Enantioseparation of Dioxopromethazine Hydrochloride in Urine with Liquid–Liquid Extraction by CE-ECL Detection

Capillary electrophoresis coupled with electrochemiluminescence detection was developed for the separation and determination of dioxopromethazine hydrochloride (DPZ) enantiomers. Performance parameters of the proposed method were evaluated. An improved separation of DPZ enantiomers could be achieved after adding boric acid to buffer. The enantiomers were completely separated with running buffer of 16.5 mM β-CD in 25 mM tris-H₃PO₄–40 mM H₃BO₃ at pH 2.5. The proposed method was successfully applied to the separation and determination of DPZ enantiomers in human urine with a liquid–liquid extraction procedure.     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

Colorimetric determination of reactive primary amino groups of macro- and microsolid supports

The several factors that could affect the sensitivity and the accuracy of the determination of solid-supported amino groups using 2-iminothiolane (Traut's reagent) and 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) are described. The authors found that by using 0.1M phosphate buffer, pH 8.0, instead of ethanol as solvent for the reaction of the solid supports with the 2-iminothiolane, using 0.1M phosphate buffer adjusted at pH 7.27 instead of 8.0 as diluent of 5,5′-dithiobis-(2-nitrobenzoic acid), and selecting carefully the concentration of the latter reagent, it was possible to produce a very sensitive assay capable of quantitatively determining the surface amino groups of very different types of samples. The assay is well adapted for quantitative determination of amino-carrying plastic beads, permitting the determination of nanomolar quantities. In addition, the assay is well suited for microparticulated solid supports (e.g., AH-Sepharose).     

Analysis of Atorvastatin and Related Substances by MEKC

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM sodium dodecyl sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100–1,200 μg mL^?1 for AT and 1.0–12.5 μg mL^?1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity, limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination of AT and purity evaluation of bulk drug and formulated products.     

Voltammetric determination of boron by using Alizarin Red S

Cordycepin is the main active metabolite of Cordyceps militaris extracts; according to recent studies it has interesting therapeutic activities. A new capillary electrophoresis (CE) procedure with UV detection at 254 nm for determination of cordycepin was developed and optimized. Optimal conditions found were 20 mM sodium borate buffer with 28.6% methanol, pH 9.5, separation voltage 20 kV, hydrodynamic injection time 10 s and temperature 25 °C. Linearity was found over the 20-100 μg/mL concentration ranges of cordycepin. The developed method has been applied for determination of cordycepin in various pharmaceutical products. A comparison was made between CE and a high performance liquid chromatography (HPLC) method. Both of these methods gave comparable results. The shorter analysis time and low running cost are the main advantages of CE method.     

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis

In this work the suitability of micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary electrophoresis (CE) to the analysis of the primary oxidation products of linoleic acid was studied with uncoated fused-silica capillaries. The primary autoxidation products of linoleic acid are the four hydroperoxide isomers 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid, 13-hydroperoxy-trans-9, trans-11-octadecadienoic acid, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid, 9-hydroperoxy-trans-10, trans-12-octadecadienoic acid. Addition of a surfactant such as sodium dodecyl sulfate (SDS) or sodium cholate (SC) into the running buffer (20-30 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or ammonium acetate, pH 9.5-11) was required to enhance the water solubility of the sample and selectivity of the separation. MEKC proved to be a promising new technique for the separation of the primary oxidation products of lipids giving results comparable to high performance liquid chromatography (HPLC). Partial separation of hydroperoxide isomers was also achieved using nonaqueous CE with methanol-acetonitrile-sodium cholate as running buffer.     

Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

Determination of hydroxy acids in cosmetics by chemometric experimental design and cyclodextrin‐modified capillary electrophoresis

A CD‐modified CE method was established for quantitative determination of seven hydroxy acids in cosmetic products. This method involved chemometric experimental design aspects, including fractional factorial design and central composite design. Chemometric experimental design was used to enhance the method's separation capability and to explore the interactions between parameters. Compared to the traditional investigation that uses multiple parameters, the method that used chemometric experimental design was less time‐consuming and lower in cost. In this study, the influences of three experimental variables (phosphate concentration, surfactant concentration, and methanol percentage) on the experimental response were investigated by applying a chromatographic resolution statistic function. The optimized conditions were as follows: a running buffer of 150 mM phosphate solution (pH 7) containing 0.5 mM CTAB, 3 mM γ‐CD, and 25% methanol; 20 s sample injection at 0.5 psi; a separation voltage of ?15 kV; temperature was set at 25°C; and UV detection at 200 nm. The seven hydroxy acids were well separated in less than 10 min. The LOD (S/N = 3) was 625 nM for both salicylic acid and mandelic acid. The correlation coefficient of the regression curve was greater than 0.998. The RSD and relative error values were all less than 9.21%. After optimization and validation, this simple and rapid analysis method was considered to be established and was successfully applied to several commercial cosmetic products.     

Application of capillary zone electrophoresis with large-volume sample stacking to the sensitive determination of sulfonamides in meat and ground water

A CZE method with UV-Vis detection has been established and validated for the determination of nine sulfonamides: sulfapyridine, sulfamethazine, sulfamerazine, sulfamether, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfachlorpyridazine, and sulfamethizole. Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using a buffer containing 45 mM sodium phosphate and 10% methanol at pH 7.3, with temperature and voltage of 27 degrees C and 25 kV, respectively. p-Aminobenzoic acid was used as an internal standard . Taking into account the lack of sensitivity of the UV-Vis detection, the application of an on-line preconcentration methodology, such as large-volume sample stacking with polarity switching has been proposed. This procedure combined with a solvent extraction/SPE method applied for off-line preconcentration and cleanup provides a significant improvement in the LODs, ranging from 2.59 to 22.95 mug/L for the studied compounds; the quantification of these residues being possible below the levels established by EU legislation in animal food products, such as meat. Satisfactory recoveries were also obtained in the analysis of these compounds in ground water.     

Determination of muscimol and ibotenic acid in mushrooms of Amanitaceae by capillary electrophoresis

In this study, the CZE method for rapid quantitative and qualitative determination of ibotenic acid and muscimol in Amanita mushrooms naturally grown in Poland was developed. The investigations included the species of A. muscaria, A. pantherina, and A. citrina, collected in southern region of Poland. The studied hallucinogenic compounds were effectively extracted with a mixture of methanol and 1 mM sodium phosphate buffer at pH 3 (1:1 v/v) using ultrasound‐assisted procedure. The obtained extracts were separated and determined by CZE utilizing a 25 mM sodium phosphate running buffer adjusted to pH 3 with 5% content of acetonitrile v/v. The calibration curves for both analytes were linear in the range of 2.5–7000 μg/mL. The intraday and interday variations of quantitative data were 1.0 and 2.5% RSD, respectively. The recovery values of analyzed compounds were over 87%. The identities of ibotenic acid and muscimol were confirmed by UV spectra, migration time, and measurements after addition of external standard.     

Direct chiral resolution of lactic acid in food products by capillary electrophoresis

Chiral resolution of native DL-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-beta-cyclodextrin with an effective voltage of -30 kV at 16 degrees C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, D- and L-lactic acids in food products were analyzed successfully.     

High efficiency joint CZE determination of sugars and acids in vegetables and fruits

In this work, an improved CE method for the medium-throughput determination of main organic acids (oxalate, malate, citrate), the amino acid glutamate and the sugars fructose, glucose and sucrose in several food matrices is described. These compounds have been identified as key components in the taste intensity of fruit and vegetable crops. Using a running buffer with 20 mM 2,6-pyridine dicarboxylic acid pH 12.1 and 0.1% hexadimethrine bromide, replacing it every 5 h to avoid pH decrease, and optimizing capillary conditioning between runs with 58 mM SDS during 2 min at 20 psi, it is possible to effectively quantify these compounds while increasing medium throughput repeatability. This procedure resolves problems such as increases in migration time and reduction of resolution between problematic peaks (malate/citrate and fructose/glucose) detected in a previous method. The new procedure even considerably reduced time analysis down to 12 min. Under optimal conditions, a large number of injections (200) could be administered without any disturbances in the same capillary. The reliability of the proposed method was further investigated with several food matrix samples, including tomato, pepper, muskmelon, winter squash, and orange. This method is recommended for routine analysis of large number of samples typical of production quality systems or plant breeding programs.     

Determination of oxalic acid in urine by co-electroosmotic capillary electrophoresis with amperometric detection

Co-electroosmotic capillary electrophoresis with amperometric detection at a Cobalt phthalocyanine (CoPC) modified carbon paste electrode was evaluated for the determination of oxalic acid in urine. The running buffer consisted of 10 mM phosphate (pH=5.70) and 0.25 mM Cetyltrimethylammonium bromide. Under the optimum conditions, a detection limit of 0.12 muM was achieved for oxalic acid. The response was linear between 0.5 and 1000 muM with a correlation coefficient of 0.9995. Applications of the method to real urine samples were described.     

Chiral CE of aromatic amino acids by ligand-exchange with zinc(II)-L-lysine complex

A novel method of chiral ligand-exchange CE was developed with either L- or D-lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4) x 7H(2)O and 6 mM L-Lys at pH 7.6, unlabeled D,L-tryptophan, D,L-phenylalanine, and D,L-tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2-4 mM and running buffer pH 7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L-Lys to D-Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L-isomers eluted before or after D-isomers in excessive D- or L-Lys, respectively.     

Separation and determination of the anthraquinones in Xanthophytum attopvensis pierre by nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoresis (NACE) method with direct on-column UV detection has been developed for the separation of the pharmaceutically important anthraquinones from the total grass of Xanthophytum attopvensis pierre extract. The separation of three main anthraquinones (1-hydroxy-2-methoxy-3-hydroxymethyl-9, 10-anthraquinone-1-O-β-d-glucoside (1), rubiadin- 1-methylether (2) and 1-methoxy-2-formyl-3-hydroxy-9, 10-anthraquinone (3)) was optimized with respect to concentration of sodium cholate (SC) and acetic acid, addition of acetonitrile (ACN), and applied voltage. Baseline separation was obtained for the three analytes within 5 min using a running buffer containing 50 mM sodium cholate (SC), 1.0% acetic acid and 40% ACN in methanol. The method of NACE for the separation and determination of bioactive ingredient in traditional Chinese medicines was discussed.     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.     

Determination of egg white lysozyme by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis - capillary zone electrophoresis method for the determination of lysozyme in selected food products is described. The optimized electrolyte system consisted of 10 mM NH(4)OH + 20 mM acetic acid (leading electrolyte), 5 mM epsilon -aminocaproic acid +5 mM acetic acid (terminating electrolyte), and 20 mM epsilon -aminocaproic acid +5 mM acetic acid +0.1% m/v hydroxypropylmethylcellulose (background electrolyte). A clear separation of lysozyme from other components of acidic sample extract was achieved within 15 min. Method characteristics, i.e., linearity (0-50 micrograms/mL), accuracy (recovery 96+/-5%), intra-assay (3.8%), quantification limit (1 microgram/ml), and detection limit (0.25 microgram/mL) were determined. Low laboriousness, sufficient sensitivity and low running costs are important attributes of this method. The developed method is suitable for the quantification of the egg content in egg pasta.     

Optimization of Capillary Isotachophoretic Method for Histidine Determination in Protein Matrices

The capillary isotachophoretic method was optimized and used for histidine determination in food samples. The optimum conditions for histidine separation and determination were found on the experimental conditions such as: selectivity, separation speed, pH, concentration of the leading and terminating electrolytes, and electroosmotic flow additives. The optimum electrolytes composition [leading electrolyte: 7 mM NH₄OH + 15 mM 2-(N-morpholino)ethanesulfonic acid + 1% hydroxyethylcellulose; pH = 6.10 and terminating electrolyte: 15 mM aminocaproic acid +5 mM acetic acid +40% methanol; pH = 5.10] and conditions of analysis were adopted for histidine determination in food samples (meat and fish products). The proposed electrolyte system was characterized by linearity (10–100 and 100–430 mg · L^?1 with R² = 0.9976 and 0.9991), accuracy (99.5% and 98%), intra-assay of the relative step height (1.40% for standard and 3.20% for food samples analysis), inter-assay of the relative step height (3.65% and 6.30%) and satisfactory quantification and detection limits. The obtained results were compared to a chromatographic method (reversed-phase (RP)-HPLC) for determination of histidine. The average concentrations of histidine in the samples assayed by both methods were statistically comparable. It should be noted that the proposed histidine determination method can be considered as a contribution to Green Analytical Chemistry.     

Fourier transform infrared spectroscopy with a sample deposition interface as a quantitative detector in size-exclusion chromatography

Micellar electrokinetic capillary chromatography was developed to analyze plant hormones including gibberellic acid, abscisic acid, indole-3-acetic acid, alpha-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin-6-furfurylaminopurine and N6-benzyladenine. The influences of some crucial parameters including buffer concentration, pH value, micelle concentration and applied voltage on electrophoretic separation were investigated. Under optimum conditions (50 mM borate as the running buffer containing 50 mM sodium dodecylsulfate, pH 8.0; separation voltage: -15 kV; injection: hydrodynamic injection, 5 s at 50 mbar; temperature: 25 degrees C), a complete separation of seven plant hormones was accomplished within 30 min. Emphasis was placed on improving detection sensitivity in order to detect small amounts of hormones in plant tissue. Multiple wavelength detection and expanded bubble cell capillary were used with enrichment factors of 2 and 3, respectively. In addition, an on-line concentration method of large volume sample stacking was designed. Enrichment factors of up to approximately 10-600 were achieved for these hormones with detection limits down to 0.306 ng/ml. The method was successfully applied to analyzing abscisic acid in flowers of transgenic tobacco.     

Online dynamic pH junction–sweeping for the determination of benzoic and sorbic acids in food products by capillary electrophoresis

A simple, low-cost, and efficient online focusing method that combines a dynamic pH junction and sweeping by capillary electrophoresis with polymer solutions was developed and optimized for the simultaneous determination of benzoic acid (BA) and sorbic acid (SA). A sample solution consisting of 2.5 mM phosphate at pH 3.0 and a buffer solution containing 15 mM tetraborate (pH 9.2), 40 mM sodium dodecyl sulfate, and 0.100 % (w/v) poly(ethylene oxide) were utilized to realize dynamic pH junction–sweeping for BA and SA. Under the optimized conditions, the entire analysis process was completed in 7 min, and a 900-fold sensitivity enhancement was achieved with limits of detection (S/N?=?3) as low as 8.2 and 6.1 nM for BA and SA, respectively. The linear ranges were between 20 nM and 20 μM for BA and 20 nM and 10 μM for SA, with correlation coefficients greater than 0.992. The recoveries of the proposed method ranged from 90 to 113 %. These satisfactory results indicate that this method has the potential to be an effective analytical tool for the rapid screening of BA and SA in different food products.

Open image in new window

Figure An online focusing strategy combining dynamic pH junction and sweeping for sensitive determination of benzoic and sorbic acid in food products using capillary electrophoresis wit polymer solutions