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Liquid chromatographic determination of aflatoxins
Microchemical Journal 73 (2002) 39–46
Liquid chromatographic determination of aflatoxins ´ H-Ottaa,*, Gyula Zaray ´ a, Emil Mincsovicsb Eszter Pappa, Klara a
¨ ¨ University, H-1518 Budapest, P.O. Box 32, Department of Chemical Technology and Environmental Chemistry, L. Eotvos Hungary b OPLC-NIT Ltd, H-1119 Budapest, Andor u. 60, Hungary
Abstract The difurancoumarin derivatives known as aflatoxins are highly toxic fungi metabolites belonging to the vast class of mycotoxins, which can contaminate foods and feeds when storage conditions favor fungal growth. Because of potential health hazards for humans, levels of aflatoxins are monitored throughout the world. During the past two decades several chromatographic and other methods were developed for identification and determination of aflatoxins in agricultural and food products. This paper is a review of the overpressured-layer chromatographic (OPLC) and high performance liquid chromatographic methods most often used for the analysis of aflatoxins. However, emphasis is placed on summarizing the OPLC methods developed for determination of aflatoxins in maize, wheat, fish meat, peanut samples, rice and sunflower seeds spiked with aflatoxins B1 , B2 , G1 and G2 in concentration of 2–10 mgy cm3, which were developed in our laboratory. The results of the proposed validation procedure, whose development was based on the guideline of the International Conference on Harmonization (ICH) for pharmaceutical products (1994, Brussels), for the determination of the above-mentioned aflatoxins in wheat samples are also presented. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Aflatoxin; Food; Liquid chromatography; Overpressured-layer chromatography; Validation
1. Introduction Mycotoxins (aflatoxins, ochratoxin, fumonisines, trichotecenes, zearalenone, cyclopiasonic acid) are toxic compounds produced by certain types of fungi. Up to now, there have been known about 200 different mycotoxins. These compounds are acutely toxic to humans and to animals. Illnesses caused by mycotoxins are known for a long time. During the World War II many Russian *Corresponding author. Tel.: q36-1-209-0555; fax: q36-1209-0602. E-mail address: [email protected] (K. H-Otta).
people died after having eaten grain poorly stored and highly contaminated with mycotoxins w1x. Most of the mycotoxins were identified after cases of poisoning in live-stock or the population at large scale. In the UK, in 1969, more than 100 000 turkeys died of Turkey ‘X’ disease that was finally traced to peanuts—a component of their feed. It turned out that the consumed peanuts were contaminated with Aspergillus flavus fungi whose metabolites, later called as aflatoxins, were responsible for the occurrence of the above-mentioned disease. In the word aflatoxin, the first syllable ‘a’ was derived from the genus Aspergillus, the second one, ‘fla’, from the species flavus
0026-265X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 2 . 0 0 0 4 8 - 6
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E. Papp et al. / Microchemical Journal 73 (2002) 39–46
Fig. 1. Chemical structures of aflatoxins B1, B2, G1 and G2 produced by Aspergillus flavus.
and the term, ‘toxin’, came from the adjective ‘toxic’. In the aflatoxin group, about 16 compounds are known, but only aflatoxin B1, B2, G1, G2 and M1 are routinely monitored. Aflatoxin B1, B2, G1 and G2 are chemical derivatives of difurancoumarin (Fig. 1) and they are most often analyzed in cereals, nuts, spices, cocoa beans, meat, milk products and eggs. Aflatoxin B1 is the prevalent compound in the samples and, at the same time, is acutely toxic and most carcinogenic amongst them. Aflatoxin M1, which can be found in milk and dairy products, is just as toxic as aflatoxin B1 w2x. Aflatoxins are known to be mutagen, carcinogen and teratogen compounds. The intake of these toxins over a long period of time in very low concentrations may be highly dangerous. These compounds can enter the food chain, mainly, by ingestion through the dietary channel of humans and animals. Absorption by inhalation is likely to occur under particular circumstances, for example, in workplaces where contaminated foodstuffs are treated or handled. These researches focusing on the determination of mycotoxins in biological fluids greatly contribute to clarify the mechanism of health impairment attributable to these toxic com-
pounds w3x After ingestion, aflatoxin B1 is metabolized by enzymes to generate a reactive 8,9-epoxide metabolite that can be bound to DNA as well as to serum albumin forming aflatoxin-N 7guanine and lysine adducts, respectively. Covalent binding to DNA is considered to be a critical step in aflatoxin hepatocarcinogenesis. Determination of these metabolites were solved by developing enzyme linked immunosorbent assay (ELISA) methods w4,5x. Nayak et al. w5x detected and quantified this adduct from urine and liver tissue samples of people having maize predominant diet by sensitive indirect competitive ELISA. Aflatoxin–albumin adducts were measured in vegetarian and non-vegetarian persons from Thailand w6x. The vegetarians were more frequently exposed to aflatoxins. The increased exposure might be the result of consumption of various vegetables, grains, soybean in which aflatoxins had been shown to occur w6 x . The biosynthesis of aflatoxins is induced by sugars. The induction is associated with the transcriptional activation of the pathway genes and the pathway regulatory gene, aflR. The regulation of aflatoxin biosynthesis had been examined by manipulating the transcription of aflR. Studies
E. Papp et al. / Microchemical Journal 73 (2002) 39–46
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Table 1 Limits of aflatoxins for various foodstuffs in different countries Country
Germany France Switzerland USA (FDA) Hungary
Concentration (ngycm3) Aflatoxin B1
Total aflatoxin (8B1B2G1G2)
Aflatoxin M1
2.0 5.0 1.0a – 2.0
4.0 – 5.0 20.0 5.0
0.05 – 0.05–0.25b 0.5c –
a
2.0 ngycm3 for maize, corn. Only for dairy products. c For milk. b
concerning this topic showed that constitutive overexpression of the pathway transcriptional regulatory gene aflR led to higher transcript accumulation of pathway genes and increased aflatoxin production w7x. The presence of fungi does not necessarily imply the presence of toxins. The fungi species can produce aflatoxins on commodities in the field under stress conditions or in storage when conditions such as high moisture and warm temperature (25–30 8C) are encountered. Because of potential health hazards for humans, worldwide monitoring of aflatoxins in various commodities has been indicated and regulatory levels (Table 1) have recently been documented w1x. It should be mentioned that the limit prescribed by the World Health Organization (WHO) for aflatoxin B1 in various foodstuffs is 5 ngycm3 and the total aflatoxin level expressed by summing the concentrations of aflatoxins B1, B2, G1 and G2 in foodstuffs cannot exceed 10 ngycm3. In Germany, even the aflatoxin B1 and the total aflatoxin level in baby-food products are regulated in values that cannot be greater than 20 and 50 pgycm3, respectively. For the same baby-food products, the Swiss regulations are even more severe allowing only 10 pgycm3 value for the total aflatoxin level and 20 pgycm3 for aflatoxin M1 in milk samples w1x. 2. Analytical techniques for determination of aflatoxins 2.1. General considerations A wide variety of food and feed matrices have been shown to be contaminated by aflatoxins and
it is, therefore, important to have available, simple and quantitative methods for aflatoxin analysis. In order to achieve this requirement one possibility is spiking of ‘aflatoxin-free’ samples with aflatoxin standard solutions in known concentrations followed by their extraction, clean-up, separation and quantitative determination. However, Yong and Cousin w8x inoculated maize and peanut samples with spores of Aspergillus parasiticus, incubated them under controlled conditions (temperature, humidity) and monitored the synthesized aflatoxins after the apparition of moulds in the samples by ELISA. Aflatoxins are most often analyzed in nuts, for example peanut w9–18x, almond w11,16x, pistachio w1,11,16,19x, cotton-seed w9,12,18x, chili w20–22x, ginger w21,22x, pepper w21–23x, paprika w21,22x, feed w9,12,24–28x, corn w9–12,14–18,27– 31,48,55,56x and wheat w27,56,59x samples. Some reports indicated natural contamination with aflatoxins B1 and G1 mainly from countries well known for their warm and humid climate. Thus, Mexican and Brazilian beer w32x, Venezuelan wheat w33x samples, peanuts collected in Argentina w34x, India Chili powders and pods w20x, cardamon, cayenne pepper, chili, cloves, cumin, curry powder, ginger, mustard, nutmeg, paprika, saffron and white pepper samples selected from supermarkets and ethnic shops in Lisbon w21x proved to be naturally contaminated with aflatoxins. Also, there are some reports from Kuwait w35x and Central Italy w36x about natural contamination of cow milk with aflatoxin M1. Micco et al. w37x determined the content of aflatoxin B1 and ochratoxin A in poultry liver samples of broilers and
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laying hens that had been fed with feedstuff containing the mentioned toxins. Neither clinical signs, nor mortality were noted among the investigated species, however, the results indicated that the two toxins interact synergistically, which was proved by the increasing toxin content and detoxification time. The most widespread methods for quantitative determination of aflatoxin content in different samples are thin-layer chromatography (TLC) w10,12,13,23,26,38x and high performance liquid chromatographic (HPLC) w1,2,9,11,14–16,19– 32,35,36,39,48x. Aflatoxins can be separated and detected using either normal- or reversed-phase HPLC methods mainly with fluorescence spectrometric detection. The use of two-dimensional thin-layer chromatography (2D-TLC) w26,40x and high performance thin-layer chromatography (HPTLC) for the determination of aflatoxins has been reported w12,17x. The use of gel permeation chromatography has not been extensively utilized for aflatoxin analysis w27,41x. Hetmanski and Scudamore w41x used a column packed with Bio-Beads S-X3 to clean-up extracts of cereals and animal feedstuffs prior to HPLC analysis. Other analytical techniques, which may be used for aflatoxin analysis are the following: gas chromatographic–mass spectrometry w7x, electrophoresis w30,42x and ELISA w6,7,20,43x. 2.2. Aflatoxin analyses by HPLC In the analytical procedures of aflatoxin analysis by HPLC, there are three steps: extraction, purification or clean-up and quantitative determination. The most common solvent system used for extraction are mixtures of chloroform and water w1,13,26,55x, methanol–water w10,12,14,15,22,28, 43,55,56x or acetonitrile–water w11,16,55,56,59x. Whatever extraction method is used, the resulted extract still contains, besides aflatoxins, various impurities (lipids, pigments) requiring further clean-up steps. The most commonly used extraction technique is the solid-phase extraction (SPE), which replaced the traditional use of column chromatography and liquid–liquid partition for clean-up. The most popular stationary phases of the SPE columns used
are the following: silica gel w12,44x, C18 bondedphase w6,12,26,45x and magnesium silicate commercialized as Florisil w12,26,46x. Multi-functional clean-up w16,17,47x and antibody affinity w14,19,21,22,24,25,30,33,35,38,48,49x SPE columns are also widely used. Considering the complexity of the matrices, the use of silica gel and C18 bonded-phase are necessary for removing the above-mentioned compounds from extracts, which interfere in the determination of target analytes w26x. Yen and Bidasee w28x extracted the aflatoxins from animal feeds and feed components with methanol–water mixture. An aliquot of the filtrated extract was defatted with petrolether, and then the aflatoxins were partitioned into chloroform. The chloroform extract was purified on silica gel column, aflatoxins were derivatized with trifluoroacetic acid and were determined by reversedphase HPLC. Cohen and Lapointe w49x performed the extraction step with acetonitrile–water mixture for determination of aflatoxins in corn. In this case, aflatoxins were also partitioned into chloroform. The resulted solution was cleaned by silica gel SPE, and the determination was carried out by HPLC. Van Egmond et al. w26x recommended a method for adoption to the European Community (EC), which involved purification of the chloroform extract of samples with Sep-Pak Florisil cartridges and Sep-Pak C18 cartridges before analyses. HPLC with I2 post-column derivatization or 2D-TLC had been applied as determinative step. Multi-functional clean-up columns, like the ones commercialized as Isolute SPE cartridges, work in just opposite way to other ones providing rapid one-step extract purification. These columns are designed to allow compounds of interest to pass through, while retaining compounds that create interference in most of the analytical methods. Wilson and Romer w47x purified different extracts of agricultural products using multi-functional columns prior to HPLC analysis. Akiyama et al. w16x developed a simple and reproducible method for nuts and corn samples, which consisted of extraction with a mixture of acetonitrile and water (9:1) and clean-up on a multi-functional Isolute SPE cartridge. The quantitative determination required pre-column derivatization with trifluoroacetic acid
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anhydride, for the HPLC analysis with fluorescence detection. Spices are particularly difficult to analyze for aflatoxins, because of their highly colored contaminating compounds that are co-extracted with aflatoxins. Garner et al. w22x analyzed a variety of powdered spices. Clean up on immunoaffinity column for the investigated samples proved to be specific and sensitive. The aflatoxin content of spice samples was quantified by HPLC, and they also developed a new method for post-column derivatization with pyridinium bromide perbromide (PBPB). As it has already briefly mentioned, the HPLC analysis using fluorescence spectrometric detection requires pre- or post-column derivatization in order to enhance detection. The most frequently used compounds for pre-column derivatization are trifluoroacetic acid w1,15,16,28,32x or its anhydride w16x, while iodine was reported as a post-column derivatizing agent w14,26,48x. 2.3. Overpressured-layer chromatography for aflatoxin analyses Overpressured-layer chromatography (OPLC) ´ et al. w50,51x, combines the developed by Tyihak advantages of HPLC and HPTLC methods. The first commercially available OPLC instrument (Chrompress 10) was a completely off-line system. In that case an external pressure was applied to the surface of the sorbent layer by means of a cushion system, forcing the eluent to flow (by overpressure) through the sorbent layer. All the principal chromatographic steps, such as sample application, separation, quantitative evaluation, and preparative isolation, were performed independently w52x. The second-generation instrument (Chrompress 25) is suitable for both off-line and on-line separations. Separated components are evaluated quantitatively by densitometric determination of the separated spots of the plate. If the eluent outlet of the chamber is connected to a flow cell detector, eluting solutes can be detected online, and fractions can be collected. The new automated OPLC system called the Personal OPLC Basic System 50 is suitable for analytical and semipreparative separations. The automatic microprocessor-controlled system ensures rapid and
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reproducible isocratic and stepwise gradient separations as well. A key step in either column or layer liquid chromatography, is the sample processing for obtaining clean analytes. The increasing complexity of samples frequently makes direct analysis difficult. A new opportunity of sample clean-up is provided by applying OPLC as a planar version of a special SPE system where the purification step is managed on layer shaped sorbent bed w53x. The layers make possible the application of uncleaned samples as well. In spite of that, in many cases, samples must be cleaned before analyzing. However, it should be mentioned that the plates are not reusable. Aflatoxin analysis by OPLC separation and densitometric quantitation has been performed by ´ w54x in 1985. In our laboratory, OPLC Gulyas methods have been developed for the determination of aflatoxins in different food and feed matrices w55,56x. The aim of our work was the development of a suitable TLC method for determination of aflatoxins. The first task was the development of methods for aflatoxin determination in corn samples like maize and wheat samples, because the most likely natural contamination in Hungary would occur during the storage of these cereals. To achieve this, ‘aflatoxin-free’ maize samples were spiked with standard solutions in concentration of 2–10 ngyg B1, B2, G1 and G2 aflatoxins. The developed method can be summarized as follows: (1) After the combination and simplification of the existing TLC and HPLC methods, the best extracting solvent mixture consisted of acetonitrile:water in 9:1 vyv% ratio. At the same time, the interfering compounds of the spiked maize samples remained mainly in the extraction residue compared to the use of other investigated solvent mixtures. (2) By employing the OPLC technique, the separation of 6–7 aflatoxins containing samples was achieved after a simple pre-washing procedure with diethyl-ether:n-hexane (1:1) mixture on a sole plate. For the development, a solvent mixture of chloroform:toluene:tetrahydrofuran (10:15:1) was found to be the most suitable. The method developed for maize samples had to be modified
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for the determination of the aflatoxins of wheat sample because of the low recovery values and poor repeatability. The modification consisted of pre-washing with diethyl-ether:n-hexane mixture (1:1) in reverse direction and the composition of the mobile phase was also slightly changed to chloroform:toluene:tetrahydrofuran (15:15:1). (3) The sensitivity of the OPLC method was increased by immersing the plate after the development in the paraffin oilyn-hexane mixture for approximately 5 min followed by densitometric detection. (4) After the modification of the extracting solvent mixture developed for maize samples, the OPLC method was applied for other samples (fish meat, peanut, soybean, rice and sunflower seeds). The determination of samples containing vegetable oil differed only in the composition of the extracting mixture. Namely, the acetonitrile:water (9:1) extracting solvent mixture was changed to chloroform:water (45:5), because the acetonitrile:water mixture was not able to extract non-polar compounds and aflatoxins might remain in the oil phase. Nowadays, the reliability of analytical methods has a primary importance. Quality assurance and the need for validated methods in mycotoxin analysis are receiving more and more attention w57x. For that reason, the next step of our work was the validation of the method and the statistical assessment of the obtained results. In pharmaceutical investigations, methods are characterized in accordance with international regulations, meanwhile food industry requirements are less uniform and strict. In pharmaceutical analysis, standardization of the validation process is stipulated by the guidelines of the International Conference on Harmonization (ICH) in which the different aspects of analytical validation are clearly defined w58x. Those guidelines served as starting point in validating the OPLC method developed by us for the determination of aflatoxins in wheat samples w59x. The results of the validation procedure based on statistical assessment of the obtained results were the following: a. Aflatoxins were separated with good selectivity and specificity, the retention factor, the asym-
metric factor and the resolution were 0.24–0.55, 0.87–1.05 and 1.74–2.35, respectively. b. For performing linearity test, wide and narrow ranges were chosen. Linearity of the calibration curve was proved in the 1–10 ng range. c. The recovery values of the measurements for accuracy were in the range of 84.55–105.77%. d. The RSD values of the measurements for repeatability and for intermediate precision were 4.35– 9.25% and 3.55–8.42%, respectively. The data of the F statistical probe were convenient. e. The detection limits and the quantification limits of the method were 0.018–0.15 ng and 0.27– 0.36 ng, respectively. f. According to the results of robustness test, the factors having the most important and least significant influence on the separation were the quality of the plate and the volumes of tetrahydrofuran of the eluent, respectively. 3. Conclusion By comparing the OPLC method for aflatoxin analysis for various foodstuffs (maize, wheat, peanut, fish meat, rice and sunflower seeds) developed in our laboratory, it can be stated that they are as competitive as the more popular HPLC methods applied for aflatoxin determination. The developed method permits the simultaneous determination of up to seven samples, is more rapid and has a lower cost than the HPLC methods. However, it should be emphasized that the OPLC technique can not replace the more reproducible HPLC technique, but can complement it. Validation of the method developed for determination aflatoxin B1, B2, G1 and G2 of wheat samples, based on the statistical evaluation of analytical data obtained, could be successfully achieved, which is very promising for validation of the other aflatoxin determining OPLC methods. References w1x R. Schuster, G. Marx, M. Rathaupt, Analysis of Mycotoxins by HPLC with Automated Confirmation by Spectral Library HP Application Note, Pub. NR. 125091-8692 E (1993). w2x C. Micco, C. Brera, M. Miraglia, R. Onori, Food Addit. Contam. 4 (1987) 407–414.
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Liquid chromatographic determination of aflatoxins ´ H-Ottaa,*, Gyula Zaray ´ a, Emil Mincsovicsb Eszter Pappa, Klara a
¨ ¨ University, H-1518 Budapest, P.O. Box 32, Department of Chemical Technology and Environmental Chemistry, L. Eotvos Hungary b OPLC-NIT Ltd, H-1119 Budapest, Andor u. 60, Hungary
Abstract The difurancoumarin derivatives known as aflatoxins are highly toxic fungi metabolites belonging to the vast class of mycotoxins, which can contaminate foods and feeds when storage conditions favor fungal growth. Because of potential health hazards for humans, levels of aflatoxins are monitored throughout the world. During the past two decades several chromatographic and other methods were developed for identification and determination of aflatoxins in agricultural and food products. This paper is a review of the overpressured-layer chromatographic (OPLC) and high performance liquid chromatographic methods most often used for the analysis of aflatoxins. However, emphasis is placed on summarizing the OPLC methods developed for determination of aflatoxins in maize, wheat, fish meat, peanut samples, rice and sunflower seeds spiked with aflatoxins B1 , B2 , G1 and G2 in concentration of 2–10 mgy cm3, which were developed in our laboratory. The results of the proposed validation procedure, whose development was based on the guideline of the International Conference on Harmonization (ICH) for pharmaceutical products (1994, Brussels), for the determination of the above-mentioned aflatoxins in wheat samples are also presented. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Aflatoxin; Food; Liquid chromatography; Overpressured-layer chromatography; Validation
1. Introduction Mycotoxins (aflatoxins, ochratoxin, fumonisines, trichotecenes, zearalenone, cyclopiasonic acid) are toxic compounds produced by certain types of fungi. Up to now, there have been known about 200 different mycotoxins. These compounds are acutely toxic to humans and to animals. Illnesses caused by mycotoxins are known for a long time. During the World War II many Russian *Corresponding author. Tel.: q36-1-209-0555; fax: q36-1209-0602. E-mail address: [email protected] (K. H-Otta).
people died after having eaten grain poorly stored and highly contaminated with mycotoxins w1x. Most of the mycotoxins were identified after cases of poisoning in live-stock or the population at large scale. In the UK, in 1969, more than 100 000 turkeys died of Turkey ‘X’ disease that was finally traced to peanuts—a component of their feed. It turned out that the consumed peanuts were contaminated with Aspergillus flavus fungi whose metabolites, later called as aflatoxins, were responsible for the occurrence of the above-mentioned disease. In the word aflatoxin, the first syllable ‘a’ was derived from the genus Aspergillus, the second one, ‘fla’, from the species flavus
0026-265X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 2 . 0 0 0 4 8 - 6
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Fig. 1. Chemical structures of aflatoxins B1, B2, G1 and G2 produced by Aspergillus flavus.
and the term, ‘toxin’, came from the adjective ‘toxic’. In the aflatoxin group, about 16 compounds are known, but only aflatoxin B1, B2, G1, G2 and M1 are routinely monitored. Aflatoxin B1, B2, G1 and G2 are chemical derivatives of difurancoumarin (Fig. 1) and they are most often analyzed in cereals, nuts, spices, cocoa beans, meat, milk products and eggs. Aflatoxin B1 is the prevalent compound in the samples and, at the same time, is acutely toxic and most carcinogenic amongst them. Aflatoxin M1, which can be found in milk and dairy products, is just as toxic as aflatoxin B1 w2x. Aflatoxins are known to be mutagen, carcinogen and teratogen compounds. The intake of these toxins over a long period of time in very low concentrations may be highly dangerous. These compounds can enter the food chain, mainly, by ingestion through the dietary channel of humans and animals. Absorption by inhalation is likely to occur under particular circumstances, for example, in workplaces where contaminated foodstuffs are treated or handled. These researches focusing on the determination of mycotoxins in biological fluids greatly contribute to clarify the mechanism of health impairment attributable to these toxic com-
pounds w3x After ingestion, aflatoxin B1 is metabolized by enzymes to generate a reactive 8,9-epoxide metabolite that can be bound to DNA as well as to serum albumin forming aflatoxin-N 7guanine and lysine adducts, respectively. Covalent binding to DNA is considered to be a critical step in aflatoxin hepatocarcinogenesis. Determination of these metabolites were solved by developing enzyme linked immunosorbent assay (ELISA) methods w4,5x. Nayak et al. w5x detected and quantified this adduct from urine and liver tissue samples of people having maize predominant diet by sensitive indirect competitive ELISA. Aflatoxin–albumin adducts were measured in vegetarian and non-vegetarian persons from Thailand w6x. The vegetarians were more frequently exposed to aflatoxins. The increased exposure might be the result of consumption of various vegetables, grains, soybean in which aflatoxins had been shown to occur w6 x . The biosynthesis of aflatoxins is induced by sugars. The induction is associated with the transcriptional activation of the pathway genes and the pathway regulatory gene, aflR. The regulation of aflatoxin biosynthesis had been examined by manipulating the transcription of aflR. Studies
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Table 1 Limits of aflatoxins for various foodstuffs in different countries Country
Germany France Switzerland USA (FDA) Hungary
Concentration (ngycm3) Aflatoxin B1
Total aflatoxin (8B1B2G1G2)
Aflatoxin M1
2.0 5.0 1.0a – 2.0
4.0 – 5.0 20.0 5.0
0.05 – 0.05–0.25b 0.5c –
a
2.0 ngycm3 for maize, corn. Only for dairy products. c For milk. b
concerning this topic showed that constitutive overexpression of the pathway transcriptional regulatory gene aflR led to higher transcript accumulation of pathway genes and increased aflatoxin production w7x. The presence of fungi does not necessarily imply the presence of toxins. The fungi species can produce aflatoxins on commodities in the field under stress conditions or in storage when conditions such as high moisture and warm temperature (25–30 8C) are encountered. Because of potential health hazards for humans, worldwide monitoring of aflatoxins in various commodities has been indicated and regulatory levels (Table 1) have recently been documented w1x. It should be mentioned that the limit prescribed by the World Health Organization (WHO) for aflatoxin B1 in various foodstuffs is 5 ngycm3 and the total aflatoxin level expressed by summing the concentrations of aflatoxins B1, B2, G1 and G2 in foodstuffs cannot exceed 10 ngycm3. In Germany, even the aflatoxin B1 and the total aflatoxin level in baby-food products are regulated in values that cannot be greater than 20 and 50 pgycm3, respectively. For the same baby-food products, the Swiss regulations are even more severe allowing only 10 pgycm3 value for the total aflatoxin level and 20 pgycm3 for aflatoxin M1 in milk samples w1x. 2. Analytical techniques for determination of aflatoxins 2.1. General considerations A wide variety of food and feed matrices have been shown to be contaminated by aflatoxins and
it is, therefore, important to have available, simple and quantitative methods for aflatoxin analysis. In order to achieve this requirement one possibility is spiking of ‘aflatoxin-free’ samples with aflatoxin standard solutions in known concentrations followed by their extraction, clean-up, separation and quantitative determination. However, Yong and Cousin w8x inoculated maize and peanut samples with spores of Aspergillus parasiticus, incubated them under controlled conditions (temperature, humidity) and monitored the synthesized aflatoxins after the apparition of moulds in the samples by ELISA. Aflatoxins are most often analyzed in nuts, for example peanut w9–18x, almond w11,16x, pistachio w1,11,16,19x, cotton-seed w9,12,18x, chili w20–22x, ginger w21,22x, pepper w21–23x, paprika w21,22x, feed w9,12,24–28x, corn w9–12,14–18,27– 31,48,55,56x and wheat w27,56,59x samples. Some reports indicated natural contamination with aflatoxins B1 and G1 mainly from countries well known for their warm and humid climate. Thus, Mexican and Brazilian beer w32x, Venezuelan wheat w33x samples, peanuts collected in Argentina w34x, India Chili powders and pods w20x, cardamon, cayenne pepper, chili, cloves, cumin, curry powder, ginger, mustard, nutmeg, paprika, saffron and white pepper samples selected from supermarkets and ethnic shops in Lisbon w21x proved to be naturally contaminated with aflatoxins. Also, there are some reports from Kuwait w35x and Central Italy w36x about natural contamination of cow milk with aflatoxin M1. Micco et al. w37x determined the content of aflatoxin B1 and ochratoxin A in poultry liver samples of broilers and
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laying hens that had been fed with feedstuff containing the mentioned toxins. Neither clinical signs, nor mortality were noted among the investigated species, however, the results indicated that the two toxins interact synergistically, which was proved by the increasing toxin content and detoxification time. The most widespread methods for quantitative determination of aflatoxin content in different samples are thin-layer chromatography (TLC) w10,12,13,23,26,38x and high performance liquid chromatographic (HPLC) w1,2,9,11,14–16,19– 32,35,36,39,48x. Aflatoxins can be separated and detected using either normal- or reversed-phase HPLC methods mainly with fluorescence spectrometric detection. The use of two-dimensional thin-layer chromatography (2D-TLC) w26,40x and high performance thin-layer chromatography (HPTLC) for the determination of aflatoxins has been reported w12,17x. The use of gel permeation chromatography has not been extensively utilized for aflatoxin analysis w27,41x. Hetmanski and Scudamore w41x used a column packed with Bio-Beads S-X3 to clean-up extracts of cereals and animal feedstuffs prior to HPLC analysis. Other analytical techniques, which may be used for aflatoxin analysis are the following: gas chromatographic–mass spectrometry w7x, electrophoresis w30,42x and ELISA w6,7,20,43x. 2.2. Aflatoxin analyses by HPLC In the analytical procedures of aflatoxin analysis by HPLC, there are three steps: extraction, purification or clean-up and quantitative determination. The most common solvent system used for extraction are mixtures of chloroform and water w1,13,26,55x, methanol–water w10,12,14,15,22,28, 43,55,56x or acetonitrile–water w11,16,55,56,59x. Whatever extraction method is used, the resulted extract still contains, besides aflatoxins, various impurities (lipids, pigments) requiring further clean-up steps. The most commonly used extraction technique is the solid-phase extraction (SPE), which replaced the traditional use of column chromatography and liquid–liquid partition for clean-up. The most popular stationary phases of the SPE columns used
are the following: silica gel w12,44x, C18 bondedphase w6,12,26,45x and magnesium silicate commercialized as Florisil w12,26,46x. Multi-functional clean-up w16,17,47x and antibody affinity w14,19,21,22,24,25,30,33,35,38,48,49x SPE columns are also widely used. Considering the complexity of the matrices, the use of silica gel and C18 bonded-phase are necessary for removing the above-mentioned compounds from extracts, which interfere in the determination of target analytes w26x. Yen and Bidasee w28x extracted the aflatoxins from animal feeds and feed components with methanol–water mixture. An aliquot of the filtrated extract was defatted with petrolether, and then the aflatoxins were partitioned into chloroform. The chloroform extract was purified on silica gel column, aflatoxins were derivatized with trifluoroacetic acid and were determined by reversedphase HPLC. Cohen and Lapointe w49x performed the extraction step with acetonitrile–water mixture for determination of aflatoxins in corn. In this case, aflatoxins were also partitioned into chloroform. The resulted solution was cleaned by silica gel SPE, and the determination was carried out by HPLC. Van Egmond et al. w26x recommended a method for adoption to the European Community (EC), which involved purification of the chloroform extract of samples with Sep-Pak Florisil cartridges and Sep-Pak C18 cartridges before analyses. HPLC with I2 post-column derivatization or 2D-TLC had been applied as determinative step. Multi-functional clean-up columns, like the ones commercialized as Isolute SPE cartridges, work in just opposite way to other ones providing rapid one-step extract purification. These columns are designed to allow compounds of interest to pass through, while retaining compounds that create interference in most of the analytical methods. Wilson and Romer w47x purified different extracts of agricultural products using multi-functional columns prior to HPLC analysis. Akiyama et al. w16x developed a simple and reproducible method for nuts and corn samples, which consisted of extraction with a mixture of acetonitrile and water (9:1) and clean-up on a multi-functional Isolute SPE cartridge. The quantitative determination required pre-column derivatization with trifluoroacetic acid
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anhydride, for the HPLC analysis with fluorescence detection. Spices are particularly difficult to analyze for aflatoxins, because of their highly colored contaminating compounds that are co-extracted with aflatoxins. Garner et al. w22x analyzed a variety of powdered spices. Clean up on immunoaffinity column for the investigated samples proved to be specific and sensitive. The aflatoxin content of spice samples was quantified by HPLC, and they also developed a new method for post-column derivatization with pyridinium bromide perbromide (PBPB). As it has already briefly mentioned, the HPLC analysis using fluorescence spectrometric detection requires pre- or post-column derivatization in order to enhance detection. The most frequently used compounds for pre-column derivatization are trifluoroacetic acid w1,15,16,28,32x or its anhydride w16x, while iodine was reported as a post-column derivatizing agent w14,26,48x. 2.3. Overpressured-layer chromatography for aflatoxin analyses Overpressured-layer chromatography (OPLC) ´ et al. w50,51x, combines the developed by Tyihak advantages of HPLC and HPTLC methods. The first commercially available OPLC instrument (Chrompress 10) was a completely off-line system. In that case an external pressure was applied to the surface of the sorbent layer by means of a cushion system, forcing the eluent to flow (by overpressure) through the sorbent layer. All the principal chromatographic steps, such as sample application, separation, quantitative evaluation, and preparative isolation, were performed independently w52x. The second-generation instrument (Chrompress 25) is suitable for both off-line and on-line separations. Separated components are evaluated quantitatively by densitometric determination of the separated spots of the plate. If the eluent outlet of the chamber is connected to a flow cell detector, eluting solutes can be detected online, and fractions can be collected. The new automated OPLC system called the Personal OPLC Basic System 50 is suitable for analytical and semipreparative separations. The automatic microprocessor-controlled system ensures rapid and
43
reproducible isocratic and stepwise gradient separations as well. A key step in either column or layer liquid chromatography, is the sample processing for obtaining clean analytes. The increasing complexity of samples frequently makes direct analysis difficult. A new opportunity of sample clean-up is provided by applying OPLC as a planar version of a special SPE system where the purification step is managed on layer shaped sorbent bed w53x. The layers make possible the application of uncleaned samples as well. In spite of that, in many cases, samples must be cleaned before analyzing. However, it should be mentioned that the plates are not reusable. Aflatoxin analysis by OPLC separation and densitometric quantitation has been performed by ´ w54x in 1985. In our laboratory, OPLC Gulyas methods have been developed for the determination of aflatoxins in different food and feed matrices w55,56x. The aim of our work was the development of a suitable TLC method for determination of aflatoxins. The first task was the development of methods for aflatoxin determination in corn samples like maize and wheat samples, because the most likely natural contamination in Hungary would occur during the storage of these cereals. To achieve this, ‘aflatoxin-free’ maize samples were spiked with standard solutions in concentration of 2–10 ngyg B1, B2, G1 and G2 aflatoxins. The developed method can be summarized as follows: (1) After the combination and simplification of the existing TLC and HPLC methods, the best extracting solvent mixture consisted of acetonitrile:water in 9:1 vyv% ratio. At the same time, the interfering compounds of the spiked maize samples remained mainly in the extraction residue compared to the use of other investigated solvent mixtures. (2) By employing the OPLC technique, the separation of 6–7 aflatoxins containing samples was achieved after a simple pre-washing procedure with diethyl-ether:n-hexane (1:1) mixture on a sole plate. For the development, a solvent mixture of chloroform:toluene:tetrahydrofuran (10:15:1) was found to be the most suitable. The method developed for maize samples had to be modified
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for the determination of the aflatoxins of wheat sample because of the low recovery values and poor repeatability. The modification consisted of pre-washing with diethyl-ether:n-hexane mixture (1:1) in reverse direction and the composition of the mobile phase was also slightly changed to chloroform:toluene:tetrahydrofuran (15:15:1). (3) The sensitivity of the OPLC method was increased by immersing the plate after the development in the paraffin oilyn-hexane mixture for approximately 5 min followed by densitometric detection. (4) After the modification of the extracting solvent mixture developed for maize samples, the OPLC method was applied for other samples (fish meat, peanut, soybean, rice and sunflower seeds). The determination of samples containing vegetable oil differed only in the composition of the extracting mixture. Namely, the acetonitrile:water (9:1) extracting solvent mixture was changed to chloroform:water (45:5), because the acetonitrile:water mixture was not able to extract non-polar compounds and aflatoxins might remain in the oil phase. Nowadays, the reliability of analytical methods has a primary importance. Quality assurance and the need for validated methods in mycotoxin analysis are receiving more and more attention w57x. For that reason, the next step of our work was the validation of the method and the statistical assessment of the obtained results. In pharmaceutical investigations, methods are characterized in accordance with international regulations, meanwhile food industry requirements are less uniform and strict. In pharmaceutical analysis, standardization of the validation process is stipulated by the guidelines of the International Conference on Harmonization (ICH) in which the different aspects of analytical validation are clearly defined w58x. Those guidelines served as starting point in validating the OPLC method developed by us for the determination of aflatoxins in wheat samples w59x. The results of the validation procedure based on statistical assessment of the obtained results were the following: a. Aflatoxins were separated with good selectivity and specificity, the retention factor, the asym-
metric factor and the resolution were 0.24–0.55, 0.87–1.05 and 1.74–2.35, respectively. b. For performing linearity test, wide and narrow ranges were chosen. Linearity of the calibration curve was proved in the 1–10 ng range. c. The recovery values of the measurements for accuracy were in the range of 84.55–105.77%. d. The RSD values of the measurements for repeatability and for intermediate precision were 4.35– 9.25% and 3.55–8.42%, respectively. The data of the F statistical probe were convenient. e. The detection limits and the quantification limits of the method were 0.018–0.15 ng and 0.27– 0.36 ng, respectively. f. According to the results of robustness test, the factors having the most important and least significant influence on the separation were the quality of the plate and the volumes of tetrahydrofuran of the eluent, respectively. 3. Conclusion By comparing the OPLC method for aflatoxin analysis for various foodstuffs (maize, wheat, peanut, fish meat, rice and sunflower seeds) developed in our laboratory, it can be stated that they are as competitive as the more popular HPLC methods applied for aflatoxin determination. The developed method permits the simultaneous determination of up to seven samples, is more rapid and has a lower cost than the HPLC methods. However, it should be emphasized that the OPLC technique can not replace the more reproducible HPLC technique, but can complement it. Validation of the method developed for determination aflatoxin B1, B2, G1 and G2 of wheat samples, based on the statistical evaluation of analytical data obtained, could be successfully achieved, which is very promising for validation of the other aflatoxin determining OPLC methods. References w1x R. Schuster, G. Marx, M. Rathaupt, Analysis of Mycotoxins by HPLC with Automated Confirmation by Spectral Library HP Application Note, Pub. NR. 125091-8692 E (1993). w2x C. Micco, C. Brera, M. Miraglia, R. Onori, Food Addit. Contam. 4 (1987) 407–414.
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