Chiral separation of dioxopromethazine in eye drops by CZE with charged cyclodextrin

Capillary zone electrophoresis (CZE) with carboxyethyl-beta-cyclodextrin (CE-beta-CD) dissolved in the operating buffer was used for the separation and determination of enantiomers of phenothiazine antihistaminic, dioxopromethazine, in commercial pharmaceutical preparation, eye drops. This chiral selector, negatively charged under given separating conditions (20 mmol/l epsilon -aminocaproic acid, acetic acid, pH 4.5), was effective in enantioresolution of the antihistamine even at its low concentrations (3-6 mg/ml) in the buffer solution. CZE identification and quantitation of the relevant constituents present in the preparation (dioxopromethazine enantiomers, phenylephrine) were based on the response of photometric absorbency detector, operating at a 275 nm detection wavelength. Changes in pH, type and concentration of chiral selector were studied in relation to chiral resolution. Acceptable validation criteria for sensitivity, precision, linearity and repeatability are included.     

胆汁酸类化合物的毛细管区带电泳法分析

目的建立毛细管区带电泳法分离胆汁酸类化合物。方法缓冲液含硼砂126 mmol·L^-1、磷酸氢二钠43 mmol·L^-1,18%甲醇,磷酸调至pH 9.3。毛细管柱570 mm×0.05 mm(有效长度500 mm),操作电压30 kV,温度30℃,检测波长200 nm,压力进样,进样量5 kPa×8 s。结果30 min内一次性分离了8种结合型和游离型胆汁酸。加样回收率89%～107%。结论本法简便,可用于熊胆中胆汁酸的测定。     

PEG conjugates in clinical development or use as anticancer agents: An overview

During the almost forty years of PEGylation, several antitumour agents, either proteins, peptides or low molecular weight drugs, have been considered for polymer conjugation but only few entered clinical phase studies. The results from the first clinical trials have shared and improved the knowledge on biodistribution, clearance, mechanism of action and stability of a polymer conjugate in vivo. This has helped to design conjugates with improved features. So far, most of the PEG conjugates comprise of a protein, which in the native form has serious shortcomings that limit the full exploitation of its therapeutic action. The main issues can be short in vivo half-life, instability towards degrading enzymes or immunogenicity. PEGylation proved to be effective in shielding sensitive sites at the protein surface, such as antigenic epitopes and enzymatic degradable sequences, as well as in prolonging the drug half-life by decreasing the kidney clearance. In this review PEG conjugates of proteins or low molecular weight drugs, in clinical development or use as anticancer agents, will be taken into consideration. In the case of PEG-protein derivatives the most represented are depleting enzymes, which act by degrading amino acids essential for cancer cells. Interestingly, PEGylated conjugates have been also considered as adjuvant therapy in many standard anticancer protocols, in this regard the case of PEG-G-CSF and PEG-interferons will be presented.     

Quantitative analysis of fosinopril sodium by capillary zone electrophoresis and liquid chromatography

A capillary zone electrophoresis (CZE) method was developed for the quantitative analysis of fosinopril sodium. Validation parameters of the CZE method were evaluated and compared to an existing LC method. In terms of precision and sensitivity, LC performance was superior to that of the CZE method for this application. The CZE method achieved better selectivity for several degradants of interest within a much shorter analysis time than did the LC method. Effects of detection wavelength, applied voltage and buffer concentration on optimization of the CZE method are presented. Effects of diluent composition on capillary loading and peak behaviour are also discussed.     

Choice of capillary electrophoresis systems for the impurity profiling of drugs

In order to develop a strategy for the impurity profiling of drugs, the possibilities of some capillary electrophoresis systems were investigated. A mixture containing a drug and some of its possible impurities has been used as a model problem. The test compounds were investigated by capillary zone electrophoresis (CZE) and by micellar electrokinetic chromatography (MEKC). The pH of the CZE buffer was varied, but the two stereoisomers could not be separated. Moreover, CZE is not suitable for neutral compounds. In MEKC, two different types of surfactants, sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB), have been used and the effect of type and concentration modifier on the separation and the elution window was studied. In the SDS system, both the resolution and the elution window could be increased considerably by the addition of modifier. The use of two MEKC systems of different selectivity seems to be a combination with high potential for the impurity profiling of drugs.     

Characterization of recombinant human granulocyte colony stimulating factor (rHuG-CSF) by capillary zone electrophoresis, capillary isoelectric focusing electrophoresis and electrospray ionization mass spectrometry

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) is a hematopietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils. Glycosylated and non-glycosylated forms of rHuG-CSF cannot be distinguished by traditional biological assays. In addition, it is very difficult to characterize impurities of the same molecular weight in biologicals. In this study, non-glycosylated rHuG-CSF, two glycosylated rHuG-CSF isoforms and their commercial dosages were successfully separated by capillary zone electrophoresis (CZE) using 50mM Tricine containing 20mM NaCl and 2.5mM 1,4-diaminobutane (DAB) at pH 8.0, which could be employed for the qualitative discrimination assay of rHuG-CSF related products. CZE, capillary isoelectric focusing electrophoresis (CIEF), and mass spectrometry (MS) were used to effectively characterize non-glycosylated rHuG-CSF. It was found that proteins in the samples with different pIs in the CIEF profile could not be detected by CZE, while no difference was observed between these proteins and rHuG-CSF. Further analysis by electrospray ionization mass spectrometry with the resolution of 2000 showed that the components with different pIs in the non-glycosylated rHuG-CSF bulk sample are nearly equal in molecular weight. Therefore, it is necessary to combine several modern analytical techniques for quality control to get well-characterized biologicals.     

Capillary zone electrophoresis characterization of low molecular weight heparin binding to interleukin 2

A method based on capillary zone electrophoresis (CZE) was used to study the interaction between low molecular weight heparin (LMWH) and interleukin 2 (IL-2). The results showed that the increase of the concentration of LMWH led to the decrease of the peak height and the increase of the peak width of IL-2, but the peak areas were kept constant. The binding constant of IL-2 with LMWH was calculated as 1.2 x 10(6)M(-1) by Scatchard analysis, which is in good agreement with the results found in the references using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the interaction between IL-2 and LMWH is of fast on-and-off kinetic binding reaction. CZE might be used to study not only slow on-and-off rates interactions, but also fast on-and-off rates ones. The binding constant can be calculated easily, and the method can be applied to study a wide range of heparin-protein interactions.     

How to evaluate the separation efficiency of CZE methods?

In trying to estimate the separation efficiency of Capillary Electrophoresis (CE) methods, the resolution (RS), the number of theoretical plates (N) and the peak-to-valley ratio (p/v) are often used assessment criteria. This study demonstrates that these criteria are not as suitable to describe the separation efficiency in case of Capillary Zone Electrophoresis (CZE) methods as they are for Liquid Chromatography (LC) methods. The investigations were performed by means of a validated CZE method for the evaluation of tetracyclines and their related substances. Four impurities of tetracycline hydrochloride are described in the European Pharmacopoeia. Three were found in the sample used for our investigations, i.e. epi-tetracycline formed by keto-enol-tautomerism, anhydrotetracyclin and epi-anhydrotetracyline. It could be shown that higher values of these assessment criteria like RS do not necessarily represent better separation. Thus, a discussion on the usefulness of separation selectivity and efficiency as assessment criteria for capillary electrophoresis as well as on the introduction of additional parameters is needed.     

重组人粒细胞集落刺激因子肽谱分析方法初探

建立检测重组人粒细胞集落刺激因子肽谱的方法。方法：采用ＲＰ－ＨＰＬＣ法及ＣＺＥ法。结果：检测ｒｈＧ－ＣＳＦ经胰蛋白酶解后的肽段,发现九源公司生产的各批产品的一级结构具有一致性。结论本法适用于基因工程药物的肽谱分析。     

Potential of capillary electrophoresis for the monitoring of the stability of placental alkaline phosphatase

Alkaline phosphatase (AP) is a potential therapeutic agent in the treatment of sepsis. In this paper the potential of capillary zone electrophoresis (CZE) for the monitoring of the degradation of placental alkaline phosphatase (PLAP) was investigated. To induce degradation PLAP samples were exposed to high temperatures, low and high pH and freeze-drying. The samples were then analyzed by CZE and enzymatic activity assay. Upon exposure to temperatures above 65 degrees C, PLAP lost its activity exponentially over time, while CZE revealed both a linear decrease of the area of the main peak and a rise of degradation products. At acidic pH the enzyme appeared to lose its activity. CZE revealed a decrease of the area of the main peak, but no degradation products could be detected. At pH 12 the enzymatic activity and the area of the main peak both decreased linearly over time and, in addition, formation of degradation products could be detected by CZE. Activity and CZE profile of PLAP remained unchanged upon freeze-drying in the presence of inulin. Prolonged storage of freeze-dried samples at room temperature caused a slight decrease of enzymatic activity, while the potential formation of oligomers was revealed by CZE analysis. The examples in this study show that, in combination with activity assays, CZE can provide useful complementary information, especially on the status of the protein and the presence of degradation products.     

头孢呋辛对映体的毛细管区带电泳分离方法研究

目的 :建立以毛细管区带电泳法分离头孢呋辛对映体的方法。方法 :采用熔融毛细管柱 (570mm×75μm ) ,缓冲液为加入羟丙基 -β-环糊精 (0 04mmol/L)的磷酸氢二钠溶液 (37 5mmol/L) ,pH为6 0 ,操作电压为15kV ,温度为25℃ ,检测波长为280nm ,压力进样 (进样量为5kPa×6s)。结果 :头孢呋辛对映体在10min内分离 ,方法加样回收率为90 %～106 %。结论 :本法操作简便 ,可用于测定头孢呋辛对映体含量。     

重组人胰岛素V8酶肽图谱的毛细管区带电泳分析

目的：建立重组人胰岛素Ｖ８酶肽图谱的毛细管电泳分析方法。方法：利用毛细管区带电泳分离模式,采用熔凝石英毛细管柱,检测了重组人胰岛素经Ｖ８蛋白酶解所产生的５个肽片段;比较了重组与天然人胰岛素的Ｖ８蛋白酶肽图谱;鉴定了毛细管区带电泳和反相高效液相色谱的肽图谱中各峰的对应关系。结果：重组人胰岛素与天然人胰岛素的分子结构具有同一性;毛细管区带电泳和反相高效液叮色谱２种方法具有不同的分离机理。结论：毛细管区     

Analysis and determination of biological activity of short-chain peptides from porcine brain hydrolysate

Gel permeation chromatography fractions of short-chain peptides from a hydrolysate product, which in turn was from the purified porcine brain through enzyme hydrolysis, were tested for their biological activities. The results showed that the fractions A4 and A5 had significant biological activities. The two fractions were analyzed with analytical techniques such as high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preliminary results showed that the main components of these two fractions were short-chain acidic peptides with a relative molecular mass (M(r)) of less than 2400.     

Capillary electrophoresis: a new analytical tool for forensic toxicologists

In capillary electrophoresis, electrophoretic or electrokinetic separations are carried out in tiny capillaries at high voltages (10-30 kV), thus achieving high efficiency (N > 105), resolution power, and mass sensitivity (down to 10(-18)-10(-20) moles). The main characteristics of capillary electrophoresis are versatility of application (from inorganic ions to large DNA fragments), use of different separation modes with different selectivity, low demands on sample volume, negligible running costs, possibility of interfacing with different detection systems including mass spectrometry, and the ruggedness and simplicity of the instrumentation. Capillary electrophoresis applications in the forensic sciences are now rapidly growing, particularly in forensic toxicology. The present paper briefly describes the basic principles of capillary electrophoresis and presents a selected review of its main applications to the analysis of illicit/controlled drugs in biologic samples. An original analytical approach to the determination of carbohydrate deficient transferrin, a new marker of chronic alcohol abuse, based on capillary electrophoresis is also described. It is concluded that the peculiar separation mechanisms and the high complementarity of capillary electrophoresis to chromatography make it a new powerful tool of investigation in the hands of forensic toxicologists.     

通宣理肺丸毛细管电泳指纹图谱研究

目的建立通宣理肺丸（TXLFP）毛细管区带电泳指纹图谱。方法以色谱分离数（TZ）、指纹分离信息量指数（RI）作为评价的目标函数选取最优试验条件。以系统指纹定量法评价14批TXLFP的质量,并与HPLC条件下试验结果比较。结果结果 2种方法均可有效的判定TXLFP质量。结论毛细管电泳可作为HPLC方法的补充,为TXLFP的质量判定提供一种新方法 。     

用SDS—填充胶毛细管电泳法测定蛋白质分子量

目的：建立蛋白质分子量测定的毛细管电泳方法。方法：以葡聚糖为分离介质,线性聚丙烯栈胺涂渍毛细管柱为支持介质,柱上２１４ｎｍ检测,进行蛋白质分离。结果：被分离的蛋白质分子量的对数与其相对迁移率之间具有良好的线性关系（ｒ＝０．９９７）;涂渍毛细管柱可以使用多达１００次面未出现分离效率的降低;同一毛细管柱和不同毛细管柱迁移时间的ＲＳＤ分别小于１．０％和１．３％;用十二烷基硫酸钠－毛细管胶电泳（ＳＤＳ－Ｃ     

三氟乙酰伯氨喹及其合成前体伯氨喹的高效毛细管电泳分析

目的：建立适合抗疟新药三氟乙酰伯氨喹及其合成前体伯氨喹的高效毛细管电泳定量检测方法。方法：在Ｂｉｏ－ＲａｄＨＰＥ－１００毛细管电泳仪上,以自由区带电泳分离模式探讨电泳体系各因素对伯氨喹及三氟乙酰伯氨喹分离的影响;并选择甲基麻黄素为内标,柱上１０ｎｍ检测,进行定性定量分析。结果：伯氨喹、三氟乙酰伯氨喹在测定浓度范围内,峰经与被测物浓度的线性关系良好。重现性考察结果表明,６次测量迁移时间的相对标准差小     

Capillary electrophoretic separation, immunochemical recognition and analysis of the diastereomers quinine and quinidine and two quinidine metabolites in body fluids

The capillary electrophoretic separation and immunochemical recognition of the two naturally fluorescing, cationic diastereomers quinine (QN) and quinidine (QD), their hydroderivatives and two major QD metabolites (3-hydroxyquinidine and quinidine-N-oxide) was investigated. Plain aqueous phosphate buffers and an alkaline buffer containing dodecyl sulfate micelles are shown to be incapable of resolving the two diastereomers. However, incorporation of an additional chemical equilibrium (with β-cyclodextrin) in the case of capillary zone electrophoresis (CZE) and the presence of a small amount of an organic solvent as buffer modifier (2-propanol) in dodecyl sulfate based micellar electrokinetic capillary chromatography (MECC), were found to provide separation media which lead to complete resolution of QN, QD and the other compounds of interest. Furthermore, for MECC- and CZE-based immunoassay formats, a commercially available antibody against QD was found to be a perfect discriminator between QD and QN. It was determined to recognize QD and the two QD metabolites (cross reactivity of 20–30%) but not QN. MECC and CZE with laser induced fluorescence (LIF) detection are shown to be suitable to determine QD and metabolites in urine and plasma (quinidine-N-oxide only) collected after single dose intake of 50 mg QD sulfate and of QN in urine, saliva and serum samples that were collected after self-administration of 0.5 l of quinine water (25 mg of QN). With direct injection of a body fluid, MECC with LIF was found to provide 10 ng/ml detection limits for QD and QN. This ppb sensitivity is comparable to that obtained in HPLC assays that are based upon drug extraction. Furthermore, MECC and CZE assays with UV detection are shown to provide the ppm sensitivity required for therapeutic drug monitoring and clinical toxicology of QD and QN.     

水和非水毛细管电泳-电导检测法分离测定水杨酸类药物

目的建立水和非水毛细管电泳-电导法分离水杨酸类药物。方法用未涂层石英毛细管柱(55 cm×50 μm),以10 mmol·L^-1 Tris-30 mmol·L^-1 H₃BO₃(pH 8.0)为运行缓冲液,分离电压为24 kV,进样时间10 s,电导检测法。结果在非水实验条件下,水杨酸(SA)、乙酰水杨酸(ASA)和磺基水杨酸(SSA)得到很好的分离。SA,ASA和SSA的线性范围分别为0.05～100 mg·L^-1,5.0～250 mg·L^-1,0.08～100 mg·L^-1,r均大于0.995。结论应用于阿斯匹林制剂中水杨酸和乙酰水杨酸含量的测定,结果令人满意。与水介质相比,乙醇介质具有更高的灵敏度和分离效率。     

Novel Eco-Friendly Stability Indicating Capillary Zone Electrophoresis Method for Determination of Aripiprazole in Tablet Dosage form: DoE Directed Optimization,Development and Method Validation

In this work, a novel environment-friendly stability indicating capillary zone electrophoresis (CZE) method has been developed and validated for assaying the aripiprazole (ARP) in tablet dosage form. The separation of ARP from its degradation products and internal standard was achieved using a fused silica capillary column (30.2 cm x 75 μm ID), a background electrolyte containing 6 mmol L^?1 ammonium formate buffer (pH 3) with 5% methanol under a potential of 15 kV and detection at 214 nm. The stability indicating ability of the method was investigated by analyzing ARP after being subjected to acidic, alkaline, thermal, photolytic, and oxidative stress conditions, according to ICH guidelines. Design of experiments was used during forced degradation and method optimization. Oxidation was the main degradation pathway among those evaluated. The drug was separated from its oxidative degradation products in less than 4 min. CZE method was linear between 60 – 140 μg mL^?1, R² = 0.9980, precise (intra-day 0.88% and inter-day 1.30%). The average recovery was 100.93 ± 0.77%. This is the first method in the literature for quantification of ARP in the presence of its related degradation products with high separation efficiency, low operation cost and minimum solvent consumption. This method could be helpful in the routine quality control analysis in the pharmaceutical industries with least harmful effect on the environment. CZE is considered an eco-friendly alternative of conventionally HPLC methods.