胶束电动毛细管色谱法分析头孢哌酮与其S-异构体等杂质

以十二烷基硫酸钠(SDS)胶束为准固定相,考察了头孢哌酮、头孢哌酮S-异构体、头孢哌酮杂质A及其他未知杂质在胶束电动毛细管色谱(MECC)分离模式下的分离行为。研究了运行缓冲液的pH值、磷酸盐浓度、SDS浓度、甲醇体积分数、分离电压、分离温度等因素对头孢哌酮、S-异构体、头孢哌酮杂质A及其他杂质的迁移时间、分离度以及可分离出的杂质个数的影响。结果发现,这些因素对头孢哌酮与诸杂质间的分离及检测有显著的影响,尤以pH值为最。它不仅影响它们的迁移时间和分离效率,还直接影响头孢哌酮及其杂质峰的检测。优化后的分离条件:运行缓冲液为70 mmol/L磷酸盐-100 mmol/L SDS (pH 6.5),分离电压为15 kV,分离温度为25 ℃。在此条件下,用非涂渍石英毛细管51.0 cm×75 μm(有效长度42.5 cm),压力进样5 kPa×5 s,在254 nm波长下进行检测,可分离出28个杂质,诸杂质彼此间及与头孢哌酮间可得到有效分离。并将该方法成功地用于测定注射用头孢哌酮钠的含量和有关物质,结果令人满意。     

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30 mM sodium phosphate buffer, pH 3.0, containing 40 mg/mL sulfated β-CD and 7 mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23 kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.     

On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens

An improved technique, pressurized capillary electrochromatography (pCEC) coupling with end-column amperometric detection (AD), was developed and used for the separation and determination of estrogens. The effects of pH value, composition of mobile phase, concentration of the surfactant sodium dodecyl sulfate (SDS) and applied voltage on separation were investigated. The electrochemical oxidation of diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX) could be reliably monitored with a carbon electrode at 0.9 V (vs. Ag/AgCl). The pCEC analyses were performed on a capillary separation column packed with 3 microm C18 particles with an acetonitrile/water (31%: 69%) mobile phase containing Tris buffer (5 mmol/L, pH 4.5) and 4 mmol/L SDS. High voltage up to 12 kV reduced the retention time dramatically and still provided a baseline resolution. In addition, supplementary pressure prevented bubble formation and provided reliability and reproducibility of the pCEC performance. The detection limits for the three estrogens ranged from 1.2 to 2.2x10(-7) mol/L, about 10 20-fold lower than those obtained with pCEC-UV detection. To evaluate the feasibility and reliability of this system, the proposed pCEC-AD method was further demonstrated with fish muscle samples spiked with estrogens.     

Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography

In this paper, the pressurized capillary electrochromatography (pCEC) with UV detection was utilized for the separation and determination of three structurally related estrogens, such as diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DE), which were difficult to be separated by capillary electrophoresis (CE) and HPLC due to their similarity in the structure and charge-to-mass ratios. Experiments were carried out in a commercially available pCEC instrument using a capillary column packed with 3 microm octadecyl silica (ODS). Surfactant sodium dodecyl sulfate (SDS) was introduced in the mobile phase to enhance the speed of analysis. The effective factors on the retention time and separation resolution, such as the applied voltage, supplementary pressure, the pH and the concentration of the buffer solution, the concentration of SDS, and the content of acetonitrile in the mobile phase, were evaluated. Based on the investigation, 31% (v/v) acetonitrile and 69% (v/v) of 10 mmol/L phosphate buffer (pH 6.5) containing 1.0 mmol/L SDS at an applied voltage of -12 kV and a supplementary pressure of 1000 psi were found to be the optimal conditions for pCEC to separate the three estrogens. The method also had been applied to the analysis of fish muscle samples spiked with estrogens.     

加压毛细管电色谱在建立中药黄柏指纹图谱中的方法学研究

以加压毛细管电色谱（pCEC）为技术平台,对其在建立中药黄柏指纹图谱中的方法学进行了研究．通过对提取溶剂、流动相中有机相种类、盐溶液等条件的优化,发现1％盐酸的甲醇溶液为提取溶液,20mmol／LNH4C1溶液．乙腈为流动相梯度洗脱,紫外检测波长为230nm时对其分离效果最好．并通过在色谱柱上施加不同的电压,详细地阐明了pCEC的双重分离机制对分离选择性的影响,发现黄柏中的主要成分药根碱、巴马汀和小檗碱在pCEC模式中随电压的不同,有不同的出峰顺序．当电压为0—4kV时出峰顺序为药根碱、巴马汀和小檗碱,当电压为8—14kV时出峰顺序为药根碱、小檗碱和巴马汀．对此原因进行了详细讨论,同时与微径液相色谱模式进行了比较,说明pCEC可以为复杂样品的分离提供更多更好的分离途径．     

Separation of peptides by pressurized capillary electrochromatography

A pressurized electrochromatography (pCEC) instrument with gradient capability was used in this work for separation of peptides. Three separation modes, namely, pCEC, high-performance liquid chromatography and capillary electrophoresiscan be carried out with the instrument. In pCEC mode, the mobile phase is driven by both electroosmotic flow and pressurized flow, facilitating fine-tuning in selectivity of neutral and charged species. A continuous gradient elution can be carried out conveniently on this instrument, which demonstrates that it is more powerful than isocratic pCEC for separation of complicated samples. The effects of applied voltage, supplementary pressure and ion-pairing agents on separation of peptides in gradient pCEC were investigated. The effects of flow-rate of the pump and the volume of the mixer on resolution were also evaluated.     

亚微米C18键合硅胶固定相加压毛细管电色谱法同时拆分3种手性三唑类农药

采用改良Stöber法制备420 nm亚微米单分散二氧化硅微球,采用C18硅烷化修饰后装填成毛细管色谱柱。采用该色谱柱,在加压毛细管电色谱平台上成功地实现了3对手性三唑类农药烯效唑、烯唑醇和丙环唑的同时拆分和分离。考察了各因素对手性分离效果的影响,优化后的色谱条件为：流动相为乙腈-20 mmol/L磷酸盐缓冲液（pH=6.8）（45：55,v/v）,其中缓冲液中含20 mmol/L羟丙基-γ-环糊精（HP-γ-CD）;泵流速为0.04 mL/min;施加电压-9.4 kV;检测波长220 nm。在上述条件下,烯效唑、烯唑醇和丙环唑3种对映体同时得到拆分和分离,相邻两峰之间的分离度依次为4.20、12.9、4.41、4.09、1.70,分离时间仅为12 min,柱效最高达到310000 plates/m。该研究为手性三唑类农药的同时分离提供了新的分离分析思路。     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.     

多肽的反相梯度加压毛细管电色谱分离

以C18为固定相，采用电压和压力联合驱动流动相，研究反相加压毛细管电色谱分离多肽；考察了加压电色谱中，电压对带电和中性物质迁移的影响，实现了梯度加压毛细管电色谱分离6种多肽；结果表明，加压电色谱可以很好地抑制气泡形成，实验结果准确，重复性好；梯度加压毛细管电色谱在复杂样品的分离分析中，具有很大的潜力。     

Rapid separation and determination of microcystins using monolithic columns in isocratic elution mode by pressurized capillary electrochromatography

As a new wave of technology, polymethacrylate-based monolithic column was prepared and its application in the separation of three kinds of microcystins (MCs) in pressurized capillary electrochromatography with ultraviolet detection was studied. The key factors affecting the separation performance, such as monolithic column, pressure of the pump, component and concentration of mobile phase and the voltage, were investigated and optimized in detail. A baseline separation could be achieved in less than 6 min using a 5 mM borate buffer with a pH of 9.6 and 10% acetonitrile as the mobile phases in isocratic elution, under a voltage of +13 kV and a supplementary pressure of 7.5 MPa. The calibration curves were linear with correlation coefficient r>0.998 over a range of 0.10-25.00 mg/L. The LODs for the three MCs were in the range of 0.03-0.09 mg/L. This method was successfully applied to separate MCs from other compounds in spiked tap water after solid-phase extraction. The lower LODs for MC-LR, MC-YR and MC-RR were obtained to be 0.10, 0.13, 0.16 microg/L, respectively. These results make it clear that this proposed system is accurate and robust enough to be used as a fast separation tool for routine monitoring of MCs in real water samples.     

Separation and determination of six catechins in tea by pressurized capillary electrochromatography

An efficient pressurized capillary electrochromatography (pCEC) method has been successfully developed for the determination of six catechins in tea. The separation was performed on a reversed-phase EP-100-20/45-3-C18 capillary column (total length of 45?cm, effective length of 20?cm, diameter of 100?μm, ODS packing inside for 3?μm). The mobile phase ratio of organic phase, the concentration of phosphate buffer and sodium heptanesulfonate, separation voltage, and other experimental conditions were investigated and optimized. The mobile phase was 15?mM NaH₂PO₄ and 12?mM sodium heptanesulfonate (pH 3.0)/methanol (64:36) at a flow rate of 0.04?mL/min. Under optimal conditions including applied voltage of ?4?kV and a UV detection wavelength of 230?nm, the six catechins in the tea were well separated. The calibration curves for the analytes had good linearity in the range of 8.02?μg/mL–202.13?μg/mL with a correlation coefficient of 0.9928–0.9997. The limits of detection (LOD) for the six catechins were 4.62?μg/mL–11.63?μg/mL (S/N?=?3). The recoveries of the six catechins were 96.2%–108.4% with a relative standard deviation (RSD) between 0.78% and 4.51%. The method has been used for the determination of six catechins in tea samples with good results.     

Development of a quality evaluation method for Fructus schisandrae by pressurized capillary electrochromatography

A pressurized CEC (pCEC) method with postcolumn detection cell had been developed for quantifying the lignans from Fructus schisandrae extracts. The effects of different experimental conditions, such as the ACN content of the mobile phase, the concentration and pH of the buffer, the applied voltage, and the supplementary pressure were studied. Five lignans (schisandrin, gomisin A, schisantherin C, deoxyschizandrin, schisandrin B) were baseline separated using a mobile phase of ACN-phosphate buffer (pH 5.4; 5 mM) (40:60 v/v) under -4 kV applied voltage. The method showed the satisfactory retention time and peak area repeatability. The calibration curves were linear in the range 50.0-1000.0 microg/mL for schisandrin, 20.0-500.0 microg/mL for gomisin A, 10.0-200.0 microg/mL for schisantherin C, 20.0-500.0 microg/mL for deoxyschizandrin, and 20.0-500.0 microg/mL for schisandrin B. The correlation coefficients were between 0.9978 and 0.9989. With this pCEC system, fingerprints of F. schisandrae were preliminarily established to distinguish two members S. chinensis (Turcz.) Baill. and S. sphenanthera Rehd. Et Wils. of F. schisandrae by characteristic peaks, and evaluate the quality of various sources of raw materials by determining the contents of the five lignans.     

Pressurized CEC coupled with QTOF‐MS for urinary metabolomics

Pressurized CEC (pCEC) coupled with ESI‐QTOF‐MS using a sheathless interface was applied for metabolomics to develop an alternative analytical method for metabolic profiling of complex biofluid samples such as urine. The hyphenated system was investigated with mixed standards and pooled urine samples to evaluate its precision, repeatability, linearity, sensitivity, and selectivity. The applied voltage, mobile phase, and gradient elution were optimized and applied for the analysis of urinary metabolites. Multivariate data analysis was subsequently performed and used to distinguish lung cancer patients from healthy controls successfully. High separation efficiency has been achieved in pCEC due to the EOF. For metabolite identification, the pCEC‐MS separation mechnism was helpful for discriminating the fragment ions of glutamine conjugates from co‐eluted metabolites. Three glutamine conjugates, including phenylacetylglutamine, acylglutamine C8:1, and acylglutamine C6:1 were identified among 16 differential urinary metabolites of lung cancer. Receiver‐operating‐characteristic analysis of acylglutamine C8:1 resulted in an area‐under‐curve value of 0.882. Overall, this work suggests that this pCEC‐ESI‐QTOF‐MS method may provide a novel and useful platform for metabolomic studies due to its superior separation and identification.     

亲水作用毛细管整体柱的制备及其用于奶制品中三聚氰胺的加压毛细管电色谱分析

以甲基丙烯酸丁酯(BMA)和3-［N,N-二甲基-［2-(2-甲基丙-2-烯酰氧基)乙基］铵］丙烷-1-磺酸内盐(SPE)为单体,制备了新型的亲水作用毛细管整体柱,并通过三聚氰胺在此柱上的保留行为证明其具有亲水性。以加压毛细管电色谱(pCEC)技术为平台,优化了整体柱基于亲水作用分离分析奶制品中三聚氰胺的色谱条件。当流动相中乙腈与10 mmol/L磷酸盐缓冲液的体积比为80:20, pH为3.0,电压为3 kV,检测波长为215 nm时,三聚氰胺能获得很好的分离。方法学考察结果表明,合成的亲水整体柱具有良好的重现性和渗透性,建立的pCEC分析方法的检出限为0.05 mg/L。该方法简单方便,回收率较高,而且流动相中无需添加离子对试剂,适合于奶制品中三聚氰胺的定量测定。     

Development and validation of a capillary electrophoresis method for the determination of escitalopram and sensitive quantification of its enantiomeric impurity in formulations

A rapid capillary zone electrophoresis method has been developed capable of quantifying 0.05% of R-enantiomer and assaying the main component in escitalopram formulations. Many parameters influencing enantioseparation were investigated, which include chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature, and rinsing procedure. Optimal separation conditions were obtained by using a 25 mM phosphate buffer at pH 7.0, containing 1.6% (w/v) sulfated-β-cyclodextrin with short-end injection at 0.5 psi for 5 s. Online UV detection was performed at 205 nm. A voltage of -20 kV was applied and the capillary temperature was kept at 25°C. Separation was achieved in less than 2 min. The method was further validated, including robustness, stability of the solution, selectivity, linearity (escitalopram from 0.25 μg/mL to 600 μg/mL, y = 1528.3 × +1812.9; R2 = 0.9999), LOD and LOQ (0.08 and 0.25 μg/mL, respectively), precision and accuracy. The proposed method was then applied to the quality control of the bulk sample and tablets of escitalopram (10 mg).     

Purity assessment of recombinant human granulocyte colony‐stimulating factor in finished drug product by capillary zone electrophoresis

Current methods for determination of impurities with different charge‐to‐volume ratio are limited especially in terms of sensitivity and precision. The main goal of this research was to establish a quantitative method for determination of impurities with charges differing from that of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF, filgrastim) with superior precision and sensitivity compared to existing methods. A CZE method has been developed, optimized, and validated for a purity assessment of filgrastim in liquid pharmaceutical formulations. Optimal separation of filgrastim from the related impurities with different charges was achieved on a 50 μm id fused‐silica capillary of a total length of 80.5 cm. A BGE that contains 100 mM phosphoric acid adjusted to pH 7.0 with triethanolamine was used. The applied voltage was 20 kV while the temperature was maintained at 25°C. UV detection was set to 200 nm. Method was validated in terms of selectivity/specificity, linearity, precision, LOD, LOQ, stability, and robustness. Linearity was observed in the concentration range of 6–600 μg/mL and the LOQ was determined to be 0.3% relative to the concentration of filgrastim of 0.6 mg/mL. Other validation parameters were also found to be acceptable; thus the method was successfully applied for a quantitative purity assessment of filgrastim in a finished drug product.     

咖啡因及其9种类似物的胶束电动毛细管分析研究

 以十二烷基硫酸钠 (SDS)胶束为准固定相 ,考察了咖啡因及其 9种类似物在胶束电动毛细管 (MECC)分离模式下的分离行为。研究了运行缓冲液的 pH值、磷酸盐浓度、SDS浓度、甲醇体积分数、分离电压、分离温度等因素对这 10种化合物的迁移时间和分离效果的影响。结果发现 ,这些因素对上述 10种化合物的分离有显著的影响 ,尤以pH值为最。它不仅影响化合物的迁移时间和分离效率 ,还改变其出峰顺序 ,这与碱性条件下化合物仲胺基上氢的电离有关。优化后的分离条件 :运行缓冲液为 2 0mmol/L磷酸盐 2 0mmol/LSDS(pH 11 0 ) ,分离电压为2 5kV。     

Development and validation of a robust capillary electrophoresis method for impurity profiling of calcium levofolinate including the (6R,2'S)-diastereomer using statistical experimental design

A chiral capillary electrophoresis assay for the simultaneous determination of the optical purity and of related substances of calcium levofolinate has been developed and validated. Using 2,6-dimethyl-beta-cyclodextrin as chiral selector at a concentration of 20 mg/mL, the method was optimized using a full factorial design with four factors including pH and concentration of the background electrolyte, column temperature and separation voltage. Optimized conditions were a 40 mM sodium tetraborate buffer, pH 9.9, a capillary temperature of 16 degrees C, and an applied voltage of 21 kV. Methotrexate was used as internal standard to compensate for injection errors and fluctuations of the migration times. A multiple linear regression model was also used to test the robustness of the method. Validation was performed with respect to specificity, linearity, range, limit of quantification and detection, precision, and accuracy. The assay allowed the detection and determination of related substances including the diastereomeric (6R,2'S)-impurity of levofolinic acid at the 0.1% level, the identification threshold of impurities for orally administered drugs for human use defined by the International Conference on Harmonization guidelines as well as the European Pharmacopoeia.     

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.     

1 μm硅胶颗粒固定相的加压电色谱分离性能

制备了1 μm无孔硅胶颗粒。通过电动填充法得到总长度为45 cm(固定相填充长度为20 cm)、内径为100 μm的毛细管色谱柱。以乙腈-水体系作为流动相,详细考察了碱性化合物在该色谱柱上的加压电色谱(pCEC)分离性能,讨论了流动相比例、缓冲液浓度、pH值及操作电压等因素对分离的影响。实验结果表明,裸硅胶柱在乙腈-水体系分离碱性样品中表现出典型的反相色谱分离性能;缓冲液浓度的改变则对分离影响不大。当pH值改变时,碱性化合物的解离程度发生变化,它们与固定相之间的作用力发生变化,使得分离度发生相应的变化。分离柱效随施加电压的增加而增加,在1 kV电压下,裸硅胶柱对邻甲苯胺的柱效为35000理论塔板/m。