structural determination of a nivalenol glucoside and development of an analytical method for the...

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Structural Determination of a Nivalenol Glucoside and Development of an Analytical Method for the Simultaneous Determination of Nivalenol and Deoxynivalenol, and Their Glucosides, in Wheat Tomoya Yoshinari,* ,Shohei Sakuda, Kazuo Furihata, Hiroko Furusawa, Takahiro Ohnishi, Yoshiko Sugita-Konishi, § Naoto Ishizaki, § and Jun Terajima National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan § Department of Food and Life Science, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan * S Supporting Information ABSTRACT: Trichothecene mycotoxins such as nivalenol and deoxynivalenol frequently contaminate foodstus. Recently, several trichothecene glucosides have been found in trichothecene-contaminated foods, and information about their chemistry, toxicity, and occurrence is required. In this study, a glucoside of nivalenol was isolated from nivalenol-contaminated wheat and was identied as nivalenol-3-O-β-D-glucopyranoside. Analytical methods using a multifunctional column or an immunoanity column have been developed for the simultaneous determination of nivalenol, nivalenol-3-O-β-D-glucopyranoside, deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyranoside in wheat. The methods were validated in a single laboratory, and recovery from wheat samples spiked at four levels ranged between 86.4 and 103.5% for the immunoanity column cleanup. These mycotoxins in contaminated wheat samples were quantitated by the validated method. Nivalenol-3-O-β-D-glucopyranoside was detected in the nivalenol-contaminated wheat, and the percentage of nivalenol-3-O-β-D-glucopyranoside to nivalenol ranged from 12 to 27%. This result indicates that the analytical method developed in this study is useful for obtaining data concerning the state and level of food contamination by nivalenol, deoxynivalenol, and their glucosides. KEYWORDS: nivalenol, glucoside, wheat INTRODUCTION Trichothecenes are a large group of mycotoxins produced by various species of Fusarium, Myrothecium, Trichoderma, and Trichothecium. They are divided into four types (A-D) on the basis of their structures. The majority of trichothecenes found in cereals belong to type A, including T-2 toxin and HT-2 toxin, and type B, including deoxynivalenol (1), 3-acetyldeoxynivale- nol, 15-acetyldeoxynivalenol, and nivalenol (2). 1 Deoxynivale- nol and nivalenol, which are mainly produced by Fusarium graminearum and Fusarium culmorum, are frequently detected in agricultural staples such as wheat, barley, and maize. 2-4 Adverse eects of these compounds have been shown by in vitro and in vivo experiments. 5-7 Acute oral exposure causes vomiting, nausea, diarrhea, and gastroenteritis, whereas chronic eects at low dietary concentrations include growth retardation, reduced food consumption, and immunosuppression. Contamination of agricultural products with these compounds has caused several outbreaks of intoxications in humans and animals. 8 Acute human mycotoxicoses have occurred in Asian countries such as Japan, China, and India. The outbreak in India was associated with the consumption of bread made from mold-damaged wheat. Some trichothecenes, including deoxynivalenol, nivale- nol, and T-2 toxin, were contaminated in the wheat. To reduce the daily intake of deoxynivalenol, many countries have established regulatory limits or guidance concentrations for deoxynivalenol in foods and feeds. In Japan, a provisional regulatory limit of 1.1 mg/kg was set for deoxynivalenol in wheat in 2002. In the European Union (EU), the maximum limits are set between 200 μg/kg for processed cereal-based foods and baby foods for infants and young children and 1750 μg/kg for unprocessed durum wheat and oats and unprocessed maize. On the other hand, no country regulates nivalenol to date. However, the Food Safety Commission in Japan assessed the risk of nivalenol in 2010 and established a tolerable daily intake (TDI) for nivalenol of 0.4 μg/kg body weight per day based on decreased white blood cell counts observed in a 90 day rat study. 9 Subsequently, the European Food Safety Authority (EFSA) provided a scientic opinion on the risks for nivalenol and established a TDI of 1.2 μg/kg body weight per day. 10 Consequently, concern about the risk of nivalenol has also risen worldwide. Recently, a new group of mycotoxin derivatives has been detected in naturally contaminated cereals. As it is dicult to detect them by conventional analytical methods, they have been termed masked mycotoxins. 11 A glucoside derivative of deoxynivalenol, deoxynivalenol-3-O-β-D-glucopyranoside (3), was isolated from deoxynivalenol-treated wheat and charac- terized by NMR. 12 It is known that some plants infected with DON-producing fungi are capable of transforming the Received: October 29, 2013 Revised: January 14, 2014 Accepted: January 16, 2014 Published: January 16, 2014 Article pubs.acs.org/JAFC © 2014 American Chemical Society 1174 dx.doi.org/10.1021/jf4048644 | J. Agric. Food Chem. 2014, 62, 1174-1180

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Page 1: Structural Determination of a Nivalenol Glucoside and Development of an Analytical Method for the Simultaneous Determination of Nivalenol and Deoxynivalenol, and Their Glucosides,

Structural Determination of a Nivalenol Glucoside and Developmentof an Analytical Method for the Simultaneous Determination ofNivalenol and Deoxynivalenol, and Their Glucosides, in WheatTomoya Yoshinari,*,† Shohei Sakuda,‡ Kazuo Furihata,‡ Hiroko Furusawa,† Takahiro Ohnishi,†

Yoshiko Sugita-Konishi,§ Naoto Ishizaki,§ and Jun Terajima†

†National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan‡Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan§Department of Food and Life Science, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan

*S Supporting Information

ABSTRACT: Trichothecene mycotoxins such as nivalenol and deoxynivalenol frequently contaminate foodstuffs. Recently,several trichothecene glucosides have been found in trichothecene-contaminated foods, and information about their chemistry,toxicity, and occurrence is required. In this study, a glucoside of nivalenol was isolated from nivalenol-contaminated wheat andwas identified as nivalenol-3-O-β-D-glucopyranoside. Analytical methods using a multifunctional column or an immunoaffinitycolumn have been developed for the simultaneous determination of nivalenol, nivalenol-3-O-β-D-glucopyranoside,deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyranoside in wheat. The methods were validated in a single laboratory,and recovery from wheat samples spiked at four levels ranged between 86.4 and 103.5% for the immunoaffinity column cleanup.These mycotoxins in contaminated wheat samples were quantitated by the validated method. Nivalenol-3-O-β-D-glucopyranosidewas detected in the nivalenol-contaminated wheat, and the percentage of nivalenol-3-O-β-D-glucopyranoside to nivalenol rangedfrom 12 to 27%. This result indicates that the analytical method developed in this study is useful for obtaining data concerningthe state and level of food contamination by nivalenol, deoxynivalenol, and their glucosides.

KEYWORDS: nivalenol, glucoside, wheat

■ INTRODUCTION

Trichothecenes are a large group of mycotoxins produced byvarious species of Fusarium, Myrothecium, Trichoderma, andTrichothecium. They are divided into four types (A−D) on thebasis of their structures. The majority of trichothecenes foundin cereals belong to type A, including T-2 toxin and HT-2 toxin,and type B, including deoxynivalenol (1), 3-acetyldeoxynivale-nol, 15-acetyldeoxynivalenol, and nivalenol (2).1 Deoxynivale-nol and nivalenol, which are mainly produced by Fusariumgraminearum and Fusarium culmorum, are frequently detected inagricultural staples such as wheat, barley, and maize.2−4 Adverseeffects of these compounds have been shown by in vitro and invivo experiments.5−7 Acute oral exposure causes vomiting,nausea, diarrhea, and gastroenteritis, whereas chronic effects atlow dietary concentrations include growth retardation, reducedfood consumption, and immunosuppression. Contamination ofagricultural products with these compounds has caused severaloutbreaks of intoxications in humans and animals.8 Acutehuman mycotoxicoses have occurred in Asian countries such asJapan, China, and India. The outbreak in India was associatedwith the consumption of bread made from mold-damagedwheat. Some trichothecenes, including deoxynivalenol, nivale-nol, and T-2 toxin, were contaminated in the wheat.To reduce the daily intake of deoxynivalenol, many countries

have established regulatory limits or guidance concentrationsfor deoxynivalenol in foods and feeds. In Japan, a provisionalregulatory limit of 1.1 mg/kg was set for deoxynivalenol in

wheat in 2002. In the European Union (EU), the maximumlimits are set between 200 μg/kg for processed cereal-basedfoods and baby foods for infants and young children and 1750μg/kg for unprocessed durum wheat and oats and unprocessedmaize. On the other hand, no country regulates nivalenol todate. However, the Food Safety Commission in Japan assessedthe risk of nivalenol in 2010 and established a tolerable dailyintake (TDI) for nivalenol of 0.4 μg/kg body weight per daybased on decreased white blood cell counts observed in a 90day rat study.9 Subsequently, the European Food SafetyAuthority (EFSA) provided a scientific opinion on the risksfor nivalenol and established a TDI of 1.2 μg/kg body weightper day.10 Consequently, concern about the risk of nivalenolhas also risen worldwide.Recently, a new group of mycotoxin derivatives has been

detected in naturally contaminated cereals. As it is difficult todetect them by conventional analytical methods, they have beentermed “masked mycotoxins”.11 A glucoside derivative ofdeoxynivalenol, deoxynivalenol-3-O-β-D-glucopyranoside (3),was isolated from deoxynivalenol-treated wheat and charac-terized by NMR.12 It is known that some plants infected withDON-producing fungi are capable of transforming the

Received: October 29, 2013Revised: January 14, 2014Accepted: January 16, 2014Published: January 16, 2014

Article

pubs.acs.org/JAFC

© 2014 American Chemical Society 1174 dx.doi.org/10.1021/jf4048644 | J. Agric. Food Chem. 2014, 62, 1174−1180

Page 2: Structural Determination of a Nivalenol Glucoside and Development of an Analytical Method for the Simultaneous Determination of Nivalenol and Deoxynivalenol, and Their Glucosides,

toxin.13,14 Because deoxynivalenol-3-O-β-D-glucopyranosideinhibited protein synthesis of wheat ribosomes in vitro muchmore weakly than deoxynivalenol, it appeared that glycosylationof deoxynivalenol is a detoxification process in plants.15

The Joint FAO/WHO Expert Committee on Food Additives(JECFA) in 2010 evaluated the health risks of 3-acetyldeox-ynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyranoside as well as deoxynivalenol. The Committeedecided to set a group provisional maximum tolerable dailyintake (PMTDI) at 1.0 mg/kg of body weight per day fordeoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxyni-valenol, but deoxynivalenol-3-O-β-D-glucopyranoside was notincluded in the group because data on the toxicity andoccurrence were insufficient at that time.16 However, recently,the occurrence of deoxynivalenol-3-O-β-D-glucopyranoside invarious foods has been reported since its analytical standardbecame commercially available.17−19 In addition, deoxynivale-nol can be released from deoxynivalenol-3-O-β-D-glucopyrano-side by some human intestinal bacteria.20,21 On the basis ofthese backgrounds, not only deoxynivalenol acetates but alsodeoxynivalenol-3-O-β-D-glucopyranoside are considered topose potential risks to humans.In addition to deoxynivalenol-3-O-β-D-glucopyranoside,

some masked trichothecene mycotoxins, such as glucosides ofnivalenol, T-2 toxin, and HT-2 toxin, were detected inartificially contaminated cereals by MS analysis.22−24 Becausethere is a possibility that these compounds exert their toxiceffects after the release of aglycone during digestion,information about the occurrence of the glucosides in foodproducts is important for conducting risk assessment of theparent mycotoxins. However, the lack of commercially availablestandards prevents the quantitative analysis of the glucosides.The aims of this study were two-fold. The first was to isolate

a glucoside of nivalenol from naturally contaminated wheat andto determine the chemical structure by NMR analysis.Information about a glucoside of nivalenol is as important asthat for deoxynivalenol-3-O-β-D-glucopyranoside because niva-lenol is frequently detected in various cereals. Second, methodsfor the simultaneous determination of nivalenol, deoxynivale-nol, and their glucosides in wheat were developed to verify theiroccurrence.

■ MATERIALS AND METHODSChemicals. Solid crystals of deoxynivalenol and nivalenol and a

standard solution of deoxynivalenol-3-O-β-D-glucopyranoside (50 ng/mL in acetonitrile) were purchased from Sigma-Aldrich (St. Louis,MO, USA). Liquid chromatography grade acetonitrile and water andother chemicals were purchased from Wako Pure Chemicals (Osaka,Japan).Q-TOF LC-MS Conditions. The LC system consisted of an Agilent

1200 series (Agilent Technologies, Palo Alto, CA, USA). The LCconditions were as follows: mobile phase, 10 mM ammonium acetate/acetonitrile; hold at 5% acetonitrile for 3 min, linear gradient of 5−90% acetonitrile for 10 min, hold at 90% acetonitrile for 6 min, lineargradient of 90−5% acetonitrile for 1 min, followed by equilibration at5% acetonitrile for 20 min before the next injection. The flow rate was0.2 mL/min, and the column used was a 150 mm × 2.1 mm i.d., 3 μm,InertSustain C18 (GL Sciences Inc., Tokyo, Japan). The column ovenwas held at 40 °C, and the autosampler tray was maintained at 4 °C.The HPLC system was connected to an Agilent 6530 Q-TOF massspectrometer. An electrospray ionization (ESI) interface with AgilentJet Stream Technology in the negative mode was used. The instrumentwas calibrated in the high-resolution mode (4 GHz) with a standardmass range (m/z < 3200). Reference masses at m/z 119.0363 and980.0164 were continually introduced along with the LC stream for

accurate mass calibration. The drying gas (nitrogen) temperature wasset at 325 °C, drying gas flow at 10 L/min, nebulizer pressure at 30 psi,and capillary voltage (Vcap) at 3500 V. A centroid datum within themass range m/z 100−1000 was acquired at a 1 spectrum/s rate with aMassHunter workstation (Agilent). Peak identification was performedusing Qualitative Analysis software version B.04.00 (Agilent).

Purification of Nivalenol-3-O-β-D-glucopyranoside fromNaturally Contaminated Wheat. Naturally contaminated groundwheat (12 kg) was extracted with 48 L of acetonitrile/water (85:15, v/v). Extraction was performed on a horizontal shaker for 120 min at100 rpm. The filtrate was collected by vacuum filtration using an A-3Saspirator (Tokyo Rikakikai Co., Tokyo, Japan), 1.2 kg of silica gel 60(63−200 mm particle size) (Merck KGaA, Darmstadt, Germany) wasadded to the filtrate, and the mixture was dried by removing thesolvent on a rotary evaporator. Fifty grams of the dried silica gel waspacked in a column (50 cm × 3 cm i.d.) and eluted with n-hexane (400mL), ethyl acetate (400 mL), and ethyl acetate/methanol (80:20, v/v,600 mL), successively. This process was repeated until all of the driedsilica gel had been treated. The ethyl acetate/methanol fractioncontaining nivalenol-3-O-β-D-glucopyranoside was evaporated todryness, and the residue was suspended in water. The suspensionwas extracted twice with ethyl acetate. The remaining water layer wasevaporated to dryness, and the residue (40 g yield) was suspended in400 mL of water. After centrifugation (3000g, 10 min), 2 mL of thesupernatant was subjected to an HF Mega BE-C18 (5 mg) cartridge(Agilent), which was preequilibrated with 20 mL of water containing10 mM ammonium acetate. The column was washed with 20 mL ofwater containing 10 mM ammonium acetate, and nivalenol-3-O-β-D-glucopyranoside was eluted from the cartridge with 20 mL of 10%acetonitrile in water containing 10 mM ammonium acetate. Thisprocess was repeated until all of the supernatant had been treated. Theeluate was evaporated to dryness, and the residue (3.6 g yield) wassubjected to an LC-20A series HPLC system (Shimadzu Corp., Kyoto,Japan). The column used was a 250 mm × 10 mm i.d., 5 μm, InertsilODS-3 (GL Sciences). Isocratic elution with 5% acetonitrile in waterat a flow rate of 4.0 mL/min with detection at 220 nm was used toobtain fraction A (18.5−21 min retention time, 100 mg yield).Fraction A was purified by reverse-phase HPLC equipped with thesame column. Isocratic elution with water containing 0.1% trifluoro-acetic acid from 0 to 5 min and then gradient elution of 0−5%acetonitrile in 0.1% trifluoroacetic acid from 5 to 50 min at a flow rateof 4.0 mL/min with detection at 220 nm was used to obtain fraction B(45 min retention time, 16.9 mg yield). Fraction B was purified byreverse-phase HPLC equipped with the same column. Isocratic elutionwith water from 0 to 2 min and then gradient elution of 0−4%acetonitrile in water from 2 to 74 min at a flow rate of 4.0 mL/minwith detection at 220 nm was used to obtain nivalenol-3-O-β-D-glucopyranoside (65 min retention time, 9.2 mg (19%) yield): [α]D

20

−5.5 (c 0.25, H2O); HRESI-TOF/MS m/z 474.1743 (calcd forC21H30O12, 474.1737);

1H and 13C NMR, see Table 1.D-Glucose Oxidase Digestion. Five hundred micrograms of

nivalenol-3-O-β-D-glucopyranoside was mixed with 4 M HCl (1 mL)and then hydrolyzed in a sealed test tube at 100 °C for 4 h. Thereaction solution was dried on a rotary evaporator and lyophilized for12 h. The resulting residue was dissolved in 400 μL of water and mixedwith 50 μL of a 0.5 M potassium phosphate buffer (pH 6.0) and 100 Uof D-glucose oxidase (Sigma-Aldrich) in 50 μL of water. The reactionsolution was incubated at 35 °C for 1 h, and the solution was directlyinfused into the LC-MS. D-Glucose oxidase digestion resulted in thedisappearance of the signal corresponding to glucose (m/z 179 [M −H]−), which originated from nivalenol-3-O-β-D-glucopyranoside, andthe appearance of a new signal corresponding to gluconolactone (m/z177 [M − H]−).

Analytical Method with the Multifunctional Column.Twenty-five grams of the wheat sample was extracted with 200 mLof acetonitrile/water (85:15, v/v). The extraction was performed on ahorizontal shaker for 30 min at 180 rpm, and then the extract wasfiltered through a filter paper (Toyo Roshi Kaisha, Tokyo, Japan). Aportion (10 mL) of the filtered extract was transferred into an InertSepVRA-3 multifunctional column (GL Sciences). The first 4 mL of eluate

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was discarded, and the next 4 mL of eluate was collected andevaporated to dryness. The residue was dissolved in 1 mL of 10%acetonitrile in water.Analytical Method with the Immunoaffinity Column.

Twenty-five grams of the wheat sample was extracted with 100 mLof distilled water and then shaken for 30 min. The extract wastransferred to a 50 mL centrifuge tube and centrifuged at 1410g for 10min. Aliquots of 10 mL of the supernatant were diluted with 50 mL ofphosphate-buffered saline (PBS). After the diluted extract was filteredthrough a glass fiber filter GA-55 (Toyo Roshi Kaisha), 6 mL of thefiltrate (equivalent to 0.25 g sample matrix) was applied to a DON-NIV WB immunoaffinity column (VICAM, Milford, MA, USA). Theimmunoaffinity column had a capacity of approximately 1 μg ofnivalenol, deoxynivalenol, and their glucosides, respectively. When thetoxins in the highly contaminated wheat samples (no. 1, 3, 4, and 5 inTable 3) were analyzed, the amount of the filtrate applied to thecolumn was decreased to 1 mL (equivalent to 0.042 g of samplematrix). The column was washed with 10 mL of PBS followed by awash with 10 mL of distilled water. Toxins were eluted using 0.5 mL ofmethanol followed by 1.5 mL of acetonitrile. The eluate was driedunder nitrogen at 40 °C. The residue was dissolved in 0.25 mL of 10%acetonitrile in water.LC-MS/MS Conditions. LC-MS/MS analyses were performed

with a 3200 Q TRAP LC-MS/MS system (AB Sciex, Foster City, CA,USA) equipped with an ESI source and an LC-20A series high-performance LC system (Shimadzu Corp.). The column used was a150 mm × 2.1 mm i.d., 3 μm, Inertsil ODS-3 (GL Sciences).Chromatographic separation was achieved at 40 °C, using a gradientelution of 5−73% acetonitrile in water from 0 to 8 min and thenisocratic elution of 90% acetonitrile in water from 8 to 10 min at a flowrate of 0.2 mL/min. The sample was centrifuged at 10000g for 5 minbefore analysis. The injection volume was 10 μL. The ESI source wasoperated at 400 °C in the negative ionization mode. Other MSparameters were as follows: curtain gas at 10 psi, nebulizer gas (GS1)at 50 psi, turbo heater gas (GS2) at 50 psi, collision-activated

dissociation gas at 5 (arbitrary units), multiple reaction monitoring,dwell time of 250 ms, and a 5 ms pause between mass ranges. Thefollowing multiple reaction monitoring transitions were used:nivalenol, 371 [M + CH3COO]

− to 281 (collision energy, −18 eV);nivalenol-3-O-β-D-glucopyranoside, 533 [M + CH3COO]

− to 263(collision energy, −28 eV); deoxynivalenol, 295 [M − H]− to 265(collision energy, −12 eV); deoxynivalenol-3-O-β-D-glucopyranoside,517 [M + CH3COO]

− to 427 (collision energy, −24 eV).Method Validation. The method was validated with deoxyniva-

lenol-negative wheat (Trilogy Analytical Laboratory Inc., Washington,MO, USA) spiked at four concentrations, 10, 50, 250, and 1000 μg/kg,with six replicates for each analyte. When using the DON-NIV WBimmunoaffinity column, the amount of the filtrate applied to thecolumn was 6 mL for the samples spiked at 10, 50, and 250 μg/kg and1 mL for the sample spiked at 1000 μg/kg. Detected concentrationswere calculated on a peak area basis using Analyst version 1.5.1software (AB Sciex). Limits of detection (LODs) and limits ofquantification (LOQs) were calculated on the basis of signal-to-noiseratios of 3:1 and 10:1, respectively.

■ RESULTS AND DISCUSSIONDetection of a Nivalenol Glycoside in Naturally

Contaminated Wheat. Naturally contaminated wheat was

extracted with 85% acetonitrile in water, and the extract wasanalyzed by Q-TOF LC-MS. Compounds having estimatedmolecular formulas of C15H20O7 and C21H30O12 (compound 4)were observed at retention times of 7.5 and 8.0 min,respectively. The former compound was identified as nivalenol.The molecular formula of 4 was larger than that of nivalenol byC6H10O5. As a result of MS/MS fragmentation analysis,common fragment ions (m/z 191, 217, 233, 281) were found

Table 1. NMR Spectroscopic Data for Nivalenol-3-O-β-D-glucopyranoside

position δCa δH

a (J in Hz) HMBCb

2 80.2 3.94, d (4.4) 4, 5, 11, 123 86.7 4.33, dd (4.4, 3.7) 4, 1′4 78.7 4.64, d (3.7) 3, 5, 6, 125 49.86 54.57 74.4 4.88, s 5, 6, 8, 11, 158 202.79 138.510 137.8 6.67, d (5.1) 6, 8, 11, 1611 71.4 4.63, d (5.1) 7, 10, 12, 1512 66.213 47.0 3.15, d (5.0) 2, 5, 12

3.19, d (5.0)14 8.2 1.05, s 4, 5, 6, 1215 60.8 3.82, d (12.7) 5, 6, 7, 11

3.85, d (12.7)16 15.4 1.82, s 8, 9, 101′ 103.3 4.74, d (7.8) 3, 2′, 3′2′ 73.8 3.38, dd (9.3, 7.8) 1′, 3′3′ 76.7 3.53, dd (9.3, 8.0) 2′4′ 70.3 3.465′ 76.9 3.476′ 61.4 3.74, dd (12.0, 4.6)

3.89, d (12.0)a150 MHz (δC) and 500 MHz (δH) in D2O. Dioxane δC 67.5 and DSSδH 0.00 were used as external standards. bFrom protons stated to theindicated carbon.

Figure 1. Structures of deoxynivalenol, nivalenol, and their glucosides.

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in nivalenol and 4 product ion spectra. From these results, 4was suspected to be a nivalenol monoglycoside.Isolation and Structure Determination of a Nivalenol

Glycoside. Compound 4 was extracted from acetonitrile/water(85:15, v/v) from wheat. The extracts were fractionated by

silica gel column chromatography, followed by liquid−liquidextraction. Further purification by C18 cartridges and reverse-phase HPLC afforded 4 in a yield of 9.2 mg from 12 kg ofwheat. The 1H and 13C NMR spectra of 4 confirmed that it is amonoglycoside of nivalenol (Figure 2a,b). The sugar present in

Figure 2. 1H NMR spectrum (a), 13C NMR spectrum (b), and HMBC spectrum (c) of nivalenol-3-O-β-D-glucopyranoside.

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4 was identified as glucose from NMR data, which aresummarized in Table 1. A long-range coupling between C-3 ofthe nivalenol moiety and C-1′ of glucose observed in theHMBC spectrum of 4 showed that 4 was a nivalenol-3-O-glucopyranoside (Figure 2c). The JH‑1′,H‑2′ value (7.8 Hz)indicated a β-glycosidic linkage. Treating the acid hydrolysateof 4 with D-glucose oxidase showed the D configuration of theglucose residue of 4. The structure of 4 was thus assigned asnivalenol-3-O-β-D-glucopyranoside (Figure 1).Analytical Methods for Determination of Nivalenol,

Deoxynivalenol, and Their Glucosides. Two purificationmethods were examined for the simultaneous determination ofnivalenol, nivalenol-3-O-β-D-glucopyranoside, deoxynivalenol,and deoxynivalenol-3-O-β-D-glucopyranoside. The first is amethod using a multifunctional column, InertSep VRA-3. The

column, which contains ion exchange and reverse phase resins,is designed for purifying mycotoxins and agricultural chemicalsfrom cereals. Previously, our group has reported a purificationmethod using InertSep VRA-3 for the identification ofdeoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivale-nol, and deoxynivalenol-3-O-β-D-glucopyranoside in corn-based products.25 The reported method was slightly modifiedto obtain good recoveries for nivalenol and nivalenol-3-O-β-D-glucopyranoside. The second is a method using a commercialimmunoaffinity column. Some immunoaffinity columns that aredesigned for simultaneous purification of deoxynivalenol andnivalenol have recently been developed. In a preliminary study,deoxynivalenol, nivalenol, and their glucosides could berecovered from an immunoaffinity column, DON-NIV WB.Quantitation of the mycotoxins was performed by LC-MS/MS.A typical chromatogram is shown in Figure 3. To validate thetwo methods, they were applied to the analysis of spiked wheatsamples containing nivalenol, nivalenol-3-O-β-D-glucopyrano-side, deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyrano-side at four levels in six replicates. Recoveries in the InertSepVRA-3 method were 81.5−91.3% for nivalenol, 69.8−75.3% fornivalenol-3-O-β-D-glucopyranoside, 65.8−71.7% for deoxyniva-lenol, and 68.3−85.3% for deoxynivalenol-3-O-β-D-glucopyr-anoside, whereas recoveries in the DON-NIV WB method were87.1−93.3% for nivalenol, 94.1−103.5% for nivalenol-3-O-β-D-glucopyranoside, 86.4−96.5% for deoxynivalenol, and 95.0−100.1% for deoxynivalenol-3-O-β-D-glucopyranoside (Table 2).The LOQs for all analytes were within the range of 1−3 μg/kg.This result showed that both methods could be used todetermine the four mycotoxins and that the recoveries obtainedby the immunoaffinity column method were better than thoseobtained by the multifunctional column method.The concentrations of nivalenol, nivalenol-3-O-β-D-glucopyr-

anoside, deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyr-anoside in wheat samples were quantitated by the analyticalmethod using an immunoaffinity column (Table 3). Nivalenol

Figure 3. LC-MS/MS chromatogram of a standard solution (30 ng/mL concentrations of each analyte). Peaks: deoxynivalenol (1) (m/z295 → 265); nivalenol (2) (m/z 371 → 281); deoxynivalenol-3-O-β-D-glucopyranoside (3) (m/z 517 → 427); nivalenol-3-O-β-D-glucopyranoside (4) (m/z 533 → 263).

Table 2. Limits of Quantitation (LOQ), Spiking Concentrations, and Recovery of Toxins for Validation of the Methods

recovery (%)

InertSepVRA-3

DON-NIVWB

analyte LOQ (μg/kg) spiking concn (μg/kg) mean RSD mean RSD

nivalenol 2 10 85.9 11.3 92.9 5.950 85.3 3.1 93.3 3.2250 81.5 4.0 87.1 3.91000 91.3 2.5 87.4 1.1

nivalenol-3-O-β-D-glucopyranoside 3 10 75.3 12.5 96.9 6.450 73.6 4.9 103.1 5.5250 69.8 1.7 94.1 2.01000 73.0 0.8 103.5 3.7

deoxynivalenol 2 10 71.1 9.0 94.8 12.050 66.1 3.7 96.5 4.5250 65.8 3.0 86.4 1.01000 71.7 1.2 88.6 4.4

deoxynivalenol-3-O-β-D-glucopyranoside 1 10 68.3 8.6 99.3 4.650 77.2 5.8 100.1 4.3250 77.0 2.6 95.0 2.31000 85.3 1.7 98.7 3.9

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and nivalenol-3-O-β-D-glucopyranoside were detected in allsamples, and the percentages of nivalenol-3-O-β-D-glucopyrano-side to nivalenol ranged from 12 to 27%. Deoxynivalenol anddeoxynivalenol-3-O-β-D-glucopyranoside were also found in thesamples, and the percentage of deoxynivalenol-3-O-β-D-glucopyranoside to deoxynivalenol was within the range of5−14%.In this study, a new derivative of nivalenol was isolated from

nivalenol-contaminated wheat and was identified as nivalenol-3-O-β-D-glucopyranoside. Methods for the simultaneous deter-mination of nivalenol, deoxynivalenol, and their glucosides inwheat were successfully developed and validated. Co-contam-ination of nivalenol-3-O-β-D-glucopyranoside with nivalenol,deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyranosidewas observed in wheat, and the ratio of nivalenol-3-O-β-D-glucopyranoside to nivalenol was equivalent to that ofdeoxynivalenol-3-O-β-D-glucopyranoside to deoxynivalenol re-ported previously.17 Because glycosylation is considered to be adetoxification process, it seems likely that nivalenol-3-O-β-D-glucopyranoside shows lower toxicity than nivalenol. However,as with deoxynivalenol-3-O-β-D-glucopyranoside, nivalenol-3-O-β-D-glucopyranoside may be hydrolyzed by intestinal bacteriaand toxic nivalenol may be produced in the body. Therefore,nivalenol-3-O-β-D-glucopyranoside can be an important con-tributor to dietary exposure to trichothecene mycotoxins andshould be monitored together with nivalenol, deoxynivalenol,and deoxynivalenol-3-O-β-D-glucopyranoside in foodstuffs.

■ ASSOCIATED CONTENT

*S Supporting InformationHPLC chromatograms of the three steps of purification ofnivalenol-3-O-β-D-glucopyranoside. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*(T.Y.) Phone: +81-3-3700-1141. Fax: +81-3-3700-9852. E-mail: [email protected].

FundingThis study was funded by The Tojuro Iijima Foundation forFood Science and Technology.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We thank Dr. T. Nakajima (Ministry of Agriculture, Forestryand Fisheries) for donating wheat samples.

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Table 3. Concentrations of Nivalenol, Nivalenol-3-O-β-D-glucopyranoside, Deoxynivalenol, and Deoxynivalenol-3-O-β-D-glucopyranoside in Wheat

wheatno.

nivalenol(mg/kg)

nivalenol-3-O-β-D-glucopyranoside

(mg/kg)

nivalenol-3-O-β-D-glucopyranoside/nivalenol

(mol %)deoxynivalenol

(mg/kg)

deoxynivalenol-3-O-β-D-glucopyranoside

(mg/kg)

deoxynivalenol-3-O-β-D-glucopyranoside/deoxynivalenol

(mol %)

1 0.37 0.15 27 10 1.5 102 0.54 0.22 27 0.67 0.15 143 1.5 0.54 24 3.6 0.27 54 1.7 0.30 12 5.6 0.62 75 15 4.0 18 0.23 0.05 14

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