Method for determining the content of vomitoxin and / or its acetylation products in wheat

Abstract

The present invention provides a method for determining the content of deoxynivalenol and / or its acetylated product in wheat, wherein the acetylated product of deoxynivalenol includes one or two selected from 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol, and the method includes using high performance liquid chromatography-tandem mass spectrometry to detect a wheat sample solution to be tested, and the chromatographic column used in the method is YMC CHIRAa ART Cellulose-sc 3.0*250mm s-3um. The method of the present invention is comprehensive, highly sensitive, simple and rapid, and can simultaneously qualitatively and quantitatively analyze vomitoxin and its acetylated product in wheat, and realizes complete separation of 3-ADON and 15-ADON on the chromatography.

Description

Technical Field

[0001] The invention belongs to the technical field of mycotoxin determination, and specifically relates to a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for determining the content of vomitoxin and its acetylated product in wheat. Background Art

[0002] Deoxynivalenol, also known as deoxynivalenol (DON), is the fungal toxin with the highest contamination rate and pollution level in the world. It is widely found in grains such as wheat, rice, corn and their products. The health effects of DON are divided into acute, short-term and long-term toxicity. DON can be converted into two acetylated products in animals and plants, namely 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON). Studies have shown that compared with the toxicity of DON, 3-ADON and 15-ADON have similar or even higher toxicity. These two toxins can be deacetylated to form DON in animals, potentially increasing the intake of DON. Due to the strong toxicity of the two acetylated products, in 2010, the Joint Expert Committee on Food Additives (JECFA) stipulated that the Provisional Maximum Tolerable Daily Intake (PMTDI) of DON+ADONs is 1μg / kg·bw / day. Studies have shown that hidden toxins ingested by animals can be converted into toxin monomers under the action of hydrolases in the body and exert toxic effects. Therefore, it is of great significance to establish a rapid, simple and efficient detection method for DON toxins in grains, which can provide a reference for the later formulation of limit standards and risk assessment of vomitoxin and its acetylated products in raw grains.

[0003] Methods for determining type B trichothecenes in food using liquid chromatography-tandem mass spectrometry (LC-MS / MS) have been reported. Most methods use solid-liquid extraction, followed by sample analysis after purification using a multifunctional purification column or immunoaffinity column. These methods are not only costly and cumbersome to operate, but also 3-ADON and 15-ADON are isomers that cannot be accurately separated on a chromatographic column using conventional HPLC methods. Therefore, the application of these methods is greatly limited.

[0004] The present invention requires a simple, rapid, quantitative and accurate method for determining vomitoxin and its acetylated products in wheat. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for determining vomitoxin and its acetylated products in wheat with a wide range of applications, high sensitivity, simplicity and rapidity. The present invention preferably selects the most effective separation column for high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) analysis, and the sample pretreatment method is rapid, simple and inexpensive. The method of the present invention has the advantages of being simple, rapid, quantitative and accurate, and is suitable for the determination of toxin content in large quantities of samples, and can accurately separate 3-ADON and 15-ADON.

[0006] Specifically, the present invention provides a method for determining the content of deoxynivalenol and / or its acetylated product in wheat, wherein the acetylated product of deoxynivalenol includes one or two selected from 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol, and the method includes using high performance liquid chromatography-tandem mass spectrometry to detect a wheat sample solution to be tested, and the chromatographic column used in the method is a chiral chromatographic column with silica gel coated with cellulose-tris(3,5-dichlorophenylcarbamate) as a filler.

[0007] In one or more embodiments, the chromatography column is YMC CHIRAa ART Cellulose-sc3.0*250mm s-3um.

[0008] In one or more embodiments, the mobile phase A of the chromatography is 5±0.5 mM ammonium acetate in water, and the mobile phase B is methanol.

[0009] In one or more embodiments, the elution program for chromatography is isocratic elution, 0 min 25-30% A, >13 min 25-30% A.

[0010] In one or more embodiments, the liquid chromatograph model is Thermo Fisher DIONEX UltiMate3000.

[0011] In one or more embodiments, the column temperature is 25 ± 5 °C.

[0012] In one or more embodiments, the injection volume is 10±5 μL.

[0013] In one or more embodiments, the flow rate is 0.3 ± 0.05 mL min -1 .

[0014] In one or more embodiments, the mass spectrometry conditions of the method include:

[0015] (1) For deoxynivalenol, the adduct ion is [DON+CH3COO] -, the precursor ion is 355.1m / z, the product quantitative ion is 295.0m / z, the product qualifier ion is 247.0m / z, the retention time is 6.38min, the collision energy is 17eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 77.5; for 15-acetyldeoxynivalenol, the adduct ion is [15-ADON+NH4] + , the precursor ion is 356.1m / z, the product quantitative ion is 320.9m / z, the product quantitative ion is 136.9m / z, the retention time is 8.81min, the collision energy is 12eV, and the peak area ratio of the product qualitative ion to the product quantitative ion is 76.4; for 3-acetyldeoxynivalenol, the adduct ion is [3-ADON+CH3COO] - , the precursor ion is 397.0m / z, the product quantitative ion is 307.1m / z, the product qualifier ion is 173.1m / z, the retention time is 10.12min, the collision energy is 16eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 40.6; or

[0016] (2) For deoxynivalenol, the adduct ion is [DON+CH3COO] - , the precursor ion is 355.1m / z, the product quantitative ion is 295.0m / z, the product qualifier ion is 247.0m / z, the retention time is 6.38min, the collision energy is 17eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 77.5; for 15-acetyldeoxynivalenol, the adduct ion is [15-ADON+H] + , the precursor ion is 339.1m / z, the product quantitative ion is 137.1m / z, the product qualifier ion is 261.1m / z, the retention time is 8.80min, the collision energy is 18eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 47.7; for 3-acetyldeoxynivalenol, the adduct ion is [3-ADON+H] + , the precursor ion was 339.1 m / z, the product quantitative ion was 137.1 m / z, the product qualifier ion was 261.1 m / z, the retention time was 10.06 min, the collision energy was 18 eV, and the peak area ratio of the product qualifier ion to the product quantitative ion was 47.7.

[0017] In one or more embodiments, the mass spectrometry conditions of the method include one or more of the following features:

[0018] The mass spectrometer model is Thermo Fisher TSQ VANTAGE;

[0019] The ionization mode was electrospray ionization in positive and negative ion switching mode;

[0020] The spray voltage was ±3.0 ± 0.5 kV;

[0021] The capillary temperature is 325±25℃;

[0022] Atomization temperature is 225±25℃;

[0023] Sheath gas pressure was 28 ± 2 psi;

[0024] The auxiliary gas pressure is 18±2psi.

[0025] In one or more embodiments, the wheat sample solution to be tested is prepared by the following method:

[0026] For each gram of wheat flour, add 5-10 mL of a mixed solvent with a volume ratio of 4:1 to 3:1 of acetonitrile: water to the wheat flour, disperse evenly, centrifuge, take the supernatant, add 100-200 mg of anhydrous MgSO4, then add 1±0.5 mL of n-hexane, centrifuge, remove the n-hexane layer, take the remaining supernatant, blow dry with liquid nitrogen, and then dissolve it with 200-1000 μL of a mixed solvent with a volume ratio of 1:4 to 1:1 of acetonitrile: water, pass through a filter membrane with a diameter of 0.22±0.02 μm, and if it is turbid, pass it through the membrane again to obtain the wheat sample solution to be tested.

[0027] In one or more embodiments, the method comprises: determining the content of deoxynivalenol and / or its acetylated product in the wheat to be tested by a standard curve method according to the peak area of ​​deoxynivalenol and / or its acetylated product detected in the wheat sample solution to be tested.

[0028] In one or more embodiments, the standard curve method uses a standard curve that is established with the peak area of ​​each toxin as the ordinate and the corresponding concentration of each toxin as the abscissa.

[0029] In one or more embodiments, the standard curve is a curve of the relationship between the peak area of ​​each toxin detected from a standard solution containing different concentrations of toxins and the content of each toxin in each standard solution using the high performance liquid chromatography-tandem mass spectrometry method described in any embodiment of the present invention, and the standard solution is prepared by diluting an acetonitrile solution of deoxynivalenol and / or its acetylated product with a concentration of 0.5 to 1 mg / L using a mixed solvent with a volume ratio of acetonitrile: water of 1:4 to 1:1, or the standard solution is prepared by diluting an acetonitrile solution of deoxynivalenol and / or its acetylated product with a concentration of 0.5 to 1 mg / L using a mixed solvent with a volume ratio of acetonitrile: water of 1:4 to 1:1, and then diluting with a blank wheat matrix extract, wherein the blank wheat matrix extract is prepared from sterilized wheat flour by the method for preparing a wheat sample solution to be tested described in any embodiment of the present invention.

[0030] In one or more embodiments, the wheat to be tested in the method is wheat infected by Fusarium graminearum, and the Fusarium graminearum is preferably a strain that does not produce 15-ADON, such as F. asiaticum.

[0031] In one or more embodiments, the method comprises determining the 15-acetyldeoxynivalenol content in the wheat, and optionally or preferably further comprises determining the deoxynivalenol and / or 3-acetyldeoxynivalenol content in the wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 These are chromatograms of the separation of DON, 3-ADON and 15-ADON using different chromatographic columns, including: (A) Watersuplc BEH amidel 2.1*100mm 1.7μm, (B) YMC-TRIART diol-Hilic100*3.0 1.9μm, (C) YMCCHIRAa ART Cellulose-sc 2.0*100mm s-3μm, and (D) YMC CHIRAa ART Cellulose-sc 2.0*100mm s-3μm.

[0033] Figure 2 The extracted ion chromatograms of the working solutions of DON, 15-ADON and 3-ADON standards are shown in Figure 1, where (A) is method A, (B) is method B, and (C) is method C.

[0034] Figure 3 These are the extracted ion chromatograms of DON, 15-ADON and 3-ADON in wheat samples infected with PH-1, where (A) is method A, (B) is method B, and (C) is method C.

[0035] Figure 4 These are the extracted ion chromatograms of DON, 15-ADON and 3-ADON in wheat samples infected with F-1, where (A) is method A, (B) is method B, and (C) is method C.

[0036] Figure 5 The contents of DON, 3-ADON and 15-ADON in wheat samples under the conditions of three detection methods (μg / kg), including (A) wheat samples infected with PH-1, and (B) wheat samples infected with F-1. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used in this document are the common meanings understood by those skilled in the art for the present invention. When there is a conflict, the definition in this specification shall prevail. The theories or mechanisms described and disclosed herein, whether right or wrong, should not limit the scope of the present invention in any way, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0038] Herein, all features such as quantity, content and concentration defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions). Herein, unless otherwise specified, ratio refers to mass ratio, percentage refers to mass percentage, and part refers to mass part.

[0039] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0040] The invention provides a method for determining the content of deoxynivalenol (DON) and / or its acetylated product in wheat, wherein the acetylated product of deoxynivalenol comprises one or both of 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON), and the method comprises using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to detect a wheat sample solution to be detected.

[0041] In some embodiments, the method of the present invention comprises using HPLC-MS / MS to detect the wheat sample solution to obtain an extracted ion current chromatogram corresponding to each toxin (DON, 3-DON and / or 15-DON).

[0042] In some embodiments, the method of the present invention comprises measuring the content of 15-ADON in wheat. In some embodiments, the method of the present invention comprises measuring the content of 15-ADON and 3-ADON in wheat. In some embodiments, the method of the present invention comprises measuring the content of DON, 15-ADON and 3-ADON in wheat.

[0043] In the present invention, the chromatographic column used is a chiral chromatographic column, and its filler is silica gel (such as high-strength macroporous silica gel) coated with a polysaccharide derivative cellulose-tris (3,5-dichlorophenylcarbamate) on the surface, which has good separation ability and life. Examples of chromatographic columns suitable for the present invention include YMC CHIRAa ART Cellulose-sc (3.0*250mm s-3um). The present invention finds that the use of a chiral chromatographic column with a polysaccharide derivative cellulose-tris (3,5-dichlorophenylcarbamate) bonded to a silica gel matrix, such as a YMC CHIRAa ARTCellulose-sc (3.0*250mm s-3um) column, can achieve a thorough separation of 3-ADON and 15-ADON, as well as accurate qualitative and quantitative analysis of DON, 3-ADON and 15-ADON.

[0044] The chromatographic conditions used in the present invention may be: mobile phase A is 5±0.5 mM ammonium acetate aqueous solution, mobile phase B is methanol; the elution procedure is isocratic elution, 0 min 25-30% A, ≥13 min (e.g. 16±3 min) 25-30% A; the liquid chromatograph model is Thermo Fisher DIONEX UltiMate 3000; the column temperature is 25±5°C; the injection volume is 10±5 μL; the flow rate is 0.3±0.05 mL min -1 .

[0045] The mass spectrometry conditions used in the present invention include using the precursor ion / product ion pairs and corresponding parameters as shown in Method B or Method C in Table 1.

[0046] Table 1: Mass spectrometric parameters of DON and its acetylated products

[0047]

[0048] Note: 1 Quantitative ion, 2 Qualifier ions, 3 The peak area ratio of the qualifier ion to the quantifier ion.

[0049] The mass spectrometry conditions used in the present invention may also include: the mass spectrometer model is Thermo Fisher TSQ VANTAGE; the ionization mode is electrospray ionization in positive and negative ion switching mode; the spray voltage is ±3.0±0.5kV; the capillary temperature is 325±25°C; the nebulization temperature is 225±25°C; the sheath gas pressure is 28±2psi; and the auxiliary gas pressure is 18±2psi.

[0050] In the present invention, the wheat sample solution to be tested and the blank wheat matrix extract can be prepared by the following method: for each gram of wheat flour, 5 to 10 mL of acetonitrile: water volume ratio of 4: 1 to 3: 1 mixed solvent is added to the wheat flour, after uniform dispersion, centrifugation, the supernatant (e.g., 2 ± 0.5 mL) is taken, 100 to 200 mg of anhydrous MgSO4 is added, and then 1 ± 0.5 mL of n-hexane is added, centrifugation, the n-hexane layer is removed, the remaining supernatant is taken, and after liquid nitrogen is blown dry, 200 to 1000 μL of acetonitrile: water volume ratio is 1: 4 to 1: 1 mixed solvent is used to redissolve, and the filter membrane with a diameter of 0.22 ± 0.02 μm is passed, and if it is turbid, the membrane is passed again to obtain the wheat sample solution to be tested. Wherein, for the wheat sample solution to be tested, wheat containing deoxynivalenol and / or its acetylated product is used, such as wheat infected by Fusarium graminearum. For the blank wheat matrix extract, wheat that does not contain deoxynivalenol and / or its acetylated products (toxin content is below the detection limit), such as sterilized wheat, is used.

[0051] In the present invention, the standard curve method can be used to determine the content of deoxynivalenol and / or its acetylated product in the wheat to be tested according to the peak area of ​​deoxynivalenol and / or its acetylated product detected from the wheat sample solution to be tested. The standard curve method can use a standard curve formulated with the peak area of ​​each toxin as the ordinate and the concentration corresponding to each toxin as the abscissa. Herein, the peak area refers to the peak area corresponding to each toxin in the extracted ion current chromatogram.

[0052] The standard curve applicable to the present invention can be a curve of the relationship between the peak area of ​​each toxin detected from the standard solution containing different concentrations of toxins and the content of each toxin in each standard solution using the high performance liquid chromatography-tandem mass spectrometry method described herein. In the present invention, the standard solution can be prepared by serially diluting an acetonitrile solution of deoxynivalenol and / or its acetylated product with a concentration of 0.5 to 1 mg / L using a mixed solvent with a volume ratio of acetonitrile: water of 1:4 to 1:1, or by diluting an acetonitrile solution of deoxynivalenol and / or its acetylated product with a concentration of 0.5 to 1 mg / L using a mixed solvent with a volume ratio of acetonitrile: water of 1:4 to 1:1, and then diluting it with a blank wheat matrix extract. In the present invention, the blank wheat matrix extract can be prepared from sterilized wheat flour by the method for preparing a wheat sample solution to be tested described herein.

[0053] The present invention also provides an HPLC-MS / MS method for determining the content of vomitoxin and its acetylated product in wheat, comprising: preparing a standard solution and a wheat sample solution to be tested; detecting the standard solution and the wheat sample solution to be tested by HPLC-MS / MS respectively, and obtaining extracted ion flow chromatograms respectively; processing the obtained extracted ion flow chromatograms, respectively obtaining the concentrations of DON, 3-ADON and 15-ADON in the wheat sample solution to be tested, and then calculating the contents of the three toxins in wheat. The method of the present invention is comprehensive, highly sensitive, simple and rapid, and can simultaneously qualitatively and quantitatively analyze the vomitoxin and its acetylated product in wheat, and realizes the complete separation of 3-ADON and 15-ADON on the chromatography.

[0054] In some embodiments, in the present invention, the method for preparing a standard solution includes: using a mixed solvent with a volume ratio of acetonitrile to water of 1:4 to 1:1 to serially dilute an acetonitrile solution of deoxynivalenol and / or its acetylated product with a concentration of 0.5 to 1 mg / L. For example, DON, 15-ADON and / or 3-ADON solid powders can be weighed and prepared into a standard stock solution with a concentration of 0.5 to 1 mg / mL using acetonitrile; the standard stock solution is diluted with a mixed solvent with a volume ratio of acetonitrile to water of 1:4 to 1:1 (e.g., 1:1) to prepare a standard intermediate solution; the standard intermediate solution is diluted with a mixed solvent with a volume ratio of acetonitrile to water of 1:4 to 1:1 (e.g., 1:4) to obtain a standard working solution. Both the standard intermediate solution and the standard working solution can be used as a standard solution. In the present invention, the standard solution, the standard stock solution, the standard intermediate solution and the standard working solution can be stored at -20±5°C. Each solution is preferably prepared as needed.

[0055] In some embodiments, the method of the present invention includes infecting wheat with Fusarium graminearum. Fusarium graminearum suitable for the present invention may be a PH-1 (Fusarium graminearum) strain and / or an F-1 strain (F.asiaticum). The method of infecting wheat with Fusarium graminearum may include: weighing 25±5g of wheat grains per bag in a ziplock bag, irradiating with an irradiation dose of 8±2kGy, placing the grains in a sterile conical flask in a clean bench after irradiation, adding 10±2mL of sterile water, inoculating each bottle with Fusarium graminearum blocks of the same size after activation (e.g., 3±2 blocks), placing the samples in a 25±5°C incubator for 21±5 days, and then placing the samples in a 55±10°C oven for drying. In some embodiments, the Fusarium graminearum used in the present invention is a strain that does not produce 15-ADON, such as an F-1 strain. The F-1 strain does not produce 15-ADON. When the method of the present invention is used to detect barley infected by the F-1 strain, 15-ADON will not be detected, while the conventional method will misdetect 15-ADON.

[0056] The wheat sample solution to be tested and the blank wheat matrix extract suitable for the present invention can be prepared by the following method: for each gram of wheat flour, 5 to 10 mL of a mixed solvent with a volume ratio of 4:1 to 3:1 of acetonitrile: water is added to the wheat flour, vortexed for 30±5 min, oscillated and ultrasonicated for 20±5 min, centrifuged, the supernatant (e.g., 2.0±0.5 ml) is placed in a centrifuge tube, 150±50 mg of anhydrous MgSO4 is added, and then 1±0.5 ml of n-hexane (for removing lipid substances in plant tissues) is added. After vigorous vortex centrifugation, the n-hexane layer (upper layer) is removed, and the remaining supernatant is transferred to a centrifuge tube, dried with liquid nitrogen (45±5° C.), and then re-dissolved with 200 to 1000 μl (e.g., 500 μl) of a mixed solvent with a volume ratio of 1:4 to 1:1 of acetonitrile: water, and the sample is filtered through a 0.22±0.02 μm filter membrane (e.g., a nylon filter membrane).

[0057] In some embodiments, the present invention also includes the step of optimizing mass spectrometry conditions. The optimization method of mass spectrometry conditions can be: first determine the product ion range of the three toxins, respectively put the respective toxin standard solutions (about 1 μg / mL) into the injection bottle, replace the chromatographic column with a double pass, and the mobile phase isocratic elution, the volume ratio of methanol: water is about 1:1, and the automatic sampler is about 1 μL injection; scan by precursor ion scanning under positive ion mode scanning, first find the precursor ion of each target compound, and further scan to determine the two product ions with the highest response value of each target compound; select the multiple reaction monitoring mode optimization voltage by the precursor ion and product ion pairs determined by each target object, as the setting voltage increases from low to high, select the voltage of the highest abundance daughter ion in the chromatogram as the most suitable voltage, and determine the quantitative ion and qualitative ion respectively according to the highest abundance of the product ion, and then determine the voltage on the basis of continuing to optimize the precursor ion and product ion, through two rounds of optimization, establish the best precursor ion / product ion pair and voltage. Precursor ion / product ion pairs and corresponding mass spectrometry parameters suitable for the present invention are shown in Table 1.

[0058] The present invention has the following advantages:

[0059] The present invention establishes a new HPLC-MS / MS method for determining vomitoxin and its acetylated products in wheat. After the wheat sample is extracted and purified with acetonitrile-water solution, it is diluted isocratically with methanol + 5±0.5mmol / L ammonium acetate solution as the mobile phase, separated by YMC CHIRAa ART Cellulose-sc (3.0*250mm s-3um), and analyzed by multi-stage reaction monitoring (MRM) and positive and negative ion mode scanning. The results show that the standard curve of vomitoxin and its acetylated products has good linearity in the range of 4 to 2000μg / L, and the correlation coefficients are all greater than 0.995. The average recovery rate of the sample at three spiked concentrations of 50, 500 and 1000μg / L is between 83.5% and 118.8%, and the relative standard deviation (RSD) is between 0.28% and 18.8%. This determination method has the advantages of being rapid, economical, highly sensitive, and highly practical. It can simultaneously realize the qualitative and quantitative detection of vomitoxin and its acetylated products, and can be used for the detection and analysis of vomitoxin and its acetylated products in wheat foods.

[0060] The present invention is further described below in conjunction with specific examples. The scope of the present invention is not limited by the contents of the following examples. The protection scope of the present invention is limited only by the claims, and any omission, substitution or modification made by those skilled in the art on the basis of the embodiments disclosed in the present invention will fall within the protection scope of the present invention.

[0061] The following examples use conventional instruments and equipment in the art. The experimental methods and detection methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercially available products. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.

[0062] The following instruments and reagents were used in the examples:

[0063] Instruments: HPLC-MS / MS: TSQ VANTAGE-U3000, equipped with electrospray ionization (ESI); LegendMicro 17 centrifuge; MD 200 nitrogen blowdown device; SDLA-B-1301 ultrapure water machine; KQ-500E ultrasonic cleaner; Tissuelyser-48 grinder; DMT-2500 vortex mixer; BSA822-CW analytical balance.

[0064] Reagents: Deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) were purchased from Sigma; methanol, acetonitrile, n-hexane and formic acid were all chromatographically pure and purchased from Amp; ultrapure water was used in the experiment.

[0065] Standard stock solution: Accurately weigh DON, 15-ADON and 3-ADON solid powders, respectively, and prepare standard stock solutions with a concentration of 1 mg / mL with chromatographic grade acetonitrile, and store at -20°C.

[0066] Standard intermediate solution: dilute the standard stock solution with a mixed solution of acetonitrile / water (50 / 50, v / v) to prepare a standard intermediate solution containing a certain concentration of DON, 15-ADON and / or 3-ADON, wherein the mixed standard intermediate solution contains 1 μg / mL of DON, 1 μg / mL of 3-ADON and 2 μg / mL of 15-ADON and is stored at -20°C.

[0067] Standard working solution: Use acetonitrile / water (20 / 80, v / v) mixed solution to gradiently dilute the standard intermediate solution and prepare it before use.

[0068] Example 1: Selection of chromatographic column

[0069] For the simultaneous detection of vomitoxin and its acetylated product, since the compounds have similar structural formulas and chemical properties, such as 3-ADON and 15-ADON are isomers with the same precursor ions and product ions, high requirements are placed on the separation degree, so the selection of chromatographic column is very critical.

[0070] The traditional C18 column cannot achieve a complete separation of 3-ADON and 15-ADON on the chromatography. This example uses different chromatographic columns to perform separation experiments on these two substances. The results are shown in Table 2. Figure 1 and Figure 2 The chromatographic conditions used in the experiment are as follows:

[0071] Liquid phase model: DIONEX UltiMate 3000, Thermo Fisher;

[0072] Chromatographic column: as shown in Table 2;

[0073] Mobile phase: A: 5 mM ammonium acetate aqueous solution, B: methanol;

[0074] Column temperature: 30°C;

[0075] Injection volume: 10 μL;

[0076] The elution program was: isocratic elution, 30% A at 0 min, 30% A at 16 min.

[0077] As can be seen from Table 2, the YMC CHIRAa ART Cellulose-sc (3.0*250mm, s-3um) column achieves complete separation of 3-ADON and 15-ADON on the chromatography, and under the same conditions, the YMC CHIRAa ART Cellulose-sc (3.0*250mm, s-3um) column has a larger particle size, a smaller relative pressure, a stronger noise resistance to complex matrices, and a longer service life. Therefore, the present invention selects the YMC CHIRAa ART Cellulose-sc (3.0*250mm, s-3um) column for separation.

[0078] Table 2: Separation effect of different chromatographic columns on 3-ADON and 15-ADON

[0079]

[0080]

[0081] Note: 1 , 3-ADON; 2 , 15-ADON.

[0082] Example 2: Establishing the Optimal Precursor Ion / Product Ion Pair

[0083] Determine the product ion range of the three toxins: put the standard intermediate solutions of DON, 15-ADON and 3-ADON (concentration of 1 μg / mL) into the injection bottle respectively, replace the chromatographic column with a double pass, elute with isocratic mobile phase, the volume ratio of methanol: water is 1:1, and 1 μL is injected by the automatic sampler; scan the precursor ions in the positive ion mode to find the precursor ions of each target compound first, and further scan to determine the two product ions with the highest response values ​​of each target compound; select the optimized voltage of the multiple reaction monitoring mode through the precursor ion and product ion pairs determined by the respective target compounds, and as the setting voltage increases from low to high, select the voltage of the highest abundance product ion in the chromatogram as the optimal voltage, and determine it as the quantitative ion and the qualitative ion according to the highest abundance of the product ion, respectively. Continue to optimize the precursor ions and product ions on the basis of the determined voltage. Through two rounds of optimization, the best precursor ion / product ion pair is established, and the mass spectrometry parameters shown in Table 1 are obtained.

[0084] Example 3: Comparison of three detection methods

[0085] Method A (known detection method), method B (detection method of the present invention) and method C (detection method of the present invention) were used to perform HPLC-MS / MS detection on vomitoxin and its acetylated product. The chromatographic conditions and mass spectrometry conditions of the three detection methods are as follows:

[0086] (1) Chromatographic conditions

[0087] Liquid phase model: DIONEX UltiMate 3000, Thermo Fisher;

[0088] Chromatographic columns: Method A: Agilent Extend-C18 (3.0 mm*150 mm, 3.5 μm); Method B and Method C: YMCCHIRAa ART Cellulose-sc (3.0*250 mm s-3 um);

[0089] Mobile phase: A: 5 mM ammonium acetate aqueous solution, B: methanol;

[0090] Column temperature: 30°C;

[0091] Injection volume: 10 μL;

[0092] The elution procedure is:

[0093] Method A: linear gradient elution, 0 min 20% A, 1 min 20% A, 5.5 min 90% A, 6.5 min 90% A, 7 min 20% A, 8.5 min 20% A, 12 min 20% A.

[0094] Method B and Method C: isocratic elution, 30% A at 0 min, 30% A at 16 min;

[0095] Flow rate: 0.35 mL min -1 ;

[0096] (2) Mass spectrometry conditions

[0097] Mass spectrometer model: TSQ VANTAGE, Thermo Fisher;

[0098] Ionization mode: electrospray ionization in positive and negative ion switching mode;

[0099] Spray voltage: ±3.5kV;

[0100] Capillary temperature: 350℃;

[0101] Atomization temperature: 250℃;

[0102] Sheath gas pressure: 30psi;

[0103] Auxiliary gas pressure: 20psi;

[0104] Mass spectrometry parameters: The mass spectrometry parameters of method A, method B and method C are shown in Table 1.

[0105] The extracted ion chromatograms of the three detection methods for DON, 15-ADON and 3-ADON standard working solutions are shown in Figure 1 The concentration of DON standard working solution is 8 ng / mL, the concentration of 3-ADON standard working solution is 8 ng / mL, and the concentration of 15-ADON standard working solution is 16 ng / mL, which are obtained by diluting a DON standard intermediate solution with a concentration of 1 μg / mL, a 3-ADON standard intermediate solution with a concentration of 1 μg / mL, and a 15-DON standard intermediate solution with a concentration of 2 μg / mL with an acetonitrile / water (20 / 80, v / v) mixed solution.

[0106] The literature Zhao, Z. et al., J Chromatogr B Analyt Technol Biomed Life 2014, 963, 75-82. shows that when method A is used, [M+H] + When it is the parent ion, the abundance ratio of 3-ADON and 15-ADON product ions is less than 10%, which greatly reduces the sensitivity of the method. Therefore, for method A, ammonium acetate needs to be added to increase the product ion ratio (>25%), thereby improving the sensitivity. In addition, during the optimization of method A, it is necessary to avoid using nonspecific product ions as qualitative and quantitative ions.

[0107] The method of the present invention can select [15-ADON+NH4] + and [3-ADON+CH3COO] - As the adduct ion (method B), [15-ADON+H] + and [3-ADON+H] + As an adduct ion (method C), it is simpler, faster and more accurate.

[0108] Example 4: Wheat sample pretreatment

[0109] The dried wheat was ground into powder with a grinder, 1 g was added into 10 mL of acetonitrile: water (80 / 20, V / V) extraction solvent, vortexed for 30 min, oscillated and ultrasonicated for 20 min, centrifuged, 2.0 ml of the supernatant was placed in a 50 mL centrifuge tube, 150 mg of anhydrous MgSO4 was added, and then 1 ml of n-hexane (for removing lipid substances in plant tissues) was added. After vigorous vortexing and centrifugation, the n-hexane layer (upper layer) was removed, and the remaining supernatant was transferred to a 10 mL centrifuge tube, dried with liquid nitrogen (45°C), and then re-dissolved with 500 μl of acetonitrile: water (20 / 80, V / V). The sample was filtered through a 0.22 μm nylon filter membrane before sampling and analysis.

[0110] Example 5: Linear Relationship, Limit of Detection and Limit of Quantitation

[0111] Wheat sterilized by irradiation and high pressure sterilization (toxin content < detection limit) was pretreated according to the method in Example 4 to obtain a blank wheat matrix extract.

[0112] The standard intermediate solutions of the three toxins (concentration of 2 μg / mL) were diluted with blank wheat matrix extracts, and the detection was carried out using method A, method B and method C in Example 3. In this paper, the peak area average value (Y) of each toxin response value was used as the ordinate, and the mass concentration (X, μg·kg -1 ) was used as the horizontal axis to develop a standard curve, and the detection limit (LOD) of the method was determined to be 3 times the signal-to-noise ratio (S / N), and the limit of quantification (LOQ) was determined to be 10 times the signal-to-noise ratio (S / N). The specific results of the correlation coefficient, linear range, LOD and LOQ of the three toxins are shown in Table 3.

[0113] As shown in Table 3, the linear range of the three toxins obtained by methods B and C was good in the range of 4 to 2000 μg / L, the correlation coefficients were all greater than 0.995, and the LOD range was 4-8 μg·kg -1 The LOQ is between 8 and 16 μg kg -1 between.

[0114] Example 6: Matrix Effects

[0115] In chromatography-mass spectrometry, the matrix effect of the sample will affect the accuracy and sensitivity of the detection. Therefore, the present invention evaluates the matrix effect of the sample. Take the standard intermediate solution of 3 toxins (concentration is 2μg / mL), respectively use the blank wheat matrix extract or blank solvent (acetonitrile: water (20 / 80, V / V)) in Example 5 for gradient dilution to a concentration range of 4-2000μg / L, and use method A, method B and method C in Example 3 for detection. The matrix effect is calculated by the following formula: SSE (%) = (slope of the calibration curve corresponding to the matrix) / calibration curve corresponding to the blank solvent) * 100, SSE is in the range of 80-120%, indicating that there is no obvious matrix effect. The matrix effect results are shown in Table 3. In detection methods B and C of the present invention, the matrix effect of the wheat sample is 81.4%-95.2%, indicating that there is no obvious matrix effect.

[0116] Example 7: Precision and recovery

[0117] The recovery test was conducted by spike test. The intermediate solutions of the three toxin standards were diluted with the blank wheat matrix extract in Example 5 to high, medium and low concentrations (high, medium and low concentrations were 50, 500 and 1000 μg / kg, respectively), and tested using Method A, Method B and Method C in Example 3. In this article, precision was calculated by repeatability and reproducibility. Repeatability (intraday relative standard deviation, RSD r) by spiking blank samples at three concentration levels and then analyzing them on the same day; reproducibility (inter-day relative standard deviation, RSD R ) by spiking blank samples at 3 concentration levels and then repeating the above steps for 3 days. RSD r and RSD R The results are shown in Table 3. The recovery results are shown in Table 4. The recovery and relative standard deviation should meet the standards of EU 401 / 2006 / EC. For analytes for which standards have not yet been established, the recovery should be 70-120% and RSD < 20%.

[0118] The results in Tables 3 and 4 show that the average recoveries of the three toxins at three spike levels were 83.5% to 118.8%, and the relative standard deviations (RSDs) were 0.28% to 18.8%, meeting the requirements of EU 401 / 2006 / EC for recovery and precision.

[0119] Table 3: Linear range, correlation coefficient, matrix effect, detection limit, quantification limit and precision of toxins detected by three detection methods

[0120]

[0121] Table 4: Recovery rates of toxins by three detection methods

[0122]

[0123]

[0124] Example 8: Sample Analysis

[0125] The Fusarium graminearum strains used in this embodiment are PH-1 (Fusarium graminearum) and F-1 (F.asiaticum) strains. 25g of wheat grains were weighed in each bag and placed in a ziplock bag for irradiation. The irradiation dose was 8kGy. After irradiation, the grains were placed in a 250mL sterile conical flask in a clean bench, 10mL of sterile water was added, and each bottle was inoculated with 3 pieces of Fusarium graminearum fungi (PH-1 or F-1 strains) of the same size after activation. After being placed in a 25°C incubator for 21 days, the samples were placed in a 55°C oven for drying. Each strain was repeated for 5 bottles, and the experiment was repeated 3 times. The dried samples were pretreated according to the pretreatment method in Example 4 to obtain wheat sample extracts infected with PH-1 or F-1 strains, and the contents of DON, 3-ADON and 15-ADON were detected by method A, method B and method C in Example 3, respectively.

[0126] Figure 3The extracted ion chromatograms of DON, 15-ADON and 3-ADON in wheat samples infected with PH-1, where (A) is method A, (B) is method B, and (C) is method C.

[0127] Figure 4 The extracted ion chromatograms of DON, 15-ADON and 3-ADON in wheat samples infected with F-1, where (A) is method A, (B) is method B, and (C) is method C.

[0128] The mixed standard intermediate solution containing the three toxins was diluted with the blank wheat matrix extract in Example 5, and the standard curve was prepared with the peak area average value (Y) of each toxin response value as the ordinate and the mass concentration (X, μg / kg) corresponding to each toxin as the abscissa, and a one-variable equation about Y and X was obtained as the calculation formula. The contents of DON, 3-ADON and 15-ADON in the wheat samples infected with PH-1 and F-1 strains detected by the three methods were calculated by the formula, respectively. The results are as follows: Figure 5 As shown, (A) is a wheat sample infected with PH-1, and (B) is a wheat sample infected with F-1.

[0129] Figure 5 The results showed that the DON toxin production of the two strains was very high, and the detection results of the three methods were basically consistent. The PH-1 strain produced 15-ADON and 3-ADON on the irradiated wheat matrix, and the detection results of the three methods were basically consistent. When detected using method A, a small amount of 15-ADON was detected in the F-1 strain. When detected using methods B and C, no 15-ADON was detected in the F-1 strain. Since the F-1 strain is a 3-ADON chemical type, methods B and C are more accurate for the detection results of DON, 3-ADON and 15-ADON content of wheat samples infected by the F-1 strain, especially for the 15-ADON content.

[0130] The above results show that the method of the present invention can simultaneously achieve qualitative and quantitative detection of vomitoxin and its acetylated products in wheat. The detection method has the advantages of rapidity, economy, high sensitivity, strong practicality, etc., and is fully capable of meeting the detection requirements of vomitoxin and its acetylated products in wheat food, and has far-reaching significance for wheat food safety detection.

Claims

1. A method for determining the content of deoxynivalenol and / or its acetylated product in wheat, characterized in that: The acetylated product of deoxynivalenol includes one or two selected from 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol. The method includes using high performance liquid chromatography-tandem mass spectrometry to detect the wheat sample solution to be tested. The chromatographic column used in the method is a chiral chromatographic column with silica gel coated with cellulose-tris (3,5-dichlorophenylcarbamate) as a filler; Wherein, the chromatographic conditions of the method include the following characteristics: The chromatographic column is YMC CHIRAL ART Cellulose-sc 3.0*250mm s-3um; Mobile phase A was 5 ± 0.5 mM ammonium acetate in water, and mobile phase B was methanol; The elution program was isocratic, 25–30% A at 0 min, and 25–30% A at ≥13 min; The liquid chromatograph model was Thermo Fisher DIONEX UltiMate 3000; Column temperature was 25 ± 5 °C; The injection volume was 10 ± 5 μL; Flow rate: 0.3 ± 0.05 mL min -1 ; The mass spectrometry conditions of the method include: (1) For deoxynivalenol, the adduct ion is [DON+CH3COO] - , the precursor ion is 355.1 m / z, the product quantitative ion is 295.0 m / z, the product qualifier ion is 247.0 m / z, the retention time is 6.38 min, the collision energy is 17 eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 77.5; for 15-acetyldeoxynivalenol, the adduct ion is [15-ADON+NH4] + , the precursor ion is 356.1 m / z, the product quantitative ion is 320.9 m / z, the product qualifier ion is 136.9 m / z, the retention time is 8.81 min, the collision energy is 12 eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 76.4; for 3-acetyldeoxynivalenol, the adduct ion is [3-ADON+CH3COO] - , the precursor ion is 397.0 m / z, the product quantitative ion is 307.1 m / z, the product qualifier ion is 173.1 m / z, the retention time is 10.12 min, the collision energy is 16 eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 40.6; or (2) For deoxynivalenol, the adduct ion is [DON+CH3COO] - , the precursor ion is 355.1 m / z, the product quantitative ion is 295.0 m / z, the product qualifier ion is 247.0 m / z, the retention time is 6.38 min, the collision energy is 17 eV, and the peak area ratio of the product qualifier ion to the product quantitative ion is 77.5; for 15-acetyldeoxynivalenol, the adduct ion is [15-ADON+H] + , the precursor ion was 339.1 m / z, the product quantitative ion was 137.1 m / z, the product qualifier ion was 261.1 m / z, the retention time was 8.80 min, the collision energy was 18 eV, and the peak area ratio of the product qualifier ion to the product quantitative ion was 47.7; for 3-acetyldeoxynivalenol, the adduct ion was [3-ADON+H] + , the precursor ion was 339.1 m / z, the product quantitative ion was 137.1 m / z, the product qualifier ion was 261.1 m / z, the retention time was 10.06 min, the collision energy was 18 eV, and the peak area ratio of the product qualifier ion to the product quantitative ion was 47.7; The mass spectrometry conditions of the method also include the following features: The mass spectrometer model is Thermo Fisher TSQ VANTAGE; The ionization mode was electrospray ionization in positive and negative ion switching mode; The spray voltage was ±3.0 ± 0.5 kV; The capillary temperature is 325±25℃; Atomization temperature is 225±25℃; Sheath gas pressure was 28 ± 2 psi; The auxiliary gas pressure is 18 ± 2 psi.

2. The method according to claim 1, characterized in that The wheat sample solution to be tested is prepared by the following method: For each gram of wheat flour, add 5-10 mL of a mixed solvent with a volume ratio of 4:1-3:1 of acetonitrile: water to the wheat flour. After uniform dispersion, centrifuge, take the supernatant, add 100-200 mg of anhydrous MgSO4, then add 1 ± 0.5 mL of n-hexane, centrifuge, remove the n-hexane layer, take the remaining supernatant, blow dry with liquid nitrogen, and then re-dissolve with 200-1000 μL of a mixed solvent with a volume ratio of 1:4-1:1 of acetonitrile: water. Pass through a filter membrane with a diameter of 0.22±0.02 μm. If it is turbid, pass through the membrane again to obtain the wheat sample solution to be tested.

3. The method according to claim 1, characterized in that The method comprises: determining the content of deoxynivalenol and / or its acetylated product in the wheat to be tested by a standard curve method according to the peak area of ​​deoxynivalenol and / or its acetylated product detected from the wheat sample solution to be tested.

4. The method according to claim 3, characterized in that The standard curve method uses a standard curve that is drawn up with the peak area of ​​each toxin as the ordinate and the corresponding concentration of each toxin as the abscissa.

5. The method according to claim 4, characterized in that The standard curve is a curve of the relationship between the peak area of ​​each toxin detected from a standard solution containing different concentrations of toxins and the content of each toxin in each standard solution using the high performance liquid chromatography-tandem mass spectrometry method described in claim 1, wherein the standard solution is prepared by diluting an acetonitrile solution of deoxynivalenol and / or its acetylated product with a concentration of 0.5-1 mg / L using a mixed solvent with a volume ratio of acetonitrile: water of 1:4-1:1, or the standard solution is prepared by diluting an acetonitrile solution of deoxynivalenol and / or its acetylated product with a concentration of 0.5-1 mg / L using a mixed solvent with a volume ratio of acetonitrile: water of 1:4-1:1, and then diluting with a blank wheat matrix extract, wherein the blank wheat matrix extract is prepared from sterilized wheat flour by the method for preparing a wheat sample solution to be tested described in claim 2.

6. The method according to claim 1, characterized in that In the method, the wheat to be tested is wheat infected by Fusarium graminearum.

7. The method according to claim 6, characterized in that The Fusarium graminearum is a strain that does not produce 15-ADON.

8. The method according to claim 7, characterized in that The Fusarium graminearum is F. asiaticum .

9. The method according to claim 1, characterized in that The method comprises determining the content of 15-acetyldeoxynivalenol in wheat, and optionally further comprises determining the content of deoxynivalenol and / or 3-acetyldeoxynivalenol in wheat.

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