Capillary Electrophoresis Procedure Research Articles

A rapid and simple capillary electrophoresis procedure for quantification of vanillylmandelic acid in urine samples

Aim: To develop a one-step vortex-assisted liquid-liquid extraction (VALLE) method, without the need for evaporation and reconstitution steps, to establish a rapid and straightforward treatment procedure based on capillary electrophoresis-diode array detection (CE-DAD) for the determination of vanillylmandelic acid (VMA) in human urine. Methodology: Optimization of VALLE and CE-DAD procedures were studied in detail. The effects of various experimental parameters, such as the type of the extraction solvent, sample pH, salt addition, and extraction time were investigated. Also, CE separation conditions including background electrolyte type, concentration, and pH, injection time and separation voltage were optimized as well. Results: A successful separation of VMA was achieved in less than 6 min using a basic background electrolyte composed of 60 mmol·L-1 acetate/ACN (acetonitrile) 50% (v/v) (final apparent pH is 5.73). The linear response was obtained over the concentration range from 1.0 to 14 μg·mL-1. The limit of detection and quantification were 0.30 and 1.0 μg·mL-1, respectively. The intra- and inter-day precisions were found to be less than 4.3%. The extraction recoveries of VMA were between 95%-97%. Conclusion: The developed method is found to be a simple, rapid, and reliable method for quantitative analysis of urinary VMA.

Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes

Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.

Comparative study of atenolol in human plasma by high performance liquid chromatography and capillary zone electrophoresis

Atenolol is a beta-blocker that is cardioselective, meaning it only affects beta receptors. It is used to treat angina pectoris and high blood pressure. The HPLC and Capillary Zone Electrophoresis analytical technique was developed for the purpose of detecting and quantifying Atenolol in human plasma, according to the study paper you're reading right now. The internal standard and atenolol were recovered from the solution after being extracted from plasma using the Liquid-Liquid Extraction method. A mobile phase of 10mM sodium hydrogen phosphate, 7.3mM Sodium Lauryl sulphate (pH=3), methanol, and acetonitrile (40:57:3, v:v:v) is used, with a flow rate of 1.0ml/min. A fluorescence detector was used to detect the isolated materials, which had an excitation wavelength of 229 nm and an emission wavelength of 298 nm. With this in view, Atenolol's and the internal norm's survival times are observed to be 5.4 and 8.3 minutes, respectively. The linear correlation coefficient (R20.9992) was found in the Atenolol calibration curve. The recovery rate for atenolol and an internal norm was estimated to be between 76 and 87 percent. Solid-phase extraction was performed on an uncoated silica capillary with a diameter of 58.5 cm 75 m, and detection was performed at 194 nm in the Capillary Zone Electrophoresis procedure. For an electrolyte solution containing 50mM H3BO3 and 50mM Na2B4O7 (50:50 V/V), atenolol was determined to be present in the solution in less than 3 minutes. Energized with a voltage of 25kV and injected with a hydrodynamic configuration for 4S. Under various conditions, this method was used to assess the stability and capability of measuring Atenolol in human plasma.

Determination of glucosamine content in nutraceuticals by capillary electrophoresis using in-capillary OPA labeling techniques

Capillary electrophoresis (CE) procedure was developed for determining glucosamine content in nutraceuticals. Glucosamine was labeled with o-phthalaldehyde (OPA) by mixing with the OPA reagent (4.5 mM OPA, 4.5 mM 2-mercaptoethanol dissolved in 20 mM borate buffer, pH 9.3). The fluorescent OPA-glucosamine adduct was separated and detected at A340 within 3 min. The pre-capillary labeling reaction can be performed by sequentially injecting OPA/sample/OPA segments into the separation capillary; the in-capillary labeling technique simplified the sample pretreatment and improved its reproducibility. The linear dynamic range of the in-capillary labeling CE method was 0.1-30 mM. No sample pretreatment procedure was required for tablets containing mainly starch or other sugar as the vehicles.

Determination of benzoate derivatives in soy sauce by capillary electrophoresis and in-capillary microextraction procedure

Benzoate derivatives in soy sauce were analyzed by capillary zone electrophoresis (CZE) coupled with a microextraction technique. The analytes were acidified and extracted with ethyl acetate. The organic extract was directly injected (30 mbar × 1 sec) into a fused silica capillary (75 μm i.d. × 34 cm) and separated in 20 mM borax buffer (pH 9.2) with 10 kV working voltage. Separation was completed in less than 10 min as monitored by A225. Hippuric acid was used as internal standard to improve the reproducibility for quantification (CV = 4.5%, n = 12).

On-Line Electrokinetic Supercharging and Sweeping for the Preconcentration and Determination of Nucleosides and Related Compounds by Capillary Electrophoresis

Nucleosides and related compounds have vital physiological and pharmacological activities. In this study, on-line preconcentration using electrokinetic supercharging (EKS)-sweeping method was proposed to enhance the efficiency of the capillary electrophoresis separation of the nucleosides and related compounds. The separation conditions, including the pH of the borate buffer (terminating electrolyte), the concentration and pH of the phosphate buffer (leading electrolyte), the concentration of sodium dodecyl sulfate (SDS) and the electrokinetic injection (EKI) time, were investigated in detail. The optimum EKS-sweeping conditions were as follows: 40 mM sodium phosphate buffer at pH 2.0 containing 50 mM SDS as the BGE, 120 mM sodium phosphate buffer at pH 2.0 as the leading electrolyte, 30 mM borate buffer at pH 12.0 as the terminating electrolyte, and EKI of the sample at −10 kV for 30 s. The separation was performed by micellar electrokinetic chromatography at −5 kV. Under these conditions, the sensitivity values of five analytes were enhanced from 26 to 109-fold compared to a standard capillary zone electrophoresis (CZE) procedure. The intra- and inter-day repeatability values (as the relative standard deviation) were less than 1.1% and 5.2% for the migration time and lower than 4.4% and 4.8% for the peak areas. Finally, the developed method was successfully applied for the determination of nucleosides and related compounds in Chinese cordyceps samples.

Analytical verification and quality assessment of the Tosoh HLC-723GX HbA1c analyzer

ObjectivesIon-exchange high-performance liquid chromatography (IE-HPLC) has long been used as a reproducible and versatile analytical tool for HbA1c measurement.In this study, we performed analytical verification and quality assessment of the recently introduced small IE-HPLC Tosoh HLC-723GX HbA1c analyzer, and a comparison of results to immunoassay (IA) and capillary electrophoresis (CE). Design and methodsThe total imprecision of Tosoh HLC-723GX was verified according to CLSI EP15-A2 protocol using commercial control materials (C-QC) and pooled human whole blood samples (HWB). The Sigma metric was used for the evaluation of quality targets. HbA1c results were compared to automated CE (MiniCap Flex Piercing, Sebia, France) and IA (Tina-quant HbA1c Gen 2, Cobas Integra 400+, Roche Diagnostics, USA) procedures. ResultsThe total imprecision of Tosoh HLC-723GX-HbA1c for IFCC(mmol/mol) and NGSP(%) units was: 1.91/1.25% (HbA1c=31mmol/mol/5.0%) and 0.51/0.63% (HbA1c=84mmol/mol/9.8%) for C-QC, and 0.39/0.2% (HbA1c=47mmol/mol/6.5%) and 0.77/0.46% (HbA1c=94mmol/mol/10.8%) in HWB samples, respectively. Bland-Altman analysis did not reveal any deviation of the results between Tosoh HLC-723GX and CE: mean difference 0.0% (95%CI: −0.02927 to 0.02653%), while the mean HbA1c difference against IA was −0.07% (95%CI: −0.1039 to −0.02765). At the selected HbA1c clinical decision level (48mmol/mol/6,5%), six sigma analysis gave σ value of 3.91, within a desirable classification of performance. ConclusionThe analytical performance of the Tosoh HLC-723GX complies with the rigorous quality criteria for clinical use of HbA1c, with the results comparable to the CE procedure. Tosoh HLC-723GX provides a plausible analytical choice for reliable HbA1c measurement in low-volume laboratories.

Capillary electrophoresis coupled with microdialysis for continuous monitoring of free metoprolol in rabbit blood

A microdialysis method followed by a capillary electrophoresis (CE) procedure has been performed for the determination of metoprolol in rabbit blood. A microdialysis probe was inserted into the ear vein for blood sampling. The electrophoresis conditions were: 47 (40) cm × 50 μm silica capillary, 50 mM acetate buffer solution (pH 4.0), 10 s hydrodynamic load, detection wavelength 214 nm, run voltage 18 kV, temperature 25°C. The calibration was linear within the range of 1.0–100 μg/mL (r > 0.999). The limit of detection (LOD) was 0.5 μg/mL. Intra-day and inter-day repeatabilities with relative standard deviations (RSDs) ≤ 5.1% were obtained. The method was then applied to pharmacokinetics profiling of metoprolol in the blood following oral administration of metoprolol to rabbits.

Determination of Shikonin and Rosmarinic Acid in Echium vulgare L. and Echium russicum J.F. Gmel. by Capillary Electrophoresis

A simple and rapid capillary zone electrophoresis (CZE) procedure for determination of shikonin and rosmarinic acid in roots of Echium vulgare L. and Echium russicum J.F. Gmel. is described. The separation was achieved in less than 4 min. The analysis was performed in fused silica capillary (i.d. 75 µm, 48.5 cm total length) using 50 mM borate, at pH 9.5 as the run buffer with applied voltage of 30 kV and constant temperature (27 °C). The method ensures good linearity and repeatability with high correlation coefficients (0.994 and 0.998 for shikonin and rosmarinic acid, respectively). The compounds were detected by UV absorption at 218 (shikonin) and 202 nm (rosmarinic acid). The limits of detection (LOD) for shikonin and rosmarinic acid were 0.603 ppm and 0.270 ppm, respectively. The method was used for estimation of both components in Echium plants. We found out that the content of shikonin and rosmarinic acid were 4.4–4.8 mg/kg air-dry material and 20.0–54.2 mg/kg air-dry material, respectively, in the Echium species analyzed.

A capillary electrophoresis procedure for the screening of oligosaccharidoses and related diseases.

The most widely used method for the biochemical screening of oligosaccharidoses is the analysis of the urinary oligosaccharide pattern by thin-layer chromatography on silica gel plates. However, this method is not always sensitive enough, and it is extremely time-consuming and laborious. In this work, the analysis of the urine oligosaccharide pattern was standardized for the first time by using capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection (Beckman P/ACE MDQ) with a 488-nm argon ion laser module. All of the analyses were conducted using the Carbohydrate Labeling and Analysis Kit (Beckman-Coulter), which derivatizes samples with 8-aminopyrene-1,3,6-trisulfonate. Urine samples from 40 control subjects (age range, 1 week to 16 years) and from ten patients diagnosed with eight different lysosomal diseases (six of them included in the Educational Oligosaccharide Kit from ERNDIM EQA schemes) were analyzed. Two oligosaccharide excretion patterns were established in our control population according to age (younger or older than 1 year of age). Abnormal peaks with slower migration times than the tetrasaccharide position were observed for fucosidosis, α-mannosidosis, GM1 gangliosidosis, GM2 gangliosidosis variant 0, Pompe disease, and glycogen storage disease type 3. In conclusion, the first CE-LIF method to screen for oligosaccharidoses and related diseases, which also present oligosacchariduria, has been standardized. In all of the cases, the urine oligosaccharide analysis was strongly informative and showed abnormal patterns that were not present in any of the urine samples from the control subjects. Only urine from patients with aspartylglucosaminuria and Schindler disease displayed normal results.

Direct nitrate sensing in water using an array of copper-microelectrodes from flat flexible cables

Nitrate determination plays a very important role in the analysis of environmental samples. In this paper, an activation procedure for nitrate sensing using a modified copper electrode made from flat flexible cables in presence of a low amount of chloride is reported. The proposed method exhibits enhanced electrocatalytic behaviour in the reduction of nitrate compared to a bare copper electrode. The proposed method showed linear response for nitrate in the concentration range 10–1070μmolL−1, with a regression coefficient R2=0.999 (n=12) and a limit of detection of 1.8μmolL−1. According to the procedure based on the standard addition technique, the recovery values obtained were 93–103%, showing the accuracy of the proposed method. In addition, the proposed sensor was applied to the determination of nitrate in mineral water samples and the results are in good agreement (for a 95% confidence level according to the Student's t-test) with those obtained using the capillary electrophoresis procedure.

Simultaneous Determination of Pyridine-Triphenylborane Anti-Fouling Agent and Its Degradation Products in Paint-Waste Samples Using Capillary Zone Electrophoresis with Field-Amplified Sample Injection

We proposed a capillary zone electrophoresis (CZE) procedure using field-amplified sample injection (FASI) for the simultaneous determination of pyridine-triphenylborane (PTPB) and its degradation products: diphenylborinic acid (DPB), phenylboronic acid (MPB), and phenol. The LODs for PTPB, DPB, MPB, and phenol were, respectively, 0.85, 0.88, 44, and 28 μg L(-1). The RSDs (n = 4) for the analytes listed above were in respective ranges of 6.2 - 14, 5.9 - 10, and 0.49 - 0.62% for the peak area, peak height, and migration time. The compounds were extracted from paint-waste samples collected from shipyards using a siliga-gel column. The extract was dissolved with acetonitrile containing 1% (v/v) pyridine. The samples were then analyzed using CZE, revealing respective concentrations of 0.076 - 0.53, 0.015 - 0.36, 1.7 - 22, and 1.2 - 13 μg g(-1). The proposed FASI-CZE method is a simple and promising procedure that is expected to be useful for the determination of PTPB and its degradation products in paint wastes.

A highly sensitive capillary electrophoresis method using p-nitrophenyl 5′-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities

A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length×75.5μm (id)), electrokinetic injection for 60s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of −80μA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (Km values for NPP1 and NPP3, Ki values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.

Rapid determination of sodium in milk and milk products by capillary zone electrophoresis

A capillary zone electrophoresis method for the determination of Na in milk and milk products was developed and compared with an International Organization for Standardization/International Dairy Federation standard method that is based on flame atomic absorption spectrometry. The adoption of a background electrolyte consisting of 10mM imidazole adjusted to pH 3.75 by the addition of oxalic acid allowed baseline separation of Na from other milk cations and from Li ion, which was adopted as an internal standard. Method validation was performed and the results for linearity, precision, limit of detection, limit of quantification, and recovery are presented. The procedure was tested on commercial milk samples differing in fat content (whole, semiskimmed, and skimmed) and processing conditions (pasteurization, UHT sterilization, and microfiltration). The reliability of the method was confirmed for different varieties of cheese and other milk products. The method enables the routine measurement of Na content by a rapid and accurate capillary zone electrophoresis procedure.

Optimization and development of capillary electrophoresis for separating and identifying wheat low molecular weight glutenin subunits

We focus on capillary electrophoresis (CE) procedure. For the first time, CE procedure is successfully used for wheat LMW-GS analysis. The CE analysis of LMW-GS is used for wheat cultivar identification. We demonstrated the application of this procedure for wheat quality improvement. The CE method can be used as a new method complimentary to the existing ones.

A Convenient Approach to Simultaneous Analysis of a Pharmaceutical Drug and Its Counter-Ion by CE Using Dual-Opposite End Injection and Contactless Conductivity Detection

Capillary electrophoresis (CE) coupled to a capacitively coupled contactless conductivity detector (C4D) was used for the determination in a single analysis of a pharmaceutical drug and its counter-ion. Dual-opposite end injection (DOI) was used to introduce hydrodynamically the analytes at each end of the capillary. No modification of the commercial apparatus is required. After applying the voltage, the cations and anions migrate from each end of the capillary in opposite directions toward the detector placed near the cathode outlet. The electrophoretic conditions were initially developed with three test drugs (chlorpheniramine maleate, metoprolol tartrate, clomiphene citrate) and then applied to two Vinca alkaloids (catharanthine sulfate, vinorelbine ditartrate). The 10 mM histidine–50 mM acetic acid buffer (pH 4.1)–methanol 90:10 (v/v) electrolyte was suitable for the analysis of these high or medium mobile anions by CE–C4D due to its low conductivity background and high buffer capacity. Finally, the CE procedure developed was successfully validated for catharanthine sulfate. The method developed herein is fast (<10 min) and accurate (repeatability on migration time < 0.6% and peak areas < 1.3%, n = 6).

Rapid monitoring of mono- and disaccharides in drinks, foodstuffs and foodstuff additives by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis (CE) procedure with contactless conductivity detection (C 4D) has been developed for monitoring of neutral mono- and disaccharides in drinks and foodstuffs. The separation of a mixture of seven neutral saccharides (glucose, fructose, galactose, mannose, ribose, sucrose and lactose) employed a quartz capillary, 5 μm i.d., with an effective length of 18.3 cm, and 75 mM NaOH (pH 12.8) as the background electrolyte (BGE). The limit of detection (LOD) values obtained lied within a range from 0.4 μmol L −1 for lactose to 0.9 μmol L −1 for ribose, with a separation time shorter than 140 s. The procedure was successfully applied to determinations of saccharides in fruit juices, Coca-Cola, milk, red and white wines, yoghurts, honey and a foodstuff additive.

Validation of a Capillary Electrophoresis Method for the Simultaneous Determination of Amlodipine Besylate and Valsartan in Pharmaceuticals and Human Plasma

A rapid, simple, and sensitive capillary electrophoresis (CE) method was developed and validated for the simultaneous determination of amlodipine (AML) and valsartan (VAL) in pharmaceuticals and human plasma using a UV photodiode array detector. Electrophoretic conditions were optimized to improve separation, sensitivity, and rapidity. The optimal conditions were 25 mM phosphate buffer at pH 8.0, injection time 10.0 s, voltage 25 kV, and column temperature 25 degrees C, with detection at 214 nm. The method was found to be linear in the range of 1.0-35 and 1.0-350 mg/L, with weighted regression 0.9999 and 0.9994, for AML and VAL, respectively. Validation of the method showed acceptable intraday and interday accuracy (85.5-95.3%) and precision (RSD 1.64-4.2%) in pharmaceutical formulation and human plasma analysis. The sensitivity of the method was enhanced by both optimization of the CE procedure and preconcentration performed by liquid-liquid extraction. The LOD for both AML and VAL was 0.03 mg/L, which allows analysis at the level of the drugs possibly found in human plasma. Therefore, the proposed method is suitable for QC in pharmaceutical laboratories and therapeutic drug monitoring in clinical laboratories.

Field-Amplified Sample Stacking for Rapid and Sensitive Determination of Aconitine Alkaloids by CE Using an Ionic Liquid Electrolyte System

A new capillary electrophoresis procedure with field-amplified sample stacking (FASS) concentration for analysis of three aconitine alkaloids has been established using 1-butyl-3-methylimidazolium-based ionic liquids as the electrolyte solution. The use of a short water plug at the column inlet is essential for improving the reproducibility of FASS with electrokinetic injection, and the water plug injection time affects the sensitivity significantly. With this stacking procedure, the concentration sensitivity was 5–142 fold higher than for pressure injection. After optimization of the separation and concentration conditions, the three alkaloids could be separated within 6 min and quantified with high sensitivity. The detection limits were 0.10 μg mL−1 for aconitine, 0.021 μg mL−1 for hypaconitine and 0.56 μg mL−1 for mesaconitine. The method was used for quantitative analysis of the three aconitine alkaloids in two Aconitum plants with recoveries of 95.8–105.4%.

Determination of pheniramine enantiomers in eye drop by capillary electrophoresis using hydroxypropyl-β-cyclodextrin as chiral selector

A capillary zone electrophoresis procedure has been developed for the chiral determination of pheniramine in eye drop. Native and derivative cyclodextrins (CDs) including γ-CD, β-CD, hydroxypropyl-β-CD and dimethyl-β-CD were tested as chiral selectors. Using 30 mM hydroxypropyl-β-CD in 50 mM phosphate buffer (pH 3.0), the acceptable resolution value (R = 1.55) was obtained. The assay was validated for linearity (3.3 × 10−6–5.0 × 10−4 M; R2 = 0.9996), limit of detection (3.3 × 10−6 M), limit of quantification (8.5 × 10−6 M), analytical precision by terms of intra- and inter-day variability (RSD ≤ 2.57%), and accuracy (recovery ≥ 89.3%). The content of pheniramine in eye drop obtained by the proposed method was in good agreement with the declared value. The results indicated that pheniramine in the eye drop was present as the racemate.