Non-aqueous capillary electrophoresis chiral separations with sulfated β-cyclodextrin

This paper reports the application of an anionic cyclodextrin (CD), sulfated β-cyclodextrin with a degree of substitution of four (β-CD-(SO₄⁻)₄, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between β-CD-(SO₄⁻)₄–analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of β-CD-(SO₄⁻)₄ was needed due to the fact that the electrostatic attraction caused stronger binding between β-CD-(SO₄⁻)₄ and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.     

Simultaneous chiral separation of tramadol and methadone in tablets,human urine,and plasma by capillary electrophoresis using maltodextrin as the chiral selector

The stereoselective analysis and separation of racemic drugs play an important role in pharmaceutical industry to eliminate the unwanted isomer and find the right therapeutic control for the patient. Present study suggests a maltodextrin‐modified capillary electrophoresis method for a single‐run chiral separation of two closely similar opiate pain relief drugs: tramadol (TRA) and methadone (MET). The best separation method possible for the both enantiomers was achieved on an uncoated fused‐silica capillary at 25°C using 100 mM phosphate buffer (pH 8.0) containing 20% (w v^?1) maltodextrin with dextrose equivalent of 4–7 and an applied voltage of 16 kV. Under optimal conditions, the baseline resolution of TRA and MET enantiomers was obtained in less than 12 minutes. The relative standard deviations (n = 3) of 20 μg mL^?1 TRA and MET were 2.28% and 3.77%, respectively. The detection limits were found to be 2 μg mL^?1 for TRA and 1.5 μg mL^?1 for MET. This method was successfully applied to the measurement of drugs concentration in their tablets, urine, and plasma samples.     

Optimization of lambda-carrageenan as a chiral selector in capillary electrophoresis separations

Lambda-carrageenan, a linear high molecular weight sulfated polysaccharide, was employed as a chiral selector in capillary electrophoresis for the separation of enantiomers of weakly basic pharmaceutical compounds. In order to improve the utility of the chiral selector, the purity and concentration of the lambda-carrageenan and other important capillary electrophoresis method parameters were investigated. The results indicated that the purity and concentration of the lambda-carrageenan, ionic strength of the buffer, and temperature were critical to successful enantioseparation. These new method conditions were then applied to previously investigated beta-blockers (such as propranolol HCl and pindolol) and racemic tryptophan derivatives. These studies were successful in identifying important method conditions for the improved enantioselectivity with lambda-carrageenan.     

Measurement of chloramphenicol by capillary zone electrophoresis following end-column amperometric detection at a carbon fiber micro-disk array electrode

Capillary zone electrophoresis was employed for the measurement of chloramphenicol using end-column amperometric detection with a carbon fiber micro-disk array electrode, at a constant potential of −1.00 V vs. saturated calomel electrode. The effect of oxygen in the buffer has been investigated. It is found that when the area of the carbon fiber electrode is smaller than 1.1 mm², the interference of oxygen can be overcome. In this procedure deoxygenation is not necessary. The effect of pH, the concentration of the buffer and the high separation voltage across the capillary on the migration time, electrophoretic peak current and separation efficiency has been studied. The optimum conditions of separation and detection are 8.4×10⁻⁴ mol/l HOAc–3.2×10⁻³ mol/l NaOAc for the buffer solution, 20 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, respectively. The calibration plot was found to be linear in the range 5×10⁻⁶ to 1×10⁻³ mol/l and the limit of detection is 9.1×10⁻⁷ mol/l or 1.4 fmol (S/N=2). The relative standard deviation is 1.1% for the migration time and 2.3% for the electrophoretic peak current. The method was applied to the determination of chloramphenicol in human serum.     

Separation of eleven central nervous system drugs by capillary zone electrophoresis

Several strategies to improve the separation of 11 central nervous system drugs (antipsychotics and antidepressants) with capillary zone electrophoresis were applied: the variation of the pH of the buffering background electrolyte, its ionic strength, addition of inclusion-complex forming β-cyclodextrin or polyvinylpyrrolidone (PVP), respectively, as a replaceable, soluble, polymeric pseudo-stationary phase. Best separation was achieved at pH 2.5 and 35 mmol/l ionic strength (phosphate buffer), with 0.5% (w/v) PVP.     

Analysis of trace amino acid neurotransmitters in hypothalamus of rats after exhausting exercise using microdialysis

A simple but effective coupling of microdialysis and capillary electrophoresis with laser induced fluorescence detection technique was applied to analysis of amino acid neurotransmitters in the hypothalamus of rats after acute exhausting exercise. The separation of amino acids was achieved using an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 10 mM disodium tetraborate at pH 10 and an applied voltage of 12.5 kV. The detection limit was 10⁻¹⁰ M for each amino acid. It is sufficiently sensitive and rapid for the determination of amino acids in a 5-μl Microdialysate. In comparison to pre-exercise, a significant increase in the levels of six hypothalamic amino acids (arginine, glycine, lysine, glutamic acid, alanine, γ-amino-n-butyric acid) was found after exercise. These results demonstrate that the increase of metabolic amino acids in the hypothalamus of rats can be induced by exhausting exercise and suggests that amino acid neurotransmitters may play functional roles in the central effects of exercise.     

Simultaneous determination of phenylethanoid glycosides and aglycones by capillary zone electrophoresis with running buffer modifier

Although the separation efficiency of capillary electrophoresis (CE) is much higher than that of other chromatographic methods, it is sometimes difficult to adequately separate the complex ingredients in biological samples. This article describes how one effective and simple way to develop the separation efficiency in CE is to add some modifiers to the running buffer. The suitable running buffer modifier β-cyclodextrin (β-CD) was explored to fast and completely separate four phenylethanoid glycosides and aglycones (homovanillyl alcohol, hydroxytyrosol, 3,4-dimethoxycinnamic acid, and caffeic acid) in Lamiophlomis rotata (Lr) and Cistanche by capillary zone electrophoresis with ultraviolet (UV) detection. It was found that when β-CD was used as running buffer modifier, a baseline separation of the four analytes could be accomplished in less than 20 min and the detection limits were as low as 10⁻³ mg L⁻¹. Other factors affecting the CE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage, and sample injection time, were investigated extensively. Under the optimal conditions, a successful practical application on the determination of Lr and Cistanche samples confirmed the validity and practicability of this method.     

Enantioselective determination of metoprolol and major metabolites in human urine by capillary electrophoresis

The enantiomeric separation of metoprolol and its metabolites in human urine was undertaken using capillary electrophoresis (CE). Resolution of the enantiomers was achieved using carboxymethyl-β-cyclodextrin (CM-β-CD) as the chiral selector. A 100-mM acetate buffer (pH 4.0) containing 5% 2-propanol and 10 mM CM-β-CD resulted in the optimum separation of the metoprolol enantiomers and its acidic metabolite in human urine. Following a single metoprolol oral administration of 100 mg racemic metoprolol tartrate, stereoselective pharmacokinetic analysis showed that urinary acidic metabolite 3 of metoprolol accounted for 62.3% of the dose with an R/S ratio of 1.23 and urinary unchanged metoprolol 1 accounted for 6.3% of the dose with an R/S ratio of 0.72.     

Resolution and Properties of Two High Affinity Cyclic Adenosine 3':5'-monophosphate-Binding Proteins from Wheat Germ

A high affinity cAMP-binding protein (cABP II) was purified to homogeneity from wheat germ. The apparent molecular weight of cABP II, as determined from gel exclusion chromatography, is 5.2 × 10⁵ (at low ionic strength) and 2.8 × 10⁵ (at high ionic strength). One polypeptide subunit (molecular weight, 80,000) was resolved by polyacrylamide gel electrophoresis of cABP II under subunit dissociating conditions. The purification protocol employed resolves cABP II from a distinct, less acidic cAMP-binding protein (cABP I). The K_d values for cAMP are about 10⁻⁶ molar and 10⁻⁷ molar for cABP II and cABP I, respectively. The cAMP-binding sites of cABP I and cABP II have a marked adenine-analog specificity, binding adenine, adenosine, adenine-derived nucleosides and nucleotides and a variety of adenine derivatives having cytokinin activity. While cABP II is phosphorylated in reactions catalyzed by endogenous protein kinases, there is no evidence for modulation of these cABP II-protein kinase interactions by cAMP.     

High-performance affinity capture-removal of bacterial pyrogen from solutions

Synthetic peptide S3Δ has high affinity for bacterial endotoxin or lipopolysaccharide (LPS). Under tested conditions of pH 5–9 and 0–0.4 M NaCl, the affinity constant, K_D ranged from 2·10⁻⁶ to 2·10⁻⁹ M⁻¹. A novel affinity matrix based on peptide S3Δ was developed for removal of LPS from solutions such as: water; buffers with a wide range of ionic strength and pH; medium for cell culture; and protein solutions under optimized conditions. At a starting LPS of ≈100 EU/ml, a post-purification level below 0.005 EU/ml was achieved.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Application of Maltodextrin as Chiral Selector in Capillary Electrophoresis for Quantification of Amlodipine Enantiomers in Commercial Tablets

Maltodextrin was investigated as a chiral selector in capillary electrophoresis (CE) analysis of amlodipine (AM) enantiomers. For development of a stereoselective CE method, various effective parameters on the enantioseparation were optimized. The best results were achieved on an uncoated fused silica capillary at 20 °C using phosphate buffer (100 mM, pH 4) containing 10% w/v maltodextrin (dextrose equivalent value 4–7). The UV detector was set at 214 nm and a constant voltage of 20 kV was applied. The range of quantitation was 2.5–250 µg/mL (R² > 0.999) for both enantiomers. Intra‐ (n = 5) and interday (n = 3) relative standard deviation (RSD) values were less than 7%. The limits of quantitation and detection were 1.7 µg/mL and 0.52 µg/mL, respectively. Recoveries of R(+) and S(?) enantiomers from tablet matrix were 97.2% and 97.8%, respectively. The method was applied for the quantification of AM enantiomers in commercial tablets. Also, the enantioseparation capability of heparin was evaluated and the results showed that heparin did not have any chiral selector activity in this study. Chirality 26:394–399, 2014. © 2014 Wiley Periodicals, Inc.     

Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis

A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm × 75 μm I.D.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25°C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10⁵-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.     

Pine nut peroxidase immobilized on chitosan crosslinked with citrate and ionic liquid used in the construction of a biosensor

A biosensor based on the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI·Tf₂N) and a novel source of peroxidase (tissue from the pine nuts of Araucaria angustifolia) was constructed. This enzyme was immobilized on chitosan crosslinked with citrate and the biosensor used for the determination of rosmarinic acid by square-wave voltammetry. The peroxidase in the presence of hydrogen peroxide catalyzes the oxidation of rosmarinic acid to quinone and the electrochemical reduction of the product was obtained at a potential of +0.15 V vs. Ag/AgCl. Different analytical parameters influencing the biosensor response, that is, peroxidase units, pH, hydrogen peroxide concentration and parameters for the square-wave voltammetry (frequency, pulse amplitude and scan increment), were investigated. The best performance was observed for the biosensor under the following conditions: 1000 units mL⁻¹ peroxidase, pH 7.0 and 8.3 × 10⁻⁴ mol L⁻¹ hydrogen peroxide with a frequency of 30 Hz, pulse amplitude of 100 mV and scan increment of 5.0 mV. The biosensor gave a linear response to rosmarinic acid over the concentration range of 9.07 × 10⁻⁷ to 4.46 × 10⁻⁶ mol L⁻¹ with a detection limit of 7.25 × 10⁻⁸ mol L⁻¹. The recovery of rosmarinic acid in plant extracts ranged from 97.0% to 109.6% and the determination of this substance in these samples using the biosensor compared favorably with that using the capillary electrophoresis method.     

Continuous extraction of xylanase from Penicillium janthinellum with reversed micelles using experimental design mathematical model

Xylanase recovery from Penicillium janthinellum with a reversed micellar system consisting of a cationic surfactant using continuous process was evaluated. A statistical approach was applied to the results and showed that the highest xylanase recovery (140%), indicated by the model, was attained at an ionic strength of 7.5 mS cm^–1 and volumetric flow of 0.6 ml min^–1. A new xylanase extraction was performed under these conditions to test the model and showed a close similarity between the experimental result and the value predicted by the model.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C₁₈ cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l⁻¹ and detection limits were between 1.1 and 2.4 mg l⁻¹.     

Ultra‐performance convergence chromatography with tandem mass spectrometry‐assisted method for rapid enantioseparation and determination of fluazifop‐butyl in tobacco and soil

A quick, green, and sensitive method for chiral separation and determination of fluazifop‐butyl enantiomers in tobacco and soil was established by ultra‐performance convergence chromatography with tandem mass spectrometry (UPC²‐MS/MS). The baseline separation was obtained on an ACQUITY UPC² Trefoil CEL2 column in 4 minutes with CO₂ and methanol as mobile phase. Column temperature, auto back pressure regulator pressure (ABPR), and modifier solvent were optimized to obtain the best separation efficiency. Under the optimal conditions, the recoveries of both enantiomers were 82.8% to 99.5% with relative standard deviations (RSDs) less than 5.5% at three different concentration levels in two matrices. Good coefficients of determination (R² ≥ 0.9976) were achieved over the concentration range of 10 to 500 ng/mL. The limits of detection (LODs) for all enantiomers in the two matrices varied from 1.6 to 2.1 μg/kg, and the limits of quantification (LOQs) did not exceed 7.0 μg/kg. The proposed method was then successfully applied to analyze authentic samples, confirming that it was a green, convenient, and reliable strategy for the analysis of fluazifop‐butyl enantiomers in tobacco and soil.     

Bovine serum albumin-hemoglobin fractionation: significance of ultrafiltration system and feed solution characteristics

This work investigates the fractionation of similar molecular weight proteins bovine serum albumin (69 kD) and bovine hemoglobin (67 kD) by ultrafiltration. Three different membranes, viz. regenerated cellulose, poly(sulfone) and surface modified poly(acrylonitrile), each with a nominal molecular cutoff rating of 100 kD, were examined. The experiments were conducted in dead end, crossflow and vortex flow filtration modes and the separation was studied as a function of feed pH and ionic strength. Under similar system hydrodynamics, the surface modified poly(acrylonitrile) membrane displayed the highest resolution with minimum membrane fouling. The separation could be improved further by operating at low applied pressure (40 kPa) and high mass transfer (> 20 × 10^–6 m/s) in a vortex flow module. Under these conditions, the highest separation factor of 40 was obtained at the pI of hemoglobin.