Development and validation of a chiral capillary electrophoresis method for assay and enantiomeric purity control of pramipexole

A rapid method for the enantioseparation of pramipexole and its R‐enantiomer has been developed by capillary electrophoresis. The influence of chemical and instrumental parameters was investigated including the type and concentration of chiral selectors, buffer composition and pH, co‐ions, applied voltage, capillary length and temperature. Optimal separation conditions were obtained using a 50 mM phosphate buffer (pH 2.8) containing 25 mM carboxymethyl‐β‐cyclodextrin on a fused‐silica capillary. Online UV detection was performed at 262 nm. A voltage of 25 kV was applied, and the capillary temperature was kept at 25°C. Hydrodynamic injection was performed at 3.45 kPa for 5.0 s. The separation of enantiomers was achieved in <6.5 min. The method was further validated in terms of stability of solutions, selectivity, linearity (both pramipexole and R‐enantiomer, R²>0.995), LOD and LOQ (0.91 and 2.94 μg/mL, respectively), repeatability (RSD<1.5%) and accuracy (pramipexole, 100.4%; R‐enantiomer, 100.5%). The proposed method was then applied to two kinds of pramipexole dihydrochloride monohydrate commercially available tablets, immediate release tablets (1.50 and 0.125 mg) and sustained release tablets (0.52 mg), to quantify the main component in the tablets. The amount of distomer could be quantified in bulk sample materials.     

毛细管电泳-柱端安培检测测定莲子心中荷叶碱、芦丁和金丝桃甙的含量

采用毛细管电泳-柱端安培检测测定莲子心中荷叶碱、芦丁和金丝桃甙的含量．研究了检测电位、运行缓冲液浓度和pH值,分离电压和进样时间对分离和检测的影响．以微碳圆盘电极（Ф=0.5ram）为工作电极,检测电位为＋0．95V（vs．Ag／AgCl）,pH为7．25的50mmol／L Na2B4O7和100mmol／L NaH2PO4缓冲液为运行液,当分离电压为15kV时,3种分析物在15min内完全分离．荷叶碱、芦丁和金丝桃甙的检出限（S／N=3）分别为0．02μg／mL、0．05μg／mL和0．04μg／mL．该方法已成功地应用于莲子心中上述3种活性成分的测定．     

微芯片毛细管电泳快速测定盐酸洛美沙星

采用微芯片毛细管电泳非接触电导检测法快速测定了盐酸洛美沙星胶囊中盐酸洛美沙星的含量。探讨了缓冲液类型、浓度,添加剂种类、浓度及分离电压、进样时间等因素对分离检测的影响。实验采用5.0mmol/L HAc(pH=2.5)+5%乙醇为缓冲溶液,分离电压3.0 kV,在1 min内实现了盐酸洛美沙星的快速分离测定。优化条件下盐酸洛美沙星的线性范围为20.0～250.0μg/mL,检出限为10.0μg/mL(S/N=3),RSD=2.0%,加标回收率为98.6%～103%。     

Simultaneous analysis of biologically active pyridines in pharmaceutical formulations by capillary zone electrophoresis

A capillary zone electrophoretic method using UV detection is developed for the analysis of four biological active pyridines [i.e., nicotine (NIC), cotinine (COT), nicotinic acid (NA), and nicotinamide (NM)]. The separation of the pyridines is achieved in 25 mM sodium dihydrogen phosphate (pH 2.1) using a fused-silica capillary with an effective length of 56 cm and an inner diameter of 50 μm (extended light path), hydrodynamic injection at 50 mbar for 10 s, a temperature of 25°C, applied voltage of 30 kV, and UV detection at 260 nm. These conditions provide baseline separation of all the analytes [resolution (R(s)) > 3.6] in 9.4 min with good linearity (r(2) > 0.998, in ranges of 50-600 μg/mL for NIC, 8-160 μg/mL for NM, and 10-200 μg/mL for COT and NA), precision (relative standard deviation <2.04%), recovery (96.4-101.6%), limits of detection (<3.0 μg/mL), and quantitation (<10 μg/mL). The method is robust upon the alterations of pH of BGE, separating voltage, and injection time [the RSDs of the relative migration time (migration time of the analyte/migration time of the internal standard) and resolution <3.26%]. The method is efficient, reliable, and simple for the routine analysis of NIC, NA, and NM in various products such as gum and tablets and can be applied to determine COT in thermal degradation of NIC gum.     

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.     

Assay and stability‐indicating micellar electrokinetic chromatography method for the simultaneous determination of valacyclovir,acyclovir and their major impurity guanine in pharmaceutical formulations

A micellar electrokinetic chromatography (MEKC) method for the simultaneous determination of the antiviral drugs acyclovir and valacyclovir and their major impurity, guanine, was developed. The influences of several factors (surfactant and buffer concentration, pH, applied voltage, capillary temperature and injection time) were studied. Using tyramine hydrochloride as internal standard, the analytes were all separated in about 4 min. The separation was carried out in reversed polarity mode at 28°C, 25 kV and using hydrodynamic injection (15 s). The separation was effected in a fused‐silica capillary 100 μm × 56 cm and a background electrolyte of 20 mM citric acid–1 M Tris solution (pH 2.75), containing 125 mM sodium dodecyl sulphate and detection at 254 nm. The method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 0.1–1 μg/mL (guanine) and from 0.1 to 120 μg/mL for both valacyclovir and acyclovir. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 5.0%. The proposed method was successfully applied to the determination of the analytes in tablets and creams. From the previous study it is concluded that the stability‐indicating method developed for acyclovir and valacyclovir can be used for analysis of the drug in various stability samples. Copyright © 2009 John Wiley & Sons, Ltd.     

电堆积在线扫集胶束电动色谱法测定苦参中的生物碱

建立了电堆积在线扫集胶束电动色谱法测定苦参碱和槐定碱的新方法.考察了pH值、磷酸二氢钠浓度、CTAB浓度、电压、有机溶剂和进样时间对分离效果的影响.采用未涂层熔融石英毛细管,以20mmol/L磷酸二氢钠-0.8 mmol/L CTAB-100mmol/L Tris(含10%异丙醇,pH 8.9)为缓冲液,在进样电压-1...     

Capillary electrophoretic enantioselective determination of zopiclone and its impurities

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL?1 for each zopiclone enantiomer, 0.8-1.6 μg mL?1 for 2-amino-5-chloropyridine and 0.4-0.8 μg mL?1 for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.     

Multivariate optimization of a capillary zone electrophoresis assay method for simultaneous quantification of metformin and vildagliptin from a formulation

A rapid, accurate, precise, and optimized capillary zone electrophoresis assay was established and validated for the simultaneous quantification of metformin and vildagliptin in tablets. The electrophoretic separation was achieved on an untreated bonded silica capillary with a background electrolyte comprising 25 mM of borate buffer at pH 7.5 at 207 nm. The concentration of the buffer and the pH of BGE were optimized using the multivariate optimization method for determining the retention time and peak area. Furthermore, the sample injection time, capillary oven temperature, and applied voltage were optimized. The capillary zone electrophoresis technique was validated for all required parameters as per the International Conference on Harmonization recommendations. The linearity ranged in the concentrations of 5–500 µg/mL and 5–100 µg/mL with the limit of detections of 0.22 µg/mL and 0.40 µg/mL for metformin and vildagliptin, respectively. In addition, the percent relative standard error for repeatability and inter-day precision was within the acceptable range. The mean recoveries determined by the capillary zone electrophoresis method were 99.2% and 100.4% for metformin and vildagliptin, respectively. Finally, the capillary zone electrophoresis process was effectively used for the assays of metformin and vildagliptin in their solid dosage form, and statistical outcomes were in agreement with the outcomes of the previously validated RP-HPLC method.     

Determination of preservatives by integrative coupling method of headspace liquid-phase microextraction and capillary zone electrophoresis

An integrative coupling method of headspace liquid-phase microextraction (HS-LPME) and capillary zone electrophoresis (CZE) was proposed in this paper. In the method, a separation capillary was used to create a microextraction droplet of the running buffer solution of CZE, hold the droplet at the capillary inlet, extract analytes of sample solutions in the headspace of a sample vial, inject concentrated analytes into the capillary and separate the analytes by CZE. The proposed method was applied to determine the preservatives of benzoic acid and sorbic acid in soy sauce and soft drink samples, in which the running buffer solution of 50 mmol/L tetraborate (pH 9.2) was directly used to form the acceptor droplet at the capillary inlet by pressure, and the preservatives in a 6-mL sample solution containing 0.25 g/mL NaCl were extracted at 90°C for 30 min in the headspace of a 14-mL sample vial. Then the concentrated preservatives were injected into the capillary at 10 cm height difference for 20 s and separated by CZE. The enrichment factors of benzoic acid and sorbic acid achieved 266 and 404, and the limits of detection (LODs) were 0.03 and 0.01 μg/mL (S/N=3), respectively. The recoveries were in the range of 88.7-105%. The integrative coupling method of HS-LPME and CZE was simple, convenient, reliable and suitable for concentrating volatile and semi-volatile organic acids and eliminating matrix interferences of real samples.     

Determination of metolcarb in food by capillary electrophoresis immunoassay with a laser-induced fluorescence detector

A capillary electrophoresis immunoassay (CEIA) was developed for the determination of trace metolcarb (MTMC) in food. The method was based on the competitive reactions between fluorescently labeled MTMC tracer and free MTMC with a limited amount of anti-MTMC antibody and the separation and determination by CE with LIF detector. A fluorescent reagent, FITC was labeled on MTMC to construct an immunofluorescent probe. CEIA experimental parameters such as the pH value and concentration of the running buffer and separation voltage as well as incubation time were systematically investigated. Under the optimized conditions, fluorescently labeled antigen and antibody bound could be well separated within 3 min using Na?B?O?/NaH?PO? buffer (20:10 mmol/L, pH 9.0) for background electrolyte, 20 kV for the separation voltage, and 20°C for the column temperature. The linear range of the method was 0.25-50.0 μg/L with LOD 0.07 μg/L. The RSD for relative migration time and relative fluorescence intensity ratio were 2.90% (intraday) and 4.73% (intraday), respectively. The proposed method has been applied to determine the residue of MTMC in food samples with the satisfactory recovery.     

毛细管电泳-电致化学发光法测定兔血浆中的羟考酮

基于稀土Eu(Ⅲ)掺杂的类普鲁士蓝膜修饰的铂电极为工作电极,建立了测定羟考酮的毛细管电泳-电致化学发光分析方法。考察了检测电位、运行缓冲溶液的酸度及浓度、分离电压、进样条件等对电泳分离效果及检测灵敏度的影响。在最佳的实验条件下,羟考酮可在4 min内得到分离,其ECL强度值与羟考酮的质量浓度在7.0×10-2～7.0μg/mL和7.0～70.0μg/mL范围内呈良好的线性关系,检出限为4.2×10-2μg/mL(3σ),峰高和迁移时间的相对偏差分别为3.6%和0.48%(n=6)。方法用于兔血浆中羟考酮含量的检测,加标回收率在99.7%～101.0%之间。     

Simultaneous determination of chlordiazepoxide and selected antidepressants using CZE

A simple CE method was developed and validated for the simultaneous determination of chlordiazepoxide (CHL), amitriptyline, and nortriptyline (mixture I) or the determination of CHL and imipramine (mixture II) using the same BGE. Sertraline and amitriptyline were used as internal standards for the first and second mixtures, respectively. The method allows amitriptyline to be completely separated from its impurity and main metabolite nortriptyline, which can be quantified from 0.2 μg/mL. The separation was achieved using 20 mM potassium phosphate buffer pH 5 containing 12 mM β‐cyclodextrin and 1 mM carboxymethyl‐β‐cyclodextrin. UV detection was performed at 200 nm and a voltage of 15 kV was applied on an uncoated fused‐silica capillary at 25°C. These experimental conditions allowed separation of the compounds to be obtained in 7 min. Calibration graphs proved the linearity up to 40 μg/mL for CHL, up to 100 μg/mL for amitriptyline and imipramine, and up to 5 μg/mL for nortriptyline. The accuracy and precision of the method have been determined by analyzing synthetic mixtures and pharmaceutical formulations. The analytical results were quite good in all cases indicating that the method was linear, sensitive, precise, accurate, and selective for both mixtures.     

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

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

微芯片毛细管电泳快速测定加替沙星注射液中加替沙星的含量

研究了用微芯片毛细管电泳非接触电导检测系统快速测定加替沙星注射液中加替沙星的方法。对缓冲液的类型、浓度、分离电压以及进样时间等因素进行了优化。最佳条件为:缓冲液5.0 mmol/L HAc,分离电压2.0 kV,进样时间15.0 s。在该条件下,可在1.0 min内实现加替沙星的快速含量测定。线性范围为4.0～150μg/mL,检出限为1.0μg/mL,加标回收率为95.7%～101%,可成功测定注射液中加替沙星的含量。     

Enantiomeric separation and determination of the enantiomeric impurity of armodafinil by capillary electrophoresis with sulfobutyl ether-β-cyclodextrin as chiral selector

A selective capillary electrophoresis method using sulfobutyl ether-β-cyclodextrin as a chiral selector was developed and validated for the determination of the enantiomeric impurity of (R)-modafinil, i.e., armodafinil. Several parameters were optimized for a satisfactory enantioresolution, including the type and concentration of chiral selector and organic modifier, pH of background electrolyte (BGE), capillary temperature. The finally adopted condition was: 20 mmol/L phosphate buffer at pH 7.5, containing 20 mmol/L sulfobutyl ether-β-cyclodextrin and 20% methanol, at temperature of 25 °C. A good resolution of 3.3 for the two enantiomers of modafinil was achieved by applying the optimal conditions. The limit of detection (LOD) and limit of quantification (LOQ) of (S)-modafinil were 1.25 μg/mL and 2.50 μg/mL, respectively. The established method was also proven to display good selectivity, repeatability, linearity and accuracy. Finally, the method was used to investigate the enantiomeric purity of armodafinil in bulk samples.     

Analysis of vancomycin and related impurities by micellar electrokinetic capillary chromatography. Method development and validation

A fast and highly selective micellar electrokinetic capillary chromatography (MEKC) method for quantitative analysis of vancomycin and related impurities is described. Among the tested surfactants, cetyltrimethylammonium chloride (CTAC) offered the best selectivity. Another important parameter, which strongly influenced the selectivity, was buffer pH. It was found that the selectivity increased with buffer pH decreasing from 9 to 5. Using Tris-phosphate buffer containing CTAC, satisfactory separation could be obtained in the pH range from 5.0 to 5.5. Excellent repeatability in terms of migration time and peak area could be obtained when the capillary was carefully washed between two runs. In order to obtain optimal conditions and to evaluate the method robustness, a central composite experimental design was carried out. The optimal conditions were: 44 cm length of fused-silica capillary with 50 microm ID, 120 mM Tris-phosphate buffer (pH 5.2) containing 50 mM CTAC, -15 kV applied voltage, UV detection at 210 nm, and a column temperature of 25 degrees C. Under the optimal conditions, more than 20 peaks could be separated within 8 min. The method has a linearity range from 0.004 to 1.2 mg/ml (concentration of vancomycin B, active component). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.4 microg/mL vancomycin, equivalent to 0.3 microg/mL vancomycin B (0.04%) and 1.1 microg/mL vancomycin, equivalent to 0.9 microg/mL vancomycin B (0.1%), respectively.     

Development and validation of a capillary electrophoresis method for the characterization of sulfoethyl cellulose

To characterize sulfoethyl cellulose el samples, a capillary electrophoresis method was developed and validated sulfoethyl cellulose el was hydrolyzed, and the resulting d ‐glucose derivatives were analyzed after reductive amination with 4‐aminobenzoic acid using 150 mM boric acid, pH 9.5, as background electrolyte at 20°C and a voltage of 28 kV. Peak identification was derived from capillary electrophoresis with mass spectrometry using 25 mM ammonia adjusted to pH 6.2 by acetic acid as electrolyte. Besides mono‐, di‐, and trisulfoethyl d ‐glucose small amounts of disaccharides could be identified resulting from incomplete hydrolysis. The linearity of the borate buffer‐based capillary electrophoresis method was evaluated using d ‐glucose in the concentration range of 3.9–97.5 μg/mL, while limits of detection and quantification derived from the signal‐to‐noise ratio of 3 and 10 were 0.4 ± 0.1 and 1.2 ± 0.3 μg/mL, respectively. Reproducibility and intermediate precision were determined using a hydrolyzed sulfoethyl cellulose el sample and ranged between 0.2 and 8.8% for migration times and between 0.3 and 10.4% for peak area. The method was applied to the analysis of the degree of substitution of synthetic sulfoethyl cellulose el samples obtained by variation of the synthetic process and compared to data obtained by elemental analysis.     

Development of a capillary electrophoresis method for the enantioselective estimation of primaquine in pharmaceutical formulations

A capillary electrophoresis (CE) method has been developed that allows the separation and estimation of primaquine enantiomers using hydroxypropyl-gamma-cyclodextrin (HP-gamma -CD) as a chiral selector. The influence of chemical and instrumental parameters on the separation, such as type and concentration of CD, buffer concentration, buffer pH, applied voltage, capillary temperature, and injection time, were investigated. Good separation of the racemic mixture of primaquine was achieved using a fused-silica capillary (52.5 cm effective length x 50 microm id) and a background electrolyte composed of tris-phosphate buffer solution (50 mM, pH 2.5) containing 15 mM HP-gamma-CD as a chiral selector. The recommended applied voltage, capillary temperature, and injection time were 15 kV, 25 degrees C, and 6 s, respectively. Within-day and interday reproducibility of peak area and migration time gave relative standard deviation values ranging from 1.05-3.30%. Good recoveries (range of 96.8-104.9%) were obtained from the determination of placebos that were spiked with 0.25-1.00 mg/L primaquine. The proposed CE method was successfully applied to the assay of primaquine diphosphate in pharmaceutical formulations (tablets).     

Simultaneous separation of 14 antiarrhythmic drugs and determination of mexiletine and flecainide by capillary zone electrophoresis

A method using capillary zone electrophoresis was developed for the simultaneous separation of 14 antiarrhythmic drugs belonging to various classes. The drugs are separated on a fused-silica capillary, 90 cm x 75 microm (72 cm effective length), with phosphate and acetate buffers as background electrolytes and UV detection at 217 nm. The effects of buffer pH, temperature, and applied voltage on the migration of the drugs were studied. The pH was found to be the most significant factor determining effective separation. The antiarrhythmic compounds are completely separated within a relatively short time (< 7 min) by using 70 mM phosphate buffer at pH 7.91, an applied voltage of 28 kV, and a temperature of 32 degrees C. Mexiletine (MEX) and flecainide (FLE) were quantified under conditions of the optimum separation. The calibration graphs were constructed over the concentration range of 4.0-14.0 microg/mL for both drugs with good correlation (r > or = 0.9999). Detection and quantitation limits were found to be 0.5 and 1.5 microg/mL for FLE and 0.7 and 2.1 microg/mL for MEX, respectively. The proposed method was used for the determination of both drugs in their commercial forms with satisfactory precision (relative standard deviations of 0.36-1.21% for FLE and 0.78-1.66% for MEX) and accuracy (relative standard errors of 0.13-1.17% for FLE and 0.35-1.18% for MEX).