Food Contaminants - American Chemical Society

Chapter 14

Determination of Fumonisin B in Botanical Roots 1

Mary W. Trucksess, Carolyn J. Oles, Carol M. Weaver, Kevin D. White, and Jeanne I. Rader

Downloaded by YORK UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch014

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740

Fumonisins are toxins produced mainly by the molds Fusarium verticillioides, F. proliferatum, and several other Fusarium species that grow on agricultural commodities in the field or during storage. More than ten types of fumonisins have been isolated and characterized. Fumonisin B (FB ), B (FB ), and B (FB ) are the major fumonisins produced. FB is the most prevalent and most toxic. Recently, fumonisins have been found in botanical roots. Fumonisins have produced liver damage and changes in the levels of certain classes of lipids, especially sphingolipids, in all animals studied. 1

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Many analytical methods for determining fumonisins in foods have been published. Among the most common of these methods are liquid chromatographic (LC) separation with fluorescence detection, enzyme-linked immunosorbent assay (ELISA) and LC-mass spectrometry (MS). In our laboratory, the botanical roots of ginseng, ginger, turmeric and kava-kava were extracted with a mixture of methanol and water, followed by cleanup on an immunoaffinity column (IAC). FB was then derivatized, separated, and determined by LC with fluorescence detection. Recoveries of FB added to ginseng, ginger, turmeric, and kava-kava roots at levels ranging from 0.05 to 2 μg/g, were >75% except for turmeric at a spiked level of 0.05 μg/g. ELISA was also applied to screen these roots for FB . 1

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© 2008 American Chemical Society

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


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Numerous dietary supplements are commercially available worldwide to promote health, increase longevity, or lose weight (1). Dietary supplement sales in the U.S. were approximately twenty billion dollars in 2005 (2). Some botanical supplements such as garlic, ginger, and turmeric have been used as food and condiments for centuries. Botanicals are generally regarded as safe for consumption, however, they might contain chemical contaminants such as heavy metals, pesticides, and mycotoxins. Fumonisins have been found in medicinal wild plants in South Africa (3) and also in herbal tea and medicinal plants in Turkey (4). Fumonisins are a group of structurally related compounds produced by several species of Fusarium', Fusarium verticilliodes, F. proliferatum and F. nygami are the main fumonisin producing strains (5). This group of mycotoxins is characterized by a 19-20 carbon aminopolyhydroxy-alkyl chain which is diesterifled with two tricarballylic acid groups. Since 1988 about 28 fumonisin analogs have been characterized and classified into four main groups: A, B, C, and P series (6). The fumonisin B analogs (FBs), comprising toxicologically important F B FB , and FB , are the most abundant naturally occurring fumonisins, with F B i usually being found at the highest levels in naturally contaminated grains (7). FBs have been associated with health problems in animals such as cancer in rodents (8), equine leukoencephalomalacia (ELEM) in horses (9), and pulmonary edema in swine (10). Epidemiology studies have correlated human consumption of fumonisin contaminated corn and esophageal cancer (//). For these reasons the International Agency for Research on Cancer (IARC) has classified FBs as possible carcinogens (72). In 2001, the US Food and Drug Administration (FDA) issued a guidance document to industry recommending a maximum allowable level of FBs in corn used in the production of human foods and animal feeds (13). The non-mandatory guidelines range from 2 \igjg to 4 \xg/g depending on the corn product. The most commonly used analytical method for determining FBs in a wide variety of matrices is LC with precolumn fluorescence derivatization of fumonisins with o-phthaldialdehyde (OPA)/mercaptoethanol (ME). ELISA methods based on monoclonal and polyclonal antibodies specific for fumonisins are being used as a cost effective high throughput screening technique. LC-MS methods have been used for quantitation as well as for validation of results obtained using LC or ELISA methods. The objectives of this study are to determine FB¡ in botanical roots using all three techniques. l5

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Materials and Methods Finely ground plant materials for the recovery study - ginseng (Panax quinquefolius), kava-kava, and turmeric - were purchased from Schumacher



268 Ginseng (Marathon, WI). Finely ground ginger (Zingiber officinale) for the recovery study was purchased from McCormick (Baltimore, MD). FBi with purity >95% (based on data obtained with LC, nuclear magnetic resonance (NMR),and LC-MS analyses) was isolated from fungal cultures and purifed in our laboratory. All solvents were suitable for LC analysis and were purchased from Baker Chemicals. Glacial acetic acid and sodium bicarbonate were obtained from Fisher Scientific; phosphate buffered saline (PBS) 10 mM, OPA, ME, and Tween 20 were from Sigma-Aldridge (St. Louis, MO). Orbital shaker (VWR DS-500E, VWR International, Bridgeport, NJ); centrifuge (Allegra X - 22R, VWR International, Bridgeport, NJ); glass microfibef filter paper, 11 cm (Whatman 934AH, Whatman Inc., Clifton, NJ); and immunoaffinity columns (G1008, wide bore Fumonitest columns, Vicam Corp., Watertown, MA) were used. The LC system consisted of a Waters 600 E pump, Waters 717 plus injector, Waters 2475 fluorescence detector (set at excitation 335 nm and emission 440 nm), Empower 2 control and data system (Waters Corp., Milford, MA), and a CI8 IP LC column (Beckman 235335, Utrasphere, 4.6 x 250 mm, 5 urn, Beckman Instruments, Inc., Fullerton, CA). The limit volume inserts were from Waters Corp. ELISA kits - Veratox for fumonisins HS - were from Neogen Corp. (Lansing, MI). The LC-MS system consisted of an Agilent 1100 series LC (Agilent Technologies, Santa Clara, CA) connected to an API 5000 triple-quadrupole mass spectrometer via a turbospray ion source (Applied Biosystems, Framingham, MA) and a Waters Y M C L-80 C18 LC column, 2 x 250 mm, 4 \im particle size. Extractions: A 5 g test portionfromeach plant material was extracted using 25 mL methanol-acetonitrile-water (25:25:50, v/v/v) with shaking for 10 min at 400 rpm. The mixture was centrifuged and 7 mL of the supernatant was mixed with 28 mL 10 mM PBS containing 1% Tween 20. The mixture was filtered through microfiber paper and 25 mL of filtrate was collected in a 25 mL graduated cylinder. Immunoaffinity column (IAC) chromatography: The column was preconditioned with 5 mL PBS followed by the addition of the 25 mL filtrate. The column was then washed with 10 mL PBS. FB! was eluted with two 1.0 mL portions of methanol-water (8:2, v/v). The eluate was collected into a 4 mL vial and evaporated to dryness in a SpeedVac evaporator. Liquid chromatography (LC): The dried eluate was dissolved in 200 \iL acetonitrile-water (1:1, v/v). The stock standard solution was prepared by dissolving 10 mg FBi in acetonitrile-water (1:1, v/v). The working standard solutions were prepared by diluting the stock solution to 2, 1, 0.5, 0.25, 0.125 and 0.625 ug/mL with the same solvent. OPA reagent was prepared by dissolving 50 mg OPA in 1 mL methanol then adding 49 mL 0.06 M sodium tetraborate and mixing. Before use, 20 \xL M E was added to 5 mL of OPA reagent and mixed. The LC column was equilibrated with a mobile phase of acetonitrile-water-acetic acid (950:1050:20, v/v) at a flow rate of 1.0 mL/min.



269 The autosampler was programmed to transfer 70 JLIL derivatization reagent into a 400 nL limited volume insert containing 30 test sample extract; 100 uL of the mixture was drawn into the syringe at 5 \iL/s; the mixture was dispensed back into the sample vial. The drawing and dispensing were performed 3 times; then 30 uL of the mixture was injected onto the LC column. The retention time for FB! was about 12 min. Calculation of FBj concentration in test samples was based on peak areas compared with those of the standards. ELISA assay: A l l reagents were included in the commercial kit. The assay procedure was provided by the manufacturer and is briefly described below. The test sample was extracted with 70% methanol and the mixture was shaken and centrifiiged. A portion of the supernatant was diluted with water. The diluted extract was mixed with an equal volume of antibody-enzyme conjugate solution and then added to the wells coated with antibodies. After incubating at room temperature the wells were emptied, washed with water, additional substrate added, incubated again, stopping reagent added and then the results were read with an ELISA reader. LC-MS/MS analysis: Eluates from the immunoaffinity column were not driedfor this analysis but were transferred to autosampler vials and injected directly into the LC-MS/MS system. The mobile phase used was the same as for the LC method above. The flow rate was 200 nL/min and the column heater was set at 30 °C. The mass spectrometer was operated in positive electrospray ionization (ESI*) mode and ions indicative of FBi were observed using multiple reaction monitoring (MRM). High purity nitrogen was used for the curtain gas, ESI nebulizing gas and the collision gas for collision activated dissociation (CAD). The ion spray voltage was set at 5 kV and the source block and desolvation temperatures were 140 and 450 °C, respectively. The two fragment ions of reactions monitored resulted from the CAD of the protonated molecular ion at m/z 722 to m/z 352 and to m/z 334 respectively. Other instrumental conditions and settings included: CAD gas 6; Curtain gas 15; nebulizing gas 30; desolvation gas 30; declustering potential (DP) 110 V; exit potential (EP) 10 V; collision energy (CE) 40 V for m/z 722 > 352 and 30 V for m/z 722 > 334.

Results and Discussion LC chromatograms of added FBj in ginger roots and FBi standards are shown in Figure 1. Chromatograms of the other roots were similar to those of the ginger roots. The OPA reagent reacts with all primary amines present in the sample extract and can lead to interferences that are difficult to separate by LC. Often modification of mobile phase is necessary. In general, the reduction of the acetonitrile concentrations to about 46% resolves some of the problem. The derivatization reagent composition and extract to reagent ratio were modified from the AOAC International Official Method (14). The concentrations of OPA,



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Figure 1. Chromatogram ofaddedfumonisin B¡ in ginger roots at 0.1 [ig/g.

sodium tetraborate and ME in the derivatization reagent were much lower and the extract to reagent ratio was changed from 1:5 to 1:2. The LC chromatogram of reagent blank showed much less response for reagent peaks at retention times near the FB, peak area than the AOAC International Official Method, M E must be added to die OPA reagent daily before use due to its volatility and the small volume of ME in the derivatization reagent. Ginseng, ginger, kava-kava, and turmeric found to contain 75% except kava-kava at an added level of 0.05 ug/g. The lowest level in our recovery study was 0.05 ug/g and this is considered as the limit of determination. The use of OPA as derivatization reagent has some drawbacks: the mixing of test extract before injection onto the L C column and the stability of the derivatives. A dead volume of extract in the sample vial is required because the openings of the needles of the injectors are on the side instead of at the end. Two different types of LC autoinjectors were investigated using FBi standards. The first injector mixed the derivatizing reagent and test solution by repetitive drawing and dispensing the entire amount of extract and reagent mixture in and out of the syringe before injection. The type 1 autoinjector withdrew reagent, then test solution and reagent again into the injector loop; mixing was by diffusion with delayed injection. For the type 1 autoinjector various extract and


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Table I. Recoveries of Fumonisin B (pg/g) Added to Botanical Roots by the immunoaffinity Column/Liquid Chromatography Method


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Roots'

Added

Recovery %

SD

RSDr

Ginseng

0.05 0.10 0.25 0.50 1.00 2.00

89 90 94 98

13 5 8 3

14 5 9 3

Ginger

0.05 0.10 0.25 0.50 1.00 2.00

84 110 110 96 75 89

21 9 9 6 5 2

25 8 8 6 6 3

Turmeric

0.05 0.10 0.25 0.50 1.00 2.00

92 101 98 110 96 85

8 4 5 8 15 1

9 4 5 7 15 1

Kava-kava

0.05 0.10 0.25 0.50 1.00 2.00

60 77 110 111 95 84

26 25 26 8 15 6

43 33 24 7 15 7

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All roots were found to contain

Food Contaminants - American Chemical Society

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