CEC-MS applications

Although the majority of chiral CEC—MS applications still involve packed columns, few reports on chiral OT-CEC-MS are found in recent literature. The feasibility of coupling OT-CEC (using a short Chirasil-Dex-coated capillary column) to MS and MS/MS for trace analysis of hexobarbital enantiomers in biological fluids was reported by Schurig and Mayer. More recently, Kamande et al. investigated polyelectrolyte multilayer (PEM) coating as a new medium for the separation of chiral analytes, and PEM-coated capillaries were successfully coupled to ESI/MS for the stereoselective analysis of five /1-blockers. 

The great majority of CEC—MS applications is run in the reversed-phase mode using alkyl-bonded silica stationary phases [12,14,24,26,38,45,81,82,98-104], The dual functionality concept is represented in these stationary phases by the alkyl chains, most frequently octadecyl chains, that constitute the top retentive layer, and residual silanol groups on the surface, that dissociate at pH values higher than 3-4 and... 

The need of column configurations and surface chemistries especially designed for CEC is now generally appreciated and novel approaches to improve the column technology for CEC—MS applications include the use of monolithic stationary phases [109,110], open-tubular capillary columns [86] and chip technology [111]. These configurations are currently under detailed investigation and the future will have to prove their applicability in routine analysis. 

The ionspray (ISP, or pneumatically assisted electrospray) LC-MS interface offers all the benefits of electrospray ionisation with the additional advantages of accommodating a wide liquid flow range (up to 1 rnl.rnin ) and improved ion current stability [536]. In most LC-MS applications, one aims at introducing the highest possible flow-rate to the interface. While early ESI interfaces show best performance at 5-l() iLrnin, ion-spray interfaces are optimised for flow-rates between 50 and 200 xLmin 1. A gradient capillary HPLC system (320 xm i.d., 3-5 xLmin 1) is ideally suited for direct coupling to an electrospray mass spectrometer [537]. In sample-limited cases, nano-ISP interfaces are applied which can efficiently be operated at sub-p,Lmin 1 flow-rates [538,539]. These flow-rates are directly compatible with micro- and capillary HPLC systems, and with other separation techniques (CE, CEC). 

The coupling of a mass spectrometer to CE and CEC provides a powerful system for the analysis of pharmaceuticals and complex biological mixtures. This can replace or complement other conventional detection methods such as UV, electrochemical, or LIE that provide less structural information. The use of mass spectrometer as a detector enhances the usefulness of the CE and CEC and allows an efficient separation and identification of complex mixtures, obtaining structure and/or molecular mass information. The choice of mass analyzers used in CE/CEC-MS depends on factors such as sensitivity, mass resolution, requirement for structural elucidation, and the type of application (Table 5). The analyzers that have been used in CEC analysis include time-of-flight (TOE), quadrupole (Q), ion-trap (IT), fourier... 

Paterson, C. J., Boughtflower, R. J., Higton, D., and Palmer, E. (1997). An investigation into the application of capillary electrochromatography-mass spectrometry (CEC-MS) for the analysis and quantification of a potential drug candidate in extracted plasma. Chromatographia 46, 599-604. 

CEC—MS has now been applied to the analysis of a wide range of species, from small ions to biomolecules. Since CEC—MS is a less familiar technique and instrumentation and column technology still needs to be improved, the first wave of reports are those investigating the general applicability of the technique to solve analytical problems. Yet, applications of CEC—MS to routine, qualitative and quantitative analysis are relatively few. Gradually, however, more real problems are being examined and the number of reports describing such real application is expected to increase rapidly. 

The analysis of compounds of pharmaceutical relevance is one of the most promising application areas for CEC—MS, because it offers high sensitivity, high selectivity and structural information [38,98,99,105], Paterson et al. [105] utilized CEC—ESI-MS for the analysis of potential drug candidates down to the 1 ng/ml level in solid-phase extracts of plasma samples. Relative standard deviations of retention time and peak area of 0.4-1.7% and 2.6-10.7%, respectively, were achieved, which proves, that the method is also applicable to quantitative analysis. The analysis of a pair of... 

CEC is a miniaturized separation technique that combines capabilities of both interactive chromatography and CE. In Chapter 17, the theory of CEC and the factors affecting separation, such as the stationary phase and mobile phase, are discussed. The chapter focuses on the preparation of various types of columns used in CEC and describes the progress made in the development of open-tubular, particle-packed, and monolithic columns. The detection techniques in CEC, such as traditional UV detection and improvements made by coupling with more sensitive detectors like mass spectrometry (MS), are also described. Furthermore, some of the applications of CEC in the analysis of pharmaceuticals and biotechnology products are provided. 

Dedicated applications of capillary zone electrophoresis (CZE) coupled to MS are discussed, particularly in the field of drug analysis. Development of other capillary-based electrodriven separation techniques such as non-aqueous capillary electrophoresis (NACE), micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC) hyphenated with MS are also treated. The successful coupling of these electromigration schemes with MS detection provides an efficient and sensitive analytical tool for the separation, quantitation, and identification of numerous pharmaceutical, biological, therapeutic, and environmental compounds. 

The present chapter will review instrumental aspects for successful coupling of CE with MS, regarding interfaces, ionization sources, and analyzers. Practical considerations concerning different CE modes such as CZE, NACE, MEKC, and CEC coupled with MS will also be discussed and illustrated with a focus on recent pharmaceutical applications. Additionally, quantitative CE-MS will be presented and various methodologies used to achieve sensitive and repeatable analysis will be discussed. Finally, the final section of this chapter will give an overview on new devices (i.e., microchips), hyphenated to MS, in terms of fabrication methods, microchip designs, MS interfacing, and applications. 

In view of the increasing level of research activities associated with the application of HP-CEC for peptide analysis, it can be confidently expected that new miniaturized, high-throughput separation systems will become available over the next several years, fully integrated with MS/MS instrumentation and other types of spectrometers capable of determining structural characteristics of the resolved peptides at the low nanomole level. 

Recently, the principal applicability of CEC to the separation of nucleotides trans-fer-RNAs has been demonstrated using an octadecyl-sulfonated mixed mode silica stationary phase. Although UV absorbance was used for detection of the analytes, it can be expected that such a separation system will be coupled to ESI-MS in the near future. Presently, our laboratory is working on the development of monolithic capillary columns for ion-pair reversed-phase CEC—ESI-MS of nucleic acids. 

The main advantage of OT CEC is that separation efficiency can be doubled using this type of column. The trade-off is that the OT columns can easily be overloaded and therefore require a sensitive detection system. The small diameter of these columns precludes the use of UV detection, and fluorometric detection or mass spectrometry (MS) needs to be used. The use of fused silica capillaries with a bubble cell at the detection window has been reported as an alternative to employ UV detection. This features limit, to a certain extent, the range of practical applications of OT CEC. 

An excellent demonstration of the concept of niche application in CEC was offered by Lurie et al. in a study of CEC feasibility for analysis of seized drugs [66], In (he past, standard separation methods such as GC, GC/MS, HPLC, and CE have been employed in the analysis of cannabinoids, weakly acidic... 

There are many different types of detectors used for pharmaceutical applications in CEC. They vary from indicating just the presence of a sample [fluorescence (FL)], to giving some qualitative information about a sample [photodiode array UV/vis detection (PDA)], to absolute sample determination of the analyte (MS). The methods can be on-column, off-column, and end-column. With on-column, the solutes are detected while stiU on the capillary, in off-column, the solute is transported from the outlet of the capillary to the detector, and end-column is done with the detector placed right at the end of the capillary. Some modes of detection used in CEC are as follows ... 

In the references to the application of CEC to biopolymers, most of the work discusses CEC-electrospray ioni-zation(ESI)-MS, much less to direct CEC-UV/FL methods. However, much of the work has evolved from the use of commercially available, prepacked capillaries, such as Cig or ion exchange or a mixed mode containing both ion exchange and reversed phase. There are very few articles that have actually attempted to develop new phases specifically for biopolymers. 

Recently, a variety of peptides and proteins were analyzed by CE-MS techniques, which include CZE-MALDI-TOF-MS, CZE-ESI-MS, CIEF-ESI-MS, and CEC-ESI-MS. Their applications are well summarized in a recent review paper [50],... 

Although the number of papers reporting CEC-ESI-MS is not very many, CEC-ESI-MS has been applied to the analysis of a wide range of species from small ions to biomolecules. Several typical examples of applications are selected for review in the following. 

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