**5. Extraction and cleanup**

422 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

Carbohydrates are the main energy source for humans. Carbohydrates are classified according to the number of monomers they contain as monosaccharides (simple sugars), oligosaccharides, or polysaccharides. Carbohydrate metabolism in plants has been reviewed (Colowick & Kaplan, 1951; Ochoa & Stern, 1952; and Horecker & Mehler, 1955). Carbohydrate levels in soybean seed are highest at growth stage R 5.5, or when the seed is half-developed (Wilson, 2004). A significant portion of the carbohydrate produced by

The most common simple sugars are glucose and fructose. Disaccharides consist of two covalently bound sugar molecules. Sucrose, for example, is a disaccharide consisting of glucose and fructose. Sugars have a role in energy, carbon transport molecules, hormonelike signaling factors, and as the source for building proteins, polysaccharides, oils and woody materials (Halford *et al*., 2010). Plant genotype and environment greatly affect the

Sucrose

Complex carbohydrates (polysaccharides) are polymers of the simple sugars . Starch is the

Starch

photosynthesis is respired in the plant roots. (Lambers *et al*., 1996).

**3. Simple sugars** 

levels found in plants (Halford *et al*., 2010).

**4. Complex carbohydrates** 

principal polysaccharide used by plants to store glucose.

(**n** is the number of repeating glucose units and ranges in the 1,000's)

The methods used for isolating carbohydrates depend on the carbohydrate type, matrix, and purpose or type of analysis. However, some extraction procedures are commonly used for isolating carbohydrates from other classes of compounds in plants and foods. As an example, foods are usually dried under vacuum to prevent thermal degradation, ground to a fine powder to enhance extraction efficiency, and then remove the fats using appropriate solvent extraction.

A commonly used method for extracting low molecular weight carbohydrates from foods is to boil a sample with a 70-80% alcohol solution (Hall 2003, Asp 1993, Smith 1973.). Monosaccharides and oligosaccharides are soluble in alcohol solutions; however, most proteins, polysaccharides and dietary fiber are insoluble. The soluble components can then be separated from the insoluble components by filtering, soluble portion passes through the filter and the insoluble part retained by the filter. The two fractions can then be dried using lyophilization or nitrogen blow down techniques. In addition, monosaccharides and oligosaccharides and various other small molecules (e.g. organic acids, amino acids) may be present in the alcoholic extract. It is usually necessary to remove those components prior to carrying out a carbohydrate analysis, for example, with clarifying agents or by elution through one or more ion-exchange resins.

Water extracts of many foods contain substances that are colored or produce turbidity, and may interfere with analyses of carbohydrates; as a result, clarifiers may be needed. The most commonly used clarifying agents are heavy metals (e.g. lead acetate) which form insoluble complexes with interfering substances that can't be removed by either filtration or centrifugation. Ion-exchange is another method for removing interfering components prior to analysis. Many monosaccharides and polysaccharides are polar non-charged molecules and can therefore be separated from charged molecules by passing samples through an ionexchange column. By using a combination of cationic and anionic resins it may be possible to remove most charged contaminants. Non-polar molecules can be removed by eluting through a column with a non-polar or hydrophobic stationary phase. Proteins, amino acids, organic acids, and hydrophobic compounds can be potentially removed from the carbohydrates in this manner prior to analyses.

Before analysis of the carbohydrates, residual alcohol (or other organic solvents) can be removed, if necessary, from the solution by evaporating under nitrogen or under vacuum using a rotary evaporator. For aqueous solutions, the sample can be concentrated using lyophilization.

Solid phase extraction (SPE) has also been reported for the cleanup and quantification of sugars and organic acids in herbal dry extracts. A three step SPE sequence was used for the

Extraction and Analysis of Inositols and Other Carbohydrates from Soybean Plant Tissues 425

monosaccharides prior to silylation in order to prevent the formation of enol-TMS ethers. These derivatives are unstable and complicate the analyses by giving rise to multiple

In most instances, the silylating reagent is an adequate solvent. However, sometimes an additional solvent is required in the reaction. The selection of that solvent is critical to the success of the derivatization process. Any active hydrogens, including those present in the solvent, may be silylated. Pyridine has been found to be an ideal solvent for silylation reactions due to the increased solubility of the carbohydrates and their derivatives in that

One of the earliest reagents used for silylation was hexamethyldisilazane (HMDS). Usually, there is no need for additional solvents when HMDS is used. Recently, Ruiz-Matute et al. (2010) reviewed derivatization techniques of carbohydrates for GC and GC/MS analyses. Included in the discussion were derivatization of common sugars through the formation of ethers and esters, oximes, alditol acetates, aldononitriles, and dithoacetals (Evershed 1993). Another silylating reagent is trimethylsilylimadazole (TMSI). Garland et al. (2009) analyzed soybean roots for pinitol using GC/MS (see Figs. 1-3). Roots were extracted in methanol and derivatized using TMSI. In this example a DB-5 capillary column was used in the splitless mode. The column eluents were analyzed by a double-focusing, four-sector mass spectrometer in the electron-ionization mode. Accurate mass measurements were also performed to determine the elemental composition of the parent and fragment ions. Under these conditions, a pinitol standard produced a single peak in the total ion chromatogram with a retention time of 9.18 min as shown in Fig. 1. Although several peaks appeared,

The mass spectrum of TMSI-derivatized pinitol in Fig. 2 shows the major ion fragments detected from this, the most common carbohydrate in soybeans (Garland et al, 2009). In this example, the base ion is m/z 260. A comparison of the extracted ion plots of the soybean extract is shown in Fig. 3. A vertical, solid black line was added to each at the retention time of derivatized pinitol as determined from the standard. In the extracted ion plot of the soybean root, Figure 4 shows the total ion chromatogram of a TMSI-derivatized sugar beet extract. In this example no significant peaks appeared at the retention time of pinitol. The sugar beet root extract also showed no substantial peaks with the m/z 260 mass fragment. The concentration of pinitol in soybean roots was approximately 4% of the soybean root's dry mass using a dry/fresh weight ratio of 54.5 mg DW/g FW (which is similar to 73.6 mg DW/g FW reported for alfalfa by Fougere, *et al.* (1991). The methanol extraction method appears to be effective for removing pinitol from the root tissue of soybean plants. The extent of extraction at the cost of time was encountered as well by Streeter and Strimbu's simultaneous extraction and derivatization method (Streeter & Strimbu 1998). Although they were able to reduce processing time, they were unable to extract as much pinitol from fibrous plant tissues in pyridine in 1 h when compared to complete extraction with ethanol

Another benefit to using methanol extraction and TMSI derivatization is the relative simplicity of the resulting chromatograms. Eleven peaks were observed in the soybean extract chromatogram in Fig. 1, with pinitol clearly defined near 9.18 min. This compares with only 6 major peaks from sugar beet (Fig. 4) and 10 from snap bean roots (Fig. 5). The simplicity of the chromatograms is an indicator that pinitol and a small amount of other compounds are present in the methanol extract, which reduces the likelihood of coelution or some other interfering matrix effect with pinitol. This also provides support for the possible

solvent (Evershed 1993). Heating slightly is often utilized to aid in efficient silylation,

products that can't be prepared quantitatively (Halket 1993).

pinitol's peak at 9.18 min was well-resolved.

for 24 h before derivatization (Streeter & Strimbu 1998).

separation of sugars from the other components. A hydrophobic cartridge was used as the first cartridge followed ion and cation exchange cartridges (Schiller et al., 2002).
