**8. Other analytical techniques**

Another technique for the separation and analysis of carbohydrates is liquid chromatography (LC). The column used in LC to provide the separation depends on whether the carbohydrates have been derivatized or not. Underivatized carbohydrates are commonly separated using ion exchange resins with water as an eluent and refractive index (RI) for detection. Refractive index detectors are, however, typically low in sensitivity, so samples need to be concentrated for quantitative analyses. The concentration of the carbohydrate must be in the percent range, and the RI detector can only be used with isocratic elution (Martens & Frankenberger 1990).

Other alternative detectors including both UV/visible absorbance and fluorescence require either pre-column or pre-detection derivatization of sugars, due to the fact that carbohydrates do not have a chromophore. Evaporative light scattering (ELS) is a detection technique used in high performance chromatography (HPLC) and supercritical fluid chromatography (SFC). It has been used for the analysis of carbohydrates and can act as a qualitative or quantitative detector (Wei & Ding 2000 ; Karlsson et al., 2005). The ELS is limited to solutes of low volatility. With the ELS, the column effluent is passed through a nebulizer and then into a heated drift tube; the solvent is evaporated leaving behind a particulate or aerosol form of the target compound. Light striking the dried particles that exit the drift tube is scattered and the photons are detected by a photodiode or photomultiplier tube at a fixed angle from the incident light. (LaPosse & Herbtreteau 2002).

Fig. 4. Total ion chromatogram of derivatized sugar beet extract. Conditions were those of the chromatogram in Figure 1. Pinitol (retention time 9.2 min.) was not detected, as

Another technique for the separation and analysis of carbohydrates is liquid chromatography (LC). The column used in LC to provide the separation depends on whether the carbohydrates have been derivatized or not. Underivatized carbohydrates are commonly separated using ion exchange resins with water as an eluent and refractive index (RI) for detection. Refractive index detectors are, however, typically low in sensitivity, so samples need to be concentrated for quantitative analyses. The concentration of the carbohydrate must be in the percent range, and the RI detector can only be used with

Other alternative detectors including both UV/visible absorbance and fluorescence require either pre-column or pre-detection derivatization of sugars, due to the fact that carbohydrates do not have a chromophore. Evaporative light scattering (ELS) is a detection technique used in high performance chromatography (HPLC) and supercritical fluid chromatography (SFC). It has been used for the analysis of carbohydrates and can act as a qualitative or quantitative detector (Wei & Ding 2000 ; Karlsson et al., 2005). The ELS is limited to solutes of low volatility. With the ELS, the column effluent is passed through a nebulizer and then into a heated drift tube; the solvent is evaporated leaving behind a particulate or aerosol form of the target compound. Light striking the dried particles that exit the drift tube is scattered and the photons are detected by a photodiode or photomultiplier tube at a fixed angle from the incident light. (LaPosse & Herbtreteau 2002).

confirmed by MS analysis (Garland, *et al.,* 2009).

isocratic elution (Martens & Frankenberger 1990).

**8. Other analytical techniques** 

Fig. 5. Total ion chromatogram of derivatized snap bean root extract. Peak 5 was at a similar retention time to that of pinitol in Fig 1 (9.2 min.), but MS analyses were unable to detect pinitol in snap bean root extract (Garland, *et al.,* 2009).

Another detector commonly used is a pulsed amphoteric detector (Lee 1996; Johnson et al., 1993).

One derivatization procedure for carbohydrates to provide a chromophore for LC analysis involves a reaction with p-nitrobenzoyl chloride and pyridine. The reaction replaces the active hydrogens with a nitrobenzoyl group. The method was applicable to mono-, di-, and trisaccharides except fructose (Nachtmann & Budna 1977; Nachtmann 1976). Many of the derivatization reactions for carbohydrates are discussed by Knapp (1979). In addition, other derivatization techniques have been discussed (Meulendijk & Underberg 1990).

Mass spectrometry can also be coupled with LC. Examples are LC/MS and capillary electrophoresis/MS. Many of the LC techniques allow carbohydrates to be analyzed without prior derivatization as is necessary in GC and GC/MS analyses.

It should be noted that there is not one LC column that has been reported to separate every carbohydrate. Togami et al. (1991) discussed the separation of carbohydrates using cation-

Extraction and Analysis of Inositols and Other Carbohydrates from Soybean Plant Tissues 431

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

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;

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;

Inositols (Fig. 6) are polyols of cyclohexane with the empirical formula C6H12O6. There are potentially 9 stereoisomers of inositol but only five are naturally occurring (structure shown below). They are *myo*-inositol, *chiro*-inositol, *scyllo*-inositol, *muco*-inositol, and *neo*-inositol. Of these, *myo*-inositol is the precursor of the other four. *myo*-Inositol is synthesized from

The synthesis of *myo*-inositol uses the enzyme L-*myo*-inositol 1-phosphate synthase to catalyze the reaction which produces L-*myo*-inositol-1-phosphate from D-glucose 6 phosphate (Hoffmann-Ostenhof and Pittner, 1982). The L-*myo*-inositol-1-phosphate is then dephosphorylated through inositol monophosphate to produce *myo-*inositol (Loewus & Murthy, 2000). The enzyme that catalyzes this step is L-*myo*-inositol 1-phosphate synthase

phase II metabolites.

Reinhold et al., 1990).

Campbell *et al.,* 2001).

**10. The inositols** 

glucose.

.

exchange columns. Richmond et al. (1991) separated carbohydrates in dairy products. Henderson and Berry (2009) have utilized Zorbax columns for the separation of carbohydrates in Stevia sweetener. Romano (2007) discussed carbohydrate analysis in food products emphasizing column chemistries and detection. Several vendors offer LC columns for carbohydrate separation. Wilcox et al. (2001) also discussed several column types used for carbohydrate separation. Hydrophilic interaction chromatography (HILIC) has also been reported as a method for analyzing ionic or polar compounds, particularly biomolecules and drug metabolites (http://www.laboratoryequipment.com/article-is-hilic-in-your-futurect92.aspx). Simple carbohydrate separations can also be performed on functionalized silica or resin-based columns (http://www.labnews.co.uk/feature\_archive. php/4000/5/just-juice).

The separation of mono- and oligosaccharides are also performed using capillary electrophoresis. Different formats are capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), and micellar electrokinetic chromatography (MEKC). These techniques are summarized in a review by Thibault and Honda (2003).
