Glycogen: A novel branched polysaccharide chiral selector in CE

Various chiral selectors have been employed in CE and among them linear polysaccharides exhibited powerful enantioselective properties. Different from linear polysaccharides, the use of branched polysaccharides as chiral selectors in CE has not been reported previously. In this study glycogen belonging to the class of branched polysaccharides was used as a novel chiral selector for the enantiomeric separations for the first time. Since glycogen is electrically neutral, the method is applicable to ionic compounds. Eighteen chiral compounds including 12 basic drugs and six acidic drugs have been tested to demonstrate the potential of this chiral selector. BGE and selector concentrations and buffer pH were systematically optimized in order to obtain successful chiral separations. Among the tested compounds, the enantiomers of ibuprofen, which is an acidic drug, were successfully recognized by 3.0% w/v glycogen with 90 mM Tris‐H₃PO₄ buffer (pH 7.0). The enantiomers of basic drugs such as citalopram, cetirizine and nefopam were also baseline‐resolved with 50 mM Tris‐H₃PO₄ buffer (pH 3.0) containing 3.0% glycogen. Amlodipine belonging to basic compound only gave partial enantioseparation under the above‐mentioned condition.     

Investigation of chondroitin sulfate D and chondroitin sulfate E as novel chiral selectors in capillary electrophoresis

Various chiral selectors have been utilized successfully in capillary electrophoresis (CE); however, the number of polysaccharides used as chiral selectors is still small and the mechanism of enantiorecognition has not been fully elucidated. Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), belonging to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. In this paper, they were investigated for the first time for their potential as chiral selectors by CE. The effect of buffer composition and pH, chiral selector concentration, and applied voltage were systematically examined and optimized. A variety of drug enantiomers were resolved in the buffer pH range of 2.8–3.4 using 20 mM Tris/H₃PO₄ buffer with 5.0 % CSD or CSE and 20 kV applied voltage. A central composite design was used to validate the optimized separation parameters and satisfactory uniformity was obtained. As observed, CSE allowed satisfactory separation of the enantiomers of amlodipine, laudanosine, nefopam, sulconazole, and tryptophan methyl ester, as well as partial resolution of citalopram, duloxetine, and propranolol under the optimized conditions. CSD allowed partial or nearly baseline separation of amlodipine, laudanosine, nefopam, and sulconazole. The results indicated that CSE has a better enantiorecognition capability than CSD toward the tested drugs.

Fast Determination of Clenbuterol and Salbutamol in Feed and Meat Products Based on Miniaturized Capillary Electrophoresis with Amperometric Detection

The fast separation capability of a novel miniaturized capillary electrophoresis with an amperometric detection (μCE‐AD) system was demonstrated by determining clenbuterol and salbutamol in real samples. The effects of several factors such as the acidity and concentration of the running buffer, the separation voltage, the applied potential and the injection time on CE‐AD were examined and optimized. Under the optimum conditions, the two β‐agonists could be baseline separated within 60 s at a separation voltage of 2 kV in a 90 mmol/L H₃BO₃‐Na₂B₄O₇ running buffer (pH 7.4), which was not interfered by ascorbic acid and uric acid. Highly linear response was obtained for above compounds over three orders of magnitude with detection limits ranging from 1.20×10^?7 to 6.50×10^?8 mol/L (S/N=3). This method was successfully used in the analysis of feed and meat products with relatively simple extraction procedures.     

Application of ionic liquid as additive in determination of three β‐agonists by capillary electrophoresis with amperometric detection

CE coupled with amperometric detection method was developed using ionic liquid 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (EMImBF₄) as additive for the simultaneous detection of clenbuterol (CLB), terbutaline (TER), and ractopamine (RAC) in feed. The effects of detection potential, concentration of EMImBF₄, pH, and concentration of the running buffer, separation voltage as well as injection time on the separation and detection of these three β‐agonists were investigated in detail. Under the optimum conditions: the detection potential at 1.05 V, 50 mmol/L Tris‐HAc at pH 8.0 with 0.6% (v/v) EMImBF₄, electrokinetic injection 6 s at 16 kV and separation voltage at 16 kV, a baseline separation for these three analytes could be achieved within 11 min. Introduction of EMImBF₄ into the running buffer resulted in significant improvement in separation selectivity and enhancement in peak currents for those β‐agonists, especially for TER and RAC, which could not be separated in the running buffer without additive. The method exhibited wide linear range with LOD (S/N = 3) of 2, 1, and 2 nmol/L for CLB, TER, and RAC, respectively. The precision was determined in both intraday (n = 5) and interday (n = 3) assays, and the RSDs for both migration time and peak current were less than 6%. The proposed method was also applied to analyze β‐agonists in feed sample.     

Boron‐chelating fluorescent probe (BOPB) in the red region combined with CE‐LIF for the detection of NO in mice liver

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8‐(3’,4’‐Diamino phenyl)‐3,5‐(2‐hydroxyphenyl)‐dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE‐LIF detection. Therefore, BOPB was used in CE‐LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB‐T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H₃BO₃–NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10^?9–5.0 × 10^?7 M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization‐based CE‐LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.     

Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods

Chiral separation of 12 pairs of basic analyte enantiomers including oxybutynin, bambuterol, tradinterol, clenbuterol, clorprenaline, terbutaline, tulobuterol, citalopram, phencynonate, fexofenadine, salbutamol, and penehyclidine was conducted by capillary electrophoresis using a single‐isomer anionic β‐cyclodextrin derivative, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector. Parameters influencing separation were studied, including background electrolyte pH, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin concentration, buffer concentration, and separation voltage. A background electrolyte consisting of 50 mM Tris‐H₃PO₄ and 6 mM heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin at pH 2.5 was found to be highly efficient for the separation of most enantiomers, with other conditions of normal polarity mode at 10 kV, detection wavelength of 210 nm using hydrodynamic injection for 3 s. Under the optimal conditions, baseline resolution (>1.50) for 11 pairs of enantiomers and somewhat lower resolution for penehyclidine enantiomers (1.17) were generated. Moreover, the possible mechanism of separation of clenbuterol, oxybutynin, salbutamol, and penehyclidine was investigated using a computational modeling method.     

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.     

Separation of Anthraquinones by Capillary Electrophoresis and High‐Performance Liquid Chromatography

The separation and determination of twelve anthraquinones, viz. anthraquinone 1, chrysphanol 2, aloe‐emodin 3, alizarin 4, anthraquinone‐2‐carboxylic acid 5, purpurin 6, sennoside B 7, sennoside A 8, emodin 9, quinalizarin 10, rhein 11, and anthraflavic acid 12, were achieved by capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). Detection at 260 nm with a buffer solution containing 30 mM sodium borate (adjusted to pH = 10.56 with 0.05N NaOH) and acetonitrile (9 : 1) in CE or with a linear gradient elution containing 20 mM KH₂PO₄ with 0.05% phosphoric acid (pH = 2.91) and methanol in HPLC was found to be the most suitable approach for this separation. Contents of six components (2, 3, 7, 8, 9, 11) in crude Rhei Rhizoma extract could easily be determined within 39 min by CE or 63 min by HPLC. The effects of buffers on this separation and the validation of the two methods were studied.     

Rapid and cost‐effective method for the simultaneous quantification of seven alkaloids in Corydalis decumbens by microwave‐assisted extraction and capillary electrophoresis

A rapid and cost‐effective method based on microwave‐assisted extraction followed by capillary electrophoresis was developed for simultaneous quantification of seven alkaloids in Corydalis decumbens for the first time. The main parameters affecting microwave‐assisted extraction and capillary electrophoresis separation were investigated and optimized. The optimal microwave‐assisted extraction was performed at 40°C for 5 min using methanol/water (90:10, v/v) as the extracting solvent. Electrophoretic separation was achieved within 15 min using an uncoated fused‐silica capillary (50 μm internal diameter and 27.7 cm effective length) and a 500 mM Tris buffer containing 45% v/v methanol (titrated to pH^* 2.86 with H₃PO₄). The developed method was successfully applied to the quantification of seven alkaloids in Corydalis decumbens obtained from different regions of China. The combination of microwave‐assisted extraction with capillary electrophoresis was an effective method for the rapid analysis of the alkaloids in Corydalis decumbens .     

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.     

Development of a fast and efficient CE enzyme assay for the characterization and inhibition studies of α‐glucosidase inhibitors

The inhibition of the α‐glucosidase enzyme plays an important role in the treatment of diabetes mellitus. We have established a highly sensitive, fast, and convenient CE method for the characterization of the enzyme and inhibition studies of α‐glucosidase inhibitors. The separation conditions were optimized; the pH value and concentration of the borate‐based separation buffer were optimized in order to achieve baseline separation of p‐nitrophenyl‐α‐d ‐glucopyranoside and p‐nitrophenolate. The optimized method using 25 mM tetraborate buffer, pH 9.5, was evaluated in terms of repeatability, LOD, LOQ, and linearity. The LOD and LOQ were 0.32 and 1.32 μM for p‐nitrophenyl‐α‐d ‐glucopyranoside and 0.83 and 3.42 μM for p‐nitrophenolate, respectively. The value of the Michaelis–Menten constant (K_m) determined for the enzyme is 0.61 mM, which is in good agreement with the reported data. The RSDs (n = 6) for the migration time was 0.67 and 1.83% for substrate and product, respectively. In the newly established CE method, the separation of the reaction analytes was completed in <4 min. The developed CE method is rapid and simple for measuring enzyme kinetics and for assaying inhibitors.     

Application of a multivariate approach for analyte focusing by micelle collapse‐micellar electrokinetic chromatography for analyzing sunscreen agents in cosmetics

The operating parameters that affect the performance of the online preconcentration technique “analyte focusing by micelle collapse‐MEKC (AFMC‐MEKC)” were examined using a multivariate approach involving experimental design to determine the sunscreen agents in cosmetics. Compared to the single‐variable approach, the advantage of the multivariate approach was that many factors could be investigated simultaneously to obtain the best separation condition. A fractional factorial design was used to identify the fewest significant factors in the central composite design (cCD). The cCD was adopted for evaluating the location of the minimum or maximum response in this study. The influences of the experimental variables on the response were investigated by applying a chromatographic exponential function. The optimized condition and the relationship between the experimental variables were acquired using the JMP software. The ANOVA analysis indicated that the Tris pH value, SDS concentration, and ethanol percentage influenced the separation quality and significantly contributed to the model. The optimized condition of the running buffer was 10 mM Tris buffer (pH 9.5) containing 60 mM SDS, 7 mM γ‐CD, and 20% v/v ethanol. The sample was prepared in 100 mM Tris buffer (pH 9.0) containing 7.5 mM SDS and 20% v/v ethanol. The SDS concentration in the sample matrix was slightly greater than the CMC value that makes the micelle be easily collapsed and the analytes be accumulated in the capillary. In addition, sunscreen agents in cosmetics after 1000‐fold dilution were successfully determined by AFMC‐MEKC.     

A new UPLC approach for the quantitation of ephedrine and guaifenesin in a syrup formulation using multivariate optimization strategy

A new ultraperformance liquid chromatography (UPLC) method with photodiode array detection was developed for the quantitative analysis of a commercial syrup formulation containing ephedrine (EPH) and guaifenesin (GUA). In the development of UPLC method, experimental chromatographic conditions, flow rate, column temperature, and percentage of 0.1?M H₃PO₄ in mobile phase, were optimized using chemometric multivariate strategy. From the application of a 3³ full factorial design, the optimal chromatographic conditions were obtained as the flow rate of 0.29?mL/min, column temperature of 36.4°C, and 56.9% of 0.1?M H₃PO₄ in the mobile phase. The optimal conditions gave us a good chromatographic separation of the analyzed drugs with short analysis runtime within 3?min. Calibration curves for EPH and GUA in the linear working range of 4–64 and 6–96?µg/mL, respectively, were obtained using peak areas detected at 215?nm. Performance and validity of the optimized UPLC method were estimated by analyzing independent binary mixtures, inter-day and intra-day samples, and standard addition solutions containing EPH and GUA substances. It was concluded that the proposed method was a promising approach for the quantitative determination and routine analysis of a commercial syrup formulation of the titled substances.     

Ultra high-performance liquid chromatography of porphyrins

An ultra high‐performance liquid chromatographic (UHPLC) system was developed and optimized for the separation of porphyrins of clinical interest. Optimum conditions for the simultaneous separation of uroporphyrin, hepta‐, hexa‐, penta‐carboxylic acid porphyrins and coproporphyrin and their type I and III isomers on a Thermo Hypersil BDS C₁₈ column (2.4 µm particle size, 100 × 2.1 mm i.d.) using a gradient elution with 10% (v/v) acetonitrile in 1.0 m ammonium acetate buffer (pH 5.16) and 10% (v/v) acetonitrile in methanol at a flow‐rate of 0.4 mL/min. The effect of mobile phase buffer molarity on the sensitivity of fluorescence detection and resolution of porphyrin isomers was investigated. The method was successfully applied to the analysis of porphyrins extracted from the urine and faeces of patients with various human porphyrias. Copyright © 2011 John Wiley & Sons, Ltd.     

Capillary electrophoresis coupled with electrochemiluminescence detection for the separation and determination of thyreostatic drugs in animal feed

A rapid, simple, and practical method for the determination of four of the most used thyreostatic drugs (methimazole, 2‐thiouracil, 6‐methyl‐2‐thiouracil, and 6‐propyl‐2‐thiouracil) using CE coupled to electrochemiluminescence detection has been established, based on the electrochemiluminescence enhancement of tris(2,2‐bipyridyl)ruthenium(II) with these analytes. Parameters that affect separation and detection were optimized. Under the optimum experimental conditions, the four analytes could be well separated within 11 min at the separation voltage of 16 kV in a running solution containing 20 mM phosphate buffer (pH 9.0) and 1.0 × 10^?4 M Ru(bpy)₃²⁺, with a solution of 20 mM phosphate buffer (pH 12.0) containing 1.0 × 10^?4 M Ru(bpy)₃²⁺ in the electrochemiluminescence detection cell. The detection limits for methimazole, 6‐methyl‐2‐thiouracil, 6‐propyl‐2‐thiouracil, and 2‐thiouracil were 0.1, 0.05, 0.05, and 0.01 μM, respectively. The proposed method was applied to analyze these drugs in spiked animal feed samples. The recoveries were 88.2～99.0 and 86.4～98.7% for the intraday and interday analyses, respectively. The RSDs were 2.7～4.8 and 1.8～5.0% for the intraday and interday analyses, respectively. The results demonstrate that the proposed method has promising applications in the detection of thyreostatic drugs in animal feeds.     

Simultaneous determination of flavonoids in chrysanthemum by capillary zone electrophoresis with running buffer modifiers

Despite the separation efficiency of capillary electrophoresis (CE) is much higher than other chromatographic methods, it is sometimes difficult to perfectly separate the complex ingredients in biological samples. One possible and simple way to develop the separation effect in CE is to add some modifiers in the running buffer. In this paper, the suitable running buffer modifiers were explored to simultaneously separate and detect six typical flavonoids (apigenin, luteolin, kaempferol, quercetin, (+)-catechin and (−)-epicatechin) which are the main active ingredients in chrysanthemum by capillary zone electrophoresis with amperometric detection (CZE-AD). It was found that when β-cyclodextrin (β-CD) and the mixture of methanol and ethanol were used as running buffer modifiers, a baseline separation of the six analytes could be accomplished in less than 20 min and the detection limits were as low as 10⁻⁷ or 10⁻⁸ g ml⁻¹. Other factors affecting the CZE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage and sample injection time were extensively investigated. Under the optimum conditions, a successful practical application on the determination of chrysanthemum samples confirmed the validity and practicability of this method.     

Simultaneous determination of atenolol,chlorthalidone and amiloride in pharmaceutical preparations by capillary zone electrophoresis with ultraviolet detection

Capillary zone electrophoresis methods for the simultaneous determination of the β‐blocker drugs, atenolol, chlorthalidone and amiloride, in pharmaceutical formulations have been developed. The influences of several factors (buffer pH, concentration, applied voltage, capillary temperature and injection time) were studied. Using phenobarbital as internal standard, the analytes were all separated in less than 4 min. The separation was carried out in normal polarity mode at 25°C, 25 kV and using hydrodynamic injection (10 s). The separation was effected in an uncoated fused‐silica capillary (75 μm i.d. × 52 cm) and a background electrolyte of 25 mm H₃PO₄ adjusted with 1 m NaOH solution (pH 9.0) and detection at 198 nm. The method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 1–250 μg/mL for atenolol and chlorthalidone and from 2.5–250 μg/mL for amiloride. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of atenolol, chlorthalidone and amiloride in various pharmaceutical tablets formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of unconjugated aromatic acids in urine by capillary electrophoresis with dual electrochemical detection – Potential application in fast diagnosis of phenylketonuria

A novel method of CE coupled with dual electrochemical detection has been developed for the determination of pathological metabolites of phenylalanine in urine samples. Factors influencing the separation and detection were examined and optimized. Five aromatic acid metabolites and a major coexisting interfering compound uric acid could be well separated within 23 min at a separation voltage of 16 kV using a 35 mmol/L SDS/60 mmol/L H₃BO₃‐Na₂B₄O₇ running buffer (pH 8.2). Highly linear response was obtained for these five biomarker compounds over three orders of magnitude with detection limits ranging from 6.6 to 0.064 μg/mL (S/N=3). The average recovery and RSD were within the range of 92.6–121.0 and 1.0–12.0%, respectively. The proposed method has been used to detect the unconjugated aromatic acids simultaneously in urine samples with the advantages of obtaining more information about target analytes and avoiding redundant measurements and high assay cost, thus could find potential applications involving assays of biomarker compounds for the purpose of fast diagnose of some metabolic diseases including phenylketonuria.     

Determination of partition coefficient and analysis of nitrophenols by three‐phase liquid‐phase microextraction coupled with capillary electrophoresis

A three‐phase hollow fiber liquid‐phase microextraction method coupled with CE was developed and used for the determination of partition coefficients and analysis of selected nitrophenols in water samples. The selected nitrophenols were extracted from 14 mL of aqueous solution (donor solution) with the pH adjusted to pH 3 into an organic phase (1‐octanol) immobilized in the pores of the hollow fiber and finally backextracted into 40.0 μL of the acceptor phase (NaOH) at pH 12.0 located inside the lumen of the hollow fiber. The extractions were carried out under the following optimum conditions: donor solution, 0.05 M H₃PO₄, pH 3.0; organic solvent, 1‐octanol; acceptor solution, 40 μL of 0.1 M NaOH, pH 12.0; agitation rate, 1050 rpm; extraction time, 15 min. Under optimized conditions, the calibration curves for the analytes were linear in the range of 0.05–0.30 mg/L with r²>0.9900 and LODs were in the range of 0.01–0.04 mg/L with RSDs of 1.25–2.32%. Excellent enrichment factors of up to 398‐folds were obtained. It was found that the partition coefficient (K_a/d) values were high for 2‐nitrophenol, 3‐nitrophenol, 4‐nitrophenol, 2,4‐dinitrophenol and 2,6‐dinitrophenol and that the individual partition coefficients (K_org/d and K_a/org) promoted efficient simultaneous extraction from the donor through the organic phase and further into the acceptor phase. The developed method was successfully applied for the analysis of water samples.     

Determination of galanthamine in Bulbus Lycoridis Radiatae by coupling capillary electrophoresis with end‐column electrochemiluminescence detection

A novel method for the determination of galanthamine (GAL) in Bulbus Lycoridis Radiatae has been developed based on coupling CE with an end‐column tris(2,2′‐bipyridyl)ruthenium(II) electrochemiluminescence (ECL). Parameters affecting CE separation and ECL detection were investigated and optimized. Baseline separation of GAL from other components in the Bulbus Lycoridis Radiatae sample was achieved with an 18 mmol/L phosphate running buffer at pH 9.0. Under the optimized conditions: 12 kV CE‐separation voltage, ECL detection potential at 1.25 V with 5 mmol/L and 50 mmol/L phosphate buffer at pH 7.5 in the detection reservoir, the linear range of GAL concentration was from 0.8 ng/mL to 2 μg/mL, whereas the detection limit was 0.25 ng/mL (S/N=3). The proposed method was successfully demonstrated for the determination of GAL in Bulbus Lycoridis Radiatae.