Micellar Electrokinetic Capillary Chromatography Method Research Articles

Determination of five coumarins in Radix Glehniae by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography method with ultraviolet detection was developed for the simultaneous determination of psoralen, xanthotoxin, isoimpinellin, bergapten and scopoletin in Radix Glehniae. The separation was performed on an uncoated fused silica capillary column (50.2 cm x 75 microm x 40 cm) with 20 mmol x L(-1) borax solution (pH 9.6) containing 16 mmol x L(-1) sodium dodecylsulfate (SDS) and 15% acetonitrile as running buffer at applied voltage of 22 kV. The detection wavelength was 214 nm. The effects of concentrations of borax solution, sodium dodecylsulfate (SDS), and organic modifier, voltage, temperature on the separation and sensitivity were investigated. The five active constituents were completely separated within 7 min. The linear ranges of psoralen, xanthotoxin, isoimpinellin, bergapten and scopoletin were 9.91-82.6, 37.2-162, 2.23-18.6, 2.73-22.3 and 2.89-20.1 mg x L(-1), respectively. And the average recoveries were 98.9%, 98.4%, 101.3%, 99.1% and 98.0%, respectively. This simple and rapid method provided a new basis for assessment on quality of Radix Glehniae.

Analysis and stability study of temozolomide using capillary electrophoresis

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the analysis of temozolomide (TMZ) and its degradants, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and 5-amino-imidazole-4-carboxamide (AIC) has been studied. Using short-end injection, the analysis of TMZ and its degradants could be performed within 1.2 min. The obtained precision of migration times was better than 1.6 RSD%, and the limit of quantitation (LOQ) was 0.31–0.93 μg/mL. The therapeutic concentration of TMZ in blood samples can be determined after direct sample injection and conventional on-capillary UV detection. The proposed MEKC method was applied to study the stability of TMZ in water and serum at different pH values. It was established that the half-life of the TMZ in vitro serum at room temperature was 33 min, close to the half-life (28 min) obtained in water at pH 7.9.

Field‐amplified sample injection‐micellar electrokinetic capillary chromatography for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers and derivatives in canned soft drinks

Conditions were established for the separation and analysis of bisphenol A, bisphenol F, and their diglycidyl ethers by micellar electrokinetic capillary chromatography (MECC). Good resolution was obtained for all compounds, although in order to achieve the separation of ortho-ortho, ortho-para, and para-para isomers of bisphenol F diglycidyl ether (BFDGE), BFDGE x 2H(2)O and BFDGE x 2HCl, it was necessary to use a 25 microm id fused silica capillary. To increase sensitivity, a field-amplified sample injection (FASI)-MECC method was developed using 10 mM SDS solution as injection matrix and a 75 microm id fused silica capillary. Instrumental quality parameters such as LODs (<55 microg/L with standards), linearity (r(2)>0.999), and run-to-run and day-to-day precisions (RSD values lower than 12.5%) were determined. Finally, the suitability of the FASI-MECC method for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers in canned soft drinks was evaluated. Quantitation was performed by matrix-matched calibration using a plastic-bottled isotonic drink as matrix. The results showed that FASI-MECC is an economic method for the screening and quantitation of these kinds of compounds in soft drink beverages, with no loss of reproducibility, and effective at concentrations lower than the specific migration level values established by the European Union.

Simultaneous Determination of Phenolic Additives in Cosmetics by Micellar Electrokinetic Capillary Chromatography with Electrochemical Detection

AbstractA micellar electrokinetic capillary chromatography method with electrochemical detection (MECC‐ED) has been developed for the simultaneous determination of eight phenolic additives, including propyl gallate (PG), tert‐butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) in cosmetic products. Method development involved optimization of the working electrode, the pH value of running buffer, the concentration of sodium dodecyl sulfate (SDS), the separation voltage, and the sample injection time. Under the optimum conditions, all analytes can be well separated within 26 min at the separation voltage of 18 kV in a 9 mmol·L−1 sodium dodecyl sulfate (SDS) −60 mmol·L−1 borate running buffer (pH 8.0). A 300 μm diameter carbon disk electrode generated good response at +0.90 V (vs. SCE) for all analytes. Linearity of the present method was over three orders of magnitude of analyte concentration with detection limits (S/N=3) ranging from 1.1×10−7 to 1.2×10−6 g·mL−1 for all analytes. This proposed method has been successfully applied to the simultaneous determination of the above additives in commercial cosmetics, and the assay results were satisfactory.

Simultaneous Determination of Ezetimibe and Simvastatin in Pharmaceutical Preparations by MEKC

A micellar electrokinetic capillary chromatography method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical preparations. The influence of buffer concentration, buffer pH, sodium dodecyl sulphate (SDS) concentration, organic modifier, capillary temperature, applied voltage, and injection time was investigated, and the method validation studies were performed. The optimum separation for these analytes was achieved in less than 10 min at 30 degrees C with a fused-silica capillary column (56 cm x 50 microm i.d.) and a 25mM borate buffer at pH 9.0 containing 25mM SDS and 10% (v/v) acetonitrile. The samples were injected hydrodynamically for 3 s at 50 mbar, and the applied voltage was +30.0 kV. Detection wavelength was set at 238 nm. Diflunisal was used as internal standard. The method was suitably validated with respect to stability, specificity, linearity, limits of detection and quantification, accuracy, precision, and robustness. The limits of detection and quantification were 1.0 and 2.0 microg/mL for both ezetimibe and simvastatin, respectively. The method developed was successfully applied to the simultaneous determination of ezetimibe and simvastatin in pharmaceutical preparations.

Fast Analysis of Statins in Pharmaceuticals by MEKC

A universal micellar electrokinetic capillary chromatographic (MEKC) method with diode-array detection for the simultaneous and short-time analysis of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin was introduced. Base hydrolysis was used to open lactone ring of lovastatin and simvastatin, administered as lactone prodrugs, in order to transform these compounds to the corresponding β-hydroxyl acid forms before MEKC analysis. This approach offered shorter analysis time due to a decrease of the migration times of negatively charged statin drugs in comparison to neutral lactone forms. Optimized conditions were found to be a 25 mM borate buffer pH 9.5 with 25 mM sodium dodecyl sulphate and 10% methanol added as an organic modifier, an applied voltage of 23 kV and a separation temperature of 30 °C. Ketoprofen was used as an internal standard. The linearity of the detector response for each statin was within the concentration range from 10 to 100 μg mL−1 with a correlation coefficient greater than 0.9994. Analyses of six statin drugs in pharmaceutical samples were carried out in only 5 min. The interference of the tablet sample matrix was not observed. The recovery values were in the range of 98.04–100.80%.

Determination of Hepatotoxic Pyrrolizidine Alkaloids in Gynura segetum by MEKC

A novel, rapid and sensitive micellar electrokinetic capillary chromatography method was developed for the separation and determination of two hepatotoxic pyrrolizidine alkaloids in Gynura segetum (Lour.) Merr. (Jusanqi) within 8 min. The method was successfully applied to the simultaneous determination of seneciphylline and senecionine in a Jusanqi sample.

Characterization of molds isolated from smoked paprika by PCR-RFLP and micellar electrokinetic capillary electrophoresis

Molds are common contaminants of paprika meat products. The drying and storage stages of paprika processing are critical because they can provide molds with the conditions particularly appropriate for their growth and proliferation. Thus, an efficient and accurate characterization of the toxigenic molds of paprika is necessary. An RFLP analysis of the rRNA genes was performed by using a TaqI restriction enzyme. In addition, a micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by mold strains commonly found in paprika. This study was confirmed with a 5.8S-ITS region sequence analysis. A total of 31 isolates were identified by RFLP and MECC analysis. These showed stable RFLP profiles that were clearly different for the different genera and species, and were grouped into clusters together with the profiles of the 16 reference strains. MECC analysis provided additional characteristic peak patterns for the characterization of the mold species present. The characterized isolates were species of the genera Fusarium spp., Aspergillus spp., Penicillium spp., Cladosporium spp., Mucor spp. and Phlebia spp. The identifications were confirmed by the 5.8S-ITS region sequence analysis and by a BLAST search of the GenBank database. RFLP patterns with TaqI restriction enzyme and MECC profiles, either singly or combined, could be of great interest to distinguish molds in paprika.

Analysis of Atorvastatin and Related Substances by MEKC

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM sodium dodecyl sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100–1,200 μg mL−1 for AT and 1.0–12.5 μg mL−1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity, limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination of AT and purity evaluation of bulk drug and formulated products.

Analysis of Flavonoids in Propolis and Ginkgo biloba by Micellar Electrokinetic Capillary Chromatography

A micellar electrokinetic capillary chromatography (MEKC) method has been developed for simultaneous determination of 10 bioactive flavonoids: rutin, apigenin, luteolin, eriodictyol, kaempferol, chrysin, acacetin, flavanone, flavone, and fisetin. The effect of several parameters, such as UV detection wavelength, buffer pH, buffer concentration, sodium dodecyl sulfate (SDS) concentration, beta-cyclodextrin (beta-CD) concentration, separation voltage, and injection time on the separation of these flavonoids were systematically investigated. The 10 flavonoids were successfully separated within 18 min in 20 mM Na(2)B(4)O(7)-10 mM NaH(2)PO(4) buffer (pH 9.7) containing 100 mM SDS and 16 mM beta-CD at a separation voltage of 19 kV, with UV detection at 254 nm. Regression analysis revealed a good linear relationship between the peak area of each analyte and its concentration with detection limits (S/N = 3), ranging from 0.15 to 1.36 microg mL(-1). This method could simultaneously quantify the 10 flavonoids and be used in the quality control of functional foods containing propolis and Ginkgo biloba.

Determination of gaseous and particulate carbonyls in air by gradient‐elution micellar electrokinetic capillary chromatography

A new continuous-flow gradient-elution micellar electrokinetic capillary chromatography method is developed for the determination of airborne carbonyls after derivatization with 2,4-dinitrophenylhydrazine. A total of 16 carbonyls can be determined with detection limits ranging from 0.94 to 8.50 mg/L, working range from 4.72 to 346 mg/L, and repeatabilities (relative standard deviation, n=5) from 1.23 to 4.6% or 3.93 to 7.6% for migration time and peak area, respectively. Coupling with denuder-filter sampling, a preliminary survey has been conducted to determine gaseous and particulate carbonyls from air sampled at a roadside station. The method is shown to have sufficient sensitivity for 1-h sampling of ambient carbonyls with detection limits ranging from 0.045 to 1.2 microg/m3 and working range from 0.11 to 43.3 microg/m3 at a flow rate of 10 Lpm. The method requires minimal modification of commercially available capillary electrophoresis equipment and can differentiate gaseous and particulate carbonyls to provide essential information and objective data for adopting effective measures to combat the discharge of carbonyl compounds to the atmosphere.

Type and concentration of redox reagents influencing nitrile formation upon myrosinase ( Brassica carinata)-catalyzed hydrolysis of glucosibarin

The ratio of isothiocyanates (ITCs) to nitriles formed in the myrosinase-catalyzed hydrolysis of glucosinolates is a key factor determining the physiological effect of glucosinolate containing plants and materials. In this context, the mechanism by which nitrile formation occurs is not well understood. In the present paper we have studied the effect of three redox reagents – Fe 2+, glutathione (GSH) and ascorbic acid – on the profile of products obtained upon the hydrolysis of a model glucosinolate (glucosibarin ((2 R)-2-hydroxy-2-phenylethylglucosinolate)) catalyzed by Brassica carinata myrosinase. A Micellar Electrokinetic Capillary Chromatography method that allows following on-line the hydrolysis of the glucosinolate, the formation of the degradation products and the oxidation of GSH was used. Increasing the concentration of Fe 2+ and GSH (from 0.25- to 2-fold molar excess with respect to the glucosinolate) increased the ratio of nitrile ((2 R)-2-hydroxy-2-phenylethylcyanide) to oxazolidine-2-thione ((5 S)-5-phenyloxazolidine-2-thione), whereas increasing the concentration of ascorbic acid decreased this ratio. Low concentrations of ascorbic acid favored nitrile formation. A mechanism for nitrile formation involving a disulfide bond in the myrosinase complex is proposed.

A simple micellar electrokinetic capillary chromatographic method for the quantitative analysis of organic expectorants

A simple and rapid micellar electrokinetic capillary chromatographic method is described for the separation and quantification of five expectorant drugs, including ambroxol (AMB), bromhexine (BRM), carbocysteine, guaiacol and guaifenesin. The drugs were separated in a mixed solution of phosphate buffer (35 mM; pH 3.90) and acetonitrile (75:25, v/v) with sodium dodecylsulfate (120 mM) as the micellar source, and the separated drugs were directly monitored with UV detector (200 nm). Several key parameters affecting the separation and analysis of the drugs were studied and optimized. Based on the corrected peak-area ratios of the drugs to an internal standard (7-hydroxy-4-methyl-coumarin) versus the concentration of the drugs, the established method is applicable to quantify AMB and BRM each over 20-150 microM, carbocysteine over 100-1500 microM, guaiacol and guaifenesin each over 10-150 microM. The detection limits (S/N=3; 0.5 psi, 5 s injection) of the method for the analytes are in the range of 3.0-5.0 microM (except that of carbocysteine at 30 microM). The precision (relative standard deviation) and accuracy (relative error) of the method for the intra-day (n=3) and inter-day (n=5) analyses of the analytes at three levels are all below 4%. The method is speedy with a run time of about 6 min for the analysis of the five analytes. Application of this method to the analysis of AMB and BRM in pharmaceutical preparations or AMB in the urine of a dosed subject proved simple and effective.

Development and validation of an MEKC method for determination of nitrogen‐containing drugs in pharmaceutical preparations

A fast and accurate micellar electrokinetic capillary chromatography method was developed for quality control of pharmaceutical preparations containing cold remedies as acetaminophen, salicylamide, caffeine, phenylephrine, pseudoephedrine, norephedrine and chlorpheniramine. The method optimization was realized on a Beckman P/ACE System MDQ instrument. The baseline separation of seven analytes was performed in an uncoated fused silica capillary internal diameter (ID)=50 microm using tris-borate (20 mM, pH=8.5) containing sodium dodecyl sulphate 30 mM BGE. On line-UV detection at 214 nm was performed and the applied voltage was 10 kV. The operating temperature was 25 degrees C. After experimental conditions optimization, the proposed method was validated. The evaluated parameters were: precision of migration time and of corrected peak area ratio, linearity range, limit of detection, limit of quantification, accuracy (recovery), ruggedness and applicability. The method was then successfully applied for the analysis of three pharmaceutical preparations containing some of the analytes listed before.

Determination of Betamethasone and Dexamethasone in Human Urine and Serum by MEKC After an Experimental Design

A simple and reliable micellar electrokinetic capillary chromatography method has been presented for the simultaneous determination of betamethasone (BM) and its epimer dexamethasone (DM) in human urine and serum. A three level full factorial experimental design was employed to search for the optimum conditions. Rapid and baseline separation of BM and DM was obtained within 7 min with the optimum conditions of 30 mM borax buffer, 30 mM sodium dodecyl sulfate at pH 10.0, separation voltage at 18 kV, injection time 15 s at a height of 10 cm, using sodium sorbate as internal standard. The proposed method was validated with respect to stability, precision, linearity and accuracy. Good relationship between peak area ratio and analyte concentration was linear over 30–1,000 µg mL−1 for BM and DM with correlation coefficients ≥0.9993. Relative standard deviations of the method were all less than 4.50% in the intra-day and inter-day analysis. The developed method was applied to assay spiked human urine and serum samples containing both compounds with recoveries in the range of 97.5–100.5%.

Comparative study of robustness between micellar electrokinetic capillary chromatography and high-performance liquid chromatography using one-variable-at-a-time and a new multi-variable-at-a-time approach

Micellar electrokinetic capillary chromatography and reverse-phase liquid chromatography methods were developed in order to perform robustness testing to determine the caffeine content in beverages. Both methods were fully validated and two different robustness approaches were applied. One-variable-at-a-time (OVAT) approach at eleven levels (0; ±1; ±2; ±3; ±4; ±5 units) was carried out and compared with multi-variable-at-a-time (MVAT) approach at three levels (±1 unit per investigated parameter). Four analysts in two laboratories on two capillary electrophoresis and two RPLC equipments have tested the samples. Robustness was statistically analyzed using peak area, migration or retention time, symmetry, and resolution of caffeine and sulfacetamide as internal standard, and presented as R.S.D. values. The RPLC method was found to be more sensitive than the MEKC method. Both methods showed acceptable robustness level for OVAT approach, whilst MEKC method was more robust when the determination of real samples coming from different beverages was based on the MVAT approach.

Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography

Arnica montana preparations have been used in Europe for centuries to treat skin disorders. Among the biologically active ingredients in the flower heads of the plant are sequiterpenes, flavonoids and phenolic acids. For the simultaneous determination of compounds belonging to the latter two groups a micellar electrokinetic capillary chromatography (MEKC) method was developed and validated. By using an electrolyte solution containing 50 mM borax, 25 mM sodium dodecyl sulfate and 30% of acetonitrile the separation of seven flavonoids and four caffeic acid derivatives was feasible in less than 20 min. The optimized system was validated for repeatability ( σ rel ≤ 4.4%), precision (inter-day σ rel ≤ 8.13%, intra-day σ rel ≤ 4.32%), accuracy (recovery rates from 96.8 to 102.4%), sensitivity (limit of detection (LOD) ≤ 4.5 μg mL −1) and linearity ( R 2 ≥ 0.9996), and then successfully applied to assay several plant samples. In all of them the most dominant flavonoid was found to be quercetin 3- O-glucuronic acid, whereas 3,5-dicaffeoylquinic acid was the major phenolic acid; the total content of flavonoids and phenolic acids varied in the samples from 0.60 to 1.70%, and 1.03 to 2.24%, respectively.

Fe2+-Catalyzed Formation of Nitriles and Thionamides from Intact Glucosinolates

The ratio of isothiocyanates to nitriles formed upon the hydrolysis of glucosinolates is a key factor determining the physiological effect of glucosinolate-containing plants and materials. A micellar electrokinetic capillary chromatography (MECC) method was used to study the nonenzymatic Fe2+-catalyzed transformation of glucosinolates. At room temperature, pH 5, and in the presence of only 2 molar excess of Fe2+ all glucosinolate was degraded in 24 h. At all molar excess Fe2+ tested, nitriles were the major compounds formed. Thionamides were also formed from glucosinolates that contained a side chain hydroxylated at C-2; in this case, trace amounts of oxazolidine-2-thione were also detected. The presence of Fe3+ had no effect. The nonenzymatic Fe2+-catalyzed transformation of glucosinolates involves the binding of Fe2+ to the glucosinolate to form a complex.

Separation and measurement of desmosine and isodesmosine in vascular tissue hydrolysates by micellar electrokinetic capillary chromatography with a mixed micelle system

A micellar electrokinetic capillary chromatography (MEKC) method with a mixed micelle system for separation and measurement of desmosine (DES) and isodesmosine (IDES) in vascular tissue hydrolysates is described. The mixed micelle system was composed of a zwitterionic surfactant named 3-( N, N-dimethylhexadecylammonium)propanesulfonate (PAPS) and a nonionic surfactant polyethylene glycol dodecyl ether (Brij 35). By using 50 mM Tris–H 3PO 4 (pH 2.5) containing 40 mM PAPS and 0.5% (m/v) Brij-35 as the optimal running buffer, DES and IDES were baseline separated from each other and from other hydrolyzed components of the vascular tissue. The limit of quantitation (LOQ) for DES and IDES were 3.00 × 10 −6 mol/L and 2.75 × 10 −6 mol/L, respectively. The relative standard deviation (RSD) of migration times and corrected peak area in terms of the inter-day and the intra-day repeatability were less than 1.7%. Hydrolysate samples of vascular tissues of rats were analyzed with the MEKC method with satisfied results.

Simultaneous Determination of Nimesulide and Valdecoxib by Micellar Electrokinetic Capillary Chromatography Method

A micellar electrokinetic capillary chromatography (MEKC) method for the analysis of nimesulide and valdecoxib, using celecoxib as internal standard, has been developed and validated. The MEKC method was carried out on a fused silica capillary (50 µm I.D., effective length 56 cm). The background electrolyte consisted of 35 mM borate buffer and 35 mM of anionic detergent SDS (pH 9.75)/acetonitrile (95:5, V/V). The capillary temperature was maintained at 35°C, the applied voltage was 30 kV, and the injection was performed using the pressure mode at 50 mbar for 5 s, with detection at 234 nm using a photodiode array detector. Method validation investigated parameters such as the linearity (r2=0.9999), range, precision, accuracy, robustness, and specificity, giving results within the acceptable range. The detection limit calculated for nimesulide and valdecoxib was 0.25 and 0.86 µg mL−1, respectively, and the quantitation limit evaluated experimentally was 2 µg mL−1 for both the compounds. The proposed method was successfully applied for the quality control analysis of pharmaceutical products and the results compared to the liquid chromatography method, showing non‐significant difference (P>0.05).