Capillary Electrophoresis Method For Determination Research Articles

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin.

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30mM sodium phosphate buffer, pH 3.0, containing 40mg/mL sulfated β-CD and 7mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.

Inside Front Cover: Development of a microchip capillary electrophoresis method for determination of the purity and integrity of mRNA in lipid nanoparticle vaccines

DOI: 10.1002/elps.202100272 The inside cover picture shows a method to measure the purity of messenger RNA (mRNA) in lipid nanoparticle (LNP) vaccines by microchip capillary electrophoresis. An LNP containing mRNA is depicted in the top left which undergoes treatment to solubilize the LNP and release mRNA, as shown in the bottom left. The mRNA is introduced into a separation channel containing a gel-sieving matrix that separates the mRNA by size, as the smaller molecules travel faster than larger molecules, followed by fluorescence detection, as shown in the bottom right. An electropherogram of the separation of just over one minute duration is shown in the top right. The mRNA purity is calculated as the percent peak area of the main peak (∼49 s).

DEVELOPMENT OF CAPILLARY ELECTROPHORESIS METHOD FOR DETERMINATION OF AMLODIPINE IN HUMAN PLASMA

Abstract: Background: Amlodipine, a calcium channel blocker, is commonly prescribed to treat high blood pressure. In Vietnam, there is no method of quantifying amlodipine in plasma by capillary electrophoresis. Therefore, the thesis was carried out with the objective: Developing and validating a method for quantifying amlodipine in human plasma by capillary electrophoresis. Materials and methods: Human plasma samples contain amlodipine. After optimizing the process, the method was validated according to the guidelines for validation of bioanalytical methods of US-FDA and EMA. Results: Amlodipine and nifedipine as internal standard (IS) were alkalized with sodium hydroxide and extracted from human plasma by liquid - liquid extraction technique with ethyl acetate. Electrophoresis conditions: Fused-silica capillary column (effective column length: 48.5 cm, total column length 57.5 cm, outside diameter: 75 μm, internal diameter: 75 μm), background electrolyte: 50 mM borate buffer at pH 8.6 containing 20 mM sodium dodecyl sulfate, voltage: +25 kV, current: 300 μA, column temperature: 25 ± 1 oC, sample injection: 35 mBar - 7s, detection at 237 nm. The method was validated and met US-FDA and EMA requirements. Conclusion: The method can serve as a basis in bioequivalence studies of medicinal products containing amlodipine. Key words: Amlodipin, human plasma, capillary electrophoresis

Development of a microchip capillary electrophoresis method for determination of the purity and integrity of mRNA in lipid nanoparticle vaccines.

Messenger RNA (mRNA)‐based vaccines are advantageous because they can be relatively quicker and more cost efficient to manufacture compared to other traditional vaccine products. Lipid nanoparticles have three common purposes: delivery, self‐adjuvanting properties, and mRNA protection. Faster vaccine development requires an efficient and fast assay to monitor mRNA purity and integrity. Microchip CE is known to be a robust technology that is capable of rapid separation. Here, we describe the development and optimization of a purity and integrity assay for mRNA‐based vaccines encapsulated in lipid nanoparticles using commercial microchip‐based separation. The analytical parameters of the optimized assay were assessed and the method is a stability indicating assay.

Capillary Electrophoresis Method for Determination of Escitalopram Oxalate in Urine Samples and Different Dosage Forms.

Application of capillary electrophoresis (CE) has become a rapidly growing analytical technique for the estimation of drugs in pharmaceutical dosage forms and biological fluids. In this study, an effective and sensitive method was developed for the determination of escitalopram oxalate (ESC-OX) by CE with diode-array detection at 200nm. The separation was achieved by a fused silica capillary with 40cm effective length (48.5cm total, 75μm i.d.). The background electrolyte was composed of 15mM phosphate buffer (pH2.5). The applied potential was 22.5kV, and the samples were injected at 50mbar pressure for 10s. Metoprolol was used as an internal standard (IS). The migration time under these optimum conditions was 6.51±0.07 and 6.73±0.08min for ESC-OX and IS, respectively, with total run time 7min. The method was validated for linearity, precision, accuracy, specificity and sensitivity. The limit of detection was calculated as 3.85 and 5.07ngmL-1 for standard and urine samples, respectively. The developed method was employed successfully for the determination of ESC-OX in different pharmaceutical dosage forms and spiked human urine after simple liquid-liquid extraction with good recovery.

Quality by Design-Based Development of a Chiral Capillary Electrophoresis Method for the Determination of Dextrodropropizine and 1-Phenylpiperazine as Impurities of Levodropropizine

Based on a quality by design approach, a capillary electrophoresis method for the simultaneous determination of dextrodropropizine and the achiral precursor 1-phenylpiperazine in levodropropizine was developed. The analytical target profile was defined as a method allowing the simultaneous determination below the 0.5% level within a maximum analysis time of 20 min with an acceptable precision and accuracy. Based on scouting experiments, sulfated β-cyclodextrin in a 25 mM potassium phosphate buffer, pH 7.0, was selected. Subsequently, cyclodextrin concentration, propan-2-ol concentration, capillary temperature and applied voltage were identified as critical process parameters using a full factorial design followed by a central composite face-centered design for the final optimization of cyclodextrin concentration and temperature. The design space was obtained by Monte Carlo simulations. The final method comprised a 40/50.2 cm effective/total length, 75 µm inner diameter fused-silica-capillary, 25 mM potassium phosphate buffer, pH 7.0, containing 23.5 mg mL−1 sulfated β-cyclodextrin and 10% (v/v) propan-2-ol as background electrolyte, 16.3 °C capillary temperature and an applied voltage of 16.5 kV. The robustness was assessed using a Plackett–Burman design followed by method validation. Finally, the method was applied to the analysis levodropropizine reference substance of the European Pharmacopoeia and a liquid dosage form.

Fast capillary electrophoresis method for determination of docosahexaenoic and eicosapentaenoic acids in marine oils omega-3 supplements

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential omega-3 fatty acids (FA) in human nutrition. Dietary supplements containing these FA are available in the market and there is a need to assess their quality to prevent frauds. The purpose of this work was to optimize and validate a fast capillary electrophoresis (CE) method to determine EPA and DHA content in marine oils omega-3 supplements. Sample preparation comprised only a saponification step and analysis time was 8 min. Validation was performed according to limits of detection and quantification, linearity, accuracy and repeatability. Ten real samples of marine oil supplements were analyzed by CE and compared to conventional gas chromatography method. No significant differences between both methods were found within 95% confidence interval. Overall, CE method was successful to FA quantification and it could be used for fast FA monitoring during omega-3 supplement manufacture, final product quality assurance and labelling.

Capillary Electrophoresis Method for the Chiral Purity Determination of Pregabalin Derivatized with Dansyl Chloride

A capillary electrophoresis method for the determination of the chiral purity of pregabalin upon derivatization with dansyl chloride was developed using design of experiment methodologies. A D-optimal design was used for the identification of the critical process parameters, while a central composite face centered design and Monte Carlo simulations were employed for defining the design space of the method. Final working conditions consisted of a background electrolyte composed of a 100 mM sodium phosphate buffer, pH 2.5, containing 40 mg mL−1 heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, a separation voltage of 15 kV and a capillary temperature of 25 °C. The analyses were carried out in a 40/50.2 cm fused-silica capillary with an inner diameter of 50 µm. Upon testing the robustness using a Plackett–Burman design, the method was validated according to the International Council on Harmonization guideline Q2(R1). The method allowed the detection of the (R)-enantiomer at the 0.015% level with a limit of quantitation at the 0.05% level with regard to a sample containing 1.59 mg mL−1 pregabalin. The method was subsequently applied to the determination of the stereochemical purity of the drug in commercial capsules.

An On-Column Enzyme Mediated Fluorescence-Amplification Method for Plasma Total Cholesterol Measurement by Capillary Electrophoresis with LIF Detection

Total cholesterol monitoring in human plasma is one of the most important routine analysis performed in the clinical laboratory. Here we report a new capillary electrophoresis method for determination of plasma total cholesterol based on on-column enzymatic assay and laser-induced fluorescence detection. In this method, the capillary is used not only as a separation medium but also as the reaction chamber which minimizes the amount of samples and the consumption of the reagents. The whole procedure can be automated which minimizes the possible errors and saves experimental time. The analytical characteristics of the proposed method, such as linearity and repeatability of the peak area and the migration time, were evaluated. The RSD of the migration time and peak area were 1.0 and 3.5 % (intraday), and 2.2 and 2.9 % (interday), respectively. The obtained LOQ values were at the nanomolar concentration level, therefore the developed method is sufficient for determination and quantification of plasma total cholesterol.

Development and Validation of a Micellar Capillary Electrophoresis Method for Determination of IFNβ-1b in Lyophilized Formulation of a Biosimilar Product.

Human interferons (IFNs) are key cytokines secreted by immune system. They have several effects such as antiviral and anti tumors activity, activating immune cells and healing of multiple sclerosis. The type IFNs present in humans are α ,β and Υ. IFN β is a polypeptide, normally produced by fibroblasts and seems to be more species-specific than IFN. Structural properties of IFNs are important for their biologic effects. There are a few analytical techniques for separation, identification and determination of IFNs in its formulations such as mass spectroscopy, RP-HPLC and capillary electrophoresis (CE). In this study we used Micellar Electrokinetic Chromatography (MEKC) as a unique mode of CE because of its capability to separate neutral as well as charged solutes. We used sodium tetraborate (Borax) as buffer without any modifier and sodium dodecyl sulfate (SDS) as surfactant. The optimum MECK running buffer consisted of Borate 50 Mm; SDS 20 mM pH =9.6. The validated method was used for determination of the IFN β-1b formulation which is manufactured in Iran. From 9 collected different batches, all of them had acceptable potency as claimed on their label with average 102.25 ±10.030 %. This is the first time that a MEKC method is introduced for quantification of IFN β-1b in its pharmaceutical dosage forms. The method is reliable safe, rapid and accurate.

Development and validation of a capillary electrophoresis method for determination of enantiomeric purity and related substances of esomeprazole in raw material and pellets

A capillary electrophoresis method using CDs for quality control of esomeprazole (ESO) in terms of enantiomeric purity and related substances in raw material and pellets was developed. ESO is the S-enantiomer of omeprazole (OMZ). Several parameters were evaluated, including type and concentration of buffer and CD, concentration of additives and electrolyte pH. Resolution between the enantiomers of OMZ obtained for each parameter tested was calculated and the presence of the main related substance such as OMZ sulfone was carefully monitored. The optimized system consisted of 100 mM Tris-phosphate buffer pH 2.5 with 20 mM 2-hydroxypropyl-β-CD, 1 mM sodium dithionite, temperature at 15°C, voltage at 28 kV, and UV detection at 301 nm. Once optimized, the electrophoretic system was validated according to ICH guidelines. The limits of detection and quantification for R-OMZ were 0.6 μg/mL (0.06% w/w of ESO) and 2.0 μg/mL (0.2% w/w of ESO), respectively. A mean concentration of R-OMZ <0.2% limit established by the United States Pharmacopeia (USP) was found in the raw material and six-pellet samples of ESO. No other impurities were found in the samples under these conditions. Therefore, the developed method was found to be appropriate not only for enantiomeric quality control of ESO but also for the analysis of ESO and the main related substance in raw material and pharmaceutical formulations as well as for stability indicating studies.

Development and application of a capillary electrophoresis method for the determination of ellagic acid in E. globulus wood and in filtrates from E. globulus kraft pulp

A capillary zone electrophoresis (CZE) method for simple and rapid determination of ellagic acid (EA) in Eucalyptus globulus wood and in the filtrate from unbleached kraft pulp has been developed. This is the first application of CZE for the detection of EA in industrial streams from cellulosic pulp production. The EA determinations in wood extractives and in pulp filtrates were succeeded only after sample acidification. This new CZE analytical procedure allowed reliable determinations of EA in E. globulus wood (1.1 ± 0.6 g kg−1 of dry wood) and in the filtrates from unbleached kraft pulp (98 ± 0.7 mg L−1). Gas chromatography–mass spectrometry was used as a reference method for the quantification of EA in industrial samples.

Development of a fast capillary electrophoresis method for determination of carbohydrates in honey samples

In this study, the determination of fructose, glucose and sucrose by capillary electrophoresis (CE) was investigated. The tendency of the analyte to undergo electromigration dispersion and the buffer capacity were evaluated using the Peakmaster® software and considered in the optimization of the background electrolyte, which was composed of 20mmolL−1 sorbic acid, 0.2mmolL−1 CTAB and 40mmolL−1 NaOH at pH 12.2. Under optimal CE conditions, the separation of the substances investigated was achieved in less than 2min. The detection limits for the three analytes were in the range of 0.022 and 0.029gL−1 and precision measurements within 0.62–4.69% were achieved. The proposed methodology was applied in the quantitative analysis by direct injection of in honey samples to determine the main sugars presents. The samples were previously dissolved in deionized water and filtered with no other sample treatment. The mean values for fructose, glucose and sucrose were in the ranges of 33.65–45.46g 100g−1, 24.63–35.06g 100g−1 and <0.22–1.32g 100g−1, respectively. The good analytical performance of the method makes it suitable for implementation in food laboratories for the routine analysis of honey samples.

Development and validation of a capillary electrophoresis method for the enantiomeric purity determination of RS86017 using experimental design

A selective capillary electrophoresis method for determination of enantiomeric purity of RS86017, a new antiarrhythmic agent with two chiral centers, was developed and validated using sulfobutyl ether-β-cyclodextrin as chiral selector. The concentration of the chiral selector and organic modifier, pH of background electrolyte (BGE), capillary temperature, and applied voltage were systematically optimized by using orthogonal design and concentration of chiral selector was further optimized. The optimal conditions included 25 mM phosphate buffer at pH 8.0, containing 28 mg/mL sulfobutyl ether-β-cyclodextrin and 20% acetonitrile as running buffer, an applied voltage of 22 kV, and a temperature of 20 °C. The detection wavelength was 206 nm. The obtained method was capable of separating RS86017 from its potential chiral impurities, the S,R-enantiomer, the R,R-diastereomer and the S,S-diastereomer with a short analysis time of 10 min. The separation was validated with respect to its selectivity, repeatability, linearity, precision, accuracy, limits of detection (LOD), limits of quantitation (LOQ) and robustness testing. The LODs and LOQs were 0.8 μg/mL and 2.5 μg/mL for all isomers of RS86017, respectively. Finally, the method was used to investigate the chiral purity of RS86017 in bulk samples.

Validation of Capillary Electrophoresis Method for Determination of N-Methylpyrrolidine in Cefepime for Injection

The present study relates to a new capillary electrophoresis method for the determination of N-methylpyrrolidine, an impurity considered to be toxic and also potential degradation impurity in cefepime hydrochloride drug substance. The newly developed capillary electrophoresis method for determining the content of N-methylpyrrolidine in cefepime for injection has been validated as per International Conference on Harmonization (ICH) guidelines to prove the selectivity, sensitivity, suitability, robustness, and ruggedness of the method. This simple, efficient, and rapid methodology may be used by pharmaceutical industry for routine analysis as well as during stability studies. The newly developed capillary electrophoresis method to determine the content of N-methylpyrrolidine in cefepime for injection requires 10 min for data acquisition, and uses an indirect UV photometry method to detect the analyte signal at 240 nm against the reference signal at 210 nm. The electrophoretic system is optimized to get stable base line, higher signal to noise ratio and peaks with narrow peak width. The method employs bare fused silica capillary with extended light path, effective length of capillary is 56 cm and inner diameter of capillary is 50 μm, 5 mmole of imidazole buffer adjusted to pH 5.1 with 3 molar acetic acid solution is used as background electrolyte. The sample is introduced in hydrodynamic mode employing pressure of 50 mbar for 5 s, and the desired separation is achieved with constant applied voltage of 25 kV at ambient temperature (~25°C).

Development of a fast capillary electrophoresis method for determination of creatinine in urine samples

The aim of this work was to develop a fast method using capillary electrophoresis for the determination of creatinine in human urine samples. The pH and constituents of the background electrolyte were selected by inspection of effective mobility of creatinine and candidate urine interferents versus pH curves. The tendency of the analyte to undergo electromigration dispersion and the buffer capacity were evaluated by the Peakmaster software and considered in the optimization of the background electrolyte, composed by 10 mmol L −1 tris(hydroxymethyl)aminomethane and 20 mmol L −1 2-hydroxyisobutyric acid (HIBA) at pH 3.93. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 μm I.D.), with short-end injection configuration and direct UV detection at 215 nm. The migration time of creatinine was only 22 s. A few figures of merit of the method are as follows: good linearity in the concentration interval of 5–70 mg L −1 ( R 2 > 0.99), limit of detection of 0.5 mg L −1, inter-day precision better than 2.7% ( n = 9) and recovery in the range 99.0–103.7% at three concentration levels (50, 100 and 150 mg L −1). Urine samples were prepared by deproteination with acetonitrile (1:3 sample:acetonitrile, v/v), centrifugation and dilution of a deproteinated aliquot with 12.5 mmol L −1 HIBA (1:4, v/v). Creatinine concentrations between 489 and 1063 mg L −1 were obtained in the urine of four healthy volunteers.

Capillary electrophoresis method for determination of D‐serine and its application for monitoring of serine racemase activity

Serine racemase (SR) is an enzyme responsible for the biosynthesis of D-serine, the coagonist of the N-methyl-D-aspartate receptor, in the brain. Therefore, it has been suggested as a possible therapeutic target for the treatment of various neurodegenerative diseases. To develop a potent inhibitor of SR, a simple, sensitive, fast, and robust assay is needed. In this paper, a new CE method for the determination of D-serine is described. Serine enantiomers are resolved in the form of o-phthaldialdehyde (OPA)/2-mercaptoethanol (2-ME) derivatives in an alkaline BGE composed of 50 mM sodium tetraborate, pH 9.7, and containing 40 mM 2-hydroxypropyl-gamma-CD as a chiral selector. The problem of time-limited stability of OPA/2-ME derivatives has been overcome by employing in-capillary derivatization of the sample, i.e., the derivatization reaction was carried out directly in the separation capillary in the first phase of the CE run. UV-absorption detection at 230 nm allowed concentration detection limit of 3 microM. Baseline resolution of D- and L-serine derivatives was achieved in less than 10 min. This fact, together with the simple sample pretreatment, allowed application of the method to medium-throughput monitoring of SR activity, such as the screening of potential SR inhibitors. A good agreement was achieved between the developed CE method and the previously established HPLC method for determination of the inhibition constant, K(i), of a new SR inhibitor, L-erythro-3-hydroxyaspartate.

Capillary electrophoresis method development for determination of impurities in sodium cysteamine phosphate samples

A capillary electrophoresis method for determination of impurities in sodium cysteamine phosphate—an alternative drug to use in place of cysteamine (Cystagon ®, Mylan Laboratories Inc.) in the treatment of cystinosis—was developed. The administration of cysteamine, divided in four doses due to the short half-life of this drug, is a helpful treatment, but several patients show intolerance, due to the very unpleasant odor and taste of cysteamine. Sodium cysteamine phosphate is less organoleptic aversive and also has a larger active time, allowing the compression of the doses to 2 per day, increasing the acceptance of the drug. In the developed method the two main decomposition products of sodium cysteamine phosphate, cystamine and cysteamine, can be determined with LOQs of 30 μg/ml (0.2%) and 16 μg/ml (0.1%), respectively. The background electrolyte is 15 mM ammonium acetate (pH 8.85) with 10% methanol and the separation takes less than 4 min. UV detection is performed at 195 nm. This volatile method was developed with the purpose of further hyphenation to a mass spectrometer.

Validation of a Capillary Electrophoresis Method for Analysis of Rabeprazole Sodium in a Pharmaceutical Dosage Form

Rabeprazole sodium is an antisecretory agent that inhibits the enzyme H+/K+ ATPase present in the stomach parietal cells. There are few data about its quantitative determinations in laboratorial routines. Capillary electrophoresis is a method being used increasingly for analysis of pharmaceutical compounds, the main advantages of which are the simplicity of instrumentation, low consumption of sample and reagents, and fast analysis. The aim of this study was to develop and validate a capillary electrophoresis method for determination of rabeprazole sodium in coated tablets. The conditions used were a bare fused silica capillary with 48.0 cm length (39.5 cm effective) and 75 microm id; a 10mM, pH 9.0, sodium tetraborate run buffer; a diode array detector set at 291 nm; hydrodynamic injection (50 mbar/5 s); and a voltage of 20 kV. HP Chemstation CE rev. A.06.03 software was used for system control, data acquisition, and analysis. The method was demonstrated to be linear in the concentration range of 5.0-40.0 microg/mL (r = 0.9993), precise (interday relative standard deviation = 0.49), accurate (mean recovery = 103.1%), and specific. The limits of detection and quantitation were 1.29 and 3.91 microg/mL, respectively.

A high performance capillary electrophoresis method for determination of methotrexate in serum

目的 建立快速检测人血清中氨甲蝶呤药物浓度的方法.方法用高效毛细管电泳技术,选用直径75 μm,总长60 cm,有效长度50.5 cm熔融石英毛细管,以75 mmol/L,pH 7.4的磷酸盐为电泳缓冲液,检测波长为306 nm,在25 kV电压下的快速分离检测氨甲蝶呤,并对此方法进行方法学评价.结果氨甲蝶呤的出峰时间在10 min内,线性范围在1.1～1 100.0 μmol/L间,最低检出浓度为0.55 μmol/L,回收率为88.2%～98.2%,批间精密度为5.4%,批内精密度为4.2%.结论高效毛细管电泳方法具有快速、准确且成本低的特点,为临床和科研中测定血清氨甲蝶呤提供了一种有效的手段。