Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

Another method has been developed for the separation of amino acids (1 min derivatization plus 22 min separation) by micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection. Interestingly enough, such work has never been performed on essential amino acids derivatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Fifteen L-amino acid standards were labelled with NBD-F at 60 degrees C for 1 min, and separated in a buffer system containing 20 mM borate, 25 mM sodium cholate, 10 mM Brij 35 and 2.5% methanol. Methanol was employed to expand the MECC migration time window; whereas Brij 35 was used to improve the fluorescence intensity of amino acid derivatives. This method also indicates that bile salt is effective for MECC separation of ionic analytes. Surprising though, improvements in resolution, sensitivity and speed for amino acids analysis are obtained in this work, which are not initially apparent in just employing another derivatizing reagent. Under optimal conditions, 15 amino acids were separated in a short 22 min analysis time, the shortest ever reported, and detection limits of nanomolar concentration and attomole mass were obtained. Furthermore, RSDs of migration time and peak height are better than 1% and 1.8%, respectively, again the smallest ever reported in the literature.     

磷脂的分离纯化及高效毛细管电泳分析

采用溶剂提取和柱色谱法分离纯化市售大豆粉末磷脂(卵磷脂含量14.05%),得到高纯度的卵磷脂产品(纯度92.80%)。重点建立了磷脂的胶束电动毛细管色谱(MECC)分离分析方法。以分离度和峰面积为优化指标,对表面活性剂及其浓度、电泳缓冲液pH、有机改性剂及其含量、缓冲液浓度、温度等条件进行优化,确定了最优化电泳条件:电泳缓冲液为35 mmol/L脱氧胆酸钠-1 mmol/L 硼砂缓冲液/正丙醇(体积比为57∶43)(pH 8.30),柱温44 ℃,操作电压25 kV,检测波长200 nm;内加法定性磷脂组分;外标法定量卵磷脂。结果表明,MECC法能有效分离5种磷脂组分;0.1～1 g/L的质量浓度范围内卵磷脂的线性关系良好(r=0.9990),平均回收率为98.0%,日内、日间精密度分别为1.36%和3.27%,定性结果与薄层色谱法、红外光谱法的定性结果相符。     

Identification and determination of active components in Angelica dahurica Benth and its medicinal preparation by capillary electrophoresis

A micellar electrokinetic capillary chromatography (MECC) method for the separation of three active components, imperatorin (IM), isoimperatorin (IO) and scopoletin (SC), in Angelica dahurica Benth and its preparation was developed. The optimum separation for these analytes was achieved using 10 mM borate, 20 mM SDS and 10%(v/v) acetonitrile, with applied voltage of 22 kV. All experiments were performed using a 50.0 cm (42.5 cm effective length)x75 mum I.D. fused-silica capillary. The linear calibration range was 12.0-800.0 mug ml(-1) for IM, 10.0-850.0 mug ml(-1) for IO and 3.5-600.0 mug ml(-1) for SC. The recoveries of three constituents were from 91.3 to 108.7%. Also, the dissociation constant (pKa) of SC was determined by the CE method established in this paper.     

不同剂型药用抑肽酶纯度的胶束电动毛细管测定

 以十六烷基三甲基溴化铵 (CTAB)为阳离子表面活性剂 ,用胶束电动毛细管 (MECC)分别对抑肽酶粉针剂和抑肽酶注射液进行纯度测定。实验中选择了最佳缓冲液 (含 4mmol/LCTAB的 80mmol/LNa2 HPO4 H3 PO4,pH 7 0 0 ) ,考察了进样量与样品中高浓度盐对分离的影响。并对毛细管区带电泳、MECC和高效液相的分离效果加以比较 ,表明MECC的分离效果最佳。     

胶束电动毛细管色谱法测定消糖灵中的优降糖

 以甲氧苄氨嘧啶为内标,采用胶束电动毛细管色谱法分离测定了新药消糖灵中的优降糖。电泳条件：以25 mmol/L硼砂-30 mmol/L十二烷基硫酸钠（pH 9.0）为电泳介质,未涂层石英毛细管（50 μm i.d.×39.5 cm,有效分离长度34.8 cm）为分离通道,压力进样(68.95 kPa.s),17 kV恒压电泳（28 ℃）,检测波长228 nm。优降糖在14 min内与其他成分得到很好分离,且质量浓度为25 mg/L～275 mg/L时,优降糖可进行定量分析,加标回收率为(100.6±1.4)%。方法简便、快速,结果准确,重现性好,可用于优降糖复方制剂的质量控制。     

Examination of colour inkjet printing inks by capillary electrophoresis

The possibility of comparing inkjet printing inks by micellar electrokinetic capillary electrophoresis (MECC) with diode array detection was studied. An analytical procedure was designed and successfully applied to discriminate between the electrophoretic profiles of inks (extracted from paper) produced by five well-known manufacturers. The separation process was conducted in a polyimide-coated fused silica capillary (ID 50 μm, 60 cm total/50 cm effective length) with +30 kV high voltage applied. Background electrolyte was used of the following optimum composition: 40 mM sodium borate buffer, 20 mM sodium dodecyl sulphate(IV) (SDS) and 10% (v/v) acetonitrile (pH 9.56). The experimental conditions were adjusted in terms of resolution and analysis time. The best results were obtained at 10 and 25 °C storage and capillary temperature, respectively, using 25 dots (ø 0.8 mm) cut from printouts as the sample and BGE diluted with water (1:99, v/v) as the injecting solution. The MECC separation of main printing ink components by the proposed method showed excellent precision - the RSD value of the migration time calculated for each of the investigated peaks did not exceed 3.3%. The optimized method was applied to group identification and differentiation of: (a) three colours of printing inks, (b) inks from different manufacturers (Hewlett-Packard, Epson, Brother, Lexmark and Canon) and (c) inks from different printer models. In all these cases, inks were successfully differentiated on the basis of position (migration time) and shape of their characteristic peaks.     

Validation and Further Optimisation of a Cyclodextrin-Modified Micellar Electrokinetic Capillary Chromatography Method for Urine Profiling

Metabonomic studies require efficient and high-resolution analytical probes to monitor changes in the ‘metabolic fingerprint’. The advantageous characteristics of Capillary Electrophoresis, enabling highly efficient separations of diverse components present in minute sample volumes, may therefore prove a useful tool in biofluid analysis. This paper describes the optimisation and validation of a sulphated β-cyclodextrin-modified MECC method for urine profiling. Cyclodextrin substitution, experimental conditions including capillary length, injection mode and time, applied voltage, temperature and capillary pre-conditioning procedures were investigated and optimised. Precision, linearity, sensitivity and robustness of the method were assessed, as well as urine stability. The validated sulphated β-cyclodextrin-modified MECC method allows for the separation of over 80 urinary analytes in under 25 min, using a sodium borate/SDS/sulfated β-cyclodextrin (25/75/6.25 mM) electrolyte and an 18 kV applied voltage in a 40 cm (effective length), 50 μm i.d. fused silica capillary at 20 °C, pre-conditioned with HCl for 5 min and BGE for 1 min, and UV diode array detection (190–600 nm). Such methodology should prove invaluable in the rapid comparison of urine profiles and indication of metabolic disorders or abnormalities.     

Simultaneous Separation and Determination of Benzoic Acid Compounds in the Plant Medicine by High Performance Capillary Electrophoresis

A simple and inexpensive high performance capillary electrophoresis (HPCE) was applied to separate five benzoic acid compounds simultaneously. The investigation was carried out by micellar electrokinetic capillary chromatography (MECC). To avoid a time‐consuming and tedious procedure, orthogonal experimental design OA₉ (3⁴) for separation experiments was applied to find the optimal conditions in terms of the resolution and analytical time. The best conditions for separation were obtained using a 20 mM borax and 30 mM sodium dodecyl sulfate (SDS) buffer (pH 9.8) containing 2 mM β‐CD and 4% methanol (v/v). Online UV detection was performed at 250 nm. A voltage of 16 kV was applied and the temperature was controlled at 25 °C. Injection was performed for 5 s. The method was validated for the quantification of benzoic acid, salicylic acid and ortho‐aminobenzoic acid in Radix Isatidis, a traditional plant medicine with removal of endotoxin. The separation and determination were satisfactory and quick.     

胶束电动毛细管色谱法测定海洋沉积物中的苯、甲苯和二甲苯

 建立了用胶束电动毛细管色谱法（MECC）对海洋沉积物中的苯、甲苯和二甲苯进行 分析测定的方法 。用45 cm（到检测窗口40 cm）×75　μm i.d.毛细管柱,以75　mmol/L SDS-2　mmol/L 四硼酸钠溶液（外加体积分数为20%的甲醇）为缓冲液（pH 9.16）。当电压为25 kV、检测 波长为200 nm时,苯、甲苯和二甲苯在20 min内得到了良好的分离。用峰面积定量,线性范 围为4～50 mg/L,相对标准偏差在6.2%以内。测得海洋沉积物中的这些苯系物的质量比范围 为3.79～17.36 μg/g。     

Determination of flavour components in natural vanilla extracts and synthetic flavourings by mixed micellar electrokinetic capillary chromatography

The development of a mixed micellar electrokinetic capillary chromatography (MECC) method for the qualitative and quantitative determination of key components, including vanillin, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid and 3-methoxybenzaldehyde, that contribute to vanilla flavour was investigated. The micellar phase consisted of sodium dodecyl sulfate (SDS) and sodium cholate (SC). The percent relative standard deviation (R.S.D.%) for migration time was <1 over six runs. The R.S.D.% for peak areas ranged between 0.85-1.96% over six runs. Peak efficiencies were excellent with theoretical plate numbers typically in the range of 130,000-200,000 per column (52 cm effective length). The limits of detection (LOD) were between 5-10 μg/ml. The quantitative data was verified by high performance liquid chromatography (HPLC) and gas chromatography (GC). The mixed MECC method was successfully applied to a number of natural vanilla extracts, nature identical extracts and synthetic flavourings.     

Determination of antibacterial flomoxef in serum by capillary electrophoresis

A determination method of flomoxef (FMOX) concentration in serum by capillary electrophoresis is developed. Serum samples are extracted with acetonitrile. After pretreatment, they are separated in a fused-silica capillary tube with a 25 mM borate buffer (pH 10.0) as a running buffer that contains 50mM sodium dodecyl sulfate. The FMOX and acetaminophen (internal standard) are detected by UV absorbance at 200 nm. Linearity (0-200 mg/L) is good, and the minimum limit of detection is 1.0 mg/L (S/N = 3). The relative standard deviations of intra- and interassay variability are 1.60-4.78% and 2.10-3.31%, respectively, and the recovery rate is 84-98%. This method can be used for determination of FMOX concentration in serum.     

Analysis of some cytokinins in coconut (Cocos nucifera L.) water by micellar electrokinetic capillary chromatography after solid-phase extraction

Micellar electrokinetic capillary chromatography (MECC) was developed for the separation of cytokinins including trans-zeatin, trans-zeatin-O-glucoside, dihydrozeatin, dihydrozeatin-O-glucoside, meta-topolin riboside, N6-isopentenyladenine and N6-benzylaminopurine. Under the optimum conditions, i.e. a combination of 10 mM phosphate and 10 mM borate as the running buffer containing 50 mM sodium dodecyl sulphate at pH 10.4, the separation of seven cytokinin standards was accomplished within 11 min. The C18 solid-phase extraction (SPE) method was used to pre-concentrate the putative cytokinins present in the coconut water. Following which, the eluate was further purified using mixed mode Oasis MCX SPE columns and this additional step helps to reduce matrix interference during MECC. After the two solid-phase extraction steps, the optimized MECC method was able to screen for certain cytokinins (zeatin-O-glucoside and dihydrozeatin-O-glucoside) present in coconut water. After this screening, the presence of zeatin-O-glucoside and dihydrozeatin-O-glucoside in coconut water was further confirmed by independent high-performance liquid chromatography and liquid chromatography-mass spectrometry experiments.     

Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis

A new capillary zone electrophoresis (CZE) method for the analysis of dipicolinic acid, a specific component found in spores but not in vegetative cells, was used to determine spore concentration in Bacillus thuringiensis according to the relationship between the spore concentration and the content of dipicolinate. The quantitative relationship was established by using purified spores. Electrolyte conditions that affected the separation efficiency of dipicolinate and the reproducibility were investigated. With 10 mM phosphate, 10 mM ethylenediaminetetraacetic acid and 0.25 mM tetradecyltrimethylammonium bromide at pH 6.2 as the carrier electrolyte, dipicolinate can be determined within 8 min at an applied voltage of -25 kV (anode at detector) and a capillary temperature of 25 degrees C. The method has a high separation efficiency with which the number of theoretical plates is above 300,000 plates m(-1). The relative standard deviations for migration time and peak area are less than 0.5% and 2.0%, respectively. The detection limit for dipicolinate was 10 ng ml(-1), which corresponds to 7.2 x 10(5) spores ml(-1). The method was used to determine spores in fermentation broths, and the results obtained agreed well with the values obtained by plate counting.     

Separation and determination of isoflavones in red clover by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method has been developed for the determination of the four isoflavones, i.e. biochanin A, formononetin, genstein and daidzein in red clover (Trifolium Pratense L.). The effect of running buffer pH and concentration were investigated. An electrolyte composed of 30 mm borate, 20 mm sodium dodecyl sulfate (SDS) and 4 mg/mL HP-beta-CD containing 5% (v/v) ethanol at pH 10.1 provides a satisfactory separation for all the analytes. The applied voltage was 25 kV, and the capillary temperature was kept constant at 25 degrees C with a UV detection at 254 nm. The relative standard deviations (RSD) of the migration time and peak area were less than 1.73 and 3.94% (intra-day), and 2.29 and 4.38% (inter-day), respectively, under the optimized separation conditions. Regression equations revealed a good linear relationship between the peak area of each compound and its concentration. The contents of the four compounds in red clover were successfully determined with satisfactory repeatability and recovery.     

寡糖的毛细管电泳分析

多种寡糖经α－萘胺衍生化后，用硼砂作为电泳介质，实现了高效毛细管电泳分离。比较了毛细管区带电泳和胶束毛细管电动色谱分离寡糖α－萘胺衍生物的电泳行为，对影响分离度的诸因素进行了考察，选择了最佳分离条件。     

Complimentary Role of Micellar Electrokinetic Capillary Chromatography and High Performance Liquid Chromatography in the Separation of Plant Phenolics

Abstract

A comparison was made between the efficiency of micellar electrokinetic capillary chromatography (MECC) and reversed phase high performance liquid chromatography (RP-HPLC) to separate a mixture of plant phenolics. Of a range of buffers 6 mM borate/10 mM phosphate/100 mM SDS at pH 8.5 was the most effective in separating a complex mixture of phenolics using MECC. Using this buffer the elution order and resolution was different from that obtained by HPLC using a reversed phase C18 column. These results illustrate how MECC and RP-HPLC are complimentary when examining complex mixtures such as those obtained from plant extracts. MECC using these conditions was applied to the examination of phenolics from leaf tissue of Eucalyptus margmata (jarrah).     

HPLC-MS and MECC analysis of coumarins

Summary MECC separation of 11 coumarins has been achieved by use of a running electrolyte at pH 10.4 prepared from 50 mM boric acid, 10 mM sodium tetraborate and 100 mM sodium hydroxide. The buffer solution contained 50 mM SDS and, as organic modifier 1%n-propanol. The applied voltage was 25 kV and the temperature of the capillary was kept constant at 20°C. HPLC baseline separation of the coumarin mixture was obtained by use of a reversed-phase column and an acetonitrile-water solvent gradient. UV detection was performed at 205 nm. Peak assignment and purity control were achieved by HPLC-mass spectrometry with either an electrospray interface or an atmospheric-pressure chemical-ionization interface. Compounds were detected in either negative- or positive-ion modes. These MECC and HPLC-MS methods are suitable for ‘fingerprint’ analysis of a number of coumarin-containing plants, e.g. Fr.Ammi visnagae, Rd.Scopoliae and Rd.Imperatoriae.     

胶束电动毛细管色谱-间接紫外检测法测定糖蜜酒精废液中的有机酸

建立了一种利用胶束电动毛细管色谱-间接紫外检测法同时测定丙二酸、甲酸、酒石酸、苹果酸、琥珀酸、戊二酸、乙酸、乳酸和谷氨酸的新方法。以7.5 mmol/L邻苯二甲酸氢钾-1.5 mmol/L十六烷基三甲基溴化铵(用0.1 mol/L氢氧化钠调pH至6.50)混合液作为电泳介质,检测波长为300 nm,参比波长为210 nm,未涂层弹性石英毛细管(50 μm i.d.×64 cm)为分离通道,在6 min内实现了9种酸的完全分离。9种有机酸的浓度与其峰面积在一定的范围呈良好的线性关系,检出限均低于1.5 mg/L,迁移时间和峰面积的日内、日间相对标准偏差均小于6%。该法用于糖蜜酒精废液中有机酸的测定,结果令人满意,9种有机酸的样品加标回收率均在93%以上。     

胶束电动毛细管色谱法用于中成药原料银杏浸膏中黄酮的质量控制

研究建立了胶束电动毛细管色谱法测定中药银杏浸膏(GBE)中黄酮的方法。以SDS作表面活性剂，在 25kV电压下，考察了不同缓冲体系的pH值及浓度对黄酮的3个水解产物槲皮素、山奈酚、异鼠李素分离度的影响。结果表明，选择25mmol/L硼砂-25mmol/L磷酸二氢钾—1％(V／V)甲醇电解液，15min之内槲皮素、山奈酚、异鼠李素可得到很好分离。把MECC定量分析的结果与反相高效液相色谱进行了对比，表明所建立的MECC法用于中药中的黄酮测定是可靠的。     

Direct automatic determination of biogenic amines in wine by flow injection-capillary electrophoresis-mass spectrometry

A capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) method for the separation and determination of nine biogenic amines is proposed. Operational variables, such as the voltage, temperature, sheath liquid composition, flow-rate, and MS parameters, were optimized. Samples are injected in the hydrodynamic mode into a 75 cm x 50 microm ID coated capillary and separated by using 25 mM citric acid at pH 2.0. Heptylamine is used as internal standard. The experimental setup includes a flow manifold coupled to the CE system for automatic insertion of samples into the CE vials. The proposed method allows amines to be determined with limits of detection from 0.018 to 0.09 microg x mL(-1) and relative standard deviation (RSD) values from 2.4% to 5.0% (except 6.8% for histamine). The method was successfully used to determine biogenic amines in red and white wines.