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ANALYTICAL METHOD CHALLENGES FOR MEASURING NUTRIENTS AND ANTINUTRIENTS IN PLANTS SYMPOSIUM |
* Crop Analytics, Monsanto Company, St. Louis, MO 63167;
and
Applied Molecular Breeding Center, Monsanto Company, Ankeny, IA 50021; and
and
Calgene Campus, Monsanto Company, Davis, CA 95616
Abstract
In this paper, the status of the analytical technologies for assaying animal antinutritional compounds, such as raffinose oligosaccharides, pentosans, phytic acid, and glucosinolates, is reviewed in terms of selectivity, sensitivity, and sample throughput. The implementation of simplified sample preparation schemes, use of novel separation approaches, and alternate detector technologies are discussed. The challenges and opportunities posed by these assays are highlighted along with the recommendations for best analytical practices.
Key Words: Glucosinolates • Pentosans • Phytate • Raffinose • Stachyose
Introduction
Raffinose and stachyose are the two main 
-linked oligosaccharides in soy (1 to 4%) proven responsible for causing flatulence in monogastric animals. These nonreducing sugars contain fructose, glucose, and galactose as 3 and 4 units, respectively (Figure 1
) and, when ingested, remain unabsorbed because of lack of -1-6-galactosidase enzyme in the intestinal mucosa of pigs and poultry. The metabolizable energy of the soy animal feeds is thus reduced because of their unavailability (Coon et al., 1988). Pentosans are composed of arabinogalactans with a galactan backbone and arabinose side residues and arabinoxylans with a linear xylan backbone with single arabinose side residues (Figure 2). Wheat-based diets contain high levels of pentosans (1 to 3%) that cannot be digested by enzymes secreted into the pig’s gastrointestinal tract. Pentosans are shown to increase gut viscosity and slow the digesta transit time and are linked to depressed weight gain and reduced feed conversion in poultry (Choct and Annison, 1990). Phytate is usually a mixture of calcium/magnesium/potassium salts of inositol hexaphosphoric acid (Figure 3) and is present in soy and corn at 1 to 2% levels. Phytate is the primary source of phosphorus in soy and corn and is shown to adversely impact mineral bioavailability and protein solubility when present in animal feeds (Liener, 1994). Glucosinolates are a class of about 100 naturally occurring thioglucosides that are characteristic of the Brassicaceae family, such as rapeseed. They generally consist of a sugar entity, ß-thioglucose, with an ester bond to an organic aglycone that is an alkyl group (Figure 4). These molecules (Table 1) are readily dissociated upon hydrolysis during animal digestion, leading to the production of toxic compounds, such as thiocyanates and nitriles. These are known to interfere with the thyroid, damage vital organs, and are shown to decrease growth and cause liver and kidney lesions in animals (Fenwick et al., 1983). In this paper, the status of the analytical methods and the analytical challenges are reviewed for the antinutrients discussed above along with appropriate recommendations.
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Present Methods
The most widely used methods for the analysis of oligosaccharides in soy can be classified under the five categories described in the following sections. Enzymatic Assay. In this approach, the oligosaccharides are hydrolyzed to galactose, glucose, and fructose using -galactosidase and invertase. The glucose is then determined using glucose oxidase/peroxidase reagent. The method does not distinguish between raffinose and stachyose, but rather, measures these as a group. Because 1 mol of each of the raffinose-series oligosaccharides contain 1 mol of glucose, the concentrations are presented on a molar basis. Free sucrose and glucose in sample extracts are determined concurrently. This assay kit can be purchased from MegaZyme International (Megazyme Int. Ireland Ltd., Bray, Ireland.
HPLC Separation on an Amino Column.
The oligosaccharides are separated on an amino column such as Spherisorb NH2 column (Waters Corp., Milford, MA) using an acetonitrile/water mobile phase using refractive index (RI) detection. However, the performance and ruggedness of amino columns in chromatography are always in question.
HPLC Separation on Polymeric Gel Column.
Alltech recently introduced a novel Prevail Carbohydrate ES column (Alltech Associates, Inc., Deerfield, IL) that is packed with a rugged hydrophilic polymeric gel. This column—with its high efficiency, excellent stability, good reproducibility, and long column lifetime—reaches full potential when used with evaporative light scattering detection (ELSD) as illustrated below (Figure 5).
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Ion Exchange Chromatography-Pulsed Amperometric Detection (IEC-PAD)
. This is the most sensitive technique available for the quantification of the sucrose, raffinose, and stachyose. The principle of separation is based on the separation of carbohydrates as anions using a highly alkaline mobile phase followed by detection at a gold working electrode using pulsed amperometric detection. The ground soy samples without defatting are extracted with 50% EtOH/water mixture and directly analyzed by anion exchange chromatography on a Dionex Carbopac 1 column (Dionex Corp., Sunnyvale, CA) using 150 mM NaOH as mobile phase in isocratic mode. All three sugars are baseline-resolved and separated in under 16 min (Figure 6). The advantages of this approach include not only greater selectivity but also enhanced sensitivity compared to the RI detection by almost two orders of magnitude. This technique is ideally suited for the analysis of single or partial seeds and for the screening of elite germplasm lines.
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Recommendations
For highest sensitivity and best resolution, the IEC-PAD approach is recommended. For normal, routine use, HPLC separation on polymeric gel column with ELSD or RI detection is quite satisfactory. If only total raffinose oligosaccharide content is of interest, the MegaZyme (Megazyme International Ireland Ltd.) enzyme assay kit is an option worth considering.
Analysis of Pentosans
Present Methods
Colorimetric Methods.
The pentosan content in wheat is determined by colorimetric methods using either orcinol•HCl (Hashimoto et al., 1987) or phloroglucinol (Rouau and Surget, 1994). The sample is first hydrolyzed with HCl at 100°C and then neutralized with sodium carbonate followed by treatment with yeast. The supernatant is mixed with ferric chloride and orcinol, heated in a boiling water bath, and cooled; the absorbance was measured at 670 nm.
Gas Chromatography Approach.
This is based on the hydrolysis and dehydration of pentosan sugars to furfural and extraction with dibutylether (Folkes, 1980). Alternatively, the hydrolyzed pentoses are derivatized to alditol acetates (Englyst et al., 1992) or converted to trimethylsilyl esters (Bradbury et al., 1981) and determined using flame ionization detection.
Ion Exchange Chromatography-Pulsed Amperometric Detection (IEC-PAD).
In this method (Houben et al., 1997), the polysaccharides were first hydrolyzed to xylose, arabinose, and galactose using HCl and were then characterized by anion exchange chromatography using Carbopac 1 (Dionex Corp.) column, and 1 mM NaOH mobile phase and pulsed amperometric detection. Interference by the high concentration of starch-derived glucose was eliminated by conversion to gluconic acid using glucose oxidase enzyme. However, for the removal of strongly held gluconic acid, the column had to be flushed with 500 mM NaOH before equilibration with 1 mM NaOH again. To maintain high detector sensitivity, the post column effluent was mixed with 250 mM NaOH before the electrochemical detection. We made three major improvements to this method to make it more rugged (in terms of reproducibility and reliability), more user-friendly, and less complicated to set up. These improvements are as follows: 1) change of mobile phase to 15 mM NaOH, 2) elimination of post column addition of NaOH, and 3) deletion of glucose oxidase treatment. The use of 15 mM NaOH not only provided adequate resolution for all four sugars but also enhanced the detector sensitivity, which in turn allowed the direct determination without further addition of NaOH (Figure 7). Because xylose and galactose are well resolved from the high concentration of glucose without the need for its enzymatic removal, a gradient step of column cleaning with 500 mM NaOH is also avoided. This resulted in the elimination of equilibration step and avoidance of baseline disturbance.
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Recommendations
For the determination of wheat pentosans, the IEC-PAD method is ideally suited because it offers excellent sensitivity, provides compositional information, and requires no prior derivatization. The colorimetric methods for pentosans are tedious, interfered by the starch-derived glucose, and do not provide the compositional information of individual sugars. The gas chromatographic methods require prior derivatization and do not provide good sensitivity.
Analysis of Phytate
Present Methods
There are a number of assay options available for the determination of phytate in soy.
Colorimetric Methods.
These are based on either the precipitation of iron-phytate complex and measurement of the iron in the supernatant (Velickovic et al., 1999) or the selective extraction of the phytate using dilute HCl, followed by ion exchange cleanup using EDTA/NaOH, subsequent elution of phytate with dilute HCl, digestion with a mixture of HNO3/H2SO4 and determination of phosphate released as phosphomolybdate (Harland and Oberleas, 1986).
Enzymatic Assay.
This is based on the spectrophotometric determination of inorganic phosphate with vanadate/molybdate reagent after the enzymatic hydrolysis of phytic acid with phytase from Asperigillus ficum (March et al., 1995).
Reverse-Phase HPLC.
This method involves the extraction of phytate using trichloroacetic acid and separation on uBondapak C18 (Waters Corp.) using sodium acetate mobile phase and RI detection (Tangendjaja et al., 1980).
Inductively Coupled Plasma (ICP)-Atomic Emission Spectroscopy (AES) Analysis as Phosphorus.
In one approach, the phytic acid after acid extraction and ion exchange clean-up was directly determined by ICP-AES as phosphorus. In another approach, the phytic acid was precipitated with FeCl3 and the iron complex was treated with NaOH to release the phytate, which is then determined by ICP-AES. In both instances, the difficult acid digestion and the spectrophotometric determination of P by traditional methods are eliminated. (Plaami and Kumpulainen, 1991).
Ion Chromatography.
The most promising and well-researched approach for the determination of phytic acid thus far has been using ion exchange chromatography. In one approach, phytic acid and other inositol phosphates were separated on a polystyrene-based strong anion exchange column and detected using FeCl3 post column chemistry (Rounds and Nielsen, 1993). In another method, the phytic acid was separated on a Dionex mixed-mode Omnipac PAX column with NaOH mobile phase under gradient conditions using suppressed conductivity detection (Talamond et al., 1998). Both these elegant separations are illustrated below (Figures 8 and 9).
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Analytical Challenges
Phytate is a strong chelating agent and requires careful handling in the sample-preparation process, in metal-free containers to prevent sample losses. This is also the reason for the long extraction times and the need for use of acidic solvents and decomplexing agents. Chromatography methods operating under acidic conditions have to be reexamined to ensure the absence of degradation phytic acid to lower inositol phosphates. Since phytate also does not possess any strong chromophoric groups, post column detection approaches are often needed. Precipitation methods are nonselective and labor intensive; reverse-phase HPLC methods have poor sensitivity, whereas ion exchange sample cleanups remain tedious.
Recommendations
If other myo-inositols are absent or present at very low concentrations or not of a concern, enzymatic treatment that is followed by phosphate determination by ion chromatography or by spectrophotometry as molybdate complex is preferred. The advantages of the ion chromatography approach include rapid extraction, fast enzymatic hydrolysis, selective phosphate determination, free P quantification by direct injection, and high sensitivity resulting from the conversion of 1 mol of phytate into 6 mol of phosphate and the use of highly sensitive conductivity detection (Figure 10). For the information on inositol species distribution and precise quantification of phytic acid, ion exchange chromatography with PCR detection is recommended.
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Present Methods
The analytical methods practiced for the analysis of glucosinolates in rapeseed and canola fall under five categories.
Reverse-Phase HPLC.
This is by far the most widely used, thoroughly tested, and well accepted by the analytical community. This is essentially a slightly modified version of the Ak 1-92 method recommended by AOCS (AOCS, 1998) and is based on the aqueous extraction of glucosinolates followed by purification and desulfation on micro-ion exchange columns. A typical HPLC separation of glucosinolates in rapeseed is illustrated in Figure 10
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The total glucosinolate content, expressed in micromoles per gram of dry matter, is equal to the sum of the contents of each glucosinolate. The response factors for various glucosinolates are shown in the Figure 11 inset. They were determined experimentally and fixed by consensus among several laboratories that collaborated. Reference samples of rapeseed desulfoglucosinolates are obtained from Community Bureau of Reference, Rue de la Loi 200, B-1049, Brussels, Belgium.
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GC Analysis of Desulfoglucosinolates.
A rapid procedure for the quantitation of intact glucosinolates, including the indole glucosinolates, in rapeseed and canola meal involves the formation of trimethylsilyl derivatives of the desulfoglucosinolates and separation on a glass capillary column by GLC within 10 min (Sosulski and Dabrowski, 1984).
Enzymatic Assay.
Glucosinolates in ground rapeseed were hydrolyzed with commercially available myrosinase (0.5 to 1.5 h, 39°) enzyme. The length of the hydrolysis could be reduced by a 100°C preliminary heat treatment. Glucose generated during hydrolysis was converted to gluconate 6-phosphate in coupled enzymic reactions (hexokinase, glucose 6-phosphate dehydrogenase), with formation of NADPH, which was determined spectrophotometrically at 340 nm (Gardrat and Pivot, 1987).
Total Glucosinolates by X-ray Fluorescence.
This is the fastest analytical method available and is based on the nondestructive assay of total sulfur in the ground seed. Calculation of total glucosinolate content is performed by comparison with values of reference samples with certified sulfur content (Wathelet et al., 1991)
Analytical Challenges
There are major challenges for the complete characterization of glucosinolates. These are a diverse class of S- and N-containing species (over 100), with closely resembling structures, and are prone to easy degradation. The extraction procedures and sample clean-up operations are very complex and require the skills of an experienced analyst in this area. The chromatography resolution profiles are tight, and one has to keep in mind the varying response factors for the various glucosinolates in making the final calculations.
Recommendations
For the characterization of individual species distribution, prior conversion of glucosinolates into their corresponding desulfo- derivatives followed by reverse-phase HPLC/UV detection is preferred. For peak authenticity and for analysis of complex matrices, HPLC/atmospheric chemical ionization-mass spectrometry should be considered. For rapid total glucosinolate screening, X-ray fluorescence provides quality results. For high throughput, analysis of total glucosinolates as S, elemental analyzers based on combustion should be considered.
Implications
We have superior sample extraction methods, better characterization tools, and more sensitive methods now for the analysis of antinutritionals in plant materials than a decade ago. In the light of these developments, the levels of many antinutritional species that were established in feed matrices several years ago might have to be reexamined. Because the selective biological effect of individual species in a class of compounds such the oligosaccharides, pentosans, and glucosinolates is not well understood in terms of animal nutrition; in many instances, simple total assays for the specific functional group might be adequate. It would be extremely beneficial to develop high throughput, near-infrared methods that allow screening thousands of conventional and/or transgenic seeds, foods, and feeds based on the primary assays described in this article.
1 Correspondence: 800 N. Lindbergh Blvd., Mail Zone S4A (phone: 314-694-3674; e-mail: dvvinj{at}monsanto.com).
Received for publication December 4, 2002. Accepted for publication August 26, 2003.
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