**6. Analysis**

Once the carbohydrate fraction has been isolated from other components of the plant, either the total carbohydrate content can be determined, or individual carbohydrates can be isolated, identified and quantified. The analysis of carbohydrates can be performed using any of several different methods. Two of these techniques include gas chromatography (GC) and liquid chromatography (LC). There are also spectral methods available including nuclear magnetic resonance (NMR), infrared (IR) and Raman spectroscopy. In this review, our focus is on the chromatographic and mass spectrometric methods.

### **7. Derivatization for GC or GC/MS analyses**

The most prevalent method used for analyzing carbohydrates is probably GC and GC coupled with mass spectrometry (MS) due to the high resolution of GC and definitive nature of MS. Since carbohydrates are nonvolatile, it is necessary to hydrolyze the sugars and then derivatize them to increase their volatility so they can elute through a GC column for analysis. Methods involving the formation of methylated glycosides, acetates, acetals, trimethylsilyl ethers, and more volatile alditol acetate derivatives of monosaccharides have been widely used (McInnes et al., 1958; Bishop & Cooper 1960; Bishop 1964; Lehrfeld 1981; Blakeney et al., 1983). More recently, trimethylsilyl (TMS) derivatives of carbohydrates have been used principally due to their relative ease of preparation and increased volatility. (Sweeley et al. 1963; Sullivan & Schewe 1977; Honda et al., 1979; Li et al., 1983; Twilley 1984). Different structural forms of carbohydrates can complicate their chromatograms due to the production of several (as many as 5) peaks for each monosaccharide. Formation of the corresponding oxime TMS-derivative reduces the number of potential peaks (Decker & Schweer 1982; Al-Hazmi & Stauffer 1986; Long & Chism 1987). Dmitriev et al. (1971) prepared the aldononitrile acetate derivatives with the oxime intermediate. Churms (1990) found the derivatization process was not affected by the presence of water in the reaction mixture, helping to minimize processing steps. Methods for the separation of neutral sugars in gums have also been reported using similar methods (Al-Hazmi & Stauffer, 1986).

Silylation is a versatile technique to increase the volatility of various analytes, including carbohydrates, making them amenable to GC and GC/MS analyses. There are several practical considerations that should be addressed prior to derivatization of a sample by this method. One major disadvantage of silylation derivatives is that they are susceptible to hydrolytic attack by any moisture present in the sample, resulting in incomplete silylation. However, the trimethylsilylation of aqueous samples of hydroxyl compounds has been achieved using a large excess of derivatizing reagent (Valdez 1985). Evershetd (1993) discussed another problem associated with silylation of carbohydrates, the existence of multiple reaction products, resulting in complicated chromatograms. The multiple products result from the formation of anomers and interconversion between pyranose and furanose rings. Interconversion of the anomers occurs via the open chain form of the sugar, while mutarotation results from the opening and closing of the ring. The interconversions can be minimized by the use of rapid and mild derivatization conditions. If silylation is the method of choice for derivatization, it may be desirable to protect the keto group of the

separation of sugars from the other components. A hydrophobic cartridge was used as the

Once the carbohydrate fraction has been isolated from other components of the plant, either the total carbohydrate content can be determined, or individual carbohydrates can be isolated, identified and quantified. The analysis of carbohydrates can be performed using any of several different methods. Two of these techniques include gas chromatography (GC) and liquid chromatography (LC). There are also spectral methods available including nuclear magnetic resonance (NMR), infrared (IR) and Raman spectroscopy. In this review,

The most prevalent method used for analyzing carbohydrates is probably GC and GC coupled with mass spectrometry (MS) due to the high resolution of GC and definitive nature of MS. Since carbohydrates are nonvolatile, it is necessary to hydrolyze the sugars and then derivatize them to increase their volatility so they can elute through a GC column for analysis. Methods involving the formation of methylated glycosides, acetates, acetals, trimethylsilyl ethers, and more volatile alditol acetate derivatives of monosaccharides have been widely used (McInnes et al., 1958; Bishop & Cooper 1960; Bishop 1964; Lehrfeld 1981; Blakeney et al., 1983). More recently, trimethylsilyl (TMS) derivatives of carbohydrates have been used principally due to their relative ease of preparation and increased volatility. (Sweeley et al. 1963; Sullivan & Schewe 1977; Honda et al., 1979; Li et al., 1983; Twilley 1984). Different structural forms of carbohydrates can complicate their chromatograms due to the production of several (as many as 5) peaks for each monosaccharide. Formation of the corresponding oxime TMS-derivative reduces the number of potential peaks (Decker & Schweer 1982; Al-Hazmi & Stauffer 1986; Long & Chism 1987). Dmitriev et al. (1971) prepared the aldononitrile acetate derivatives with the oxime intermediate. Churms (1990) found the derivatization process was not affected by the presence of water in the reaction mixture, helping to minimize processing steps. Methods for the separation of neutral sugars

in gums have also been reported using similar methods (Al-Hazmi & Stauffer, 1986).

Silylation is a versatile technique to increase the volatility of various analytes, including carbohydrates, making them amenable to GC and GC/MS analyses. There are several practical considerations that should be addressed prior to derivatization of a sample by this method. One major disadvantage of silylation derivatives is that they are susceptible to hydrolytic attack by any moisture present in the sample, resulting in incomplete silylation. However, the trimethylsilylation of aqueous samples of hydroxyl compounds has been achieved using a large excess of derivatizing reagent (Valdez 1985). Evershetd (1993) discussed another problem associated with silylation of carbohydrates, the existence of multiple reaction products, resulting in complicated chromatograms. The multiple products result from the formation of anomers and interconversion between pyranose and furanose rings. Interconversion of the anomers occurs via the open chain form of the sugar, while mutarotation results from the opening and closing of the ring. The interconversions can be minimized by the use of rapid and mild derivatization conditions. If silylation is the method of choice for derivatization, it may be desirable to protect the keto group of the

first cartridge followed ion and cation exchange cartridges (Schiller et al., 2002).

our focus is on the chromatographic and mass spectrometric methods.

**7. Derivatization for GC or GC/MS analyses** 

**6. Analysis** 

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

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

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

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

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

Extraction and Analysis of Inositols and Other Carbohydrates from Soybean Plant Tissues 427

Fig. 2. Mass spectrum of TMSI-derivatized pinitol. From Garland, *et al.* (2009).

Fig. 3. Extracted ion plot of TMSI-derivatized soybean root extract. The labels on the vertical axis indicate the fragment mass of each extracted ion chromatogram. The chromatograms were spaced for easier representation. All peaks are on the same scale relative to their baselines. A vertical black line was inserted at the retention time of pinitol for reference.

From Garland, *et al.* (2009).

use of methanol extraction as a first step in the purification of pinitol from soybean root tissue.

The mass spectrum of the derivatized pinitol shown in Figure 2 is very similar to that reported previously (Savidge & Forster, 2001). Identification of pinitol by mass spectrometry is made exceedingly easy by the presence of a high-intensity m/z 260 fragment ion. The fragment ion at m/z 260 appears to be a unique ion associated with pinitol and the other Omethylinositols compared with the other sugars observed using this analytical procedure. This allows for a high probability of quantitative results even in the event of another analyte coeluting with pinitol. The elemental composition obtained from accurate mass measurements for m/z = 260 was determined to be C11H24O3Si2, which was matched within 4.3 millimass units (mmu).

We have also extracted roots using 80% ethanol rather than methanol. This led to the extraction of a greater variety of inositols and O-methylinositols from several plant roots (unpublished data).

Permethylation is another derivatizing method for the analysis of carbohydrates. The methods using permethylation initially provided relatively long retention times. Some of the reactions to form permethylated derivatives include the use of methyl iodide/silver oxide (Gee & Walker 1962; Walker et al., 1962; Kircher, 1960) methylsulfinylcarbanion/methyl iodide (Hakomori 1964; Corey & Chaykovsky 1962; Moor & Waight 1975), and potassium/liquid ammonia/methyl iodide (Muskat 1934a; Muskat 1934b). Permethylation has also become very popular in the LC/MS analysis of carbohydrates.

Fig. 1. Total ion chromatogram of an extract of soybean roots. Peak 4 was determined to be pinitol. From Garland, *et al* (2009).

use of methanol extraction as a first step in the purification of pinitol from soybean root

The mass spectrum of the derivatized pinitol shown in Figure 2 is very similar to that reported previously (Savidge & Forster, 2001). Identification of pinitol by mass spectrometry is made exceedingly easy by the presence of a high-intensity m/z 260 fragment ion. The fragment ion at m/z 260 appears to be a unique ion associated with pinitol and the other Omethylinositols compared with the other sugars observed using this analytical procedure. This allows for a high probability of quantitative results even in the event of another analyte coeluting with pinitol. The elemental composition obtained from accurate mass measurements for m/z = 260 was determined to be C11H24O3Si2, which was matched within

We have also extracted roots using 80% ethanol rather than methanol. This led to the extraction of a greater variety of inositols and O-methylinositols from several plant roots

Permethylation is another derivatizing method for the analysis of carbohydrates. The methods using permethylation initially provided relatively long retention times. Some of the reactions to form permethylated derivatives include the use of methyl iodide/silver oxide (Gee & Walker 1962; Walker et al., 1962; Kircher, 1960) methylsulfinylcarbanion/methyl iodide (Hakomori 1964; Corey & Chaykovsky 1962; Moor & Waight 1975), and potassium/liquid ammonia/methyl iodide (Muskat 1934a; Muskat 1934b). Permethylation

Fig. 1. Total ion chromatogram of an extract of soybean roots. Peak 4 was determined to be

has also become very popular in the LC/MS analysis of carbohydrates.

tissue.

4.3 millimass units (mmu).

pinitol. From Garland, *et al* (2009).

(unpublished data).

Fig. 2. Mass spectrum of TMSI-derivatized pinitol. From Garland, *et al.* (2009).

Fig. 3. Extracted ion plot of TMSI-derivatized soybean root extract. The labels on the vertical axis indicate the fragment mass of each extracted ion chromatogram. The chromatograms were spaced for easier representation. All peaks are on the same scale relative to their baselines. A vertical black line was inserted at the retention time of pinitol for reference. From Garland, *et al.* (2009).

Extraction and Analysis of Inositols and Other Carbohydrates from Soybean Plant Tissues 429

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

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

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

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

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-

derivatization techniques have been discussed (Meulendijk & Underberg 1990).

prior derivatization as is necessary in GC and GC/MS analyses.

pinitol in snap bean root extract (Garland, *et al.,* 2009).

1993).

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