Simple HPLC Determination of Aflatoxins B1, B2, G1 and G2 in Nuts and Corn (Received February 7, 1996)

Transcription

1 ugust Simple HPLC Determination of flatoxins 1, 2, G1 and G2 in Nuts and Corn (Received February 7, 1996) Hiroshi KIYM, Dayl CHEN, Makoto MIYHR, Masatake TOYOD and Yukio SITO (National Institute of Health Sciences: , Kamiyoga, Setagaya-ku, Tokyo 158, Japan) rapid, sensitive and simple method was developed for the analyses of aflatoxins 1, 2, G1 and G2 in nuts and corn. The method consists of extraction with a mixture of acetonitrile and water (9: 1), clean-up on a multifunctional column, derivatization with trifluoroacetic anhydride, and determination of aflatoxins using HPLC with a fluorescence detector. The established method make it possible to analyze aflatoxins wihout using chloroform. Recoveries of aflatoxins 1, 2, G1 and G2 spiked in peanuts, various other nuts and corn at the level of 1 or 10 ng/g were all in the range of % with low coefficients of variation (2-7%). The minimum detectable concentration for aflatoxin 1 in peanuts was 0.01ng/g. Key words: aflatoxins; solid phase extraction; fluorocarbon column; nuts Introduction flatoxins are strongly carcinogenic mycotoxins produced by spergillus flavus which may contaminate crops such as peanuts and corny. Permissible levels of these compounds in foods are regulated tightly in many countries. In Japan, a TLC method similar to an OC official method has been officially adopted for analysis of aflatoxins in peanuts. In addition, a minicolumn chromatographic method coupled with TLC or HPLC is used for analysis of aflatoxins in food commodities2)-4). However, these methods use chloroform for the extraction of the toxins. Recently, there has been pressure to replace chloroform with other solvents such as methanol and acetonitrile on environmental and toxicity grounds5). Furthermore, the value of multifunctional clean-up columns which contain both lipophilic and charged active sites for clean-up of aflatoxins was reported in a collaborative study6)7). This paper deals with an HPLC procedure with fluorescence detection for determination of individual aflatoxins after non-chloroform extraction from nuts and corn, followed by cleanup on a multifunctional column. Materials and Methods pparatus The HPLC system consisted of Hitachi L-7100 pumps, a Tosoh S-8020 autoinjector and a Hitachi F-1000 fluorescence detector. guard column, Inertsil ODS-3 (3 cm X 4.6 mm, 5m, GL Science), was placed between the autoinjector and the separative column, Inertsil ODS-3 (15 cm X 4.6 mm, 5m, GL Science). The integrator was a Hitachi D ISOLUTE MULTIMODE (International Sorbet Technology), ond-elut Certify (Varian) and ond-elut Certify II (Varian) columns were examined for clean-up of extracts from peanuts. coffee mill (SCM-40, Shibata) was used for grinding nuts and corn samples. column for qualitative analysis, Fluofix 120 E (15 cm X 4. 6 mm, 5 tcm, Neos) was purchased from Neos Co., Ltd. Reagents Organic solvents of HPLC grade were pur-

2 196 J. Food Hyg. Soc. Japan Vol. 37, No. 4 methanol: water (8:2) acetonitrile : water (9:1) Fig. 1. Typical chromatograms of blank peanut extract with methanol-water (8: 2) [] and with acetonitrile-water (9:1) [] chased from Merck-Cica. Water was distilled and deionized. Trifluoroacetic anhydride (TF) was purchased from Wako. The aflatoxin 1, 2, G1 and G2 standards were purchased from Sigma Chemical Co. Stock standard solutions of aflatoxins at a concentration of l ig/ml were prepared in methanol and stored at 4C in the dark. Samples Peanuts was supplied through the courtesy of the Center for Inspection of Imported Foods and Infectious Diseases in the Yokohama Quarantine Station. Other samples were purchased from markets in Tokyo. Extraction and purification of aflatoxins test sample (20 g) was ground in a coffee mill and extracted with 40 ml of a mixture of acetonitrile and water (9: 1) by shaking vigorously in a 200 ml glass flask for 15 min. The solution was filtered through filter paper (Whatman No. 5). 2 ml portion of the filtrate was loaded on an ISOLUTE MULTIMODE column and passed through at a flow rate of 2 ml/min. The 3 ml capacity column had previously been washed with 5 ml of methanol. The aflatoxins were eluted with 5 ml of a mixture of acetonitrile and water (9: 1). 1 ml portion of the eluate was evaporated to dryness at 50C in a glass centrifuge tube and the residue was used for derivatization. Precolumn derivatization For derivatization of aflatoxins, 0.1 ml of TF solution was added to the residue from a sample extract or aflatoxin working standard and the mixture was allowed to stand at room temperature for 15 min in the dark. Then 0.9 ml of a mixture of acetone and water (1: 9) was added to the tube. 20l aliquot of the sample or standard solution in the tube was subjected to HPLC. HPLC conditions The mobile phase was acetonitrile-methanolwater (50: 150: 300), degassed by sonication. Inertsil ODS-3 (4.6 mm X 150 mm) and Inertsil ODS-3 (4.6 mm X 250 mm) columns were connected for separation, and maintained at 40C. The flow rate of the mobile phase was 1.0 ml/ min. The peaks were monitored with a fluorescence detector at the excitation and emission wavelengths of 365 nm and 450 nm, respectively. The injection volume was 20cl. For qualitative analysis, the mobile phase was methanol-water (150:350). Fluofix 120 E (4.6 mm i. d. X 250 mm) and Fluofix 120 E (4.6 mm i.d. X 150 mm) were connected for separation, and maintained at 40C. The flow rate of the mobile phase was 0.8 ml/min. flatoxins were detected as described above. The injection volume was 20, 1.

3 ugust 1996 Simple HPLC Determination of flatoxins in Nuts and Corn 197 Table 1. Effects of Sorbent Mass in ISOLUTE MULTIMODE Column on Recoveries from Peanuts Spiked with 10 ng/g of flatoxins 1, 2, G1 and G2 pre-conditioning methanol 5 ml acetonitrile-water (9: 1) 5 ml Scheme 1. Procedure for analysis of aflatoxins 1, 2, G1 and G2 in peanuts, various other nuts and corn Results and Discussion Selection of extracting solvent It was reported that acetonitrile-water (9: 1) at a 2: 1 ratio of solvent to sample and methanolwater (8: 2) at a 3: 1 ratio of solvent to sample were effective for recovery of aflatoxins from peanuts5). We found that extraction with a mixture of acetonitrile and water (9: 1) at a 2: 1 ratio of solvent to sample gave better chromatograms after multifunctional column clean-up, as shown in Fig. 1. Recoveries of aflatoxins 1, 2, G1 and G2 spiked in peanuts were 89.2%, 92.4%, 93.5% and 87.5% with acetonitrilewater (9: 1) at a 2: 1 ration of solvent to sample, while those with methanol-water (8: 2) at a 3: 1 ratio of solvent to sample were 95.2%, 96.3%, 93.4% and 98.2%. The above results are consistent with those reported by Wilson and Romer6). Comparison of multifunctional columns Several commercial multifunctional columns have been studied for clean-up of aflatoxins from foods. We examined ond-elut Certify, ond-elut Certify II and ISOLUTE MULTI- MODE columns. These multifunctional columns4) retain interfering components, such as fats, proteinaceous compounds and carbohydrates extracted from foods, but allow aflatoxins to pass through. ll the columns gave recoveries of more than 80% of aflatoxins from peanuts, but the eluate from the ISOLUTE MULTIMODE column gave the lowest level of interfering peaks on the subsequent HPLC. Optimum sorbent mass of multifunctional column Using an applied volume of 2 ml, commercial ISOLUTE MULTIMODE columns of 300 mg, 500 mg and 1000 mg were examined. Table 1 shows the recovery results. The 300 mg and

4 198 J. Food Hyg. Soc. Japan Vol. 37, No. 4 Fig. 2. Typical chromatograms of blank peanut extract () and extract of peanuts spiked with 10 ng/g of aflatoxins 1i 2, G1 and G2 () 2 aflatoxin 1 Fig. 3. Typical chromatograms of blank pistachio nut extract () and extract of pistachio nuts spiked with 10 ng/g of aflatoxins 1, 2, G1 and G2 () 500 mg columns gave recoveries of more than 85% of each aflatoxin from peanuts. The largest column showed a slightly lower recovery, so the 500 mg column was selected. Examination of applied volume to ISOL UTE MULTIMODE column pplied volumes from 1.5 ml to 4.0 ml were tested. With 1.5 ml and 2.0 ml applied volume, excellent chromatograms were obtained, but at more than 3.0 ml, interfering peaks appeared before the peak of derivatized aflatoxin G1.Therefore, we selected 2 ml as the optimum applied volume. Thus, the procedure shown in Scheme 1 was established for clean-up from peanuts. Examination of HPLC conditions Various HPLC conditions were examined. The mobile phase of acetonitrile-methanolwater (50: 150: 300) afforded satisfactory separation of peaks of aflatoxins, derivatized aflatoxins and impurities. mong several columns examined, the most suitable was Inertsil ODS-3 (4.6 mm X 150 mm, S LIm). However, in some samples, such as corn, it was difficult to separate the peak of derivatized aflatoxin G1 from an unknown peak derived from the extract. There-

5 ugust 1996 Simple HPLC Determination of flatoxins in Nuts and Corn 199 Fig. 4. Typical chromatograms of blank macadamia nut extract () and extract of macadamia nuts spiked with 10 ng/g of aflatoxins 1, 2, G1 and G2 () Fig. 5. Typical chromatograms of blank corn extract () and extract of corn spiked with 10 ng/g of aflatoxins 1, 2, G1 and G2 () fore, we connected an Inertsil ODS-3 (4.6mm x 150 mm, 5m) column and an Inertsil ODS-3 (4.6 mm X 250 mm, 5m) column in tandem. This arrangement gave sufficient peak separation. The detection wavelength used was based on the reported method7). Recovery tests from nuts and corn The developed method was applied to the determination of aflatoxins in peanuts, pistachio nuts, almonds, cashew nuts, macadamia nuts, walnuts, hazelnuts, giant corn and corn. Figure 2-5 showed HPLC chromatograms of cleaned-up extracts from unspiked and spiked samples of peanuts (Fig. 2), pistachio nuts (Fig. 3), macadamia nuts (Fig. 4) and corn (Fig. 5). The peaks were clearly resolved from the background peaks of blank samples. However, it was difficult to separate completely the peak of aflatoxin G1 and the unknown peak in the extract from corn. Therefore, we examined another HPLC column as described in the next section. Table 2 shows that the recoveries of aflatoxins 1, 2, G1 and G2 spiked in peanuts, pistachio nuts, almonds, cashew nuts, and corn at the level of 10ng/g were all in the range of %, and the coefficients of variation were low, ranging from 2 to 7%. Repeatability for spiked samples was good. The detection limits for aflatoxins 1, 2, G1 and G2 were approximately 0.01 ng/g in

6 200 J. Food Hyg. Soc. Japan Vol. 37, No. 4 Table 2. Recoveries of flatoxins 1, 2, G1 and G2 from Spiked Peanuts, Pistachio Nuts, lmonds, Cashew Nuts and Corn Each value represents the mean+sd from triplicate analysis. Fig. 6. Typical chromatograms of blank corn extract () and extract of corn spiked with 10 ng/g of aflatoxins 1, 2, G1 and G2 () using a fluorocarbon bonded silica-gel column peanuts (signal-noise ratio, 3: 1) and these values are clearly better than the value of below 0.5g/g reported by Wilson and Romer6) using a Mycosep multifunctional clean-up column, showing the high sensitivity of our proposed method. The analysis of naturally contaminated commodities with this method is in progress. Examination of qualitative analysis using a different HPLC column In the analyses of aflatoxins from foods, many samples are contaminated at a very low level. If the retention time of a trace unknown peak from an extract is close to that of aflatoxins and derivatized aflatoxins, misidentification is possible. Thus, we tried to develop a qualitative assay using a different HPLC column (reversedphase polymer column or fluorocarbon bonded silica-gel column). The reversed-phase polymer column gave poor results, but the fluorocarbon bonded silicagel column fully separated the peak of derivatized aflatoxin Gl and the unknown peak in corn (Fig. 6). The presence of aflatoxins could thus be confirmed by using two different columns. This is simpler and faster than the official TLC method. It should also be possible to obtain further confirmation by LC/ MS, if necessary.

7 ugust 1996 Simple HPLC Determination of flatoxins in Nuts and Corn 201 Conclusion This proposed method using the ISOLUTE MULTIMODE multifunctional clean-up column is a rapid (within 2 hr), simple and reproducible method for the analysis of aflatoxins in nuts and corn without the use of chloroform. Recoveries of aflatoxins 1, 2, G1 and G2 spiked in nuts and corn at the level of 1 or 10 ng/g were more than 80%. The detection limit in peanuts was 0.01 ng/g. cknowledgments We are indebted to Mr. Hisashi Takeda, Center for Inspection of Imported Foods and Infectious Diseases in Yokohama Quarantine Station, for providing peanuts. part of this study was supported by a Grant for Scientific Research Expense for Health and Welfare Programs from the Japanese Government. References 1) utler, W. H., are, J. M.: rit. J. Cancer 17, (1963) 2) Kamimura, H., Nishijima, M., Yasuda, M., Ushiyama, H., Tabata, S., Matsumoto, S.: J. ssoc. Off. nal. Chem. 68, (1993). 3) Tabata, S., Kamimura, H., Ibe,., Hashimoto, H., Iida, M., Tamura, Y., Nishima, T.: ibid. 76, (1993). 4) OC Official Methods of nalysis: Chapter 49, Natural Toxins, p (1995). 5) Cole, R. J., Dorner, J. W.: J. ssoc. Off. nal. Chem. 77, 1,509-1,511 (1994). 6) Wilson, T. J., Romer, T. R.: ibid. 74, (1991). 7) Trucksess, M. W., Stack, M. E., Nesheim, S., lbert, R., H., Romer, T. R.: ibid. 77, 1,512-1,521 (1994).