On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

Determination of copper and iron using [S,S']-ethylenediaminedisuccinic acid as a chelating agent in wood pulp by capillary electrophoresis.

A capillary electrophoresis (CE) method is described for the simultaneous determination of copper and iron after complexation with a readily biodegradable chelating agent, [S,S']-ethylenediaminedisuccinic acid (EDDS), in wood pulp. CE separation was performed in a fused-silica capillary (50 microm i.d.; total length, 65 cm) with an electrolyte containing 25 mM borate buffer and 0.5 mM CTAB at pH 7.0 and an applied voltage of -25 kV. The samples were introduced by applying a 50 mbar pressure for 2 s, and detection of the complexes was monitored at 245 nm. The methodology performance of the methods was evaluated in terms of the linearity, limit of detection (LOD), limit of quantitation (LOQ) and reproducibility. The applicability of the method was demonstrated for the analysis of copper and iron in wood pulp.     

Determination of cyclodextrins and their derivatives by capillary electrophoresis with indirect UV and conductivity detection

A fast and simple capillary electrophoretic method suitable for the determination of native alpha-, beta-, gamma-cyclodextrins, their randomly substituted tert-butyl derivatives (average degree of substitution 3.8-4.4), heptakis (2,6-di-O-methyl)- and heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin was developed. Naphthyl-2-sulfonic acid (2-NSA), 3-iodobenzoic acid (3-IBA) and (1S)-1-phenylethylamine (PHEA) were tested as selective complex forming and UV absorbing background electrolyte additives. The composition of optimized background electrolyte for the separation of uncharged cyclodextrins and their derivatives was: 15 mM 3-iodobenzoic acid titrated with tris[hydroxymethyl]aminomethane to pH 8.0, 5% (v/v) of acetonitrile. A complete resolution of mono-2-O-, mono-3-O- and mono-6-O-carboxymethyl-beta-cyclodextrin regioisomers was achieved in the optimized background electrolyte system: 40 mM PHEA titrated with 2-[N-morpholino]ethanesulfonic acid to pH 5.6. In addition to indirect UV detection a contactless conductometric detector was successfully utilized.     

Rapid capillary electrophoretic method for the determination of clozapine and desmethylclozapine in human plasma

A rapid and sensitive high-performance capillary electrophoretic method for the determination of clozapine and its main metabolite desmethylclozapine in human plasma was developed. The separation of the two analytes was carried out in an untreated fused-silica capillary [33 cm (8.5 cm effective length) x 50 microm I.D.] filled with a background electrolyte at pH 2.5 containing beta-cyclodextrin. Baseline separation of clozapine and desmethylclozapine was recorded in less than 3 min. An accurate sample pretreatment by means of solid-phase extraction and subsequent concentration allows for reliable quantitation of clozapine in the plasma of schizophrenic patients under treatment with the drug. The method showed good precision (mean RSD = 4.0%) as well as satisfactory extraction yields (approximately 88%) and a good sensitivity (limit of quantitation = 0.075 microg ml(-1), limit of detection = 0.025 microg ml(-1)).     

Determination of cyclodextrins and their derivatives by capillary electrophoresis with indirect UV and conductivity detection

A fast and simple capillary electrophoretic method suitable for the determination of native α-, β-, γ-cyclodextrins, their randomly substituted tert-butyl derivatives (average degree of substitution 3.8 – 4.4), heptakis (2,6-di-O-methyl)- and heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin was developed. Naphthyl-2-sulfonic acid (2-NSA), 3-iodobenzoic acid (3-IBA) and (1S)-1-phenylethylamine (PHEA) were tested as selective complex forming and UV absorbing background electrolyte additives. The composition of optimized background electrolyte for the separation of uncharged cyclodextrins and their derivatives was: 15 mM 3-iodobenzoic acid titrated with tris[hydroxymethyl]aminomethane to pH 8.0, 5% (v/v) of acetonitrile. A complete resolution of mono-2-O-, mono-3-O- and mono-6-O-carboxymethyl-β-cyclodextrin regioisomers was achieved in the optimized background electrolyte system: 40 mM PHEA titrated with 2-[N-morpholino]ethanesulfonic acid to pH 5.6. In addition to indirect UV detection a contactless conductometric detector was successfully utilized.     

Determination of haloalkane dehalogenase activity by capillary zone electrophoresis

A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 microm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 25 degrees C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.     

Determination of 5-aminosalicylic acid related impurities by micellar electrokinetic chromatography with an ion-pair reagent

A micellar electrokinetic chromatographic (MEKC) method was developed for the quantification of mesalazine or 5-aminosalicylic acid (5-ASA) and its major impurities 3-aminosalicylic acid, salicylic acid, sulfanilic acid and 4-aminophenol. The optimisation of the experimental conditions was carried out considering some important requirements: resolution, reproducibility, detection limits of 0.1% (m/m) or less, short total analysis time. Preliminary investigations employing sodium dodecyl sulfate (SDS) as surfactant did not lead to the necessary resolution of the studied compounds; the addition of tetrabutylammonium bromide (TBAB) to the SDS micellar system resulted in the complete separation of all the compounds. The effects on the separation by several parameters such as TBAB concentration, SDS concentration, background electrolyte pH and concentration, were evaluated. Using a fused-silica capillary (8.5 cm effective length) complete analysis was obtained in a very short time. Under the optimised final conditions [120 mM 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer, pH 10.20, 65 mM SDS in the presence of 55 mM TBAB and 5% methanol] the method was validated for specificity, precision, linearity, limits of detection and quantitation, and robustness: the 5-ASA related impurities can be quantified at least at the 0.1% (m/m) level.     

Enantiomeric purity determination of ketoprofen by capillary electrophoresis: development and validation of the method

A simple, fast capillary electrophoresis method for determining the total ketoprofen content in an oral pharmaceutical formulation is proposed. The addition of 75 mM of heptakis(tri- O-methyl)-beta-cyclodextrin to the background electrolyte allows the quantitation of the enantiomeric impurity of ( R)-(-)-ketoprofen contained in the formulation. A relative limit of detection is proposed as a measure of the lowest detectable enantiomeric impurity and the results show that the method can detect the minor enantiomer at levels as low as 0.04% in ( S)-(+)-ketoprofen. The chiral method was validated following ICH recommendations and the quality parameters obtained show the suitability of the proposed method. The analysis of samples examined during the course of a stability study under chiral and achiral conditions revealed that the total ketoprofen content did not change significantly with time and the enantiomeric impurity range was 0.1-0.4%.     

Determination of egg white lysozyme by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis - capillary zone electrophoresis method for the determination of lysozyme in selected food products is described. The optimized electrolyte system consisted of 10 mM NH(4)OH + 20 mM acetic acid (leading electrolyte), 5 mM epsilon -aminocaproic acid +5 mM acetic acid (terminating electrolyte), and 20 mM epsilon -aminocaproic acid +5 mM acetic acid +0.1% m/v hydroxypropylmethylcellulose (background electrolyte). A clear separation of lysozyme from other components of acidic sample extract was achieved within 15 min. Method characteristics, i.e., linearity (0-50 micrograms/mL), accuracy (recovery 96+/-5%), intra-assay (3.8%), quantification limit (1 microgram/ml), and detection limit (0.25 microgram/mL) were determined. Low laboriousness, sufficient sensitivity and low running costs are important attributes of this method. The developed method is suitable for the quantification of the egg content in egg pasta.     

Simultaneous analysis by capillary electrophoresis of five amyloid peptides as potential biomarkers of Alzheimer's disease

We report here a CE method for the separation and quantitation of five amyloid peptides (Abeta1-42, 1-40, 1-39, 1-38, and 1-37) considered as potential biomarkers of Alzheimer's disease. These amyloid peptides have very similar structures. Sample preparation and storage conditions are critical parameters to ensure their solubility and to avoid the aggregation process in particular for Abeta1-42. Their solubility was found fully dependent on the NH(4)OH concentration that was employed initially to dissolve the lyophilized amyloid peptides. Conditions to achieve a full separation of these peptides were found using a dynamic coating with 1,4-diaminobutane (DAB). The linear decrease of their electrophoretic mobility highlighted an ion-pairing phenomenon between the peptides and DAB. The optimal background electrolyte was a 40 mM borate buffer, pH 9 containing 3 mM of DAB. Under these conditions, resolutions ranged from 1.3 to 2.4 with theoretical plates reaching 300,000. Under the retained conditions, we showed that adsorption of peptides to silica was negligible (recovery over 94.5%) and depletion effect of the background electrolyte was overcome. The method was finally validated in terms of linearity and repeatability and the limits of detection for the five Abeta peptides were estimated. The inter-day repeatability of the migration times was very satisfactory with RSDs less than 1.55%. The RSDs of the peak areas were below 5%. With this CE-UV method, limits of detection of the peptides ranged from 300 to 500 nM. We finally demonstrated that this method can be applied to real biological samples such as CSF.     

Determination of fosfomycin in pus by capillary zone electrophoresis

A method is described for the determination of fosfomycin in pus by capillary zone electrophoresis with reversed electroosmotic flow, and indirect UV absorbance detection. Sample pre-treatment is limited to removal of proteins and cell debris by adding the double volume of methanol, followed by vortexing for few seconds, and centrifugation at 15,000 x g for 2 min. The supernatant is directly injected into the instrument. Fosfomycin is separated from sample constituents with a background electrolyte at pH 7.25 (25 mM benzoate buffer with 0.5 mM hexadecyltrimethylammonium bromide added, adjusted to pH with tris(hydroxymethyl)-aminomethane (TRIS)). Separation is carried out in a capillary with 50 microm I.D., 64.5 cm total length, 56.0 cm to the detector, at 25 degrees C with -25 kV voltage applied. Due to the low absorbance of the analyte, indirect UV detection was performed at 254 nm using a bubble cell capillary. Sample was injected by pressure (450 mbar s). Repeatability for fosfomycin in spiked pus (from 8 or 10 consecutive injections of three different series at concentrations of 100 microg/mL of the antibiotic) was between 2.4 and 8.2% relative standard deviation (RSD). Accuracy (expressed as recovery of fosfomycin determined by three independent analysis at 10, 100 and 300 microg/mL fosfomycin added to plain pus) was between 75 and 102%. Intermediate reproducibility (n = 9 at three different days) was between 2 and 12% RSD. Limit of detection and limit of quantitation were 4.5 and 15 microg/mL, respectively. The concentration of fosfomycin in pus of patients treated with the antibiotic ranged up to 240 microg/mL. The concentration of other anionic pus constituents identified beside chloride (acetate, succinate, lactate, phosphate) ranged between 20 and 7800 microg/mL.     

Determination of anthracycline antibiotics doxorubicin and daunorubicin by capillary electrophoresis with UV absorption detection.

Sweeping preconcentration and electrokinetic injection was used for the capillary electrophoretic analysis of trace amounts of biologically active anthracyclines with UV absorption detection. Phosphate buffer (100 mM), pH 2.5, with addition of 40% v/v methanol was used as background electrolyte (BGE). Sodium dodecyl sulfate (150 mM) was added to BGE in the inlet vial as the sweeping agent. The system enables effective separation of anthracyclines as well as cleanup from matrix impurities. Sweeping preconcentration of sample provides an excellent detection limit (1 x 10(-9) mol L(-1)). The method was applied for the determination of therapeutic levels of doxorubicin in real plasma samples.     

Simple in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity method for the determination of colistin

An in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity detection method was developed for the direct measurement of colistin in pharmaceutical samples. The flow injection and capillary electrophoresis systems are connected by an acrylic interface. Capillary electrophoresis separation is achieved within 2 min using a background electrolyte solution of 5 mM 2‐morpholinoethanesulfonic acid and 5 mM histidine (pH 6). The flow‐injection section allows for convenient filling of the capillary and sample introduction without the use of a pressure/vacuum manifold. Capacitively coupled contactless conductivity detection is employed since colistin has no chromophore but is cationic at pH 6. Calibration curve is linear from 20 to 150 mg/L, with a correlation coefficient (r²) of 0.997. The limit of quantitation is 20 mg/L. The developed method provides precision, simplicity, and short analysis time.     

Determination of fumaric acid in apple juice by on-line coupled capillary isotachophoresis-capillary zone electrophoresis with UV detection

An on-line coupled capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) method for the determination of the fumaric acid content in apple juice is presented. A clear separation of fumaric acid in real samples is achieved within 20 min. The leading, terminating and background electrolyte of the employed system consist of 10 mM HCl+beta-alanine+5 mM beta-cyclodextrin+0.05% hydroxypropylmethylcelullose (HPMC), pH 3, 10 mM citric acid and 20 mM citric acid+beta-alanine+5 mM beta-cyclodextrin+0.1% HPMC, pH 3.3, respectively. The linearity, recovery, repeatability and detection limit of the developed method are 25-1000 ng/ml, 1.07%, 95.4+/-3.5 (+/-s)% and 10 ng/ml, respectively. Low laboriousness (no sample pretreatment), sufficient sensitivity and low running cost are the important attributes of the cITP-CZE method which was successfully applied to analyses of real samples of apple juices.     

On-column complexation capillary electrophoretic separation of Fe2+ and Fe3+ using 2,6-pyridinedicarboxylic acid coupled with large-volume sample stacking

On-column complexation of Fe2+ and Fe3+ with 2,6-pyridinedicarboxylic acid (2,6-PDCA) formed anionic complexes, which were then separated by capillary zone electrophoresis with direct UV detection at 214 nm. To achieve reasonable separation selectivity and on-column complexation, the conditions such as pH, the concentration of 2,6-PCDA and the EOF modifiers in the electrolyte were examined. The electrolyte contained 5.0 mM 2,6-PDCA, 0.25 mM tetradecyltrimethlammonium bromide (TTAB) and 5% (v/v) acetonitrile at pH 4.0 was optimised for on-column complexation and the separation of Fe[PCDA]2(2-) and Fe[PCDA]2(-). To enhance the detection sensitivity, large-volume sample stacking (LVSS) was used for the on-line preconcentration of Fe[PCDA]2(2-) and Fe[PCDA]2(-). Under the optimised conditions, satisfactory working ranges (0.5-50 microM), lower detection limits (less than 0.1 microM) and good repeatability of the peak areas (R.S.D.: 5.2-7.8%, n = 5) was achieved using LVSS (300 s). With LVSS, the detection sensitivity was enhanced more than 50-fold compared to conventional hydrodynamic injection. The proposed method was used successfully for the determination of Fe2+ and Fe3+ in water samples.     

Analysis of colistin sulfate by capillary zone electrophoresis with cyclodextrins as additive

A method for the quantitative analysis of colistin sulfate by capillary zone electrophoresis is described. Since colistin components have five free amino groups, they tend to adsorb onto the capillary wall and cause peak tailing. It was found that triethanolamine (TEA)-phosphate buffer at pH 2.5 was useful to reduce such adsorption. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found necessary for selectivity enhancement. In order to optimize the separation parameters and predict the method robustness, a central composite design was performed including three variables, namely concentration of M-beta-CD, TEA, and IPA. The effects of capillary length and applied voltage on separation were also investigated. The optimal conditions established were: 140 mM TEA-phosphate buffer containing 5 mM M-beta-CD and 6% v/v IPA, a capillary with 55 cm total length (50 microm inner diameter, 47 cm from inlet to detection window) and 24 kV applied voltage. The method was found to be robust when the variables were changed in the following range: 4-6 mM M-beta-CD, 5-7% v/v IPA, and 130-150 mM TEA. Further, the linearity, limit of detection (LOD), and limit of quantitation (LOQ), as well as repeatability for both colistin A and B were examined and three commercial samples were quantitatively analyzed.     

Electro membrane extraction combined with capillary electrophoresis for the determination of amlodipine enantiomers in biological samples

Electro membrane extraction as a new microextraction method was applied for the extraction of amlodipine (AM) enantiomers from biological samples. During the extraction time of 15 min, AM enantiomers migrated from a 3 mL sample solution, through a supported liquid membrane into a 20 μL acceptor solution presented inside the lumen of the hollow fiber. The driving force of the extraction was 200 V potential, with the negative electrode in the acceptor solution and the positive electrode in the sample solution. 2-Nitro phenyl octylether was used as the supported liquid membrane. Using 10 mM HCl as background electrolyte in the sample and acceptor solution, enrichment up to 124 times was achieved. Then, the extract was analyzed using CD modified CE method for separation of AM enantiomers. Best results were achieved using a phosphate running buffer (100 mM, pH 2.0) containing 5 mM hydroxypropyl-α-CD. The range of quantitation for both enantiomers was 10-500 ng/mL. Intra- and interday RSD (n=6) were less than 14%. The limits of quantitation and detection for both enantiomers were 10 and 3 ng/mL respectively. Finally, this procedure was applied to determine the concentration of AM enantiomers in plasma and urine samples.     

Determination of Total Radiochemical Purity of [18F]FDG and [18F]FET by High-Performance Liquid Chromatography Avoiding TLC Method

The goal of this work was to present two high-performance liquid chromatography (HPLC) method that could be applied for the determination of the total radioactive purity of 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) and O-(2-[¹⁸F]fluoroethyl)-L-tyrosine ([¹⁸F]FET). The separation of [¹⁸F]fluoride ions, [¹⁸F]FET and [¹⁸F]FET intermediate was accomplished on LiChrosper RP-18, 250?×?4 mm, 5 µm (Merck) analytical column. For mobile phase 10 mM potassium dihydrogen phosphate buffer at pH7 (A) and acetonitrile (B) was used: 0–2 min: 15% B; 2–12 min: 85% B; 12–15 min: 15% B, respectively. Analysis of [¹⁸F]FDG was performed using LiChrosper 100 NH₂, 250?×?4.5 mm, 5 µm (Merck) analytical column. The initial mobile phase composition was 10 mM KH₂PO₄ buffer (pH7) and acetonitrile (15:85, v/v) and the acetonitrile ratio was decreased to 15% at 2 min after the sample injection and held for 5 min. Complete elution of [¹⁸F]fluoride ions from stationary phases could be achieved by adding 10 mg/mL K[¹⁹F]F to radioactive samples in a ratio 1:1 during the sample preparation. Recovery of [¹⁸F]fluoride ions ranged from 99.5 to 100.6%. The validation of the developed methods showed good results for linearity (r²?=?0.9981–0.9996), specificity (R_S?=?3.7–10.2), repeatability (%Area RSD_%?=?1.2–4.3%) and limit of quantitation (LOQ?=?1.6–4.5 kBq). During the cross-validation similar radiochemical purity values were obtained by the novel HPLC methods and thin layer chromatography performed according to the recommendations of the Ph. Eur. monographs.

     

Quantitative analysis of aglycone quercetin in mulberry leaves (Morus alba L.) by capillary zone electrophoresis.

A capillary zone electrophoresis method was established for analysis of aglycone quercetin in mulberry leaves (Morus alba L.). The influence of, e.g., background electrolyte concentrations and pH, surfactant concentrations, organic solvents, temperature, and voltage on the separation of aglycone quercetin, rutin, quercitrin, kaempferol, catechin, and gallic acid was systematically investigated. The optimum condition providing baseline separation of all compounds within 16.5 min was obtained in 150 mM boric acid (pH 10.0) using a fused-silica capillary with an effective length of 42.5 cm (50 microm inner diameter), temperature of 32 degrees C, and voltage of 15 kV. Method assessment was performed by standard addition method using rutin as an internal standard. Linearity of the method was excellent (r(2) > 0.999) over the concentration tested (40-160 microg/mL). The relative standard deviations (%RSDs) from injection, intraday, and interday precision were less than 2.5%. Recoveries were good (asymptotically equal to 100.0%,%RSD = 0.8%) with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.86 and 3.16 microg/mL (%RSD = 1.8%), respectively. The aglycone quercetin found in the mulberry leaves was 0.452 g/100 g (%RSD = 0.6%) on dry weight.     

Capillary electrophoresis analysis of gentamicin sulphate with UV detection after pre-capillary derivatization with 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid

A selective, sensitive, and rapid pre-capillary derivatization method for determination of the multicomponent aminoglycoside antibiotic gentamicin is described. The derivatization reagents 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid were used and the thioisoindole derivative was UV detected at 330 nm. A central composite experimental design was performed to optimize selectivity and derivatization conditions. Baseline separation of gentamicin C1, C1a, C2, C2a, C2b, sisomicin and several minor components was achieved with a background electrolyte containing 30 mM sodium tetraborate, 7.5 mM beta-cyclodextrin and 12.5% (v/v) methanol at pH 10. Quantitative analysis was performed and illustrated the potential use of capillary electrophoresis for the identification and quantitation of gentamicin as an alternative to methods prescribed in the United States Pharmacopeia and European Pharmacopoeia.