Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.     

Application of a capillary‐assembled microfluidic system for separation of cephalosporins

This paper demonstrates a simple and easy setting up of a fused‐silica capillary‐assembled microfluidic system (μCE). This system incorporates a split‐flow pressure injection of the sample into a microfluidic system made from PDMS and a short (～20 cm) length of fused‐silica capillary as a separation unit. The on‐capillary detection was carried out by fiber optic spectrometry. A mixture of six cephalosporin antibiotics was separated in the μCE system and the obtained results were compared to those achievable by conventional CE. The six components could be separated within 8.5 min with the number of theoretical plates around 10 000.     

Effects of electrolyte modification and capillary coating on separation of glycoprotein isoforms by capillary electrophoresis

The capillary electrophoresis (CE) running electrolyte composition was optimized for the separation of selected glycoproteins. A good separation of the ovalbumin (OV) and alpha-acid glycoprotein (AAG) isoforms was achieved in 20 mmol x L(-1) N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) at pH 7.0, in 20 mmol x L(-1) phosphate, pH 7.0, or in 25 mmol x L(-1) borate, pH 7.9. Various ways of suppression of the glycoprotein adsorption onto the capillary wall were compared. Alpha, omega-diamine alkanes and bis(aminoalkyl) amines were added to the CE buffers, the optimized concentration being 1 mmol x L(-1) in 20 mmol x L(-1) phosphate buffer. The OV and AAG isoforms could be separated using all the alpha,omega-diamine alkanes or bis(2-aminoethyl)amine. The length of the alkyl chain in the diaminoalkane did not influence the separation. The separation of the isoforms of pollen allergens was also tested. The effects of modification of the capillary wall by succinyl-poly-L-lysine and hydrophilic CElect-P1 capillary were compared. A decrease in the glycoprotein and protein adsorption resulted in an improved separation of the isoforms.     

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.     

Miniaturization in carbohydrate analysis

Recent progress of microchip electrophoresis (ME) of carbohydrates is overviewed. Carbohydrate analysis by ME encounters difficulties such as lack of electric charge and deficiency of a chromophore/fluorophore in analyte molecules, however, it benefits from the accumulated knowledge of capillary electrophoresis (CE) and rapid separation of simple sugars also by ME, with high column efficiency comparable to CE, has become possible. Analysis at high pH, with electrochemical detection, is a promising approach because carbohydrates can be ionized by weak dissociation of the hydroxyl groups and the in situ formed ionic species can be effectively separated by the zone electrophoresis mode. The separated species can be sensitively monitored by electrochemical detection on a gold or copper electrode. Ionization as borate complexes and refractometric detection is also possible, though sensitivity is lower. Introduction of UV-absorbing or fluorescent tags is potentially useful but the time-consuming derivatization processes sacrifice the rapidity of ME. Examples of ME of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone (PMP; for simple mono- and oligosaccharides with UV detection), 8-aminopyrene-1,3,6-trisulfonate (APTS; for oligosaccharides ladders with LIF detection), and 4-nitro-2,1,3-benzoxadiazole (NBD-F; for amino sugars and aminoalditols with LIF detection) derivatives are presented, with details of the analytical conditions. Since ME in a short separation channel enables rapid analysis within 1 min, it presents an ideal tool for clinical analysis, as shown in a few papers reporting protocols for specific blood glucose assay. Finally, the usefulness of microfluidic reactors and microarrays for enzyme-assisted carbohydrate analysis as well as glycan profiling is pointed out.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Rapid enantioseparation of 1-aminoindan by microchip electrophoresis with linear-imaging UV detection.

Chiral separations of 1-aminoindan (AI) by cyclodextrin electrokinetic chromatography (CDEKC) were investigated on microfluidic quartz chips. By using a microchip electrophoresis (MCE) instrument equipped with a linear-imaging UV detector, the separation process of the enantiomeric compounds was observed. When sulfated beta-cyclodextrin was employed as a chiral selector, the baseline separation of AI could be achieved within 1 min with a high repeatability. The relative standard deviation of the migration time was less than 6%. The fastest separation was achieved in 14 s, utilizing a separation length of only 6.1 mm. These results show that the MCE analysis employing a linear imaging UV detector has a significant potential for fast chiral analysis.     

Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate-based buffers

This study focused on optimizing phosphate-based buffers and other capillary electrophoresis (CE) parameters for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), emmer (Triticum dicoccum, AABB, 2n = 4x = 28) and Aegilops tauschii (DD, 2n = 2x = 14). The fast and high-resolution separation of HMW-GS was achieved using 0.1 M phosphate-glycine buffer (pH 2.5, containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose) at 12.5 kV and 40 degrees C with 25 microm inside diameter (ID)x27 cm uncoated fused-silica capillary. In general, one sample separation can be analyzed in 15 min. The good run-to-run repeatable separation of HMW-GS could be obtained with a relative standard deviation of less than 1% when capillaries were rinsed with 1 M phosphoric acid for 2 min, followed by separation buffer for 2 min after each separation. The HMW-GS from some bread wheat cultivars as well as tetraploid and diploid accessions was separated by the CE method described above, and all subunits detected were well characterized and readily identified. Some HMW-GS showed reversed mobilities and elution order compared to the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-CE. Particularly, most of the HMW-GS analyzed with the CE buffer used were separated into multiple peaks, generally a high peak plus a minor peak. CE appears to be capable of separating and characterizing HMW-GS with fast and high-resolution features, therefore it is expected to be useful for specific germplasm screening and desirable HMW-GS identification in wheat quality improvement.     

毛细管电泳法分析藏红花植物细胞多糖中单糖组成

以对甲氧基苯胺为衍生试剂,采用毛细管电泳法分析了藏红花植物细胞多糖中的单糖组成。对衍生条件进行了优化,并对毛细管分离条件进行了系统的研究。衍生反应在醋酸含量9.5%(v/v)、80 ℃下反应2 h的衍生产率最大,衍生产物紫外检测波长234 nm。在优化的毛细管电泳分离条件(未涂层熔融石英毛细管柱(60 cm(有效长度50 cm)×50 μm),柱温25 ℃,电压20 kV,使用350 mmol/L硼酸电解液(pH 10.21),压力(3.4475 kPa)进样5 s)下,基线分离了11种结构相近的醛糖(来苏糖、木糖、核糖、葡萄糖、甘露糖、半乳糖、鼠李糖、纤维二糖、麦芽糖、乳糖)、酮糖(果糖)的衍生产物。应用该方法定量检测了藏红花植物细胞多糖水解物中糖的成分,各糖的回收率为94.3%～105.4%,相对标准偏差为3.3%～4.6%。     

Extremely highly efficient on-line concentration and separation of gold nanoparticles using the reversed electrode polarity stacking mode and surfactant-modified capillary electrophoresis

In this study, gold nanoparticles (Au NPs) were separated using the reversed electrode polarity stacking mode (REPSM) of a capillary electrophoresis (CE) system for on-line enhancement prior to performing surfactant-modified CE separation. Under optimized conditions [running electrolyte buffer, sodium dodecyl sulfate (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (10 mM) at pH 10.0; applied voltage, 20 kV; operating temperature, 25°C; REPSM strategy for sample on-line concentration; REPSM applied prior to initializing separation], two parameters were varied to further enhance the concentration and separation of the Au NPs: (i) the rate of polarity switching (from -20 to +20 kV) between the REPSM and surfactant-modified CE separation modes and (ii) the length of the capillary column. At a polarity switching rate of 1333 kV min(-1) and a column length of ca. 83.5 cm, the resolution of the separation of a mixture of 5.3- and 40.1-nm Au NPs was greater than 19; in addition, the numbers of theoretical plates for the 5.3- and 40.1-nm-diameter Au NPs were greater than 15,000 and up to 1.15×10(7), respectively-the latter being extremely high. Thus, this CE-based method for separating Au NPs provided high performance in terms of separation resolution and the number of theoretical plates, both of which were improved by greater than fivefold relative to those published previously. Notably, the sensitivity enhancement factors for the 5.3- and 40.1-nm-diameter Au NPs were improved (by ca. 20- and 500-fold, respectively) relative to those obtained using conventional surfactant-modified CE separation.     

Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis

We report on microbore liquid chromatography (microLC) and capillary electrophoresis (CE) separation of glycopeptides and high-mannose-type oligosaccharides, digested from recombinant phospholipase C, expressed in Pichia pastoris. The glycopeptides were subject to microLC/electrospray ionization/mass spectrometry (ESI-MS) and microLC/ESI-tandem MS (MS/MS) analysis that revealed high-mannose structure size variation between Man(7)GlcNAc(2) and Man(14)GlcNAc(2). Then, high-performance CE was applied to identify possible positional isomers of the high-mannose structures. For the CE experiments, the oligosaccharides were released from the glycoproteins by peptide-N-glycosidase F and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS). Excellent separation of the possible positional isomers was attained, suggesting one for Man(9)GlcNAc(2), two for Man(10)GlcNAc(2), three for Man(11)GlcNAc(2), Man(12)GlcNAc(2), and Man(13)GlcNAc(2), and two for Man(14)GlcNAc(2). The CE results provided complementary information to the microLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.     

Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.     

Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

CE is a high‐resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ≥100 fold excess of fluorophore reagent, 8‐aminopyrene‐1,3,6‐trisulfonic‐acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:≥100 ratio) maintained the >95% derivatization yield at 55°C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8‐aminopyrene‐1,3,6‐trisulfonic‐acid simplified the removal of excess reagent, important in both CE‐LIF (electrokinetic injection bias) and CE‐MS (ion suppression). Coupling CE‐ ESI‐MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.     

Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine

A novel capillary zone electrophoresis method is described for the determination of taurine in plasma. The method is rapidly executed and is highly selective for taurine as separation is based on the difference in ionisation of this amino acid from that of other amino acids. Following addition of homotaurine as internal standard, plasma proteins were precipitated with acetonitrile and the supernatant was derivatised with fluorescamine in the presence of a borate buffer. Capillary electrophoresis (CE) separations were carried out in reverse polarity mode at 27.5 kV on a Beckman P/ACE MDQ CE instrument, equipped with a diode array detector (DAD) set at 266 nm. The sample tray was cooled to 5 degrees C and separations were carried out at 20 degrees C. The fused-silica capillary was 50.2 cm in length (40.2 cm to detector) with an internal diameter of 75 microm. A capillary conditioning solution was applied daily in order to suppress the residual electroosmotic flow (EOF). The method, which was validated using feline plasma as the blank matrix, was shown to be linear and reproducible over the concentration range 2.5-100 microg/mL. The coefficients of variation (CVs) of replicate analyses were less than 4.5% at 1 microg/mL taurine in feline plasma and less than 3% for 2.5 microg/mL in human plasma. Recovery was estimated at 99.2% with a CV of 4.85%. It has been demonstrated that quantitation in aqueous solution yields similar results to those obtained by interpolation on a plasma calibration curve provided that subtraction for the taurine peak in unspiked plasma is carried out and that a suitable internal standard is employed.     

Sensitive determination of carbohydrates labelled with p-nitroaniline by capillary electrophoresis with photometric detection using a 406 nm light-emitting diode

p-Nitroaniline was explored as a derivatising reagent for UV absorbance detection of carbohydrates after separation by CE. This derivatising agent has three advantages: first, it has excellent water solubility; second, it has high molar absorptivity; and third, it is possible to obtain sensitive detection using a UV or blue light-emitting diode (LED) as the light source. The labelling reaction took less than 30 min to complete with high reaction yield. The separation process was modelled and optimised using an artificial neural network. Nine carbohydrates were separated by a CE system within 16 min using a 0.17 M boric acid buffer at pH 9.7. On-column LED detection at 406 nm allowed the detection of carbohydrates with good detection limits (<1.1 microM or 8.8 fmol) and reproducible quantification in the concentration range of 2.6-200 microM. This method was applied successfully to the determination of component carbohydrates in some food samples.     

A microchip electrophoresis system with integrated in-plane electrodes for contactless conductivity detection

We present a new approach for contactless conductivity detection for microchip-based capillary electrophoresis (CE). The detector integrates easily with well-known microfabrication techniques for glass-based microfluidic devices. Platinum electrodes are structured in recesses in-plane with the microchannel network after glass etching, which allows precise positioning and batch fabrication of the electrodes. A thin glass wall of 10-15 microm separates the electrodes and the buffer electrolyte in the separation channel to achieve the electrical insulation necessary for contactless operation. The effective separation length is 34 mm, with a channel width of 50 microm and depth of 12 microm. Microchip CE devices with conductivity detection were characterized in terms of sensitivity and linearity of response, and were tested using samples containing up to three small cations. The limit of detection for K+ (18 microM) is good, though an order of magnitude higher than for comparable capillary-based systems and one recently reported example of contactless conductivity on chip. However, an integrated field-amplified stacking step could be employed prior to CE to preconcentrate the sample ions by a factor of four.     

Separation and characterization of human high-density apolipoproteins using a nonaqueous modifier in capillary electrophoresis-mass spectrometry

The separation and characterization of human apolipoproteins and their isoforms was investigated using capillary electrophoresis (CE) in combination with mass spectrometry (MS). The focus of these analyses was the major protein constituents of plasma high-density lipoproteins, apolipoprotein A-I and A-II. Using aqueous buffers in CE, no separation between apolipoprotein A-I and A-II was observed. With the addition of 10-20% acetonitrile, however, the two species could be separated. Furthermore, multiple peaks for each of the apolipoprotein species were observed under these CE conditions. In order to identify and characterize the components, these separations were then coupled with online mass spectrometric detection (CE-MS). Our CE-MS results suggest that the multiple components observed in the acetonitrile-containing CE separation appear to be oxidized forms of the proteins in addition to native forms of the apolipoprotein A-I and A-II. These data are in agreement with previous reports that the methionine residues of the high-density lipoproteins (HDLs) are sensitive to oxidation, which in turn, alters their lipid binding characteristics and secondary structure. In addition to oxidized forms of the proteins, apolipoprotein A-II contained additional components, which varied in mass by 128 Da. The structural differences between these components were determined by proteolytic digestion and tandem MS. Using these techniques, we determined that these components were due to truncation of the C-terminal glutamine amino acid residue on apolipoprotein A-II. These results demonstrate that CE in combination with MS is a promising technique for screening and characterizing isomers of plasma apolipoproteins.     

Separation of negatively charged carbohydrates by capillary electrophoresis

Capillary electrophoresis (CE) has recently emerged as a highly promising technique consuming an extremely small amount of sample and capable of the rapid, high-resolution separation, characterization, and quantitation of analytes. CE has been used for the separation of biopolymers, including acidic carbohydrates. Since CE is basically an analytical method for ions, acidic carbohydrates that give anions in weakly acid, neutral, or alkaline media are often the direct objects of this method. The scope of this review is limited to the use of CE for the analysis of carbohydrates containing carboxylate, sulfate, and phosphate groups as well as neutral carbohydrates that have been derivatized to incorporate strongly acidic functionality, such as sulfonate groups.     

Cover Picture: Electrophoresis 14'2011

Issue no. 14 is a regular issue consisting of 17 contributions distributed over 3 distinct parts. Part I has 7 articles describing studies on proteins and proteomics including measuring protein mobility using laser Doppler electrophoresis, high sensitivity protein analysis by FESI‐CE‐MALDI‐MS, protocol for SDS‐PAGE separation of myosin heavy chain isoforms, proteomics analysis of a spring wheat cultivar in response to prolonged cold stress, analysis of changes in the 20S proteasome, treatment of acute lymphoblastic leukemia with L‐asparaginase, and improved method for immunostaining of mucin. Part II is on nucleic acids and has 4 contributions on multiplex PCR‐CE analysis, copy number variation, ethnic difference, SNPs, CE‐SSCP, allelic ladder and SNaPshot technique. Part III has 6 articles on various fundamentals and methodologies, including nanofluidics, nanopore sensing, sequential injection setup for CIEF combined with MS detection, determination of imatinib mesylate in chronic myeloid leukemia patients by CE, UV detection of plasma malondialdehyde using CE‐FASI, enantioseparation of basic drugs by CE using clarithromycin lactobionate chiral selector and CE determination of bioactive constituents in Herba Houttuyniae. Featured articles include: Advances in the measurement of protein mobility using laser Doppler electrophoresis – the diffusion barrier technique (( 10.1002/elps.201100108 )) Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure (( 10.1002/elps.201000608 )) Strategy for high‐fidelity multiplex DNA copy number assay system using capillary electrophoresis devices (( 10.1002/elps.201100093 )) Electrokinetic particle translocation through a nanopore containing a floating electrode (( 10.1002/elps.201100050 )) Sequential injection setup for capillary isoelectric focusing combined with MS detection (( 10.1002/elps.201100012 ))     

Simultaneous determination of amphoteric surfactants in detergents by capillary electrophoresis with indirect UV detection

A simple, rapid and simultaneous determination of four types of amphoteric surfactants, i.e., C8, C10, C12, C14, C16 and C18-homologues of alkyldimethylamine N-oxide (AO), alkylamidopropylamine N-oxide (APAO), alkylbetaine (Bt) and alkylamidopropylbetaine (APB), was performed by using capillary electrophoresis (CE) with indirect UV detection. To optimize the separation conditions, effects of pH of background solution (BGS), organic modifier and chromophore for indirect UV detection on the CE separation of the amphoteric compounds were investigated. Addition of 50% (v/v) acetonitrile to the BGS under a lower pH condition brought a good separation performance due to the suppression of micelle formation for the analytes and the adsorption onto the inner surface of the capillary. Under an optimal condition, the 24 amphoteric analytes were completely separated in a single run within 17min. The relative standard deviation of the migration time was ranging from 0.20 to 0.23% and the limit of detection values for AO, APAO, Bt and APB homologues were 10-20, 20, 20-50 and 50microg/mL, respectively. Furthermore, the developed method can provide a high resolution separation of the amphoteric surfactants in commercially available detergents and shampoo without any sample pretreatments.