Detection of N-(1-deoxy-d-fructos-1-yl) Fumonisins B2 and B3 in Corn by High-Resolution LC-Orbitrap MS

Matsuo, Yosuke; Takahara, Kentaro; Sago, Yuki; Kushiro, Masayo; Nagashima, Hitoshi; Nakagawa, Hiroyuki

doi:10.3390/toxins7093700

开放存取Article

检测，检测N-（1-deoxy-d-fructos-1-yl）Fumonisins B₂and B₃in Corn by High-Solution LC-Orbitrap MS

^1个

National Agriculture and Food Research Organization（NARO），National Food Research Institute，2-1-12 Kannon-dai，Tsukuba-shi，Ibaraki305-8642，Japan

²

Thermo Fisher Scientific，C-2F，3-9 Moriya-cho，Yokohama-shi，Kanagawa221-0022，Japan

^*

Author to whom correspondence should be addressed。

Toxins 2015单击功能区上，7（9），3700-3714；https://doi.org/10.3390/toxins7093700

Submission received:30June2015/Revised:3September 2015/Accepted:7 September 2015/Published:16 September 2015

（This article belongs to the Collection）Fusarium Toxins–Relevance for Human and Animal Health）

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Abstract

：

热塑性钢管₂（FB₂）and fumonisin B₃（FB₃）in corn powder was confirmed for the first time。These“bound-fumonisins”（FB₂and FB₃bound to glucose were identified asN-（1-deoxy-d-fructos-1-yl）fumonisin B₂（NDfrc-FB₂）andN-（1-deoxy-d-fructos-1-yl）fumonisin B₃（NDfrc-FB₃）respectively，based on the accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometry（LC-Orbitrap MS）analysis。Treatment on NDfrc-FB₂and NDfrc-FB₃with theo-phthalaldehyde（OPA）reagent also supported thatd-glucose binding to FB₂and FB₃molecules occurred to their primary amine residues。

Keywords:

LC-Orbitrap MS打开；fumonisin打开；Fusarium打开；corn打开；N-（1-deoxy-d-fructos-1-yl）fumonisin

Graphical Abstract

1.Introduction

Fusariumfungi are known as plant pathogen infecting cereals such as wheat，barley，and corn，and some of these fungi produce mycotoxins（e.g.，trichothecenes，zearalenone，and fumonisins）[1个]。In Japan，Fusariumfungi infection is occasionally serious，as these crops are usually planted through the rainy season.AmongFusarium本地化mycotoxins，fumonisins are a group of naturally-occurring mycotoxins which are typically produced byFusarium verticillioidesand，andF.proliferatum单击功能区上2]。The most abundant fumonisin is fumonisin B_1个（FB_1个），followed by fumonisin B₂（FB₂），and fumonisin B₃（FB₃）。FB_1个isa causative compound of equine leukoencephalomalacia[3]和porcine pulmonary oedema syndrome[4个]，and has also been confirmed to be hepatoxic and hepatocarcinogenic in rats and mice[5单击功能区上，6个]。Fumonisins are widely distributed geographically，and their natural occurrence in maize has been reported in various regions throughout the world[6个]。A particular concern regarding fumonisins involves the higher concentrations occasionally found in maize produced and consumed by some subpopulations，such as subsistence farmers[6个]。Considerable annual variations in contamination have been noted.Fumonisins also occur infrequently in other foods，including sorghum，asparagus，rice beer，and mung beans.In2002，the FAO/WHO Joint Expert Committee on Food Additives（JECFA）established a provisional maximum tolerday intake（PMI）^-1bw day^-1for FB_1个，FB₂，and FB₃，either alone or in combination[6个]。The European Committee concluded the establishment of a group PMTDI of 2μg kg^-1bw day^-1for FB_1个，FB₂，and FB₃，combined[7]。

Recently，aglucosylated derivative of deoxynivalenol（DON），DON-3-glucoside（DON3 Glc）was found in cereal grain and beer[8个单击功能区上，9]，和similar compounds have also been found for several other mycotoxins[10单击功能区上，11]。Because these glucosylated derivatives are not detected by conventional analytical methods due to their high er polarity[12单击功能区上，13]，they are referred to asked（modified）mycotoxins”。Hydrolysis of masked mycotoxins to their aglycons has also been reported[14单击功能区上，15]，and it has been suggested that they present an additional potential risk to consumers.In the case of fumonisins，the presence of“bound-fumonisin”has been suggested by several researchers[16单击功能区上，第十七节：]。For instance，N-（1-deoxy-d-fructos-1-yl）fumonisin B_1个（NDfrc-FB_1个）was found in corn，并向回水弯头d-glucose binding reaction to the primary amine residue of the FB_1个molecule[18]。Although proven under laboratory conditions，the NDfrc-FB_1个occurrence at significant levels in processed samples is still controversial[19]。Inaddition，in vivo稳定度为this conjugate has not yet been definitively proven[第十七节：]。In order to understand the total fumonisin foods and feeds correctly，there is a need to clarify the presence of these series of fumonisin conjugates.In this paper，authors report the existence of new glucose conjugates derived from type B fumonisins（FB₂and FB₃在工作空间的边缘火焰1）。These species were detected and identified via high resolution liquid chromatography-Orbitrap mass spectrometry（LC-Orbitrap MS），in combination with treatment using a specific reagent（o-phthalaldehyde，OPA）。

Figure1。Chemical structures of FBs and their glucose conjugates。

2.Materials and Methods

2.1。Chemicals

FB_1个and FB₂were purchased from Wako pure chemical Industries Ltd.（Osaka，Japan）.FB₃was purchased from PROMEC（Tygerberg，South Africa）.All other chemicals used were commercially available and of a chemically pure grade.OPA and D-glucose（>98%of chemically pure grade）were obtained from Wako。Acetonitrile（LCMS grade）was from Fisher Scientific（Waltham，MA，USA），and distilled water（LCMS grade）was obtained from Kanto Chemical（Tokyo，Japan）.Ammonium acetate（chemically pure grade）was from Kanto，and acetic acid（>99.9%of chemically pure grade，not glacial）was from Wako。

2.2。Corn Powder Sample Contaminated with Fumonisins

ycotoxin reference material of corn powder（batch number MTC-9999C）was purchased from Trilogy Co.Ltd（Washington，MO，USA）.This material was contaminated with FB_1个，FB₂，FB₃，aflatoxin B_1个，aflatoxin B₂，aflatoxin G_1个单击功能区上，DON，zearalenone，HT-2toxin，and T-2toxin.The origin of this corn was from the USA.This was a crop naturally contaminated with the above toxins.There was no inoculation of any of the toxins in this sample.The manufacturer-warranted concentrations of FB_1个，FB₂，and FB₃were19.8±4.8mg/kg^-1（FB_1个），6.6±2.1mg/kg^-1（FB₂），and2.2±1.6mg/kg^-1（FB₃），respectively.This material was stored at-20°C in the dark until analysis。

2.3。预定位和工作解决方案

FB_1个and FB₂obtained as powder were accurately weighed on an aluminum boat on a micro scale，placed in a 10ml brown volumetric flask，and dissolved in acetonitrile/water（1:1，v/v）。FB₃，obtained asa crystalline sample，was directly dissolved in acetonitrile/water（1:1，v/v）。The concentration was normally adjusted to 100-200mg·L^-1，and stored in brown glass containers at4°C as the stock solutions。工作solutions，each stock solution was taken in brown volumetric flasks，diluted appropriately with acetonitrile/water/acetic acid（5:94:1，v/v/v），and stored at4°C。

2.4。我的成本和泵送

Corn powder（8g），40mL of methanol/water（75:25，v/v），and 0.4 mL of acetic acid（>99.9%）were homogenized with a POLYTRON PT3100 homogenizer（Kinematica AG.，Lucerne，Switzerland）ata rate of7000rpm for 5min，and centrifuged at 2000×gfor10min.A portion of the supernatant（5mL）was loaded onto a strong anion exchange column（Sep-Pak Accell Plus QMA Short Cartridge（360mg），Waters，Milford，MA，USA）with no conditioning.Then5mL methanol/water（3:1，v/v）and5mL methanol were successively loaded on the column for washing。FBs were eluted with5mL of a solution methanol/acetic acid（98:2，v/v）。All of the eluent was collected in a glass tube，and the solvent was evaporated under a nitrogen gas stream at50°C。The residue was dissolved in 0.25 mL of acetonitrile/water/acetic acid（5:94:1，v/v/v）and subjected to LC-MS analysis。

2.5。Synthesis of N-（1-deoxy-d-fructos-1-yl Fumonisins（NDfrc-FBS）

NDfrc-FBS was chemically prepared essentially following the procedure reported by Polinget al。单击功能区上18]。aglassware amber vial（2mL volume），0.1mg of each fumonisin（stock solution of each was appropriately taken and evaporated），40mg ofd-glucose，five or six beads of molecular sieve（pore size；0.4nm）（Millipore，Darmstadt，Germany），and2mL of methanol were taken，mixed，and further heated in an incubator with shaking（120rpm）at60°C overnight。After the reaction，the solvent was evaporated under a nitrogen gas stream at40°C。Theresidue was re-dissolved in 2.5 mL of acetonitrile/water/acetic acid（5/94/1，v/v/v），and cleaned using a solid phase extraction column according to the reported procedure，with a slight modification.The re-dissolved residue（2.5mL）was loaded on an OASIS HLB（3cc）column（Waters），that was conditioned in advance with3mL of methanol and 3mL of acetonitrile/water/acetic/94（94），v/v/v），successively.The column was washed with6mL of acetonitrile/water/acetic acid（5/94/1，v/v/v），and further eluted with3mL of methanol.This eluate was collected and evaporated to dryness under nitrogen gas at 40℃，re-dissolved in 0.25mL of acetonitrile/water/acetic acid（5/94/1，v/v/v），and subjected to LC-MS analysis。

2.6。LC-MS Analysis

FB-glucose conjugate was conducted in accordance with author’s previous studies with the LC-Orbitrap MS instrument，“Exactive”（Thermo Fisher Scientific）[20]。LCwas performed byusing 0.5mM ammonium acetate and 0.1%acetic acid aqueous solution as solvent A and 0.1%acetic acid in acetonitrile as solvent B[11]。The gradient profile used was10%B（0-3.0min），90%B（18.0-22.0min），and10%B（22.1-29.0min）。The flow rate was set to 0.3 mL/min and the column temperature was maintained at40°C。The chromatographic separation was carried out on a HyPURITY C18column（250×3mm i.d.，5μm particle size）（Thermo Fisher）with an injection volume of 0.02 mL.The Exactive mass spectrometer was operated in negative mode with a heated electrospray ionization source（HESI-II）and a spravoy of 50 k。同类型pical and common fragment ion of fumonisins，ketene form of tricarballylic acid（TCA）ion[TCA-H₂O-H]^-什么（TCAKion[TCAK-H]^-什么）was detected with higher sensitivity in negative mode than positive mode。The ion of[TCAK-H]^-什么is often selected as the primary fragment for the detection of fumonisins.The capillary and the heater temperature was350℃and 300℃，respectively.The sheath gas and the auxiliary gas flow rate was adjusted as40and5（in arbitrary units），respectively.The system was operated in the range of 150–1100m/z100000FWHM（full width at half maximum）（m/z200）with an accurate mass/high resolution（AM/HR）full scan（scan event1）and allion fragmentation spectrum acquisition with collision energy in a single run。Fragmentation was achieved with optional CID（collision-induced dissociation）equipment，using a collision energy of 60eV（scan event2），联邦调查局_1个.The external mass axis calibration without the use of specific lock mass was employed。For the mass accuracy estimation，the mass value observed asan abundant ion extracted at the apex of the chromatographic peak was used.The exact mass values（calculated and observed）of the analysts’ions are summarized inTable1单击功能区上，Table2and，andTable3.The mass deviation is expressed either in terms of millimass units（mmu）or parts per million（ppm）。The latter is calculated with the equation:ppm=10^6个×Δm/m打开；whereΔm（calculated）and observed mass，andmis the mass.In accordance with the European Commission guideline[21]，mass deviation<5ppm from the calculated value was used as the criterion for compound identification.LC-Orbitrap MS is a special type of ion trap[22]，and achieves a mass resolving power of up to 100000FWHM（m/z200）and maintains mass accuracy（<5ppm）even without the use of continuous internal mass correction。Therefore，it can detect and identify the various chemical compounds based on their accurate mass values calculated from the corresponding compositional formula even if those chemical standards are not available。

Table1。完全负荷B_1个（FB_1个）and fumonisin B₂（NDfrc-FB_1个）and relative fragmentions（calculated and observed）at negative polarity。

**Table1。**完全负荷B_1个（FB_1个）and fumonisin B₂（NDfrc-FB_1个）and relative fragmentions（calculated and observed）at negative polarity。
Ion	FB_1个（RT:15.09min）				NDfrc-FB_1个（RT:14.87min）
Ion	公式，公式	Cal.Mass（m/z）^a	Obs.Mass（m/z）^b	错误（ppm）	公式，公式	Cal.Mass（m/z）^a	Obs.Mass（m/z）^b	错误（ppm）
[TCA-H₂O-H]^-什么（TCAK-H]^-什么）	C、C_6个H_6个O₅	157.0142	157.0137^c	-0.59（-3.76）	C、C_6个H_6个O₅	157.0142	157.0140^c	-0.28（-1.81）
[TCA-H₂O-H]^-什么（TCAK-H]^-什么）	C、C_6个H_6个O₅	157.0142	157.0135	-0.70（-4.44）	C、C_6个H_6个O₅	157.0142	157.0138	-0.41（-2.59）
[M-Glc-2 TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₂H₄₇否₅	404.3381	404.3378^c	-0.32（-0.78）
[M-Glc-2 TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₂H₄₇否₅	404.3381	404.3383	0.20（0.50）
[M-2TCAK-H]^-什么	C、C₂₂H₄₇否₅	404.3381	404.3379^c	-0.29（-0.71）	C、C₂₂H₅₁否₅	408.3695	-是的	-是的
[M-2TCAK-H]^-什么	C、C₂₂H₄₇否₅	404.3381	404.3380	-0.16（-0.41）	C、C₂₂H₅₁否₅	408.3695	-是的	-是的
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₈H₅₃否₁₀	562.3597	562.3607^c	0.99（1.67）
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₈H₅₃否₁₀	562.3597	562.3607	0.99（1.67）
[M-TCAK-H]^-什么	C、C₂₈H₅₃否₁₀	562.3597	562.3597^c	0.01（0.02）	C、C₃₄H₆₃否₁₅	724.4125	724.4136^c	1.14（1.58）
[M-TCAK-H]^-什么	C、C₂₈H₅₃否₁₀	562.3597	562.3600	0.32（0.56）	C、C₃₄H₆₃否₁₅	724.4125	724.4137	1.20（1.66）
[M-Glc-H]^-什么	-是的	-是的	-是的	-是的	C、C₃₄H₅₉否₁₅	720.3812	720.3824^c	1.19（1.65）
[M-Glc-H]^-什么	-是的	-是的	-是的	-是的	C、C₃₄H₅₉否₁₅	720.3812	720.3823	1.13（1.57）
[M-H]^-什么	C、C₃₄H₅₉否₁₅	720.3812	720.3805^c	-0.70（-0.97）	C、C₄₀H₆₉否₂₀	882.4340	882.4349^c	0.92（1.04）
[M-H]^-什么	C、C₃₄H₅₉否₁₅	720.3812	720.3813	0.09（0.13）	C、C₄₀H₆₉否₂₀	882.4340	882.4362	2.20（2.50）

^a主汽化基础设施；^bass values detected with the allions fragmentation with collision energy（scan event2）；^cass values detected by full scan（scan event1）。

2.7。Fumonisins and FBs-Glucose Conjugate by OPA Reagent

For confirmation of the D-glucose binding position in the fumonisin molecule structures，treatment with the OPA reagent was performed.Since OPA reacts specifically with primary amines，this reagent is often used for the derivatization of fumonisin molecules，when they are analyzed by the conventional method with HPLC23]。The OPA reagent was composed of 8mg of OPA，successively dissolved in 0.2mL of methanol，0.01mL of 2-mercaptoethanol，and1mL of 100mM sodium tetraborate aqueous solution.The reagent was freshly prepared each week and stored in brown glass containers at4°C for protection freshly prepared each week and stored in brown glass containers at。The corn powder extract（0.05 mL）was reacted with OPA reagent（0.05 mL）by mixing，and immediately subjected to LC-MS analysis。

Table2。FB的Exact mass values of₂and NDfrc-FB₂and relative fragmentions（calculated and observed）at negative polarity。

**Table2。**FB的Exact mass values of₂and NDfrc-FB₂and relative fragmentions（calculated and observed）at negative polarity。
Ion	FB₂（RT:16.78min）				NDfrc-FB₂（RT:16.40min）
Ion	公式，公式	Cal.Mass（m/z）^a	Obs.Mass（m/z）^b	错误（ppm）	公式，公式	Cal.Mass（m/z）^a	Obs.Mass（m/z）^b	错误（ppm）
[TCAK-H]^-什么	C、C_6个H_6个O₅	157.0142	157.0140^c	-0.28（-1.81）	C、C_6个H_6个O₅	157.0142	157.0141^c	-0.16（-1.04）
[TCAK-H]^-什么	C、C_6个H_6个O₅	157.0142	157.0138	-0.44（-2.79）	C、C_6个H_6个O₅	157.0142	157.0140	-0.27（-1.72）
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₂H₄₇否_4个	388.3432	388.3430^c	-0.22（-0.55）
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₂H₄₇否_4个	388.3432	388.3439	0.70（1.80）
[M-2TCAK-H]^-什么	C、C₂₂H₄₇否_4个	388.3432	-是的	-是的	C、C₂₂H₅₁否_4个	392.3745	-是的	-是的
[M-2TCAK-H]^-什么	C、C₂₂H₄₇否_4个	388.3432	388.3434^c	0.18（0.47）	C、C₂₂H₅₁否_4个	392.3745	-是的	-是的
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₈H₅₃否₉	546.3648	546.3652^c	0.48（0.88）
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₈H₅₃否₉	546.3648	546.3655	0.78（1.44）
[M-TCAK-H]^-什么	C、C₂₈H₅₃否₉	546.3648	546.3650^c	0.23（0.43）	C、C₃₄H₆₃否₁₄	708.4176	708.4188^c	1.24（1.76）
[M-TCAK-H]^-什么	C、C₂₈H₅₃否₉	546.3648	546.3657	0.97（1.77）	C、C₃₄H₆₃否₁₄	708.4176	708.4183	0.70（0.98）
[M-Glc-H]^-什么	-是的	-是的	-是的	-是的	C、C₃₄H₅₉否₁₄	704.3863	704.3875^c	1.17（1.66）
[M-Glc-H]^-什么	-是的	-是的	-是的	-是的	C、C₃₄H₅₉否₁₄	704.3863	704.3879	1.60（2.27）
[M-H]^-什么	C、C₃₄H₅₉否₁₄	704.3863	704.3865^c	0.20（0.28）	C、C₄₀H₆₉否₁₉	866.4391	866.4410^c	1.88（2.17）
[M-H]^-什么	C、C₃₄H₅₉否₁₄	704.3863	704.3878	1.54（2.18）	C、C₄₀H₆₉否₁₉	866.4391	866.4411	2.00（2.31）

^a主汽化基础设施；^bass values detected with the allions fragmentation with collision energy（scan event2）；^cass values detected by full scan（scan event1）。

Table3。FB的Exact mass values of₃and NDfrc-FB₃and relative fragmentions（calculated and observed）at negative polarity。

**Table3。**FB的Exact mass values of₃and NDfrc-FB₃and relative fragmentions（calculated and observed）at negative polarity。
Ion	FB₃（RT:16.09min）				NDfrc-FB₃（RT:15.68min）
Ion	公式，公式	Cal.Mass（m/z）^a	Obs.Mass（m/z）^b	错误（ppm）	公式，公式	Cal.Mass（m/z）^a	Obs.Mass（m/z）^b	错误（ppm）
[TCAK-H]^-什么	C、C_6个H_6个O₅	157.0142	157.0139^c	-0.33（-2.11）	C、C_6个H_6个O₅	157.0142	157.0142^c	-0.03（-0.16）
[TCAK-H]^-什么	C、C_6个H_6个O₅	157.0142	157.0139	-0.38（-2.40）	C、C_6个H_6个O₅	157.0142	157.0143	0.02（0.13）
[M-Glc-2 TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₂H₄₇否_4个	388.3432	388.3437^c	0.49（1.25）
[M-Glc-2 TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₂H₄₇否_4个	388.3432	388.3444	1.13（2.91）
[M-2TCAK-H]^-什么	C、C₂₂H₄₇否_4个	388.3432	-是的	-是的	C、C₂₂H₅₁否_4个	392.3745	-是的	-是的
[M-2TCAK-H]^-什么	C、C₂₂H₄₇否_4个	388.3432	388.3430^c	-0.21（-0.55）	C、C₂₂H₅₁否_4个	392.3745	-是的	-是的
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₈H₅₃否₉	546.3648	546.3655^c	0.78（1.44）
[M-Glc-TCAK-H]^-什么	-是的	-是的	-是的	-是的	C、C₂₈H₅₃否₉	546.3648	546.3652	0.42（0.77）
[M-TCAK-H]^-什么	C、C₂₈H₅₃否₉	546.3648	546.3657^c	0.97（1.77）	C、C₃₄H₆₃否₁₄	708.4176	708.4200^c	2.40（3.39）
[M-TCAK-H]^-什么	C、C₂₈H₅₃否₉	546.3648	546.3658	1.03（1.88）	C、C₃₄H₆₃否₁₄	708.4176	708.4192	1.61（2.27）
[M-Glc-H]^-什么	-是的	-是的	-是的	-是的	C、C₃₄H₅₉否₁₄	704.3863	704.3880^c	1.72（2.44）
[M-Glc-H]^-什么	-是的	-是的	-是的	-是的	C、C₃₄H₅₉否₁₄	704.3863	704.3887	2.39（3.40）
[M-H]^-什么	C、C₃₄H₅₉否₁₄	704.3863	704.3871^c	0.81（1.14）	C、C₄₀H₆₉否₁₉	866.4391	866.4417^c	2.55（2.94）
[M-H]^-什么	C、C₃₄H₅₉否₁₄	704.3863	704.3879	1.66（2.36）	C、C₄₀H₆₉否₁₉	866.4391	866.4420	2.91（3.36）

^a主汽化基础设施；^bass values detected with the allions fragmentation with collision energy（scan event2）；^cass values detected by full scan（scan event1）。

3.Results

3.1。FB检测_1个and NDfrc-FB_1个

联邦调查局对Authors first confirmed the existence of FB_1个and NDfrc-FB_1个in the corn powder extract，based on the full scan results using the calculated masses.In the full scan data（scan event1），peaks corresponding to the monitor ions[FB_1个-H]^-什么（720.3812）and[NDfrc-FB_1个-H]^-什么（882.4340）were detected at15.09min and 14.87min，respectively.The same peaks were observed when standard FB_1个and authentic NDfrc-FB_1个were injected into the LC-MS system.Regarding detection of NDfrc-FB_1个，abundant[NDfrc-FB_1个-H]^-什么（882.4349）ion was detected with a deviation of 0.92mmu（1.04ppm）（火焰2B）.In addition，the fragmentions[NDfrc-FB_1个-TCAK-H]^-什么（724.4136），[NDfrc-FB_1个-Glc-H]^-什么（720.3824），[NDfrc-FB_1个-Glc-TCAK-H]^-什么（562.3607），and[NDfrc-FB_1个-Glc-2 TCAK-H]^-什么（404.3378）were observed，with deviations of 1.14mmu（1.58ppm），1.19mmu（1.65ppm），0.99mmu（1.67ppm）and-0.32mmu（-0.78ppm），respectively（火焰2B，C）.During the scan event2，the latter two fragmentions，as well as[TCAK-H]^-什么（157.0138）provided dual peaks（at15.11min and 14.88min）（火焰2A，Table1），FB预应力混凝土基础隔墙布置_1个and NDfrc-FB_1个.Although[NDfrc-FB_1个-2TCAK-H]^-什么（408.3695）水suggested as a fragment of NDfrc-FB_1个（Table1），NDfrc-FB的无噪声检测_1个（chemically synthesized or contained in corn extract）。从以下位置开始Table1。

3.2。NDfrc-FB检测与识别₂and NDfrc-FB₃

Figure3NDfrc-FB为screening for shows the results of screening₂and NDfrc-FB₃in the corn powder extract.Using the same procedure as adopted for NDfrc-FB_1个，the existence of FB₂and FB₃was first confirmed based on the full scan results（scan event1）using the calculated mass of[FB₂-H]^-什么（704.3863）。A major peak corresponding to[FB₂-H]^-什么was detected at 16.78 min，as shown at the top ofFigure3A，and the same peak was found for the FB₂standard.As fragment ions of FB₂，[FB]₂-TCAK-H]^-什么，[FB]₂-2TCAK-H]^-什么，and[TCAK-H]^-什么were observed（Table2）。“NDfrc-FB₂-H]^-什么（866.4391），a major peak was detected for[NDfrc-FB₂-H]^-什么at16.40min（Figure3A），and abundant[NDfrc-FB₂–H]^-什么（866.4410）was detected with a mass deviation of 1.88 mmu（2.17 ppm）（Figure3B）.In addition，the fragmentions[NDfrc-FB₂–TCAK-H]^-什么（708.4188），[NDfrc-FB₂-Glc-H]^-什么（704.3875），[NDfrc-FB₂-Glc-TCAK-H]^-什么（546.3652），[NDfrc-FB₂-Glc-2 TCAK-H]^-什么（388.3430）were observed with deviations of 1.24mmu（1.76ppm）、1.17mmu（1.66ppm）、0.48mmu（0.88ppm）、and-0.22mmu（-0.55ppm）、respectively（Figure3B，Table2）。Due to the low intensities of the signals，several fragments（Figure3C）were observed only in the magnified spectra.Two peaks at 16.79min and 16.39min were detected for the monitor ions[NDfrc-FB₂-Glc-TCAK-H]^-什么（546.3648）and[NDfrc-FB₂-Glc-2 TCAK-H]^-什么（388.3432）with scan event2（Figure3A），suggesting that similar fragmentation was occurring for FB₂and NDfrc-FB₂.During scan event2，fragments corresponding to[NDfrc-FB₂-TCAK-H]^-什么and[NDfrc-FB₂-Glc-2 TCAK-H]^-什么were observed（Table2）。Therespective mass values of these fragments were708.4183mmu and 388.3439mmu，with mass deviations from the calculated values of 0.70mmu（0.98ppm）and 0.70mmu（1.80ppm），respectively.In the case of screening for NDfrc-FB₃，a major peak for[NDfrc-FB₃-H]^-什么was observed at 15.68 min（scan event1）（Figure3A），and a fragmentation pattern similar to that of NDfrc-FB₂水上管道连接Table3）。FB of the fragmentation patterns in the was no difference in the fragmentation patterns of FB₂and FB₃，as[FB₃-TCAK-H]^-什么，[FB]₃-2TCAK-H]^-什么，and[TCAK-H]^-什么were observed as the corresponding fragmentions.Based on the data described above，authors were convinced that both NDfrc-FB₂and NDfrc-FB₃were contained in the corn powder extract。

Figure2。NDfrc-FB检测和识别_1个.Mass chromatogram with scan results（scan events1and2）（A）；full mass spectrum obtained at 14.87 min（scan event1）（B）；全半径测量m/z：400–410）obtained at 14.87 min（scan event1）（C、C）。

Figure3。NDfrc-FB检测和识别₂.Mass chromatogram with scan results（scan events1and2）（A）；full mass spectrum obtained at 16.40 min（scan event1）（B）；全半径测量m/z：385-395）obtained at 16.40 min（scan event1）（C、C）。

3.3。NDfrc-FB结构₂and NDfrc-FB₃

火焰4shows the LC-MS chromatograms of the NDfrc-FB₂（NDfrc-FB₃）and FB₂（FB₃）detected in the corn powder extract before and after treatment with OPA reagent（scan event1）。NDfrc-FB₂and NDfrc-FB₃NDfrc-FB_1个（glucose bound to the primary amine of the fumonisin molecule），it was considered that OPA would not reat with these species.As shown in火焰4单击功能区上，FB of the signal intensities of₂and FB₃were decreased by the OPA treatment，whereas those of NDfrc-FB₂and NDfrc-FB₃were not.The peak area ratios of NDfrc-FB₂（NDfrc-FB₃），before to after the OPA reaction，were1.44-1.46.On the other hand，these ratios of FB₂（FB₃），before to after the OPA reaction were281.4-1372.1.These results indicate that the primary amine residue was occupied by glucose conjugation in the molecules of NDfrc-FB₂（NDfrc-FB₃）。The slight shift observed for the fumonisin peaks was attributed to the increase in methanol concentration in the samples following the OPA treatment。

Figure4。NDfrc-FB₂（NDfrc-FB₃）and FB₂（FB₃）in corn powder extract before and after the treatment with OPA reagent（scan event1）。

NDfrc-FB的内部控制时间和fragmentation profiles of NDfrc-FB₂and NDfrc-FB₃during the LC-Orbitrap MS analysis，these compounds were chemically synthesized with the standards of FB₂and FB₃with reference to the Polinget al。单击功能区上18report。火焰5NDfrc-FB shows the chromatograms of₂and NDfrc-FB₃in the corn powder extract and for chemically synthesized species.The mass fragmentation profiles of the synthesized NDfrc-FB₂（NDfrc-FB₃）保密协议with those of NDfrc-FB₂（NDfrc-FB₃）detected in the corn sample extract（Figure S1）。These results indicate that NDfrc-FB2（and NDfrc-FB₃）appear to be formed though anon-enzymatic reaction between FB₂（and FB₃）and D-glucose。Inaddition，these synthesized NDfrc-FB₂and NDfrc-FB₃did not reat with the OPA reagent（details not shown）。Because the synthesized NDfrc-FB₂（and NDfrc-FB₃）were not sufficiently pure，和连接回电联轴节₂（and FB₃），they could not be used for performing the quantitative analysis。

Figure5。NDfrc-FB₂and NDfrc-FB₃in corn powder extract in comparison with the chemically synthesized species（scan event1）。

4.Discussion

In2002，Polinget al。单击功能区上18reported that NDfrc-FB_1个was formed through anon-enzymatic reaction between FB_1个and glucose.Hence，authors presumed that FB₂and FB₃炉渣回填withd-glucose non-enzymatically to form glucose conjugates such as NDfrc-FB_1个.In the current study，the existence of FB₂-glucose and FB₃-glucose conjugates（suggested to be NDfrc-FB）₂and NDfrc-FB₃）in corn powder extract was confirmed by LC-Orbitrap MS.In order to confirm that the glucose conjugation occurred at the amine residue of the FB_1个molecule，Luet al。单击功能区上第二十四节：]treated the FB_1个-glucose conjugate with OPA reagent。In the same manner，NDfrc-FB₂（and NDfrc-FB₃）was treated with OPA in the current study。When analyzed by LC-Orbitrap MS，the peaks suggested to be NDfrc-FB₂and NDfrc-FB₃were scarcely reduced after OPA treatment，whereas those of FB₂and FB₃were reduced substantially.Based on these observations，accompanied with the high-resolution MS spectrum data described above，authors became convinced that the conjugates detected in this study should correctly correspond to NDfrc-FB₂and NDfrc-FB₃.Additionally，NDfrc-FB₂and NDfrc-FB₃（MTC-9999A and MTC-9999E）Figure S2）。

内部设备，several trichothecene glucosides（O-glucoside conjugates）were detected in a corn reference material sample of the same line from Trilogy Co.Ltd（although the batch number was different）[11单击功能区上，20单击功能区上，22]。Therefore，authors initially screened for the presence ofO联邦调查局_1个–FB₃.After treating the corn powder extract with the OPA reagent，the full MS scan data was scrutinized with the calculated mass values of C₅₀H₇₇NSO₂₁（FB_1个-是的O-glucoside-OPA）and C₅₀H₇₇NSO₂₀（FB₂（FB₃）-O-glucoside-OPA），respectively.However，no peaks were detected.Based on these results，it appears that fumonisins are not enzymatically glucosylated，but chemically bound to glucose in-plant，which differs from the case of trichothecenes。

Among the fumonisomers，there are several different groups，such as fumonisin A（FA）[25]and fumonisin C（FC）[26in addition to fumonisin B（FB_1个–FB₃）。It is suggested that FA hardly reacts withd-glucose，because its amine residue is acetylated.In contrast，FC appears to reat withd-glucose non-enzymatically via the free primary amine harbored in its structure。NDfrc-FB_1个was found from the cooked maize with heat[27]，whereas NDfrc-FB_1个，NDfrc-FB₂，and NDfrc-FB₃were found in the corn powder（not cooked sample）used in this study。FB₂and FB₃）and glucose，as reported for FB_1个单击功能区上18]。Therefore，it seems likely that high temperature（around the cooking conditions）is not indispensable for the formation of NDfrc-FBs。Once the corn grains are harvested，they are normally dried，stored at the keeping place，and ground if necessary.In the drying process，if the corn grains containing fumonisins were subjected to some heat（for promoting to eliminate the moisture），NDfrc-FB might be formed.From the standpoint of toxicity，NDfrc-FB_1个has been reported as less toxic，compared to FB_1个单击功能区上28]。NDfrc-FB₂and NDfrc-FB₃降落伞to be lower than FB₂and FB₃with reference to NDfrc-FB_1个.It was also reported that NDfrc-FB_1个was partly converted back to FB_1个in the gastrointestinal tract of rats[29]。On the other hand，Cirliniet al。单击功能区上30reported that NDfrc-FB_1个was not reduced to FB_1个 in vitrodigestion model[30]。可供使用的factor reducing NDfrc-FB_1个to FB_1个单击功能区上，the involvement of rat microbiota is inferred.Although crucial microbes reducing NDfrc-FB_1个are unknown，the microbiota in the gastrointestinal tract is greatly different amongst host species，area，and age[31单击功能区上，32]。Another important question concerns the amount of these fumonisin conjugates that are present.However，in the current study，it was not possible to estimate the amounts of these compounds due to a lack in the pure（purified）chemical standards。

5.Conclusions

联调局，联调局，联调局₂and FB₃（NDfrc-FB₂and NDfrc-FB₃）were detected for the first time in the corn sample in this study。FB₂and FB₃）and glucose。Although these reactions are similar to Maillard reaction，NDfrc-FBS are not reduced to FBs in contrast to the conjugate reaction of amino acid and D-glucose[33]even if they are treated with alkali。Considering that NDfrc-FB seems to be less toxic than FB，some food processing procedures（for example，promotion of Maillard reaction）can be suggested to mitigate the fumonisin toxicity，as examined previously[19]。The existence of acyl-fumonisin B_1个单击功能区上第十七节：]和fumonisins bound to starch（hidden fumonisins）[34has also been reported by other researchers。Although the use of the hydrolyzed FBs（HFBs）obtained with either alkaline or enzymatic treatment has been proposed for quantitation of the total fumonisins in starch[第十七节：]，NDfrc-FBS should not be determined with these methods.Since the PMTDI value was designated as2μg·kg^-1bw·day^-1in the JECFA[6个]，the existence of those fumonisin conjugates may be taken into account for the establishment of this value.In order to estimate the potential risk of fumonisins，further studies on the prevalence of these and other conjugates in foods，as well as their relevance for human health is needededed。

辅助材料

Supplementary materials can be accessed at：https://www.mdpi.com/2072-6651/7/9/3700/s1。

Acknowledgments

Apart of this work was supported by a grant from the Ministry of Agriculture，Forestry and Fisheries of Japan（research project for improving food safety and animal health）。

Author Contributions

Yosuke Matsuo，Hiroyuki Nakagawa，and Masayo Kushiro conceived and designed the experiments.Yosuke Matsuo and Yuki Sago prepared all kinds of samples from the crop extract，and Masayo Kushiro administrated the OPA treatment experiments for the structure determination of fumonis conjugates.Yosuke Higa， and Kentaro Takahara conducted the LC-Orbitrap MS experiments and analyzed the data.Yosuke Matsuo and Hiroyuki Nakagawa mainly constructed the manuscript under the support of all co-authors.Masayo Kushiro，Hitoshi Nagashima and Hiroyuki Nakagawa supervised the work and revised the mancript。

Conflicts of Interest

The authors declare no conflicts of interest。

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MDPI and ACS Style

Matsuo，Y.；Takahara，K.；Sago，Y.；Kushiro，M.；Nagashima，H.；Nakagawa，H。检测，检测N-（1-deoxy-d-fructos-1-yl）Fumonisins B₂and B₃in Corn by High-Solution LC-Orbitrap MS。Toxins 2015单击功能区上，73700-3714。https://doi.org/10.3390/toxins7093700

AMA Style

Matsuo Y，Takahara K，Sago Y，Kushiro M，Nagashima H，Nakagawa H。检测，检测N-（1-deoxy-d-fructos-1-yl）Fumonisins B₂and B₃in Corn by High-Solution LC-Orbitrap MS。Toxins.2015；7（9）：3700-3714。https://doi.org/10.3390/toxins7093700

Chicago/Turabian Style

Matsuo，Yosuke，Kentaro Takahara，Yuki Sago，Masayo Kushiro，Hitoshi Nagashima，and Hiroyuki Nakagawa。2015.“Detection ofN-（1-deoxy-d-fructos-1-yl）Fumonisins B₂and B₃in Corn by High-Solution LC-Orbitrap MS“Toxins7，no.9:370-3714。https://doi.org/10.3390/toxins7093700

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检测，检测N-（1-deoxy-d-fructos-1-yl）Fumonisins B₂and B₃in Corn by High-Solution LC-Orbitrap MS

Abstract

1.Introduction