CN103969364A - Method for measuring seven fungaltoxin in fodder and cereal through liquid chromatogram tandem mass spectrometry - Google Patents

Abstract

The invention relates to a liquid chromatography tandem mass spectrometry method for determining seven kinds of mycotoxins in feed and grain. The mycotoxins involved are: DON, D3G, NIV, 3ADON, 15ADON, FUSX and DOM and other toxins. The method uses an improved dispersive solid-phase extraction method to remove lipids and other interfering substances in the sample, and then the processed sample is qualitatively and quantitatively analyzed by liquid chromatography in series with a triple quadrupole mass spectrometer. The method of the invention has the advantages of simple, rapid and economical sample pretreatment, short analysis time, high sensitivity and recovery rate, good reproducibility and specificity, and is applicable to the detection of multi-component mycotoxins in feed and grain samples. To provide effective technical support to ensure the safety of feed and grain in our country.

Description

Determination of 7 Mycotoxins in Feed and Grain by Liquid Chromatography Tandem Mass Spectrometry

technical field

The invention relates to a method for determining mycotoxins in feed and grain, in particular to a method for measuring seven mycotoxins in feed and grain by liquid chromatography tandem mass spectrometry.

Background technique

Mycotoxins are secondary metabolites produced by fungi. Their pollution in crop products, food and feed is becoming more and more serious, causing great harm to human and animal health. Among them, type B trichothecenes are the most common type of mycotoxins in the world. It is produced by Fusarium and widely pollutes wheat, barley, corn and other grains and their products. Such toxins mainly include 5 kinds: deoxynivalenol (DON), nivalenol (NIV), 3-acetyl deoxynivalenol (3ADON), 15-acetyl deoxynivalenol Fusarenol (15ADON) and Fusarone-X (FUSX).

DON can cause symptoms such as food refusal, vomiting, nausea, growth retardation, neuroendocrine disturbance and immunosuppression in animals. At the same time, it also has strong cytotoxicity (inhibition of DNA, RNA, protein synthesis), embryotoxicity (embryo death, fetal growth retardation, functional hypoplasia), certain teratogenicity, weak carcinogenicity, and affects the immunity of humans and animals. system. 3ADON and 15ADON are acetylated compounds of DON. These two toxins have similar or even higher toxicity compared to that of DON. Studies have shown that these two toxins can be deacetylated in animals to form DON, potentially increasing DON intake. At present, there are few toxicological studies on NIV and FUSX. However, a small number of studies have found that broiler chickens fed 1 mg/kg feed containing NIV can have slight pathological changes, while feeding 5 mg/kg feed containing DON has no obvious pathological changes.

Under the action of enzymes in plants, some mycotoxins can bind some highly polar substances, such as sugars, amino acids, sulfates, etc. These bound toxins are often undetectable by conventional analytical methods and are therefore referred to as cryptic mycotoxins. Deoxynivalenol-3-glucoside (D3G) is the most reported hidden toxin in recent years, and it is widely found in grains and beer fermentation liquid. Studies have shown that D3G has low toxicity, but after ingestion by humans and animals, it can be hydrolyzed into DON toxin monomer under the action of microorganisms in the body to exert toxic effects. At present, the contamination of concealed DON in feed has not been reported. Due to the potential health risks of D3G, it has attracted great attention. In addition, deepoxy-deoxynivalenol (DOM) is the main metabolite of DON in vivo. It has not been found in natural cereals. Considering that tissues of animal origin may be added to feed as a protein source, DOM testing is also necessary.

In 2006, the European Union promulgated the recommended limit values of DON in pig feed and feed grains as 900 μg/kg and 8000 μg/kg respectively; The allowable amount of nivalenol stipulates that the maximum limit of pig compound feed, calf compound feed and lactating animal compound feed is 1000μg/kg, and the maximum limit of cattle compound feed and poultry compound feed is 5000μg/kg. In addition, at the 72nd meeting held in 2010, the Food and Agriculture Organization of the United Nations/WHO Joint Expert Committee on Food Additives (JECFA) proposed to formulate a limit standard including the total amount of DON, 3ADON and 15ADON toxins. At the same time, D3G was considered by JECFA as a potential source of DON intake. For NIV, due to the lack of relevant toxicological studies, FUSX has not yet formulated relevant limit standards.

At present, the detection methods of class B trichothecenes and D3G in grains and foods have been reported: "Journal of agricultural and food chemistry", 2012, 46, 11638 and "Mycotoxinresearch", 2012, 28, 181 through multifunctional Determination of DON, D3G, 3ADON and 15ADON in grains, corn and their products by liquid chromatography tandem mass spectrometry after MycoSep226 or 225 column purification. The pretreatment operation of this method is cumbersome and costly, and the recovery rate of D3G is low (50%-60%); "Foodadditives and contaminants: Part A", 2009, 26, 507, after the sample was extracted, the unpurified direct liquid phase series Determination of DON and D3G content in wheat and corn by mass spectrometry. Although the pretreatment of this method is simple and quick, the interference substances such as lipids contained in the sample seriously reduce the detection sensitivity, and long-term use causes great damage to the mass spectrometry ion source.

At present, the contamination of concealed DON and B trichothecene mycotoxins in feed has not been reported. At the same time, the existing methods for detecting such mycotoxins mainly focus on food and grains, excluding animal feed. In order to ensure feed safety and animal production efficiency, it is necessary to establish effective and reliable analytical methods to monitor its occurrence and contamination.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, and provide a simple, economical, fast, sensitive and accurate liquid phase tandem mass spectrometry method for the determination of B-type trichothecenes in feed and grains Methods of class and hidden type DON.

In order to achieve the above object, the present invention adopts the following technical solutions, specifically comprising the following steps:

(1) Accurately weigh 2 g of the sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 8 mL of acetonitrile/water solution (84/16, v/v). After vortexing for 2 min, ultrasonic extraction was performed for 0.5 h. Then centrifuge at 4000r/min for 5min, and take 2mL supernatant to be purified.

(2) Transfer 2 mL of the supernatant to a 15 mL conical centrifuge tube, then add 100 mg of anhydrous magnesium sulfate and 1 mL of n-hexane, vortex vigorously for 1 min, and centrifuge at 4000 r/min for 5 min to remove the n-hexane layer. The remaining solution was dried under nitrogen at 40°C, and 500 μL of ammonium acetate aqueous solution (5 mM) was added to vigorously vortex, and finally the sample solution was passed through a 0.22 μm nylon filter membrane for analysis.

(3) Analyzing the sample to be detected obtained in step (2) by liquid chromatography tandem mass spectrometer, and obtaining the results of B group trichothecenes and D3G residues respectively.

(4) The liquid phase analysis conditions used in the step (3) are as follows:

Chromatographic column: Agilent Extend-C18column (150mm×3.0mm, 3.5μm), column temperature: 40°C, flow rate: 0.3mL/min, injection volume: 5μL;

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

Gradient elution program: 0-1min, 20%B; 1-5min, 90%B, 5-6min90%B, 6-6.5min, 20%B, 6.5-8min20%B;

(5) The mass spectrometry conditions used in step (3) are as follows:

Ion source mode: positive and negative ion mode (ESI ⁺ and ESI ^- ); spray voltage: 4.5kv (ESI ⁺ ) and -3.5ky (ESI ^- ); heating block temperature: 400°C; ion transfer tube temperature: 250°C; atomization Gas and drying gas flow rates: 3mL/min and 15mL/min; the mass spectrometry parameters of the seven mycotoxins are shown in Table 1.

Table 1 Mass spectrometry parameters of each mycotoxin

Note; *, Quantitative ion; ^a , Quantitative ion abundance and quantitative ion abundance percentage;

Compared with the prior art, the present invention has the following technical advantages and effects: the improved dispersion solid-phase extraction method is adopted for pretreatment. Compared with the common solid-phase extraction method, this method is simpler and faster, saves the time and cost of sample processing, and reduces the consumption of organic solvents. At the same time, the processed samples are very clean, the sensitivity will not change significantly after continuous sample injection, and the ion source of the triple quadrupole mass spectrometer remains clean. There was satisfactory recovery (85%) for D3G. The quantitative limits of different mycotoxins in different matrices in the method of the present invention are: DON, 5.0-7.8 μg/kg; NIV, 8.4-12.6 μg/kg; D3G, 5.5-8.4 μg/kg; 3ADON, 5.3-8.8 μg /kg; 15ADON, 5.3-10.0μg/kg; FUSX, 8.3-13.6μg/kg; the recovery rate was between 80%-118%, and the relative standard deviation was between 2.73%-18.39%. It can be concluded that the sensitivity and precision are significantly higher than those of the reported analytical methods.

Description of drawings

Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail

Fig. 1 is the selective reaction monitoring (SRM) pattern of 7 kinds of mycotoxin standard solutions (DON, NIV, 3ADON, 15ADON and FUSX, concentration is 100ng/mL; DOM and D3G, concentration is 75ng/mL);

Fig. 2 is the selected reaction monitoring (SRM) collection of patterns of positive sample;

Fig. 3 is the impact (b) of different purification methods on the purification effect (a) and recovery rate of 7 kinds of mycotoxins;

Figure 4 shows the matrix effect (a) of seven mycotoxins in different matrices and the matrix effect (b) after isotope internal standard calibration.

Detailed ways

The following examples are given to further illustrate the present invention. The present invention is described in conjunction with the best embodiment, but after reading the examples of the present invention, those skilled in the art can understand and make many changes in the disclosed implementation to obtain the same or similar results, which all belong to the concept of the present invention and range. More specifically, there are reagents that can be substituted for the reagents disclosed herein to achieve the same or similar results. All similar substitutions or modifications are considered to be within the concept and scope of the present invention, and all the above-mentioned equivalent forms belong to the scope defined by the claims of the present invention.

Example 1

1. Materials and methods

1.1 Main instruments and reagents

Liquid chromatography tandem mass spectrometer (LC-MS/MS) (Thermo Company, USA);

High-speed pulverizer (Beijing Yanshan Zhengde Machinery Equipment Co., Ltd.);

Vortex instrument (Haimen Qilin Bell Company), ultrasonic oscillator (Ningbo Xinzhi Company);

High-speed centrifuge (Changsha Xiangyi Company), nitrogen blowing instrument (Shanghai Quandao Company);

Milli-Q ultrapure water machine (Millipore, Germany);

1.2 Standards and reagents

Deoxynivalenol (DON), Nivalenol (NIV), 3-Acetyldeoxynivalenol (3ADON), 15-Acetyldeoxynivalenol (15ADON) And fusarium-X (FUSX) (Sigma, USA); deoxynivalenol-3-glucoside (D3G), de-epoxy-deoxynivalenol (DOM) and isotope internal standard [ ¹³ C ₁₅ ]-DON (Austria Romerlab Company); graphitized carbon black (GCB), N-propylethylenediamine adsorbent (PSA), cleanert silica gel, C18 and other adsorption materials (Tianjin Agela Company).

2. Experimental method

2.1 Sample pretreatment method

(1) Accurately weigh 2 g of the sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 8 mL of acetonitrile/water solution (84/16, v/v). After vortexing for 2 min, ultrasonic extraction was performed for 0.5 h. Then centrifuge at 4000r/min for 5min, and take 2mL supernatant to be purified.

(2) Transfer 2 mL of the supernatant to a 15 mL conical centrifuge tube, then add 100 mg of anhydrous magnesium sulfate and 1 mL of n-hexane, vortex vigorously for 1 min, and centrifuge at 4000 r/min for 5 min to remove the n-hexane layer. The remaining solution was blown dry under nitrogen at 40°C, first added 480 μL ammonium acetate aqueous solution (5 mM) and vigorously vortexed, then added 20 μL isotope internal standard [ ¹³ C ₁₅ ]-DON (1 μg/mL), and finally the sample solution was passed through 0.22 μm Nylon filter membrane to be analyzed.

(3) The obtained samples to be detected are analyzed by liquid chromatography tandem mass spectrometer, and the results of B group trichothecenes and D3G residues are respectively obtained.

2.2 Liquid Chromatography Mass Spectrometry Conditions

(1) Liquid phase conditions: the chromatographic column is Agilent Extend-C18column (150mm×3.0 mm, 3.5μm); the mobile phase is 5mM ammonium acetate aqueous solution (A) and methanol (B), and the flow rate is 0.3mL/min. Gradient elution, the elution program is shown in Table 2 below:

Table 2 Mobile phase gradient elution program

(2) Mass spectrometry conditions: ion source mode: positive and negative ion mode (ESI ⁺ and ESI ^- ); spray voltage: 4.5kv (ESI ⁺ ) and -3.5ky (ESI ^- ); heating block temperature: 400 ° C; ion transfer tube temperature : 250°C; nebulizing gas and drying gas flow rates: 3mL/min and 15mL/min; the mass spectrometry parameters of the seven mycotoxins are shown in Table 3.

Table 3 Mass spectrometry parameters of each mycotoxin

Note: *, Quantitative ion; ^a , Quantitative ion abundance and quantitative ion abundance percentage;

3. Results and discussion

3.1 Optimization of mass spectrometry conditions for different mycotoxins

The mass spectrometry parameters of each mycotoxin were optimized under different mobile phase conditions (phase A: 0.05% ammonia solution or 5 mM ammonium acetate solution). The result shows that under 0.05% ammoniacal liquor condition, the relative ion ratio (qualitative ion abundance and quantitative ion abundance percentage) of NIV and 15ADON is extremely low (as shown in table 4), and this has greatly influenced detection sensitivity; On the contrary, in Under the condition of 5mM ammonium acetate solution, the ion ratios of all mycotoxins were greater than 25, indicating that the addition of ammonium acetate in the aqueous solution greatly improved the sensitivity of each mycotoxin. Therefore, 5mM ammonium acetate solution and methanol were selected as the final mobile phase. In addition, 3ADON and 15ADON are isomers. The two compounds cannot be separated by existing chromatographic equipment, and the two compounds produce the same molecular ion during ionization. In order to better distinguish these two compounds, positive and negative ion fast switching mode was used. The results showed that this method can distinguish and quantify 3ADON and 15ADON well.

The mass spectrometry parameters of each mycotoxin under the condition of 0.05% ammonia water in table 4

3.2 Optimization of pre-processing method

The method of the present invention investigates the purifying effects of different disperse solid phase extractants (GCB, PSA, cleaner silica, C18, etc.) and n-hexane on samples. The results show that the anhydrous MgSO ₄ +GCB adsorbent can remove the pigment in the sample and make the sample cleaner, but the recoveries of NIV, DON and D3G are low (<70%). In addition, the sensitivity of anhydrous MgSO ₄ +PSA (or cleanert silica, C18) to various mycotoxins in different matrices is greatly improved, but the recovery rate of D3G is not ideal (<60%). Therefore, anhydrous MgSO ₄ +n-hexane was selected as the best purification combination.

3.3 Matrix effect

The method of the present invention investigated the matrix effect of 7 mycotoxins in different matrices (Fig. 4). At the same time, the matrix effect was calibrated using the isotope internal standard ([ ¹³ C ₁₅ ]-DON), and it was found that 3ADON and 15ADON still had serious matrix effects (about 40%) in pig feed and corn after calibration by the internal standard method. Therefore, calibration curves corresponding to the matrices were used for accurate quantification of mycotoxins in feed and grain samples.

3.4 Linear relationship and limit of detection, limit of quantification

Standard working solutions of 7 kinds of mycotoxins were prepared with blank matrix (pig feed, chicken feed and corn). Concentration levels are shown in Table 5. The linear range, the limit of detection (LOD) and the limit of quantification (LOD) were investigated under the optimal experimental conditions. Correlation linearity and LOD, LOQ values are shown in Table 5

Table 5 The linear relationship, detection limit and quantification limit of 7 kinds of mycotoxins in different matrices

3.4. Recovery rate and precision experiment

Recovery rate test: a mixture of seven mycotoxins at high, medium and low levels was added to the blank matrix (concentrations of D3G and DOM were 400 μg/kg, 200 μg/kg, 20 μg/kg; DON, NIV, 3ADON, 15ADON and FUSX concentrations were 1000 μg/kg, 500 μg/kg, 50 μg/kg). Treat according to the method 2.1, each parallel 3 copies, the addition recovery results are shown in Table 6. Intra-day precision (RSDr) test: the mixed standard of 7 kinds of mycotoxins at three concentration levels of high, medium and low was added to the blank matrix (adding concentration, same as recovery test). On the same day, according to the pretreatment method in 2.1, each level was repeated 3 times. Day-to-day precision (RSDR) test: the mixed standard of 7 kinds of mycotoxins at three concentration levels of high, medium and low were added to the blank matrix (the addition concentration was the same as above). In 5 consecutive days, according to the method 2.1, each level was repeated 3 times.

The specific results of precision are shown in Table 6

The recovery rate and precision of 7 kinds of mycotoxins in different matrices in table 6

4 Conclusion

The invention uses an improved dispersive solid phase extraction method and a liquid phase tandem mass spectrometry method to simultaneously measure the contents of seven mycotoxins in feed and grain. The method is simple, economical and reliable, and has high sensitivity and stability. It solves the technical problems of the reported methods and provides a good technical support for ensuring feed safety in my country.

Claims (6)

1. a liquid chromatography tandem mass spectrometry method for measuring 7 kinds of mycotoxins in feed and grain, is characterized in that the method comprises the steps: (1) Weigh the pulverized feed or grain sample, add the extraction solution, vortex and ultrasonically oscillate to extract, centrifuge, and take the supernatant to be purified; (2) The supernatant was transferred to a centrifuge tube containing anhydrous magnesium sulfate and a degreasing solvent, and after vortex oscillation, the n-hexane layer was removed; the remaining solution was blown dry with nitrogen and made to volume; (3) The samples after constant volume were analyzed qualitatively and quantitatively by liquid chromatography tandem mass spectrometer for 7 kinds of mycotoxins. 2. the method for measuring 7 kinds of mycotoxins in a kind of liquid chromatography tandem mass spectrometry as claimed in claim 1 is characterized in that in described step (1), feed and grain sample are: pig feed, chicken feed and corn. 3. liquid chromatography tandem mass spectrometry as claimed in claim 1 measures the method for 7 kinds of mycotoxins in feed and grain, it is characterized in that the extraction solution in described step (1) is 84% acetonitrile-water solution, and methanol- aqueous solution. 4. a kind of liquid chromatography tandem mass spectrometry as claimed in claim 1 measures the method for 7 kinds of mycotoxins in feed and grain, it is characterized in that in described step (2), degreasing agent is to comprise normal hexane, sherwood oil, and other An organic solvent that can remove lipids. 5. the method for measuring 7 kinds of mycotoxins in a kind of liquid chromatography tandem mass spectrometry as claimed in claim 1 is characterized in that the mycotoxins measured in the described step (3) comprises: deoxynivalene alcohol (DON), nivalenol (NIV), 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15ADON) and fusarone-X (FUSX), deoxynivalenol-3-glucoside (D3G) and deepoxy-deoxynivalenol (DOM). 6. a kind of liquid chromatography tandem mass spectrometry as claimed in claim 1 measures feed and the method for 7 kinds of mycotoxins in grain, it is characterized in that in described step (3) in liquid phase tandem mass spectrometry, chromatographic column is for including but not limited to C ₁₈ , C ₈ chromatographic column, the mobile phase includes but not limited to methanol, acetonitrile or aqueous solution.