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In this work, we demonstrate the applicability of electroextraction (EE) to urine metabolites. To investigate which urine metabolite classes are susceptible to EE, off-line EE experiments were carried out with a prototype device, in which urine metabolites were electroextracted from ethyl acetate into water. The obtained extracts were examined with direct infusion MS and the results demonstrated that several compound classes could be extracted, amongst which amino acids and acylcarnitines. Acylcarnitines were selected for evaluation of the performance of EE. For this, the EE setup was adapted to capillary EE (cEE) to be able to analyze large urine sample series, and it was coupled online to LC-MS. cEE-LC-MS of acylcarnitines was optimized and characterized. The recovery, linearity, repeatability, and detection limit of the cEE-LC-MS method was good to excellent. To demonstrate the versatility of EE for sample preparation in analytical procedures, extracts were injected into a CZE-MS system, resulting in detection of the acylcarnitines along with more than 100 presumed metabolite peaks. The results presented here indicate that EE can be used as a fast sample preconcentration technique of low abundant urine metabolites, in combination with both LC and CZE., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this research paper, we show that capillary electroextraction (cEE) is capable of fast online peptide concentration and that it can be coupled online to LC-MS to result in a fast and sensitive method. Electroextraction takes place when an electrical field is applied in a two-phase liquid-liquid system. Sample molecules in the organic phase migrate very fast into the aqueous phase and are concentrated in a small zone. In this work, cEE of peptides is developed and coupled online to LC-MS via a switching valve. Comparison of 10 min of cEE-LC-MS with a normal LC-MS injection showed more than 100-fold increased peak heights. Of five model peptides, good calibration curves in the range of 0.05-5 μmol/L were obtained. The linearity was good (R(2) values between 0.984 and 0.996) and RSD between 5% at the highest to 25% at the lowest concentration (n=3). The LOD of bradykinin, angiotensin I-converting enzyme inhibitor and angiotensin I was in the low nmol/L range. Analysis of a tryptic digest of eight model proteins resulted in more than 170 peptides, without bias for pI or hydrophilicity. Urine analysis is demonstrated, resulting in an LOD around 0.04 μmol/L urine for tryptic cytochrome C peptides spiked to urine and an increase of 42% in the number of chromatographic peaks compared with the conventional LC-MS. In summary, cEE-LC-MS is a fast electrophoresis-driven sample preconcentration technique that is quantitative, able to extract a wide peptide range and applicable to bioanalysis.

In this study, the potential of poly(methylmethacrylate) (PMMA, Plexiglas) and polyether ether ketone (PEEK) tubing for CE-ESI-MS separations of anions at high pH values was examined. A set of model compounds of biological interest was used to investigate the main operational parameters for CE-ESI-MS, such as the sheath-flow interface design, the polarity of the ionization voltage, the use of ammonia-based separation electrolytes, and the sheath liquid composition. Optimum separations and detection sensitivities in negative ESI mode were obtained using a running electrolyte of 75 mM of ammonia at pH 11 and a sheath liquid of 60:40 v/v or 75:25 v/v isopropanol/water with 0.5% v/v of ammonia. At these experimental conditions, PMMA and PEEK capillaries show good hydrolytic stabilities and lower EOF values than fused-silica columns. Better separation resolutions were obtained with PMMA capillary, but this plastic rapidly swelled and bled because of its limited chemical resistance to the sheath liquid. PMMA columns equipped with a fused-silica tip were used for a safer exposure to the sheath liquid, but the inner surface of the fused-silica tips had limited stability at pH 11. On the other hand, good separations and reproducibility on migration times and peak areas were obtained using PEEK capillaries without capillary column deterioration.

We investigated the potential of CE coupled to electrospray MS (CE-ESI-MS) in metabolite profiling of human urine without any sample prefractionation step. A heterogeneous mixture of biologically relevant compounds covering a broad range of physicochemical properties was used to optimize separation conditions in fused-silica capillaries. A running electrolyte containing 50 mM of acetic acid and 50 mM of formic acid at pH 2.5 was used for the CE separations. A sheath-flow electrospray interface was employed for CE-ESI-MS analysis. Sheath liquids containing 80:20 v/v methanol/water with 0.1% v/v of acetic acid or 60:40 v/v isopropanol/water with 0.5% v/v of ammonia were selected for optimum detection in positive and negative ESI modes, respectively. Reproducibility and sensitivity were studied, and strategies for identification of the separated urinary compounds are suggested. We report major advantages and disadvantages of CE-ESI-MS for metabolite profiling of human body fluids. This work may be regarded as a first step in the use of CE-ESI-MS for reliable differential analysis of body fluids from healthy and diseased individuals.

Proteolytic (18)O labeling is a very powerful tool for differential analysis applied to proteome studies. However, it is a relatively new technique and the optimization of the labeling process still needs some attention. We found that the two-step post-proteolytic labeling should be favored over the conventional digestion of proteins in H(2) (18)O, since the former allows for higher sample concentrations and thus more favorable kinetics. It was demonstrated that the inhibitory effect of urea on (18)O incorporation could be compensated by the use of higher sample concentrations. Furthermore, it was shown that heat-deactivation of trypsin prevents (18)O/(16)O back-exchange. In addition, no non-specific hydrolysis of the peptides could be observed as a result of the heating. Heat inactivation of trypsin opens the way for the use of capillary electrophoresis as a separation technique in proteolytic labeling studies, as it abolishes the need for use of detrimental additives. Analysis of a labeled protein digest by capillary isoelectric focusing/mass spectrometry showed the applicability of the method. No back-exchange was observed across the entire electropherogram., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Capillary isoelectric focusing (CIEF) is a high-resolution separation technique for the analysis of peptides and protein digests. When coupled to ion trap-mass spectrometry (CIEF-MS) the unique separation mechanism is combined with a highly efficient detection system. In an earlier report, we described aspects of separation and interfacing in connection to the analysis of a digest of set of standard proteins. Now, we report on different aspects of the process of protein identification. Sequest software parameters were optimized by using a standard protein digest. These settings were used for the analysis of periplasmic proteins from Escherichia coli. Since in CIEF peptides are focused according to their pI values, the mobilization time of a particular peptide is dependent on its pI value. Based on this relation, the identification of some peptides was facilitated. Furthermore, the Sequest settings that were used could be evaluated. In total, 159 proteins were identified in a single run.

We report on capillary isoelectric focusing-mass spectrometry (CIEF-MS) of complex peptide mixtures in the absence of carrier ampholytes. Furthermore, the use of low concentrations of carrier ampholytes as mere spacers is investigated. Carrier ampholytes are complex mixtures of amphoteric compounds with high buffering capacity. Since all peptides are amphoteric compounds by themselves, the use of carrier ampholytes may be superfluous to establish a stable pH gradient in CIEF analysis of protein digests. Our research showed that when carrier ampholytes are omitted, the analyte ions are not focused at their isoelectric point. The analytes are charged, leading to electrophoretic mobility uncharacteristic for CIEF. The method was tested for a five-protein-mixture at 0.02 mg/mL per protein and 0.05 mg/mL per protein. At the lower concentration, the analytes were stacked during the focusing process in only a limited length of the capillary. Therefore, the higher concentration led to better separation efficiency. It was found that at low concentration (0.20%) the carrier ampholytes could work as spacers. Though it led to sensitivity losses of 15-45%, this was compensated by the higher separation efficiencies seen. The method was evaluated with an eight-protein-mixture, of which all could be identified after performing MS/MS.

Capillary separations of proteins using carrier ampholytes are performed between an anolyte and a catholyte of same pH (pH 3). Depending upon the concentration of carrier ampholytes used, two different separation processes take place. At a 10% concentration, the high-resolution separation of six model proteins is achieved, which can be described as a transient capillary isoelectric focusing (cIEF) system moving isotachophoretically. The isotachophoretic (ITP) behaviour of the system is evidenced by the influence of the catholyte concentration on the separation. The separation is neither pure cIEF nor pure cITP and the migration order of the proteins results from the influence of both their isolelectric points and their mobilities.

The on-line combination of capillary electrophoresis and electrospray ionisation mass spectrometry was applied for the determination of some basic histones from calf thymus. The separation performance of those basic proteins was significantly improved by coating the capillaries with hydroxypropylmethylcellulose. The coating appeared to mask effectively the underlying silanol groups thus avoiding undesirable adsorption of the histones onto the capillary walls, while it was also shown to be an effective way to avoid contamination of the mass spectrometer. Finally, capillary electrophoresiselectrospray ionisation mass spectrometry with coaxial sheath liquid was successfully applied to the analysis of histones using a simple dialysis step of the sample as sample pretreatment.

Platinum adducts are supposed to be the cytotoxic lesions in DNA after platinum-containing anticancer therapy. Various adducts are formed upon interaction of platinum complexes with nucleotides, but contribution of individual adducts to antitumor activity and toxicity of platinum complexes still remains to be examined. A capillary zone electrophoresis (CZE) method is described that is suitable to separate individual platinum adducts. We investigated the formation of adducts following the reaction of cis-diamminedichloroplatinum (II) (cisplatin) with various DNA nucleotides. Baseline separation of unmodified and modified nucleotides (adducts) was achieved using uncoated fused-silica capillaries and basic separation buffers. In order to elucidate the observed peak pattern, a coupled CZE-electrospray ionization-mass spectrometry (ESI)-MS approach was applied. After incubation of mononucleotides with cisplatin, monochloro, monoaqua and bifunctional adduct species were detected. Consequently, the migration order of nucleotides and individual platinum adducts could be determined. Moreover, the time-dependent conversion from monochloro to monoaqua and subsequently to bifunctional adducts was monitored. In conclusion, individual platinum adducts were separated by CZE and identified by CZE-ESI-MS. Formation and conversion of distinct species were confirmed. Potential applications comprise studies of novel platinum complexes, investigations of platinum-adduct formation with DNA, and determination of platinum-DNA adducts in cells.

A method for the in-line preconcentration and enantioseparation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV absorbance detection (transient ITP-CZE-UV) has been developed. It implies the use of dimethyl-beta-cyclodextrin as chiral selector and the application of a hydrodynamic counterflow during the ITP step. ITP is used to focus the sample constituents prior to CE whereas a counterpressure counterbalances the electrophoretic migration of the compounds. The sample is then focused and kept stationary in the proximity of the capillary inlet before CZE separation, leading to an extended-volume ITP-CZE system. A new strategy for the fast optimization of the counterpressure has been developed which implies the measurement of the hydrodynamic and electrophoretic velocities of the analyte during ITP. The in-line preconcentration and enantioseparation of clenbuterol selected as model compound was optimized using this method. Salbutamol was chosen as internal reference in order to check the reproducibility of the method. A 173-nl volume of aqueous ample solution was injected which implies an improvement of the injection volume of about 16 and a resolution of 4.8 was obtained for the clenbuterol enantiomers. A concentration detection limit of 10(-6) mol/l was readily achieved for clenbuterol and salbutamol using only 3 min ITP preconcentration in in-line counterflow transient ITP-CZE-UV. Thanks to its fast optimization, the method is applicable to any enantioseparation by means of only five very short preliminary measurements.

A sheathless and electrodeless nanospray interface has been used to interface a polycarbonate capillary electrophoresis (CE) chip to a mass spectrometer (MS). The chip was made of two flat polycarbonate plates which were bolted together. Channels were imprinted in one of the plates with metal wires, using a hydraulic press. A short tapered capillary connected to the chip was used as the nanospray emitter. The advantage of this electrodeless interface is that it was not necessary to apply a electrospray voltage to the chip or the nanospray emitter. Instead, the CE voltage already applied to the buffer compartment on the chip, to drive the electrophoresis, was used to generate the spray also. A low conductivity buffer of 1.25 mmol/L ammonium acetate in 80% methanol was used to obtain a large electric field across the buffer channel. The performance of the device was evaluated by analyzing a mixture of three beta-agonists Relative standard deviation (RSD) values obtained were between 4.8 and 5.0%. A sample concentration of 40 nmol/L resulted in a signal-to-noise ratio of 2 to 5 for the different components. Compared to a conventional CE analysis in a fused silica capillary with UV detection, only a minor loss of resolution was observed, which can be attributed to the design of the chip., (Copyright 2000 John Wiley & Sons, Ltd.)

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection. The on-line coupling of this system with ESI-MS was successfully realised for three model proteins (myoglobin, carbonic anhydrase I and beta-lactoglobulin B)., (Copyright 2000 John Wiley & Sons, Ltd.)

The utility of isotachophoresis-capillary zone electrophoresis (ITP-CZE) and high-performance size-exclusion chromatography (HPSEC) was investigated for determination of dimeric and monomeric recombinant human interleukin-6 (rhIL-6). Using ITP-CZE heterogeneity of dimeric rhIL-6 could be revealed resolving two peaks in the electropherograms, while with HPSEC dimeric rhIL-6 eluted as one homogeneous fraction. Both protein forms were monitored during incubation of monomeric rhIL-6 at different pH and temperature. The selectivity of counterflow ITP-CZE in conjunction with the low concentration determination limits enabled reanalysis of HPSEC fractions for identification of the dimer in the electropherograms. Both ITP-CZE and HPSEC were shown to be suitable to monitor the dimerization of rhIL-6, similar monomer-to-dimer peak area ratios were obtained throughout the incubation. Dimer formation kinetics increased with decreasing pH and with increasing temperature, it was entirely suppressed at neutral pH and room temperature. In contrast to HPSEC, ITP-CZE enabled separation of further still unidentified artifacts apparently formed during incubation of rhIL-6. CZE analysis in conjunction with electrospray ionization mass spectrometry revealed the non-covalent binding character of the dimeric rhIL-6 complex and facilitated interpretation of the electropherograms.

Purpose: Capillary zone electrophoresis with isotachophoretic sample preconcentration (ITP-CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection and on-line coupling to electrospray-ionization mass spectrometry were investigated for their potential to separate and identify fragments of recombinant human interleukin-6 formed during acidic stress of the parent protein., Results: Based on the orthogonal separation principles governing ITP-CZE and RP-HPLC, different peak patterns were observed using both methods. The selectivity of ESI-MS allowed identification of several co-migrating compounds. Data obtained by on-line ESI-MS were compared to results from off-line investigations by MALDI-TOF-MS performed with single fractions collected from the RP-HPLC system. Cleavage of the protein backbone occurred preferably at acid-labile Asp-sites. The total amount of rhIL-6 needed for ITP-CZE-ESI-MS identification of all fragments was only in the upper femtomole range, while RP-HPLC required amounts of protein three orders of magnitude higher. On the other hand, the low CE sample volume opposes the collection of fractions to perform off-line analysis., Conclusions: Growing acceptance of CE with on-line MS detection for pharrmaceutical quality control of proteins is expected.

A capillary zone electrophoretic method for the quantification of (E)-5-(2-bromovinyl)-2'-deoxyuridine in plasma has been developed and validated. Separation was performed with a 25 mmol/l borate buffer, pH 9.0, after an initial rinsing step with sodium hydroxide. The rinsing step was necessary for reproducible analyses of aqueous samples and plasma extracts obtained by C18 solid-phase extraction after deproteination with perchloric acid. No interferences with plasma compounds were observed. The calibration graph was linear over the range of 30 to 3000 ng/ml using 5-fluorouracil as external standard. The limit of quantification was 24 ng/ml. The CZE method is fast, reproducible, linear and is therefore a good alternative for the already established HPLC methods.

A method for the determination of 1,2,6-inositol trisphosphate (IP3) and derivatives in plasma by capillary zone electrophoresis with (indirect) UV detection has been developed. The sample pretreatment is based on the selective isolation after complexation of inositol phosphates with iron(III) loaded on an adsorbent. Plasma protein denaturation was performed with sodium dodecyl sulfate. The selectivity of the method is demonstrated with the analysis of phenylacetate-IP3. The recoveries amount to 65% and 88% in plasma and in water, respectively.