Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane

A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.     

Analysis of metacycline by capillary electrophoresis

The development and validation of an optimized capillary electrophoresis method for the determination of metacycline in the presence of its related substances by capillary electrophoresis is shown. The influence of methanol as organic modifier, buffer pH, buffer concentration, capillary length, column temperature, Triton X-100 and methyl-beta-cyclodextrin was investigated. A central composite design was performed in order to optimize the method. The optimal separation conditions were: uncoated fused-silica capillary (39 cm total length, 31 cm effective length, 50 microm ID); as background electrolyte a solution of 160 mM sodium carbonate and 1 mM EDTA (pH 10.35)/methanol (89:13 v/v); temperature, 15 degrees C; voltage, 12 kV. The method showed good selectivity, repeatability, linearity, and sensitivity. The limits of detection and quantitation are 0.024% and 0.06%, respectively, relative to a 2.5 mg/mL solution. Six commercial samples were analyzed quantitatively.     

Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists

A capillary zone electrophoretic (CZE) method was optimized for the separation of five angiotensin II receptor antagonists (Losartan, Irbesartan, Valsartan, Telmisartan and Eprosartan) and two of their metabolites (EXP 3174 and Candesartan M1) by means of experimental design methodologies. The aim of this study was to define rapidly experimental conditions under which the analytes can be resolved for quantitation. The effects of the buffer (pH, concentration and composition), the organic modifier and voltage were studied. Critical factors were identified in a screening design (fractional factorial design) and sequentially an optimization design (central composite design) was used to choose optimal conditions for separation. The most favorable electrophoretic conditions were found by setting the resolution at a threshold value (Rs < or = 1.5) and minimizing, if possible, analysis time. Successful results were obtained with a 50 mM potassium dihydrogen phosphate:boric acid (25:75 v/v) buffer at pH 5.5 in the presence of 5% methanol and application of a 25 kV voltage. Analysis time was 8 min in a conventional fused-silica capillary (50 cm effective length) in a normal cationic mode (anode at the inlet and cathode at the outlet) after hydrostatical sample injection for 30 s.     

The analysis of human amniotic fluid using capillary electrophoresis

This study has investigated the composition of amniotic fluid (AF) using capillary electrophoresis (CE). A detailed optimisation investigation was undertaken to obtain the best resolution of the major peaks in amniotic fluid. In the final method, capillary zone electrophoresis (CZE) of AF was performed on a Hewlett Packard3D CE instrument using a fused-silica capillary of 44 cm total length (36 cm to the detector) with in internal diameter of 50 microm. The background electrolyte was 20 mM sodium tetraborate containing 0.8 mM EDTA adjusted to pH 9.0. AF was diluted 1 plus 1 with deionised water prior to hydrodynamic injection for 3 s at 50 mbar. The separation was performed at +22.5 kV and resulted in a current of 65 microA. The capillary temperature was 28 degrees C. Using this CZE method, some eight peaks were consistently resolved in AF samples and several other more transient peaks have been separated from AF in less than 10 min. A scheme for the identification of peaks once they had been separated was also developed. Four peaks have been identified as proteins, i.e., gamma-globulin, alpha1-antitrypsin, transferrin and albumin. Surprisingly, one major peak was shown to be the purine catabolite, xanthine.     

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

Capillary electrophoresis analysis of fosfomycin in biological fluids for clinical pharmacokinetic studies

A feasible capillary zone electrophoresis (CZE) method with indirect UV and contactless conductivity detection was developed for the determination of fosfomycin, an antibiotic, in human plasma and microdialysis samples. Samples were collected from test persons during a clinical trial. The background electrolytes used consisted of 25 mM benzoic acid and 0.5 mM hexadecyltrimethylammonium bromide, adjusted with tris(hydroxymethyl)aminomethane solution to pH 6.95 for plasma, and to pH 8.05 for microdialysis samples. CZE separations of the anionic analyte were carried out with reversed electroosmotic flow directed towards the anode. The limit of detection was between 0.6 and 2 microg/mL, depending on the matrix and the detection method. No sample preparation was needed for microdialysis samples; for plasma samples, proteins were precipitated with methanol (1+2, v+v), and the supernatant was analyzed. The yield determined with spiked samples was about 100%, the reproducibility of the entire method, expressed by the RSD% of three independent determinations of fosfomycin in triplicate after spiking Ringer's solutions and plasma samples, respectively, was better than 8%. The method is thus well-suited for clinical studies for the determination of the antibiotic in biological fluids.     

Capillary Zone Electrophoresis of Eleven Priority Phenols with Amperometric Detection

A scheme for separation and detection of eleven priority phenols using capillary zone electrophoresis (CZE) coupled with amperometric detection is described. With a capillary of I.D. 50 μm and length 62.5 cm at 9 kV and an electrophoretic buffer of 20 mM CHES (pH 10.1), complete separation of the eleven compounds was achieved in less than 17 min. Amperometric detection was carried out using a carbon fiber microelectrode of diameter 9 μm inserted into the end of the detection capillary. Linearity over two orders of magnitude was generally obtained for the eleven priority phenols. With an electrode potential+1.10 V (vs. Ag/AgCl reference), the concentration limits of detection were in the sub-ppm (10^?6 M) level. This method was successfully applied to analysis of priority phenols in industrial waste water.     

Online preconcentration by electrokinetic supercharging for sensitive determination of berberine and jatrorrhizine in biological samples

Electrokinetic supercharging, a convenient and powerful online preconcentration technique in capillary electrophoresis, was introduced and evaluated for the determination of two alkaloids, berberine and jatrorrhizine, in mice fecal samples for the first time. The method depended on using a bare fused silica capillary (50 cm × 50 μm i.d.) and applying the voltage of 25 kV with UV detection at 205 nm. Parameters that affect the separation and preconcentration efficiency have been optimized. The optimum conditions used were as follows: background electrolyte consisting of 40mM sodium dihydrogenphosphate containing 30% methanol (v/v); hydrodynamic injection of 20mM KCl (50 mbar × 150 s) as the leading electrolyte; electrokinetic injection of the sample (+15 kV, 120 s) followed by the hydrodynamic injection of 30mM dodecyl trimethyl ammonium chloride (50 mbar × 12 s) as the terminating electrolyte. The results showed that the detection sensitivity of berberine and jatrorrhizine was, respectively, improved up 2740- and 2928-fold compared with normal injection, providing limits of detection lower than 3 ng/mL with good repeatability in areas (relative standard deviation < 3%). In summary, the developed method proved its ability in analyzing trace alkaloids in complicated biological samples.     

Determination of the beta-blocker atenolol in plasma by capillary zone electrophoresis

A capillary zone electrophoretic method was optimised for the determination of the beta-blocker atenolol in plasma. Separation was performed in an uncoated silica capillary of 58.5 cm (effective length 50 cm) x 75 microm I.D., and detection was at 194 nm. The effects of the buffer (concentration and pH), the injection time, the voltage applied and the plasma clean-up procedure were studied. The determination of atenolol was achieved in less than 3 min, using an electrolyte of 50 mM H3BO3-50 mM Na2B4O7 (50:50, v/v) pH 9, injected hydrodynamically for 4 s at 50 mbar and applying a voltage of +25 kV. This method was applied to the determination of atenolol in plasma of nine hypertensive patients (male and female, aged from 39 to 73 years). Atenolol concentrations found vary from 30 to 585 ng/ml.     

逍遥丸的毛细管电泳指纹图谱的建立

采用毛细管区带电泳法建立了逍遥丸(Xiaoyao Pill, XYP)的毛细管电泳指纹图谱(CEFP)。运用正方形优化法,以色谱指纹图谱分离量指数(RF)为优化的目标函数,对建立指纹图谱的实验条件进行了优化,确定了最佳背景电解质(BGE)溶液50 mmol/L硼砂-50 mmol/L磷酸氢二钠-150 mmol/L磷酸二氢钠-50 mmol/L碳酸氢钠(1:1:1:5, v/v/v/v; pH 7.40)、紫外检测波长228 nm、运行电压12 kV、重力进样25 s (高度14 cm)的分离检测条件。采用未涂层石英毛细管(70 cm×75 μm,有效分离长度57 cm)分离,以咖啡酸色谱峰为参照,确定13批逍遥丸样品的21个共有指纹峰。通过聚类分析确定用其中10批样品生成对照CEFP,以此为标准用系统指纹定量法鉴别13批逍遥丸的质量,结果显示: S3号样品的化学成分数量和分布比例不合格,S10和S12号样品含量明显偏高,其余批次质量均合格。所建立的正方形优化法操作简便,适用于中药的毛细管区带电泳BGE的选择;所建立的逍遥丸CEFP具有较好的精密度和重现性,可以为逍遥丸的质量控制提供新的参考。     

Analysis of the aconitine alkaloids in traditional Chinese medicines by nonaqueous capillary electrophoresis using a new recording mode

The determination of three aconitine alkaloids (hypaconitine, aconitine, mesaconitine) in five traditional Chinese medicines including two Tibetan medicines, Chuanwu, Caowu, Fuzi, Aconitum Tanguticum Maxim and Aconitum Gymnandrum Maxim by non-aqueous capillary electrophoresis using a new recording mode is described. The dissociation constants of aconitine, mesaconitine and hypaconitine have also been determined by CZE and were 7.71, 6.60 and 6.25, respectively. The separation was achieved by optimizing the applied voltage, the pH and the concentration of the buffer. The electrophoretic medium was 20 mM borax-70% (v/v) methanol (pH 8.5) and an uncoated capillary (50 cm x 75 microm i.d.) was used. Detection was carried out with a UV monitor at 214 nm. The total time for separation and determination was under 13 min.     

SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: comparison of direct injection to SPE-MEKC

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco LC-8, LC-18, LC-SCX, and LC-WCX, as well as Strata-X and X-C). DOX was determined on a 56 cm (effective length 50 cm) x 50 microm id fused-silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 sx50 mbar), and the temperature and voltage were 25 degrees C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 microg/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80 mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s x 50 mbar; 25 degrees C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition.     

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.     

毛细管区带电泳测定D1蛋白酶的活性及其抑制剂先导化合物的筛选

建立了毛细管区带电泳技术快速测定D1蛋白酶活性及其抑制剂先导化合物的筛选方法。实验选用未涂层熔融石英毛细管(43 cm 5mm)和磷酸盐缓冲溶液(50 mmol/L, pH 3.0)作为分离介质,运行电压18 kV,测定了D1蛋白酶的活性并对部分抑制剂先导化合物进行了筛选。结果表明, ITP26和ITP21两种异噁唑噻唑哌啶类先导化合物对蛋白酶活性具有抑制作用,抑制率分别为26%和13%。     

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.     

Study of aldehyde oxidase by micellar electrokinetic chromatography separation of O6‐benzylguanine and 8‐oxo‐O6‐benzylguanine

A separation method for O⁶‐benzylguanine (O⁶‐BG) and 8‐oxo‐O⁶‐benzylguanine (8‐oxo‐O⁶‐BG) is developed by using MEKC. This study includes the optimization of separation and incubation parameters for both off‐line and on‐line procedures. The BGE consisted of 25 mM sodium phosphate buffer‐methanol (70:30, v/v), apparent pH 7.4, in which SDS and methyl‐β‐cyclodextrin were dissolved yielding final concentrations of 50 and 15 mM, respectively. Separations were performed at 15 kV using an untreated fused‐silica capillary (40 cm length, effective length is 30 cm) with the detection wavelength at 195 nm. The capillary was kept at 15°C. Good performances were demonstrated for the repeatability and linearity. The LOQ was determined to be 14 μM for 8‐oxo‐O⁶‐BG (S/N = 10). The accuracy values showed a bias of +7.9% for 50 μM and –7.0% for 100 μM. Premix and transverse diffusion of laminar flow profiles (TDLFP) methods were used for on‐line mixing and reaction of the substrate O⁶‐BG with aldehyde oxidase. Both procedures were successful in mixing as well as subsequent separation of the substrate and the metabolite, while the repeatability of TDLFP (14.7% (n = 3)) was much better than the premix technique.     

Analysis of colistin sulfate by capillary zone electrophoresis with cyclodextrins as additive

A method for the quantitative analysis of colistin sulfate by capillary zone electrophoresis is described. Since colistin components have five free amino groups, they tend to adsorb onto the capillary wall and cause peak tailing. It was found that triethanolamine (TEA)-phosphate buffer at pH 2.5 was useful to reduce such adsorption. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found necessary for selectivity enhancement. In order to optimize the separation parameters and predict the method robustness, a central composite design was performed including three variables, namely concentration of M-beta-CD, TEA, and IPA. The effects of capillary length and applied voltage on separation were also investigated. The optimal conditions established were: 140 mM TEA-phosphate buffer containing 5 mM M-beta-CD and 6% v/v IPA, a capillary with 55 cm total length (50 microm inner diameter, 47 cm from inlet to detection window) and 24 kV applied voltage. The method was found to be robust when the variables were changed in the following range: 4-6 mM M-beta-CD, 5-7% v/v IPA, and 130-150 mM TEA. Further, the linearity, limit of detection (LOD), and limit of quantitation (LOQ), as well as repeatability for both colistin A and B were examined and three commercial samples were quantitatively analyzed.     

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.     

Monitoring folding transitions of synthetic,branched-chain polypeptides by capillary zone electrophoresis

The coil/helix transition of a synthetic, branched-chain polymeric polypeptide (poly (Lys(Glu(1)-DL-Ala(3))EAK), 50-Lys residues long in the backbone, as a function of increasing molarities of methanol in solution, is here studied by both, circular dichroism (CD) and capillary zone electrophoresis. CD spectra showed that, at 75% v/v methanol, the transition from random coil to fully helical structure was obtained, in a pH 1.1 HCI solution in the presence of 20 mM NaCI. CZE studies, run in parallel, exhibited the classical unfolding to folding sigmoidal transition, with mid-point at 60% v/v methanol concentration, plateauing at ca. 80% v/v organic solvent. Surprisingly, though, such unfolding to folding transition was accompanied by an expansion, rather than a contraction, of the resulting ordered polypeptide. As the charge of the polypeptide (a pure polycation at a pH of 2.1 in CZE) was kept rigorously constant, a plot of the radius of the polymer along the sigmoidal transition clearly showed that the radius of gyration of the helical, structured polypeptide was in fact larger than that of the random coil. Such results were confirmed by molecular dynamics simulations, which indicated that the dimensions of such polypeptide, in alpha-helix configuration, were 8.5 nm (in length) and 3.2 nm (in diameter), whereas those of the corresponding random coil were 7.2 nm (in length) and 5.1 nm (length of shorter axis). It would thus appear that the randomized structure assumes the shape of a more compact object, roughly resembling a "rugby ball".     

Enantioselective determination of the novel antidepressant mirtazapine and its active demethylated metabolite in human plasma by means of capillary electrophoresis

Mirtazapine is a recent noradrenergic and specific serotonergic antidepressant drug. A capillary electrophoretic method has been developed for the enantioseparation and analysis of mirtazapine and its main active metabolite, N-desmethylmirtazapine, in human plasma. For method optimisation several experimental parameters were investigated, such as type and concentration of the chiral selector, buffer pH and capillary temperature. Baseline enantioseparation of the analytes was achieved in 2.5 min in a fused silica capillary (50 microm i.d.; 48.5 cm total length; 8.5 cm effective length) using carboxymethyl-beta-cyclodextrin, dissolved in a background electrolyte consisting of 50 mM phosphate buffer at pH 2.5, as the chiral selector. UV detection was set at 205 nm. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with hydrophilic-lipophilic balance cartridges (60 mg, 3 mL), eluting the sample with methanol, then concentrating it 37.5 times before injection. Extraction yield values are very satisfactory, being the average 89% for mirtazapine and 73% for N-desmethylmirtazapine. Application of the method to some human plasma samples has given satisfactory results.