Strategies for enantioseparations of catecholamines and structurally related compounds by capillary zone electrophoresis using sulfated beta-cyclodextrins as chiral selectors

Strategies for simultaneous enantioseparations of three catecholamines (DL-norepinephrine, DL-epinephrine, and DL-isoproterenol) and three structurally related compounds (DL-octopamine, DL-synephrine, and DL-norephedrine) by CZE using sulfated beta-CDs as chiral selectors were investigated. Four different separation modes were attempted: (I) using randomly sulfate-substituted beta-CD (MI-S-beta-CD) at relatively low concentrations in a high-concentration phosphate buffer at low pH in the normal polarity mode, (II) using MI-S-beta-CD at high concentrations at low pH in the reversed polarity mode, (III) using MI-S-beta-CD at moderately high concentrations in a phosphate buffer at neutral pH in the normal polarity mode, and (IV) using the single isomer heptakis(2,3-dihydroxy-6-O-sulfo)-beta-CD (SI-S-beta-CD) at low to moderately high concentrations in a high-concentration BGE at low pH in the normal polarity mode. Among them, enantioseparation of these cationic solutes was best achieved under the conditions of mode (II). In mode (II) and mode (III), temperature is an important factor affecting the enantioresolution of norepinephrine. In mode (I) and mode (IV), the use of a high-concentration BGE (150-200 mM) is crucial for effective enantioseparation of these cationic solutes with sulfated beta-CDs. Comparative studies of enantioseparations of these cationic solutes with MI-S-beta-CD and SI-S-beta-CD reveal that the sulfate substituents of MI-S-beta-CD located at the C(2)- position interact strongly with the diol moiety of catecholamines.     

Simultaneous quantification of energetically important metabolites in various cell types by CZE

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field‐enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on‐line pre‐concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.     

Influence of Mobile Phase Composition on the Enantioseparation of Methoxyl Flavanones with Self-prepared CDMPC Column and Chiral Recognition Mechanism

Chiral stationary phases (CSPs) based on the cellulose derivatives have proven to be one type of the most useful CSPs in high-performance liquid chromatography because of their versatility, durability and loadability1, 2. Flavanone was often used as a standard chiral compound for evaluating cellulose derivative for chiral columns1, 3. Krause and Galensa4 reported the enantioseparation of flavanone and its seven derivatives on six kinds of commercial chiral column. 4-Methoxyl flavanone, 5-…     

Enantioseparation of atropine by capillary electrophoresis using sulfated beta-cyclodextrin: application to a plant extract

A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Determination of Substance P by capillary zone electrophoresis in rat brain

In the complex neuronal network, chemical messengers like neuropeptides play a key role in signaling. To understand the mechanism of signaling, it is necessary to analyze the levels of neuropeptides from biological sources, which is important for neuroscience research. In the present work, a detailed investigation of the capillary zone electrophoresis (CZE) method was carried out to detect and quantify Substance P (SP), a bioactive neuropeptide, in rat brain tissues. The method involves specifically, a combination of solid phase extraction and immunoprecipitation prior to the CZE quantification. In this procedure, antibodies are used to capture the analyte of interest before the separation by CZE. Different separation parameters like buffer type, concentration, pH and applied voltage were the steps taken to study and achieve high efficiency CZE separation. CZE analysis was performed in an untreated fused-silica capillary column (35 cm×75 μm i.d.) and 185 nm wavelength using 100 mM phosphate buffer (pH 2.5) as a separation buffer. Electrophoresis in acidic mode and successive washing procedures solved the adsorption problem. The method provides a rapid analysis time of less than 15 min with 3.91% of RSD. Simultaneously, SP was quantified by Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) and compared with CZE data. Starting from milligram amounts of brain tissue, the method allowed the detection of low picomole amounts of SP and the combined use of CZE and MALDI-TOF-MS was a success in quantification in this study.     

Original paper application of cyclodextrins as modifiers in electrophoretic separation of metalloporphyrins

Several metallocomplexes of tetrakis-carboxyphenylporphyrin (TCPP) were separated on fused-silica capillary using CZE with UV-VIS detection. Metalloporphyrins of Co(II), Cu(II), Mn(II), Ni(II), and Zn(II) were formed directly in TCPP solution with addition of Cd(II) to increase the formation reaction rate. The composition of BGE, its concentration, and pH were optimized to ensure the stability of complexes and proper resolution. In particular, the problem of signals' shape was investigated and discussed. The presence of beta-CD in borate buffer significantly improved separation efficiency and signal shapes due to formation of inclusion complexes. Under the best separation conditions (50 mM borate running buffer at pH 9 with addition of 2 mM beta-CD, 30 kV applied voltage) a separation of metal complexes with TCPP was accomplished in 16 min.     

Combined use of ionic liquid and sulfobutylether‐β‐cyclodextrin as electrolyte additives for separation and determination of camptothecin alkaloids by CZE

This work reported that ionic liquid (IL) ([Bmim] [PF₆]) and sulfobutylether‐β‐CD (SBE‐β‐CD) were used as electrolyte additives for the separation and determination of camptothecin (CPT) alkaloids by CZE. Separation parameters such as the buffer type, pH, and concentration of the running buffer, the concentration of SBE‐β‐CD and IL, temperature, and separation voltage were all investigated in order to achieve the maximum possible resolution. The four analytes were baseline separated within 10 min in capillary at the separation voltage of 15 kV with a running buffer consisting of 20 mM borate buffer, 20 mM IL, and 100 mM SBE‐β‐CD at pH 9.0. Under such conditions, good linearity about two orders of magnitudes of peak areas was achieved for the investigated CPT alkaloids with the correlation coefficients ranging from 0.9946 to 0.9985. For all analytes, detection limits (S/N = 3) and quantitation limits (S/N = 10) range from 0.05 to 0.92 μg/mL and 0.17 to 3.06 μg/mL, respectively. The proposed method has not only been successfully applied to the separation and determination of CPT alkaloids but also showed that IL seemed to be a promising additive in CZE separation.     

Development of a capillary zone electrophoresis method for the separation of a furan combinatorial library

Nine component mixtures of a furan library were simultaneously separated by capillary zone electrophoresis (CZE) using a phosphate buffer as a background electrolyte at low pH. The effects of buffer concentration, buffer pH, type and concentration of organic solvents on the electrophoretic mobility, resolution, and analysis time were systematically investigated. Resolution and efficiency of furan library components were further improved using cyclodextrin (CD)-modified CZE. Under optimum conditions, eight of the nine furans were baseline-resolved in less than 10 min at 30 kV using 50 mM phosphate buffer, 10% v/v acetonitrile (ACN), pH 2.0, with 5 mM gamma-CD.     

Determination of berberine and strychnine in medicinal plants and herbal preparations by pressurized liquid extraction with capillary zone electrophoresis.

A simple and rapid method for the determination of berberine and strychnine in medicinal plants and herbal preparations for regulatory purposes using a home-made pressurized liquid extraction (PLE) system with capillary zone electrophoresis (CZE) using ultraviolet detection at 254 nm was developed. The effects of pH, concentration of buffer, and organic modifiers in the electrophoretic separation were investigated. The buffer used for CZE contained 50 mM ammonium acetate, pH 3.1. The effect of temperature on the extraction efficiency of strychnine in medicinal plants by PLE was demonstrated. Comparable or higher extraction efficiency was achieved with PLE for strychnine in medicinal plants and berberine in herbal preparations compared to soxhlet extraction. The effect of matrix interference in medicinal plants and herbal preparations containing a number of medicinal plants samples using CZE was investigated by standard additional experiments. The reproducibility of the method using PLE with CZE was found to vary between 2.4 and 10.7% (n = 5/6) for different types of samples on different days.     

Separation and Migration Behavior of Dichlorophenols in β‐Cyclodextrin‐Modified Capillary Zone Electrophoresis

The separation and migration behavior of six isomeric dichlorophenols (DCPs) in cyclodextrin‐modified capillary zone electrophoresis (CD‐CZE) using a phosphate‐borate buffer at alkaline pH with β‐CD and hydroxypropyl‐β‐CD (HP‐β‐CD) as electrolyte modifiers were investigated. The influence of buffer pH and the concentration of β‐cyclodextrins were examined. The results indicate that baseline separation of six isomeric DCPs can be achieved with addition of β‐CD concentration in the range of 2.0‐10 mM or HP‐β‐CD concentration in the range of 4.0‐10 mM at pH 10.0. Binding constants of DCPs to β‐CDs were evaluated for a better understanding of the interaction of DCPs with β‐CDs.     

Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature

The use of capillary electrochromatography (CEC) for the separation by isocratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary phase was prepared from silanized fused-silica capillaries of 75 microm I.D. by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the presence of propanol and formamide as the porogens. The chloromethyl groups at the surface of the porous monolith were reacted with N,N-dimethylbutylamine to form a positively charged chromatographic surface with fixed n-butyl chains. Results of studies on the influence of temperature and mobile phase composition on the retention and selectivity of separation by CEC demonstrated the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degrees C and 75 kV/m with hydro-organic mobile phases containing acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and proteins plots of logarithmic k'cec against acetonitrile concentration were nonlinear, whereas Arrhenius plots for the mobilities were nearly linear. Comparison of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CEC is unique and leads to a chromatographic profile different from that obtained by CZE.     

Separation of chlorophenoxyacetic acids and chlorophenols by using capillary zone electrophoresis

In this study, the choice of electrolyte systems for the separation and detection of a range of chlorophenoxyacetic acids and chlorophenols by means of capillary zone electrophoresis (CZE) is discussed. A series of acetate buffers over the buffering capacity pH range 4.03-5.5 were initially chosen for the separation. It was found that chlorophenoxyacetic acids could be separated at pH 4.03 and 4.5 but the most satisfactory separation of chlorophenols was obtained at pH 5.5. The factors affecting separation selectivity, including the addition of organic modifiers, was also studied. The use of 25% 2-butanol, 5% ethylene glycol and 10% acetonitrile as organic solvents resulted in the total separation of both classes of these compounds but poor peak shape of chlorophenols resulted and a number of chlorophenoxyacetic acids were not well separated. A borate-phosphate buffer gave improved peak shape of chlorophenols. Further improved separation of the components of the mixture was obtained by the addition of 2 mM fully methylated-beta-cyclodextrin to the 35 mM borate- 60 mM phosphate buffer at pH 6.5, maintaining good peak shape. In this case, separation of the two compound classes, chlorophenoxyacetic acids and chlorophenols, is achieved, with complete resolution of individual compounds in less than 5 min with high efficiency (of the order of 150,000 plates for the ca. 40 cm column). The method is applied to a commercial 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide mixture.     

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.     

Capillary electrophoretic determination of the constituents of Artemisiae Capillaris Herba

Two capillary electrophoretic methods, a micellar electrokinetic electrophoretic (MEKC) one and a capillary zone electrophoretic (CZE) one, were developed for the separation of 12 constituents in Artemisiae Capillaris Herba. Detection at 254 nm with 20 mM sodium dodecyl sulfate and 20 mM sodium borate buffer (pH 9.82) in MEKC or with 25 mM sodium borate and 6.75 mg/ml 2,3,6-tri-O-methyl-beta-cyclodextrin buffer in CZE was found to be the most suitable approach for this analysis. Within 42 min, the MEKC method could successfully separate 12 authentic constituents, whereof chlorogenic acid, however, appeared as a broad and split peak, and capillarisin and chlorogenic acid overlapped partially with other coexisting substances in crude extract of the herb. The CZE method could completely overcome these problems and was used to determine the amounts of capillarisin, chlorogenic acid, scopoletin and caffeic acid in the extract. The effect of buffers on the constituent separation and the validation of the two methods were discussed.     

Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Uses of ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate as a good separation electrolyte for direct electrophoretic separation of quaternary ammonium herbicides

This paper describes a new method for the direct separation of paraquat and diquat by CZE with ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate employed as reliable electrolyte. Several factors that affect the separation efficiency were investigated in detail. The experimental results indicated that the optimal running buffer consisted of 50 mM 1‐butyl‐3‐methylimidazolium hexafluorophosphate and 10% ethanol (pH 5.0), applied voltage was 15 kV, and temperature was kept at 30°C and baseline‐separation was achieved within 18 min for the analytes. The proposed method would be very useful and have wide use to monitor the residual level of such pollutants when combined with high‐sensitive detector and an excellent sample preconcentration technique with high enrichment factor in the future.     

Rapid determination of aristolochic acid I and II in medicinal plants with high sensitivity by cucurbit[7]uril-modifier capillary zone electrophoresis

Aristolochic acids (AAs) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end-stage renal failure. Hence, quantitative analysis and quality control for the plants containing AAs is of great importance. In this study, a novel macrocylcic molecule, cucurbit[7]uril (CB[7]) was employed as a modifier in capillary zone electrophoresis (CZE) for rapid determination of aristolochic acid I and II in medicinal plants. In similarity to other macrocyclic molecules, such as cyclodextrins (CDs), CB[7] can be used to manipulate selectivity in CE because it can form inclusion complexes with a variety of guest molecules. During the running process, CB[7] bears a positive charge in the pH range of 2.5-7.5 and can be adsorbed onto the inner wall of a fused-capillary, leading to a reversal of the electroosmotic flow (EOF). By applying a negative polarity, a rapid separation of AA-I and AA-II was achieved within 7min using 100mM phosphate buffer (pH 7.5) containing 3mM CB[7] and 10% acetonitrile (v/v) as modifiers, due to the same directions of the EOF and the electrophoretic mobilities of the analytes. By applying electrokinetic injection with field-enhanced sample stacking, two kinds of aristolochic acids in four medicinal plants were successfully determined with high sensitivity, high separation efficiency, repeatability and recovery. The proposed method was also used to determine AA-I and AA-II in two slimming pills with complex matrix.     

Optimization for quantitative determination of four flavonoids in Epimedium by capillary zone electrophoresis coupled with diode array detection using central composite design

Herba Epimedii (family Berberidaceae), Ying-Yang-Huo in Chinese, is a famous Chinese herbal medicine. Flavonoids are thought to be the major active components in it. A capillary zone electrophoresis (CZE) separation were developed for simultaneous determination of four flavonoids including icariin, epimedin A, epimedin B and epimedin C in Epimedium. The effects of the experimental variables on CZE had been optimized by using central composite design (CCD). The best separation of four flavonoids could be obtained using 50 mM borate buffer (pH 10.0) containing 22% acetontrile as modifier, while separation voltage was 15 kV and temperature was at 25 degrees C. The method developed is accurate, simple and reproducible, which could be used for quality control of Epimedium and its medical preparations.     

Capillary electrophoretic separation of phenolic diterpenes from rosemary

The major phenolic diterpenes responsible for the antioxidant properties of rosemary extracts, namely carnosol and carnosic acid, were separated by capillary zone electrophoresis (CZE) using a 56 cm long uncoated fused-silica capillary and a 50 mM disodium tetraborate buffer of pH 10.1. The effect of the buffer type, pH and concentration, and the capillary length on the separation, was studied. Carnosol and carnosic acid were identified in the electrophoregrams of rosemary extracts through their migration times and UV spectra obtained by CZE analysis of pure compounds isolated from a rosemary extract by HPLC fractionation. The CZE method had good reproducibility (relative standard deviation less than 5%) and was applied to compare the contents of carnosol and carnosic acid in solid and oil-dispersed commercial extracts of rosemary and in rosemary leaves. The separation of carnosol and carnosic acid was accomplished in less than 11 min.