Capillary Electrophoretic Method Research Articles

Capillary electrophoretic analysis of hydroxyl radicals produced by respiring mitochondria

Here, we report the use of a capillary electrophoretic method with laser-induced fluorescence detection to evaluate hydroxyl radicals produced by respiring mitochondria. The probe, hydroxyphenylfluorescein (HPF), is separated from the product, fluorescein, in under 5 min with zeptomole and attomole limits of detection for fluorescein and HPF, respectively. Purification of the probe with a C-18 SPE column is necessary to reduce the fluorescein impurity in the probe stock solution from 0.4% to less than 0.001%. HPF was responsive to hydroxyl radicals produced by isolated mitochondria from L6 cells, and this signal was blunted when DMSO was added to scavenge hydroxyl radicals and when carbonyl cyanide m-chlorophenylhydrazone was added to depolarize the mitochondria. The method was used to compare hydroxyl radical levels in mitochondria isolated from brown adipose tissue of lean and obese mice. Mitochondria from obese mice produced significantly more hydroxyl radicals than those from lean mice.

Simultaneous Trace Level Determination of Potentially Genotoxic Hydrazine, Methylhydrazine and Alkylamines in Pharmaceutical Substances by CE Using Indirect Photometric Detection

A simple, fast, and sensitive capillary electrophoretic method was developed for determining two genotoxic impurities hydrazine and methylhydrazine and eight alkyl amines at trace levels in pharmaceutical substances using indirect photometric detection. The method development involved a systematic screening of various cationic visualizing reagents and optimization of separation conditions to obtain the best resolution and sensitivities. The optimized method was validated for specificity, precision, linearity, and accuracy. Linear calibration curves (R > 0.99) were obtained for all analytes in the range LOQ–200 % of nominal concentrations. The developed CE method was effectively implemented for estimating hydrazine and alkylamines in several active pharmaceutical ingredients (APIs).

DETERMINATION OF ZEARALENONE BY THE CAPILLARY ZONE ELECTROPHORESIS-UV DETECTION AND ITS APPLICATION TO POULTRY FEED AND CEREALS

A capillary zone electrophoretic method for the determination of zearalenone is described in this study. In the separation, a run buffer consisting of 20 mM sodium tetraborate at pH 9.0 with 15% acetonitrile applying 15 kV, injecting 10 s at 50 mbar was utilized. Phenobarbital was a suitable internal standard and the signals were recorded at 254 nm. Zearalanon and internal standard appeared at 5.46 min (RSD% 0.20) and 6.35 min (RSD% 0.18), respectively. The repeatability results are in the range of 0.01–1.58 for inter-day. The linearity was investigated in the range of 5.20 × 10−7 M to 7.86 × 10−5 M ZEA and it was linear fitting to the equation of [rPN = 244803.8 C(M) −0.0348; r = 0.9999]. The LOD and LOQ were calculated to be 2.70 × 10−8 M (8.25 µg/L) and 8.17 × 10−8 M (25 µg/L), respectively. Then, the method was successfully applied to the analysis of ZEA in poultry feeds, flour of maize, grain, and certain cereal samples such as fibrous biscuit, popcorn, and rice crisp.

Capillary electrophoretic and extraction conditions for the analysis of Catha edulisFORKS active principles

A capillary electrophoretic method, which allowed the detection and separation of the active principles of Catha edulis, i.e. cathinone, cathine and phenylpropanolamine, was developed. A suitable internal standard (nicotinamide), which permitted the quantification of the analytes reducing the variability of the migration times due to EOF changes, was identified. The analytical method was validated, assessing linearity, sensitivity and repeatability, showing optimal features for the analysis of the vegetable material. Moreover extraction conditions were investigated to achieve the exhaustion of the plant material in the fastest and most efficient way to meet the requirements of the Court.

Oroxylum indicum Seeds—Analysis of Flavonoids by Micellar Electrokinetic Chromatography

Flavonoids are bioactive constituents in Oroxylum indicum seeds, an Asian traditional remedy used for the treatment of respiratory infections. In this study the first capillary electrophoretic method for their determination is presented. By using a 25 mM borax buffer at pH 9.2 containing 10 mM SDS as detergent, the determination of seven flavonoids was feasible in only 13 min. Method validation confirmed that the assay is in accordance with ICH requirements in respect to linearity, selectivity, sensitivity, accuracy and precision. Quantitative results revealed that baicalein-7-O-gentiobioside is the most abundant flavonoid in the drug (1.19 to 5.33%), followed by other baicalein derivatives (7-O-glucoside, 7-O-glucuronide). These observations were in good qualitative and quantitative agreement with LC-MS results.

Principles of micellar electrokinetic capillary chromatography applied in pharmaceutical analysis.

Since its introduction capillary electrophoresis has shown great potential in areas where electrophoretic techniques have rarely been used before, including here the analysis of pharmaceutical substances. The large majority of pharmaceutical substances are neutral from electrophoretic point of view, consequently separations by the classic capillary zone electrophoresis; where separation is based on the differences between the own electrophoretic mobilities of the analytes; are hard to achieve. Micellar electrokinetic capillary chromatography, a hybrid method that combines chromatographic and electrophoretic separation principles, extends the applicability of capillary electrophoretic methods to neutral analytes. In micellar electrokinetic capillary chromatography, surfactants are added to the buffer solution in concentration above their critical micellar concentrations, consequently micelles are formed; micelles that undergo electrophoretic migration like any other charged particle. The separation is based on the differential partitioning of an analyte between the two-phase system: the mobile aqueous phase and micellar pseudostationary phase. The present paper aims to summarize the basic aspects regarding separation principles and practical applications of micellar electrokinetic capillary chromatography, with particular attention to those relevant in pharmaceutical analysis.

Assay of Citrus Flavonoids, Troxerutin, and Ascorbic Acid in Food Supplements and Pharmaceuticals by Capillary Zone Electrophoresis

Capillary electrophoretic method suitable for the assay of citrus flavonoids (hesperidin, diosmin, and rutin), troxerutin, and ascorbic acid in food supplements and pharmaceutical formulations was devised and validated. The separation was carried out at 25 kV in uncoated fused-silica capillary (50 μm i.d.; total length, 32 cm (21 cm to detector); and spectrophotometric detection at 280 nm) maintained at 25 °C with 40 mM sodium tetraborate buffer of pH 9.5 containing 25 % (v/v) of methanol as the background electrolyte. The samples were injected hydrodynamically (50 mbar, 6 s). The calibration curves were linear (correlation coefficients r = 0.9994–0.9998) for 0.05–0.50 mg/mL of hesperidin, diosmin, rutin, and troxerutin and 0.10–1.00 mg/mL of ascorbic acid when using cinnamic acid (0.05 mg/mL) as internal standard (IS). The LOQ values (S/N = 10) ranged between 0.02 and 0.04 mg/mL of an analyte. The intra- and interday repeatability of migration times and corrected peak areas was characterized by RSD ≤ 3.6 %. Single CE analysis of a standard mixture containing all five analytes and the IS took less than 11 min. Accuracy of the method was evaluated by the added/found analyte recovery experiments (recoveries, 95.5–99.8 %). The method was applied to the analysis of five commercially available food supplements and pharmaceuticals.

Effective Pre-Concentration and Analysis of Heavy Metals Using Ligand Step Gradient Focusing in Combination with Isotachophoresis

A capillary electrophoretic method for the pre-concentration of metals based on selective focusing of metal chelates with subsequent online ITP analysis was developed and verified. The selected ions of heavy metals (Pb2+ and Cd2+) were subjected to pre-concentration from the mixture and analysed. Focusing of the metals was carried out in a ligand step gradient, which was created by the addition of a convenient ligand agent to the regular stationary pH step gradient. The metal ions were continuously dosed into the column, where they were selectively trapped (with the exception of Na+) on the stationary ligand step gradient in the form of non-moving zones of citrate complexes with an effective charge of zero. After a detectable amount of analyte was accumulated, the dosing was stopped and the accumulated zones were mobilized to the analytical column, where they were analysed by the ITP method with conductivity or photometric detection. The electrolyte systems for the dosing (mode IEF), mobilizing (mode MBE), and analytical steps (mode ITP) were developed and tested. The cLOD was decreased by approximately two orders of magnitude using an acceptable dosing time of 25 min.

Capillary Electrophoresis of Clenbuterol Enantiomers and NMR Investigation of the Clenbuterol/Carboxymethyl- -cyclodextrin Complex

A capillary electrophoretic method has been established for the separation of the enantiomers of clenbuterol. The effects of pH value, composition of the background electrolyte, concentration of carboxymethyl-β-cyclodextrin (CM-β-CD), capillary temperature and running voltage have been investigated. The two enantiomers were separated in an uncoated capillary with phosphate buffer (50 mmol/L, pH 3.5) containing 10 mmol/L CM-β-CD. The capillary temperature was at 15°C and applied voltage was at 20 kV. The inclusion complex of CM-β-CD and clenbuterol was synthesized and characterized by two-dimensional rotating frame spectroscopy (2D ROESY). Based on the 2D ROESY analysis, an inclusion structure of the clenbuterol/CM-β-CD complex was proposed, in which clenbuterol penetrated CM-β-CD in a tilted manner due to the interaction of intermolecular hydrogen bonds between clenbuterol and CM-β-CD.

Development of Capillary Electrophoretic Method Combined with Accelerated Solvent Extraction for Simultaneous Determination of Residual Sulfonamides and Their Acetylated Metabolites in Aquatic Products

A rapid and effective method was developed in the first time for the determination of four sulfonamides (SAs) and their N4-acetylated metabolites in aquatic products using capillary electrophoresis with accelerated solvent extraction (ASE). The eight residues were extracted with acetonitrile at 70 °C under 10.3 MPa pressure and two static cycles with a static time of 5 min. The eight analytes can be baseline separated with 6 min. The standard curves for the determination of eight residues by the capillary electrophoretic method have good linearity (r2 > 0.999). The method limits of quantification (LOQs) for N4-acetylsulfadiazine, sulfadiazine, sulfamerazine, sulfamethoxazole, sulfamethazine, N4-acetylsulfamethazine, N4-acetylsulfamerazine, and N4-acetylsulfamethoxazole in aquatic products were 26.4, 33.0, 33.0, 33.0, 39.6, 370, and 1,076 μg kg−1, respectively. Intra- and inter-day precision (RSD) were 0.4–2.2 % and 1.4–3.5 %, respectively. Their average recoveries with three spiked levels ranged from 83 % to 116 % with the RSDs less than 4 %. The proposed method provides a rapid and simple extraction procedure with high sensitivity and recoveries, and can permit routine detection of the studied SAs residues in aquatic products at the maximum residue level.

Field-amplified sample stacking–sweeping of vitamins B determination in capillary electrophoresis

A capillary electrophoretic method for determination of five water soluble vitamins B along with baclofen as an internal standard has been developed and assessed in context of precision, accuracy, sensitivity, freedom from interference, linearity, detection and quantification limits. On-line preconcentration technique, namely field-amplified sample stacking (FASS)–sweeping, has been employed in respect to obtain more sensitive analysis. Separation conditions received after optimization procedure were as following background electrolyte (BGE), 10mM NaH2PO4, 80mM SDS, (pH 7.25); sample matrix (SM), 10mM NaH2PO4 (pH 4.60); uncoated fused silica capillary (50μm i.d.×67cm length); UV spectrophotometric detection at 200nm; injection times: 10s and 30s at 3.45kPa; applied voltage 22kV; temperature 22°C. Validation parameters, namely precision, accuracy and linearity, were considered as satisfactory. Under the optimized conditions, it has been also successfully applied for vitamins B determination in bacterial growth medium and commercially available Ilex paraguariensis leaves.

Separation and determination of chiral composition in penicillamine tablets by capillary electrophoresis in a broad pH range

A chiral capillary electrophoretic method with nearly full pH window was explored for the separation and determination of dl-penicillamine. A facile one-pot labeling technique was coupled in the method for introduction of chromophore and charge groups onto the analytes to facilitate the electromigration and sensitive detection. By using simply a cost-effective neutral β-cyclodextrin as chiral selector, baseline separation of the dl-penicillamine was achieved from pH 2.0 to over pH 10. Quantification of standard d- and l-penicillamines was demonstrated by taking pH 4.5, 7.4, and 9.7 as the representatives of acidic, neutral, and basic conditions. The working curves were constructed between peak area and concentration, having linear ranges of 8.56-8.56 × 10(2) μg/mL for pH 4.5 and 8.56-1.71 × 10(3) μg/mL for pH 7.4 and 9.7, with correlation coefficients all better than 0.999. The limit of detection (S/N = 3) was 2.58 μg/mL in acidic and neutral conditions or 1.41 μg/mL in basic condition. The method was further validated by assaying the commercial penicillamine tablets, applicable to quantification of the effective enantiomer and the trace impurity of l-penicillamine at a content of down to 0.2, 0.6, and 2.0% for pH 9.7, 4.5, and 7.4, respectively. The recovery determined by spiking technique was in a range from 93.1 to 105 %. The method is easily extendable to the analysis of other chiral amines or amino acids.

Capillary electrophoretic enantioselective determination of zopiclone and its impurities

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL⁻¹ for each zopiclone enantiomer, 0.8-1.6 μg mL⁻¹ for 2-amino-5-chloropyridine and 0.4-0.8 μg mL⁻¹ for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.

Application of a new capillary electrophoretic method for the determination of carbohydrates in forensic, pharmaceutical, and beverage samples

A new capillary electrophoresis method dedicated to the analysis of neutral underivatized carbohydrates was recently developed by our group. It involved a background electrolyte composed of 98mM NaOH and 120mM NaCl, and direct UV detection via the formation of an absorbing intermediate in the detection window by photooxidation. This article focuses on the validation of this method for the determination of fructose, glucose, lactose, and sucrose in forensic, pharmaceutical, and beverage samples. Intermediate precisions were about 2.3% for normalized corrected peak areas and 1.8% for normalized migration times using naphthalenesulfonate as internal standard. Limits of detection varying from 5μM for sucrose and lactose to 7μM for glucose and 10μM for fructose were obtained. Potential matrix effects were statistically studied for soil, cloth, plastic, cotton, red wine, and with simulated iron, calcium, and sucrose-based matrices, containing various inorganic anions and cations, sometimes at high levels. No significant matrix effect was observed. Finally, analyses of real post-explosion residues, smoke device, cough syrup, red wine, and apple juice were successfully performed.

Stereoselective analysis of endomorphin diastereomers: Resolution of biologically active analogues by capillary electrophoresis applying cyclodextrins as mobile phase additives

Seven diastereomer pairs of tetrapeptide analogues (TP) of endomorphin-1 and -2 were synthesized. A stereoselective capillary electrophoretic method was developed for controlling stereoisomeric ratio or purity. The isoelectric points of the tetrapeptides were between 8.3 and 8.9 as predicted and measured. A few of the analytes could be resolved without selectors due to the difference in their mobility. Neutral and anionic cyclodextrins (CDs) were applied in order to improve resolution. Stability constants as well as the mobilities of complexes were determined. Contributions of differences in the mobilities of free analytes and in the mobilities and stabilities of their complexes formed by CDs were equally important in the efficient resolution and migration order of diastereomers. As a result of the optimization of the pH of buffers and the concentration of the CD derivatives each diastereomer pair was resolved at baseline at least or better.

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

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

High-performance capillary electrophoretic separation of double-stranded oligonucleotides using a poly- (ethylpyrrolidine methacrylate-co-methyl methacrylate)-coated capillary

Here we describe a capillary electrophoretic method for the separation of double-stranded oligonucleotides (ds-ODNs) ranging from 16-20 bp with 2 bp resolution using a low concentration of poly(ethylpyrrolidine methacrylate-co-methyl methacrylate) (PEPyM-co-PMMA) copolymer physically adsorbed to a capillary surface. Contrary to traditional DNA separations, we show that the ds-ODN with the highest molecular size eluted first and propose that this phenomena is due to a screening effect by the PEPyM-co-PMMA coating on the smaller ds-ODNs negative charge during elution. Key to the performance of this separation was a sample preparation time of less than 1 h and analysis time of 40 min. Repeatability of intraday migration time for the mixtures was typically < 1% relative standard deviation (n = 3). In addition, we demonstrate that the coating has an acceptable capillary lifetime of over 70 injections.

Determination of five priority haloacetic acids by capillary electrophoresis with contactless conductivity detection and solid phase extraction preconcentration

A sensitive capillary electrophoretic separation method with contactless conductivity detection (C4D) for analysis of five priority haloacetic acids (HAA5) is presented. The analytes were baseline separated in an electrolyte composed of 20 mM 2-(N-Morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (HIS), and 30 μM cetyltrimethylammonium bromide (CTAB) at pH 6.0 in less than 4 min. A simplified solid-phase extraction (SPE) preconcentration procedure on highly cross-linked polystyrene-divinylbenzene (PS-DVB) type sorbent was developed and optimized with respect to short preconcentration time. HAA5 from a 25-mL sample aliquot of tap and swimming pool water could be preconcentrated in less than 5 min using an in-house made SPE column with recoveries ranging from 23 to 98%. Combining the SPE preconcentration procedure with capillary electrophoretic analysis, the attained limits of detection were between 6.1 and 12.2 μg/L with total analysis time of less than 10 min.

Capillary electrophoretic methods in the development of metal‐based therapeutics and diagnostics: New methodology and applications

In recent years, capillary electrophoresis (CE) has matured to a standard method in medicinal inorganic chemistry. More and more steps of the drug discovery process are followed by CE. However, not only the number of applications has steadily increased but also the variety of used methodology has significantly broadened and, as compared to a few years ago, a wider scope of separation modes and hyphenated systems has been used. Herein, a summary of the newly utilized CE methods and their applications in metallodrug research in the timeframe 2006-2011 is presented, following related reviews from 2003 and 2007 (Electrophoresis, 2003, 24, 2023-2037; Electrophoresis 2007, 28, 3436-3446). Areas covered include impurity profiling, quality control of pharmaceutical formulations, lipophilicity estimation, interactions between metallodrugs and proteins or nucleotides, and characterization and also quantification of metabolites in biological matrices and real-world samples.

Accelerated solvent extraction combined with capillary electrophoresis as an improved methodology for simultaneous determination of residual fluoroquinolones and sulfonamides in aquatic products

A rapid and effective accelerated solvent extraction (ASE)–capillary electrophoretic method was developed for the determination of fluoroquinolone and sulfonamide residues in aquatic products. Seven residues were extracted with acetonitrile at 80 °C under 10.3 MPa pressure in less than 25 min. The standard curves for the determination of seven residues by the capillary electrophoretic method have good linearity (r2 > 0.999). The method limits of quantification (LOQs) were 70–100 μg kg−1. Their average recoveries with three spiked levels ranged from 83% to 106%. Intra- and inter-day precision (RSD) were 2.3–4.9% and 3.1–6.5%, respectively. The method provides a rapid and simple extraction procedure with high recoveries, and has been applied for the determination of 4 fluoroquinolone and 4 sulfonamide residues in aquatic products with satisfactory results.