SCREENING AND QUANTIFICATION OF AFLATOXINS AND OCHRATOXIN A IN DIFFERENT CEREALS CULTIVATED IN ROMANIA USING THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY C. BRAICU1,4, C. PUIA2, E. BODOKI3 and C. SOCACIU1 1 Department of Chemistry and Biochemistry University of Agricultural Sciences and Veterinary Medicine 400372 Cluj-Napoca, Romania 2
Department of Plant Protection University of Agricultural Sciences and Veterinary Medicine 400372 Cluj-Napoca, Romania 3
Department of Analytical Chemistry University of Medicine and Pharmacy “Iuliu Hatieganu” 400349, Cluj-Napoca, Romania Accepted for Publication July 24, 2007
ABSTRACT The main focus of our study was to implement a rapid, inexpensive and reliable method that could be utilized to check the cereals for safety (i.e., screening for total aflatoxins, as well as individual B1, B2, G1, G2 aflatoxins and ochratoxin A). We developed a protocol by which we were able to isolate mycotoxins from cereals collected from different regions of Romania. After extraction in chloroform, the mycotoxins were separated by thin-layer chromatography (TLC) and quantified using densitometry. Forty-three samples of different cereals (wheat, maize, rye and Triticale) were analyzed. Twenty-five of the 43 samples (58.14% of the total number) were found to be contaminated with different mycotoxins in various concentrations: aflatoxin B1 (1.6–5.7 mg/kg), aflatoxin B2 (0.89–4 mg/kg), aflatoxin G1 (1.2–5.76 mg/kg), aflatoxin G2 (0.96–3.4 mg/kg) and/or 4.3–30 mg/kg ochratoxin A. The concentration of total aflatoxin contamination ranged from 11.2 to 10.8 mg/kg. Among the different cereals, the highest number of contaminated samples was found to be in the wheat samples (62.5%). The method outlined in this study has been adopted already by our laboratory for current analyses of mycotoxins. The results obtained using this method is in 4
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Journal of Food Quality 31 (2008) 108–120. All Rights Reserved. © 2008, The Author(s) Journal compilation © 2008, Blackwell Publishing
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compliance with the strict regulatory guidelines developed both in Romania, as well as in the European Union.
PRACTICAL APPLICATIONS Thin-layer chromatography (TLC) is a rapid, inexpensive and convenient method that can be used routinely to screen for mycotoxins. This article describes the detailed procedures for the extraction, purification and quantification of aflatoxins and ochratoxin A, using TLC. Using this method one can identify concomitantly five different mycotoxins and by coupling it with densitometry a quantitative determination is also possible. Therefore, this could become a routine technique in regional laboratories responsible for checking agrifood safety.
INTRODUCTION Mycotoxins were first identified for more than 40 years as contaminants frequently found in a variety of food products (seeds in particular) (Bennett and Klich 2003; Castells et al. 2005). The aflatoxins are the most extensively studied subclass of mycotoxins, and they can be identified in peanuts, pistachios, figs, paprika and corn, just to name a few of the most relevant foods (Richard et al. 1993; Czerwiecki et al. 2005; Krska et al. 2005). Aflatoxins are produced as secondary metabolites by the fungus Aspergillus flavus and Aspergillus parasiticus and have been proven to be highly toxic, carcinogenic, mutagenic and immunosuppressive agents (Mahfoud et al. 2002). Among the 20 known aflatoxins, four (designated as AB1, AB2, AG1 and AG2) are the most frequently found in foods (Krska et al. 2005), and are carcinogenic in both humans and animals (Smith et al. 1995; Bennett and Klich 2003; Castells et al. 2005). Ochratoxin A is produced by some species of Penicillium and Aspergillus in cereal grains, but occurs naturally as well in a variety of plant products such as cereals, coffee beans, beans, pulses and dried fruit (Bauer and Gareis 1987; Czerwiecki 1994). It is also found in kidney, liver and blood from mammals by absorption from food. Investigations of the frequency and levels of occurrence of ochratoxin A in food and human blood samples indicate that foodstuffs are frequently contaminated (Czerwiecki et al. 2005). Ochratoxin A possesses carcinogenic, nephrotoxic, teratogenic and immunotoxic effects (Pohland et al. 1992; Czerwiecki et al. 2005). It is also linked to nephropathy in humans (Bauer and Gareis 1987; Pohland et al. 1992).
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According to the European Commission’s Regulation, No 472/2002, amending (EC) Regulation No 466/2001, the maximum level accepted (MLA) for total aflatoxins is 4 mg/kg (for aflatoxin B1 is 2 mg/kg) and for ochratoxin A is 3–5 mg/kg. Different screening methods and advanced techniques for detection and quantification of mycotoxins were reported in the last 20 years (Bauer and Gareis 1987; Richard et al. 1993; Aziz et al. 1998; Castro and Vargas 2001). Thin-layer chromatography (TLC) has been the most widely used method since its development in the early 1960s, probably because of its low analysis costs and accessibility (Stubblefield et al. 1967). This method is still recommended by the Association of Official Analytical Chemists for mycotoxin analysis (Grabarkiewicz-Saczesna et al. 1985; Castro and Vargas 2001). Initially, this technique was used as a qualitative method, but coupling with densitometry increased its application for quantitative analysis. Several advanced methods have been used during the last years for the mycotoxin quantitative evaluation in food and cereals. High Performance Liquid Chromatography (HPLC) separation is usually achieved on C18 reversed-phase columns with methanol/water mixtures as mobile phase (Hassan et al. 2001; Czerwiecki et al. 2005; Krska et al. 2005). However, the technique is still expensive and it needs qualified personnel to perform it (Amezqueta et al. 2004). HPLC is used more as a reference method for mycotoxins quantification. Enzyme-linked immunosorbent assays (ELISA) have recently been developed also for quantitative analysis of some individual aflatoxins and have been used also as a rapid screening technique. Cleanup is minimal, since these methods are being used also for their quantification in less complex matrices (Abouzied et al. 1991), but it is limited by the unavailability and the high price of specific mycotoxin antibodies. These advanced techniques require investments and expertise, and because of their high cost, they are not recommended for screening and routine analysis. In comparison with HPLC and ELISA, previous studies have shown that TLC is easy to perform, robust in its application and reliable in its results (Stubblefield et al. 1967; Grabarkiewicz-Saczesna et al. 1985; Castro and Vargas 2001). On this basis, it can be considered a more suitable method. Coupled with densitometry, TLC can be optimized for a rapid screening of mycotoxins found in cereals. The purification, separation and quantification of four aflatoxins (AB1, AB2, AG1, AG2) and ochratoxin A, followed by quantitative TLC analysis, was previously reported since 1967 and is still actual until now (Stubblefield et al. 1967; Grabarkiewicz-Saczesna et al. 1985; Castro and Vargas 2001; Braicu et al. 2005).
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There are only a few articles concerning the mycotoxin’s incidence in Romania. The first study that investigates the natural occurrence of mycotoxins in feeding stuff was presented by Curtui et al. (1998). The present studies are focused on using rapid, inexpensive and reliable methods for isolation and screening of aflatoxin B1 (AB1), aflatoxin B2 (AB2), aflatoxin G1 (AG1), aflatoxin G2 (AG2) and ochratoxin A in cereals (for feed and bakery) randomly chosen from different regions of Romania. This is a screening study for the occurrence of aflatoxins and ochratoxin A in cereals, based on a TLC method developed in our laboratory (Braicu et al. 2005). Mycotoxins were extracted by specific protocols in chloroform, separated by TLC and quantified using densitometry (Stubblefield et al. 1967; Braicu et al. 2005).
MATERIALS AND METHODS Cereal Samples A total of 202 different random samples of grains (wheat – 130, maize – 60, Triticale – 6 and rye – 6) were collected from seven districts of our country, Romania (year: 2005). Each sample (1 kilo) was put in a sterilized bag and was divided into two parts in the laboratory. One part was used for mycological analysis and the second part was kept for mycotoxin analysis (stored at 4C until the TLC analysis was performed). The mycological analysis was conducted in five repetitions, using the blotting paper method (Raicu and Baciu 1978) and the Ulster method on agarised plates (Hulea et al. 1982) using 10 grains from each sample in three or five repetitions. We used the Czapek–Dox agar medium with Streptomycin (Sigma, Bucharest, Romania) as a bacteriostatic, added after sterilization (Hulea 1969), and the identification of fungi was carried out according to Raicu and Baciu (1978) and Domsch and Gams (1972). Aliquots of 50 g from each sample were grounded and taken for analysis. Some of the contaminated samples, as determined by mycological analyses, were screened for the mycotoxins (aflatoxins and ochratoxin A) quantification (43 samples). Each sample was analyzed in triplicate and the results were expressed as an average of three repetitions. Reagents Standards: Total Aflatoxin Standard 1,000 ng/mL, containing 250 ng/mL of each aflatoxin (AB1, AB2, AG1, AG2) and Ochratoxin A Standard 250 ng/mL from Diagnostic (R-Biopharm Rhone, Ltd., Bucharest, Romania).
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Solvents provided from Merck GmbH (Bucharest, Romania) (chloroform, acetonitrile, acetone, toluene, ethyl acetate, anhydrous ethyl ether). The reagents, formic acid, anhydrous sodium sulphate, sulphuric acid, paper filter: Whatman 4 were provided by Merck GmbH. Hamilton microsyringes were used for sample applications on the chromatographic plate (in 1 cm bands). Simultaneous Extraction and Purification of Mycotoxins The extraction was performed using a method described by Braicu et al. (2005). (1) Aliquots of 10 g cereal grains were finely ground and weighed into a suitable flask; (2) Extracted with 50 mL chloroform at 22–25C and then sonicated for 10 min; (3) Then filtered under vacuum through paper filter Whatman 4; (4) The chloroform extract was washed by distilled water and dried over 20 g anhydrous sodium sulphate; (5) The extracts were concentrated almost to dryness on a rotary evaporator and the residue was resolved in 1 mL chloroform. Known quantities of each standard were added to samples in order to verify the percentage of mycotoxin’s recovery during the extraction. TLC Silica gel F254 10 ¥ 20 cm TLC plates with a thickness of 0.25 mm were used (Merck). Samples (30 mL) of chloroform extracts and standard solution of aflatoxins and ochratoxin were spotted in 1 cm bands. Three successive developing solvent systems were used: chloroform : acetone (9:1, v/v) for first, and toluene : ethyl acetate: 80% formic acid (6:3:1, v/v/v) for second and third migration in a mobile phase vapor saturated chromatographic tank. This is a method as described by Braicu et al. (2005). TLC plates were examined under UV light at 366 nm before quantification. Chemical tests to confirm mycotoxin presence were performed in order to avoid mistakes. Several parameters were tracked in order to establish the presence of mycotoxins: the Rf value, the fluorescence color (lexcitation = 366 nm) and its change after treatment with spray reagents (AlCl3 and H2SO4) and heated 10 min at 110C. When the plates were sprayed with 20% AlCl3 solution or 20% sulfuric acid and heated 10 min at 110C the fluorescence intensity increased. This was only applied to the confirmation of mycotoxins, and not for the densitometric
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analysis, because postchromatographic derivatization potentially could have given rise to errors in quantitative evaluation. Densitometric Analysis Desaga CD6 TLC Densitometer (Bucharest, Romania) equipped with data acquisition and processing software was used for quantitative evaluation of the plates. The separated mycotoxins were detected and quantified in fluorescence mode (lexcitation = 365 nm, slit of 0.06 ¥ 4 mm for aflatoxins, and lexcitation = 336 nm, slit of 0.2 ¥ 4 mm for ochratoxin A), with a scanning resolution of 0.025 mm and 16 readouts/point. The applied quantities of standards on the TLC plate ranged between 0.25–7.5 ng aflatoxin (AB1, AB2, AG1, AG2) and 1–30 ng for ochratoxin A. The linear calibration curves were established representing the peak areas in function of the applied quantities of separated mycotoxin standards. Calculation of Mycotoxin Concentration in Cereals Samples The concentration of mycotoxins was calculated according the formula:
c p = cs( vf vs )(1 m s ) where cp – Concentration of mycotoxin (mg/kg) in the cereal sample cs – Quantity determined by densitometry (ng mycotoxin/spot) Vf – Final volume of the extract after concentration (1,000 mL) vs – Volume of extract spotted on TLC plate (30 mL) ms – Sample weight (10 g) RESULTS AND DISCUSSION Several pioneering studies of mycotoxins have been carried out using the TLC method well before the general availability of HPLC and immunological techniques. Nevertheless, not all laboratories are equipped with HPLC or GC, particularly in the developing parts of the world. Therefore, there is an increased need to monitor the level of mycotoxins in food (especially intended for export). The TLC method provides reliable results for aflatoxins and ochratoxin A screening from different matrices. So, in conclusion TLC can be a good alternative to expensive techniques such as HPLC or ELISA. Standards Curves of Five Mycotoxins and Densitometric Analysis Fluorescent spots of pure aflatoxins and ochratoxin A standards were visible under UV light (366 nm) used as a quantification method by densito-
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FIG. 1. SAMPLE TLC ANALYSIS IN PARALLEL WITH STANDARDS A 1:1.25 ng from each aflatoxin (AB1, AB2, AG1, AG2); 2:2.5 ng from each aflatoxin (AB1, AB2, AG1, AG2); 3:3.75 ng from each aflatoxin (AB1, AB2, AG1, AG2), 10 ng ochratoxin A; 4:5 ng from each aflatoxin (AB1, AB2, AG1, AG2), 15 ng ochratoxin A; 5:20 ng ochratoxin A; 6:25 ng ochratoxin A; 7, 8, 9: wheat samples. This figure appears in color in the online version of the article [DOI:10.1111/j.1745-4557.2008.00187.x].
metry. In Fig. 1, the TLC separation of aflatoxin standards (AB1, AB2, AG1 and AG2) and ochratoxin A bands, used for the calibration curve, are presented parallel with three wheat samples. The most important problem was the overlapping of bands with the interference compounds on the TLC plate. Using a multiple developing solvent system, we were able to separate all four aflatoxins and ochratoxin A without interference compounds. The first developing system (chloroform : acetone, 9:1, v/v) proved to be very useful for the separation of mycotoxins. The second and third migration offered a good separation of aflatoxins without overlap while interference compounds were moved to higher Rf’s relative to aflatoxins. The Rf values of aflatoxins were: 0.278 for AG2, 0.324 for AG1, 0.348 for AB1, 0.402 for AB2 and 0.584 for ochratoxin A. Quantification by densitometry is based on peak area measurements and calibration with standards for samples. The most difficult part of this procedure was to find the right adjustment of the parameters necessary to obtain the maximum intensity of fluorescence peaks, with minimal background noise. A typical densitogram of aflatoxin standards was recorded, showing a good baseline separation for each peak. The calibration curves were established based on densitograms and considering the peak areas in function of the applied quantities of separated mycotoxin standards on the following domain (0.25– 6.25 ng/spot for aflatoxins and 1–30 ng/spot for ochratoxin A). Data concerning the calibration curve for the five mycotoxins are presented in Table 1. The linear calibration curves show a high correlation between the analytical signal and the increasing quantities of mycotoxins (R2 > 0.993). The method’s calculated limits of detection (ng/plate) were 0.14 ng AB1, 0.30 ng AB2, 0.10 ng AG1, 0.13 ng AG2 and 0.88 ng ochratoxin A, respectively. The calculated limits of quantification (ng/plate) are 0.47 ng AB1, 1.01 ng AB2, 0.33 ng AG1, 0.44 ng AG2 and 2.96 ng ochratoxin A, respectively. Individual
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TABLE 1. REPORTING SLOPE, INTERCEPT AND R2 VALUES USED FOR QUANTIFICATION OF AB1, AB2, AG1, AG2 OCHRATOXIN A STANDARDS Nr.
Mycotoxin
Slope
Intercept
R2
1 2 3 4 5
AB1 AB2 AG1 AG2 Ochratoxin A
62.525 120.89 45.251 104.1 26.689
-1.9821 9.9426 -0.6142 13.897 -4.5405
0.9988 0.9966 0.9984 0.9931 0.9971
recovery values for densitometric analysis varied from 83.4 to 105.5% for all aflatoxins and from 67.5 to 101% for ochratoxin A. The recovery of aflatoxins from cereals achieved in the present study is similar with the results reported by Castro and Vargas (2001) and Kamimura et al. (1985). Quantification of Mycotoxins Isolated from Cereals The mycotoxin quantification was based on TLC coupled with densitometry and is presented in Fig. 1. Quantification was accomplished by comparison to the reference standards on the TLC plate and fluorescence densitometry. The proposed method can quantify with good precision, amounts as low as 0.25 ng/spot for each of AG1, AG2, AB1, AB2 and 1 ng/spot for ochratoxin A, respectively. The lowest limit of quantification for mycotoxin contamination in cereals were 0.83 mg/kg AG1, AG2, AB1, AB2 and 3.33 mg/kg for ochratoxin A, being similar with other citations (Richard et al. 1993; Mahfoud et al. 2002). Castro and Vargas (2001) reported a similar method, using TLC/ visual and densitometric analysis, and obtained aflatoxin levels as low as 1.0 mg/kg of AB1, 0.3 mg/kg of AB2 and 0.6 mg/kg of AG2 and AG1 in maize. In Table 2 we present the natural occurrence of mycotoxins analyzed by TLC coupled with densitometry from different Romanian districts. From the 43 samples taken for analysis, 16 samples contained mycotoxin levels higher than MLA. In a total of 11 samples, we obtained higher amounts of AB1 and total aflatoxins than the MLA. In 11 samples, the concentration of B1 was higher than MLA. In a total of six samples, ochratoxin A was identified in one maize sample (lower than MLA) and two rye samples. AB1 was identified in nine wheat samples, in three maize samples and in one rye sample. AB2 was identified in nine wheat samples, in one maize sample and two rye samples. AG1 was identified in four wheat samples, in one maize sample, in four Triticale samples. AG2 was identified in four wheat samples. In general, the cereal contamination ranged between 0.93 and
3
2 3
2 4 2 2
1 1 2
1 1 2
2 2 1 4
BV
BH HD
CJ MM GR SB
SM SJ BC
BV MM SB
HD GR SM BV
SB
Triticale
Rye
2
2
1 0 0 3
0 0 2
1 0 2
0 1 0 2
1 2
3
1 4
Contaminated
TLC, thin-layer chromatography.
Limits by European Commissions of Regulation (EC) no. 472/2002 Limits by Romanian legislation
Maize
2 4
BC BN
Wheat
Analyzed
No. of samples
District
Samples
0 0 6.57 0 0 31.3 30.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4.39 0 0 0 0 0 0 25.6 24.1 3–5
