Determination of ammonia, creatinine and inorganic cations in urine using CE with contactless conductivity detection

CE with capacitively coupled contactless conductivity detection (C(4)D) was used to determine waste products of the nitrogen metabolism (ammonia and creatinine) and of biogenic inorganic cations in samples of human urine. The CE separation was performed in two BGEs, consisting of 2 M acetic acid + 1.5 mM crown ether 18-crown-6 (BGE I) and 2 M acetic acid + 2% w/v PEG (BGE II). Only BGE II permitted complete separation of all the analytes in a model sample and in real urine samples. The LOD values for the optimized procedure ranged from 0.8 microM for Ca(2+) and Mg(2+) to 2.9 microM for NH(4)(+) (in terms of mass concentration units, from 7 microg/L for Li(+) to 102 microg/L for creatinine). These values are adequate for determination of NH(4)(+), creatinine, Na(+), K(+), Ca(2+) and Mg(2+) in real urine samples.     

Recent advances in applications of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C⁴D): an update

Capillary electrophoresis with a capacitively contactless conductivity detector (CE-C?D) is becoming a significant useful technique for the analysis of analytes in various fields such as pharmaceutical, biomedical, food and environmental. This review is an update describing the recent developments in the application of CE with a C?D detector.     

Separation of perfluorocarboxylic acids using capillary electrophoresis with UV detection

A capillary electrophoretic method with UV detection for separation and quantitation of perfluorocarboxylic acids (PFCAs) from C6-PFCA to C12-PFCA has been developed. The optimization of measurement conditions included the choice of the most appropriate type and concentration of buffer in the background electrolyte (BGE), as well as the type and the content of an organic modifier. The optimal separation of investigated PFCAs was achieved with 50 mM phosphate buffer and 40% isopropanol in the BGE using direct UV detection. The optimum wavelength for direct UV detection was optimized at 190 nm. For indirect detection, several chromophores were studied. Five mM 3,5-Dinitrobenzoic acid (3,5-DNBA) in 20 mM phosphate buffer BGE and indirect UV detection at 280 nm gave the optimal detection and separation performance for the investigated PFCAs. The possibility of on-line preconcentration of solutes by stacking has been examined for indirect detection. The detection limits (LODs) determined for direct UV detection ranged from 2 microg/mL for C6-PFCA to 33 microg/mL for C12-PFCA. The LODs obtained for indirect UV detection were comparable to those obtained for direct UV detection.     

Field-amplified sample stacking for the detection of chemical warfare agent degradation products in low-conductivity matrices by capillary electrophoresis-mass spectrometry

Preconcentration of chemical warfare agent degradation products (alkylphosphonic acids and alkyl alkylphosphonic acids) in low-conductivity matrices (purified water, tap water and local river water) by field-amplified sample stacking (FASS) was developed for capillary electrophoresis (CE) coupled to ion trap mass spectrometry. FASS was performed by adding a mixture of HCOONH(4) and NH(4)OH in appropriate concentrations to the sample. This allowed to control the conductivity and the pH of the sample in order to obtain FASS performances that are independent of analyte concentration. The influence of different parameters on FASS (sample to background electrolyte (BGE) conductivity ratio, injection volume and concentration of BGE) was studied to determine the optimal conditions and was rationalized by using the theoretical model developed by Burgi and Chien. A good correlation was obtained between the bulk electroosmotic velocity predicted by this model and the experimental value deduced from the migration time of the electroosmotic flow marker detected by mass spectrometry (MS). This newly developed method was successfully applied to the analysis of tap water and local river water fortified with the analytes and provided a 10-fold sensitivity enhancement in comparison to the signal obtained without preconcentration procedure. The quite satisfactory repeatability and linearity for peak areas obtained in the 0.5-5 microg mL(-1) concentration range allow quantitative analysis to be implemented. Limits of detection of 0.25-0.5 microg mL(-1) for the alkyl alkylphosphonic acids and of 0.35-5 microg mL(-1) for the alkylphosphonic acids were reached in tap water and river water.     

Development and validation of a capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C(4) D) for the analysis of amikacin and its related substances

Amikacin is a semisynthetic aminoglycoside antibiotic derived from kanamycin A that lacks a strong UV absorbing chromophore or fluorophore. Due to the physicochemical properties of amikacin and its related substances, CE in combination with capacitively coupled contactless conductivity detection (CE-C(4) D) was chosen. The optimized separation method uses a BGE composed of 20 mM MES adjusted to pH 6.6 by l-histidine and 0.3 mM CTAB that was added as flow modifier in a concentration below the CMC. Ammonium acetate 20 mg.L(-1) was used as internal standard. 30 kV was applied in reverse polarity on a fused silica capillary (73/48 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2) = 0.9996) for amikacin base. It shows a good precision expressed as RSD on relative peak areas equal to 0.1 and 0.7% for intraday and interday, respectively. The LOD and LOQ are 0.5 mg.L(-1) and 1.7 mg.L(-1) , respectively.     

Determination of γ‐hydroxybutyric acid in saliva by capillary electrophoresis coupled with contactless conductivity and indirect UV absorbance detectors

The aim of the current study was to optimise and validate the methodology for determination of γ‐hydroxybutyric acid (GHB) in saliva by CE combined with a contactless conductivity detector (C⁴D) and indirect UV absorbance detection (λ_ABS = 210 nm). The optimized BGE, consisting of 8.5 mM maleic acid, 17 mM arginine, 255 μM cetyltrimethylammonium bromide (CTAB), and 15% acetonitrile, was evaluated for the separation of GHB in saliva within 6 min. The performance characteristics of the CE‐C⁴D‐indirect UV methodology was validated. The instrument detection and quantification limits were 0.49 and 1.6 mg/L for C⁴D, and 5.1 mg/L and 17.0 mg/L for indirect UV, respectively. The linearity was obtained over the range from 2.5 to 400 mg/L for C⁴D and from 12.5 to 400 mg/L for indirect UV. The interday precisions were within 2.3–5.7% and intraday precisions were within 1.6–9.0% for C⁴D as well as 2.1–9.3%, 5.6–10.1% for indirect UV in spiked saliva, respectively. The recoveries were within 87.2–104.4%. The matrix effects were +53.2% for small concentrations up to 25 mg/L for C⁴D and +23.6% for concentrations up to 75 for mg/L for indirect UV detection. No matrix effects were observed for higher concentration levels. In conclusion, CE‐C4D‐indirect UV can offer a rapid, accurate, sensitive, and definitive method for the determination of GHB abuse in saliva samples as a forensic screening tool.     

Capillary electrophoresis with capacitively coupled contactless conductivity detection for low molecular weight organic acids in different samples

CE with capacitively coupled contactless conductivity detection (CE-C(4)D) was explored and validated for the identification and quantification of organic acids in various types of samples. The analyses were performed under optimized conditions, using a buffer system composed of 20 mM MES-histidine (His), pH 6.0, 0.1 mM CTAB, 0.025% HP-beta-CD, and 10% methanol. The investigation included a study of the effects of buffer pH, concentration of CTAB, type and concentration of organic additives, on the migration behavior, resolution and selectivity of the organic acids. The intra- and interday RSDs (n = 6) obtained for migration time and peak area were typically in the range of 0.12-2% and 0.5-4%, respectively. Linearity, detection limits, and repeatability were evaluated. In order to evaluate the application potential of the developed method, real samples from different sources were analyzed. The results demonstrate that CE-C(4)D is a versatile tool for analyzing organic acids in beverages, Chinese herbal medicines (CHM) and plants as it allows for their detection, identification, and quantification.     

Simultaneous determination of naringenin and hesperetin in rats after oral administration of Da-Cheng-Qi decoction by high-performance liquid chromatography-tandem mass spectrometry

To quantify naringenin and hesperetin in rat plasma after oral administration of Da-Cheng-Qi decoction, a famous purgative traditional Chinese medicine, a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated. The HPLC separation was carried out on a Zorbax SB-C(18) column using 0.1% formic acid-methanol as mobile phase and estazolam as internal standard after the sample of rat plasma had been cleaned up with one-step protein precipitation using methanol. Atmospheric pressure chemical ionization in the positive ion mode and selected reaction monitoring method was developed to determine the active components. This method was validated in terms of recovery, linearity, accuracy and precision (intra- and inter-batch variation). The recoveries of naringenin and hesperetin were 72.8-76.6 and 75.7-77.2%, respectively. Linearity in rat plasma was observed over the range of 0.5-250 ng/mL (r2 > 0.99) for both naringenin and hesperetin. The accuracy and precision were well within the acceptable range and the relative standard deviation of the measured rat plasma samples was less than 15% (n = 5). The validated method was successfully applied for the evaluation of the pharmacokinetics of naringenin and hesperetin administered to six rats.     

Electromembrane extraction of heavy metal cations followed by capillary electrophoresis with capacitively coupled contactless conductivity detection

Electromembrane extraction (EME) was used as an off-line sample pre-treatment method for the determination of heavy metal cations in aqueous samples using CE with capacitively coupled contactless conductivity detection (CE-C(4) D). A short segment of porous polypropylene hollow fibre was penetrated with 1-octanol and 0.5%?v/v bis(2-ethylhexyl)phosphonic acid and constituted a low cost, single use, disposable supported liquid membrane, which selectively transported and pre-concentrated heavy metal cations into the fibre lumen filled with 100?mM acetic acid acceptor solution. Donor solutions were standard solutions and real samples dissolved in deionized water at neutral pH. At optimized EME conditions (penetration time, 5?s; applied voltage, 75?V; and stirring rate, 750?rpm), 15-42% recoveries of heavy metal cations were achieved for a 5?min extraction time. Repeatability of the EME pre-treatment was examined for six independent EME runs and ranged from 6.6 to 11.1%. Limits of detection for the EME-CE-C(4) D method ranged from 25 to 200?nM, resulting into one to two orders of magnitude improvement compared with CE-C(4) D without sample treatment. The developed EME sample pre-treatment procedure was applied to the analysis of heavy metal cations in tap water and powdered milk samples. Zinc in the real samples was identified and quantified in a background electrolyte solution consisting of 20?mM L-histidine and 30?mM acetic acid at pH 4.95 in about 3?min.     

Potential of CE for the determination of inorganic and acidic anions in cyanoacrylate adhesives

In this work, a CZE method with indirect UV detection was developed for the simultaneous determination of the inorganic and acidic anions, chloride, sulfate, nitrate, fluoride, formate, phosphate, diethylphosphate, methyl sulfonate, cyanoacetate, and methacrylate present in cyanoacrylate adhesives. Chromate was employed as the probe ion, and the EOF was reversed by incorporating CTAB into BGE. Detection limits of 0.7-4.6 microg/mL were obtained for all the anions studied. The CE method developed is a significant improvement on traditionally used chromatographic methods such as ion chromatography, as it resulted in shorter analysis times with enhanced separation efficiencies. This method was successfully employed for the analysis of inorganic and acidic anions in cyanoacrylate adhesive samples.     

Fast assay of glucosamine in pharmaceuticals and nutraceuticals by capillary zone electrophoresis with contactless conductivity detection

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K(+) in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25 degrees C), GlAm (migrating as glucosaminium cation) was well separated from K(+) that could occur in the dosage forms as excipient. The CE analysis was performed in fused-silica capillaries (50 microm i.d., 75 cm total length, 27 cm to detector) and the separation took <3 min. The calibration graphs were linear for both GlAm (100-300 microg/mL; r(2)=0.997) and K(+) (15-75 microg/mL; r(2)=0.997) when using ethanolamine (100 microg/mL) as the internal standard. The LOD values (S/N=3) were 9.3 microg/mL for GlAm and 2.9 microg/mL for K(+). The method was applied to the assay of GlAm content in various dosage forms. Intermediate precision evaluated by determining the content of GlAm in a single formulation on 3 consecutive days was characterized by RSD 2.35% (n=15). Acceptable accuracy of the CE method was confirmed by the added/found GlAm recovery experiments (recoveries 94.6-103.3%) and by statistical comparison of the results attained by the proposed CE and a reference HPLC method.     

Analysis of inorganic cations in biological samples by the combination of micro-electrodialysis and capillary electrophoresis with capacitively coupled contactless conductivity detection

Micro-electrodialysis (μED) and CE were combined for rapid pretreatment and subsequent determination of inorganic cations in biological samples. Combination of μED with CE greatly improved the analytical performance of the latter as the adsorption of high molecular weight compounds present in real samples on the inner capillary wall was eliminated. Fifty microliter of 80-fold diluted human body fluids such as plasma, serum and whole blood was used in the donor compartment of the μED system requiring less than 1?μL of the original body fluid per analysis. Inorganic cations that migrated through a cellulose acetate dialysis membrane with molecular weight cut-off value of 500?Da were collected in the acceptor solution and were then analyzed using CE-C?D. Baseline separation of inorganic cations was achieved in a BGE solution consisting of 12.5?mM maleic acid, 15?mM L-arginine and 3?mM 18-crown-6 at pH 5.5. Repeatability of the CE-C?D method was better than 0.5% and 2.5% for migration times and peak areas, respectively; limits of detection of all inorganic cations in the presence of 2?mM excess of Na(+) were around 1?μM and calibration curves were linear with correlation coefficients better than 0.998. Repeatability of the sample pretreatment procedure was calculated for six independent electrodialysis runs of artificial and real samples and was better than 11.8%. Recovery values between 96.3 and 110% were achieved for optimized electrodialysis conditions of standard solutions and real samples; lifetime of the dialysis membranes for pretreatment of real samples was estimated to 100 runs.     

Sulfation of hesperetin,naringenin and apigenin by the human cytosolic sulfotransferases: a comprehensive analysis

Abstract

Previous studies have revealed sulfation as a major pathway for the metabolism of hesperetin, naringenin and apigenin. The current study was designed to identify the human cytosolic sulfotransferase (SULT) enzyme(s) capable of sulfating these flavonoid compounds. Of the thirteen human SULTs, six (1A1, 1A2, 1A3, 1B2, 1C4, 1E1) displayed significant sulfating activity toward hesperetin, five (1A1, 1A2, 1A3, 1B2, 1C4) displayed sulfating activity towards naringenin, and four (1A1, 1A2, 1A3, 1C4) showed sulfating activity towards apigenin. Of the four human organ specimens tested, liver and intestine cytosols displayed much higher hesperetin-, naringenin- and apigenin-sulfating activity than lung and kidney cytosols. Moreover, sulfation of hesperetin, naringenin and apigenin was shown to take place in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under cultured conditions. Taken together, these results provided a biochemical basis underlying the metabolism of hesperetin, naringenin and apigenin through sulfation in humans.

     

Automated two-dimensional separation flow system with electrochemical preconcentration, stripping, capillary electrophoresis and contactless conductivity detection for trace metal ion analysis

This paper describes the automation of a fully electrochemical system for preconcentration, cleanup, separation and detection, comprising the hyphenation of a thin layer electrochemical flow cell with CE coupled with contactless conductivity detection (CE-C?D). Traces of heavy metal ions were extracted from the pulsed-flowing sample and accumulated on a glassy carbon working electrode by electroreduction for some minutes. Anodic stripping of the accumulated metals was synchronized with hydrodynamic injection into the capillary. The effect of the angle of the slant polished tip of the CE capillary and its orientation against the working electrode in the electrochemical preconcentration (EPC) flow cell and of the accumulation time were studied, aiming at maximum CE-C?D signal enhancement. After 6 min of EPC, enhancement factors close to 50 times were obtained for thallium, lead, cadmium and copper ions, and about 16 for zinc ions. Limits of detection below 25 nmol/L were estimated for all target analytes but zinc. A second separation dimension was added to the CE separation capabilities by staircase scanning of the potentiostatic deposition and/or stripping potentials of metal ions, as implemented with the EPC-CE-C?D flow system. A matrix exchange between the deposition and stripping steps, highly valuable for sample cleanup, can be straightforwardly programmed with the multi-pumping flow management system. The automated simultaneous determination of the traces of five accumulable heavy metals together with four non-accumulated alkaline and alkaline earth metals in a single run was demonstrated, to highlight the potentiality of the system.     

Determination of free cyanide and zinc cyanide complex by capillary electrophoresis

A capillary electrophoretic (CE) protocol was developed for the separation and quantification of free cyanide and zinc cyanide complex, two key species in gold cyanidation of zinc-bearing sulfidic ores. Several common carrier electrolytes were implemented in an indirect UV detection method. The effect of electric field strength, injection volume, concentration of electro-osmotic flow (EOF) modifier and UV-absorbing agent in background electrolyte (BGE) was examined while peak height, peak area and noise were considered for optimization. The best results were obtained using a BGE that contained 35 mM sodium chromate, 12 mM free cyanide and 0.45 mM hexamethonium bromide at pH 10.5. Free cyanide concentration was compared to that measured with the conventional silver nitrate titration method in solutions containing free cyanides and weak cyano-complexes. The developed CE protocol proved very robust in capturing the concentration of free cyanides (4% error) unlike the titration method which exhibited substantial sensitivity to the interfering weak cyano-complexes (38% error).     

Simultaneous determination of the flavonoid aglycones diosmetin and hesperetin in human plasma and urine by a validated GC/MS method: in vivo metabolic reduction of diosmetin to hesperetin

Diosmetin and hesperetin are the aglycones of the flavonoid glycosides diosmin and hesperidin which occur naturally in citrus fruit. A GC/MS method for the simultaneous determination of diosmetin and hesperetin in human plasma and urine has been developed and validated. The method was linear in the 2-300 ng/mL concentration range for both diosmetin and hesperetin in plasma and urine (r > 0.999). The precision of the method was better than 6.01 and 7.16% for diosmetin and hesperetin, respectively, and the accuracy was 96.76-100.40% and 95.00-105.50% for diosmetin and hesperetin, respectively. The lower limit of quantitation was found to be 2 ng/mL for both analytes in plasma and urine. Recovery of diosmetin, hesperetin and internal standard naringenin was greater than 82.5%. The method has been applied for the determination of diosmetin and hesperetin in plasma and urine samples obtained from a healthy male subject following a single oral 1000 mg dose of the flavonoid glycoside diosmin. The presence of hesperetin in plasma and urine samples indicates the metabolic reduction of diosmetin to its flavanone analogue hesperetin through reduction of the 2,3 double bond of the C-ring by the enzymes of bacteria of the intestinal microflora.     

Use of high‐conductivity sample solution with sweeping‐micellar electrokinetic capillary chromatography for trace‐level quantification of paliperidone in human plasma

Paliperidone is a new antipsychotic drug with a relatively low therapeutic concentration of 20–60 ng/mL. We established an accurate and sensitive CE method for the determination of paliperidone concentrations in human plasma in this study. To minimize matrix effect caused by quantification errors, paliperidone was extracted from human plasma using Oasis HLB SPE cartridges with three‐step washing procedure. To achieve sensitive quantification of paliperidone in human plasma, a high‐conductivity sample solution with sweeping‐MEKC method was applied for analysis. The separation is performed in a BGE composed of 75 mM phosphoric acid, 100 mM SDS, 12% acetonitrile, and 15% tetrahydrofuran. Sample solution consisted of 10% methanol in 250 mM phosphoric acid and the conductivity ratio between sample matrix and BGE was 2.0 (γ, sample/BGE). The results showed it able to detect paliperidone in plasma samples at concentration as low as 10 ng/mL (S/N = 3) with a linear range between 20 and 200 ng/mL. Compared to the conventional MEKC method, the sensitivity enhancement factor of the developed sweeping‐MEKC method was 100. Intra‐ and interday precision of peak area ratios were less than 6.03%; the method accuracy was between 93.4 and 97.9%. This method was successfully applied to the analysis of plasma samples of patients undergoing paliperidone treatment.     

Determination of gamma-hydroxybutyric acid in clinical samples using capillary electrophoresis with contactless conductivity detection

A method for the determination of gamma-hydroxybutyric acid (GHB) in urine and serum samples with capillary electrophoresis using capacitively coupled contactless conductivity detection (CE-C(4)D) was developed. The optimized separation buffer consisting of 20 mM of arginine, 10 mM of maleic acid and 30 microM of cetyltrimethylammonium bromide (CTAB) contained 5 mM vancomycin to facilitate the separation of gamma-hydroxybutyric acid from beta-hydroxybutyric acid (BHB), which is also present in clinical samples. The detection limits in the clinical samples were found to be about 2 microg/ml, which is well below the required sensitivity for the recognition of overdosage and adequate for the determination of endogenous concentrations in urine. The determination of GHB in both types of samples was carried out directly after a fourfold dilution without requiring any derivatization or extraction procedures.     

Study of the determination of inorganic arsenic species by CE with capacitively coupled contactless conductivity detection

The determination of arsenic(III) and arsenic(V), as inorganic arsenite and arsenate, was investigated by CE with capacitively coupled contactless conductivity detection (CE-C(4)D). It was found necessary to determine the two inorganic arsenic species separately employing two different electrolyte systems. Electrolyte solutions consisting of 50 mM CAPS/2 mM L-arginine (Arg) (pH 9.0) and of 45 mM acetic acid (pH 3.2) were used for arsenic(III) and arsenic(V) determinations, respectively. Detection limits of 0.29 and 0.15 microM were achieved for As(III) and As(V), respectively by using large-volume injection to maximize the sensitivity. The analysis of contaminated well water samples from Vietnam is demonstrated.     

Gas chromatographic-mass spectrometric fragmentation study of flavonoids as their trimethylsilyl derivatives: analysis of flavonoids, sugars, carboxylic and amino acids in model systems and in citrus fruits

The fragmentation patterns and quantitation possibilities of three anthocyanidins (pelargonidin, cyanidin, malvidin), one flavonol (quercetin), two flavones (apigenin, luteolin) and two flavanones (naringenin, hesperetin) have been investigated as trimethylsilyl and as trimethylsilyl (oxime) derivatives by gas chromatography-mass spectrometry. Results proved that anthocyanidins and flavanones form trimethylsilyl (oximes), while flavonol and flavones provide simple trimethylsilyl derivatives. In all cases, characteristic fragments of high masses are formed proper for quantitation purposes. Hydrolysis conditions for naringin, hesperidin and rutin have been optimized, resulting in the quantitative release of naringenin, hesperetin and quercetin together with their corresponding saccharides. These basic studies made possible the identification and quantification of the flavonoid, carboxylic-/amino acid and sugar constituents of citrus fruit juices and albedos, without any extraction/enrichment procedure. In total 33 compounds have been determined in hydrolyzed samples, such as 2 flavonoids (naringenin and hesperetin), 6 phenolic acids (trimethoxybenzoic, 4-hydroxybenzoic, vanillic, quinic, chlorogenic and rosmarinic acids), 3 aliphatic carboxylic acids (levulinic, malic, citric acids), phosphoric acid, 4 amino acids (aspartic, glutamic acids, alanine, proline), 9 monosaccharides (xylose, arabinose, rhamnose, fucose, fructose, galactose, glucose, galacturonic acid, sedoheptulose), inositol, sugarphosphate, 5 disaccharides and tocopherol. Measurements were carried out as the trimethylsilyl (oxime) ether/ester derivatives of constituents, in the concentration range of 2 x 10(-3) to 49.9%. Identification level of samples varied between 26.4 and 77.5%, expressed in dry matter content of juices and albedos.