Application of nanoscale packed capillary liquid chromatography (75 μm id) and capillary zone electrophoresis/electrospray ionization mass spectrometry to the analysis of macrolide antibiotics

Nanoscale separation techniques, nanoscale packed capillary columns (75 μm id), and capillary zone electrophoresis (CZE), on-line with electrospray mass spectrometry (ESI/MS), were applied to the separation of a series of ten macrolide antibiotics. Both techniques use sub-microliter-per-minute flow rates through the analytical column and therefore require an electrospray probe that incorporates coaxial sheath flow. Positive ion electrospray mass spectra of these compounds yielded mainly protonated molecules. Fragmentation to yield structurally significant fragment ions was achieved by collision-induced dissociation (CID) at increased skimmer voltages. Separations were achieved using both techniques, with CZE/ESI/MS showing improved peak shapes and detection limits combined with faster analysis times. Nanoscale packed capillary columns provided better chromatographic resolution and was less susceptible to peak broadening caused by overloading of the analytes.     

In-line sample trap columns with diatomite for large-volume injection in CZE–IM–MS

The analysis of low-abundant compounds with capillary zone electrophoresis–drift-tube ion mobility spectrometry–mass spectrometry (CZE–DTIMS–MS) is compromised due to the low injectable sample volumes in CZE and low duty cycle in DTIMS. Fritless packed in-line trap columns, using solid-phase extraction sorbent particles, have been used to increase injection volumes in CZE, but these columns are difficult to prepare and exhibit rapidly increasing back pressures. To provide smooth and complete filling of trap columns as well as to ensure higher and sustained flow rates though the columns, blends of cation and anion exchange particles with diatomite were used. The application of diatomite blends ensured the use of trap columns for at least 100 injections, with maximum injection volumes over 10 µl, which corresponds to an enrichment factor of more than 1000 over conventional injections in CZE–MS, enabling the detection of low nM concentrations of N-glycans with CZE–IMS–MS.     

Separation of basic compounds of pharmaceutical interest by using nano-liquid chromatography coupled with mass spectrometry

Nano-liquid chromatography-mass spectrometry (nano-LC-MS) was evaluated for the separation of basic compounds of pharmaceutical interest. The separation of selected beta-blockers, namely nadolol, oxprenolol, alprenolol and propranolol in the presence of terbutaline was performed using two 75 microm I.D. capillaries packed with two different RP18 stationary phases (SP). The best results concerning resolution and efficiency were achieved using the SP where free silanol groups were not present. As expected, this latter SP proved to be very efficient and symmetry factors were observed mainly in the case of the more retained analytes. Baseline resolution of all studied basic compounds was achieved with the Cogent bidentate C18 silica phase (CBC18) eluting analytes at 800 nL/min with a mobile phase containing 500 mM ammonium acetate pH 4.5-water-methanol (1:8:91, v/v/v). The separated basic compounds were revealed using on-column UV detector at 205 nm and electrospray-ion-trap mass spectrometer (ESI-MS). The packed capillary was connected to the MS through a commercial sheath liquid interface or a sheathless nano-spray interface and in both cases the sensitivity was studied and the results compared. Limit of detection (LOD) as low as 0.1 ng/mL was measured for nadolol using the sheathless nano-spray interface and the capillary column packed with the CBC18 stationary phase.     

Selective trace enrichment by immunoaffinity capillary electrochromatography on-line with capillary zone electrophoresis - laser-induced fluorescence

Limited by the lack of a sensitive, universal detector, many capillary-based liquid-phase separation techniques might benefit from techniques that overcome modest concentration sensitivity by preconcentrating large injection volumes. The work presented employs selective solid-phase extraction by immunoaffinity capillary electrochromatography (IACEC) to enhance detection limits. A model analyte, fluorescein isothiocyanate (FITC) biotin, is electrokinetically applied to a capillary column packed with an immobilized anti-biotin-IgG support. After selective extraction by the immunoaffinity capillary, the bound analyte is eluted, migrates by capillary zone electrophoresis (CZE), and is detected by laser-induced fluorescence. The column is regenerated and reused many times. We evaluate the performance of IACEC for selective trace enrichment of analytes prior to CZE. The calibration curve for FITC-biotin bound versus application time is linear from 10 to 300 seconds. Recovery of FITC-biotin spiked into a diluted urinary metabolites solution was 89.4% versus spiked buffer, with a precision of 1.8% relative standard deviation (RSD).     

Nanoscale separations combined with electrospray ionization mass spectrometry: Sulfonamide determination

Nanoscale capillary liquid chromatography (nCLC) and capillary zone electrophoresis (CZE) have been combined with quadrupole mass spectrometry via an electrospray ionization (ESI) interface. These methodologies have been applied to the separation and determination of a variety of sulfonamides. CZE/ESI/MS is the more rapid and sensitive technique, but nCLC/ESI/MS shows promise for the analysis of dilute samples. Ultimately, the two techniques provide complementary methods of analysis. The detection limits of these techniques in the full-scan mode are in the low picomole range. Dissociation of the sulfonamides can be induced by increasing the skimmer voltage. This provides a limited means of discriminating between compounds of identical molecular weight but, more important, provides fragments that could be used to confirm the presence of analyte within a sample.     

Comprehensive two-dimensional chromatography and capillary electrophoresis coupled with tandem time-of-flight mass spectrometry for high-speed proteome analysis

A comprehensive two-dimensional capillary liquid chromatography and capillary zone electrophoresis system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) proteomics analyzer is presented. Protein/peptide samples were separated by capillary high-performance liquid chromatography (cHPLC). The effluents from cHPLC (the first dimension) were continuously transferred into capillary zone electrophoresis (CZE, the second dimension) through a novel valve-free hydrodynamic sampling interface. The CZE effluents were mixed with alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix sheath flow via CE-MALDI interface, and then directly deposited on the MALDI target at a 3 s time-interval for further MS analysis. The high efficiency of the overall system was demonstrated by analysis of proteins in D20 (human hepatocellular carcinoma model in nude mice with high metastatic potential) liver cancer tissue. More than 300 proteins were identified, which proved the system potential for high-throughput analysis and application in proteomics.     

On-line affinity selection of histidine-containing peptides using a polymeric monolithic support for capillary electrophoresis

An on-line affinity selection method using a polymeric monolithic support is proposed for the retention of histidine-containing peptides and their subsequent separation by capillary zone electrophoresis (CZE). Monolithic capillary columns were prepared in fused-silica capillaries of 150 mum inner diameter (ID) by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iminodiacetic acid (IDA) and copper ion. Monolithic microextractors were coupled on-line near the inlet of the separation capillary (fused-silica capillary, 75 mum ID x 28 cm from the microextractor to the detector). Model peptide mixtures of histidine-containing and histidine-noncontaining peptides were assessed. Peptides were released from the sorbent by a 5 mM imidazole solution and then separated by CZE with ultraviolet detection. Relative standard deviation values for migration times and corrected peak areas were found to be lower than 5.8 and 10.5%, respectively. IDA-Cu(II) ion modified monolithic microextractors showed a chromatographic behavior and could be reused at least 25 times. The use of monolithic supports proved to be an advantageous alternative to packed particles for the preparation of microextractors.     

Enantiomeric separation of some demethylated analogues of clofibric acid by capillary zone electrophoresis and nano-liquid chromatography

The enantiomeric separation of some demethylated analogues of clofibric acid, namely 2-(6-chloro-benzothiazol-2-ylsulfanyl)-, 2-(6-methoxy-benzothiazol-2-ylsulfanyl)-, 2-(quinolin-2-yloxy)-, 2-(6-chloro-quinolin-2-yloxy)-, 2-(7-chloro-quinolin-4-yloxy)-propionic acid (compounds A-E, respectively), has been studied by CZE and nano-LC using for the first technique two beta-CD derivatives and vancomycin added to the BGE and vancomycin-modified silica particles for the second one, with the aim to find the optimum experimental conditions for the baseline resolution. The type and the concentration of the chiral selector added to the BGE, the buffer pH, the type of organic modifier and its concentration, the capillary temperature and the applied voltage played a very important role in the enantioresolution of the analysed compounds. The use of 6-monodeoxy-6-monoamino-beta-CD allowed to achieve baseline resolution of four of five clofibric acid derivatives in less than 10 min while heptakis-(2,3,6-tri-O-methyl)-beta-CD partially resolved the same compounds in their enantiomers. Employing vancomycin as the chiral selector in CZE, the counter-current partial filling method was chosen achieving baseline resolution of four analytes. All the studied compounds were enantioresolved employing a capillary column packed with vancomycin stationary phase by nano-LC, and the resolution was strongly influenced by the concentration of the organic modifier and by the pH of the mobile phase. The best results were achieved at pH 4.5 in presence of 60% of methanol (MeOH). However, longer analysis times were observed in the experiments carried out by nano-LC.     

Interfacing microbore and capillary liquid chromatography to continuous-flow fast atom bombardment mass spectrometry for the analysis of glycopeptides

This work describes a system to interface either microbore or packed capillary gradient liquid chromatography (LC) to fast atom bombardment mass spectrometry (FAB-MS). The interface incorporates on-line ultraviolet detection and post-column matrix addition to enable independent optimization of both LC and FAB-MS. The glycopeptide antibiotic teicoplanin was chosen as a model system because this group of compounds places severe demands on the chromatographic separation and is difficult to analyze by FAB-MS. For both microbore and capillary LC, high-quality mass spectra of the major components in teicoplanin were obtained; however, the increased sensitivity of the capillary system allowed spectra to be obtained at low picomole concentrations. The sensitivity and ease of use make capillary LC the preferred system for use in LC-FAB-MS.     

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.     

Analysis of synthetic chemical drugs in adulterated Chinese medicines by capillary electrophoresis/electrospray ionization mass spectrometry

Sixteen synthetic chemical drugs, often found in adulterated Chinese medicines, were studied by capillary electrophoresis/UV absorbance (CE/UV) and capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS). Only nine peaks were detected with CZE/UV, but on-line CZE/MS provided clear identification for most compounds. For a real sample of a Chinese medicinal preparation, a few adulterants were identified by their migration times and protonated molecular ions. For coeluting compounds, more reliable identification was achieved by MS/MS in selected reaction monitoring mode. Micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) provided better separation than capillary zone electrophoresis (CZE), and, under optimal conditions, fourteen peaks were detected using UV detection. In ESI, the interference of SDS was less severe in positive ion mode than in negative ion mode. Up to 20 mM SDS could be used in direct coupling of MEKC with ESI-MS if the mass spectrometer was operated in positive ion mode. Because of better resolution in MEKC, adulterants can be identified without the use of MS/MS.     

毛细管反相电色谱法分离行为的研究

对乙睛-水-磷酸二氢销体系毛细管反相电色谱分离行为进行了研究。采用柱上紫外检测,在75μmi.d.×30cm的毛细管ODS（3μm）填充柱上获得了小于2.0的折合培板高度。同时还研究了乙睛的比例、电解质的浓度和电场强度等因素对电渗流和往效的影响。     

Capillary zone electrophoresis with electroosmotic flow controlled by external radial electric field.

A new way of regulation of electroosmotic flow (EOF) in capillary zone electrophoresis (CZE) by external electric field has been developed. A set of three high-voltage power supplies is used to form a radial electric field across the capillary wall. One power supply is applied in the usual way as a driving force of CZE and EOF to the ends of the inner capillary compartment dipped into the electrode vessels and filled with background electrolyte. Two power supplies are connected to the ends of the outer low-conductivity coating of the capillary which is formed by the dispersion of copolymer of aniline and p-phenylenediamine in polystyrene matrix. The difference between electric potentials on the outer capillary surface and inside the capillary determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid-liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow rate of EOF, on the migration times of charged analytes and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine and octapeptide fragments of human insulin was evaluated. Through the EOF control by external electric field the dynamic effective length of the capillary was obtained and the speed of analysis and resolution of CZE separations of peptide analytes could be optimized.     

Comprehensive on-line RPLC-CZE-MS of peptides

Peptide standards and tryptic digests of ribonuclease B are separated by comprehensive two-dimensional reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) and detected on-line by electrospray mass spectrometry. The RPLC column is coupled to the CZE column by a transverse flow gating interface. A new rugged microelectrospray needle is described that combines high ionization efficiency, low flow rates, and a sheath flow. The result is a system combining the separation capabilities of both RPLC and CZE with on-line mass spectrometric detection, all in about 15 min.     

Comparison of gas chromatography-mass spectrometry and capillary electrophoresis in analysis of phenolic compounds extracted from solid matrices with pressurized hot water

Self-constructed pressurized hot water extraction (PHWE) equipment was used in dynamic mode to extract spiked phenolic compounds (phenol, 3-methylphenol, 4-chloro-3-methylphenol and 3,4-dichlorophenol) from sea sand and soil. Phenols were analyzed by both gas chromatography-mass spectrometry (GC-MS) and capillary zone electrophoresis (CZE) to compare the techniques and to find out if CZE is a suitable tool for analysis of phenols extracted from environmental matrix. Good recoveries of phenols spiked in sea sand were achieved at all PHWE temperatures (50, 100, 200, 300 C). GC-MS studies showed that phenols were selectively extracted from soil at 50 C but various other compounds (e.g. polyaromatic hydrocarbons) were extracted along with the phenols at 300 degrees C. In the case of CZE, phenols extracted from the soil, at 300 C were separated with good resolution at pH 9.7, and co-extracted compounds did not interfere with the analysis. The analytical values obtained by GC-MS and CZE were generally of similar magnitude.     

Up-scaling capillary zone electrophoresis separations of polydisperse anionic polyelectrolytes with preparative free-flow electrophoresis exemplified with a soil fulvic acid

A scale-up of analytical capillary zone electrophoresis (CZE) to preparative free-flow electrophoresis (FFE) is described. FFE allows fractionations based on charge densities in larger amounts than in CZE, enabling further off-line analysis of the fractions. Model compounds (carboxylic acids and polystyrene sulfonates) showed a similar behavior in FFE as in CZE. Diffusion and electrodynamic distortion effects are more pronounced in FFE than in CZE. A soil fulvic acid was analyzed by CZE and fractionated by FFE. A comparison of the FFE fractions with CZE measurements of the same sample using the effective mobility scale showed good agreement of the two methods.     

Integrated electroosmotically-driven on-line sample purification system for nanoliter DNA sequencing by capillary electrophoresis

An integrated on-line system is developed for DNA sequencing at the nanoliter scale. The technique involves the use of a nanoreactor for small-volume cycle-sequencing reaction, capillary zone electrophoresis (CZE) for purification of the sequencing fragments, and capillary gel electrophoresis (CGE) for separation of the purified DNA fragments. The nanoreactor and CZE are integrated into one capillary, where a 100-nl dye-labeled terminator cycle-sequencing reaction is carried out followed by CZE to separate excess dye-labeled terminators from the sequencing fragments. On-line electrokinetic injection of the purified DNA fragments into the CGE system is accomplished at a small-volume tee connector by which the CZE capillary is interfaced to the CGE system. The utility of the system is demonstrated in sequencing nanoliter volumes of single-stranded DNA (M13mp18) and double-stranded DNA (pGEM). The use of voltage to drive both CZE and CGE makes it feasible for automation and future adaptation of the whole system to a microchip.     

Design and performance of a low-flow capillary electrophoresis-electrospray-mass spectrometry interface using an emitter with dual beveled edge

A low-flow electrophoresis-mass spectrometry (CE-MS) interface has been developed for interfacing capillary zone electrophoresis (CZE) with electrospray- ionization-mass spectrometry (ESI-MS). The interface consists of two parallel capillary columns (a separation column and a makeup column), and an emitter with a dual beveled edge. While maintaining a relatively low optimum flow rate, the dual-beveled-edge ESI emitter allows the use of a tip with larger orifice. Therefore, this interface is less prone to column blocking in comparison with a flat tip. Primarily attributed to low sample dilution and smaller initial droplet, the interface showed better sensitivity than a conventional sheath liquid interface. Furthermore, the interface was found to be more resistant to the presence of nonvolatile salts. By using 40 mM borate and 20 mM alpha-cyclodextrin (alpha-CD) as the running buffer, four major forms of gangliosides were detected by CE-MS.     

Interlaboratory reproducibility of mobility parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

An interlaboratory pilot study was performed to determine the reproducibility of mobility parameters in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The study was performed by an intended small number of laboratories (three) that used different brands of instruments (two). The effective mobility was corrected using standards by a method that was recently introduced to obtain a more reproducible migration parameter. A test set of 20 acidic test compounds and 5 reference compounds were analyzed during five days in each laboratory using CZE and MEKC. Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). Analyses were carried out using fused-silica capillaries at an electric field strength of either 52.6 kV/m or 37.5 kV/m. The interlaboratory reproducibility (mean RSD) of the effective mobility was 3.0% for CZE and 6.7% for MEKC. After applying the correction method, these values became 3.0% for CZE and 3.3% for MEKC, which is adequate for systematic toxicological analysis (STA) applications. A significant improvement of reproducibility for the calculated corrected effective mobility mu(eff)c was observed when variations are high. Therefore, it is recommended to use the correction method in interlaboratory situations, especially when instruments and capillaries from different manufacturers are used.