Bare Fused-silica Capillary Research Articles

In-vial liquid–liquid microextraction-capillary electrophoresis method for the determination of phenolic acids in vegetable oils

An in-vial liquid–liquid microextraction method was developed for the selective extraction of the phenolic acids (caffeic, gallic, cinnamic, ferulic, chlorogenic, syringic, vanillic, benzoic, p-hydroxybenzoic, 2,4-dihydroxybenzoic, o-coumaric, m-coumaric and p-coumaric) in vegetable oil samples. The optimised extraction conditions for 20g sample were: volume of diluent (n-hexane), 2mL; extractant, methanol: 5mM sodium hydroxide (60:40; v/v); volume of extractant, 300μL (twice); vortex, 1min; centrifugation, 5min. Recoveries for the studied phenolic acids were 80.1–119.5%. The simultaneous determination of the phenolic acid extracts was investigated by capillary electrophoresis (CE). Separations were carried out on a bare fused-silica capillary (50μm i.d.×40cm length) involving 25mM sodium tetraborate (pH 9.15) and 5% methanol as CE background electrolyte in the normal polarity mode, voltage of 30kV, temperature of 25°C, injection time of 4s (50mbar) and electropherograms were recorded at 200nm. The phenolic acids were successfully separated in less than 10min. The validated in-vial LLME-CE method was applied to the determination of phenolic acids in vegetable oil samples (extra virgin olive oil, virgin olive oil, pure olive oil, walnut oil and grapeseed oil). The developed method shows significant advantages over the current methods as lengthy evaporation step is not required.

Integration of on‐column catalysis and EKC analysis: Investigation of enantioselective sulfoxidations

A novel technique is presented to investigate catalytic reactions by coupling a fused-silica capillary coated with an immobilized catalyst and a bare fused-silica capillary to achieve separation of the reaction products and to generate an electroosmotic flow, which drives the transport of the reactants and products through the catalytically active capillary. The principle of this technique is illustrated by the enantioselective sulfoxidation of benzylphenylsulfide with hydrogen peroxide to the corresponding sulfoxide in the presence of a vanadium(IV)-salen catalyst, which is immobilized to nonpolar polysiloxane and permanently bonded to the inner surface of the reaction capillary. The enantiomeric ratio of the reaction product is simultaneously determined by electrokinetic chromatography using 150 mg/mL sulfated β-cyclodextrin as chiral additive in 10 mM sodium dihydrogenphosphate background electrolyte at pH 8.3. In contrast to conventional enantioselective sulfoxidations of benzylphenylsulfide using the vanadium(IV)-salen catalyst, which give ees of up to 11%, an ee of up to 23% was achieved by this approach. Furthermore, the presented technique offers many more advantages, such as improved substrate selectivity using the nonpolar polysiloxane phase as a solvent, the feasibility to perform high-throughput kinetic measurements of substrate libraries, rapid screening and investigation of stereolabile compounds, that is, chiral sulfoxides, and screening of reactions using only minute amounts of reagents.

Development and Validation of a Simple and Rapid Capillary Zone Electrophoresis Method for Quantification of Heparin in a Pharmaceutical Product and Stability Studies

A simple and rapid capillary zone electrophoresis method has been developed and validated for determination of heparin in a pharmaceutical product and evaluation of heparin stability under different stress conditions. The best separation was achieved by a bare fused silica capillary (50 μm i.d.; 50 cm total and 41.5 cm effective length), phosphate buffer (pH=3.50, 72 mM) at 35°C, hydrodynamic injection at 50 mbar for 40 seconds and applied voltage of -30 kV. Heparin and force degradation products were detected by a photo diode array detector at 200 nm and 257 nm, respectively. The proposed method was validated in terms of linearity, accuracy, precision, limit of quantification (LOQ) and limit of detection (LOD). For evaluation of heparin stability, heparin solutions were subjected to thermal (90 ± 1°C), acidic (pH=2.00, 70 ± 1°C) and basic (pH=12.00, 70 ± 1°C) stress conditions, also sun light exposure (in the lab). The results indicated that, the method was linear in the range of 0.312 to 15.0 mg/ml with LOD of 0.078 mg/ ml and LOQ of 0.312 mg/ml, accurate (between 97.27% and 101.0%) and precise (intra-day precision of 0.28 to 1.8 and inter-day precision of 0.78 to 3.2%). The migration time of heparin under optimized conditions, was 2.39 ± 0.03 minutes and force degradation products were separated within 7 minutes. Heparin content in the pharmaceutical product quantified by the method was 99.58 ± 0.70% of the label claim. Meanwhile the method could show the changes in the heparin electropherogram, also formation of degradation products under different stress conditions. Therefore the proposed method could be applied as a fast and suitable technique for determination of heparin in pharmaceutical dosage forms and stability studies.

Flow-Through Microvial Facilitating Interface of Capillary Isoelectric Focusing and Electrospray Ionization Mass Spectrometry

A capillary electrophoresis mass spectrometry (CE-MS) interface utilizing a flow-through microvial is used to ensure the electric continuity and supply the catholyte and mobilizer solutions during the capillary isoelectric focusing (cIEF) and mobilization process. The flow-through microvial provides a stable chemical environment and helps to improve the ionization efficiency without significantly diluting the analyte. The CE-MS interface facilitates the transfer of the mobilized cIEF effluent to the site of electrospray ionization, and the gaseous ions can be detected directly by a mass spectrometer. It also allows for complete focusing and mobilization processes to be performed automatically in programmed sequences with commercial CE systems. Two different strategies, using either a part of the capillary or the flow-through microvial of the CE-MS interface as the catholyte reservoir for bare fused silica capillaries or neutral coated capillaries, respectively, were developed for automated cIEF-electrospray ionization (ESI)-MS. Reasonable separation efficiency was achieved using proper concentration of carrier ampholytes and suitable strategies of electroosmotic/electrophoretic mobilization.

A novel cationic triblock copolymer as noncovalent coating for the separation of proteins by CE

A novel noncovalent adsorbed coating for CE has been prepared and explored. This coating was based on quaternized poly(2-(dimethylamino)ethyl methacrylate)-block-poly(ethylene oxide)-block-poly(2-(dimethylamino)ethyl methacrylate) (QDED) triblock copolymer which was synthesized by atomic transfer radical polymerization (ATRP) in our laboratory. The polycationic polymer and the negatively charged fused-silica surface attracted each other through electrostatic interactions and hydrogen bonds. It was demonstrated that the coated capillaries provided an electroosmotic flow with reverse direction, and the magnitude of the electroosmotic flow can be modulated by varying the molecular mass of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) block and pH value of the buffer. The effects of the molecular mass of PDMAEMA block in QDED triblock copolymer and pH value of the buffer on the separation of basic proteins were investigated in detail. The triblock copolymer coatings showed higher separation efficiency, better migration time repeatability and would apply to wider range of pH than bare fused-silica capillary when used in separating proteins. Proteins from egg white were also separated through this QDED triblock copolymer-coated capillary. These results demonstrated that the QDED triblock copolymer coatings are suitable for analyzing biosamples.

Stability-indicating micellar electrokinetic chromatography method for the analysis of sumatriptan succinate in pharmaceutical formulations

A micellar electrokinetic chromatography method for the determination of sumatriptan succinate in pharmaceutical formulations was developed. The effects of several factors such as pH, surfactant and buffer concentration, applied voltage, capillary temperature, and injection time were investigated. Separation took about 5 min using phenobarbital as internal standard. The separation was carried out in reversed polarity mode at 20 °C, 26 kV and using hydrodynamic injection for 10 s. Separation was achieved using a bare fused-silica capillary 50 μm × 40 cm and background electrolyte of 25 mM sodium dihydrogen phosphate—adjusted with concentrated phosphoric acid to pH 2.2, containing 125 mM sodium dodecyl sulfate and detection was at 226 nm. The method was validated with respect to linearity, limits of detection and quantification, accuracy, precision and selectivity. The calibration curve was linear over the range of 100–2000 μg mL −1. The relative standard deviations of intra-day and inter-day precision for migration time, peak area, corrected peak area, ratio of corrected peak area and ratio of peak area were less than 0.68, 3.48, 3.28, 2.97 and 2.83% and 2.01, 5.50, 4.46, 4.92 and 4.07%, respectively. The proposed method was successfully applied to the determinations of the analyte in tablet. Forced degradation studies were conducted by introducing a sample of sumatriptan succinate standard solution to different forced degradation conditions using neutral (water), basic (0.1 M NaOH), acidic (0.1 M HCl), oxidative (10% H 2O 2) and photolytic (exposure to UV light at 254 nm for 2 h). It is concluded that the stability-indicating method for sumatriptan succinate can be used for the analysis of the drug in various samples.

Determination of 5′-mononucleotides in infant formula by capillary electrophoresis with ultraviolet detection

A method for the simultaneous determination of five 5′-mononucleotides in infant formula by capillary electrophoresis (CE) with ultraviolet detection was developed. The analysis was performed using a bare fused-silica capillary (75 μm, i.d. × 50.2 cm; effective length, 40 cm) with a separation buffer consisting of 0.03 mol.L−1 sodium tetraborate and 0.08 mol.L−1 hydroxypropyl-β-cyclodextrin at pH 10.00. The samples were deproteinized with 0.05 mol.L−1 acetic acid. Limits of detection were between 0.8 and 1.0 mg.L−1 with an injection volume of 96 nL. The precision of the method was in the range of between 2.0% and 5.2% (n = 7). The recoveries at the concentrations studied (5 and 20 mg.L−1) were between 87.3% and 113.5%. The method was used for the analysis of five different kinds of infant formula, and the results were compared with the HPLC method. The CE method, which had high separation efficiency, consumed no solvent and could be used as an alternative to the HPLC method.

A promising capillary electrophoresis–electrospray ionization–mass spectrometry method for carbohydrate analysis

A method for adapting widely used CE conditions for the separation of fluorescently labeled carbohydrates to permit online ESI-MS detection is presented. Reverse polarity separations were performed in bare fused-silica capillaries with an acidic BGE. Under these conditions, negatively charged 8-aminopyrene 1,3,6-trisulfonate-labeled carbohydrates migrate forward against the EOF, which is towards the capillary inlet. Therefore, the CE-MS interface must simultaneously back-fill the capillary, in order to maintain the CE circuit, and provide a stable forward flow at the sprayer tip to support the electrospray process. This was achieved using a junction-at-the-tip interface, which provides a flow of solution to the junction formed by the capillary terminus and the inner wall of the emitter needle tip. Because the flow rate required for this arrangement is much less than in conventional sheath flow interfaces, dilution of the analytes is minimized. Optimized separation conditions permit baseline resolution of glucose oligomers containing up to 15 glucose units, while longer oligomers, up to 33 glucose units, were observed as resolved peaks in the negative ion mode mass spectrum.

Polymeric ionic liquid‐coated capillary for capillary electrophoresis

A new application of the polymeric ionic liquid (PIL) in capillary electrophoresis is reported. Poly(1-vinyl-3-butylimidazolium bromide) was physically adsorbed on silica capillary as the simple and effective coating for capillary electrophoresis (CE) analysis, in which the PIL is not present in the background electrolyte. The electroosmotic flow (EOF) of the PIL-coated capillary as compared with that of the bare fused-silica capillary shows a different dependence on electrolyte pH values. The EOF is reversed over a wide pH range from 3.0 to 9.0 and shows good repeatability. It is also found that the coated capillary has a good tolerance to some organic solvents, 0.1 M NaOH and 0.1 M HCl. The PIL-coated capillary has been employed in different areas. Both the basic proteins and anionic analytes can be well separated by PIL-coated capillaries in a fast and easy way. The PIL-coated capillary is also able to separate organic acid additives in a grape juice. The results showed that this type of coating provides an alternative to the CE separation of anions and basic proteins.

Hydroxyethylcellulose‐graft‐poly(2‐(dimethylamino)ethyl methacrylate) as physically adsorbed coating for protein separation by CE

In this work, a novel graft copolymer, hydroxyethylcellulose-graft-poly(2-(dimethylamino)ethyl methacrylate) (HEC-g-PDMAEMA), used as physical coatings of the bare fused-silica capillaries, was synthesized by using ceric ammonium nitrate initiator in aqueous nitric acid solution. EOF measurement results showed that the synthesized HEC-g-PDMAEMA graft copolymer-coated capillary in this paper could suppress EOF effectively compared to the bare fused-silica capillary, and efficient separations of basic proteins were also achieved. The electrical charge of the coated capillary wall could be modulated by varying not only the pH of the running buffer, but also the grafting ratio of poly(2-(dimethylamino)ethyl methacrylate) grafts, which makes possible the analysis of basic and acidic proteins in the same capillary. The effects of poly(2-(dimethylamino)ethyl methacrylate) grafting ratio in HEC-g-PDMAEMA and buffer pH on the separation of basic proteins for capillary electrophoresis were investigated in detail. Furthermore, egg white proteins and milk powder samples were separated by the HEC-g-PDMAEMA-coated capillary. The results demonstrated that the HEC-g-PDMAEMA copolymer coatings have great potential in the field of diagnosis and proteomics.

Interactions of Proteins with the Acidic Components of the Electrolyte Solution and Their Role in the Performance of Separations by CZE

This paper reports the results of a study performed to investigate the dependence of the performance of protein separation by capillary zone electrophoresis (CZE) on the anionic component of the electrolyte solutions consisting of 20 mM N,N,N′,N′-tetramethyl-1,3-butanediamine (TMBD) titrated to either pH 4.0 or pH 6.5 with either a monoprotic or a polyprotic acid. With the exception of hydrochloric acid, the acids were selected among those commonly used as the constituents of the solutions employed for protein analysis by either HPLC or CZE. TMBD was chosen for its effectiveness at preventing the interactions of proteins with the inner wall of bare fused-silica capillaries. The performance of separations was evaluated using four basic model proteins having pI value and molecular mass ranging from 9.5 to 11.0 and from 12,400 to 25,000 Da, respectively. It is shown that the different acids used as the components of the background electrolyte solutions, all containing the same concentration of TMBD, affect to different extents both migration time and peak shape of the tested proteins. The performance displayed by the BGE containing phosphate ions is enhanced using TMBD in combination with diethylenetriamine, an aliphatic vicinal triamine having effective buffering capacity at pH 4.0 and capability at minimizing protein–capillary wall interactions. The reported experimental evidences are discussed based on the possible interactions that the phosphate ions are known to establish with both the protein molecules and the surface of bare fused-silica capillaries.

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.

Determination of muscle relaxants pancuronium and vecuronium bromide by capillary electrophoresis with capacitively coupled contactless conductivity detection

A novel capillary electrophoretic method for the separation of pancuronium (PM) and vecuronium (VM) ions utilizing capacitively coupled contactless conductivity detection was devised and validated. The separation was carried out in bare fused-silica capillaries (50 μm id, 75/45 cm) at 25°C. Optimal BGE was 50 mM borate buffer of pH 9.5 containing 12.5 mg/mL of (2-hydoxypropyl)-γ-CD. The samples were injected hydrodynamically at 1000 mbar for 3 s. Separation was performed at +30 kV. Under such conditions the PM and VM were base-line resolved and the separation took < 4 min. For quantification phenyltrimethylammonium iodide was used as internal standard. Calibration curves were linear for both pancuronium bromide (PMB) and vecuronium bromide (VMB) in the range 25-250 μg/mL with r> 0.9968. The limits of detection were 7 and 6 μg/mL for PMB and VMB, respectively. The accuracy tested by recovery experiment at three concentration levels of added PMB and VMB was satisfactory (95.7-102.7%, n =3, with RSD < 2.61%). The method was successfully applied to the assay of PMB and VMB in commercial injection solutions.

Analysis of α-lactalbumin, β-lactoglobulin A and B in whey protein powder, colostrum, raw milk, and infant formula by CE and LC

Two analytical methods, capillary electrophoresis (CE) with UV detection and high-performance liquid chromatography (HPLC) with photodiode array detection, were developed for the determination of α-lactalbumin (α-Lac), β-lactoglobulin A (β-Lg A), and β-lactoglobulin B (β-Lg B) in whey protein powder, colostrum, raw milk, and infant formula. The CE analysis was performed in a bare fused silica capillary (57 cm × 50 μm, effective length 50 cm) with a separation buffer consisting of 0.5 mol L−1 boric acid, 0.025 mol L−1 hydroxypropyl-β-cyclodextrin, and 0.8 g L−1 poly(ethylene oxide) 4,000,000 at pH 9.10. The HPLC analysis was performed on a CAPCELL PAK C8 SG 300 (200 × 4.6 mm, 5-μm particles) column with a gradient mobile phase mixture consisting of acetonitrile and water containing trifluoroacetic acid. The CE method is suitable for the simultaneous determination of α-Lac, β-Lg A, and β-Lg B in whey powder, colostrum, and raw bovine milk, whereas the HPLC method can only be used for quantitative assays of β-Lg A and β-Lg B due to the co-elution of lactoferrin with α-Lac. Recoveries for both methods over the concentration range were between 90.7% and 116.8%. Whey protein powder had the highest content of the above three proteins. The contents of α-Lac in infant formula increased with the raise of age stage. Infant formula with the same age stage but different brands and countries had significant differences in the content of α-Lac.

Validation of Capillary Electrophoresis Method for Determination of N-Methylpyrrolidine in Cefepime for Injection

The present study relates to a new capillary electrophoresis method for the determination of N-methylpyrrolidine, an impurity considered to be toxic and also potential degradation impurity in cefepime hydrochloride drug substance. The newly developed capillary electrophoresis method for determining the content of N-methylpyrrolidine in cefepime for injection has been validated as per International Conference on Harmonization (ICH) guidelines to prove the selectivity, sensitivity, suitability, robustness, and ruggedness of the method. This simple, efficient, and rapid methodology may be used by pharmaceutical industry for routine analysis as well as during stability studies. The newly developed capillary electrophoresis method to determine the content of N-methylpyrrolidine in cefepime for injection requires 10 min for data acquisition, and uses an indirect UV photometry method to detect the analyte signal at 240 nm against the reference signal at 210 nm. The electrophoretic system is optimized to get stable base line, higher signal to noise ratio and peaks with narrow peak width. The method employs bare fused silica capillary with extended light path, effective length of capillary is 56 cm and inner diameter of capillary is 50 μm, 5 mmole of imidazole buffer adjusted to pH 5.1 with 3 molar acetic acid solution is used as background electrolyte. The sample is introduced in hydrodynamic mode employing pressure of 50 mbar for 5 s, and the desired separation is achieved with constant applied voltage of 25 kV at ambient temperature (~25°C).

On‐line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV‐absorption detection

This study presents the in-capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused-silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2-propanol (10%, v/v). This BGE was suitable for monitoring both off-line and in-capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in-capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing—Electrospray ionization mass spectrometry in glycerol–water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol–water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close p I values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close p I values (4.5–5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on p I determination. After optimization of sheath liquid composition (80:20 (v/v) methanol–water + 1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57–136 nM and 11–68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF–MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.

Enantioselective analysis of ofloxacin and ornidazole in pharmaceutical formulations by capillary electrophoresis using single chiral selector and computational calculation of their inclusion complexes

A capillary electrophoretic method for the separation of the enantiomers of both ofloxacin and ornidazole is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixtures was achieved in less than 16 min with resolution factors Rs = 5.45 and 6.28 for ofloxacin and ornidazole enantiomers, respectively. Separation was conducted using a bare fused-silica capillary and a background electrolyte (BGE) of 50 mM H 3PO 4–1 M tris solution; pH 1.85; containing 30 mg mL −1 of sulfated-β-cyclodextrin (S-β-CD). The separation was carried out in reversed polarity mode at 25 °C, 18 kV, detection wavelength at 230 nm and using hydrodynamic injection for 15 s. Acceptable validation criteria for selectivity, linearity, precision, and accuracy were studied. The limits of detection (LOD) and limits of quantitation (LOQ) of the enantiomers (ofloxacin enantiomer 1 (OF-E1), ofloxacin enantiomer 2 (OF-E2), ornidazole enantiomer 1 (OR-E1) and ornidazole enantiomer 2 (OR-E2)) were (0.52, 0.46, 0.54, 0.89) and (1.59, 1.40, 3.07, 2.70) μg mL −1, respectively. The proposed method was successfully applied to the assay of enantiomers of both ofloxacin and ornidazole in pharmaceutical formulations. The computational calculations for the enantiomeric inclusion complexes rationalized the reasons for the different migration times between the ofloxacin and ornidazole enantiomers.

Simultaneous detection of genetically modified organisms by multiplex ligation‐dependent genome amplification and capillary gel electrophoresis with laser‐induced fluorescence

In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis.

Capillary electrophoresis–electrospray ionization-mass spectrometry using fused-silica capillaries to profile anionic metabolites

A capillary electrophoresis–electrospray ionization-mass spectrometry (CE–ESI-MS) method is proposed to profile anionic metabolites. This application is based on the use of a bare fused-silica capillary in two different characteristic modes, high-speed and high-resolution. The high-speed mode aims to simultaneously analyze a number of major anionic metabolites including organic acids, sugar phosphates, nucleotides and coenzymes. Using ammonium formate (pH 8.0) as the electrolyte and applying pressure-assisted flow, a standard mixture including 38 compounds can be analyzed in this way in less than 16 min. The relative standard deviations were better than 0.7 for the migration times and between 1.2 and 7.0 for the peak areas. However, the peaks of several isomers overlapped. Thus, a high-resolution mode was developed for these isomers. In this mode, a mixture of ammonium acetate (pH 10.0) and methanol as electrolyte allowed such isomers to be separately analyzed. The relative standard deviations were better than 0.9 for migration times and between 1.5 and 6.3 for peak areas. This was particularly advantageous for hexose phosphate isomers, which are difficult to separate using existing CE–MS methods. To evaluate the effectiveness of our proposed method, we performed metabolite profiling of extracts from moss. In the high-speed mode, all target metabolites except isocitrate could be determined. In the high-resolution mode, five successive peaks were obtained corresponding to the hexose phosphate isomers mannose 6-phosphate, glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and an unknown compound. These results indicate that our new method has great utility in the profiling of anionic metabolites.