Determination of lignans in Schisandra chinensis using micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) has been developed as a promising method for the determination of lignans in plant samples. The separation conditions have been optimized with respect to the different parameters including sodium dodecyl sulfate (SDS) and acetonitrile concentration, pH of the background electrolyte, separation voltage, and capillary temperature. The background electrolyte consisting of 40 mM SDS and 35% acetonitrile in 10 mM tetraborate buffer (pH 9.3) was found to be the most suitable electrolyte for this analysis. The applied voltage of 28 kV (positive polarity) and the capillary temperature 25 degrees C gave the best separation of lignans. The interday reproducibility of the peak areas and the migration times was below 2.0%. The results of MEKC analyses were compared with those obtained by capillary electrochromatography (CEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The possibilities of using this method for the determination of lignans in drug and in serum samples were also tested.     

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25 degrees C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed-phase high-performance liquid chromatography (RP-HPLC).     

Optimised separation of endogenous urinary components using cyclodextrin-modified micellar electrokinetic capillary chromatography

In this study both native and chemically modified cyclodextrins (CDs) were investigated as buffer additives to improve the micellar electrokinetic capillary chromatography (MEKC) separation of endogenous bioanalytes in human urine. The following CDs were investigated: alpha, beta, gamma-CDs; hydroxypropyl-alpha-CD, hydroxypropyl-beta-CD, methylated beta-CD, sulphated beta-CD, sulphobutyl ether-beta-CD and hydroxypropyl-gamma-CD. The separations were compared to MEKC without additives. The best improvement in peak resolution and separation of urine components was observed with the sulphated beta-CD. A four-factor three-level full factorial design study was conducted on voltage, temperature, pH and sulphated beta-CD molarity. The optimum conditions were 25 mM sodium tetraborate, pH 9.5, 75 mM sodium dodecyl sulphate (SDS) and 6.25 mM sulphated beta-CD and were able to resolve 70 peaks from a urine pool in 12 min. These optimum conditions have been successfully applied to a number of clinical samples.     

Analysis of organic components of smokeless gunpowders: high-performance liquid chromatogaphy vs. micellar electrokinetic capillary chromatography

The aim of the present study was to verify the analytical performances of high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MEKC) for the separation and qualitative determination of a selected group of organic components of smokeless gunpowders. The HPLC method was based on a gradient reversed-phase elution with a mobile phase composed of 0.17 M H(3)PO(4)/methanol; detection was performed by UV absorption at the wavelengths of 220, 254, and 270 nm. The MEKC experiments were carried out by using uncoated fused-silica capillaries (50 microm inside diameter, 50 cm effective length) and a running buffer composed of 10 mM sodium tetraborate at pH 9.24 added with 25 mM sodium dodecyl sulfate (SDS); the applied voltage was 25 kV; detection was either at a fixed wavelength UV of 214 nm or with a diode-array detector operating in the wavelength range from 190 to 350 nm. Both reversed-phase HPLC and MEKC techniques succeeded in resolving the tested standard mixtures of organic components of smokeless powders. Although the sequence of elution of the different analytes was slightly different between HPLC and MEKC, a statistical analysis based on the Spearman's rank correlation test showed that the two separation patterns were highly correlated. HPLC and MEKC were comparable in terms of elution/migration time precision, whereas MEKC showed higher reproducibility of peak areas. The interfacing of capillary electrophoresis with diode array UV detection provided distinct UV spectra of the individual analytes, thus improving, on the detection side, the analytical selectivity and identification power of capillary electrophoresis.     

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.     

Separation of isomers of dienoic acids by electromigration techniques

Different capillary electromigration techniques were employed to resolve geometrical isomers of sorbic acid, decadienoic acid, and ethyl sorbate. Since these substances differ in their polarity, shape, and size, various electromigration approaches were investigated to separate the four geometrical isomers of each compound. With capillary electrophoresis (CE) modified with a cyclodextrin (β‐CD) the four isomers of sorbic acid were separated using a buffer that consists of 60 mM tetraborate and 8 mg/mL β‐CD. The separation of decadienoic acid geometrical isomers was not possible, even at elevated tetraborate and cyclodextrin concentrations. The four isomers of decadienoic acid were successfully separated using micellar electrokinetic chromatography (MEKC) with a buffer consisting of 30 mM tetraborate and 100 mM SDS and microemulsion electrokinetic chromatography (MEEKC). Ethyl sorbate is the least polar of all the studied substances and its isomers could not be separated by MEKC or MEEKC. The resolution was improved and isomers were fully separated using capillary electrochromatography (CEC) with ODS stationary phase and a mobile phase consisting of 10 mM boric acid in 50% acetonitrile. Minor differences in the polarity and the shape of isomers and high resolving power of the applied techniques were sufficient for separation of very similar compounds. We have shown that versatile electromigration techniques can be applied for separation of geometrical isomers of dienoic acids and their esters.     

Analysis of clindamycin by micellar electrokinetic chromatography with a mixed micellar system.

This study details the development and validation of an optimized method with micellar electrokinetic chromatography for the analysis of clindamycin. The method uses a mixed micellar phase containing anionic sodium dodecylsulfate (SDS) and non ionic Brij 35 on an untreated fused-silica capillary. The influences of buffer concentration, pH, SDS, Brij 35 and organic modifier were investigated. Special attention was given to the role of the non ionic Brij 35 in the mixed micellar system. Optimization with a central composite design resulted in optimal separation conditions: background electrolyte containing 25 mM sodium tetraborate pH 7.75, 90 mM SDS, 14 mM Brij 35 and 21% acetonitrile. The applied voltage was 15 kV and the capillary temperature 15 degrees C. The method was robust and gave good linearity and repeatability. The limits of detection and quantitation were 0.05 and 0.15%, respectively, relative to a 2.5 mg/ml clindamycin solution. Two commercial bulk products were analysed with this system.     

Preliminary study on the monitoring of glutathione S-tranferase activity toward styrene oxide by electromigration methods

A new method has been developed for the monitoring of glutathione S-tranferase (GST) detoxification activity toward styrene oxide (SO). The enzymatic reaction was carried out directly in a thermostatted autosampler vial and the formation of conjugates between glutathione (GSH) and SO was monitored by sequential MEKC runs. The determinations were performed in a 50-microm fused silica capillary using 50 mM SDS in 20 mM phosphate 20 mM tetraborate buffer (pH 8.3) as a background electrolyte; separation voltage 28 kV (positive polarity), temperature of capillary 25 degrees C, and detection at 200 nm. The method is rapid, amenable to automation, and requires only small amounts of samples, which is especially important in the case of GST isoenzyme analyses.     

Rapid detection of ornithine transcarbamylase activity by micellar electrokinetic chromatography

A quantitative ultraviolet detection method for determining ornithine transcarbamylase (OTC-ase) activity using micellar electrokinetic chromatography (MEKC) is described. The method is based on the direct determination of citrulline formed upon enzymatic reaction. Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM sodium dodecyl sulfate (SDS), the peak of citrulline in the electropherogram was easily identified and integrated. This allowed us to determine the rate of formation of the reaction product and to calculate the kinetic parameter Km of the OTC-ase investigated. The capillary electrophoretic method developed was applied to the determination of OTC-ase activity in plasma samples for citrulline in the nanogram range.     

Analysis of combinatorial chemistry samples by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method has been developed that can evaluate the purity of samples generated in combinatorial chemistry libraries. This method uses an open tube capillary (27 cm x 50 microm) along with a run buffer composed of sodium dodecyl sulfate (SDS), hydroxypropyl-beta-cyclodextrin, and sodium tetraborate coupled with UV detection. Neutral compounds and compounds that were insoluble in aqueous buffers could be analyzed under these conditions in approximately 3 min. The concentration of SDS and the concentration of hydroxypropyl-beta-cyclodextrin effected the separation. The affect on selectivity resulting from the addition of an organic modifier to the run buffer was examined. The low background absorbency of the run buffer made for easy detection of compounds that absorbed at low UV wavelengths. The quick analysis time made this suitable for analysis of combinatorial chemistry samples.     

Determination of polyphenol components in herbal medicines by micellar electrokinetic capillary chromatography with Tween 20

A simple and convenient method of micellar electrokinetic capillary chromatography (MEKC) using polyoxyethylene sorbitan monolaurate (Tween 20) to form single micelle and methanol as a buffer additive was introduced for the simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% (v/v) methanol was adopted in the separation. Because rutin and esculetin were difficult to be separated by capillary zone electrophoresis (CZE) and SDS-based MEKC, Tween 20-based MEKC was adopted and the polyphenols were separated satisfactorily. The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily.     

Application of MEKC and monolithic CEC for the analysis of bioactive naphthoquinones in Eleutherine americana

Two microscale separation techniques for the analysis of bioactive naphthoquinones in Eleutherine americana were developed and validated. By MEKC four compounds (eleuthoside B, isoeleutherin, eleutherol and eleutherinoside A) could be determined in plant extracts using an aqueous electrolyte solution composed of 25 mM sodium tetraborate, 50 mM sodium cholate and 20% THF. CEC on a polymeric methacrylate‐based monolith with strong cationic properties showed promising results, as it additionally enabled the separation of two enantiomers, eleutherin and isoeleutherin. The mobile phase for CEC experiments comprised 3 mM ammonium formate in a mixture of ACN and water. At an applied voltage of ?25 kV, all five markers were baseline separated in less than 12 min. Both methods were successfully validated for linearity (MEKC: R²≥0.999; CEC: R²≥0.997), sensitivity (MEKC: LOD=4–5 μg/mL; CEC: LOD=2–8 μg/mL), accuracy (MEKC: 96.5–102.7% recovery; CEC: 97.1–103.5% recovery) and precision (MEKC: σ_rel≤2.43%; CEC: σ_rel≤2.21%). The quantitative analysis of naphthoquinone derivatives in several E. americana samples showed that both methods are suitable for practical applications, because the results were well comparable to those obtained by established techniques such as HPLC.     

Cloud point extraction combined with micellar electrokinetic capillary chromatography determination of benzophenones in cosmetic matrix

A method has been developed for the separation and determination of three hydrophobic benzophenones: 2,4-dihydroxybenzophenone (BP-1), 2,2'4,4'-tetrahydroxybenzophenone (BP-2), and 2-hydroxy-4-methoxybenzophenone (BP-3) in sunscreen by micellar electrokinetic capillary chromatography (MEKC) combined with cloud point extraction (CPE). The analytes were extracted at pH 5.0 by micelles of the nonionic surfactant polyoxyethylene-7.5-octylphenyl ether (Triton X-114). A 150 microL aliquot from the extracted surfactant-rich phase was diluted up to 500 microL with ethanol to reduce its viscosity before separation by MEKC. A background electrolyte of 25 mmol/L sodium tetraborate containing 30 mmol/L sodium dodecyl sulfate at pH 9.25 was used as the separation medium to avoid the adsorption of hydrophobic substances and Triton X-114 onto the inner surface of the separation capillary, ensuring the separation efficiency and reproducibility. Detection is performed at 290 nm. Under the optimized conditions, an enrichment factor of 20 was obtained and the determination limits of BP-1, BP-2, and BP-3 were found to be 3.90 x 10(-) (7), 3.83 x 10(-7), and 6.42 x 10(-8) mol/L, respectively. In comparison with the earlier reported methods, the LODs of this method are superior to the other methods. The presented procedure was successfully applied to the determination of BP-1, BP-2, and BP-3 in sunscreen with satisfactory results.     

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.     

On-line pretreatment and determination of parabens in cosmetic products by combination of flow injection analysis, solid-phase extraction and micellar electrokinetic chromatography

A convenient and automated method for on-line pretreatment and determination of three parabens (i.e. methyl, ethyl and propyl p-hydroxybenzoate) in cosmetic products is proposed by using flow injection analysis (FIA), solid-phase extraction (SPE) and micellar electrokinetic chromatography (MEKC). An improved split–flow interface is used to couple SPE on C₈-bonded silica with MEKC separation, which can avoid running buffer contamination and reduce buffer consumption, especially, containing expensive reagents. The analytes are loaded onto a C₈ column at 0.6 mL/min for 60 s and eluted with a mixed eluent of 40% (v/v) 10 mmol/L sodium tetraborate buffer (pH 9.3) and 60% (v/v) ethanol at 0.75 mL/min. The MEKC separation is accomplished with a running buffer of 20 mmol/L sodium tetraborate (pH 9.3) containing 100 mmol/L sodium dodecyl sulfate (SDS) at 15 kV. For butyl p-hydroxybenzoate did not be detected in the cosmetic products, it was used as an internal standard (IS) added into the real samples. This FIA–SPE–MEKC method using IS allows the sample separation within 12 min and the sample throughput of five samples per hour with the relative standard deviation (R.S.D.) less than 2.3% (n = 5). The limits of detection (LOD) are in the range from 0.07 to 0.1 μg/mL (S/N = 3 and n = 11). The proposed method has been used to determine three parabens in real cosmetic products satisfactorily.     

Determination of antitubercular drugs by micellar electrokinetic capillary chromatography (MEKC)

A method for the determination of isoniazid (ISO), pyrazinamide (PYR) and rifampicin (RIF) in pharmaceutical products, by micellar electrokinetic capillary chromatography (MEKC) with ultraviolet detection is described. The influence of pH, concentration of surfactants, buffer and organic solvents, over the separation were studied as experimental variables. The optimal separation was carried out at 30 degrees C and 20 kV, using a 40 mM borate buffer and 100 mM sodium dodecylsulphate (SDS) adjusted to pH 8.5. Under these conditions, the analysis is accomplished in about 8 min. The method was applied to the determination of these compounds in different pharmaceuticals with good results when compared with a reference liquid chromatographic (LC) method.     

不同缓冲体系对毛细管电泳法分离15种核苷类化合物效果的比较

对毛细管电泳法分离15种核苷类化合物所用的不同缓冲液体系进行了系统比较,确定不同模式毛细管电泳法分析多种核苷类化合物的最适合背景缓冲液体系(BGE)。分别以四硼酸钠、磷酸氢二钠、乙酸钠、碳酸氢钠、乙酸铵和乙二胺(DEA)为背景电解质,对毛细管区带电泳(CZE)、毛细管电泳-电喷雾飞行时间质谱(CE-ESI-TOF/MS)以及胶束电动毛细管电泳(MEKC)3种模式进行比较,并对其中几种优势缓冲体系进行了优化。结果表明,CZE模式下使用四硼酸钠和磷酸氢二钠缓冲体系无法同时分离15种核苷类化合物,因此只适用于分析核苷类化合物数量较少的样品。而使用含有2%丙酮的300 mmol/L DEA能完全分开15种核苷类化合物,且分辨率和峰形良好。MEKC模式下,以25 mmol/L磷酸氢二钠(添加70 mmol/L十二烷基磺酸钠(SDS))为缓冲盐的分离结果最佳,并且此方法能成功应用于海洋生物海葵中核苷类化合物的分离。CE-ESI-TOF/MS分析中,以20 mmol/L乙酸铵(pH 10.0)为背景电解质,正离子模式检测,15种核苷类化合物的质谱信号均良好,检测灵敏度明显优于文献中报道的使用DEA缓冲体系的结果。本研究阐明了不同缓冲体系对15种核苷类化合物分离的适用性,为毛细管电泳技术在复杂基质中多种核苷类化合物的分离方法中的应用奠定了基础。     

Sensitive and fast determination of Sudan dyes in chili powder by partial-filling micellar electrokinetic chromatography-tandem mass spectrometry

A fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in food samples was developed for the first time using partial filling micellar electrokinectic chromatography-mass spectrometry (MEKC-MS). The use of MEKC was essential to achieve the separation of these neutral analytes, while the partial filling technique was necessary to avoid the contamination of the ion source with non-volatile micelles. MEKC separation and MS detection conditions were optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Filling 25% of the capillary with an MEKC solution containing 40 mM ammonium bicarbonate, 25 mM SDS, and 32.5% (v/v) acetonitrile, a baseline separation of the four azo-dyes was obtained in 10 min. Tandem MS was investigated in order to improve the sensitivity and selectivity of the analysis. Limits of detection (LOD) values 5, 8, 15, and 29 times better were obtained for Sudan III, I, II, and IV, respectively, using partial filling MEKC-MS/MS instead of partial filling MEKC-MS. Under optimized conditions, LOD from 0.05 to 0.2 μg/mL were obtained. The suitability of the developed method was demonstrated through the fast and sensitive determination of Sudan I, II, III, and IV in spiked chilli powder samples. This determination could not be achieved by MEKC-UV due to the existence of several interfering compounds from the matrix.     

Enantioseparation of esbiothrin by cyclodextrin‐modified microemulsion and micellar electrokinetic chromatography

A comparison between chiral cyclodextrin‐modified microemulsion electrokinetic chromatography (CD‐MEEKC) and cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC) for the enantiomeric separation of esbiothrin was carried out. For both methods, the separation conditions were optimized by varying CD types and concentration, running buffer pH and compositions, organic modifiers, and temperature. The optimal CD‐MEEKC conditions were 0.8% n‐heptane, 2.3% SDS, 6.6% n‐butanol, 90.3% 10 mM sodium tetraborate containing 3% (w/v, the ratio of CD mass to microemulsion volume) methyl‐β‐cyclodextrin, pH 10, 25°C. The optimized CD‐MEKC conditions were 3.3% SDS, 96.7% 10 mM sodium tetraborate containing 5% (w/v) β‐CD, pH 10, 25°C. The difference in physicochemical properties of the buffer and CDs resulted in different optimal CD type. The competitive distribution between the microemulsion (or micelle) and chiral CD contributed to the chiral separation. Both methods provided excellent separation (R_s ～? 3) with similar migration time (ca. 15 min). CD‐MEEKC provided higher separation efficiencies (>300000) than CD‐MEKC (>200000). The LODs for CD‐MEEKC and CD‐MEKC were 4.7 μg/mL and 3.2 μg/mL, respectively. The RSDs of migration time and peak area for CD‐MEEKC were slightly higher than for CD‐MEKC. Both the demonstrated CD‐MEEKC and CD‐MEKC methods provided high efficiencies, low LODs, and reproducible enantioseparations of esbiothrin.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.