Micellar electrokinetic capillary chromatography for therapeutic drug monitoring of zonisamide

The simultaneous determination of zonisamide, a new type of antiepileptic drug, and the typical antiepileptic drugs phenobarbital, phenytoin and carbamazepine in human serum was developed using micellar electrokinetic capillary chromatography (MECC) with a diode array detector. A high correlation was revealed between the zonisamide levels in human serum obtained by MECC and those obtained by high-performance liquid chromatography (r=0.981). The serum levels of phenobarbital, phenytoin and carbamazepine determined by MECC were almost equal to those obtained by fluorescence polarization immunoassay. The reproducibility of separation and quantification with MECC analysis was appropriate for the intra- and inter-day assay coefficients. Therefore, the MECC method established here could provide a simple and efficient therapeutic drug monitoring method for antiepileptic drugs in patients, especially those treated with a combination of zonisamide and other antiepileptic drugs.     

Development and Validation of a Micellar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC.

Introduction – A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective – To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology – The extracts were analysed in an uncoated fused‐silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results – A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2 . The analytical curve in the range 10.0–50.0 µg/mL (r² = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 µg/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 ± 1.4% of recovery. Conclusions – A novel high‐performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of alginate copolymer in pharmaceutical formulations by micellar electrokinetic chromatography

A micellar electrokinetic chromatography method was developed for the determination and quantification of sodium alginate. The alginate peak migrated in the very short time of 1.33 min and calibrated easily though the polydisperse properties of alginates. The minimum detection limit (LOD) of the method was calculated as 0.393 mg/ml. This analysis method was successfully applied to the alginate quantification in an antacid pharmaceutical formulation. Precise and reproducible analysis results were obtained, with liquid formulations injected directly without any pre-separation process.     

Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by high-performance liquid chromatography

Determination of the introduced moieties into derivatized proteins is an essential step in the preparation and quality control of chemically defined immunoconjugates. For the derivatized proteins using pyridyl disulfide-containing cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), the derivatization degree (ratio of pyridyl disulfide moieties to protein) has been traditionally determined by measuring the absorbance of both the derivatized protein and 2-thiopyridone (2-TP) released from the dithiothreitol (DTT) treatment (spectrophotometric method). This method, however, causes several problems including false high and low determinations of the protein and 2-TP, respectively, low selectivity, poor reproducibility, and relatively large amounts of sample consumption. A quantitative determination method of the derivatization ratios using bovine serum albumin derivatized with SPDP and SMPT as the model system has been developed. The concentration of protein and 2-TP released from the DTT treatment of derivatized proteins was determined directly without consideration of different reagents used and their concentrations. The present HPLC method was proved to be better in terms of accuracy, selectivity, and reproducibility with micro sample consumption. Moreover, this HPLC method can be directly applied to all derivatized proteins prepared with pyridyl disulfide-containing cross-linkers.     

Sensitive determination of pheomelanin after 5-carboxyfluorescein succinimidyl ester precapillary derivatization and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the determination of pheomelanin in biological materials has been investigated. 5-Carboxyfluorescein succinimidyl ester was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic acid (HI). Various parameters affecting derivatization and separation were systematically studied. Under optimal conditions, the analytes could be separated within 18 min, and the relative standard deviations (R.S.D.) of migration time and corrected peak areas were less than 5.5%. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical detection, the 100-fold improvements in sensitivity were achieved by applying LIF detection. As a preliminary application, this method has been successfully applied to the determination of pheomelanin in two human melanoma cell cultures, black hair, melanoma tissue and urine samples of human melanoma patients with the spiked recoveries in the range of 88-96%.     

Validation of a rapid micellar electrokinetic capillary chromatographic method for the simultaneous determination of isoniazid and pyridoxine hydrochloride in pharmaceutical formulation

An efficient and reliable micellar electrokinetic capillary chromatography (MEKC) method has been developed for the simultaneous determination of isoniazid (ISO) and pyridoxine hydrochloride (PYR) in pharmaceutical formulations. A chemometric two level full factorial design approach was used to search for the optimum conditions of separation. Three parameters were selected for this study: the buffer pH, the buffer concentration and sodium dodecyl sulphate (SDS) concentrations. Resolution, peak symmetry and analysis time were established as response. The two analytes were separated within 6 min with the optimized conditions: 50 mM borate buffer, 25 mM SDS pH 7.8, 35 degrees C, at 50 mbar 4s injection and 30 kV by using a fused silica capillary (72 cm effective length, 50 microm i.d.). The detection wavelength was set to 205 nm. Meloxicam was used as internal standard. The method was validated with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity and robustness. The detection limits of the method were 1.0 microg mL(-1) for ISO and 0.40 microg mL(-1) for PYR and the method was linear at least in the range of 3.0-100 microg mL(-1) for ISO and 1.0-100 microg mL(-1) for PYR with excellent correlation coefficients (0.9995 for ISO and 0.9998 for PYR). Relative standard deviations (R.S.D.s) of the described method ranged between 0.54 and 2.27% for intra-day precision and between 0.65 and 2.69% for inter-day precision. The developed method was applied to the tablet form of ISO and PYR-containing the pharmaceutical preparations and the data were compared with obtained from the standard addition method. No statistically significant difference was found.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Determination of methanol in biodiesel by headspace solid phase microextraction

A new direct gas chromatography procedure (headspace solid phase microextraction) was developed for the quantitative determination of methanol in biodiesel. The analysis was performed by exposing a carboxen–polydimethylsiloxane SPME fiber assembly to the headspace of the biodiesel sample. The gas chromatography used a HP-5 capillary column and flame ionization detection. A polynomial relationship was observed between the methanol concentration and its peak area. This method showed good reproducibility (average relative standard deviation 7.06%) and recovery (average recovery 100.2%).     

Micellar electrokinetic capillary chromatography determination of +S and -R arotinolol in serum using UV detection and solid phase extraction.

A method for the simultaneous determination of +S and -R arotinolol in serum by micellar electrokinetic capillary chromatography is described. Stereoselective resolution of the arotinolol enantiomers was achieved using 5 mM sodium taurocholate in 10 mM sodium dihydrogen phosphate buffer of pH 2.5. A 72-cm uncoated fused-silica capillary at a constant voltage of 15 kV was used for the analysis. The analytes of interest were extracted from serum using solid phase extraction. An octadecyl cartridge gave good recoveries in excess of 87% for both +S and -R arotinolol without any interference. The calibration curves were linear over the range of 50-500 ng ml(-1) with +S propranolol as the internal standard and the coefficient of determination was greater than 0.999 (n = 3). The limit of quantitation was 50 ng ml(-1) for each enantiomer and the detection limit using 1 ml serum and a UV detection set et 220 nm was 25 ng ml(-1) (S/N = 2). Precision and accuracy of the method were in the range 0.8-2.7% and 1.2-6.4%, respectively, for +S arotinolol and 1.1-3.9% and 2.2-6.5%, respectively, for -R arotinolol.     

Development of comparative methods using gas chromatography-mass spectrometry and capillary electrophoresis for determination of endocrine disrupting chemicals in bio-solids

Two analytical separation techniques are being investigated for their potential in determining a wide range of endocrine disrupting chemicals (EDCs) in the environment. Capillary electrophoresis (CE) in the micellar mode in conjunction with a cyclodextrin (CD) modifier is shown to have potential for determination of alkylphenol breakdown products. Gas chromatography with mass spectrometric (GC-MS) detection is being utilised for validation of the CE method development and in addition as a separation technique to optimise preconcentration using solid-phase extraction. GC has demonstrated potential for the separation of 26 priority chemicals suspected as being endocrine disrupting compounds. The challenge of the method development process lies in the fact that these compounds are of differing polarities, size and charge and therefore are difficult to separate in a single run. Capillary electrophoresis in the CD-MEKC (micellar electrokinetic chromatography) mode is showing potential in this regard. Limits of determination are in the low mg/l range for CE and GC, however, using preconcentration it is possible to improve detection sensitivity with >80% recovery for some analytes and up to 100% recovery for most target species.     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C₁₈ cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l⁻¹ and detection limits were between 1.1 and 2.4 mg l⁻¹.     

A novel assay method for theanine synthetase activity by capillary electrophoresis

The determination of theanine has been performed by micellar electrokinetic capillary chromatography (MECC) using 2,4-dinitrofluorobenzene (DNFB) as a derivative reagent. To achieve the separation, a fused-silica capillary column was used with a borax buffer at 0.03 mol/L pH 9.8 (containing Brij35 and isopropanol) at 17 degrees C with detection wave length at 360 nm. The factors affecting the efficiency of the sample separation were examined simultaneously. A 40-min reaction at 35 degrees C between l-glutamate and ethylamine (with Tris-HCl buffer, pH 7.5) was investigated using the theanine synthetase from budding tea seeds. A novel method for the analysis of theanine synthetase activity based on MECC was established. The method shows mean recovery ranged from 87.1 to 105.3% and linearity ranged from 0.2 to 5.0 mmol/L.     

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.     

Determination of lormetazepam and its main metabolite in serum using micellar electrokinetic capillary chromatography with direct injection and ultraviolet absorbance detection

The use of micellar electrokinetic chromatography for the determination of lormetazepam and its metabolite, lorazepam, in serum samples at a concentration range of therapeutic interest was investigated. The separation was carried out at 30 degrees C and 25 kV, using a 15 mM borate-phosphate buffer (pH 8) with 30 mM sodium dodecyl sulfate as the separation electrolyte and 15% methanol as organic modifier. The analyses were carried out in 20 min under these conditions. Detection limits of 0.5 mg l(-1) were achieved for both benzodiazepines in serum. This method was employed for the quantitative resolution of both drugs (at different concentration ratios) in serum with very good recoveries.     

Selective photo-reduction of N-nitroamines combined with micellar electrokinetic chromatography and laser fluorimetric detection

N-nitroso compounds (NOC) are potent carcinogens. Reliable methods for the analysis of volatile carcinogenic NOC are well established; however selective and sensitive methods for routine analysis of thermally unstable, ionic or non-volatile NOC are still needed. For this purpose, a method based on micellar electrokinetic chromatography (MEKC) with laser induced fluorescence (LIF) detection is described for the simultaneous determination of a broad range of N-nitroso compounds. In this procedure, the nitroso group is photolytically cleaved from the NOC to yield the corresponding amine. The amines are then derivatized with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), identified and quantified using MEKC-LIF. For the standard mixture of NOC, this method has good sensitivity and a large dynamic range. The detection limit provided by the method is 9 ppb for N-nitrosopyrrolidine.     

Detection of a mixed-backbone oligonucleotide (GEM 231) in liver and tumor tissues by capillary electrophoresis

A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 mM, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min.     

Heart-cut two-dimensional separation method via hyphenation of micellar electrokinetic capillary chromatography and capillary zone electrophoresis using analyte focusing by micelle collapse

A novel two-dimensional (2D) separation method, which hyphenated micellar electrokinetic capillary chromatography (MEKC) and capillary zone electrophoresis (CZE), was developed for analysis of flavonoids in Leonurus cardiaca. The Leonurus cardiaca sample was separated and purified in first dimension by MEKC. Then only a selected portion of the first dimension separation was transferred into the second dimension by pressure. Finally, the zone of flavonoids was separated by CZE. As the key to successful hyphenation of MEKC and CZE, an analyte focusing by micelle collapse (AFMC) concentration method was employed between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The whole heart-cut 2D separation process can be performed in a conventional CE analyzer. The relative standard deviation of peak height, peak area and migration time were in the range of 2.3-4.2%, 1.5-3.8% and 3.6-5.5%, respectively, and detection limits (S/N=3) were 15-55 ng/mL. The new methodology was applied with success for the flavonoids separation of Leonurus cardiaca.     

抗癌药物紫杉醇的提取与分离纯化技术

本对紫杉醇的提取与分离纯化技术,包括柱层析法、薄层色谱法、沉淀法、胶束动电毛细管色普法、膜分离法、树脂吸附分离法、高速逆流色谱法、化学反应法和药理作用靶点法进行了较全面的综述。     

Non-competitive immunoassay for alpha-fetoprotein using micellar electrokinetic capillary chromatography and laser-induced fluorescence detection

A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP-antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 mM tetraborate containing 100 mM SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.     

Development and validation of a plasma assay for acyclovir using high-performance capillary electrophoresis with sample stacking

A sensitive plasma assay for acyclovir has been developed and validated. Acyclovir was separated from plasma components using Oasis HLB columns. Separation was obtained with no plasma interference using micellar electrokinetic chromatography (175 mM SDS) and hydroxypropyl-beta-cyclodextrin (100 mM) in 90 mM borate buffer (pH 8.8) containing 0.2% NaCl. High sensitivity was achieved by large volume sample introduction and stacking. The linear range was from 20 to 10000 ng/ml with a limit of quantitation of 20 ng/ml. This method is a viable alternative to HPLC because of its high separation and sensitivity, reproducibility, and adaptability to other nucleoside analogs.