Ultra-fast two-dimensional microchip electrophoresis using SDS μ-CGE and microemulsion electrokinetic chromatography for protein separations

A poly(methyl methacrylate) microfluidic chip was used to perform a two-dimensional (2-D) separation of a complex protein mixture in short development times. The separation was performed by combining sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS μ-CGE) with microemulsion electrokinetic chromatography (μ-MEEKC), which were used for the first and second dimensions, respectively. Fluorescently labeled Escherichia coli cytosolic proteins were profiled by this 2-D approach with the results compared to a similar 2-D separation using SDS μ-CGE × μ-MEKC (micelle electrokinetic chromatography). The relatively short column lengths (effective length = 10 mm) for both dimensions were used to achieve separations requiring only 220 s of development time. High spot production rates (131 ± 11 spots min⁻¹) and reasonable peak capacities (481 ± 18) were generated despite the fact that short columns were used. In addition, the use of μ-MEEKC in the second dimension was found to produce higher peak capacities compared to μ-MEKC (481 ± 18 for μ-MEEKC and 332 ± 17 for μ-MEKC) due to the higher plate numbers associated with μ-MEEKC.     

毛细管微乳液电动色谱和胶束电动色谱分离冰毒及其麻黄碱杂质的比较

通过毛细管微乳液电动色谱10 m in内同时分离了安非他明、甲基安非他明、4,5-亚甲基二氧基安非他明(MDA)和3-甲氧基-4,5-亚甲基二氧基安非他明(MDMA)4种苯丙胺类毒品及其麻黄碱、伪麻黄碱、甲基麻黄碱、甲基伪麻黄碱、去甲麻黄碱等麻黄生物碱杂质。比较了毛细管微乳液电动色谱和丁醇改进的胶束电动色谱模式对分离的影响,发现正丁醇是影响分离的最主要因素。本方法具有很好的重复性和稳定性,可实现对冰毒及其麻黄生物碱杂质的快速分析和鉴定,相对保留时间和相对峰面积的RSD分别小于1.3%和5.0%,可用于冰毒的实际来源推断。     

Systematic investigations of different capillary electrophoretic techniques for separation of methylquinolines

The migration behaviour of isoquinoline, quinoline, and methyl derivatives of quinoline in different capillary electrophoretic modes has been systematically investigated. Optimised separation conditions were established by varying the key parameters (solvent, pH, temperature, surfactant concentration, core phase) for aqueous and non‐aqueous capillary zone electrophoresis (NACE), micellar electrokinetic chromatography (MEKC) with anionic or non‐ionic micelles (SDS, Brij 35), and microemulsion electrokinetic chromatography (MEEKC) with charged or uncharged microemulsion droplets. A separation of all quinolines could be achieved by MEEKC with charged droplets, by MEKC or by formamide‐based NACE. Comparing the separations with respect to separation selectivity, substantial changes in migration order could be observed between the different techniques. Regarding separation efficiency, the number of theoretical plates and limits of detection (LOD) have been compared. The best LODs were achieved using SDS as surfactant in MEKC, followed by MEEKC.     

Capillary Electrochromatography of Pyrimidine Derivatives Using UV and Mass Spectrometric Detection

 The separation of pyrimidine derivatives by capillary electrochromatography (CEC) using either UV or mass spectrometric detection is described. For UV detection an aqueous phosphate carrier electrolyte containing acetonitrile is employed. The results are compared to the analysis of the same compounds by micellar electrokinetic chromatography in terms of selectivity, migration times, linearity, and detection limits. For the combination of CEC and mass spectrometry (MS) an inexpensive way to couple commercially available instruments is presented; the interface consists of an electrically grounded stainless steel connector (containing a stainless steel frit) serving as the electrode and coupling the CEC capillary with a fused silica transfer capillary to the MS instrument. Alternatively, a PEEK adapter combining the CEC capillary and a grounded stainless steel transfer capillary serving as the electrode is employed. To avoid the formation of hydrogen gas at the coupling piece or the transfer capillary, p-benzoquinone is added to the carrier electrolyte consisting of aqueous ammonium acetate and acetonitrile.     

Determination of doxycycline in pharmaceuticals and human urine by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) was performed at 25 °C and 30 kV (under a pressure of 15 mbar), using 30 mM borate buffer containing 60 mM sodium dodecysulfate (SDS) and 5% (v/v) methanol as background electrolyte (pH 9.0) to determine doxycycline. UV detection was at 350 nm. The method was shown to be specific, accurate (recovery was 100.3 ± 1.0%), linear over the tested range (correlation coefficient 0.9995) and precise (RSD <1.9%). The method was used to determine doxycycline in tablets, capsules and human urine after oral application.     

Microemulsion electrokinetic chromatography of proteins.

Microemulsion electrokinetic chromatography was used to separate a test mixture of proteins effectively. The separation was carried out in a 42.5 cm (to the detector) x 50 microns I.D. fused-silica capillary using a microemulsion system consisting of 80 mM heptane, 120 mM SDS, 900 mM butanol in 2.5 mM borate buffer, pH 8.5-9.5. Optimum separation conditions were investigated with respect to the running voltage, temperature, pH and the composition of microemulsion. Results were compared with those obtained in micellar electrokinetic chromatography and capillary zone electrophoresis. The examined method is practical and successfully applied to the assay of genetically engineering pharmaceuticals, recombinant human granulocyte macrophage colony stimulating factor injection and recombinant human granulocyte colony stimulating factor injection.     

Capillary Electrochromatography of Pyrimidine Derivatives Using UV and Mass Spectrometric Detection

Summary.  The separation of pyrimidine derivatives by capillary electrochromatography (CEC) using either UV or mass spectrometric detection is described. For UV detection an aqueous phosphate carrier electrolyte containing acetonitrile is employed. The results are compared to the analysis of the same compounds by micellar electrokinetic chromatography in terms of selectivity, migration times, linearity, and detection limits. For the combination of CEC and mass spectrometry (MS) an inexpensive way to couple commercially available instruments is presented; the interface consists of an electrically grounded stainless steel connector (containing a stainless steel frit) serving as the electrode and coupling the CEC capillary with a fused silica transfer capillary to the MS instrument. Alternatively, a PEEK adapter combining the CEC capillary and a grounded stainless steel transfer capillary serving as the electrode is employed. To avoid the formation of hydrogen gas at the coupling piece or the transfer capillary, p-benzoquinone is added to the carrier electrolyte consisting of aqueous ammonium acetate and acetonitrile. Received July 21, 1999. Accepted July 29, 1999     

Combination of Aqueous and Non-Aqueous Capillary Electrophoresis with Electrospray Mass Spectrometry for the Determination of Drug Residues in Water

 The work presented in this paper deals with the combination of capillary electrophoresis (CE) with electrospray mass spectrometry (MS) for the determination of drug residues in water. CE/MS methods have been developed based on either aqueous or non-aqueous ammonium acetate solutions as the carrier electrolyte for the separation of selected drugs. The different separation conditions were compared in terms of selectivity and detection limits; both aqueous and non-aqueous CE proved to be suitable for the present analytical task, exhibiting detection limits between 3 and 93 μg/dm³ (injected standard concentration) corresponding to concentrations between 5 and 19 ng/dm³ in the sample. A combination of liquid-liquid extraction and solid-phase extraction was investigated for sample pretreatment, yielding enrichment factors of 10000. The applicability of CE/MS was demonstrated for the analysis of several river water samples.     

Combination of Aqueous and Non-Aqueous Capillary Electrophoresis with Electrospray Mass Spectrometry for the Determination of Drug Residues in Water

Summary.  The work presented in this paper deals with the combination of capillary electrophoresis (CE) with electrospray mass spectrometry (MS) for the determination of drug residues in water. CE/MS methods have been developed based on either aqueous or non-aqueous ammonium acetate solutions as the carrier electrolyte for the separation of selected drugs. The different separation conditions were compared in terms of selectivity and detection limits; both aqueous and non-aqueous CE proved to be suitable for the present analytical task, exhibiting detection limits between 3 and 93 μg/dm³ (injected standard concentration) corresponding to concentrations between 5 and 19 ng/dm³ in the sample. A combination of liquid-liquid extraction and solid-phase extraction was investigated for sample pretreatment, yielding enrichment factors of 10000. The applicability of CE/MS was demonstrated for the analysis of several river water samples. Received August 25, 2000. Accepted October 17, 2000     

Simultaneous Determination of Heroin, Amphetamine and their Basic Impurities and Adulterants Using Microemulsion Electrokinetic Chromatography

Heroin and amphetamine are two most abused drugs that can be highly complex, containing various impurities, byproducts, adulterants and diluents due to differences in the agricultural and manufacturing procedures. Therefore, the comprehensive analysis of above illicit drugs seized or purchased undercover by law enforcement authorities is important for legal and intelligence gathering purposes, and clinical and pharmaceutical purposes as well1. In last decade, micellar electrokinetic chromatogr…     

Possibilities and limitations of different modes of capillary electrophoresis for the quantitative determination of catechols and caffeine in black and green tea

Methods of capillary electrophoresis (capillary zone electrophoresis, micellar electrokinetic chromatography, and microemulsion electrokinetic chromatography) were compared in determining polyphenolic antioxidants and alkaloids in black and green tea. It was shown that reverse-flow micellar electrokinetic chromatography (RF-MEKC, pH < 2.5) using reversed pola rity is most preferable for the rapid simultaneous determination of catechols and caffeine. The detection limit was 1 mg/L.     

鼠李糖脂应用于微乳毛细管电动色谱快速测定化妆品中激素类物质

采用生物表面活性剂鼠李糖脂建立了无需助表面活性剂的微乳体系,并应用于微乳毛细管电动色谱快速分析化妆品中皮质类激素泼尼松、泼尼松龙和氢化可的松。考察了pH值、鼠李糖脂浓度、离子强度、油相种类和浓度、分离温度、分离电压及进样电压和时间的影响,得出微乳体系最佳组成为0.1%(w/w)鼠李糖脂+0.8%(w/w)正庚烷+99.1%(w/w)硼砂缓冲液(80 mmol/L,pH 9.2)。分离温度20℃,分离电压20kV,电动进样10 kV×3 s,泼尼松、氢化可的松和泼尼松龙在9.4 min内可基线分离。重复进样7次,迁移时间和峰面积的RSD分别小于0.2%和5.0%。3种分析物线性范围均为5～100 mg/L;检出限分别为1.0,1.1和1.3 mg/L(S/N=3)。仅需简单萃取即可用于化妆品样品测定,回收率为81.6%～108%;RSD均小于4.8%。     

Development of a capillary electrophoretic method for the separation of diastereoisomers of a new human immunodeficiency virus protease inhibitor

A capillary electrophoretic (CE) method was developed for the separation of diastereoisomers of a new human immunodeficiency virus (HIV) protease inhibitor TMC114. In total 16 isomers of this drug have been synthesized (eight pairs of enantiomers). We succeeded in the separation of the eight diastereoisomers, but no enantiomers could be separated. Because of the high similarity and water-insolubility of these isomers, the separation is a real challenge. Different CE modes were tried out: capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), micellar electrokinetic capillary chromatography (MEKC), and microemulsion electrokinetic capillary chromatography (MEEKC). Only MEEKC offered resolution of these compounds.     

Separation of seven fluoroquinolones by microemulsion electrokinetic chromatography and application to ciprofloxacin, lomefloxacin determination in urine

A simple, reliable microemulsion electrokinetic chromatography (MEEKC) method is developed for the simultaneous separation of seven fluoroquinolones (FQs). The best separation is achieved in a carrier electrolyte containing 1% (v/v) heptane, 100 mmol/L sodium dodecyl sulfate (SDS), 10% (v/v) 1-butanol, and 8 mmol/L phosphate-sodium tetraborate buffer at pH 7.30. The proposed method was directly applied to the determination of ciprofloxacin (CPF) and lomefloxacin (LMF) in urine samples of subjects administered either with CPF or LMF.     

Quantitative determination of isoniazid in biological samples by cation-selective exhaustive injection-sweeping-micellar electrokinetic chromatography

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, infects approximately one third of the current world population. Isoniazid is one of the most frequently used first-line anti-TB drugs. In this study, we developed a sensitive cation-selective exhaustive injection–sweeping–micellar electrokinetic chromatography method (CSEI-Sweep-MEKC) for analyzing isoniazid in human plasma. Parameters including acetonitrile (ACN) percentage in the separation buffer; the injection time, and concentration of the high-conductivity buffer; sodium dodecyl sulfate (SDS) concentration; phosphate concentration in the sample matrix; and the sample injection time were all optimized to obtain the best analytical performance. The optimal background electrolyte comprised 50 mM phosphate buffer, 100 mM SDS, and 15% ACN. Non-micelle background electrolyte, containing 75 mM phosphate buffer and 15% ACN, was first injected into the capillary, followed by a short plug of 200 mM phosphate (high-conductivity buffer). Run-to-run repeatability (n = 3) and intermediate precision (n = 3) of peak area ratios were found to be lower than 8.7% and 11.4% RSD, respectively. The accuracy of the method was within 98.1–106.9%. The limit of detection of isoniazod in human plasma was 9 ng mL⁻¹. Compared with conventional MEKC, the enhancement factor of the CSEI-Sweep-MEKC method was 85 in plasma samples. The developed method was successfully used to determine isoniazid concentration in patient plasma. The results demonstrated that CSEI-Sweep-MEKC has the potential to analyze isoniazid in human plasma for therapeutic drug monitoring and clinical research.     

Separation and sweeping of flavonoids by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants

In this report, a novel means for the separation and sweeping of flavonoids (quercetin, rutin, calycosin, ononin and calycosin-7-O-β-d-glucoside) by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants as modified pseudostationary phase was presented. The optimized background electrolyte consisted of 0.5% (w/v) ethyl acetate, 2.0% (w/v) SDS, 9 mM DTAC, 4.0% (w/v) 1-butanol and 10 mM sodium borate or 25 mM phosphoric acid. We systematically investigated the separation and preconcentration conditions, including the concentrations of surfactant, types of sweeping, sample matrix, the effect of high salt or acetonitrile, and sample injection volume. It was found that the use of mixed surfactants significantly enhanced the separation efficiency through the change of the efficient electrophoretic mobility of analytes. Compared with normal sample injection, 185-508-fold sensitivity enhancement in terms of limit of detection was achieved through effective sweeping of large sample volume at 50 mbar pressure (up to 45% capillary length). At last, the proposed method was suitable for the determination of Radix Astragali sample.     

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of l-MTX and its enantiomeric impurity d-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10⁻⁷ M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of l-MTX. Furthermore, l-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples.     

蛋白质的微乳液毛细管电动色谱分离研究

考察了用微乳液毛细管电动色谱(MEEKC)分离蛋白质时微乳液组成等不同因素对分离的影响,并与胶束电动色谱进行对比,探讨了其分离机理,为蛋白质的分离鉴定提供了一种有力的工具.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.     

Application of micellar electrokinetic chromatography to pharmaceutical analysis

Electrokinetic chromatography is a new type of analytical separation method which belongs to the group of high performance capillary electrophoretic techniques but whose separation principle is based on that of chromatography. The solute distributes itself between a carrier and the surrounding medium. The carrier, which corresponds to the stationary phase in conventional chromatography, can be transported by electrophoresis with a different velocity from the surrounding medium. The separation is achieved by the differential solute distribution and the differential migration of the carrier. The charged molecules or charged molecular aggregates are employed as the carrier. Various kinds of carriers are available for electrokinetic chromatography along with different partition mechanisms. Among them, micellar electrokinetic chromatography, which employs an ionic micelle as a carrier, has become the most popular method because of its unique and attractive characteristics as well as the separating capability of electrically neutral or nonionic solutes in comparison with capillary zone electrophoresis. The present paper describes the principle, separation characteristics and its application to the analysis of pharmaceuticals.