Separation and detection of peroxynitrite and its metabolites by capillary electrophoresis with UV detection

A method for the separation and direct detection of peroxynitrite (ONOO(-)) and two of its degradation products, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), using capillary electrophoresis with ultraviolet detection is described. The separation parameters were optimized and included electrokinetic injection, a run buffer consisting of 25 mM K(2)HPO(4) 7.5 mM DTAB, pH 12, and a field strength of -323 V/cm. A diode array UV detector was employed in these studies as it allowed the determination of all three species simultaneously. Nitrate and nitrite provided the maximum response at 214 nm while peroxynitrite generated the best response at 302 nm. All three species could be detected at 214 nm, while simultaneous detection at 214 and 302 nm positively identified each peak.     

Determination of Six Analgesics by CE with an Improved Electromagnetic Induction Detector

A novel, rapid and validated capillary electrophoretic method with an improved electromagnetic induction detector has been developed for the determination of ibuprofen, acetaminophen, amantadine hydrochloride, aminopyrine, diclofenac sodium and codeine phosphate in three kinds of analgesic pharmaceutical preparations. Fabrication of the electromagnetic inductor was the same as previously described, but a brand new electric circuit was designed for the detector to lower the background noise and improve the detection sensitivity. Important factors which might influence the response of the detector, including the value of the adjustable inductor and resistor, excitation frequency and voltage, were examined. Electrophoretic parameters affecting separation efficiency, such as variety of buffer, buffer pH, buffer concentration and electroosmotic flow modifier, were also deliberately investigated. Under the optimal conditions, highly linear response was obtained for these six compounds over three orders of magnitude with detection limits ranging from 0.1 to 1.0 μg mL⁻¹ (S/N = 3). The average recovery and RSD were within the range of 97.5–101.5 and 1.1–2.3%, respectively. This simple, convenient, effective and stable method held great promise in quality control of pharmaceutical preparations.

     

Rapid analysis of clenbuterol, salbutamol, procaterol, and fenoterol in pharmaceuticals and human urine by capillary electrophoresis

Capillary electrophoresis (CE) with UV detection for the simultaneous and short-time analysis of clenbuterol, salbutamol, procaterol, fenoterol is described and validated. Optimized conditions were found to be a 10 mmoll(-1) borate buffer (pH 10.0), an separation voltage of 19 kV, and a separation temperature of 32 degrees C. Detection was set at 205 nm. Under the optimized conditions, analyses of the four analytes in pharmaceutical and human urine samples were carried out in approximately 1 min. The interference of the sample matrix was not observed. The LOD (limits of detection) defined at S/N of 3:1 was found between 0.5 and 2.0 mgl(-1) for the analytes. The linearity of the detector response was within the range from 2.0 to 30 mgl(-1) with correlation coefficient >0.996.     

Evaluation of Mobile and Stationary Phases in Reversed-Phase High-Performance Liquid Chromatography of Conjugated Bile Acids

Abstract

The reversed-phase high-performance liquid chromatographic separation of the ten major conjugated bile acids of man using isocratic conditions is described. Each component of the mobile and stationary phases was examined for its ability to influence the separation selectivity. Manipulation of pH, buffer species, organic modifier and different types of packings showed that optimal resolution was obtained with a mobile phase of methanol-0.02M sodium acetate (60:30) adjusted to pH 4.2 with phosphoric acid, on a Supelcosil LC-18-DB column. Advantages of the optimized phase system are the complete baseline separation of compounds within a short period of time, improved peak symmetry and a high rate of reproducibility. This new chromatographic method, coupled with UV detection at 205 nm, is suitable for the simultaneous determination of bile acid conjugates in routine clinical analysis.     

High Performance Liquid Chromatography of Uroporphyrin Isomers

Abstract

The separation of uroporphyrin I and III isomers by reversed-phase high performance liquid chromatography on ODS-Hypersil with ammonium acetate buffer-acetonitrile solvent systems is described. The effects of buffer concentration, pH, organic modifier proportion and different organic modifiers on the resolution are studied. The optimum conditions for the separation were 12–13% acetonitrile in 1M ammonium acetate buffer pH 5.10–5.20. The method also separated uroporphyrin I and III from the II isomers but the resolution of uroporphyrin III and IV isomers was not achieved.     

Macrocyclic polyamine as a selective modifier in a bonded-phase capillary column for the electrophoretic separation of aromatic acids.

The use of a macrocyclic polyamine, 28[ane]-N6O2, as a selective modifier in a bonded-phase capillary column for the electrophoretic separation of 14 aromatic acids is described. Parameters that affect the performance of the separations, such as the type of buffer, the pH and concentration of buffer, the applied potential and the injection mode were studied. By changing the buffer pH (4.0-5.0), buffer concentration (10-50 mM) and applied potential (-10 approximately -20 kV), optimum conditions were obtained at -20 kV, using an acetate buffer (20 mM, pH 4.5), hydrodynamic injection with a vacuum at the buffer reservoir on the detector side and detection at 220 nm. The results showed that the separation was effective under these conditions. The plate number was greater than 4 x 10(4) m-l. Due to the wide variation in the mobilities of the test compounds, injection studies suggested that a vacuum at the buffer reservoir on the detector side would produce a result that is more representative of the initial sample composition. Benzoic acid in soy sauce, salicylic acid in Salic ointment and Aspirin were sampled and analyzed using the established conditions.     

A Comparison and Clinical Applications of Ammonium Acetate and Phosphate Based Mobile Phases for the Separation of Catecholamines on Reversed Phase Columns

Abstract

A novel ion paired high performance liquid chromatographic system on reversed phase columns with ammonium acetate buffer as eluent is described for the separation of catecholamines. The advantages of using ammonium acetate buffer have been systematically studied and compared with the more widely employed phosphate buffer. The applicability of the method was demonstrated by analysis of catecholamines in clinical specimens.     

On-line concentration and separation of indolamines, catecholamines, and metanephrines in capillary electrophoresis using high concentration of poly(diallyldimethylammonium chloride)

This paper tackles a simple and efficient method for the simultaneous separation and stacking of neurotransmitters in capillary electrophoresis with UV detection. By using poly(diallyldimethylammonium chloride) (PDDAC) as a buffer additive, the high and reversed EOF are observed. Moreover, the mobility of indolamines and catecholamines decreases as the PDDAC concentration increases. Based on the difference in mobility in the presence and absence of PDDAC, the analytes were simply stacked between the boundary of the sample zone and the background electrolyte containing PDDAC. The separation of 14 analytes including indolamines, catecholamines, and metanephrines was accomplished within 33 min under optimal conditions (1.2% PDDAC and 5 mM formic acid at pH 4.0), and the values of relative standard deviation of their migration time were less than 3.1%. By applying stacking methods for fourteen analytes, we observed: (a) the sample injection volume of sample is up to 216 nL, (b) the limits of detection at signal-to-noise of 3 range from 15.4 to 122.1 nM, and (c) the sensitivity enhancements, compared to normal injection (12 nL), range from 110- to 220-fold. Under the optimal stacking conditions, the present method has been applied to analyze of vanillomandelic acid, 5-hydroxyindole-3-acetic acid, dopamine, tryptamine, and 3-indoxyl sulfate in urine samples.     

Electrokinetic characterization of superparamagnetic nanoparticle–aptamer conjugates: design of new highly specific probes for miniaturized molecular diagnostics

With the view of designing new nanoparticle (NP)–aptamer conjugates and proving their suitability as biorecognition tools for miniaturized molecular diagnostics, new maghemite–silica core–shell NP–aptamer conjugates were characterized for the first time in terms of grafting rate and colloidal stability under electrophoretic conditions using capillary electrophoresis. After the grafting rate (on the order of six to 50) of the lysozyme-binding aptamer had been estimated, the electrophoretic stability and peak dispersion of the resulting oligonucleotide–NP conjugates were estimated so as to determine the optimal separation conditions in terms of buffer pH, ionic strength and nature, as well as temperature and electric field strength. The effective surface charge density of the NPs was close to zero for pH lower than 5, which led to some aggregation. The NPs were stable in the pH range from 5 to 9, and an increase in electrophoretic mobility was evidenced with increasing pH. Colloidal stability was preserved at physiological pH for both non-grafted NPs and grafted NPs in the 10–100 mM ionic strength range and in the 15–60 °C temperature range. A strong influence of the nature of the buffer counterion on NP electrophoretic mobility and peak dispersion was evidenced, thus indicating some interactions between buffer components and NP–aptamer conjugates. Whereas an electric field effect (50–900 V cm^?1) on NP electrophoretic mobility was evidenced, probably linked to counterion dissociation, temperature seems to have an appreciable effect on the zeta potential and aptamer configuration as well. This information is crucial for estimating the potentialities of such biorecognition tools in electrophoretic systems.     

Ion Exchanger Liquid Chromatography of N-Acetylaspartic Acid and Some N-Acetylaspartyl Peptides

Abstract

A high performance liquid chromatographic (HPLC) procedure for the rapid separation of N-acetylaspartic acid, N-acetyl-aspartyl-glutamic acid and N-acetylaspartyl-glutamyl-aspartic acid is described. The procedure utilizes a pellicular ion-exchange column support, and ionic strength gradient with mobile phase solutions buffered to pH 5.0 and a UV detector operated at 210 nm. Reproducibility and quantitative capabilities are also discussed. The method has been used for a tentative estimation of N-acetylaspartic acid and N-acetylaspartyl peptides in a rat brain synaptosomal extract.     

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.     

Simultaneous determination of preservatives (benzoic acid, sorbic acid, methylparaben and propylparaben) in foodstuffs using high-performance liquid chromatography

A reversed-phased HPLC method that allows the separation and simultaneous determination of the preservatives benzoic (BA) and sorbic acids (SA), methyl- (MP) and propylparabens (PP) is described. The separations were effected by using an initial mobile phase of methanol-acetate buffer (pH 4.4) (35:65) to elute BA, SA and MP and changing the mobile phase composition to methanol-acetate buffer (pH 4.4) (50:50) thereafter. The detector wavelength was set at 254 nm. Under these conditions, separation of the four components was achieved in less than 23 min. Analytical characteristics of the separation such as limit of detection, limit of quantification, linear range and reproducibility were evaluated. The developed method was applied to the determination of 67 foodstuffs (mainly imported), comprising soft drinks, jams, sauces, canned fruits/vegetables, dried vegetables/fruits and others. The range of preservatives found were from not detected (nd)--1260, nd--1390, nd--44.8 and nd--221 mg kg(-1) for BA, SA, MP and PP, respectively.     

Simultaneous analysis of six cardiovascular drugs by capillary electrophoresis coupled with electrochemical and electrochemiluminescence detection,using a chemometrical optimization approach

CE coupled with dual electrochemical (EC) and electrochemiluminescence (ECL) detection was optimized for simultaneous analysis of six cardiovascular drugs (alprenolol, propafenone, acebutolol, verapamil, atenolol and metoprolol) via central composite design. Following this study, three critical electrophoretic factors governing the CE separation were investigated: Tris‐H₃PO₄ buffer concentration, buffer pH value and separation voltage. A modified chromatographic response was adopted for evaluating CE separation quality. Optimum conditions were achieved using Tris‐H₃PO₄ buffer 35.6 mM (pH 2.3) separated at 13.9 kV, which was employed experimentally and led to the successful simultaneous separation of the above six drugs. The good agreement of the chromatographic response was observed between predicted data and actual experimental results using these optimized conditions (RSD=3.75%). The proposed method was validated for linearity, repeatability and sensitivity, and subsequently successfully applied to determine six basic drugs in urine samples.     

Simultaneous determination of nitrite and nitrate by normal phase ion-pair liquid chromatography

Normal phase ion-pair high performance liquid chromatography has been used for simultaneous separation of nitrite and nitrate using tetraethylammonium (TEA)(+) as ion-pairing reagent. The concentration effect of (TEA)(+), buffer salt and pH of the eluent on separation is investigated. The UV detector response at various wavelengths has been optimized. The performance of the proposed method is compared with ion chromatography for quantification of the anions in potable water, wastewater and in food samples, such as spinach and lettuce.     

The separation of dimethylarsinic acid,methylarsonous acid,methylarsonic acid,arsenate and dimethylarsinous acid on the Hamilton PRP‐X100 anion‐exchange column

In order to separate the potential arsenite metabolites methylarsonous acid and dimethylarsinous acid from arsenite, arsenate, methylarsonic acid and dimethylarsinic acid, the pH‐dependent retention behaviour of all six arsenic compounds was studied on a Hamilton PRP‐X100 anion‐exchange column with 30 mM phosphate buffers (pH 5, 6, 7, 8 and 9) containing 20% (v/v) methanol as mobile phase and employing an inductively coupled plasma atomic emission spectrometer (ICP–AES) as the arsenic‐specific detector. Baseline separation of dimethylarsinic acid, methylarsonous acid, methylarsonic acid, arsenate and dimethylarsinous acid was achieved with a 30 mmol dm⁻³ phosphate buffer (pH 5)–methanol mixture (80:20, v/v) in 25 min. Arsenite is not baseline‐separated from dimethylarsinic acid under these conditions. Copyright © 1999 John Wiley & Sons, Ltd.     

Fast and simultaneous detection of heavy metals using a simple and reliable microchip-electrochemistry route: An alternative approach to food analysis

This paper reports, for the first, the fast and simultaneous detection of prominent heavy metals, including: lead, cadmium and copper using microchip CE with electrochemical detection. The direct amperometric detection mode for microchip CE was successfully applied to these heavy metal ions. The influences of separation voltage, detection potential, as well as the concentration and pH value of the running buffer on the response of the detector were carefully assayed and optimized. The results clearly show that reliable analysis for lead, cadmium, and copper by the degree of electrophoretic separation occurs in less than 3min using a MES buffer (pH 7.0, 25mM) and l-histidine, with 1.2kV separation voltage and -0.8V detection potential. The detection limits for Pb(2+), Cd(2+), and Cu(2+) were 1.74, 0.73 and 0.13microM (S/N=3). The %R.S.D. of each peak current was <6% and migration times <2% for prolonged operation. To demonstrate the potential and future role of microchip CE, analytical possibilities and a new route in the raw sample analysis were presented. The results obtained allow the proposed microchip CE-ED acts as an alternative approach for metal analysis in foods.     

Separation and Detection of Nitrophenols at Capillary Electrophoresis Microchips with Amperometric Detection

A miniaturized analytical system for the separation and amperometric detection of toxic nitrophenols, based on the coupling of a micromachined capillary electrophoresis (CE) chip with a glassy carbon detector is described. This microsystem enables a rapid (120 s/sample) simultaneous determination of five priority nitrophenolic pollutants (2‐nitrophenol, 3‐nitrophenol, 4‐nitrophenol, 2,4‐dinitrophenol, and 2‐methyl‐4,6‐dinitrophenol). These compounds can be detected down to the 1×10^?5 M level using a 15 mM phosphate buffer pH 7.2 (containing 1.3 mM α‐cyclodextrin) as running solution on 77 mm long microchannel by applying a separation voltage of 3000 V and a negative potential of ?0.7 V (vs. Ag /AgCl wire). Applicability to ground water samples was demonstrated.     

Determination of catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high‐performance liquid chromatography and electrochemical detection

A new method was developed for the simultaneous determination of three catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high‐performance liquid chromatography with electrochemical detection. Novel aminophenylboronic acid functionalized magnetic nanoparticles were prepared by multi‐step covalent modification, and characterized by transmission electron microscopy, Fourier‐transformed infrared spectroscopy, X‐ray diffraction, and vibrating sample magnetometry. With the help of the high affinity between the boronate and cis‐diol group, the particles were used for the highly selective separation and enrichment of three major catecholamines, norepinephrine, epinephrine, and dopamine. Effects of the pH of the feed solution, the extraction time, the composition of the buffer solution, the amount of the magnetic particles, the elution conditions, and the recycling of aminophenylboronic acid functionalized magnetic nanoparticles were explored. Under the optimized conditions, 13–17‐fold enrichment factors were obtained. The linear ranges were 0.01–2.0 μg/mL for the studied analytes. The limits of detection and quantification were in the range of 2.0–7.9 and 6.7–26.3 ng/mL, respectively. The relative recoveries were in the range of 92–108%, with intraday and interday relative standard deviations lower than 6.8%. This method was successfully applied to analysis of catecholamines in real urine.     

Simultaneous Determination of Atenolol and Chlorthalidone in Pharmaceutical Preparations by Capillary-Zone Electrophoresis

Abstract

A capillary zone electrophoresis (CZE) method for the simultaneous determination of the β-blocker drugs atenolol and chlorthalidone in pharmaceutical formulations has been developed. The CZE separation was performed under the following conditions: capillary temperature, 25°C; applied voltage, 25 kV; 20 mM H₃PO₄–NaOH running buffer (pH 9.0); and detection wavelength, 198 nm. Phenobarbital was used as internal standard. The method was validated and showed not only good precision and accuracy but also good robustness. The method has been successfully applied to the simultaneous determination of both atenolol and chlorthalidone in pharmaceutical tablets.     

Reversed Phase Liquid Chromatographic Separation of Lysergic Acid Diethylamide (LSD) and Lysergic Acid Methylpropylamide (LAMPA)

Abstract

A fast and convenient procedure is described for the HPLC separation of LSD and LAMPA. These compounds are separated by a reversed phase (C18) procedure using a binary solvent system of methanol and pH 3 phosphate buffer. Under these conditions all compounds are eluted in a retention volume of 27 mL or less. This procedure allows a forensic sample to be identified as LSD and to eliminate LAMPA as a possibility.