Development and validation of a capillary electrophoresis method applied to the analysis of l‐citrulline in an oral formulation for pediatric use

A simple and highly sensitive CE–UV method was applied in the determination of l ‐ctrulline, which was developed from an oral formulation for pediatric use. The novel method was based on the analysis of l ‐citrulline for direct ultraviolet detection at 198 nm. The BGE consisted of 10 mM sodium tetraborate and 50 mM SDS at pH 9, and the electrophoretic parameters were optimized. The method was validated in terms of specificity, linearity, LOD, LOQ, precision, accuracy, and robustness. The LOD and LOQ obtained were 1.36 and 4.54 μg/mL, respectively. In addition, the method offers higher sensitivity and specificity compared with the results obtained from HPLC method using UV‐detectors, in which l‐ citrulline needs to be derivatizated. Furthermore, low cost and simplicity of the system allowed the rapid and simple quantitation of l‐ citrulline in the oral formulation for quality control and stability indicated method.     

Determination of folic acid by capillary electrophoresis with chemiluminescence detection

A rapid and simple method is presented for the determination of folic acid (FA) by capillary electrophoresis (CE) with chemiluminescence (CL) detection. This method was based on enhance effect of FA on the CL reaction between luminol and BrO(-) in alkaline aqueous solution. Optimal separation and determination was obtained with an electrophoretic buffer of 35 mM sodium borate (pH 9.4) containing 0.8 mM luminol, and an oxidizer solution of 1.6 mM NaBrO in 100 mM NaCO(3) buffer solution (pH 12.0). Under the optimal conditions, the determination of FA was achieved in less than 20 min, and the detection limit was 2.0 x 10(-8) M (S/N=3). The relative standard deviations (RSDs) on peak area and migration time were in the 1.5 and 1.1%, respectively. The present CE-CL method was applied to the determination of FA in commercial pharmaceutical tablets, apple juices and human urine.     

Determination of citrulline and homocitrulline by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic method was developed for the determination of citrulline and homocitrulline using a post-column colorimetric reaction with o-phthaladehyde and N-(1-naphthyl)-ethylenediamine. Citrulline and homocitrulline were determined with no interferences from protein amino acids. The results show that the level of citrulline in the plasma of patients with uremia on intermittent hemodialysis is higher than that in healthy human plasma, and that homocitrulline is excreted into the urine of healthy adults.     

Electrochemical Behavior and Voltammetric Determination of Curcumin at Electrochemically Reduced Graphene Oxide Modified Glassy Carbon Electrode

This work presents a sensitive voltammetric method for determination of curcumin by using a electrochemically reduced graphene oxide (ERGO) modified glass carbon electrode (GCE) in 100 mM KCl‐10 mM sodium phosphate buffer solution (pH 7.40). The electrochemical behaviors of curcumin at ERGO/GCE were investigated by cyclic voltammetry, suggesting that the ERGO/GCE exhibits excellent electrocatalytic activity towards curcumin, compared with bare GCE and GO/GCE electrodes. The electrochemical reaction mechanisms of curcumin, demethoxycurcumin and bisdemethoxycurcumin at the ERGO/GCE were also investigated and discussed systematically. Under physiological condition, the modified electrode showed linear voltammetric response from 0.2 μM to 60.0 μM for curcumin, with the detection limit of 0.1 μm. This work demonstrates that the graphene‐modified electrode is a promising strategy for electrochemical determination of biological important phenolic compounds.     

Flow injection analysis system for on-line cutinase activity assay

A FIA system based on a micellar system for a substrate (p-nitrophenylbutyrate) with low stability in aqueous phase was built to monitor cutinase activity in bioprocesses. All samples were previously diluted with 50 mM phosphate buffer pH 7.0 containing 11.6 mM sodium cholate. The cutinase activity in this diluted solution enhances about 40% in relation to phosphate buffer. Furthermore, the enzyme adsorption and consequent blocking/fouling of injector and tubes of the FIA system was eliminated due to excellent properties of sodium cholate as surface active agent.The cutinase activity is based in following the hydrolysis of p-nitrophenylbutyrate to p-nitrophenol in the reaction stream through the formation of an absorbance peak at 400 nm proportional to enzyme activity. The compositions of reaction stream as well as its stability were studied in order to minimize non-enzymatic hydrolysis of p-nitrophenylbutyrate and maximize cutinase activity assay reproducibility. An excellent correlation was obtained between the FIA system and off-line method for determination of cutinase activity in the culture media, and during separation of Saccharomyces cerevisiae cells and cutinase concentration by micro and ultrafiltration, respectively.     

Analysis method of the angiotensin-I converting enzyme inhibitory activity based on micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.     

Precise method for the measurement of catalase activity in honey

An improved method is reported for the determination of catalase activity in honey. We tested different dialysis membranes, dialysis fluid compositions and amounts, dialysis temperatures, sample amounts, and dialysis times. The best results were obtained by dialysis of 7.50 g sample in a cellulose dialysis sack, using two 3 L portions of 0.015 M sodium phosphate buffer (pH 7.0) as the dialysis fluid at 4 degrees C for 22 h. As in previous methods, catalase activity was determined on the basis of the rate of disappearance of the substrate, H202, with the H202 determined spectrophotometrically at 400 nm in an assay system containing o-dianisidine and peroxidase. Trials indicated that the best solvent for the o-dianisidine was 0.2 M sodium phosphate buffer, pH 6.1; the best starting H202 concentration was 3 mM; the best HCl concentration for stopping the reaction was 6 N; and the best sample volume for catalase measurement was 7.0 mL. Precision values (relative standard deviations for analyses of 10 subsamples of each of 3 samples) were high, ranging from 0.48% for samples with high catalase activity to 1.98% for samples with low catalase activity.     

Determination of neutral carbohydrates by CZE with direct UV detection

A new CZE method relying on in-capillary reaction and direct UV detection at the wavelength 270 nm is presented for the simultaneous separation of the neutral carbohydrates xylitol, D-(-)-mannitol, sucrose, D-(+)-fucose, D-(+)-cellobiose, D-(+)-galactose, D-(+)-glucose, L-rhamnose, D-(+)-mannose, D-(-)-arabinose, D-(+)-xylose, and D-(-)-ribose. The alkaline electrolyte solution was prepared of 130 mM sodium hydroxide and 36 mM disodium hydrogen phosphate dihydrate. Separation of the sample mixture was achieved within 35 min. Calibration plots were linear in the range of 0.05-3 mM. Reproducibility of migration times was between 0.3 and 1.1%, and the detection limits for the analytes were 0.02 and 0.05 mM. The optimized method was applied for the determination of neutral monosaccharides in lemon, pineapple, and orange juices and a cognac sample. The methodology is fast since no other sample preparation except dilution is required.     

Simultaneous determination of digoxin and digitoxin by micellar electrokinetic chromatography and application to drug formulations

A simple micellar electrokinetic chromatographic method is described for simultaneous determination of digoxin and digitoxin. The simultaneous analysis of digoxin and digitoxin was performed in Tris buffer (10 mM; pH 9) with 90 mM sodium dodecyl sulfate and 10% isopropyl alcohol as an anionic surfactant and organic modifier. Under these conditions, good separation with high efficiency is achieved in short analysis times. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and sodium dodecyl sulfate. The linear range of the method for the determination of digoxin and digitoxin was over 0.01–0.3 mg/mL; the detection limit (signal to noise ratio = 3; injection 3.5 kPa 3 s) was 4 and 6 μg/mL, respectively. Application of the proposed method to the determination of digoxin in commercial tablets and in injections proved to be feasible.     

Kinetic study of gamma-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography

The use of capillary electrophoresis for the determination of gamma-glutamyltransferase (GGT) activity with gamma-glutamyl-p-nitroanilide (Glu-p-NA) as a substrate was investigated. The reaction velocity was quantified spectrophotometrically by the corrected peak area of the product p-nitroaniline (pNA) at 380 nm. Micelles composed of sodium deoxycholic acid were used in the background electrolyte in order to obtain a baseline separation between the substrate and the product. The presence of the micelles did not influence the enzymatic reaction. The electrophoretic system was used, not only for the separation and quantitation of the different reaction compounds but also for the in-capillary mixing of the enzyme and substrate plugs. This methodology is known as electrophoretically mediated microanalysis (EMMA). With the developed in-capillary activity assay an average Michaelis constant (K(M)) for GGT was calculated to be 2.09 mM (RSD = 7.3%, n = 3), a value consistent with previously reported values.     

Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis.

A fast, sensitive and direct method has been developed for the determination of glutathione peroxidase activity (both selenium- and non-selenium-dependent) in cell-free preparations. The assay is based on the separation and quantitation of reduced and oxidized glutathione by capillary electrophoresis. The electrophoretic separation buffer was 100 mM sodium tetraborate (pH 8.2) containing 100 mM sodium dodecylsulphate. A micellar electrokinetic mechanism took place under these conditions, and a total mass recovery was observed for both peptides. The reproducibility of migration times was excellent (less than 3% variability). A linear detector response range was observed in the range 5-50 U/ml, and both the reproducibility and accuracy were satisfied. Samples out of this linear range could be analysed by either increasing the reaction time or diluting the enzyme preparation. The results obtained with the new direct capillary electrophoresis assay were compared with those derived from a reversed phase high-performance liquid chromatographic and spectrophotometric coupled assay. A very good agreement was found between the two direct assay methods in all samples. Capillary electrophoresis is a versatile technique that allows the automation of the glutathione peroxidase assay in a reproducible manner and within a relatively short time with sufficient accuracy and precision.     

Micro determination of cortisol and cortisone in umbilical cord blood by chemiluminescent high‐performance liquid chromatography

A simple, sensitive and specific chemiluminescent high‐performance liquid chromatography method, based on the luminol reaction, for determination of serum cortisol and cortisone, was established. In infants, placental 11β‐hydroxysteroid dehydrogenase type 2 enzyme (11β‐HSD2) activity may affect adrenal function early after birth. The cortisol–cortisone ratio of serum concentrations in umbilical cord blood is an indicator of placental 11β‐HSD2 activity. The optimum conditions for the luminol reaction were determined to be 1.5 mM luminol, 0.6 M sodium hydroxide, 0.15 mm potassium hexacyanoferrate(III) and 200 mM potassium hexacyanoferrate (II). The calibration curves for cortisol and cortisone exhibited good linearity. The correlation coefficients of the calibration curves were 0.996. The intra‐ and inter‐day precisions were in the ranges: cortisol 7.0–12.2 and 4.4–9.2%, cortisone 5.3–7.0 and 6.2–9.9%. The recoveries of these steroids were in the ranges: cortisol 97–105%, cortisone 94–102%. The limits of detection were as follows: cortisol, 0.17 μg/dl; cortisone 0.15 μg/dl. This assay could be successfully applied to determination of the cortisol–cortiosone ratio of serum concentrations in umbilical cord bloods. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of streptomycin in eggs yolk by capillary electrophoresis

Summary A capillary electrophoresis method for the determination of streptomycin in eggs is described. Analyses were performed on an uncoated silica capillary using a buffer solution of 30 mM sodium dihydrophosphate, 5 mM boric acid and 5 mM sodium tetraborate. Analytes were detected at 200 nm an the calibration curve was linear over the range of 0.16 to 2.0 μg g⁻¹ (r=0.999). The total analysis time was 7 min. The method has been successfully applied to the quantitative determination of streptomycin in hen eggs after drug ingestion and could used for evaluation of maximum residue limits.     

Optimization and validation of an RP-HPLC method for direct determination of metformin hydrochloride in human urine and in a dosage form

A simple, selective, sensitive, accurate, and precise method was developed for determination of metformin hydrochloride (MF) in human urine using RP-HPLC. The method depends upon using an octylsilyl (C8) 5 microm particle size column at ambient temperature with mobile phase consisting of 33 mM sodium dihydrogen phosphate containing 6.38 mM hexanesulfonic acid sodium salt and adjusted to apparent pH 3.0 with phosphoric acid-acetonitrile (93 + 7, v/v) at a flow rate of 1.5 mL/min. Quantitation was achieved with UV detection at 231 nm based on peak area with a linear calibration curve over the concentration range of 0.01-50 microg/mL. The proposed method was applied to the determination of the urinary excretion pattern of MF (the cumulative amounts excreted were calculated without pretreatment of the urine sample) and for determination of the dissolution pattern of MF tablets. The proposed method was completely validated according to the U.S. Food and Drug Administration guidelines.     

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.     

Determination of catecholamines and serotonin by micellar electrokinetic chromatography with laser-induced fluorescence detection

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein, a new synthesized fluorescent reagent, was established for the first time as a label for the sensitive analysis of catecholamines (CAs) and serotonin (5-HT) by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. After careful study on the derivatization conditions such as pH value, reagent concentration, temperature and reaction time, the labeling reaction was accomplished as quickly as 7 min with stable yield. The separation parameters for the CAs and 5-HT were also optimized in detail. The derivatives were baseline separated in a running buffer containing 30 mM boric acid and 15 mM sodium dodeculsulfate at pH 9.0. The detection limits ranged from 5 x 10(-10) to 2 x 10(-9) M (signal-to-noise ratio = 3). The rapid and sensitive method was also applied to the determination of the CAs and 5-HT of urine samples.     

Determination of endo- and exogenous corticosteroids by cyclodextrin-modified micellar electrokinetic chromatography with the use of on-line preconcentration

A method was proposed for the simultaneous determination of steroids of the endo- and exogeneous origin with the use of micellar electrokinetic chromatography, which provides the determination of residual medicines in biological fluids and the control of medication efficiency at different steroidogenesis abnormalities. The introduction of a macrocyclic addition of β-cyclodextrin (from 1 to 6 mM) into a buffer electrolyte solution (25 mM phosphoric acid, 20 mM sodium dodecyl sulfate, 4.5 mM urea, pH 2.5) or into a sample solution decreases the detection limit by a factor of 20–200.     

Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in‐capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β‐nicotinamide adenine dinucleotide 2′‐phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β‐nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver–Burk equation, the Michaelis–Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.     

Buffer effect on the kinetics of ornithine carbamyl transferase by HPLC

A kinetic study of ornithine carbamyl transferase (OCT) from Streptococcus faecalis has been carried out, employing a new HPLC method based on the direct determination of citrulline. A different kinetic pattern was observed in tris(hydroxymethyl)-aminomethane (TRIS) and triethanolamine (TEA) buffers: a ping-pong or an ordered sequential mechanism are suggested, respectively. The inhibition of Tris buffer on OCT reaction has also been demonstrated. The inhibition of Tris buffer on OCT reaction has also been demonstrated.     

Simultaneous determination of water-soluble vitamins in a vitamin-enriched drink by an in-capillary enzyme reaction method

The in-capillary enzyme reaction method was used to determine riboflavin phosphate in a vitamin-enriched drink based on its conversion to riboflavin (vitamin B2) with alkaline phosphatase. Simultaneously, three water-soluble vitamins [thiamine nitrate (vitamin B2 mononitrate), pyridoxine hydrochloride (vitamin B6 hydrochloride) and nicotinamide (vitamin PP)] and anhydrous caffeine in the drink were subjected to quantitative analysis. In the system, electrophoretic migration was used to mix zones containing the substrate (riboflavin phosphate) and the enzyme (alkaline phosphatase). The reaction was then allowed to proceed in the presence of a weak electric field and, finally, the product (riboflavin) of enzyme reaction and other water-soluble vitamins migrated under the influence of an applied electric field to the detector. All the active ingredients and the formulation excipients were successfully separated by micellar electrokinetic chromatography with 135 mM sodium dodecyl sulfate. To prevent inhibition of enzyme reaction by the addition of sodium dodecyl sulfate to the reaction zone, sandwich mode injection, in which plugs of sandwich solution without sodium dodecyl sulfate were introduced into the capillary on both sides of the reaction zone, was utilized as a barrier to protect the enzyme reaction from the inhibitor. The relationship between the peak area of the product and the concentration of the substrate was calculated in the in-capillary enzyme reaction method. Excellent linearity was obtained, with correlation coefficients of 0.9999. The established method was validated and demonstrated to be applicable to the determination of the five active ingredients, including riboflavin phosphate, in a commercial vitamin-enriched drink. No interference from the formulation excipients was observed. Good linearities were obtained, with correlation coefficients above 0.999. Recoveries and precisions ranged from 99.3 to 101.8%, and from 0.1 to 2.5% RSD, respectively. Good agreement was obtained between the established method and traditional high-performance liquid chromatographic methods. These results suggest that the in-capillary enzyme reaction method can be used for the simultaneous determination of riboflavin phosphate and other water-soluble vitamins in pharmaceuticals.