Aflatoxin analytical methods for groundnuts
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Aflatoxin determination in groundnuts can be approached in several ways. Groundnuts are often contaminated with aflatoxins B1 and B2, less often with aflatoxins B1, B2, G1, and G2 so it is important to have analytical values that represent the total aflatoxin content. Some countries are only interested in B1 content and others are interested in the total aflatoxin content. It is essential to safely handle all experimental materials associated with aflatoxin analyses or the aflatoxigenic fungi. Visual screening of suspect groundnut lots, based on the presence of conidial heads of the Aspergillus flavus group, is not a chemical test and may allow aflatoxin-contaminated lots into commerce. Minicolumn screening techniques can be useful but they should always be used in conjunction with a quantitative method. Several thin layer chromatographic (TLC) and high performance liquid chromatographic (HPLC) methods are suitable for quantification and are in general use. The newer immunochemical methods such as the enzyme - linked immunosorbent assay (ELISA) or affinity column methods are being rapidly developed. ELISA methods are available for screening as well as quantification, but these methods are temperature - sensitive and they should only be used with proper controls. The affinity column method is less temperature - sensitive and can be used for either screening or quantification. The chemical and immunochemical methods are reliable if care is taken and personnel are well trained. All analytical laboratories should stress safety and include suitable analytical validation procedures.
The study of alfatoxin contamination of foods and feeds is important because aflatoxins are toxic and carcinogenic to humans and animals. This paper reviews the sampling plans and analytical methods for alfatoxins in groundnuts and groundnut products, and also refers to the safety aspects of such methods.
Guidelines for mycotoxin safety precautions are given by the Association of Official Analytical' Chemists (AOAC) in Official Methods of Analysis Chapter 26 (1984). The mycotoxin analysis publication from the International Agency for Research on Cancer (IARC) also has a good discussion of safety precautions (Stoloff et al. 1982). The safety guidelines discussed in these books are appropriate for both crude and pure aflatoxin preparations. The chemicals should only be handled with gloves and used only in properly ventilated hoods or glove boxes.
Spores and other viable propagules of Aspergillus flavus, A. parasiticus, and other fungi can cause three types of disease in humans: allergy, poisoning, and infection (Hill et al. 1985). A. flavus infection in humans is uncommon but possible. Airborne spores and dust containing propagules of the A. flavus group may cause allergies in some people and the inhaled particles may contain aflatoxins (Shotwell et al. 1981). Two thin layer chromatography (TLC) methods have been developed to measure aflatoxins in maize and grain dust (Ehrlich and Lee 1984, Shotwell et al. 1981).
Hill and co-workers (1984) found between to and 109 viable fungal propagules per m³ of air containing maize dust; air containing groundnut dust is probably equivalent. The majority of the A. flavus progagules in air samples were deposited on the stages of the Andersen sampler corresponding to the trachea, primary bronchi, and secondary bronchi in the human respiratory system (Hill et al. 1984). A. flavus spores and propagules in dust associated with inoculation, shelling, grinding, and extraction procedures are sufficiently hazardous to require safe handling procedures including gloves, masks, protective clothing, and efficient dust collection mechanisms.
Sampling is the most important contributor to the variability of analyses for aflatoxin in agricultural commodities, particularly groundnuts, because of the nonhomogenous nature of aflatoxin distribution. The first consideration in any experimental or regulatory protocol should be the sampling method. Protocols have been published on sampling techniques (Dickens and Whitaker 1986). Schuller et al. (1976) published an excellent review of sampling plans and collaboratively studied methods for aflatoxin analysis.
Alfatoxin contamination in groundnuts generally is more variable in single fields, single test plots, or single lots than aflatoxin contamination in maize and some other crops. Therefore a 22 kg sample is needed for groundnut whereas a 4.54 kg sample is usually sufficient for maize, especially when several analytical samples are averaged to approximate the true mean (Whitaker and Dickens (1983). In groundnut lots composed of mixed loads from different sources, a larger initial sample should be taken. In the United States three 22 - kg samples are taken from each groundnut lot (Dickens and Whitaker 1986). The total sample should be ground so that it passes through a 0.85 - mm sieve, thoroughly mixed or divided, and properly subsampled before analytical samples are taken. Sampling protocols for test plots must be part of the experimental design and should be tailored to meet the experimental objectives.
Criteria for aflatoxin standards (Rodricks 1973) and procedures for checking the concentration and purity of aflatoxin standards can be found in AOAC Official Methods of Analysis(1984). The use of calibrated standards in all analytical laboratories is essential. Prepared standards are available from several commercial companies at reasonable prices and analytical laboratories should, if possible, routinely use these standards. Velasco (1981) found that cyclohexane, heptane, and toluene could be substituted for benzene in standards if the solutions were not exposed to light. Analysts should take solvent composition into consideration when standards are prepared for high performance liquid chromatography (HPLC) as it has been found that solvent composition affects aflatoxin fluorescence (Chang - Yen et al. 1984).
Presumptive and Screening Methods
Some applications require only presumptive or screening tests while others require the quantification of only B. or several of the aflatoxins. Groundnuts at the buying point are visually inspected in the United States for evidence of A. flavus conidial heads and if present the suspect lots are not allowed into commerce for human consumption (Dickens and Whitaker 1986). This visual examination is not a chemical test and may result in family acceptances or rejections. The other commonly used screening technique is the application of one of several minicolumn procedures to detect aflatoxin contamination above a predetermined level (Holaday 1981, Romer et al. 1979). Shannon and Shotwell (1979) conducted a collaborative study of two minicolumn methods and found that a combination method using the Holaday extraction and the Velasco minicolumn was the most satisfactory method. Minicolumn techniques should not be used for quantitative purposes where accurate quantitative data are required. Madhyastha and Bhat (1984) recently developed a minicolumn confirmation method for aflatoxins. These workers confirmed the identity of aflatoxins on the developed minicolumn by applying 20% H2SO4, 20% HCI, or trifluoroacetic acid (TFA) in 20% HNO3. All acids changed the fluorescence from blue to yellow, with the TFA in 20% HNO3 having the lowest detection limit.
Many of the methods adopted by scientific groups and government agencies are based on TLC detection and quantification procedures that have been evaluated in collaborative studies. The HPLC methods are not more often recommended only because very few collaborative studies have been conducted on them as yet. The AOAC (1984) recommends the contamination branch (CB) and the best foods (BF) methods for aflatoxin analysis in groundnuts. Nesheim (1979) and Shotwell (1983) have also reviewed methods on aflatoxin analysis.
Thin layer chromatography (TLC) methods
The CB method (AOAC 1984) is the standard by which other methods are judged. Details can be found in Stoloff et al. (1982). Shotwell and Goulden (1977) compared the AOAC BF groundnut method and the AOAC cottonseed method with the CB method for analysis of aflatoxins in maize. The BF method for uses a methanol + water (55 + 45) extraction solution, while the cottonseed method uses an acetone+water(85+15) extraction solution. Neither of these solvents extracted aflatoxins from maize as efficiently as did the chloroform+water(250+15) extraction of the CB method. The BF method is suitable for groundnuts with aflatoxin contents below 50 µg kg-1. Lee and Catalano (1981) developed a scaled-down cleanup column as a solventsaving modification of the CB method. Laboratories which use fluorodensitometry for quantitative measurements need to be careful to avoid fading of aflatoxin spots on TLC plates; fading could be delayed by covering the layer on the TLC plate with another glass plate (Nesheim 1971).
The CB method is an excellent TLC method, but it has two major disadvantages: (1) it is expensive because it uses large amounts of solvents which create a disposal problem, and (2) the major solvent used is chloroform which may be a hazard to workers. Dantzman and Stoloff (1972) developed a modified a modified screening method in which they omitted the column chromatography step of the CB method and directly spotted the residual oil from maize extracted with CHCI3 water. Spilman (1985) modified this screening method for maize by adding benzene+acetonitrile (98+2) to the residual oil and measuring the volume to obtain quantitative TLC results. Groundnuts would have to be defatted with hexane before the CHCI3 water extraction for these screening methods.
Kamimura et al. (1985) recently described a simple rapid HPTLC method which compared favorably with the CB method. Davis et al. (1981) used a novel approach by devising a method using the fluorescence of the iodine derivative of aflatoxin B. for quantification and TLC confirmation. Josefesson and Moller (1977) developed a multi-mycotoxin screening method for detection of aflatoxins, ochratoxin, patulim, sterigmatocystin, and zearalenone, while Seitz and Mohr (1976) and Thomas et al. (1975) developed methods for aflatoxin and zearalenone determination.
The AOAC aflatoxin confirmation method is based on the TFA reaction with B1, G1, or M1 (Przybylski 1975). The TFA procedure or direct acetlylation (Cauderay 1979) can be carried out on a TLC plate before development. Trucksess et al. (1984) recently published a rapid TLC method using a disposable silicagel column for cleanup and confirmation by gas chromatography-mass spectroscopy. No matter which TLC method is used the aflatoxin identified needs to be confirmed. A review of confirmation methods has been written by Nesheim and Brumley (1981).
High performance liquid chromatography (HPLC) methods
Aflatoxin analysis using HPLC for separation and detection is quite similar to TLC because similar sampling and extraction procedures are used. The major advantages of HPLC over TLC are speed, automation, improved accuray, and precision. Both normal-phase and reverse-phase HPLC separations have been developed for aflatoxin analyses. Early experimental work by Seitz (1975) and Garner (1975) on HPLC separations revealed that aflatoxins could be separated on nominal-phase columns and detected with either a UV detector or a fluorescence detector. Seitz (1975) noted that the fluorescence detector had limited usefulness for aflatoxin B1 and B2 with normal phase separations.
Panalaks and Scott (1977) developed a silica-gel packed flow cell for fluorometric detection of B1, B2, G1, and G2 with normal phase aflatoxin separations. A silica-gel packed cell was used by Pons (1979) and Thean et al. (1980) in two different HPLC methods for determination of aflatoxins. The major disadvantage of the packed cell is lack of stability. The cell needs to be repacked often and the detector signal weakens with time. The advantages of a packed cell method are that no derivative is necessary for detection and the mobile phase can be recycled.
Reverse-phase HPLC separations of aflatoxins are more widely used than normal-phase HPLC separations. However, the fluoresence intensities of B1 and G1 are diminished in reverse-phase solvent mixtures so the derivatives B and G are diminished in reverse-phase solvent mixtures so the derivatives B and G are generally prepared before injection. Analysts should be aware that derivatives B and G are not stable in methanol, which should be used with caution, especially in the injection solvent. Acetonitrile-water mixtures do not degrade B and G rapidly and are preferred to methanol-water mobile phases.
Several reverse-phase methods have been published (Cohen and Lapointe 1981, Stubblefield and Shotwell 1977) including three with comparisons to the CB method (De Vries and Chang 1982, Hutchins and Hagler 1983, Tarter et al. 1984). Stubblefield and Shotwell (1977) found that M, and M2 as well as B1, B1, B2, G1, and G2 could be resolved and detected with a UV detector at 350 nm using reverse-phase chromatography. The methods developed by Hutchins and Hagler (1983), De Vries and Chang (1982) and Tarter et al. (1984) all use TFA derivatization and apparently compare favorably with other methods. Diebold et al. (1979) used laser fluorometry to detect aflatoxin B after reverse-phase chromatography.
Davis and Diener (1980) found that the iodine derivative of B1 could be used for confirmation and developed a reverse-phase method with fluorescence detection for this derivative. This work led to the development of a postcolumn iodination method to enhance B. and G. fluorescence after reversephase chromatography. Shepard and Gilbert (1984) investigated the conditions needed for the postcolumn iodination reaction to enhance fluorescence of aflatoxins B1 and G1.
Aflatoxin B1 in groundnuts can be determined using solid-phase radio-immunoassay (RIA). (Langone and van Vunakis 1976, Sun and Chu 1977), monoclonal affinity column immunoassay (Groopman et al. 1984), or enzyme-linked immunosorbent assay (ELISA) techniques (Chu and Ueno 1977, El-Nakib et al. 1981, Lawellin et al. 1977, Pestka et al. 1980). ELISA or affinity column techniques are more suited to field use than RIA techniques and will probably be extensively developed and utilized. The major advantages of the ELISA and affinity column methods include speed, ease of sample preparation, ease of use, and a potentially low cost per anylysis. The disadvantages include different a antibody specificities for B1 and cross reactivity with other aflatoxins. ELISA procedures are qualitative or semi-quantitative at best and are temperature sensitive. The major application for ELISA procedures at present is screening for aflatoxin B1 below a predetermined concentration. The color developed by the enzyme-mediated reaction gives an indication of the amount of B1 present. More development is needed before immunochemical techniques will be generally useful for applicatications where quantification is critical. Methods also need to be developed that will distinguish between B1, B2, G1, and G2 individually or collectively.
SELECTION OF ANALYTICAL APPROACH
Regulatory and experimental applications of methods for aflatoxin analysis do not always need to be the same. Regulatory applications need to be quantitative and legally acceptable, but acceptable methods may vary within a country or between countries. However, it is important to use validated methods for regulatory applications.
Aflatoxin analysis in experimentral work must be tailored to the objectives and method selection should be a part of the experimental design. Inexpensive minicolumn data may be sufficient for some experimental purposes whereas quantitative data on B1, B2, G1, and G2, may be required for other purposes. Costs and data requirements can sometimes seem to be at odds when quantitative data are necessary. The TLC and immunochemical methods may not always be cheaper than HPLC in the long run because HPLC requires a single large initial investment, and TLC and ELISA both use expensive disposable plates. HPLC is possibly more suitable for large analytical laboratories while TLC is more suitable for laboratories with only a few samples to be analyzed. With further development, immunochemical methods will probably become more versatile and suited to a wider variety of applications.
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Table 1. Major physico-chemical properties of aflatoxins.
Figure 1. Molecular structure of aflatoxins.
Extracted from Goto and Marabe (1989).
Citation: ICRISAT (International Crops Research Institute for the Semi-Arid Tropics).1989. Aflatoxin contamination of groundnut: proceedings of the International Workshop, 6-9 Oct 1987, ICRISAT Center, India. Patancheru, A.P. 502324, India: lCRISAT.
Figure 2. Chromatograms of aflatoxins obtained by HPLC mobile phase, 1duene-ethylacetate-formic acid-methanol (89:7:2:2) Source: IBIP, p. 177
Table 2. Comparison of performance of Enzyme-Linked Immunosorbent Assay (ELISA) kits for aflatoxin analysis
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