Liquid chromatography-tandem mass spectrometry detection method for simultaneously detecting eight fumonisins

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry detection method for simultaneously detecting eight fumonisins, and belongs to the technical field of biological detection, and the eight fumonisins are respectively fumonisins B1, fumonisins B2, fumonisins B3, hydrolytic fumonisins B1, hydrolytic fumonisins B2, hydrolytic fumonisins B3, fumonisins A1 and N-(carboxymethyl)-fumonisins B1. The method comprises the following steps: preparing a fumonisins standard substance into standard solutions with different concentrations, drawing standard curves of eight fumonisins, pre-treating a grain sample, and then carrying out liquid chromatography-tandem mass spectrometry UPLC-MS / MS detection, so that high-sensitivity quantitative simultaneous detection of the eight fumonisins in the grain is realized, single sample detection is completed within 12 minutes, the detection limit is as low as 5 ng / ml, and the trace detection requirement is met; the method has the advantages of simple operation, strong stability, batch sample analysis of corn, wheat, rice and other grains, detection efficiency improvement, and manpower and time cost reduction.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a liquid chromatography-tandem mass spectrometry method for the simultaneous detection of eight fumonisins. Background Technology

[0002] Mycotoxins cause 60% to 80% of the world's grain contamination annually, resulting in severe economic losses. Fumonisins (FBs), also known as fumonisins, are secondary metabolites produced by fungi such as *Fusarium monitiforme* and *Fusarium verticillioides* under certain temperature and humidity conditions. They readily infect cereal crops such as corn, sorghum, and rice, with corn showing the most widespread and severe contamination. In recent years, fumonisin contamination in grains has been on the rise, attracting significant attention. Random sampling of corn samples has revealed varying levels of fumonisins in all samples. This not only poses a serious food safety hazard but also causes severe economic losses for those involved in the production and processing of corn, wheat, and other grains.

[0003] Fumonisins are fungal toxins widely found in food. Currently, more than 30 different types of fumonisins have been identified. Fumonisins can be divided into four main categories: A, B, C, and P, with category B being the most common. Fumonisin B1 (FB1) is the most common type, accounting for approximately 70% of all fumonisins. FB2 and FB3 are also frequently detected alongside FB1. Fumonisins pose significant health risks and have been classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). Studies have shown that fumonisins can cause various health problems, such as pulmonary edema in pigs, white matter atrophy in horses, nephrotoxicity and hepatotoxicity in rodents, and even primary liver and esophageal cancer in humans. FB1 has excellent water solubility, readily soluble in acetonitrile-water and methanol, and also exhibits excellent thermal stability, resisting the destruction of its toxic structure by high temperatures. As research deepens, various transformation products of fumonisins, collectively known as cryptic fumonisins, have been discovered beyond the original fumonisins. These are fumonisin derivatives whose chemical structures have changed from the original fumonisins, making them difficult to detect using conventional analytical methods. Recent studies indicate that cryptic fumonisins are present in high concentrations in certain food samples and may possess toxic effects, with toxicity exceeding that of the original fumonisins. my country is a major agricultural producer, ranking first in the world in grain production. Reducing the risk of mycotoxin contamination in grains is of great significance to ensuring my country's food and food security. Therefore, researching and developing efficient and highly sensitive new methods for detecting fumonisins and their cryptic products in grains plays a crucial role in ensuring the quality and safety of my country's food at all stages of production.

[0004] Therefore, in order to achieve highly sensitive simultaneous detection of multiple fumonisins and their cryptic products in grains, this paper establishes a high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous detection of eight fumonisins and their cryptic products in grains. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides a liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) method for the simultaneous detection of eight fumonisins. This method solves the technical problems of the increasing trend of fumonisin contamination in grains, the potential for fumonisins to cause various health problems, and the difficulty in detecting fumonisin derivatives using conventional analytical methods. To achieve highly sensitive simultaneous detection of fumonisins and their cryptic products in grains, the UPLC and mass spectrometry conditions are optimized, and an analytical method based on UPLC-MS / MS for the simultaneous detection of eight fumonisins and their cryptic products in grains is established. This method is simple, efficient, and low-cost, providing technical support for the detection of biotoxins and the assessment of contamination risks during food processing, reducing food safety problems caused by biotoxin residues, improving the level of technological innovation in biotoxin contamination research, and safeguarding public health and safety.

[0006] The technical solution adopted in this invention is as follows: A liquid chromatography-tandem mass spectrometry method for the simultaneous detection of eight fumonisins includes the following steps: The first step is the preparation of standard substances: accurately measure fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1), hydrolyzed fumonisin B2 (HFB2), hydrolyzed fumonisin B3 (HFB3), fumonisin A1 (FA1), and N-(carboxymethyl)-fumonisin B1 (NCM-FB1), and dilute them with 80% acetonitrile aqueous solution to prepare a series of mixed standard solutions with mass concentrations of 2, 5, 10, 25, 50, 100, 250, and 500 ng / mL; The second step is sample pretreatment: Accurately weigh 5 g of grain sample powder into a 50 mL centrifuge tube, add 20 mL of 50% acetonitrile aqueous solution, homogenize for 2 min, centrifuge at 10000 r / min for 5 min, then accurately measure 4 mL of supernatant, transfer it to a QuEChERS EMR-Lipid purification package centrifuge tube, vortex mix, centrifuge at 10000 r / min for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, and use the filtrate for liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis; The third step is to set the conditions for liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) determination: The liquid chromatography conditions are as follows: Chromatographic column: ZORBAX 300SB C8 3.5 μm 2.1×150 mm; Mobile phase: A is 0.1% (v / v) formic acid aqueous solution, B is methanol-acetonitrile (v / v) at a ratio of 1:1; Flow rate: 0.3 mL / min; Column temperature: 40 ℃; Injection volume: 5 μL; Gradient elution program: 0–8.5 min, 50% mobile phase A; 8.5–9.0 min, 50%–10% mobile phase A; 9.0–11.5 min, 10%–50% mobile phase A; Mass spectrometry conditions are: Ion source type: ESI source; Drying gas flow rate: 10 L / min; Drying gas temperature: 350℃; Nebulizer pressure: 45psi; Sheath gas flow rate: 11 L / min; Sheath gas temperature: 300℃; Ion source temperature: 350℃; Capillary voltage: 4000 V; Scanning mode: Multiple reaction monitoring mode.

[0007] Preferably, the mass spectrometry parameters of fumonisin B1 (FB1) are as follows: retention time of 2.80 min, precursor ion of 722.2 m / z, quantitative ion of 334.3 m / z, collision voltage of 40 eV, qualitative ion of 318.3 m / z, collision energy of 40 eV, and declustering voltage of 140 V.

[0008] Preferably, the mass spectrometry parameters of fumonisin B2 (FB2) are as follows: retention time of 8.10 min, precursor ion of 706.0 m / z, quantitative ion of 336.3 m / z, collision voltage of 40 eV, qualitative ion of 352.4 m / z, collision energy of 40 eV, and declustering voltage of 140 V.

[0009] Preferably, the mass spectrometry parameters of fumonisin B3 (FB3) are as follows: retention time of 5.28 min, precursor ion of 706.0 m / z, quantitative ion of 336.2 m / z, collision voltage of 40 eV, qualitative ion of 352.4 m / z, collision energy of 40 eV, and declustering voltage of 140 V.

[0010] Preferably, the mass spectrometry parameters of hydrolyzed fumonisin B1, i.e., HFB1, are as follows: retention time of 2.21 min, precursor ion of 406.1 m / z, quantitative ion of 352.2 m / z, collision voltage of 25 eV, qualitative ion of 334 m / z, collision energy of 25 eV, and declustering voltage of 140 V.

[0011] Preferably, the mass spectrometry parameters of hydrolyzed fumonisin B2, i.e., HFB2, are as follows: retention time of 5.40 min, precursor ion of 390.0 m / z, quantitative ion of 336.0 m / z, collision voltage of 25 eV, qualitative ion of 238.1 m / z, collision energy of 25 eV, and declustering voltage of 140 V.

[0012] Preferably, the mass spectrometry parameters of hydrolyzed fumonisin B3, i.e., HFB3, are as follows: retention time of 3.84 min, precursor ion of 390.0 m / z, quantitative ion of 336.2 m / z, collision voltage of 25 eV, qualitative ion of 238.1 m / z, collision energy of 25 eV, and declustering voltage of 140 V.

[0013] Preferably, the mass spectrometry parameters of fumonisin A1 (FA1) are as follows: retention time of 5.73 min, precursor ion of 764.6 m / z, quantitative ion of 746.4 m / z, collision voltage of 24 eV, qualitative ion of 728.4 m / z, collision energy of 24 eV, and declustering voltage of 140 V.

[0014] Preferably, the mass spectrometry parameters of N-(carboxymethyl)-fumonisin B1, i.e., NCM-FB1, are as follows: retention time of 3.34 min, precursor ion of 780.3 m / z, quantitative ion of 392.1 m / z, collision voltage of 40 eV, qualitative ion of 410.1 m / z, collision energy of 40 eV, and declustering voltage of 140 V.

[0015] Preferably, in the second step, the grain is a cereal crop, which is one or more of corn, sorghum, wheat, and rice.

[0016] Explanation of the principle: This invention addresses the need for simultaneous detection of eight fumonisins in grains. It optimizes the extraction process based on the characteristics of the grain matrix, achieving efficient enrichment of the target analytes. Based on UPLC-MS / MS technology, it optimizes the chromatographic column, gradient elution mobile phase, and mass spectrometry parameters to achieve baseline separation and precise quantification of the eight fumonisins. Multiple reaction monitoring (MRM) ensures detection sensitivity and specificity, meeting the need for simultaneous detection of low-content fumonisins and their occult forms in grains.

[0017] Beneficial effects: 1. This application fills the gap in the detection of cryptic fumonisins, ensuring the integrity of food safety. Existing national standards (such as GB 5009.240-2017) only cover the prototypes of fumonisins FB1, FB2, and FB3, and there are gaps in international standards for the detection methods and limits of cryptic fumonisins, resulting in the undetectable cryptic toxins generated during grain processing, which pose a safety hazard. This invention is the first to include five cryptic fumonisins (HFB1, HFB2, HFB3, NCM-FB1, and FA1) in the detection scope. By independently preparing cryptic standard products and optimizing the detection system, it achieves simultaneous screening of eight fumonisins, which can accurately identify the full picture of toxin contamination in grains from planting to processing, avoiding food safety risks caused by missed detection of cryptic toxins, and providing technical support for the whole-chain safety management of grain "from farm to table". 2. This application improves detection efficiency and accuracy, adapting to the needs of batch sample analysis. Traditional detection methods suffer from problems such as low pretreatment efficiency, large matrix interference, and insufficient sensitivity. For example, enzyme-linked immunosorbent assay (ELISA) requires professional operation and cannot simultaneously detect multiple components, while conventional LC-MS / MS methods are only suitable for common fumonisins. This invention optimizes the extraction method, increasing the recovery rate of eight target substances to 85%-95%. At the same time, it optimizes UPLC-MS / MS parameters, enabling single-sample detection within 12 minutes, with a detection limit as low as 5 ng / ml, meeting the needs of trace detection. This method is simple to operate, highly stable, and can be adapted to batch sample analysis of various grains such as corn, wheat, and rice, significantly improving detection efficiency and reducing labor and time costs. 3. The liquid chromatography-tandem mass spectrometry method for the simultaneous detection of eight fumonisins in this application can strengthen the technical support for market supervision and promote the standardization of the industry. As a major grain producer, China faces the critical issue of fumonisin contamination affecting grain quality and international competitiveness. However, existing regulatory technologies are insufficient to meet the needs of detecting concealed toxins. The method established in this invention can be directly applied to market supervision, grain procurement, and import and export inspection and quarantine, providing accurate data for grain product sampling and contamination risk assessment, and helping to improve my country's fumonisin limit standard system (such as supplementing concealed toxin limit indicators). 4. The simplicity and low cost of this method make it suitable for application in testing institutions and grain enterprises at all levels across the country, thereby improving the overall testing technology level of the industry, enhancing my country's food security supervision capabilities, and maintaining the international influence and market reputation of a major grain producer. Attached Figure Description

[0018] Figure 1 The total ion chromatogram of the eight fumonisin standards in this application; Figure 2 This is a standard curve diagram of fumonisin B1 in this application; Figure 3This is a standard curve diagram of fumonisin B2 in this application; Figure 4 This is a standard curve diagram of fumonisin B3 in this application; Figure 5 This is the standard curve diagram of hydrolyzed fumonisin B1 in this application; Figure 6 This is the standard curve diagram of hydrolyzed fumonisin B2 in this application; Figure 7 This is the standard curve diagram of hydrolyzed fumonisin B3 in this application; Figure 8 This is a standard curve diagram of fumonisin A1 in this application; Figure 9 This is the standard curve for N-(carboxymethyl)-fumonisin B1 in this application. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the embodiments. It should be understood that the following embodiments are only for explanation and illustration of the present invention, but do not limit the present invention to these specific embodiments, and do not limit the scope of the present invention in any way.

[0020] The Agilent 1290 Infinity II UPLC system and Agilent 6470 triple quadrupole mass spectrometer used in this application, equipped with an AJS ESI ion source, were purchased from Agilent Technologies, Inc.

[0021] The Milli-Q IQ7000 ultrapure water system was purchased from Merck, Germany.

[0022] The ML304T electronic analytical balance was purchased from Mettler Toledo, Switzerland.

[0023] The QQ15-400A ultrasonic cleaner was purchased from Shanghai Qiqian Electronic Technology Co., Ltd.

[0024] The 5430R high-speed centrifuge was purchased from Eppendorf in Germany. 0.22 μm microporous filter membrane: organic system purchased from Shanghai Anpu Company.

[0025] Chromatographic column: ZORBAX 300SB C8 (3.5 μm 2.1×150 mm) purchased from Agilent Technologies, USA.

[0026] Methanol and acetonitrile: mass spectrometry grade, purity ≥99.9%, purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0027] Formic acid: chromatographic grade, purity ≥98%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0028] Fumonisin B1 (FB1), Fumonisin B2 (FB2), Fumonisin B3 (FB3), Hydrolyzed Fumonisin B1 (HFB1), Hydrolyzed Fumonisin B2 (HFB2), Fumonisin A1 (FA1), and N-(carboxymethyl)-fumonisin B1 (NCM-FB1) were purchased from Qingdao Puribang Biotechnology Co., Ltd.

[0029] Hydrolyzed fumonisin B3, or HFB3, was purchased from Tianjin Alta Technology Co., Ltd.

[0030] Example 1: A liquid chromatography-tandem mass spectrometry method for the simultaneous detection of eight fumonisins, wherein the standard curve plotting for the UPLC-MS / MS method for the simultaneous detection of eight fumonisins in cereals includes the following steps: Step 1: Preparation of standard substances: Accurately measure fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1), hydrolyzed fumonisin B2 (HFB2), hydrolyzed fumonisin B3 (HFB3), fumonisin A1 (FA1), and N-(carboxymethyl)-fumonisin B1 (NCM-FB1), and dilute with 80% acetonitrile aqueous solution to prepare a series of mixed standard solutions with mass concentrations of 2, 5, 10, 25, 50, 100, 250, and 500 ng / mL; add 100 μL of each concentration to a 2 mL sample vial for analysis; The second step is to set the conditions for liquid chromatography-tandem mass spectrometry (UPLC-MS / MS): The liquid chromatography conditions are as follows: Chromatographic column: ZORBAX 300SB C8 3.5 μm 2.1×150 mm; Mobile phase: A is 0.1% (v / v) formic acid aqueous solution, B is methanol-acetonitrile (v / v) at a ratio of 1:1; Flow rate: 0.3 mL / min; Column temperature: 40 ℃; Injection volume: 5 μL; Gradient elution program: 0–8.5 min, 50% mobile phase A; 8.5–9.0 min, 50%–10% mobile phase A; 9.0–11.5 min, 10%–50% mobile phase A; Mass spectrometry conditions are: Ion source type: ESI source; Dry gas flow rate: 10 L / min; Dry gas temperature: 350℃; Nebulizer pressure: 45psi; Sheath gas flow rate: 11 L / min; Sheath gas temperature: 300℃; Ion source temperature: 350℃; Capillary voltage: 4000 V; Scanning mode: Multiple reaction monitoring mode. The mass spectrometry parameters of the eight fumonisins are as follows: The mass spectrometry parameters of fumonisin B1 (FB1) are as follows: retention time 2.80 min, precursor ion 722.2 m / z, quantitative ion 334.3 m / z, collision voltage 40 eV, qualitative ion 318.3 m / z, collision energy 40 eV, and declustering voltage 140 V. The mass spectrometry parameters of fumonisin B2 (FB2) are as follows: retention time 8.10 min, precursor ion 706.0 m / z, quantitative ion 336.3 m / z, collision voltage 40 eV, qualitative ion 352.4 m / z, collision energy 40 eV, and declustering voltage 140 V. The mass spectrometry parameters of fumonisin B3 (FB3) are as follows: retention time 5.28 min, precursor ion 706.0 m / z, quantitative ion 336.2 m / z, collision voltage 40 eV, qualitative ion 352.4 m / z, collision energy 40 eV, and declustering voltage 140 V. The mass spectrometry parameters of hydrolyzed fumonisin B1, i.e., HFB1, are as follows: retention time 2.21 min, precursor ion 406.1 m / z, quantitative ion 352.2 m / z, collision voltage 25 eV, qualitative ion 334 m / z, collision energy 25 eV, and declustering voltage 140 V. The mass spectrometry parameters of hydrolyzed fumonisin B2, i.e., HFB2, are as follows: retention time 5.40 min, precursor ion 390.0 m / z, quantitative ion 336.0 m / z, collision voltage 25 eV, qualitative ion 238.1 m / z, collision energy 25 eV, and declustering voltage 140 V. The mass spectrometry parameters of hydrolyzed fumonisin B3, i.e., HFB3, are as follows: retention time 3.84 min, precursor ion 390.0 m / z, quantitative ion 336.2 m / z, collision voltage 25 eV, qualitative ion 238.1 m / z, collision energy 25 eV, and declustering voltage 140 V. The mass spectrometry parameters of fumonisin A1 (FA1) are as follows: retention time 5.73 min, precursor ion 764.6 m / z, quantitative ion 746.4 m / z, collision voltage 24 eV, qualitative ion 728.4 m / z, collision energy 24 eV, and declustering voltage 140 V. The mass spectrometry parameters of N-(carboxymethyl)-fumonisin B1, i.e., NCM-FB1, are as follows: retention time 3.34 min, precursor ion 780.3 m / z, quantitative ion 392.1 m / z, collision voltage 40 eV, qualitative ion 410.1 m / z, collision energy 40 eV, and declustering voltage 140 V. The third step is to place the fumonisin standard prepared in the first step into the injection tray according to the set instrument parameters, edit the injection sequence, and obtain experimental data after injection. The fourth step is to plot standard curves for the eight fumonisin standards, with the concentration of fumonisin on the x-axis and the response value on the y-axis.

[0031] Figure 2 This indicates that the linear range of FB1 is 2-500 ng / mL, and the linear equation is y=517.99x-1689.92, R0. 2 =0.9994; Figure 3 This indicates that the linear range of FB2 is 2-500 ng / mL, and the linear equation is y=310.79x-538.52, R0. 2 =0.9995; Figure 4 This indicates that the linear range of FB3 is 2-500 ng / mL, and the linear equation is y=429.37x-2220.24, R0. 2 =0.9996; Figure 5 The results indicate that the linear range of HFB1 is 2-500 ng / mL, and the linear equation is y=1141.62x-3080.50, R0. 2 =0.9998; Figure 6 The results indicate that the linear range of HFB2 is 2-500 ng / mL, and the linear equation is y=251.61x-603.36, R0. 2 =0.9998; Figure 7 The results indicate that the linear range of HFB3 is 2-500 ng / mL, and the linear equation is y=1395.00x-4409.76, R0. 2 =0.9997; Figure 8 The results indicate that the linear range of FA1 is 2-500 ng / mL, and the linear equation is y=210.06x-250.94, R0. 2 =0.9999; Figure 9 The results showed that the linear range of NCM-FB1 was 2-500 ng / mL, and the linear equation was y=162.79x-305.45, R0. 2 =0.9994.

[0032] Example 2: A liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins in grains, comprising the following steps: The first step is the preparation of standard substances: accurately measure fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1), hydrolyzed fumonisin B2 (HFB2), hydrolyzed fumonisin B3 (HFB3), fumonisin A1 (FA1), and N-(carboxymethyl)-fumonisin B1 (NCM-FB1), and dilute them with 80% acetonitrile aqueous solution to prepare a series of mixed standard solutions with mass concentrations of 2, 5, 10, 25, 50, 100, 250, and 500 ng / mL; The second step is sample pretreatment: Accurately weigh 5 g of grain sample powder into a 50 mL centrifuge tube, add 20 mL of 50% acetonitrile aqueous solution, homogenize for 2 min, centrifuge at 10000 r / min for 5 min, then accurately measure 4 mL of supernatant, transfer it to a QuEChERS EMR-Lipid purification package centrifuge tube, vortex mix, centrifuge at 10000 r / min for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, and use the filtrate for liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis; The third step involves setting the liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) determination conditions according to the technical solution of Example 1, and simultaneously detecting eight fumonisins in the grain sample powder.

[0033] Cereals are grain crops, which include one or more of the following: corn, sorghum, wheat, rice, and their products.

[0034] Example 3, determination of the recovery rate of a liquid chromatography-tandem mass spectrometry method for the simultaneous detection of eight fumonisins in grains, comprising the following steps: The first step is the preparation of standard substances: accurately measure fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1), hydrolyzed fumonisin B2 (HFB2), hydrolyzed fumonisin B3 (HFB3), fumonisin A1 (FA1), and N-(carboxymethyl)-fumonisin B1 (NCM-FB1), and dilute them with 80% acetonitrile aqueous solution to prepare a series of mixed standard solutions with a mass concentration of 50 ng / mL; The second step is sample pretreatment: Accurately weigh 5 g of crushed corn kernels into a 50 mL centrifuge tube, add 20 mL of 50% acetonitrile aqueous solution, homogenize for 2 min, centrifuge at 10000 r / min for 5 min, then accurately measure 4 mL of supernatant, transfer it to a QuEChERS EMR-Lipid purification package centrifuge tube, vortex mix, centrifuge at 10000 r / min for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, and use the filtrate for liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis; Third, referring to the technical solution of Example 1, the determination conditions of liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) were set, and the eight fumonisins in the standard solution and corn kernel sample were detected simultaneously. The peak area corresponding to each fumonisin was calculated, and the average recovery rate was calculated. The results are shown in Table 1.

[0035] Table 1. Spike recoveries of eight fumonisins in corn kernel samples (n=3) .

[0036] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins, characterized in that, Includes the following steps: The first step is the preparation of standard substances: accurately measure fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1), hydrolyzed fumonisin B2 (HFB2), hydrolyzed fumonisin B3 (HFB3), fumonisin A1 (FA1), and N-(carboxymethyl)-fumonisin B1 (NCM-FB1), and dilute them with 80% acetonitrile aqueous solution to prepare a series of mixed standard solutions with mass concentrations of 2, 5, 10, 25, 50, 100, 250, and 500 ng / mL; The second step is sample pretreatment: Accurately weigh 5 g of grain sample powder into a 50 mL centrifuge tube, add 20 mL of 50% acetonitrile aqueous solution, homogenize for 2 min, centrifuge at 10000 r / min for 5 min, then accurately measure 4 mL of supernatant, transfer it to a QuEChERS EMR-Lipid purification package centrifuge tube, vortex mix, centrifuge at 10000 r / min for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, and use the filtrate for liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis; The third step is to set the conditions for liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) determination: The liquid chromatography conditions are as follows: Chromatographic column: ZORBAX 300SB C8 3.5 μm 2.1×150 mm; Mobile phase: A is 0.1% (v / v) formic acid aqueous solution, B is methanol-acetonitrile (v / v) at a ratio of 1:1; Flow rate: 0.3 mL / min; Column temperature: 40 ℃; Injection volume: 5 μL; Gradient elution program: 0–8.5 min, 50% mobile phase A; 8.5–9.0 min, 50%–10% mobile phase A; 9.0–11.5 min, 10%–50% mobile phase A; Mass spectrometry conditions are: Ion source type: ESI source; Drying gas flow rate: 10 L / min; Drying gas temperature: 350℃; Nebulizer pressure: 45 psi; Sheath gas flow rate: 11 L / min; Sheath gas temperature: 300℃; Ion source temperature: 350℃; Capillary voltage: 4000 V; Scanning mode: Multiple reaction monitoring mode.

2. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters for fumonisin B1 (FB1) are as follows: retention time 2.80 min, precursor ion 722.2 m / z, quantitative ion 334.3 m / z, collision voltage 40 eV, qualitative ion 318.3 m / z, collision energy 40 eV, and declustering voltage 140 V.

3. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters for fumonisin B2 (FB2) are as follows: retention time 8.10 min, precursor ion 706.0 m / z, quantitative ion 336.3 m / z, collision voltage 40 eV, qualitative ion 352.4 m / z, collision energy 40 eV, and declustering voltage 140 V.

4. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters for fumonisin B3 (FB3) are as follows: retention time 5.28 min, precursor ion 706.0 m / z, quantitative ion 336.2 m / z, collision voltage 40 eV, qualitative ion 352.4 m / z, collision energy 40 eV, and declustering voltage 140 V.

5. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters of hydrolyzed fumonisin B1, i.e., HFB1, are as follows: retention time 2.21 min, precursor ion 406.1 m / z, quantitative ion 352.2 m / z, collision voltage 25 eV, qualitative ion 334 m / z, collision energy 25 eV, and declustering voltage 140 V.

6. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters of hydrolyzed fumonisin B2, i.e., HFB2, are as follows: retention time 5.40 min, precursor ion 390.0 m / z, quantitative ion 336.0 m / z, collision voltage 25 eV, qualitative ion 238.1 m / z, collision energy 25 eV, and declustering voltage 140 V.

7. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters of hydrolyzed fumonisin B3, i.e., HFB3, are as follows: retention time 3.84 min, precursor ion 390.0 m / z, quantitative ion 336.2 m / z, collision voltage 25 eV, qualitative ion 238.1 m / z, collision energy 25 eV, and declustering voltage 140 V.

8. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters for fumonisin A1 (FA1) are as follows: retention time 5.73 min, precursor ion 764.6 m / z, quantitative ion 746.4 m / z, collision voltage 24 eV, qualitative ion 728.4 m / z, collision energy 24 eV, and declustering voltage 140 V.

9. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, The mass spectrometry parameters for N-(carboxymethyl)-fumonisin B1, or NCM-FB1, are as follows: retention time 3.34 min, precursor ion 780.3 m / z, quantitative ion 392.1 m / z, collision voltage 40 eV, qualitative ion 410.1 m / z, collision energy 40 eV, and declustering voltage 140 V.

10. The liquid chromatography-tandem mass spectrometry method for simultaneous detection of eight fumonisins according to claim 1, characterized in that, In the second step, the grains are cereal crops, which include one or more of the following: corn, sorghum, wheat, and rice.

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