Methods for Detection and Quantitation of Fumonisins in Corn, Cereal Products and Animal Excreta

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1 536 Journal of Food Protection, Vol. 57, No.6, Pages Copyright, International Association of Milk, Food and Environmental Sanitarians Methods for Detection and Quantitation of Fumonisins in Corn, Cereal Products and Animal Excreta LARRY G. RICE,* and P. FRANK ROSS U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Ames, Iowa (Received August 2, 1993/Accepted March 24, 1994) ABSTRACT Fumonisins are a group of naturally occurring mycotoxins produced by strains of several different mating populations of Gibberella fujikori (Fusarium section Liseola). Fumonisins have been shown experimentally to be the causative agent of equine leukoencephalomalacia (ELEM), porcine pulmonary edema (PPE) syndrome, and to produce liver cancer in rats. Epidemiological evidence also indicates a possible correlation between the fumonisins and human esophageal cancer. The analytical method of choice for most samples has been high performance liquid chromatography (HPLC) using fluorescence detection. The present work describes the baseline resolution using an isocratic mobile phase of the ophthalaldehyde (OPA) derivatives of fumonisin BI (FBI)' fumonisin B 2 (FB 2 ) and fumonisin B) (FB). The separation of the hydrolyzed forms of FBI' partially hydrolyzed FBI (PHFB I ) and fully hydrolyzed FBI (HFB) is also described. Results of analyses of com from 1992 crop year in both Iowa (mean = 0.05 Ilg/g, N = 80) and Pennsylvania (mean = 0.37 Ilg/g, N = 91) were significantly lower than mean levels reported for 1988, 1989, 1990 and Significant levels of FB I were found in commercially prepared rat (2 Ilg/g) and horse (37 Ilg/g) feed. Levels of FBI' (0.05 to 1.2 Ilg/g) found in com meal purchased from local groceries indicated a possible source of low level exposure of humans to fumonisins. The simultaneous isocratic separation of FBI' FB 2, FB) and the hydrolysis products of FBI' PHFB I and HFB I from fecal samples indicated a possible difference in metabolism of FB I in ruminants and nonruminants. In ruminants, the hydrolyzed forms of FBI composed a significant (60 to 90%) portion of the total FB I concentration found in the feces. In nonruminants, the parent compound, FBI' was the dominant (90%) species present. Both ruminants and nonruminants showed limited excretion of FBI in their urine «1 to 7% total FBI in excreta). Key Words: Fumonisins, methods, detection, corn, cereal products The Food and Agriculture Organization estimates that at least 25% of the world's food crops are affected by mycotoxins annually (10). Contamination by Fusarium species is a major concern in maize and sorghum production worldwide. Fusarium moniliforme is the most common fungal contaminant found in maize and is distributed worldwide (11). Fusarium proliferatum is the second most common fungal contaminant of maize worldwide and both have been found to produce large amounts of fumonisins when cultured on maize (17). While Fusarium species are phytotoxic to plants (26) causing stalk rot and ear rot in maize, the consequence of Fusarium contamination that has received the most recent investigation has been the demonstration that FB 1 is the etiological agent in animal diseases, such as ELEM (8,28) and PPE (13,16). Epidemiological evidence has correlated the consumption of F. moniliforme contaminated maize with human esophageal cancer in high incidence areas of Africa and China (15) and atherogenic effects in nonhuman primates under experimental conditions (6). Based on available information the animal disease most commonly associated with fumonisins is ELEM. Originally known as moldy com poisoning, ELEM was first clinically described in the late 1800s and associated with Fusarium as early as It was not until 1988 that a group of South African researchers (4) characterized the fumonisins as diesters of tricarballylic acid and C 22 aminoalcohols and reproduced ELEM with chemically pure FBI' In addition to FBI' FB 2 and FB3 are found in field com samples and occur in consistent proportions (12). Fumonisin B 2 and FB3 contain one less hydroxyl group than FBI' The chemical identification and the fortuitous abundance of field outbreaks of ELEM and PPE in 1989 in the United States accelerated the development of analytical techniques for the qualitative and quantitative analysis of the fumonisins. While thinlayer chromatography (TLC) led to the separation and eventual identification of the fumonisins, it lacks the specificity needed for a quantitative method. With modifications by Rottinghaus et al. (19), the use of fluorescamine as the visualization reagent has led to a useful procedure for screening samples for the presence of FBI and FB 2 The first quantitative analytical procedure for the analysis of fumonisins involved a modification of a procedure by Siler and Gilchrist (23) originally developed for the determination of phytotoxins from Alternaria alternata. The maleyl derivative using HPLC with ultraviolet (UV) detection was sufficient for culture material but lacked the sensitivity and specificity needed for routine field sample analysis. High performance liquid chromatography is ideally suited for repetitive analysis of fumonisins, partly because they are water soluble. The use of OPA as the derivatizing agent (17,20) has gained wide use as the

2 RICE AND ROSS analytical procedure with sensitivity in the ng/ml range. Other investigators have used naphthalene2,3 dicarboxaldehyde (NDA) (5) and fluorescamine (24,28). While fluorescamine is used in TLC for visualization of fumonisins, its use in precolumn derivatization for HPLC is limited because it forms 2 peaks for each fumonisin. Naphthalene2,3dicarboxaldehyde forms a highly stable fluorescent product with the fumonisins but has received little attention, probably because it uses highly toxic sodium cyanide as a catalyst for the reaction. Gas chromatography/mass spectrometry (GC/MS) has been used to a limited extent in the analysis of fumonisins (7,14,17,18,24,27,28), mainly for positive confirmation of the presence of fumonisins. Plattner (14) used base hydrolysis to form the C 22 aminoakohol (HFB,) backbone of fumonisin and then analyzed the hydrolyzed backbone by GC/MS. This technique showed good sensitivity and correlation with HPLC procedures. Additional MS procedures have included the use of thermos pray and electro spray MS in the apalysis of standards and culture material (9). The production of polyclonal antibodies (1), as well as monoclonal antibodies (2), has lead to the commercial development of enzymelinked test kits and affinity columns. The usefulness of these products in the screening of large numbers of samples holds promise. The purpose of this project was to investigate the use of an HPLC method for the quantitative analysis of fumonisins in com, combased feeds and animal waste excreta. Because of the cool, short and damp growing season in the upper midwest in 1992, differences in the levels of fumonisins were predicted when compared to published results for years 1988 through 1991 (12). Because little work has been done on the possible metabolites of the fumonisins such as HFB I' the formation of the hydrolyzed products of FB I in vivo was investigated. MATERIALS AND METHODS Fumonisin B" monoester CIIaminoa!cohQl(PHFBI) and HFBI were obtained from Ron Plattner, U.S. Department of Agriculturel Agricultural Research ServicelNational Center for Agricultural Utilization Research (USDA, ARS, NCAUR), or produced in our laboratory. Com samples Whole com samples and com screenings were provided by Charles Hurburgh and Patricia Murphy, Iowa State University, Ames, IA and by Paul Nelson, Pennsylvania State University, University Park, PA. Samples from the 1988, 1989, 1990, 1991 and 1992 harvests were a random selection of com principally from the state of Iowa taken from trucksat grain elevators.a 400 g sample from the truck was ground in a Stein mill to uniform consistency. Iowa corn samples from 1988 through 1991 were the same as reported by Murphy et al. (12) and presented for comparision purposes. Combased foods and animal feeds were purchased locally or received from commercial producers around the United States. These samples were ground in a Stein Mill to uniform consistency and then analyzed as com samples. In vitro hydrolysis The PHFB, and HFB, were formed from pure standard FB, using base hydrolysis and controlling the time and temperature of the reaction. Hydrolyzed fumonisin BI was formed using I ml of the FB, solution (10 Ilg/ml), adding I ml of I N potassium hydroxide (KOH), and heating at 60~C for I h. Partially hydrolyzedfbi was formedusing 1 m1of the samefb, solution(10ilglm\), adding I ml of KOH and letting the solution sit at room temperature for 15 min. After hydrolysis, samples were acidified with I ml of 1.5 N hydrochloric acid then treated as the com extract. Fecal samples Animals were fed balanced diets mixed with culture material to obtain the levelsoffb, ranging from 50 Ilglg (sheep) to 1,000Ilg! g (rats). Fecal samples were collected from experimentally dosed cattle, sheep and rats. A IOg sample of fecal material was blended in 50 ml of 50% ACN with a handheld blender for 30 s at high speed; the supernatant was filtered and treated as the com extract. Analytical methods Analysis of samples was according to Ross et al. (18) with minormodification.briefly,10g sampleswereextractedwith50 ml of 50% acetonitrile (ACN) for at least 30 min. The supernatant was filtered through filter paper, and 2 ml of the filtered extract was diluted with 6 ml of I% potassium chloride (KC\). The diluted sample was then cleaned up using a 350mg CIAsolidphase extraction (SPE) column (Waters/Millipore, Milford, MA) by rinsing with I ml of 1% KCI and 3 ml of 15% ACN. The fumonisins and their hydrolyzed products were then eluted using 2 ml of 70% ACN. Fluorescent derivatives of the fumonisins were formed using OPA and detected using a fluorescence detector (LC240, PerkinElmer, Norwalk, CT) with an excitation wavelength of 335 nm and an emission wavelength of 440 nm. Derivatizationwas accomplishedat room temperatureusing 0.25 ml sample, 0.25 ml borate buffer (ph 8.3), 0.25 ml OPA solution (15 mg OPA and 20 III of 2mercaptoethanol in 10 ml ACN) and 0.25 ml water. Reaction time was 10 min. Ten microliters of derivatized sample (0.5 mg equivalent) was injected onto a 3cm x 4.6mm high speed, 311 packing material, CIAcolumn(PerkinElmer) with quantitation made using external standards and peak height measurements. An isocratic mobile phase of 40% ACN and 60% 50mM KH I P0 4 (ph adjusted to 3.3 using phosphoric acid) was used at a flow rate of ImUmin. Fumonisin B, and FBI was purchased from Sigma Chemical Company (St. Louis, MO). Urine samples Urine samples from experimentally dosed cattle, sheep and rats were collected and 5 ml of urine combined with 5 ml of 100% ACN. The dilutedsample was centrifugedat 2,000 rpm for 15 min, and the supernatant treated as the filtered com extract. RESULTS AND DISCUSSION Analysis The results of the analysis of com samples by Murphy et al. (12) indicated a consistent range of fumonisin concentration in com from 1988, 1989, 1990 and 1991 crop years. The mean value for com samples analyzed from Iowa in 1992 (n = 80) as shown in Fig. 1 yielded very contrasting results. Method of sample collection, transport, storage and testing for the 1992 samples were similar to previous years and therefore did not contribute to the differences found. Similar results were obtained from com samples from Pennsylvania (n = 91) with mean values and ranges significantly lower for 1992 (Table 1). Levels of FBI in 1991 Pennsylvania com were 1.4 Ilg/g (mean, N = 70) with a range of 0.0 to 9.8 Ilg/g. The range of FB, concentrations shown in Table 1 also varied greatly from the previous JOURNAL OF FOOD PROTECFION, VOL. 57, JUNE 1994

3 538 METHODS FOR DETECTION OF FUMONISINS so 7 60 % Sample TABLE 2. Fumonisin BJ and HFBJ levels (f.1g/g) in urine and feces samples. Exposure Animal level FBI Urine FBI Feces FBI Feces HFBI Rat (n = 5) Cattle (n = 5) I 14 Cattle (n = 5) Sheep (n = 6) < FB1 (mg/kg) lola '91 _ IA '92 ~ PA '91 r±m PA '92 Figure I. Mean FBI concentration in corn from Iowa elevators TABLE I. Mean FBI (ppm) of corn samples from elevators in Iowa and Pennsylvania. Year State Mean Range N 1988 Iowa Iowa Iowa Iowa Iowa Pennsylvania years. The fungal contamination of the 1992 corn crop was higher than normal, due mainly to the cool and damp growing season throughout the upper midwest. Though fungal contamination was high, fumonisin levels were abnormally low. Two factors were most likely involved in these results. The first was the previously mentioned weather conditions that led to growth of the Fusarium species but not the production of FBI' The weather conditions favored the growth of saprophytic fungi, and the corn plants were not stressed by either excessive heat or lack of moisture. The effects of stress on the corn plant would appear to be important factors in the production of the fumonisins (3). The second was the increased presence of the fungus, in this case F. moniliforme or F. proliferatum, does not indicate increased production of mycotoxin. Spore counts as a general rule are not related to levels of mycotoxin present (25). A number of combased animal feeds and human foods were analyzed (Table 2). Commercial horse feeds containing levels of FBI as high as 37 Ilg/g were found. Com screenings appeared to have approximately 10 times more FB contamination I than did whole kernel corn. Animal feeds represent a large market for corn screenings, and the concentrations found in screenings indicate a possible health risk to animals. Hydrolysis Results of heating FBI in base for I h indicated better than 98% conversion of FBI to HFBI in vitro. Under mild reaction conditions of room temperature, peak height and peak area measurements indicated two peaks (PHFB I and HFB ) of approximately equal size. The sum of the two peak~ accounted for better than 98% of the loss in FBI concentration. High performance liquid chromatography retention times of the compounds formed by this procedure agreed with samples of PHFB I and HFB, furnished by Ron Plattner, and PHFB I and HFB, were confirmed by GC/MS. Also, fecal samples positive for PHFB, and HFB, by HPLC, the two compounds were confirmed by GC/MS. Partially hydrolyzed FBI elutes before FBI and HFB, elutes after FB and are baseline resolved. It was not determined if both ~artially hydrolyzed isomers coeluted or if one of the isomers coeluted with HFB,. Results for the hydrolyzed products are thus reported as the sum of both PHFB, and HFB, peaks (Table 3). The presence of FB, was detected in the urine of both ruminants and nonruminants. Urine from rats exposed to levels of FB as high as 1,000 Ilg/g in their diet had levels,. f offb, in their urine as high as 2llg/g (Table 3). Urme rom both cattle and sheep also tested positive for FBI' but at lower values because the level of exposure was much lower (50 to 400 Ilg/g). An important difference was found between the feces of ruminants compared to nonruminants. Both cattle and sheep showed high levels of HFBJ in their feces compared to the rat feces where FB, apparently was excreted as the intact parent compound. Cattle and sheep feces also contained what appeared to be the PHFB,. This was confirmed by MS (14) and in vitro experiment with standard FB, that was slowly hydrolyzed. Typical chromatograms for a field case of ELEM, rat feces and sheep feces are shown in Fig. 2 (A, B, C). Levels found in the urine are in agreement with previous investigations indicating that FB7 is excreted primarily in the feces, with very little found in the urine (21,22). In conclusion, fumonisins have been found in each of the last five corn crop years in Iowa. The mean levels found TABLE 3. Levels of FBI (f.1g/g) in cornbased feeds and foods. Product n Range Median Popcorn 5 < Indian corn Yellow corn meal White corn meal Horse feed 5 < Rat teed Chicken feed Corn screenings

4 I. A!!! B c allowing the simultaneous cleanup of the hydrolyzed forms of fumonisin. Additional research is needed into the toxicokinetics of the fumonisins and the possible effects of low level exposure of animals and humans to these mycotoxins. Carcinogenicity of the fumonisins is being studied by the U.S. Food and Drug Administration. ACKNOWLEDMENTS The authors would like to acknowledge Dr. Ron Plattner (USDA, ARS, NCAUR, Peoria, IL) for his assistance with mass spectrometry confirmation, Dr. Ken Voss (USDA, ARS, Athens, GA) for the rat fecal and urine samples, Dr. Gary Osweiler (Iowa State University, Ames, IA) for the sheep fecal and urine samples, Drs. Patricia Murphy and Charles Hurburgh (Iowa State University, Ames, IA) for the Iowa com samples and Dr. Paul Nelson (The Pennsylvania State University, University Park, PA) for the Pennsylvania com samples. Also a special mention should go to Janice Dejong and Debra Owens for their assistance in the analysis of the samples. REFERENCES Figure 2. Chromatograms of A) corn from an ELEM field case, B) rat feces and C) sheep feces. in 1992, however, were significantly lower than those found in the previous four years. The lower values found in Iowa in 1992 were also found in 1992 Pennsylvania corn. These lower values found in 1992 might be explained by the cool, damp growing season in the upper midwest resulting in less stress on the com plants. Similar growing conditions also occurred in Pennsylvania in Cornbased foods and feeds appear to be a source of exposure to FB I especially in animal feeds produced from corn screenings. The contamination of human foods appears low and the level of human exposure correspondingly low. The results reported agree with previously reported levels for human foods (27). The effects on humans, especially at these low levels is unknown; however, atherogenic effects in nonhuman primates has been postulated from experimental data (6). Further work in this area is needed to properly assess the human health risk. Toxicokinetic studies on the fumonisins have been limited. This report would suggest the necessity of using an analytical method such as the OPA HPLC procedure since significant amounts of hydrolyzed FB I is found in the feces of ruminants. This study also suggests there may be different metabolites formed in ruminants as opposed to nonruminant animals. Quantitation limit (100 ng/m!) and percent recovery (>95%) for urine, fecal samples and animal feeds were comparable to spiked corn samples as reported earlier (18) and were determined by spiked recoveries from each matrix. The use of the SPEcolumn for cleanup greatly simplified cleanup as compared to the strong anionexchange cartridge column used by Shephard et al. (20). The use of the SPEcolumn also has the additional advantage of I. AzconaOlivera, J. 1.,M. M. Abouzied, R. D. Plattner, W. P. Norred and J. J. 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