Capillary Zone Electrophoresis Method Research Articles

Method development for quantitative monitoring of monoclonal antibodies in upstream cell-culture process samples with limited sample preparation - An evaluation of various capillary coatings.

Monoclonal antibodies (mAbs) have become an important class of biopharmaceuticals used for the treatment of various diseases. Their quantification during the manufacturing process is important. In this work, a capillary zone electrophoresis (CZE) method was developed for the monitoring of the mAb concentration during cell-culture processes. CZE method development rules are outlined, particularly discussing various capillary coatings, such as a neutral covalent polyvinyl alcohol coating, a dynamic successive multiple ionic-polymer coating, and dynamic coatings using background electrolyte additives such as triethanolamine (T-EthA) and triethylamine. The dynamic T-EthA coating resulted in most stable electro-osmotic flows and most efficient peak shapes. The method is validated over the range 0.1-10mg/ml, with a linear range of 0.08-1.3mg/ml and an extended range of 1-10mg/ml by diluting samples in the latter concentration range 10-fold in water. The intraday precision and accuracy were 2%-12% and 88%-107%, respectively, and inter-day precision and accuracy were 4%-9% and 93%-104%, respectively. The precision and accuracy of the lowest concentration level (0.08mg/ml) were slightly worse and still well in scope for monitoring purposes. The presented method proved applicable for analysing in-process cell-culture samples from different cell-culture processes and is possibly well suited as platform method.

Contribution of Capillary Zone Electrophoresis Hyphenated with Drift Tube Ion Mobility Mass Spectrometry as a Complementary Tool to Microfluidic Reversed Phase Liquid Chromatography for Antigen Discovery.

The discovery of new antigens specific to multiple myeloma that could be targeted by novel immunotherapeutic approaches is currently of great interest. To this end, it is important to increase the number of proteins identified in the sample by combining different separation strategies. A capillary zone electrophoresis (CZE) method, coupled with drift tube ion mobility (DTIMS) and quadrupole time-of-flight mass spectrometry (QTOF), was developed for antigen discovery using the human myeloma cell line LP-1. This method was first optimized to obtain a maximum number of identifications. Then, its performance in terms of uniqueness of identifications was compared to data acquired by a microfluidic reverse phase liquid chromatography (RPLC) method. The orthogonality of these two approaches and the physicochemical properties of the entities identified by CZE and RPLC were evaluated. In addition, the contribution of DTIMS to CZE was investigated in terms of orthogonality as well as the ability to provide unique information. In conclusion, we believe that the combination of CZE-DTIMS-QTOF and microfluidic RPLC provides unique information in the context of antigen discovery.

Evaluation of natamycin in commercial dairy products by a green capillary zone electrophoresis method and confirmation with a Liquid Chromatography-Mass Spectrometry

In the present study, a simple, rapid, sensitive, robust, convenient, economical and environmentally friendly Capillary Zone Electrophoresis (CZE) method based on the use of non-toxic chemicals was developed to monitor the concentrations of natamycin, which is used to prevent mold and yeast that may occur in cheese crust, cheese crumb, yogurt and ayran. Additionally, a LC-ESI-MS method was developed and used for confirmation as well. The validated methods produced excellent results in terms of linearity (R2 > 0.999) and recovery values (ranging from 99.06% to 100.54%) and successfully applied for the analysis of natamycin, according to European regulations and Turkish Food Codex in 14 different brands of ayran, 17 different brands of yogurt and 12 different brands of Kashar cheese collected from local markets. CZE demonstrated flexibility, sensitivity and selectivity, accuracy, robustness, and low cost. The developed CZE method is very advantageous because it is environmentally friendly and economical due to the analysis time and the absence of organic solvents. In addition, the reliability of the study was increased by using the internal standard and possible errors were prevented. Both CZE and LC-MS methods provided good results for natamycin analysis and these methods can be used for natamycin analysis in dairy products and possible pharmaceuticals. Finally, embracing the primary concepts of green chemistry, the proposed technique can be taken into consideration friendly to the nature, sustainable and “reagent-free”. These assays are easily applicable as quality control methods for natural products and pharmaceuticals containing natamycin.

Comparison of clonidine and cyproheptadine determination in animal-derived foods by sweeping-micellar electrokinetic chromatography and large volume sample stacking-capillary zone electrophoresis

Abstract This study establishes a method for rapid detection of clonidine and cyproheptadine in foods of animal origin. In order to obtain the best detection method, capillary zone electrophoresis (CZE), large volume sample stacking (LVSS), and sweeping-micellar electrokinetic capillary chromatography (sweeping-MEKC) were used respectively. The limits of detection (LODs) of clonidine and cyproheptadine by LVSS-CZE were 0.028 μg mL−1 and 0.034 μg mL−1, and those by sweeping-MEKC were 0.023 μg mL−1 and 0.031 μg mL−1, respectively. Compared with the CZE method, the two online pre-concentration technologies have greatly improved the detection sensitivity and achieved good enrichment results. However, compared with the sweeping-MEKC system, the LVSS system consumed a longer time and was greatly affected by the actual sample matrix. The sweeping-MEKC method was proved to be suitable for real sample analysis. Under the best sweeping-MEKC conditions, clonidine and cyproheptadine could be well separated within 8 min and good linear relationships in the range of 0.1–1.0 μg mL−1 (r 2 > 0.99) were obtained. This method was successfully applied to the determination of clonidine and cyproheptadine in animal-derived foods with the recoveries of 82.3%–90.1% and the relative standard deviations (RSDs) less than 3.11%. The sweeping-MEKC method is simple to operate and has great potential in the rapid detection of clonidine and cyproheptadine in animal-derived foods.

Novel Eco-Friendly Stability Indicating Capillary Zone Electrophoresis Method for Determination of Aripiprazole in Tablet Dosage form: DoE Directed Optimization, Development and Method Validation

In this work, a novel environment-friendly stability indicating capillary zone electrophoresis (CZE) method has been developed and validated for assaying the aripiprazole (ARP) in tablet dosage form. The separation of ARP from its degradation products and internal standard was achieved using a fused silica capillary column (30.2 cm x 75 μm ID), a background electrolyte containing 6 mmol L−1 ammonium formate buffer (pH 3) with 5% methanol under a potential of 15 kV and detection at 214 nm. The stability indicating ability of the method was investigated by analyzing ARP after being subjected to acidic, alkaline, thermal, photolytic, and oxidative stress conditions, according to ICH guidelines. Design of experiments was used during forced degradation and method optimization. Oxidation was the main degradation pathway among those evaluated. The drug was separated from its oxidative degradation products in less than 4 min. CZE method was linear between 60 – 140 μg mL−1, R2 = 0.9980, precise (intra-day 0.88% and inter-day 1.30%). The average recovery was 100.93 ± 0.77%. This is the first method in the literature for quantification of ARP in the presence of its related degradation products with high separation efficiency, low operation cost and minimum solvent consumption. This method could be helpful in the routine quality control analysis in the pharmaceutical industries with least harmful effect on the environment. CZE is considered an eco-friendly alternative of conventionally HPLC methods.

Sensitive detection of heart-failure biomarkers natriuretic peptides using multi-photon laser wave-mixing spectroscopy

Nonlinear laser wave-mixing spectroscopy is demonstrated as a fast and sensitive detection method for heart-failure biomarkers, pro-atrial natriuretic peptide (proANP) and brain natriuretic peptide (BNP). Wave mixing is an ultrasensitive optical absorption-based method and analytes can be detected in their native form or labeled with fluorophore and chromophore labels. In this study, we utilized Chromeo P540 dye to label the peptides for wave-mixing detection. The wave-mixing signal is created from the diffraction of incoming photons by the thermal grating at the capillary analyte cell. The signal beam is strong, collimated, and coherent (laser-like) and it is collected using a simple photodetector with an excellent signal-to-noise ratio. We demonstrated advantages of this technique over conventional assays including shorter analysis times, smaller sample requirements, and higher throughput. To enhance detection selectivity and sensitivity levels, wave mixing is effectively coupled to capillary zone electrophoresis (CZE) and field-amplified sample stacking (FASS) methods. We determined detection limits of 7.4 × 10−10 M or 55 zmol and 6.8 × 10−10 M or 51 zmol for proANP and BNP, respectively, and separated and detected both peptides within 2 min. Due to the challenges in the confirmatory diagnoses of heart failure, wave-mixing serves as a potentially beneficial screening tool in addition to the commonly used echocardiographic tests.

An Integration of Statistical and Anthropometric Measurement Approach Towards Improving Ergonomic Design for Production Workbench

Capillary zone electrophoresis (CZE) is a method of haemoglobin (Hb) analysis for the screening of haemoglobinopathies or variant haemoglobins. In our centre, few samples sent for Hb analysis showed Hb peak at zone 10 requiring further evaluation. This pattern was observed in patients with history of diabetes mellitus (DM); hence it was postulated the peak observed is glycated Hb (HbA1c). The objective of this study was to determine whether the expression of Hb peak at zone 10 among blood samples from DM patients correlate with HbA1c values measured using high performance liquid chromatography (HPLC). Methods: This was a cross-sectional study involving blood samples from DM patients. Samples with HbA1c values ≥ 6.5% by HPLC were selected and subsequently analysed by CZE method. Presence of Hb peak in zone 10 were correlated and analysed with the HbA1c levels measured by HPLC method. Results: A total of 131 samples were analysed. Hb peak was detected at zone 10 in 50/131(38.2%). Out of 50 samples, 47 (94%) were from patients with HbA1c level > 10%. Correlation study showed it was 18 times more likely for Hb peak to appear in zone 10 in patients with HbA1c > 10%. Cut off point for HbA1c to appear in CZE is 10.5% with AUC of 0.965. Conclusion: Hb peak detected at zone 10 of CZE was most likely to be HbA1c. However, it is recommended that for every primary method of Hb analysis used be confirmed by secondary method. Therefore, both zone 10 in CZE and P2 peak in HPLC must be correlated together to achieve final diagnosis

МЕТОДЫ ИДЕНТИФИКАЦИИ КАРБОНОВЫХ КИСЛОТ В СОСТАВЕ ЭКЗОМЕТАБОЛИТА ПРОБИОТИЧЕСКОЙ КУЛЬТУРЫ LACTOBACILUS PLANTARUM 8P-А3

Abstract. Human life in modern conditions is characterized by the constant influence of adverse environmental factors affecting the neuropsychological, physiological and metabolic parameters of the body. The compensatory capabilities of each individual, including the immune state, are largely determined by the state of the microbiota. In practical microbiology, research is relevant in the development of a new generation of drugs designed to maintain and restore normal flora, including fundamentally new probiotics - metabiotics created on the basis of microbial exometabolites. Taking into account the demand for metabiotics in the pharmaceutical market, an exometabolite complex was proposed for the design of such preparations, obtained by ultrafiltration of the culture liquid of a probiotic strain used in the production of lactobacterin. It is known that, along with bacteriocins, carboxylic acids remain among very significant components of the metabolite complexes of lactobacilli. In this regard, the purpose of this work was to choose the optimal physicochemical method for the joint determination of carboxylic acids on the model of the complex of exometabolites of the probiotic culture Lactobacilus plantarum 8P-A3.As a result of the studies, it was found that the optimal physicochemical method for identifying carboxylic acids in the composition of the metabolite complex is the method of capillary zone electrophoresis. In the composition of metabolites, such significant acids as lactic (217.7 g/l), citric and acetic (29.6 and 10.2 g/l) were identified. The simplicity and expressiveness of this method, combined with its high sensitivity in the determination of carboxylic acids, including in samples with complex matrices, is noted.

Simple, fast and reliable CE method for simultaneous determination of ciprofloxacin and ofloxacin in human urine

A simple, fast, and accurate capillary zone electrophoresis method has been developed for the determination of ciprofloxacin and ofloxacin. This method uses liquid–liquid extraction. Therefore, it is characterized by a very simple procedure of sample preparation but at the same time satisfactory precision and accuracy. The extraction process of the same urine sample was repeated three times. The extraction protocol was performed each time for 15 min with 1 mL of dichloromethane and chloroform mixture in a 3:1 volume ratio. A 0.1 mol/L phosphate-borate buffer (pH 8.40) was selected as the background electrolyte. UV detection was performed at 288 nm. The separation was carried out at a voltage of 16 kV, at a temperature of 25 °C. Experimentally evaluated LOQ values for ciprofloxacin and ofloxacin were 0.2 nmol/mL urine and 0.05 nmol/mL urine, respectively. For both analytes the calibration curves exhibited linearity over the entire tested concentration range of 1–6 nmol/mL urine. The precision of the method did not exceed 15%, and the recovery was in the range of 85–115%. The developed and validated procedure was applied to analyze human urine for the content of ciprofloxacin and ofloxacin.

Screening prolyl hydroxylase domain 2 inhibitory activity of traditional Chinese medicine by CZE-UV.

Prolyl hydroxylase domain 2 (PHD2) is a key enzyme regulating the expression of hypoxia inducible factor (HIF). Its inhibitors can improve the expression of HIF and downstream genes, which can treat hypoxia-related diseases. Therefore, the establishment of a reliable PHD2 inhibitors screening method is of great significance for the drug development of hypoxia-related diseases. In this work, an accurate, rapid, and simple screening method for PHD2 inhibitors was introduced by capillary zone electrophoresis (CZE). In order to improve the detection sensitivity, the derivative reaction of α-ketoglutaric acid (α-OG) and 1,2-diaminobenzene (OPD) was used to enhance the UV absorption of α-OG (the substrate in the enzymatic reaction). The CZE method selected 20mM Na2 B4 O7 buffer (pH9.0) as the separation buffer, +25kV as the separation voltage, 25°Cas the cartridge temperature, and 210nm as the detection wavelength. Under this condition, the analysis of a single sample can be realized within 9min. Compared with the existing reported methods, the present work can directly screen the PHD2 inhibitory activity of traditional Chinese medicine (TCM) extracts, which is of significance for the target-purification of bioactive individual compounds from TCMs. Under the optimal conditions, the PHD2 inhibitor screening platform was successfully established, and it was found that 70% methanol/water extracts of Astragali Radix and Codonopsis pilosula had good PHD2 inhibitory activity. Furthermore, the present work provides a novel approach for screening the PHD2 inhibitory activity of TCM extracts and the discovery of anti-hypoxia bioactive compounds.

Analysis of Denosumab by a Validated CZE Method and Determination of Sialic Acids by the RP-HPLC Method.

A capillary zone electrophoresis (CZE) method was developed and validated to quantitate the monoclonal antibody denosumab (DmAb) and its charge variants in pharmaceutical products, demonstrating excellent precision, linearity and accuracy. Separations were obtained with migration times of 11.3min for DmAb and the calibration curve was linear in the range of 0.95-20mg/mL. The analytical comparability of seven batches of Prolia® showed mean differences of the estimated content/potencies of 1.87% lower, and 0.84 and 1.21% higher compared with the size-exclusion and reversed-phase liquid chromatography (SE-HPLC and RP-HPLC) methods and the osteoclast antiproliferative bioassay, respectively, with non-significant differences (P > 0.05). An RP-HPLC method with fluorescence detection (RP-HPLC-F), performed on a Kinetex® EVO C18 column (5μm, 100Å, 250mm × 4.6mm), was optimized to determine the levels of sialic acids of DmAb biomolecules, giving mean concentrations of 0.16 and 0.17μg N-acetylneuraminic acid/mg DmAb for Prolia® and Xgeva® pharmaceutical products, respectively. The results demonstrated the capability of each one of the methods, and their use in combination constitutes a strategy to monitor instability, thereby assuring the quality and the batch-to-batch consistency of the biotechnology-derived medicine.

Validated Capillary Zone Electrophoresis Method for Impurity Profiling and Determination of NiII(3-OMe-Salophene)

A capillary zone electrophoresis method was developed for the determination of NiII(3-OMe-salophene), a substance with anticancer activity in vitro. A fused silica capillary (56 cm × 100 µm) was used for this purpose. The method was optimized in terms of parameters affecting the electrophoretic conditions in order to optimize separation efficiency and total time of migration. The analysis was best performed using an operating buffer of 50 mM borate, adjusted to pH 9.3, mixed with acetonitrile (50%, v/v) as organic modifier. Injections were performed hydrodynamically by applying a pressure of 50 mbar for 8 s, and a 30 kV separation voltage was selected at 25 °C. Detection was carried out at 250 nm using diode array detector (DAD). The method allowed the separation of NiII(3-OMe-salophene) from four other structurally related impurities in a total migration time (tm) of 8 min. Peak identification was achieved using the standard reference of individual impurities. The purity of the migrated NiII(3-OMe-salophene) was confirmed by Ultra-violet (UV) scan overlay depending on DAD. The linear ranges for the determination of NiII(3-OMe-salophene) was 400–20,000 ng mL−1 with limit of detection (LOD) of 120 ng mL−1. Acceptable intra-day and inter-day precisions were achieved (%relative standard deviation (RSD) results were less than 0.76% and 0.30%, respectively). The proposed method was assessed for greenness and compared to reported methodologies to prove superiority.

Inhibition of histamine accumulation by novel histamine-degrading species of Staphylococcus sp. isolated from goats and sheep milk.

Histamine is an active amine compound that occurs in various fermented foods that may cause adverse effects on the human health. Certain microorganisms are able to degrade histamine by an oxidative deamination reaction. Therefore, the present study aimed to quantify histamine‐forming and/or ‐degrading activity of the isolates derived from milk of goat and sheep herds, in Iran, by the capillary zone electrophoresis (CZE) method; and we evaluated the molecular characteristics of staphylococcal isolates. Among 243 staphylococcal isolates, 29 histamine‐degrading bacteria were identified. One of these isolates, identified as Staph. epidermidis, No. 605, exhibited the highest activity compared to others, degrading available histamine to 58.33% within 24 h. By polymerase chain reaction (PCR) analysis, the isolate, No. 605 that exhibited remarkable histamine‐degrading activity lacked the genes encoding coagulase and DNase, nor did it harbor any of the five classical enterotoxin genes. This is the first report to show that seven Staphylococcus species, including Staph. chromogenes, Staph. aureus, Staph. haemolyticus, Staph. epidermidis, Staph. pseudintermedius, Staph. agnetis, and Staph. hyicus, were able to degrade histamine, which were hitherto not known to have this capacity. Therefore, histamine‐degrading activity is a definite criterion to introduce fermenting organisms able to decrease histamine content in different food products.

Capillary zone electrophoresis of lipoarabinomannan by multi-layered concentration.

The present article describes a capillary zone electrophoresis method which relies on a multilayered water-alkali solvent stacking with online ionization to enhance detection of mannose, arabinose, and their oligosaccharides, which are used as the migration profile standards but are also the distinctive structural components of lipoarabinomannan. Lipoarabinomannan is detected in patients having tuberculosis. The capillary electrophoresis method with ionization of the whole saccharides without degradation in alkaline solution inside the capillary is based on the structural deprotonation of the molecules under ultrahigh pH conditions. The validation of the capillary electrophoresis parameters revealed that the 15-fold electrolyte-water-injection plug allowed detection of one-third lower concentrations than detected without online concentration. For the first time, the better detectability was seen especially for highly polymerized, but otherwise poorly ionized, arabinooctaose. The applicability of the method for detecting whole large biological saccharide complexes was confirmed by the glycolipid lipoarabinomannan. For the first time also, the migration of the indestructible lipoarabinomannan was detected together with oligosaccharides used representing the capping units, namely mannose, mannobiose, and mannotriose. The myo-inositol-phosphate-lipid unit was seen to migrate separately from the arabinomannan, since it was hydrolyzed from one lipoarabinomannan product under alkaline conditions in capillary electrophoresis.

Investigation of metformin hydrochloride-bovine serum albumin interaction by narrow-bore capillary zone electrophoresis.

A narrow-bore capillary (inner diameter = 2 μm) zone electrophoresis method was developed to study the interaction between metformin hydrochloride and bovine serum albumin. Free metformin hydrochloride, free bovine serum albumin, and their complex were completely separated in a volume of tens of picoliters. The association constant (Ka = 1.04 × 103 L mol-1) and the number of binding sites (n = 0.9789) were then obtained.

Evaluation of Natamycin in Commercial Dairy Products by a Green Capillary Zone Electrophoresis Method and Confirmation with a Liquid Chromatography-Mass Spectrometry

Evaluation of Natamycin in Commercial Dairy Products by a Green Capillary Zone Electrophoresis Method and Confirmation with a Liquid Chromatography-Mass Spectrometry

Application of capillary zone electrophoresis to determine second-generation H1 antihistaminic drugs, loratadine and rupatadine

The second generation of H1 antihistamines from the piperidine group are often used for treating allergic diseases due to their action on histaminic receptors, the primary mediator of allergy. Moreover, the antihistamines have anti-inflammatory action, mediated through platelet-activating factor blocking activity. A simple and rapid capillary zone electrophoresis method was developed and validated for the determination of loratadine (LOR) and rupatadine (RUP) in tablets. The analyses were carried out using a fused silica capillary of 50.2 cm (40 cm effective length), 75 µm i.d. The background electrolyte was composed of boric acid 35 mmol/L, pH 2.5. Voltage of 20 kV, hydrodynamic injection of 3447.3 Pa for 3s, temperature at 25 ºC, and UV detection at 205 nm were applied. Electrophoretic separation was achieved at 1.8 and 2.8 min for RUP and LOR, respectively. The method was linear for both drugs in a range of 50.0 to 400.0 μg/mL (r>0.99). The limits of detection and quantification were 46.37 and 140.52 μg/mL, for LOR and 29.60 and 89.69 μg/mL for RUP respectively. The precision was less than 5.0 % for both drugs. The average recovery was approximately 100 %. The proposed novel method can significantly contribute to the rapid detection of counterfeit products and in quality control of drug products containing antihistamines.

Extension of the Internal Standard Method for Determination of Thermodynamic Acidity Constants of Compounds Sparingly Soluble in Water by Capillary Zone Electrophoresis.

The paper extends applicability of the internal standard method published in 2009 (Fuguet E. et al., J. Chromatogr. A2009,1216(17), 3646). Although the original capillary zone electrophoresis method was suggested to determine thermodynamic acidity constants of compounds sparingly soluble in aqueous solutions by carrying out only runs at two different pH values (i.e., without the need to perform many experiments over the appropriate pH range including the form of a low-ionized analyte), we proved that the approach also virtually overcomes any interactions of the analyte in mixed solvents, so that the experiments can be carried out in a methanol–water buffer where the solubility is much better. Applicability of the extended method is illustrated on six selected β-blockers.

Determination of quetiapine and its metabolites in plasma by field-enhanced sample stacking.

Quetiapine is an atypical antipsychotic drug that can be used to treat mental disorders, including schizophrenia, bipolar disorder and Alzheimer's disease. Quetiapine is mainly converted into the active metabolites of norquetiapine and 7-hydroxyquetiapine by the liver enzymes CYP3A4 and CYP2D6. In this study, liquid-liquid extraction (LLE) was used as a sample pretreatment method to eliminate interferences in plasma. tert-Butyl methyl ether was chosen as the extraction solvent. Field-enhanced sample injection (FESS), an online preconcentration technique, was used to analyze quetiapine and its metabolites norquetiapine and 7-hydroxyquetiapine in plasma. The optimal separation condition was 120 mM phosphate (pH 4.0) containing 0.005% (w/v) polyvinyl pyrrolidone and 40% (v/v) methanol. The methanol plug was 0.3 psi for 6 s, the sample was electrokinetic injection by 10 kV for 60 s at positive polarity, and the separation voltage was set at 26 kV. In this experiment, quetiapine, norquetiapine and 7-hydroxyquetiapine were successfully extracted from plasma by the LLE method and stacking and separated by FESS within 15 min. The limits of detection (S/N = 3) of quetiapine, norquetiapine and 7-hydroxyquetiapine were 0.25 ng/mL, 0.50 ng/mL and 1.00 ng/mL, respectively. The linear ranges of quetiapine, norquetiapine and 7-hydroxyquetiapine were 3-120 ng/mL and the correlation coefficients were 0.999. Compared with that of the traditional capillary zone electrophoresis method, the sensitivity enrichment of analytes was 463-835-fold. The optimal experimental conditions were successfully applied to the analysis of plasma samples from patients taking quetiapine for the treatment of schizophrenia.

METHOD DEVELOPMENT FOR THE DETERMINATION OF METFORMIN IN HUMAN PLASMA BY CAPILLARY ZONE ELECTROPHORESIS

Background: Metformin is an oral antidiabetic drug from the biguanide class and it is the first-line drug chosen for the treatment of type 2 diabetes. This is a drug on the list of pharmaceutical substances required to report bioequivalence study data when registering the drug. Objectives: (1) To develope a capillary zone electrophoresis method for determining metformin hydrochloride in human plasma. (2) To validate the method. Materials and methods: Human plasma, metformin hydrochloride, ranitidine hydrochloride. The method was developed and validated according to US-FDA 2018 and EMA 2011. Results: The procedure was developed by using the Agilent 7100 CE electrophoresis system with ranitidine hydrochloride as an internal standard. Sample preparation was accomplished through protein precipitation with acetonitrile. The optimal electrophoresis conditions are as follows: uncoated fused-silica capillary column of a total length of 40 cm (31.5 cm effective length, inner diameter 100 μm), phosphate buffer solution 100 mM (pH = 4), the voltage applied to both capillary ends of 15 kV, the inlet end of capillary dipped in water before sample injection, sample injection mode of 50 mBar in 7 seconds, using a PDA detector at 232 nm. The analysis method was validated according to the requirements of the US-FDA 2018 and EMA 2011 with the following criteria: system suitability with RSD < 3%; good specificity; the calibration curves were linear (r2 ≥ 0.98) in the concentration range of 0.1 – 4.0 μg/ml for metformin in human plasma; the lower limit of quantification was 0.1 μg/ml; the intra-day and inter-day accuracy were 99.41 – 105.28% and 92.47 – 106.26%, respectively; the intra-day and inter-day precision were 1.99 – 4.03% and 3.11 – 6.05%, respectively; the mean recovery of ranitidine (internal standard) was 86.2%, the mean recoveries of metformin at three levels LQC, MQC, and HQC were 72.9%, 75.9%, and 77.4%, respectively; plasma samples were stable to analysis. Conclusions: The developed method meets the requirements of US-FDA 2018 and EMA 2011 to determine metformin in plasma. Key words: Capillary zone electrophoresis, plasma, metformin