Awalyst, January, 1979, Vol. 104, pp. 73-78 73 Polarographic Study of Aflatoxins B,, B,, GI and G, : Application of Differential-pulse Polarography to the Determination of Aflatoxin B, in Various Foodstuffs Malcolm R. Smyth, David W. Lawellin" and Janet G. Osteryoung Department of ilficvobiology, Colorado State University, Fovt Collins, Colo. 50523, USA The polarographic behaviour of aflatoxins B,, B,, G, and G, has been investi- gated and found to parallel closely that of coumarin and its derivatives. Differential-pulse polarography has been applied to the determination of aflatoxin B, in a variety of food products. Good agreement was obtained between the differential-pulse polarographic method and visual comparison of the fluorescence exhibited by the aflatoxin following thin-layer chromato- graphic separation.Electroactive interferences co-extracted from high lipid-containing foods were removed by separation on a Sepliadex LH-20 column. Keywords : _4flatoxin determination ; diffeerential-pulse polarogvaphy ; food a w l y s i s Aflatoxins are a group of toxic metabolites produced by certain strains of the common mould Aspergillus jlavus (e.g., Aspergillus parasiticus) and by various other mould genera. They have been shown to be highly carcinogenic in various animal species and hence pose a potential health risk to man. Accurate and sensitive methods of analysis are therefore required for the determination of these compounds in foodstuffs that have sustained mould growth. Aflatoxins B,, B,, G, and G, are difuranocoumarin derivatives that give rise to intense fluorescence in the blue (B, and B,) and green (G, and G2) regions of the spectrum.As a result of this characteristic fluorescence there are many published methods for the determina- tion of these compounds based on thin-layer chromatography with either visual or fluoro- densitometric measurement.l-3 Although these methods can detect concentrations of individual aflatoxins down to 1 ng per spot, the error associated with visual comparison of fluorescence intensity is usually of the order of &20-28%, whereas that associated with fluorodensitometric measurement is about & 643% .l The use of polarography for the identification of aflatoxins was first reported by Gajan ei aL4 but no attempt was made either to explain the complicated electrochemical behaviour of these compounds or to produce a working method for their determination in foodstuffs.It was decided, therefore, to study the polarographic behaviour of these compounds in greater detail and to investigate whether differential-pulse polarography could provide an accurate and sensitive method for their determination. This technique has recently found wide application for the determination of many other foreign organic compounds in biological materials. Experimental Apparatus Polarograyhic measurements were carried out using a PAR, Model 174, Polarographic Analyser in conjunction with a three-electrode cell system with a saturated calomel electrode (S.C.E.) as the reference electrode and platinum as the counter electrode. The dropping- mercury electrode (D.M.E.) used had a flow-rate of 0.82 mg s-l and a drop time of 7.9 s at a mercury head of 76 cm.* Present address : Department of Biochemistry, The Medical and Dental School, North Western Uni- versity, Chicago, Ill., USA.74 SMYTH et al. : APPLICATION OF DIFFERENTIAL-PULSE POLAROGRAPHY Analyst, VoZ. 104 Reagents Samples of aflatoxins B,, B,, G, and G, were obtained from Aldrich Chemical Co., Inc. Stock solutions of these compounds (100 pg ml-l) were prepared in AnalaR methanol and stored in the dark under refrigeration. A stock Britton - Robinson buffer solution, 0.04 M in glacial acetic acid, orthophosphoric acid and boric acid, was prepared from analytical- reagent grade compounds; buffer solutions of various pH (2-12) were prepared by the drop- wise addition of 0.2 M sodium hydroxide solution and measurement of the pH using a glass elect rode.Thin-layer chromatograms were run on silica gel G plates (0.25 mm thick) obtained from E. Merck. All other compounds and solvents used were of analytical-reagent grade. Yeast extract was obtained from Difco. Procedures Polarographic studies were carried out on solutions that had previously been de-gassed with oxygen-free nitrogen for 10 min. Current - potential curves were recorded in the direct-current, normal-pulse polarographic arid diff erential-pulse polarographic modes. The samples were blanketed with an atmosphere. of nitrogen during analysis and each solution was scanned between - 1 .O V and the potential of electrochemical reduction of the supporting electrolyte.Scan rates of 1-5 mV s-1, drop times of 1-2 s and a modulation amplitude of 100 mV (in the d.p.p. mode) were typically employed. For the thin-layer chromatographic investigations, 5 pl of various aflatoxin standard solutions (containing 1-20 pg ml-1 of the aflatoxin) and solutions containing unknown concentrations of aflatoxins were spotted on the plate and elution was achieved using a chloroform - acetone - water mixture (88 + 10 + 2). The spots were rendered visible under longwave ultraviolet light. The concentratiton of individual aflatoxins in the unknown was then determined by a visual comparison of t'he fluorescence intensities of the standards and the unknown. Aflatoxins were produced in vivo by inoculating Asfiergillus parasiticus (ATCC 1551 7) spores into yeast extract - sucrose medium (YES: 20% of sucrose, 2% of yeast extract and 88% of tap water) and whole milk and incubaked at 28 "C for 7 d.The surfaces of moistened rice, corn and pelletised rabbit feed (the last medium was chosen to represent silage) were also inoculated with Aspergillm parasiticus spores and incubated under the same conditions. In each instance, aflatoxin B, was found to be the predominant mycotoxin present following incubation. For the aflatoxin produced in YES medium, 5-ml aliquots of the medium were taken and extracted twice with chloroform -I methanol (9 + 1). The chloroform fractions were then pooled and reduced to dryness by flash evaporation. The residue was taken up in 0.5 ml of methanol and 5 pl of this extract were spotted on a thin-layer chromatographic plate.The remaining solution was diluted to 5 ml with Britton - Robinson buffer (pH 9) and the concentration of aflatoxin determined by diff erential-pulse polarography (operating conditions: scan rate, 1 mV s-1; drop time, 2 s; modulation amplitude, 100 mV). With the other growth media, 50 g were homogenisedl with 200 ml of water - methanol (1 + 1) for 15 min. Following centrifugation a t 5000 g for 15 min, the supernatant was extracted twice with butan-1-01 and three times with chloroform. The butan-1-01 and chloroform layers were then pooled and reduced to dryness by flash evaporation. The residue was taken up in 1 ml of Britton - Robinson buffer (pH 9) - methanol (9 + 1) and applied to a Sephadex LH-20 column (100 x 10 mm).Elution was carried out with the same solvent and the fraction (5ml) exhibiting fluorescence and eluting at the same retention time as aflatoxin B, was examined by differential-pulse polarography for aflatoxin content. Results and Discussion Polarographic Behaviour Aflatoxins B,, B,, GI and G, were found to exhibit similar polarographic behaviour over the pH range 4-11. The effect of pH on the E, and ilim values of the two waves exhibited by aflatoxin B, in direct-current polarography is shown in Fig. 1. At pH values below 8 both waves are dependent on pH with the first wave, i a , exhibiting a change of E+ with pH of 35 mV pH-1 and the second wave, ib, exhibiting a change of 70 mV pH-l. At pH values greater than 8 wave ib disappears and wave i a becomes independent of pH.A study onJanuary, 1979 TO THE DETERMINATION OF AFLATOXIN B, IN FOODSTUFFS 75 the variation of ilim with h and h* (where h = height of mercury column) for the two waves exhibited by aflatoxin B, in Britton - Robinson buffer (pH 7) indicated that both processes are diffusion controlled. The normal-pulse polarograms, however, exhibit a maximum, which indicates that reactant adsorption is involved in the over-all electrode process. Assuming that the aflatoxins have a diffusion coefficient (D) similar to that of coumarin, Le., 8 x cm2 s-l (ref. 6), and substituting into the Ilkovic equation, it was found that in the pH range 6-7 the total height of the two waves corresponded approximately to a two-electron process. As the heights of the individual waves are not equal, it is assumed that competing reactions are involved in the over-all electrode process.;~ t -1.2 -1.1 0.04 0.03 < 0.02 5 - .- 0.01 0 4 5 6 7 8 9 10 11 12- PH Fig. 1. Plots of E* (solid lines) and ilim (broken lines) against pH for waves i, (0) and ib (A) exhibited by aflatoxin B,. This behaviour is similar to that reported for coumarin by Polievktov and Lomadze,' who found that the first wave of coumarin in neutral aqueous alcoholic solutions corre- sponded to the formation of an anion-radical species, accompanied by dimer formation. They attributed the second wave to the reduction of the undimerised anion-radical and reported that in acidic media protonation of the molecule preceded reduction. The forma- tion of an anion-radical followed by dimerisation has also been reported by other workers in studies on the electrochemical behaviour of coumarin.6s8 As the electrochemical behaviour of the aflatoxins closely parallels that of coumarin, it is likely that the electroactivity of these compounds is associated with the coumarin moiety in their molecular structure.The main difference in behaviour lies in the fact that the reduction of the aflatoxins occurs at a potential about 300400 mV more positive than that for coumarin. This can be explained by the increased conjugation caused by the keto group in the neighbouring cyclopentanone (B, and B,) or &lactone (G, and G,) rings. Slight differences in the potential of reduction of the aflatoxins have also been observed owing to their slight differences in structure, e.g., in solutions of pH 8-10, the half-wave potentials of reduction of the various aflatoxins were found to be B, = -1.26, B, = -1.27, G, = -1.21 and G, = -1.23V (all versus S.C.E.).These potentials are in good agreement with the values quoted by Gajan et aZ.4 The mechanism of reduction of these compounds can therefore be postulated to be as shown on the next page.76 SMYTH et aE. : APPLICATION OF DIFFERENTIAL-PULSE POLAROGRAPHY Analyst, VoZ. 104 pH 8-11 2e- -+ 2H20 --dimer +20H- Choice of Wave for Analytical Purposes The best defined waves for analytical purposes were obtained in Britton - Robinson buffer of pH 9 where, although the peak current in differential-pulse polarography was slightly smaller than that obtained in solutions of pH 7-8, measurement of the peak height was made easier owing to the disappearance of the second wave.A graph of i p against con- centration for aflatoxin B, in the range 1 :K lO-'-l X 1 0 - 5 ~ was linear with a slope of 0.039 nA ng-1 ml. The limit of detection for the differential-pulse polarographic determina- tion of aflatoxin B, in pure solution was found to be about 8 x M (25 ng ml-1). The E , value (in differential-pulse polarography) was also found to be dependent on concentra- tion for each of the aflatoxins studied. This :is illustrated in Fig. 2 for the effect of concentra- tion on the E, value of aflatoxin B, in Britton - Robinson buffer (pH 9). Determination of Aflatoxin B1 in Foodstuffs Using Differential-pulse Polarography When aflatoxin B,ywas produced by Aspergillus parasiticus in YES medium, extracted I 0 200 400 6 Concentrationhg ml-' -1.27 Fig.2. Effect of concentration on E , value (in differential-pulse polarography) of aflatoxin B, in Britton - Robinson buffer (pH 9). Conditions: scan rate, 2 mVs-l; drop time, 2 s ; and modula- tion amplitude, 100 mV.January, 1979 TO THE DETERMINATION OF AFLATOXIN B, IN FOODSTUFFS 77 twice with chloroform-methanol (9 + 1) and determined by both thin-layer chromato- graphy and diff erential-pulse polarography, a good correlation was obtained between the two sets of results (Table I). The differential-pulse polarographic method did, however, permit the earlier detection of aflatoxin B, (because only 5 pl of the 0.5 ml of extract was applied to the thin-layer chromatographic plate) and gave rise to more precise results.TABLE I DETERMINATION OF AFLATOXIN B, IN YEAST EXTRACT - SUCROSE MEDIUM BY THIN-LAYER CHROMATOGRAPHY AND DIFFERENTIAL-PULSE POLAROGRAPHY Results are averages of three separate innoculations. Concentration of aflatoxin BJpg ml-1 Time of incubation/h Thin-layer chromatography Diff erential-pulse polarography 24 N.D.* 0.148 f 0.08 72 1.30 f 0.64 1.21 f 0.22 108 3.20 & 0.46 2.96 f 0.29 0 0 0 * N.D. = Not detected. When aflatoxin B, was grown on rice, corn, milk or pelletised rabbit feed, and extracted in a similar fashion, it was found that the concentration of aflatoxin B, in the extract could not be determined by diff erential-pulse polarography owing to the high level of electroactive interference (Table 11).In order to overcome this problem, aflatoxin B, grown on these foods was first extracted with butan-1-01 and then separated on a Sephadex LH-20 column. This method of purification has often been used in the analysis of aflatoxins, most recently by Josefsson and Moller.9 When the eluent was analysed by differential-pulse polarography , a good correlation was again obtained between the diff erential-pulse polarographic and thin-layer chromatographic assays (Table 11). The limit of detection for the differential- pulse polarographic method was found to be 0.15 pg ml-, for a 5-ml fraction of eluent. This permits the determination of 1-2 pg g-l of the aflatoxin in the original material when 50 g of material were taken for analysis. TABLE I1 DETERMINATION OF AFLATOXIN B, IN VARIOUS FOOD PRODUCTS BY THIN-LAYER CHROMATOGRAPHY AND DIFFERENTIAL-PULSE POLAROGRAPHY Concentration of aflatoxin BJpg nil-' A r > Diff erential-pulse polarograph y Food matrix Thin-layer chromatography A* Bt Rice .. .. .. 17.0 Pelletised rabbit feed . . 1.5 Milk .. .. .. .. 0.18 Corn .. .. .. .. 3.0 U.Q.$ 16.0 U.Q. 0. 2 U.Q. 1.3 U.Q. 2.6 * Following extraction with chloroform - methanol (9 + 1). t Following extraction with butan-1-01 and chloroform and separation on a Sephadex LH-20 $ U.Q. = Unable to quantitate. column. These results indicate, therefore, that diff erential-pulse polarography can provide an accurate and sensitive method for the determination of aflatoxin B, in various foodstuffs. However, when a mixture of aflatoxins is produced in vivo, a separation procedure, e.g., high-performance liquid chromatography, would have to be applied prior to analysis.It is suggested that diff erential-pulse polarography or a combination of high-performance liquid chromatography with electrochemical detection could provide information complimentary to that obtained with fluorescence methods for the screening of these carcinogenic myco- toxins in situations of environmental significance.78 SMYTH, LAWELLIIX AND OSTERYOUNG The authors thank Drs. Dale W. Grant and Jeffrey W. Whittaker for valuable discussions. This paper was presented in part at the 173rtl ACS National Meeting in New Orleans, March 20th-25th, 1977. Partial financial support was provided through NSF Grant No. MPS 75-00332. 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