Structural Information from Electrospray Ionization

In the previous section, the generation and processing of molecular weight data from electrospray spectra have been described. The other great strength of mass spectrometry is its ability to generate structural information from the analyte 

The implications of charge must also be considered when constant-neutral-loss spectra are obtained because no longer is the loss necessarily of a neutral species, 

Electrospray is an unusual mass spectrometry technique in that it allows the study of the three-dimensional structure of compounds, particularly proteins, in solution as it is believed that this is relatively unchanged when ions are transferred to the vapour phase. This type of application will be discussed in more detail in Chapter 5 but attention is drawn at this point to the previous comments regarding the effect that the HPLC conditions, such as pH, may have on the appearance of an electrospray spectrum and the conformational deductions that may be made from them. 

The advent of the electrospray interface has allowed the full potential of LC-MS to be achieved. It is now probably the most widely used LC-MS interface as it is applicable to a wide range of polar and thermally labile analytes of both low and high molecular weight and is compatible with a wide range of HPLC conditions. 

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Electrospray is a soft-ionization method prodncing intact molecular species and structural information is not usually available. Electrospray sources are capable of producing structural information from cone-voltage fragmentation but these spectra are not always easily interpretable. Experimentally, the best solution is to use a mass spectrometer capable of MS-MS operation but this has not inconsequential financial implications. 

MS-MS is a term that covers a number of techniques in which two stages of mass spectrometry are used to investigate the relationship between ions found in a mass spectrum. In particular, the product-ion scan is used to derive structural information from a molecular ion generated by a soft ionization technique such as electrospray and, as such, is an alternative to CVF. The advantage of the product-ion scan over CVF is that it allows a specific ion to be selected and its fragmentation to be studied in isolation, while CVF bring about the fragmentation of all species in the ion source and this may hinder interpretation of the data obtained. 

Mass spectra are used to determine molecular weights and molecular composition (from the parent or molecular ion) and to obtain structural information from the fragmentation of the molecular ion into daughter ions. Electrospray ionization (ESI-MS) and matrix-assisted laser desorption ionization (MALDI-MS) mass spectroscopy can be used to obtain structural information about macromolecules, including proteins, polymers, and drug-DNA complexes. 

Electrospray and APCI spectra consist predominately of molecular species and the ways in which structural information may be generated from analytes ionized by these techniques have been considered in some detail. 

For the last several years, mass spectrometry with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) have determined the trends in the analysis of dyes. Since 1987, various variants of ESI have been used in which droplet formation was assisted by compressed air,[1,2] temperature (e.g. Turbo Ion Spray ) or ultrasound, and they were able to handle flow rates up to 1 2 ml min This made a combination of analytical RPLC and ESI easily and widely used. The reason why it often was (and is) used instead of a traditional UV-Vis detector is the better sensitivity and selectivity of MS in comparison with spectrophotometric detection. Apart from these advantages, MS offers easily interpretable structural information. However, various... 

Mass spectrometry is a valuble tool with which an abundancy of structural information may be obtained from a minute amount of material. Capillary electrophoresis may be interfaced with mass spectrometry by electrospray ionization [124-126] or continuous-flow, fast-atom bombardment methods [127,128]. Several reviews discuss applications of the interfacing techniques, and address the attributes and disadvantages associated with these methods [129,130]. Critical parameters involved in the optimization of CE-electrospray ionization mass spectrometry analysis have been reviewed as well [131],... 

The on-line coupling of CE with electrospray ionization mass spectrometry (CE-ESI-MS) allows high separation efficiency together with high sensitivity and selectivity as well as molecular structural information. A CE-UV-ESI-MS method was developed for the analysis of hoscyamine, scopolamine, and other tropane derivatives [131]. The differentiation of hyoscyamine from littorine, commonly encountered in plant material, was demonstrated using in-source collision-induced dissociation. The developed method was applied to the analysis of these alkaloids in Belladonna leaf extract and in Datura Candida x D. awreahairy root extract. Recently, CE coupled with electrochemiluminescence detection has been used for the determination of atropine and scopolamine in Flos daturae [132]. 

More extensive structural information is obtained when using MS-MS, where information on the peptide sequence can be extracted from the fragmentation pattern. So far, CE-MS-MS has predominantly been performed with electrospray ionization (ESI)-triple quadrupole and ESI-ion trap instruments, as reviewed in Ref. 13. The advent of ESI-TOF and ESI-quadru-pole-TOF instruments is believed to have a strong impact on CE-MS. TOF instruments require an extremely short time to produce a full mass spectrum and are especially attractive as a detection device for a separation technique producing sharp peaks, as illustrated by the separation of three enkephalins in a time window of 6 s with detection by means of ESI-TOF [14],... 

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