Uncoated Fused-silica Capillary Research Articles

Determination of sixteen food additives in beverage by capillary zone electrophoresis

A capillary zone electrophoretic (CZE) method for the separation of ten synthetic colorants such as acid red 92, patent blue V, uranine, acid red 1, indigo carmine, black BN, ponceau 6R, quinoline, amaranth, lemon yellow, and six preservatives including benzoic acid, sorbic acid, methyl paraben, ethyl paraben, propyl paraben, butyl paraben in beverages was developed. The effects of separation voltage, capillary temperature, pH and concentration of running buffer on the separation were investigated. A 46 cm x 50 microm uncoated fused silica capillary was used with 70 mmol/L boric acid (pH 9.5) containing 4% (v/v) acetonitrile as background electrolyte solution. The detection wavelength was set at 220 nm, and the run voltage was 30 kV. The injection time was 5 s at 500 mPa and the temperature was 25 degrees C. The linear relationship between the mass concentration and peak area for each of these analytes was obtained in

Capillary electrophoresis fingerprints of Baizi Yangxin Wan

The capillary electrophoresis fingerprint (CEFP) of Baizi Yangxin Wan (BZYXW) was established by capillary zone electrophoresis (CZE). The chromatographic fingerprint resolution index (RF) was applied to optimize the CEFP conditions. The electrophoretic separation was performed on a 75 cm (the effective length of 57 cm) x 75 microm uncoated fused silica capillary with 50 mmol/L sodium borate-50 mmol/L Na2HPO(4)-200 mmol/L H3BO(3)-150 mmol/L NaHCO3 (7:7:1:1, v/v/v/v) containing 4% acetonitrile (pH 9.70) as the background electrolyte (BGE), and the BGE was chosen by using the triangular prism optimization method. The running voltage was set at 12 kV, and the detection wavelength at 228 nm. The sample solution was injected into the capillary by hydraulic pressure in 25 s. The CEFPs were produced by the electropherograms from 12 batches of BZYXW, and the 17 co-possessing peaks were selected as the fingerprint peaks of BZYXW's CEFP by choosing ferulic acid peak as the referential peak. According to the results of classification, the reference CEFP (RCEFP) was synthesized from 12 batches of BZYXW. Taking the RCEFP for the qualified model, the systematically quantified fingerprint method(SQFM) was applied to evaluate the quality of 17 batches of BZYXW, in which the qualities of 3 batches was evaluated as good, 1 batch as fine, 3 batches as moderate, 1 batch as common, and 4 batches as inferior. The triangular prism optimization method was suggested to be good to optimize the BGE. The results showed that the BZYXW-CEFP was established with good precision and reproducibility, which can be served as a novel reference to identify and control the quality of BZYXW.

Analysis of monosaccharides in the saffron corm glycoconjugate by capillary electrophoresis

The monosaccharides in the saffron corm glycoconjugate were separated by capillary electrophoresis (CE) coupled with pre-column derivatization. 4-Methoxyaniline was used as derivatization reagent. The derivatization and CE separation conditions were investigated. The ultraviolet detection wavelength was 234 nm. The maximum yield of this derivatization reaction was obtained under the presence of 9.5% (v/v) acetic acid at 80 degrees C for 2 h. An uncoated fused-silica capillary of 50 microm i.d. and 50/60 cm length (effective length/total length) was employed, and a pressure injection (3.4475 kPa, 5 s) was applied. The baseline separation of 11 monosaccharides and disaccharides (lyxose, xylose, ribose, glucose, mannose, galactose, rhamnose, cellobiose, maltose, lactose, fructose) was reached at 25 degrees C, 20 kV of separation voltage and with 350 mmol/L boric acid (pH 10.21) as running buffer. The developed method has been successfully applied to quantitatively determine the components of saffron corm glycoconjugate, and the results showed that the recovery of each monosaccharide was in the range of 94.3% - 105.4%, the relative standard deviation was 3.3% - 4.6%.

Simultaneous determination of chlordiazepoxide and selected antidepressants using CZE

A simple CE method was developed and validated for the simultaneous determination of chlordiazepoxide (CHL), amitriptyline, and nortriptyline (mixture I) or the determination of CHL and imipramine (mixture II) using the same BGE. Sertraline and amitriptyline were used as internal standards for the first and second mixtures, respectively. The method allows amitriptyline to be completely separated from its impurity and main metabolite nortriptyline, which can be quantified from 0.2 μg/mL. The separation was achieved using 20 mM potassium phosphate buffer pH 5 containing 12 mM β-cyclodextrin and 1 mM carboxymethyl-β-cyclodextrin. UV detection was performed at 200 nm and a voltage of 15 kV was applied on an uncoated fused-silica capillary at 25°C. These experimental conditions allowed separation of the compounds to be obtained in 7 min. Calibration graphs proved the linearity up to 40 μg/mL for CHL, up to 100 μg/mL for amitriptyline and imipramine, and up to 5 μg/mL for nortriptyline. The accuracy and precision of the method have been determined by analyzing synthetic mixtures and pharmaceutical formulations. The analytical results were quite good in all cases indicating that the method was linear, sensitive, precise, accurate, and selective for both mixtures.

Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Determination of pKavalues of a triptolide derivative and its impurities by pressure-assisted capillary electrophoresis

A pressure-assisted capillary zone electrophoresis method was developed to determine the pKa values of a triptolide derivative (LLDT-246) and its impurities. The method was performed in an uncoated fused-silica capillary under the electric voltage of 18kV and 50mbar of external pressure applied simultaneously. A series of running electrolyte buffers were used with pH ranging between 2.2 and 10.0 with the constant ionic strength of 0.05M. The values of pKa of LLDT-246 and two impurities were calculated based on the pH dependence of effective mobilities (μeff). The pKa value of LLDT-246 was in good agreement with that of determined by potentiometric titration method.

Simultaneous determination of pharmaceutical components in dietary supplements for weight loss by capillary electrophoresis tandem mass spectrometry

A simple capillary electrophoresis tandem mass spectrometry method was developed for the simultaneous determination of 20 pharmaceutical components such as cathartics and appetite suppressants in dietary supplements for weight loss. This method allowed multidrug target screening and confirmation in differing radically in chemical structure. The separation was achieved on an uncoated fused-silica capillary using 20 mM ammonium formate in 20 % v/v acetonitrile-water (pH 8.0) as the electrolyte, followed by detection mixed with a sheath liquid that consisted of a mixture of 5 mM ammonium formate and 0.1 % v/v formic acid in 50 % v/v methanol-water. Samples were hydrodynamically injected and separated at 30 kV within 25 min. The limits of detection were 1.0-750 µg/g for the target analytes. The method was successfully applied to 12 dietary supplements for weight loss and 3 non-prescription drugs. The proposed method was suitable as the determination and identification of pharmaceutical components in dietary supplements for weight loss.

Evaluation of dispersive liquid–liquid microextraction in the stereoselective determination of cetirizine following the fungal biotransformation of hydroxyzine and analysis by capillary electrophoresis

We developed a capillary electrophoresis (CE) and dispersive liquid–liquid microextraction (DLLME) method to stereoselectively analyze hydroxyzine (HZ) and cetirizine (CTZ) in liquid culture media. The CE analyses were performed on an uncoated fused-silica capillary; 50mmolL−1 sodium borate buffer (pH 9.0) containing 0.8% (w/v) S-β-CD was used as the background electrolyte. The applied voltage and temperature were +6kV and 15°C, respectively, and the UV detector was set to 214nm. Chloroform (300µL) and ethanol (400µL) were used as the extraction and disperser solvents, respectively, for the DLLME. Following the formation of a cloudy solution, the samples were subjected to vortex agitation at 2000rpm for 30s and to centrifugation at 3000rpm for 5min. The recoveries ranged from 87.4 to 91.7%. The method was linear over a concentration range of 250–12,500ngmL−1 for each HZ enantiomer (r>0.998) and 125–6250ngmL−1 for each CTZ enantiomer (r>0.998). The limits of quantification were 125 and 250ngmL−1 for CTZ and HZ, respectively. Among the six fungi studied, three species were able to convert HZ to CTZ enantioselectively, particularly the fungus Cunninghamella elegans ATCC 10028B, which converted 19% of (S)-HZ to (S)-CTZ with 65% enantiomeric excess.

Simultaneous determination of amino acids in tea leaves by micellar electrokinetic chromatography with laser-induced fluorescence detection

A rapid and effective method of micellar electrokinetic chromatography with laser-induced fluorescence detection was developed for the simultaneous determination of amino acids in tea leaves. Pre-column derivatization of the analytes used 4-chloro-7-nitrobenzofurazan (NDB-Cl). Optimal separation was achieved at +20kV using an uncoated fused silica capillary (40.0cm effective length, 50.2cm total length, 75μm internal diameter), as well as 20mM sodium borate (pH 8.5), 20mM Brij 35, and acetonitrile 10% (v/v) as running buffers. Within 11min, 15 amino acids were separated completely. The optimized method demonstrated good linearity (r2⩾0.9990), precision (⩽6.65%), accuracy (85.50–112.74%), and sensitivity (0.1ng/mL–100ng/mL). The method successfully determined the quantity of amino acids in five different tea leaves; furthermore, theanine was identified as the most abundant amino acid in teas. The proposed method showed great potential in further investigations on the biofunctions of different tea samples.

Plasma L-Ergothioneine Measurement by High-Performance Liquid Chromatography and Capillary Electrophoresis after a Pre-Column Derivatization with 5-Iodoacetamidofluorescein (5-IAF) and Fluorescence Detection

Two sensitive and reproducible capillary electrophoresis and high-performance liquid chromatography-fluorescence procedures were established for quantitative determination of L-egothioneine in plasma. After derivatization of L-ergothioneine with 5-iodoacetamidofluorescein, the separation was carried out by HPLC on an ODS-2 C-18 sperisorb column by using a linear gradient elution and by HPCE on an uncoated fused silica capillary, 50 µm id, and 60 cm length. The methods were validated and found to be linear in the range of 0.3 to 10 µmol/l. The limit of quantification was 0.27 µmol/l for HPCE and 0.15 µmol/l for HPLC. The variations for intra- and inter-assay precision were around 6 RSD%, and the mean recovery accuracy close to 100% (96.11%).

Separation of somatropin charge variants by multiple‐injection CZE with Polybrene/chondroitin sulfate A double‐coated capillaries

The performance of dynamic double-coated fused-silica capillaries with Polybrene and chondroitin sulfate A has been compared with uncoated fused-silica capillaries for the determination of recombinant human growth factor (somatropin) charge variants. The separations were carried out under the same electrophoretic conditions as described in the European Pharmacopoeia, i.e. at pH 6.0 and 30°C. The coating significantly reduced the interactions between the proteins and the surface of the fused-silica capillary. The first five separations performed in a new bare fused-silica capillary were discarded because of very poor separation performance as a result of protein-surface interactions. There was an approximate twofold increase in the interday migration time precision (%RSD ≤ 6.5%) in the double-coated capillaries. The method was successfully transferred to a multiple CZE mode where two samples were analyzed in a single electrophoretic run. The average purity of somatropin certified reference standard was 98.0% (%RSD ≤ 0.3%) determined by using uncoated and coated capillaries.

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.

Development and Validation of Electromigration Technique for the Determination of Lincomycin and Clindamycin Residues in Poultry Tissues

An analytical method to quantify lincosamide residues (lincomycin and clindamycin) in poultry tissue samples by micellar electrokinetic capillary chromatography combined with UV detection has been developed and validated. The clean-up procedure with a simple deproteinization and solid-phase extraction was successfully applied for the determination of antibiotics in different edible tissue samples of poultry. The electrophoretic separation was achieved using an uncoated fused silica capillary (57 cm × 75 μm i.d.), voltage 16 kV, temperature 22 °C, and the optimal wavelength at 200 nm. The running buffer consisting of 10 mM sodium tetraborate decahydrate (pH 9.3) and 25 mM sodium dodecylsulfate was employed. The validation of the method was performed by evaluation of the analytical parameters: linearity, specificity, precision, and accuracy with limits of detection of 13.2 and 18.5 ng/g, for clindamycin and lincomycin, respectively. Good intermediate precision was obtained with relative standard deviation values less than 12 %. The mean recoveries were 91.2 and 87.7 % for lincomycin and clindamycin, respectively. The obtained results confirm that the proposed method is capable to identify and quantify of lincosamide residues in animal tissues far below maximum residue limit values and could be suitable for routine application in commercial of poultry samples.

Determination of mangiferin in Baihe Zhimu decoction by HPCE

Objective To establish HPCE method for determining the mangiferin in Baihe Zhimu decoction.Methods Rutin was used as internal standard.An uncoated fused silica capillary (77 cm×75 μm,effective length of 70 cm) was served as separation gate.The electrolyte buffer was composed of 12 mmol/L borax-MeOH (90 ∶ 10).20 kV voltages were applied and detection wavelength was set at 318nm.Results Mangiferin was successfully separated within 20 rin,the linear response range was 0.06～1.14 mg/ml.The average recovery was 99.0％.Conclusion The method was simple,rapid and well reproducible.It could be used as a reliable tool for the quantity control of Baihe Zhimu decoction and descriptions containing mangiferin. Key words: Baihe Zhimu decoction; Mangiferin; HPCE

Combination of hollow‐fiber liquid‐phase microextraction and capillary electrophoresis for pioglitazone and its main metabolites determination in rat liver microsomal fraction

Pioglitazone (PGZ), a thiazolidinedione antidiabetic agent, is reported as a potent and selective activator of peroxisome proliferator-activated receptor γ (PPAR γ). This drug has been widely prescribed for the treatment of Type 2 diabetes mellitus. In this regard, this manuscript presents, for the first time, an alternative electrophoretic method for PGZ and its main metabolites determination in rat liver microsomal fraction. The electrophoretic analyses were performed using an uncoated fused-silica capillary of 50 μm id, 48 cm in total length and 40 cm in effective length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5). All experiments were carried out under the normal mode. The capillary temperature was set at 35°C and a constant voltage of +30 kV was applied during the analyses. Samples were introduced into the capillary by hydrodynamic injection (50 mbar, 15 s) and detection was performed at 190 nm. The sample preparation procedure, based on hollow-fiber liquid-phase microextraction, was optimized using multifactorial experiments. Next, the following optimal condition was established: sample agitation at 1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0. The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear over the concentration range of 200-25,000 ng/mL for PGZ and 200-2000 ng/mL for the metabolites. Finally, the validated method was employed to study the in vitro metabolism of PGZ using rat liver microsomal fraction.

Determination of 2-Methylimidazole and 4-Methylimidazole in Caramel Colors by Capillary Electrophoresis

The use of chemical preservative compounds is common in the food products industry. Caramel color is the most usual additive used in beverages, desserts, and breads worldwide. During its fabrication process, 2- and 4-methylimidazole (MeI), highly carcinogenic compounds, are generated. In these cases, the development of reliable analytical methods for the monitoring of undesirable compounds is necessary. The primary procedure for the analysis of 2- and 4-MeI is using LC- or GC-MS techniques. These procedures are time-consuming and require large amounts of organic solvents and several pretreatment steps. This prevents the routine use of this procedure. This paper describes a rapid, efficient, and simple method using capillary electrophoresis (CE) for the separation and determination of 2- and 4-MeI in caramel colors. The analyses were performed using a 75 μm i.d. uncoated fused-silica capillary with an effective length of 40 cm and a running electrolyte consisting of 160 mmol L(-1) phosphate plus 30% acetonitrile. The pH was adjusted to 2.5 with triethylamine. The analytes were separated within 6 min at a voltage of 20 kV. Method validation revealed good repeatability of both migration time (<0.8% RSD) and peak area (<2% RSD). Analytical curves for 2- and 4-MeI were linear in the 0.4-40 mg L(-1) concentration interval. Detection limits were 0.16 mg L(-1) for 4-MeI and 0.22 mg L(-1) for 2-MeI. The extraction recoveries were satisfactory. The developed method showed many advantages when compared to the previously used method.

Development of a simple analytical method using capillary electrophoresis-tandem mass spectrometry for product identification and simultaneous determination of free amino acids in dietary supplements containing royal jelly

A simple capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method was developed for the analysis of free amino acids in commercial royal jelly (RJ) products containing various kinds of matrices. This method required no concentration step for sample preparation, and all 16 amino acids were determined without derivatization. The CE separation was achieved in an uncoated fused-silica capillary using a 1M formic acid solution (pH 1.8) as the electrolyte, followed by MS/MS detection after mixing with a sheath liquid comprising 50% (v/v) methanol. The limits of detection (LODs) ranged from 0.61 to 10.5μg (dry weight)/g for each amino acid. The recoveries for tablets, liquid drinks, and raw materials ranged from 88.3 to 108.6%, and the relative standard deviations (RSDs) were within 10%. The method was applied to 17 commercial RJ products, and the results were compared to those for honey. The relative proportions of free amino acids were specific for each RJ product, and the method was found to be useful in distinguishing not only among the different RJ products but also between RJ and honey.

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.

Quantification of 5-aminolevulinic acid by CE using dynamic pH junction technique

An online dynamic pH junction preconcentration method was developed for quantification of 5-aminolevulinic acid (ALA) by CE with the separation time less than 6 min. The optimal dynamic pH junction of ALA was carried out between pH 9.3 borate buffer (BGE, 40 mM) and pH 2.5 phosphate buffer (sample matrix, 40 mM) when 4.1 cm of sample plug was hydrodynamically injected into an uncoated fused-silica capillary (48.5 cm in length, id of 50 μm). If a 24 kV separation voltage was applied, the calibration curve of ALA peak area (200 nm) showed good linearity (R(2) = 0.9991) ranging from 0.01 to 6.5 mg/mL. The reproducibility of this system was excellent with RSDs (n = 10) of 2.5% for peak area response and 0.6% for migration time at ALA concentration of 0.5 mg/mL. The LOD was evaluated as 1.0 μg/mL (S/N > 3). Compared to conventional CE procedure, the sensitivity was successfully improved over 50-fold. The analytical results of pharmaceutical formulations show a good agreement with those by HPLC (r = 0.94).

Application of capillary zone electrophoresis in the interaction analysis of protein C with protein C activator from Agkistrodon acutus venom

A new capillary zone electrophoresis method (CZE) has been established for the interaction analysis of protein C (PC) with a protein C activator (PCA) from Agkistrodon acutus venom. The analysis was performed on an uncoated fused-silica capillary with 75 microm i.d. and a total length of 60.2 cm (50 cm to the detector) with a buffer solution of 50 mmol/L Tris-HCl (pH 7.4) and 198 nm of wavelength. The factors which influence the separation of the PCA, such as buffer solution and ion concentration, and the interaction between the PCA and PC incubated for different times at 37.5 degrees C were studied. The linear range was from 10 to 300 mg/L. The limit of detection was 3 mg/L (S/N = 3). The relative standard deviation (RSD) for the migration time of the PCA was 0.56%. The RSD for the peak area was 3.8% (n = 6). The equal volumes of the PCA (200 mg/L) and PC (60 mg/L) were incubated for five minutes, at which their binding rate reached the maximum. And no hydrolyzed peptide chain from PC was found in the electropherogram. The PCA from Agkistrodon acutus venom could activate PC directly through changing the space conformation of PC. The method is simple, and highly sensitive with high resolution, and will provide important theoretical basis for the rapid detection of venom proteins and their activities in the future.