**9. Liquid chromatography/mass spectrometry (LC/MS) and other MS techniques**

Efficient separation methods such as high performance liquid chromatography (HPLC) and capillary electrophoresis combined with detection methods (e.g. mass spectrometry) that supply structural or compositional information is a preferred tool for the analysis of biomolecules, particularly carbohydrates. Liquid chromatography/mass spectrometry with both electrospray (ESI) and atmospheric pressure ionization (APCI) has spurred a major interest in the analysis of carbohydrates.

In ESI , the liquid containing the analyte(s) of interest is dispersed into a fine aerosol. Because the ion formation involves extensive solvent evaporation, the typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile). To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are customarily added to the solution. Large-flow electrosprays can benefit from additional nebulization by an inert gas such as nitrogen. The aerosol is sampled into the first vacuum stage of a mass spectrometer through a capillary, which can be heated to aid further solvent evaporation from the charged droplets. The ions observed by mass spectrometry may be quasimolecular ions created by the addition of a hydrogen ion and denoted [*M* + H]+, or of another cation such as sodium ion, [*M* + Na]+, or the removal of a proton, [*M* − H]−. Multiply-charged ions such as [*M* + nH]n+ are often observed (Gaskell 1997). As examples, Fountain and Grumbach (2009) used negative ion electrospray mass spectrometry for the analysis of fructose, glucose, sucrose, and lactose. Taormina et al. (2007) and Mauri et al. (2002) used flow injection techniques with mass spectrometry. Fugimoto et al. (2005) used rubidium in the mobile phase as a complexing agent for both nuclear magnetic resonance and electrospray mass spectrometry analysis. Taylor et al. (2005) utilized ESI/MS to study fragmentation patterns of carbohydrates. Schlichtherle-Cerny et al. (2003) utilized a HILIC column coupled with ESI/MS for the analysis of amino acids, peptides, glycoconjugates, and organic acids in foods without prior derivatization.

In APCI, typically the mobile phase containing eluting analyte is heated to relatively high temperatures (above 400 C), sprayed with high flow rates of nitrogen and the entire aerosol cloud is subjected to a corona discharge that creates ions. Often APCI can be performed in a modified ESI source. The ionization occurs in the gas phase, unlike ESI, where the ionization occurs in the liquid phase. A potential advantage of APCI is that it is possible to use a nonpolar solvent as a mobile phase solution, instead of a polar solvent, because the solvent and molecules of interest are converted to a gaseous state before reaching the corona discharge pin. Typically, APCI is a harder ionization technique than ESI, i.e. it generates more fragment ions relative to the parent ion.(Kostianinen et al., 2003). Kumaguai (2001) used atmospheric pressure chemical ionization mass spectrometry for the analysis of sugars and sugar alcohols without derivatization but did use methylene chloride or chloroform that was added post column to increase the sensitivity. The ions detected included (M+Cl)- . Shimadzu application note also used solvent addition post column to improve sensitivity. This application also used APCI in the negative ion mode. Keski-Hynnila et al. (2004) compared APCI, atmospheric pressure photoionization, and electrospray in the analysis of phase II metabolites.

Other types of mass spectrometers used for analysis of carbohydrates include quadrupole time-of-flight (QTOF) mass spectrometers which allow both accurate mass (elemental composition) and MS/MS studies to be performed. Another mass spectrometer very useful for the analysis of carbohydrates is the ion trap (IT) MS. Ion trap technology has been described in (March & Todd 2005a, 2005b), and its major advantage includes the capability of MSn which can provide additional structural information. Examples of glycoprotein analysis using IT have been described by (Stumpo & Reinhold 2010; Jiao et al., 2010; Reinhold et al., 1990).

Another technique that has been utilized for the analysis of carbohydrates is matrix assisted laser desorption/time-of-flight mass spectrometry (MALDI/TOFMS)(Harvey 1999, 2009)). In MALDI, the sample to be analyzed is mixed with a matrix, which in turns absorbs heat energy from irradiation with a nitrogen laser light. For example, dihydroxybenzoic acid (DHB) or ferulic acid which are commonly used as a matrices have a carboxyl group on a benzene ring. The DHB absorbs the energy and acts as a proton donor (Zenobi & Knochenmuss 1998). Time-of-flight mass spectrometry allows the majority of the ions generated throughout the mass range to be collected by the detector. MALDI has been primarily used to obtain spectra of very large polymers, biomolecules, and a variety of thermally labile materials (Hillenkamp et al., 1991, Nelson et al., 1990. We have also used MALDI/TOF for the analysis of smaller molecules (e.g. <500 amu) (Goheen *et al.,* 1997; Campbell *et al.,* 2001).
