decellularization, followed two different strategies. The decellularization solutions used in both approaches were the same and involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate and sodium dodecyl sulfate buffers for 12h each, followed by incubation in a serum medium. Both approaches involved 3 decellularization cycles. Histological analysis, biochemical and DNA quantification were performed. Cytotoxicity assay and repopulation of acellular kidneys were also applied. Histological, biochemical and DNA quantification confirmed that the 2nd approach had the best outcome regarding the kidney composition and cell elimination. Acellular kidneys from both approaches were successfully recellularized. Based on the above data, the production of kidney scaffolds with the proposed cost- effective decellularization approaches, was efficient.

Optimisation of Sporosori Purification and Protein Extraction Techniques for the Biotrophic Protozoan Plant Pathogen Spongospora subterranea.

Spongospora subterranea is a soil-borne plant pathogen responsible for the economically significant root and powdery scab diseases of potato. However, the obligate biotrophic nature of S. subterranea has made the detailed study of the pathogen problematic. Here, we first compared the benefits of sporosori partial purification utilizing Ludox® gradient centrifugation. We then undertook optimization efforts for protein isolation comparing the use of a urea buffer followed by single-pot solid-phase-enhanced sample preparation (SP3) and a sodium dodecyl sulphate (SDS) buffer followed by suspension-trapping (S-Trap). Label-free, quantitative proteomics was then used to evaluate the efficiency of the sporosori purification and the protein preparation methods. The purification protocol produced a highly purified suspension of S. subterranea sporosori without affecting the viability of the spores. The results indicated that the use of a combination of SDS and S-Trap for sample clean-up and digestion obtained a significantly higher number of identified proteins compared to using urea and SP3, with 218 and 652 proteins identified using the SP3 and S-Trap methods, respectively. The analysis of proteins by mass spectrometry showed that the number of identified proteins increased by approximately 40% after the purification of spores by Ludox®. These results suggested a potential use of the described spore purification and protein preparation methods for the proteomics study of obligate biotrophic pathogens such as S. subterranea.

Application of supercritical fluids for complete decellularization of porcine cartilage

Porcine cartilage was ground by cryomill (Retch, Germany) at –196°C. A fraction of cartilage microparticles (CMP) of size 100-250 µm was isolated. CMP was decellularized at room temperature with periodic mixing in 3 shifts of 0.1% sodium dodecyl sulfate buffer solution, containing an increasing concentration (1, 2 and 3%) of Triton X100. CMP treatment in a supercritical CO2 (sc-CO2) atmosphere was carried out at a pressure of 300 bar, T = 35°C, with a flow rate of sc-CO2 of 2.5 ±0.5 ml/min for 8-24 hours using RESS-SAS equipment (Waters Corporation, USA). Ethanol (96%) at a concentration of 10% was used as a polarity modifier. The degree of decellularization of CMP was assessed by histological methods (stained by hematoxylin and eosin) and by detection of the residual amount of DNA in samples using DNA-binding fluorescent dye DAPI. In the case of treatment with the detergents only and detergents after sc-CO2, the required degree of decellularization of CMP was not achieved. Histological analysis of the samples has shown that only a partial release of chondrocytes occurs. CMP treatment by detergents followed by sc-CO2 was more effective. Complete removal of cells can be achieved if the cartilage is first treated with surfactant, and then CO2. When ethanol was added as a polarity modifier, histological studies confirm that non-disrupted cells were almost completely absent and study with the DAPI dye has shown that more than 90% of CMP samples were completely free of DNA or contained only single whole cells.To achieve the highest possible degree of decellularization, the treatment of cartilage microparticles should be carried out first with detergent solutions followed by exposure to sc-CO2. The introduction of a polarity modifier (ethanol) at a concentration of 10% has a positive effect on the degree of decellularization and in combination with lengthy treatment time allows to reach complete decellularization of cartilage tissue.

An innovative method to obtain porous porcine aorta scaffolds for tissue engineering.

Decellularized porcine aorta (PA) is a promising biomaterial for vascular substitutes. However, decellularized PAs suffer from mechanical weakness and have less pores, which limit cellular ingrowth into the grafts and hinder the remodeling. In this study, PAs were decellularized by vacuum-freeze-thawing cycles and 0.3% of sodium dodecyl sulfate (SDS) buffer (VLS). Results showed that the application of vacuum-freeze-thawing significantly improved the decellularizationefficiency of SDS while effectively preserved the mechanical function of PA tissues, decreased residual SDS, and minimized cytotoxicity. Furthermore, scanning electron microscopy (SEM) examination demonstrated that VLS generated interconnected pores with uniform distribution. In vivo subcutaneous implantation assay further demonstrated that VLS implants had less calcification and adverse inflammatory response. Moreover, VLS treatment markedly enhanced ingrowth of myofibroblasts and endothelial cells, and thereby promoted synthesis of extracellular matrix and vascularization. These results suggest that the application of vacuum-freeze-thawing into thedecellularization process may produce a promising vascular graft candidate for tissue engineering application.

Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct.

Background: Current esophageal treatment is associated with significant morbidity. The gold standard therapeutic strategies are stomach interposition or autografts derived from the jejunum and colon. However, severe adverse reactions, such as esophageal leakage, stenosis and infection, accompany the above treatments, which, most times, are life threating. The aim of this study was the optimization of a decellularization protocol in order to develop a proper esophageal tissue engineered construct. Methods: Rat esophagi were obtained from animals and were decellularized. The decellularization process involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) buffers for 6 h each, followed by incubation in a serum medium. The whole process involved two decellularization cycles. Then, a histological analysis was performed. In addition, the amounts of collagen, sulphated glycosaminoglycans and DNA content were quantified. Results: The histological analysis revealed that only the first decellularization cycle was enough to produce a cellular and nuclei free esophageal scaffold with a proper extracellular matrix orientation. These results were further confirmed by biochemical quantification. Conclusions: Based on the above results, the current decellularization protocol can be applied successfully in order to produce an esophageal tissue engineered construct.

Toxic reagents and expensive equipment: are they really necessary for the extraction of good quality fungal DNA?

There are numerous methods for DNA extraction from fungi. Some rely on expensive commercial kits and/or equipments, unavailable for many laboratories, or make use of toxic chemicals such as chloroform, phenol and mercaptoethanol. This study clearly demonstrates that it is possible to obtain high yields of pure DNA from pigmented conidia without the use of strong and expensive cell disrupting procedures and of toxic reagents. The method herein described is simultaneously inexpensive and adequate to DNA extraction from several different types of fungi.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Variation and Correlation of Protein Molecular Weight Distribution and Semolina Quality Parameters for Durum Genotypes Grown in North Dakota

This research assessed variation of protein molecular weight distribution (MWD) parameters and their correlations with quality characteristics of semolina samples that were obtained from durum genotypes grown in North Dakota. Sodium dodecyl sulfate buffer extractable and unextractable proteins in semolina were analyzed for MWD by size‐exclusion HPLC with a microbore column. ANOVA indicated that quantitative variations of all the HPLC protein fractions were significantly (P < 0.001) influenced by growing environments. The extractable and unextractable gluten proteins correlated differently with semolina gluten characteristics. Both gluten index and mixograph classification showed positive correlations (P < 0.05) with unextractable polymeric proteins and negative correlations (P < 0.05) with extractable gliadins and polymeric proteins. Quantitative variations of gluten proteins greatly influenced spaghetti cooking characteristics. Specifically, cooked spaghetti firmness (CSF) had high and positive simple linear correlations (P < 0.001) with quantity of gluten proteins in both extractable and unextractable fractions. However, a qualitative MWD parameter, percentage of the extractable gliadins in total protein, had a negative genotypic correlation with CSF (r = –0.81, P < 0.01), whereas percentage of the unextractable polymeric proteins had a positive genotypic correlation (r = 0.75, P < 0.01). Those two MWD parameters also showed significant (P < 0.05) variations for genotypes, indicating that they might be useful for screening durum genotypes for pasta cooking quality.

Determination of HMG-CoA reductase inhibitors by micellar electrokinetic chromatography

Abstract Objective: In this study we report the development of a simple, rapid and efficient capillary electrophoresis method for the simultaneous determination of atorvastatin, fluvastatin, lovastatin and simvastin. Methods: Capillary zone electrophoresis proved to be efficient for the simultaneous separation of atorvastatin and fluvastatin, but could not resolve the determination of lovastatin and simvastatin. The simultaneous separation of all four statins was achieved by applying a micellar electrokinetic chromatographic method, after transforming lovastatin and simvastatin in β-hydroxyl acid forms through alkaline hydrolysis. The optimum electrophoretic conditions and analytical parameters were investigated and the analytical performances of the method were verified with regard to linearity, precision, accuracy, LOD and LOQ. Results: The optimum electrophoretic separation conditions were: 25 mM sodium tetraborate with 25 mM sodium dodecyl sulphate buffer electrolyte at pH 9.5, applied voltage + 25 kV, separation temperature 25 °C, injection pressure/time 50 mbar/1 minutes, UV detection at 230 nm. Using the optimized electrophoretic conditions we succeeded in the simultaneous determination of the four statins in approximately 3 minutes, the order of migration being: atorvastatin, fluvastatin, lovastatin, simvastatin. The proposed method has been applied to the determination of the analytes in pharmaceutical tablets formulations. Conclusions: The capillary electrophoretic method developed in the present work proved to be suitable for the routine analysis of statins and can be adopted as quality control protocol in pharmaceutical analysis.

Mass spectrometry data from proteomic analysis of human skin keratins after exposure to UV radiation.

A mass spectrometry (MS)-based proteomic methodology was employed to monitor oxidative modifications in keratins, the main constituents of human skin (“Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry” [1], “UV irradiation-induced methionine oxidation in human skin keratins: mass spectrometry-based non-invasive proteomic analysis” [2]). Human skin proteins were obtained non-invasively by tape stripping and solubilized in sodium dodecyl sulfate (SDS) buffer, followed by purification and digestion using the filter-aided sample preparation method. The tryptic peptides were then analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-MS, tandem MS (MS/MS), and LC/ESI-selected reaction monitoring (SRM)/MS. The MS/MS data were generated to confirm amino acid sequences and oxidation sites of tryptic peptides D290VDGAYMTK298 (P1) and N258MQDMVEDYR267 (P2), which contain the most susceptible oxidation sites (Met259, Met262, and Met296 in K1 keratin) upon UVA irradiation [2]. Subsequently, quantitative determination of the relative oxidation levels of P1 and P1 [2] was achieved by LC/ESI-SRM/MS analyses of P1 and P2 together with their oxidized forms after exposure to UVA radiation or treatment with hydrogen peroxide (H2O2).

Effect of flour polymeric proteins on dough thermal properties and breadmaking characteristics for hard red spring wheat genotypes

The aim of this research was to investigate the effect of variation of flour polymeric proteins on dough thermal properties for hard red spring (HRS) wheat genotypes grown in the U.S. state of North Dakota. Flour polymeric proteins were analyzed by size exclusion HPLC for sodium dodecyl sulfate (SDS) buffer extractable and unextractable fractions. Mixolab was employed to measure dough thermal property. The Mixolab torque profile was fitted to nonlinear equations, and envelope amplitude value was calculated by treating the torque profile as a wave. The dough tolerance to thermal weakening, as determined from the Mixolab parameters (including the constants of a nonlinear model and envelope torque amplitude values derived from the mixing torque profile during heating from 30 to 70 °C), was identified to have significant (P < 0.05) and positive correlations with flour bread loaf volume potential. Variations of high molecular weight polymeric proteins appeared to affect dough tolerance to thermal weakening and bread loaf volume for HRS wheat genotypes, and different associations with breadmaking characteristics were observed according to extractability in SDS buffer. Negative correlations with tolerance to thermal weakening and bread loaf volume were seen for SDS extractable polymeric proteins and positive correlations were observed for unextractable polymeric proteins.

Holistic analysis of seven active ingredients by micellar electrokinetic chromatography from three medicinal herbs composing Shuanghuanglian.

A simple and reliable method has been developed with a new strategy named holistic analysis of multiple constituents to evaluate the quality of the well-known traditional Chinese medicine (TCM) Shuanghuanglian (SHL) oral liquid and soft capsule. Seven main constituents of the medicine, i.e., baicalein, baicalin, chlorogenic acid, wogonin, scutellarin, forsythin and hyperin, were selected as the evaluation markers and analyzed by micellar electrokinetic chromatography. The effects of buffer pH, concentration of electrolyte, organic modifier and applied voltage on migration behavior were studied systematically. The optimum conditions for the separation were achieved in a 12.5 mM borate-10 mM sodium dihydrogen phosphate-10 mM sodium dodecyl sulfate buffer at pH 9.1 containing 10% (v/v) acetonitrile under 15 kV. The analytes were identified by their relative time with regard to para-hydroxybenzoic acid migration time used as an internal standard. The method was validated in terms of linearity, limit of detection and quantification, precision, accuracy and recoveries. The correlation coefficient ranged from 0.9962 to 0.9992. The limits of detection (S/N = 3) were from 0.15 to 3.95 μg mL(-1). Recoveries of seven analytes in the SHL samples ranged from 89.00 to 103.04%. The proposed method was successfully applied for the quality control of complicated TCM SHL.

Characteristics and Protein Subunit Composition of Flour Mill Streams from Different Commercial Wheat Classes and Their Relationship to White Salted Noodle Quality

Proximate characteristics and protein compositions of selected commercial flour streams of three Australian and two U.S. wheats were investigated to evaluate their effects on the quality of white salted noodles. Wheat proteins of flour mill streams were fractionated into salt‐soluble proteins, sodium dodecyl sulfate (SDS)‐soluble proteins, and SDS‐insoluble proteins with a sequential extraction procedure. SDS‐soluble proteins treated by sonication were subsequently separated by nonreducing SDS polyacrylamide gel electrophoresis (SDS‐PAGE). There was a substantial amount of variation in distributions of protein content and protein composition between break and reduction mill streams. SDS‐insoluble proteins related strongly to differences in protein quantity and quality of flour mill streams. The soluble protein extracted by SDS buffer included smaller glutenin aggregates (SDS‐soluble glutenin) and monomeric proteins, mainly gliadin (α‐, β‐, γ‐, and ω‐types) and albumin and globulin. SDS‐soluble proteins of different flour mill streams had similar protein subunit composition but different proportions of the protein subunit groups. Noodle brightness (L) decreased and redness (a) increased with increased SDS‐insoluble protein and decreased monomeric gliadin. Noodle cooking loss and cooking weight gain decreased with increased glutenin aggregate (SDS‐soluble glutenin and SDS‐insoluble glutenin) and decreased monomeric gliadin. Noodle hardness, springiness, cohesiveness, gumminess, chewiness, tensile strength, breaking length, and area under the tensile strength versus breaking length curve increased with increased glutenin aggregate. Monomeric gliadin contributed differently to texture qualities of cooked noodles from glutenin aggregate. Monomeric albumin and globulin were not related to noodle color attributes (except redness), noodle cooking quality, and texture qualities of cooked noodles. The results suggested that variation in protein composition of flour mill streams was strongly associated with noodle qualities.

Effect of pre-harvest sprouting on physicochemical changes of proteins in wheat

High moisture before harvest can cause sprouting of the wheat kernel, which is termed pre-harvest sprouting (PHS). The aim of this study was to examine the variation in physicochemical properties of proteins in PHS-damaged (sprouted) hard red and white spring wheat genotypes. Specifically, protein content, enzyme activity and degradation of proteins were evaluated in sound and PHS-damaged wheat. Protein contents of sprouted wheat samples were lower than that of non-sprouted samples; however, their differences were not significantly (P > 0.05) correlated with sprouting score. Sodium dodecyl sulfate (SDS) buffer extractable proteins (EXP) and unextractable proteins (UNP) were analyzed by high-performance size exclusion chromatography. PHS damage elevated endoprotease activity and consequently increased the degradation of polymeric UNP and free asparagine concentration in wheat samples. Free asparagine is known to be a precursor for formation of carcinogenic acrylamide during high heat treatment, such as baking bread. Free asparagine content had significant correlations (P < 0.01) with sprouting score, endoprotease activity and protein degradation. Genotypes with higher endoprotease activity tend to exhibit a larger degree of degradation of UNP and higher free asparagine concentration in sprouted wheat samples.

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

BackgroundArtemether-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.MethodsArtemether 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.cm-1 electric field and a detection wavelength of 214 nm.ResultsArtemether, 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.ConclusionThe 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.

Optimization and validation of a MEKC method assisted by Box–Behnken Design for fast and simultaneous determination of nitrendipine and atenolol in new antihypertensive combination tablets

In this paper, we established a micellar electrokinetic chromatography method for fast and simultaneous determination of nitrendipine and atenolol in new antihypertensive combination tablets. Buffer conditions were optimized by using a multivariate response surface methodology (RSM) established by Box–Behnken Design in terms of sodium dodecyl sulfate concentration, buffer concentration and pH of buffer. Under the optimum buffer conditions, the separation of the two drugs can be finished in 3 minutes in a 31.2 cm × 50 μm fused-silica capillary at an applied voltage of 25 kV and the temperature of 25 °C. The optimal running buffer (pH 8.9) was Na2B4O7 (7.5 mmol L−1) and NaH2PO4 (30 mmol L−1) containing 15 mmol L−1 sodium dodecyl sulfate. Good correlation coefficients were found (γ2 >0.999) at concentrations of 5–17.5 μg mL−1 for nitrendipine and 10–35 μg mL−1 for atenolol, respectively. All the RSD results of precision experiments were below 3%. The recoveries of nitrendipine and atenolol were 98.98–100.15% and 99.46–101.18%, respectively. The limits of detection of this method were 1 μg mL−1 and 0.5 μg mL−1 for nitrendipine and atenolol and the limits of quantification of this method were 2.5 μg mL−1 for nitrendipine and 1.5 μg mL−1 for atenolol. After the optimization, the method was successfully applied for the content uniformity test according to the United States Pharmacopeia.