Micellar Electrokinetic Capillary Electrophoresis Research Articles

Analysis of flavonoids in propolis by capillary electrophoresis

Flavonoid contents of nine raw and nine commercial propolis samples were analyzed by capillary electrophoresis. At first, 15 commercially available flavonoid standards including apigenin, chrysin, luteolin, galangin, kaempferol, morin, myricetin, quercetin, quercitrin, rutin, hesperetin, naringin, naringenin, daidzein and genistein were used to develop the electrophoretic conditions. At a running temperature of 25°C and detection wavelength at 214 nm, the separation of these standards was most satisfactorily achieved by a combination of capillary zone electrophoresis (CZE), utilizing 0.1 M borate buffer (pH 9.5) containing 5% methanol under a voltage of 18 kV, and micellar electrokinetic capillary electrophoresis (MECC), utilizing 0.03 M sodium borate (pH 8.5) containing 0.05 M SDS under a voltage of -15 kV. We found 12 out of the 15 flavonoids were identified in raw propolis, but only 11 were detected in commercial propolis products. The total contents of the identified flavonoids in raw and commercial propolis were 254 to 19147 ppm and 1228 to 7985 ppm, respectively, suggesting that both the origin and processing are important factors affecting the flavonoid content in propolis.

Enantioseparation of ofloxacin and its four related substances with ligand exchange-micellar electrokinetic chromatography using copper(II)-L-isoleucine complex as chiral selector.

A ligand-exchange micellar electrokinetic capillary electrophoresis system with copper(II)-L-isoleucine complexes as the chiral selector incorporated in micelles of sodium dodecyl sulfate (SDS) was developed for the enantioseparation of ofloxacin and its four related substances (impurities A, C, E, and F). The effects of important parameters affecting separation such as buffer pH, SDS concentration, chiral selector concentration, and organic additive were investigated in detail. Under optimum experimental conditions, enantioseparation of ofloxacin, impurities A, C, E, and F enantiomers was accomplished with resolutions of 4.28, 2.83, 3.40, 3.58, and 2.46, respectively. Further, simultaneous separation of impurities A, C, E, and F enantiomers was achieved using 10 mmol/L NH4 OAc as the running buffer containing 4 mmol/L copper sulfate,20 mmol/L L-isoleucine, 20 mmol/L SDS, and 5% methanol at pH 8.5. To the best of our knowledge, the simultaneous enantioseparation of four impurities of ofloxacin has not been reported previously.

Simultaneous determination of main phenolic acids and flavonoids in tomato by micellar electrokinetic capillary electrophoresis.

A methodology for the determination of tomato phenolic acids and flavonoids has been developed combining MEKC and DAD detection. The influence on polyphenol separation of pH and background electrolyte, BGE (borax, acetonitrile, methanol and SDS concentrations), was studied and optimized using response surface methodology and weighted desirability function. Separation of polyphenols was achieved within 20min at 15°C using 11.3mM borax and 11.2mM SDS adjusted to pH 8.5 as BGE. Validation was performed using standards and tomato extracts. Recoveries ranged from 77 to 106%. Acceptable repeatabilities were obtained for peak area (%RSD <3.1% and <3.7%) and migration times (%RSD <0.2% and <1.4%) for intra- and inter-day respectively. Detection limits ranged between 0.8 and 3.8mgkg-1. Five and seven of these polyphenols were determined in samples of tomato and related species. This methodology will be valuable tool in breeding programs, analyzing a large number of samples.

Separation and analysis of triazine herbcide residues by capillary electrophoresis

Triazines are widely used in agriculture around the world as selective pre- and post-emergence herbicides for the control of broad leaf and grassy weeds. With high toxicity and persistence, triazines can contaminate the environment and crops, so the development of rapid and sensitive methods for the determination of different triazines is necessary. Capillary electrophoresis comprises a group of techniques used to separate chemical mixtures. Analytical separation is based on different electrophoretic mobilities. This review focuses on the analysis of triazine herbicides with different modes of capillary electrophoresis, including capillary zone electrophoresis, micellar electrokinetic capillary electrophoresis, capillary electrochromatography and nonaqueous capillary electrophoresis. Determinations of triazines in various matrices such as surface water, groundwater, vegetables, soil and grains are emphasized.

Évaluation électrophysiologique de la perception consciente. Application à la médecine d’expertise

Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 102–104 cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs.

Development of a Multiplex PCR Method for the Detection of Patulin-, Ochratoxin A- and Aflatoxin-Producing Moulds in Foods

A multiplex PCR was developed for the simultaneous detection of patulin-, ochratoxin A-, and aflatoxin-producing moulds. The idh, otanpsPN and omt-1 genes involved in the patulin, ochratoxin A and aflatoxin biosynthesis, respectively, were used as target sequences in the multiplex PCR. The expected amplicons of 496, 373, and 289 bp for the corresponding primer pairs were detected in all tested toxigenic mould strains. These results were closely related to the mycotoxin detection by micellar electrokinetic capillary electrophoresis and high-performance liquid chromatography–mass spectrometry. The sensitivity of the method was tested on DNA from mould pure cultures and DNA from artificially contaminated food products (dry-fermented sausage “salchichon”, paprika, apple, wheat and peanut). The method was able to detect down to 1 ng of pure DNA from producing strains and from 103 to 104 cfu/g in inoculated foods. Inhibition from the food matrices was no detected in the PCR assay. The developed multiplex PCR protocol is a good alternative to traditional diagnostic methods for the simultaneous and early detection of different types of toxigenic moulds in foods.

Development of a PCR protocol to detect ochratoxin A producing moulds in food products

Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 102–104 cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs.

Development of Two Quantitative Real-Time PCR Methods Based on SYBR Green and TaqMan to Quantify Sterigmatocystin-Producing Molds in Foods

Sterigmatocystin (ST) is a mycotoxin produced by different species of molds that can grow and contaminate some foods such as dry-ripened foods, cereals, and spices. To improve food safety, the presence of ST-producing molds in foods should be quantified. In the present work, two real-time quantitative PCR (qPCR) protocols based on SYBR Green and TaqMan were developed. Twenty-eight ST-producing and non-producing strains belonging to different species usually reported in food products were used. The ST production of the reference strains was checked by micellar electrokinetic capillary electrophoresis and high-performance liquid chromatography–mass spectrometry. For development of the qPCR methods, a primer pair (FluGF1/FluGR1) and a TaqMan probe (FluGp) were designed on the basis of fluG gene, involved in the ST biosynthesis. The presence of fungal DNA was tested by using a qPCR method based on the universal fungal β-tubulin gene. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the fluG gene copies number and Ct values for the different ST producers tested. The ability to quantify ST producers of the developed SYBR Green and TaqMan assays in different artificially inoculated food samples and naturally infected samples was successful, with a minimum detection limit of 1 log cfu/g. In conclusion, both qPCR methods developed allow quantifying ST-producing molds, in raw materials/ingredients and pre-processed foods, which they would be very useful for monitoring these toxigenic molds in HACCP programs, to prevent ST accumulation in processed foods.

Development of a PCR Protocol To Detect Aflatoxigenic Molds in Food Products

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.

Duplex real-time PCR method with internal amplification control for quantification of verrucosidin producing molds in dry-ripened foods

Verrucosidin, which is a tremorgenic mycotoxin responsible for neurological diseases, has been detected in different dry-ripened foods as consequence of the growth of toxigenic molds. To improve food safety, the presence of verrucosidin producing molds in these kind foods should be quantified. The aim of this study was to design a duplex real-time PCR (qPCR) protocol based on TaqMan methodology with an internal amplification control (IAC). Eleven verrucosidin producing and 11 non producing strains belonging to different species often reported in food products were used. Verrucosidin production was tested by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair (VerF1/VerR1) and a TaqMan probe (Verprobe) were designed from the SVr1 probe sequence of a verrucosidin producing Penicillium polonicum. The conserved regions of the β-tubulin gene were used to design primers (TubF1/TubR1) and probe (Tubprobe) of the non-competitive IAC. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves which relating Ct values and DNA template of the tested verrucosidin producers using the verrucosidin and IAC primers. The ability to quantify verrucosidin producers of the developed TaqMan assay in all artificially inoculated food samples was successful, with a minimum detection limit of 1 log cfu per gram of food. This qPCR protocol including an IAC could be very useful to quantify verrucosidin producing molds in dry-ripened foods avoiding false negative results. This method should be proposed to monitor the target molds in HACCP programs to prevent the risk of verrucosidin formation and consequently avoid its presence in the food chain.

Examination of colour inkjet printing inks by capillary electrophoresis

The possibility of comparing inkjet printing inks by micellar electrokinetic capillary electrophoresis (MECC) with diode array detection was studied. An analytical procedure was designed and successfully applied to discriminate between the electrophoretic profiles of inks (extracted from paper) produced by five well-known manufacturers. The separation process was conducted in a polyimide-coated fused silica capillary (ID 50μm, 60cm total/50cm effective length) with +30kV high voltage applied. Background electrolyte was used of the following optimum composition: 40mM sodium borate buffer, 20mM sodium dodecyl sulphate(IV) (SDS) and 10% (v/v) acetonitrile (pH 9.56). The experimental conditions were adjusted in terms of resolution and analysis time. The best results were obtained at 10 and 25°C storage and capillary temperature, respectively, using 25 dots (ø 0.8mm) cut from printouts as the sample and BGE diluted with water (1:99, v/v) as the injecting solution. The MECC separation of main printing ink components by the proposed method showed excellent precision – the RSD value of the migration time calculated for each of the investigated peaks did not exceed 3.3%. The optimized method was applied to group identification and differentiation of: (a) three colours of printing inks, (b) inks from different manufacturers (Hewlett–Packard, Epson, Brother, Lexmark and Canon) and (c) inks from different printer models. In all these cases, inks were successfully differentiated on the basis of position (migration time) and shape of their characteristic peaks.

Determination of tea fermentation degree by a rapid micellar electrokinetic chromatography

A micellar electrokinetic capillary electrophoresis (MEKC) method was used to rapidly determine seven tea catechins, three xanthines, gallic acid, vitamin C, theanine and theaflavin in tea. All the compounds were successfully separated (20 kV) within 8 min of migration in the running buffer (pH 7.0) containing 10 mM phosphate, 4 mM sodium tetraborate, 45 mM SDS and 0.5% ethanol. The linear dynamic ranges for polyphenols were from 2.5 (S/N >8) to 100 μg/ml. As revealed by the electropherograms, tea catechins were found to be depleted along with the indoor withering process for making pouching tea, and the fermentation degree can be quantified. The results were compared with the redox potentials of tea infusions.

Separation and Determination of Low Abundant Flavonoids in Scutellaria Baicalensis Georgi by Micellar Electrokinetic Capillary Electrophoresis

Flavonoids are secondary metabolites in many plants with diverse biological activities. A simple and rapid micellar electrokinetic capillary electrophoresis method was developed for simultaneous determination of three low abundant flavonoids (chrysin, 5,7,4′-trihydroxy-8-methoxyflavone, and skullcapflavone II) in Scutellaria baicalensis Georgi within 12 min. The calibration curves revealed a good linear relationship between the peak areas of analytes and their concentrations (11.38 to 436.67 mg/L). The standard deviations of the migration time of the three flavonoids were determined to be 0.64%, 0.83%, and 1.17%, and peak areas were 6.14%, 7.57%, and 8.33%. Recoveries ranged between 90.37 and 109.4%.

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.

The influence of ligands on the preparation and optical properties of water-soluble CdTe quantum dots

In a systematic investigation has been found that ligands play an important role in both the water-phase preparation and optical properties of CdTe quantum dots. Experiments were performed using three typical thioalkyl acids as ligands, namely mercaptoacetic acid (MAA), l-cysteine (Cys) and reduced glutathione (GSH). The growth rate and size-distribution of CdTe quantum dots (QDs) are shown to depend on the type of ligands. A proper choice of ligand enables to make lager nanocrystals with narrower size-distribution. The effects of pH (buffer solution), illumination, heating and cations on the spectroscopic properties of CdTe QDs for the three ligands are reported. In addition, three same-size CdTe QDs were individually characterized by micellar electrokinetic capillary electrophoresis with laser-induced fluorescent detection, which proved their monodisperse size-distribution and different electric charge distribution on the surface for each of the three different type of QDs.

Effect of fermentation conditions on the antioxidant compounds and antioxidant capacity of Lupinus angustifolius cv. zapaton

To study the effect of fermentation on the antioxidant compounds and antioxidant capacity of Lupinus angustifolious cv. zapaton, two different types of fermentation processes were performed. Solid-state fermentations in cracked seeds carried out by Aspergillus oryzae, Rhizopus oryzae and Bacillus subtilis and liquid state fermentations in flour and cracked seeds carried out by the microbial population present in the seed (natural fermentation) or by L. plantarum inocula. Antioxidant compounds that were quantified included vitamin C by micellar electrokinetic capillary electrophoresis, vitamin E isomers by high performance liquid chromatography, total phenolic compounds (TPC) by spectrophotometry and reduced glutathione (GSH) by spectrofluorimetry. The antioxidant capacity was analysed by determining the superoxide dismutase-like activity (SOD-like activity), Peroxyl Radical-Trapping Capacity (PRTC) and Trolox Equivalent Antioxidant Capacity (TEAC) and by in vitro methods using unilamellar liposomes of egg yolk phosphatidylcholine (PC). In general, fermentation process produced a reduction in vitamin C, vitamin E activity, GSH and SOD-like activity, however TPC, PRTC, TEAC and inhibition of PC peroxidation increased under most of the fermentation conditions. Optimal results to obtain functional lupin products were achieved in cracked seeds fermented with B. subtilis where increases in TPC content, PRTC, inhibition of PC peroxidation and TEAC content of 490, 669, 492 and 224%, respectively, were found. Also, fermentation carried out with L. plantarum in lupin flours and naturally in cracked seeds caused smaller, although significant (P ≤ 0.05) increases in TPC, PRTC, inhibition of PC and TEAC (80–148, 50–90, 23 and 45–65%, respectively).

Determination of active components in Chinese medicinal preparations by capillary electrophoresis

AbstractThe determination of paeoniforin, paeonol, and censenoside Rg1 in traditional Chinese medicinal preparations, Tze Po San Pien pills and Liuwen Dihuang pills has been investigated by micellar electrokinetic capillary electrophoresis with borate buffer (20 mM), sodium dodecylsulfate (30 mM) and acetonitrile (20%) as background electrolytes (pH 9.30), 20 kV applied voltage and 203 nm UV detection. The effects of SDS concentration, borate, buffer pH, and organic modifier on electrophoretic behavior and separation are discussed. Regression equations revealed linear relationships between the peak-area of each component and the content with the correlation coefficients from 0.9982 to 0.9999. In addition, the levels of the active compounds in two kinds of traditional Chinese medicinal preparations were easily determined with the recoveries from 93.1% to 108.2%.

Experimental design and capillary electrophoresis for simultaneous analysis of arbutin, kojic acid and hydroquinone in cosmetics

A statistical experimental design was used to optimize one micellar electrokinetic capillary electrophoresis (MEKC) for simultaneous analysis of arbutin (AR), kojic acid (KA) and hydroquinone (HQ). Untreated fused-silica capillaries were operated using a phosphate buffer (20 mM, pH 6.5) under 20 kV and detection at 200 nm. Quantitative linear ranges were 20–200 μg/ml for AR, 20–100 μg/ml for KA and 8–80 μg/ml for HQ with correlation coefficients ≥0.9994. R.S.D. and R.E. were less than 3.0% for the intra-day and inter-day analysis, and all recoveries were greater than 99%. Our method was applied to assay commercial cosmetics. The results were within the labeled amount of 99.6–102.5%.

Instrumentation for Medium-Throughput Two-Dimensional Capillary Electrophoresis with Laser-Induced Fluorescence Detection

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.

Determination of Phosphoamino Acids by 6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein and MEKC with LIF Detection

The fluorescent reagent 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein (SAMF), has been newly synthesized for use as a label for characterization of phosphoamino acids by micellar electrokinetic capillary electrophoresis (MEKC) with laser-induced fluorescence (LIF) detection. The conditions for derivatization and separation of the phosphoamino acids, including pH, concentration of electrolyte, and Brij-35 concentration were optimized in detail. Derivatization was performed at 35 °C for 10min in borate buffer (pH 8.0). The derivatives were separated to baseline by use of running buffer containing 50 mM borate and 20 mM Brij-35 at pH 9.3. Detection limits ranged from 5 × 10−11 to 1 × 10−9 mol L−1 (signal to noise ratio = 3). The method was used for characterization of the phosphoamino acids in a sample from hydrolysis of a novel protein kinase from tobacco cells.