Coupling of capillary electrochromatography to coordination ion spray mass spectrometry, a novel detection method.

Miniaturized separation techniques such as capillary electrochromatography (CEC), pressurized capillary electrochromatography (pCEC) and capillary high performance liquid chromatography (CHPLC) have been coupled to a new detection technique: coordination ion spray mass spectrometry (CIS-MS). Electrospray ionization (ESI) has found widespread applications in mass spectrometry (MS) for the analysis of polar compounds such as peptides or nucleotides. However, for weakly polar or nonpolar substances, ESI-MS yields poor sensitivity since, in the absence of basic or acidic groups, protonation or deprotonation is not possible. CIS is a universal ionization technique capable of detecting these compounds. Through the addition of a central complexing ion, charged coordination compounds are formed, enabling the detection with good sensitivity. Using the coaxial sheath flow interface commonly employed in CE-MS coupling, we were able to separate and detect various important natural compounds such as unsaturated fatty acid methyl esters, vitamins D2 and D3, and four different estrogens. A central ion solution of 100 microg/mL AgNO3 in water was used as sheath flow liquid, resulting in the formation of positively charged coordination compounds.     

Simplifying capillary electrophoresis-mass spectrometry operation: eliminating capillary derivatization by using self-coating background electrolytes

To simplify capillary electrophoresis-mass spectrometry (CE-MS) operation, a background electrolyte (BGE) containing a polymer additive is introduced that allows the analysis of peptides and protein mixtures in underivatized fused-silica capillaries without any pretreatment, thereby increasing throughput. The most important characteristic of these polymer additives is that they do not significantly suppress the signals of the proteins and peptides under electrospray ionization, thereby allowing them to be used as an additive to common BGEs that are used for CE-MS analysis of peptide and protein mixtures. In addition, because the fused-silica capillary inner wall is continuously coated with the polymer additive, migration irreproducibility, due to the degradation of the capillary inner wall coating, under CE-MS is minimized. High sensitivity of detection, migration reproducibility, and ease of fabrication allow CE-MS analyses that require long analysis time, such as (CE-MS/MS)n, to be performed with ease. The utility of this background electrolyte has been demonstrated for the analysis of complex protein digests and intact proteins.     

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.     

CE-MSn of complex pectin-derived oligomers

As pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers, which are often analyzed by high-performance anion exchange chromatography (HPAEC). However, the high salt concentration necessary to elute pectin oligomers by HPAEC is incompatible with online mass detection. To overcome such a disadvantage, a CE-IT-MS system was set up to further elucidate the fine structure of charged oligosaccharides. An effective separation of differently substituted galacturonic acid containing oligomers was obtained by low-pH CE-LIF analysis. By adapting the buffer and capillary online MS detection was enabled. Moreover, with MS/MS it was possible to localize sugar residues' substitutions. With this combined CE-MS approach LIF electropherograms of xylogalacturonan and rhamnogalacturonan I digests could be annotated. The method was further exemplified by a complex oligomer mixture of acid hydrolyzed apple pectin, which was separated and characterized by CE-MSn. Oligomers present in low amounts could be localized by their corresponding m/z, as was demonstrated by selected mass range representation.     

Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry.

The application of capillary electrophoresis-mass spectrometry (CE-MS) to the analysis of compounds of concern to the aquaculture industry is reported. Two different approaches to coupling the CE column to an IonSpray atmospheric pressure ionization (API) interface, viz., a liquid-junction and a coaxial arrangement, are describe and compared with regard to ruggedness, ease of use, sensitivity and electrophoretic performance. The different injection modes used in three commercial capillary electrophoresis systems were also evaluated for their applicability to CE-MS. The use of CE-MS for the analysis of a variety of classes of antibiotics used in the fish aquaculture industry, such as the sulfonamides and their potentiators (e.g., trimethoprim), is demonstrated and was used to confirm the presence of these components in shellfish extracts at the low ppm level. CE-MS was also applied to the analysis of marine toxins such as saxitoxin and its analogues which are associated with paralytic shellfish poisoning, and also the toxins responsible for amnesic and diarrheic shellfish poisoning. Tandem mass spectrometry (MS-MS) was used to provide structural information on these analytes, and the ability to distinguish isomeric compounds based on their different migration and fragmentation characteristics using CE-MS-MS is demonstrated.     

Chiral capillary electrophoresis-mass spectrometry: modes and applications

A review is presented to highlight several approaches for coupling capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) for analysis of chiral compounds. A short discussion of commercially available CE-MS instruments and interface design is followed by a detail review on various modes of chiral CE-MS. In general, for each CE-MS mode, the capabilities, applications and limitations for chiral analysis have been pointed out. The first mode, chiral capillary zone electrophoresis-mass spectrometry (CZE-MS) in which neutral derivatized cyclodextrins (CDs) are used is possible using either column coupling with voltage switching or a partial-filling technique (PFT). However, some applications of direct coupling of CZE-MS mode are also reported. The second mode is a chiral electrokinetic chromatography-mass spectrometry (EKC-MS) in which a charged chiral selector such as a sulfated beta-CD or a vancomycin could be conveniently employed. This is because these chiral selectors have a significantly higher countercurrent electrophoretic mobility which prevents the entrance of these selectors into the mass spectrometer. The combination of counter-migration and PFT demonstrates that this synergism could be successfully applied to chiral analysis of a broader range of compounds. It is well-known that the on-line coupling of micellar electrokinetic chromatography to mass spectrometry (MEKC-MS) is problematic because the high surface activity and nonvolatile nature of conventional surfactant molecules lower the electrospray ionization efficiency. However, a recent report demonstrates that this hyphenation is now possible with the use of molecular micelles. Various MEKC-ESI-MS parameters that can be used to optimize both chiral resolution and ESI response are discussed. Finally, two recent examples that demonstrate the feasibility of using either open-tubular or packed chiral CEC with MS are reviewed. This survey will attempt to cover the state-of-the-art on various modes of CE-MS from 1998 up to 2002.     

Comparison of high-performance liquid chromatography — Mass spectroscopy and capillary electrophoresis— mass spectroscopy for the analysis of phenolic compounds in diethyl ether extracts of red wines

Summary Reversed-phase high-performance liquid chromatography-mass spectroscopy (HPLC-MS) and capillary electrophoresis-mass spectroscopy (CE-MS) have been compared for the analysis of phenolic compounds in diethyl ether extracts of red wines. MS was performed in the electrospray negative-ionization mode. Despite the much higher separation efficiency of CE compared with LC, LC-MS furnishes far superior information for elucidation of the structure of the constituents. LC-MS enabled the identification of twenty-four compounds whereas only thirteen were characterized by CE-MS.     

Capillary electrophoresis-mass spectrometry for impurity profiling of basic pharmaceuticals using non-volatile background electrolytes

A generic approach has been developed for coupling capillary electrophoresis (CE) using non-volatile background electrolytes (BGEs) with mass spectrometry (MS) using a sheath liquid interface. CE-MS has been applied for basic and bi-functional compounds using a BGE consisting of 100 mM of TRIS adjusted to pH 2.5 using phosphoric acid. A liquid sheath effect is observed which may influence the CZE separation and hence may complicate the correlation between CE-UV and CE-MS methods. The influence of the liquid sheath effect on the migration behavior of basic pharmaceuticals has been studied by simulation experiments, in which the BGE outlet vial is replaced by sheath liquid in a CE-UV experiment. As a consequence of the liquid sheath effect, phosphate based BGEs can be used without significant loss of MS sensitivity compared to volatile BGEs. The use of buffer constituents such as TRIS can lead to lower detection limits as loss of MS sensitivity can be compensated by better CE performance. TRIS based BGEs permit relatively high injection amounts of about 100 pmol while maintaining high resolution. The ESI-MS parameters were optimized for a generic method with maximum sensitivity and stable operation, in which the composition of the sheath liquid and the position of the capillary were found to be important. Furthermore, the nebulizing pressure strongly influenced the separation efficiency. The system showed stable performance for several days and a reproducibility of about 15% RSD in peak area has been obtained. Nearly all test compounds used in this study could be analyzed with an MS detection limit of 0.05% measured in scan mode using extracted ion chromatograms. As a result, CE-MS was found to be a valuable analytical tool for pharmaceutical impurity profiling.     

Relevance and use of capillary coatings in capillary electrophoresis–mass spectrometry

Over the last two decades, coupled capillary electrophoresis (CE)–mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS applications make use of capillaries where the internal wall is modified with surface coating agents. In CE-MS, capillary coatings are used to prevent analyte adsorption and to provide appropriate conditions for CE-MS interfacing. This paper gives an overview of the various capillary coating strategies used in CE-MS. The main attention is devoted to the way coatings can contribute to a proper CE-MS operation. The foremost capillary coating methods are discussed with emphasis on their compatibility with MS detection. The role of capillary coatings in the control of the electroosmotic flow and the consequences for CE-MS coupling are treated. Subsequently, an overview of reported applications of CE-MS employing different coating principles is presented. Selected examples are given to illustrate the usefulness of the coatings and the overall applicability of the CE-MS systems. It is concluded that capillary coatings can enhance the performance and stability of CE-MS systems, yielding a highly valuable and reproducible analytical tool.     

Protein micropatterning on bifunctional organic-inorganic sol-gel hybrid materials

Active protein micropatterns and microarrays made by selective localization are popular candidates for medical diagnostics, such as biosensors, bioMEMS, and basic protein studies. In this paper, we present a simple fabrication process of thick (approximately 20 microm) protein micropatterning using capillary force lithography with bifunctional sol-gel hybrid materials. Because bifunctional sol-gel hybrid material can have both an amine function for linking with protein and a methacryl function for photocuring, proteins such as streptavidin can be immobilized directly on thick bifunctional sol-gel hybrid micropatterns. Another advantage of the bifunctional sol-gel hybrid materials is the high selective stability of the amine group on bifunctional sol-gel hybrid patterns. Because amine function is regularly contained in each siloxane oligomers, immobilizing sites for streptavidin are widely distributed on the surface of thick hybrid micropatterns. The micropatterning processes of active proteins using efficient bifunctional sol-gel hybrid materials will be useful for the development of future bioengineered systems because they can save several processing steps and reduce costs.     

Solid-phase extraction for metabolomic analysis of high-salinity samples by capillary electrophoresis-mass spectrometry

Environmental samples such as soil solutions contain inorganic ions such as NH4(+), K(+), Na(+), NO3(-), and PO4(3-) in high concentrations, which must be removed prior to capillary electrophoresis-mass spectrometry analysis to obtain accurate results. However, the separation of these inorganic ions from ionic metabolites, which are the target compounds in capillary electrophoresis-mass spectrometry analysis, is difficult because the physicochemical properties of the inorganic ions are similar to those of the ionic metabolites. In this study, we used various solid-phase extraction (SPE) columns for the purification of the samples containing inorganic ions in high concentrations. We found that cation-exchange SPE columns successfully filtered out the inorganic ions while retaining most of the organic compounds, which were easily collected with high recovery rates. In addition, 17 cationic metabolites in the soil solution were quantified by CE-MS analysis following the SPE purification process. The results suggest that our method can be used to analyze other environmental samples containing inorganic ions in high concentrations.     

Using Taguchi experimental design to reveal the impact of parameters affecting the abrasion resistance of sol–gel based UV curable nanocomposite films on polycarbonate

This work aims at studying the abrasion resistance of differently formulated organic–inorganic hybrid coatings prepared by sol–gel method. The organic phase contained UV curable urethane acrylate oligomers and monomers having different functionalities. The inorganic phase was composed of tetraethyl orthosilicate (TEOS) and 3-methacryloxy propyltrimethoxy silane (MEMO). Through a Taguchi experimental design, the impact of influencing parameters such as molar ratio of precursors, hydrolysis ratio (R), post-curing temperature, post-curing time and weight percentage of inorganic to organic part were investigated. Very high transparency of hybrid coatings indicated that nano sized inorganic phase had formed. MEMO could facilitate the connection of two phases, preventing macro phase separation. However, high levels of MEMO lead to ‘defect structure’ in silica network as well as to decreased transparency and mechanical properties. The optimum condition in which highly transparent films with great abrasion resistance occurred was observed at equimolar ratio of water to alkoxide and TEOS: MEMO ratio being unity. Statistical analysis revealed that thermal post-curing was not significantly important.     

Capillary electrophoresis-mass spectrometry: recent advances to the analysis of small achiral and chiral solutes

We describe here the state-of-the-art development of on-line capillary electrophoresis-mass spectrometry (CE-MS) over the last two years. Technological developments included are novel designs of new interfaces and ionization sources, new capillary coatings, buffers, and micelles as well as application of various modes of CE-MS published in the recent literature. The areas of CE-MS application in analysis of small achiral and chiral solutes are covered in sections that highlight the recent advances and possibilities of each mode of CE-MS. Application areas reviewed in this paper include achiral and chiral pharmaceuticals, agrochemicals, carbohydrates, and small peptides. The separation of enantiomers using micellar electrokinetic chromatography (MEKC)-MS with molecular micelles and capillary electrochromatography (CEC)-MS using pack tapered columns appears to provide good tolerance to electrospray stability for routine on-line CE-MS. These two modes seem to be very suitable for sensitive detection of chiral pharmaceuticals in biological samples, but their use will probably increase in the near future. Overall, it seems that one mode of CE-MS, in particular capillary zone electrophoresis (CZE)-MS, is now recognized as established technique for analysis of small charged solutes, but other modes, such as MEKC-MS and CEC-MS, are still within a period of development in terms of both MS-compatible pseudostationary phases and columns as well as applications.     

Capillary electrophoresis-mass spectrometry of a new cross-linker with acrylic functionality

Analytical characterization of dimethacrylate-tyrosine-lysine-tyrosine (DMTLT, a new biodegradable acrylic cross-linker synthesized at our laboratory) is carried out using CE-MS. DMTLT is a pseudopeptide composed by tyrosine-lysine-tyrosine amino acids linked through urea bonds with two methacrylic groups, one at each end of the molecule, making this compound an excellent cross-linker for polymerization reactions and for obtaining new biodegradable materials. A new CE-MS method is developed for the characterization of DMTLT and its products of degradation after basic hydrolysis. In order to carry out an exhaustive examination of such degradation products methods based on CE coupled to IT and TOF-MS are employed. Based on CE-IT-MS results and the elemental composition of the degradation products obtained by CE-TOF-MS, conclusions on the mechanism and kinetic of hydrolysis of DMTLT are obtained confirming both the usefulness of CE-MS to characterize new biomaterials and the applicability of DMTLT for preparing new biodegradable polymers. These results are corroborated through the CE-MS detection of the identified products of degradation in a dimethyl acrylamide polymer cross-linked with DMTLT.     

Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis-mass spectrometry (CE-MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE-MS using the same porous tip capillary and an isopropanol-water-acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE-MS, detection limits were improved by a factor 6.5-20 with sheathless CE-MS. The analyte response in sheathless CE-MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary - equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE-MS of intact proteins.     

Separation of enantiomers by capillary electrophoresis-mass spectrometry employing a partial filling technique with a chiral crown ether

Enantiomer separations were performed by capillary electrophoresis-mass spectrometry (CE-MS) with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) as a chiral selector. In order to prevent the introduction of the nonvolatile chiral, selector, 18C6H4, into the nozzle of the CE-MS interface and/or the orifice plate, a partial filling technique was employed in this study. By the partial filling technique, the contamination caused by the nonvolatile chiral selector was avoided not only during the analysis but also during the washing of capillary with the separation solution prior to the run. Several racemic compounds having a primary amino group were successfully separated. Racemic 3-aminopyrrolidine and racemic alpha-amino-epsilon-caprolactam have no strong UV absorption, but such compounds were detected with a high sensitivity by MS detection. In this paper, the effects of the length of separation zone and those of the 18C6H4 concentration were described. As the length of the separation zone was longer or as the concentration of 18C6H4 was higher, the enantiomer resolution was enhanced more and more. However, the optimization of 18C6H4 concentration was practically enough to obtain the baseline separation.     

Challenges and applications of isotachophoresis coupled to mass spectrometry: A review

Electrophoretic separations are of growing interest to tackle complex analytical challenges. Nevertheless, capillary electrophoresis, as the most common mode, still suffers from insufficient detection limits due to low capillary loadability. ITP is of growing interest as preconcentration method for capillary electrophoresis and is also interesting to be applied as an independent analytical method. While mass spectrometric detection is common for capillary electrophoresis, the combination of ITP with MS is still a niche technique. In this work, we want to give an overview on isotachophoretic effects in CE-MS and ITP-MS methods, as well as coupling techniques of ITP with CE-MS. The challenges and possibilities associated with mass spectrometric detection in ITP and its coupling to capillary electrophoresis are critically discussed.     

Analysis and validation of the phosphorylated metabolites of two anti-human immunodeficiency virus nucleotides (stavudine and didanosine) by pressure-assisted CE-ESI-MS/MS in cell extracts: sensitivity enhancement by the use of perfluorinated acids and alcohols as coaxial sheath-liquid make-up constituents

A CE method utilizing triple quadrupole electrospray (ES) MS (MS/MS) detection was developed and validated for the simultaneous measurement of nucleoside 5'-triphosphate and 5'-monophosphate anabolites of the anti-HIV (human immunodeficiency virus) didanosine (ddAMP, ddATP) and stavudine (d4TMP, d4TTP), among a pool of 14 endogenous 5'-mono-, di-, and triphosphate nucleosides. These compounds were spiked and extracted from peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. An acetic acid/ammonia buffer (pH 10, ionic strength of 40 mM) was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of +30 kV and an overimposed pressure of 28 mbar (0.4 psi). The application of pressure assistance was needed to provide stable ES conditions for successful coupling. The coupling was carried out with a modified sheath-flow interface, with one uninterrupted capillary (80 cmx 50 microm id; 192 microm od) in a dimension that fits into the ESI needle to get a stable ion spray. Some CE-MS parameters such as overimposed pressure, sheath-liquid composition, sheath-liquid and sheath-gas flow rates, ES voltage, and the CE capillary position were optimized in order to obtain an optimal sensitivity. The use of perfluorinated alcohols and acids in the coaxial sheath-liquid make-up (2,2,2-trifluoroethanol + 0.2 mM tridecafluoroheptanoic acid) appeared to provide the best MS sensitivity and improve the stability of spray. The linearity of the CE-MS and CE-MS/MS methods was checked under these conditions. Validation parameters such as accuracy, intraday and interday precision, and LOQs were determined in CE-MS/MS mode. Finally, the quantitation of d4T-TP and ddA-TP was validated in this CE-MS/MS system.     

Solvent-resistant sol-gel polydimethyldiphenylsiloxane coating for on-line hyphenation of capillary microextraction with high-performance liquid chromatography

A sol-gel polydimethyldiphenylsiloxane (PDMDPS) coating was developed for capillary microextraction on-line hyphenated with high-performance liquid chromatography (HPLC). This coating was created using methyltrimethoxysilane (MTMS) as the sol-gel precursor and di-hydroxy-terminated PDMDPS as the sol-gel active polymer. The methyl and phenyl groups on the sol-gel active polymer and the methyl groups on the sol-gel precursor ultimately turned into pendant groups providing the ability to extract non-polar analytes. A 40-cm segment of 0.25mm I.D. fused silica capillary containing the sol-gel PDMDPS coating was installed as an external sampling loop in an HPLC injection port. Aqueous samples containing polycyclic aromatic hydrocarbons (PAHs), aromatic compounds, ketones, and aldehydes were passed through this capillary wherein the analytes were extracted by the sol-gel coating. The extracted analytes were then transferred to the HPLC column using isocratic or gradient elution with an acetonitrile/water mobile phase. This capillary demonstrated excellent extraction capability for non-polar (e.g., polycyclic aromatic hydrocarbons and aromatic compounds) as well as moderately polar compounds, such as aromatic amines, ketones, and aldehydes. The test results indicate that PDMDPS can be successfully immobilized into a sol-gel network and that the resulting solvent-resistant sol-gel organic-inorganic hybrid coating can be effectively used for on-line hyphenation of capillary microextraction with high-performance liquid chromatography. The test results also indicate that the sol-gel PDMDPS coated capillary is resistant to high-temperature solvents, making it suitable for applications in high-temperature HPLC. To the best of our knowledge, this is the first report on the creation of a silica-based sol-gel PDMDPS coating used in capillary microextraction on-line hyphenated to HPLC.     

Mass Spectrometric and Contactless Conductivity Detection Approaches in the Determination of Muscle Relaxants by Capillary Electrophoresis

Novel and rapid capillary electrophoresis-coupled tandem mass spectrometry (CE-MS/MS) and capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C⁴D) methods have been developed for the separation and determination of three neuromuscular blocking agents: pancuronium, vecuronium, and rocuronium. In both cases, the separation was conducted in background electrolytes based on acidic acetate-ammonium buffers to avoid possible decomposition of the analytes that are known to be unstable in alkaline media. Baseline resolution of the analytes was achieved in the presence of modified γ-cyclodextrin by CE with C⁴D detection. The two detection techniques were compared with regard to analytical figures of merit including linear dynamic range, limit of detection, limit of quantification, precision, and accuracy. The calibration curves showed good linearity for both detection methods examined (characterized by r² ≥ 0.9908). The LODs of the CE-MS/MS and the CE-C⁴D methods differed at least by two orders of magnitude considering all analytes. The differences in precision and accuracy of these methods were evaluated and discussed. The assays of pancuronium, vecuronium, and rocuronium in commercial injection solutions by CE-MS/MS and CE-C⁴D were performed and the results compared.