Towards a general approach for the impurity profiling of drugs by micellar electrokinetic chromatography

A general micellar electrokinetic chromatographic (MEKC) strategy for the impurity profiling of drugs was developed involving a sodium dodecyl sulfate (SDS) and a cetyltrimethylammonium bromide (CTAB) MEKC system. With this combination, in principle, each sample component passes the detector in at least one of the two MEKC systems provided that separation buffers of the same pH are used in both systems. In order to select the proper MEKC systems, the electroosmotic flow (EOF) and micelle migration time (t(mc)) were determined for separation buffers of several pH values, containing various amounts of surfactant and organic modifier. The selectivity of the MEKC systems was studied using a mixture of compounds with a wide range of physico-chemical properties. The final selection of two adequate MEKC systems for this approach was based on the requirements that the t(mc) (i.e., analysis time) of both systems was below 20 min and that the t(mc)/t(eof) ratio was above 3 or 2 for the SDS and CTAB system, respectively. Furthermore, the systems should provide high efficiency, exhibit differences in selectivity and use moderate concentrations of modifier and surfactant, so that, if needed, further optimization is possible. The selected MEKC systems contained 60 mM SDS or 10 mM CTAB, respectively, in a phosphate buffer (pH 7.5) with 10% acetonitrile. Some test compounds with extreme mobilities were used to demonstrate the suitability of the MEKC approach to detect each component of a sample. The potential of the proposed MEKC combination for impurity profiling was demonstrated by the analysis of fluvoxamine with several impurities at the 0.1% level.     

Modeling of anti-Langmuirian peaks in micellar electrokinetic chromatography: benzene and naphthalene

Peaks of benzene (bz) and naphthalene (np) having diffuse fronts and steep rears under overload conditions were studied quantitatively in MEKC with SDS surfactant. The retardation factors of these compounds, solubilized at microM to mM concentrations by either 10, 30, or 50 mM SDS, were determined by vacancy MEKC and frontal analysis MEKC. Isotherm coordinates were calculated from the retardation factors, and the equation for the concave upward anti-Langmuir isotherm was fit to them. Peak profiles were computed with the MacCormack algorithm from the isotherm fits and a simplified continuity equation appropriate to MEKC. These profiles were compared to ones generated in normal MEKC from samples of bz and np solubilized at muM to mM concentrations by either 10, 30, or 50 mM SDS. In all cases, the anti-Langmuir isotherm described the asymmetry of experimental peaks. For bz in 30 and 50 mM SDS and np in 10 and 50 mM SDS, good to excellent agreement was found between the experimental and predicted profiles. For bz in 10 mM SDS, the experimental profiles were more broadened than the predicted ones, although their asymmetries agreed. For np in 30 mM SDS, the experimental isotherm predicted greater peak asymmetry than was observed, and the correct anti-Langmuir isotherm for all sample concentrations and field strengths was calculated from the most asymmetrical peak by the inverse method. The relative decrease of zone velocity with increasing analyte concentration was calculated from the isotherm parameters, electrokinetic mobilities, retardation factors, surfactant concentrations, and CMC. The simplification of the continuity equation was justified.     

阳离子选择性耗尽进样-胶束电动色谱法对可非糖浆中盐酸异丙嗪与磷酸可待因的测定

在胶束电动色谱法的基础上,联用阳离子选择性耗尽进样技术,对盐酸异丙嗪和磷酸可待因同时测定的方法进行了研究。考察了pH值、有机溶剂、SDS浓度、进样时间、进样电压等实验条件对分离效果的影响。最佳实验条件为:缓冲体系16%乙腈+80 mmol/L SDS+20 mmol/L NaH2PO4(pH2.4),分离电压为-18 kV,测量波长214 nm,萃取液pH2.4,进样电压10 kV,进样时间100 s。在优化实验条件下,两种物质在8 min内出峰,峰面积RSD不大于4.6%。盐酸异丙嗪、磷酸可待因的线性范围分别为0.50~81.3、0.78~62.5μg/L,检出限分别为0.16、0.12μg/L,相关系数分别为0.998 9、0.998 8。将方法用于可非糖浆中盐酸异丙嗪与磷酸可待因的测定,回收率为96%~106%。     

Determination of hydrolyzable tannins in the fruit of Terminalia chebula Retz. by high-performance liquid chromatography and capillary electrophoresis

A RP-HPLC method for determining fourteen components (gallic acid, chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose, casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia chebula Retz. is described. The separation was achieved within 80 min using a binary gradient with mobile phases consisting of a pH 2.7 phosphoric acid solution and an 80% CH3CN solution. Capillary electrophoretic analyses were also attempted, and it was found that CZE (25 mM Na2B4O7, 5 mM NaH2PO4, pH 7.0) was an efficient method for the separation of gallotannins, while an MEKC method (25 mM Na2B4O7, 5 mM NaH2PO4, 20 mM SDS, pH 7.0, and 10% acetonitrile) provided a better separation for most of the tannins examined. The HPLC and CE methods developed were both successfully applied to the assay of tannins in commercial samples of Chebulae Fructus.     

On-line concentration by field-enhanced sample injection with reverse migrating micelles in micellar electrokinetic capillary chromatography for the analysis of flavonoids in Epimedium brevicornum Maxim

A simple and sensitive micellar electrokinetic capillary chromatography (MEKC) method was developed for the separation and determination of six flavonoids in Epimedium brevicornum Maxim. Field-enhanced sample injection with reverse migrating micelles (FESI-RMM) was used for on-line concentration of the flavonoids. An electrolyte containing 20 mM H3PO4, 100 mM SDS, 20% acetonitrile and 2% 2-propanol (pH 2.0) was chosen as the electrophoretic buffer. By optimizing the stacking conditions, about 40-360-fold improvement in the detection sensitivity was obtained for the flavonoids.     

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.     

Separation of aromatic hydrophobic sulfonates by micellar electrokinetic chromatography

Two different buffer systems for the separation of 12 aromatic hydrophobic sulfonates by micellar electrokinetic chromatography (MEKC) were developed. The following buffer systems were used: aqueous phosphate buffers containing either cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS). Eleven aromatic sulfonates were simultaneously separated in less than 35 min employing 20 mM phosphate buffer, pH 7.0 containing 50 mM SDS and 10% of acetonitrile.     

Monomeric and polymeric anionic gemini surfactants and mixed surfactant systems in micellar electrokinetic chromatography. Part I: characterization and application as novel pseudostationary phases

Sodium di(undecenyl) tartarate monomer (SDUT), a vesicle-forming amphiphilic compound possessing two hydrophilic carboxylate head groups and two hydrophobic undecenyl chains gemini surfactant, was prepared and polymerized to form a polymeric gemini surfactant (i.e., poly-SDUT). These anionic surfactant systems with carboxylate (SDUT and poly-SDUT) and sulfate (sodium dodecyl sulfate, SDS) head groups as well as mixed surfactant systems (SDS/SDUT, SDS/poly-SDUT, and SDUT/poly-SDUT) were then applied as novel pseudostationary phases in micellar electrokinetic chromatography (MEKC). The SDUT and poly-SDUT were characterized using various analytical techniques. Retention factors of 36 benzene derivatives were calculated in 20 mM phosphate buffer of each surfactant system. The retention factor values of the test solutes show that there are distinctive selectivity differences between the surfactant systems. Solute-pseudostationary phase interactions in MEKC were also examined by determining the free energy of transfer of the substituted functional groups from the aqueous buffer phase into the pseudostationary phase.     

Separation and determination of lignans from seeds of Schisandra species by micellar electrokinetic capillary chromatography using ionic liquid as modifier

In this paper, a micellar electrokinetic chromatographic (MEKC) method using ionic liquid as modifier for the quantification of the active components of lignans found in the medicinal herbs Schisandra species was developed for the first time. Preliminary investigations employing sodium dodecyl sulfate (SDS) as surfactant did not lead to the necessary resolution of the studied compounds, the addition of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) to the SDS micellar system resulted in the complete separation of all the compounds. The effects on the separation by several parameters such as BMIM-BF4 and SDS concentration, applied voltage, background electrolyte pH and concentration, were evaluated. Under the optimal conditions (5 mM borate-5 mM phosphate buffer in the presence of 20 mM SDS and 10 mM BMIM-BF4, pH 9.2, applied voltage 25 kV and detection at 254 nm), the method successfully applied to the determination of lignans in extracts of Schisandra chinensis (Turcz.) Baill. and Schisandra henryi C.B. Clarke in less than 13 min. The separation mechanism was also discussed.     

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).     

Development of an ultrasensitive stacking technique for 5‐nitroimidazole determination in untreated biological fluids by micellar electrokinetic chromatography

Cation‐selective exhaustive injection and sweeping followed by a MEKC separation is evaluated for the sensitive analysis of 5‐nitroimidazoles in untreated human serum and urine. Deproteinized serum and urine samples were diluted 76 and 143 times, respectively, in a low‐conductivity solvent (5.00 mM orthophosphoric acid containing 5.0% v/v methanol). Samples were electrokinetically injected at 9.8 kV for 632 s in a previously conditioned fused‐silica capillary (65.0 cm × 50 μm id). Separation was performed at –30 kV and 20°C using 44 mM phosphate buffer (pH 2.5), 123 mM SDS, and 8% v/v tetrahydrofurane as BGE. Signals were monitored at 276 nm and peak area was selected as analytical response. Good linearity (R² ≥ 0.988) and LODs lower than 1.5 and 1.8 μg/mL were achieved in serum and urine, respectively.     

Fast CE for combinatorial catalysis

Two solvent-modified MEKC methods were developed for the quantitative analysis of heterocyclic amines synthesised using intramolecular ring closure via catalysed hydroamination. The first method was capable of resolving six of the amines (precursors and products) with a sample-to-sample injection time of 2 min employing a 20 mM borate buffer, pH 9.2 with 20 mM SDS and 5% v/v n-butanol (n-BuOH). A second low-pH method using 20 mM phosphate buffer, 100 mm SDS, 5% v/v n-BuOH and 20% v/v iso-propanol (i-PrOH) was able to resolve an additional pair of compounds with a sample-to-sample time of 3.5 min. Application of the first method to the analysis of a sample containing catalyst as well as amines placed directly in a 96-well plate showed excellent performance, with migration time and peak height and area reproducibility being less than 0.9 and 9.6%, respectively. The quantity of conversion by catalyst was calculated to be 68 +/- 7%, which was in excellent agreement with the 67 +/- 5% obtained by more conventional (1)H NMR experiments, with the added advantage that this method is also cheaper, quicker and more amendable to high-throughput screening of combinatorial libraries.     

Micellar electrokinetic and high-performance liquid chromatographic determination of potential manufacturing impurities in pholcodine

Quantitative high-performance liquid chromatographic (HPLC) and micellar electrokinetic chromatographic (MEKC) methods have been developed for the determination of four structurally related potential manufacturing impurities, including morphine, of the opiate derivative pholcodine. Pholcodine and the four impurities were separated by MEKC in less than 14 min using a 70 cm x 75 microm I.D. uncoated fused-silica capillary (25 kV at 30 degrees C) and a running buffer consisting of 10% acetonitrile (v/v) in 20 mM borate-phosphate buffer pH 8.0 containing 40 mM sodium dodecyl sulphate (SDS). The MEKC method was compared to a HPLC method using a 5 microm Luna phenyl-hexyl column (150 x 4.6 mm I.D.) eluted with a mobile phase consisting of a mixture of 10% (v/v) acetonitrile, 7% (v/v) tetrahydrofuran in 20 mM phosphate buffer pH 8.0. Both methods were fully validated and a comparison was made regarding selectivity, linearity, precision, robustness and limits of detection and quantitation. The presence of the impurities in different samples of pholcodine drug substance was investigated using both methods.     

Analysis of flavonols ofSedum telephium L. leaves by capillary electrophoresis and HPLC-mass spectrometry

Summary Flavonol glycosides ofSedum telephium L. were analysed by MEKC. Baseline separation was achieved within 14 min using a fused silica capillary and a borate/phosphate buffer solution (35 mM, pH 5.8) containing 35 mM SDS and 4% MeOH. The applied voltage was 25 kV and the thermostating temperature was kept constant at 40°C. Injection was performed via the pressure mode for 3 s, the detection wavelength was 205 nm. The optimized MEKC method was used for the quantitative determination of flavonol glycosides in extracts ofS. telephium leaves. Analysis was also performed by HPLC-MS using an electrospray ionization (ESI) interface. The good agreement between the quantitative CE results and those obtained by LC clearly demonstrated the applicability of the methods presented.     

Comparison of micellar electrokinetic capillary chromatography and high performance liquid chromatography on fingerprint of Cnidium monnieri

In our studies, micellar electrokinetic capillary chromatography (MEKC) was employed in fingerprint analysis of Cnidium monnieri for the first time. Average chromatography of 10 batches Cnidium monnieri from Jiangsu province, China, which have long been considered as the original and genuine herbal medicine, was first established as the characteristic fingerprint. Within 25 min the major effective components were separated by 18 mM borate, 12 mM phosphate and 50 mM SDS (pH 9.2) containing 20% methanol. The relative standard deviations of migration times and peak areas were less than 5%. As a new approach of fingerprint, MKCE was compared to the conventional approach-HPLC in our experiments. The fingerprint developed by HPLC comprised 8 peaks that were collected within 40 min. Relative standard deviation (RSD) values of retention times of corresponding peaks in HPLC analysis were very small (maximum 3% and average 0.9%). In conclusion, each two methods had its advantages and disadvantages. Furthermore, besides HPLC, MEKC as a feasible method, could be used in the development of fingerprint of Cnidium monnieri.     

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.     

Simultaneous enantioseparation of cyproconazole,bromuconazole, and diniconazole enantiomers by CD‐modified MEKC

An efficient method for the simultaneous enantioseparation of cyproconazole, bromuconazole, and diniconazole enantiomers was developed by CD‐modified MEKC using a dual mixture of neutral CDs as chiral selector. Three neutral CDs namely hydroxypropyl‐β‐CD, hydroxypropyl‐γ‐CD, and γ‐CD were tested as chiral selectors at different concentrations ranging from 10, 20, 30 and 40 mM, but enantiomers of the studied fungicides were not completely separated. The best dual chiral recognition mode for the simultaneous separation of cyproconazole, bromuconazole, and diniconazole enantiomers was achieved with a mixture of 27 mM hydroxypropyl‐β‐CD and 3 mM hydroxypropyl‐γ‐CD in 25 mM phosphate buffer (pH 3.0) containing 40 mM SDS to which methanol‐acetonitrile (10%:5% v/v) was added as organic modifiers. The best separation was based on the appearance of 10 peaks simultaneously, with good resolution (R_s 1.1–15.9), and peak efficiency (N>200 000). Good repeatabilities in the migration time, peak area, and peak height were obtained in terms of RSD ranging from (0.72 to 1.06)%, (0.39 to 3.49)%, and (1.90 to 4.84)%, respectively.     

On-chip chiral and achiral separation of amphetamine and related compounds labeled with 4-fluoro-7-nitrobenzofurazane

Amphetamine and analogous compounds have been labeled with 4-fluoro-7-nitrobenzofurazane and analyzed on a microfabricated chip. Separation of norephedrine, ephedrine, cathinone, pseudoephedrine, methcathinone, amphetamine and methamphetamine is demonstrated using micellar electrokinetic capillary chromatography (MEKC) and laser-induced fluorescence (LIF) detection. Chiral separations of individual drugs were studied using neutral and negatively charged cyclodextrins (CDs) with and without the addition of an organic modifier and/or sodium dodecyl sulfate (SDS). The best results were obtained using a highly sulfated gamma-CD (HS-gamm-CD) in combination with a low concentration of SDS. To obtain complete separation of a mixture of (+/-)-norephedrine, (+/-)ephedrine, (+/-)-pseudoephedrine, (+/-)-methcathinone, (+/-)-amphetamine and (+/-)-methamphetamine it was necessary to add a small amount (1.5 mM) of SDS to the separation buffer. Optimized chiral separation was achieved within 7 min using an S-folded separation channel, a separation voltage of 8 kV and a buffer consisting of 50 mM phosphate (pH 7.35), 10 mM HS-gamma-CD and 1.5 mM SDS.     

Studies on bovine serum albumin-sodium dodecyl sulfate complexes using pyrene fluorescence probe and 5-doxylstearic acid spin probe

Interactions and characteristics of 0.1% bovine serum albumin (BSA)-sodium dodecyl sulfate (SDS) in 20 mM phosphate buffer solution were investigated by means of fluorescence spectroscopy and electron spin resonance (ESR) spectroscopy. In BSA-SDS system, the intensity ratio, Im3/Im1, of the third vibronic band of the pyrene monomer to the first vibronic band showed a small peak at about 0.1 mM SDS in the phosphate buffer below cmc. In accordance with this Im3/Im1 ratio, the intensity ratio, Ie/Im1, of fluorescence from the pyrene excimer to that from the monomer showed a pseudo-plateau (0.08-0.8 mM) and suggested the existence of micelle-like aggregates below the cmc. Temperature dependence of ln(Ie/Im1) in pyrene fluorescence in the SDS-BSA system was examined as a function of SDS concentration. The activation energy of pyrene diffusion for excimer formation in a micelle was estimated to be 19.2 kJ mol(-1) for the BSA-SDS system. ESR spectra of 5-doxylstearic acid (5-DSA) showed that the probe location is restricted at SDS concentrations above the cmc, and that the probe also is highly restricted in motion for BSA-bound SDS micelles.