A method for rapid detection of vomitoxin content in pig liver and its application

Abstract

The present invention discloses a method and application for rapid detection of vomitoxin content in pig liver, comprising the following steps: S1. crushing and mixing the pig liver sample to be tested, adding formic acid aqueous solution, vortexing, ultrasonication, centrifugation, and obtaining supernatant; S2.IPS-RT-Y53 automatic column loading, collecting eluent; S3. preparing the liquid to be tested; S4. performing high performance liquid chromatography-mass spectrometry detection, obtaining the peak area of ​​vomitoxin, and calculating the content of vomitoxin in the pig liver to be tested according to the standard curve of vomitoxin. The method of the present invention not only removes most of the matrix interference in the sample, but also improves the pretreatment efficiency, and can accurately detect the content of vomitoxin in pig liver, with a detection limit of 0.147μg / kg; the recovery rate of spiked addition is 82.8% to 99.2%, the precision is 2.8% to 5.3%, the detection limit is low, the precision is high, and it is suitable for rapid and accurate qualitative and quantitative detection of vomitoxin in pig liver.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for rapidly detecting the content of vomitoxin in pig liver and its application. Background Art

[0002] Deoxynivalenol (DON), also known as deoxynivalenol (DON), is a common mycotoxin. In recent years, vomitoxin has been found in pig liver many times, posing a huge threat to people's food health and safety. However, the extraction and detection of vomitoxin in pig liver is still blank. At the same time, due to the influence of pig liver matrix effect, conventional detection methods are difficult to quantitatively detect vomitoxin. The detection methods of DON include: high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) detection method, thin layer chromatography (TLC) and colloidal gold immunochromatography technology. Among them, the rapid test strips based on TLC and colloidal gold immunochromatography technology are qualitative detection, and the other methods are quantitative detection. However, GC analysis and HPLC currently require complex pretreatment analysis, long machine time, and cannot produce results in a short time. Therefore, it is necessary to develop a method for rapid detection of vomitoxin content in pig liver to solve the above problems. Summary of the invention

[0003] The purpose of the present invention is to provide a method for quickly detecting the content of vomitoxin in pig liver and its application.

[0004] According to one aspect of the present invention, a method for rapidly detecting the content of vomitoxin in pig liver is provided, comprising the following steps:

[0005] S1. The pig liver sample to be tested was crushed with a grinder and mixed, the sample was weighed, an aqueous formic acid solution was added, vortexed, ultrasonicated and then centrifuged to obtain a supernatant and a precipitate;

[0006] S2. Transfer all the supernatant to the IPS-RT-Y53 automatic column analyzer for sample loading and collect the eluate in a centrifuge tube;

[0007] S3. Vortex the collected eluate and filter it through a microporous membrane to obtain a test solution;

[0008] S4. The test liquid is subjected to high performance liquid chromatography-mass spectrometry detection to obtain the peak area of ​​vomitoxin, and the content of vomitoxin in the test pig liver is calculated based on the standard curve of vomitoxin.

[0009] In some embodiments, the formic acid aqueous solution in step S1 is a formic acid aqueous solution with a concentration of 0.3%.

[0010] In some embodiments, the temperature condition of the ultrasonic treatment in step S1 is 4° C., and the speed of the centrifugation in step S1 is 8000-10000 r / min.

[0011] In some embodiments, the pore size of the microporous filter membrane in step S3 is 0.22 μm.

[0012] In some embodiments, the IPS-RT-Y53 automatic column analyzer in step S3 uses a DON-IPS special column, the model of the DON-IPS special column is Puruibon IAC-030-3, the specification is 25T, the column capacity is 3mL, the column pressure is 0.2MPa, the flow rate is 2mL / min, the activation liquid is methanol + water (volume ratio of 1:1), the eluent is water, and the eluent is methanol + 25% ammonium hydroxide solution + water (volume ratio of 7:0.5:2.5).

[0013] In some embodiments, the chromatographic conditions of the liquid phase in step S4 are: chromatographic column: Acquity UPLC BEHC18-HILIC-DIOL tandem chromatographic column system, the specifications of the Acquity UPLC BEH C18 chromatographic column are: 2.1 m×100 mm, 1.7 μm; the specifications of the HILIC-DIOL chromatographic column are: 4.6 m×250 mm, 5 μm; injection volume: 5 μL; column temperature: 30°C; flow rate: 0.20 mL / min; gradient elution is performed using mobile phases A and B, mobile phase A is 0.1% formic acid methanol solution, and mobile phase B is 0.1% formic acid pure water; the gradient elution program is as follows:

[0014] 0~0.5min, mobile phase A: 95%, mobile phase B: 5%;

[0015] 0.5~1.5min, mobile phase A: decreased from 95% to 35%, mobile phase B: increased from 5% to 65%;

[0016] 1.5-4.5 min, mobile phase A: 35%, mobile phase B: 65%;

[0017] 4.5-4.6 min, mobile phase A: increased from 35% to 95%, mobile phase B: decreased from 65% to 5%;

[0018] 4.6~6.0min, mobile phase A: 95%, mobile phase B: 5%.

[0019] In some embodiments, the mass spectrometry conditions in step S4 are: ion source: electrospray ionization source (ESI); scanning mode: multiple reaction monitoring (MRM); positive and negative ion mode: positive ion mode; mode temperature: 550°C; time: 6 min; capillary voltage: 1.00 kV; ion source temperature (TEM): 550°C; desolvation gas flow rate: nitrogen, 1000 L / h; collision gas: argon; the remaining nebulizing gas, curtain gas, and auxiliary gas used in mass spectrometry detection are all high-purity nitrogen.

[0020] In some embodiments, during mass spectrometry detection in step S4, the mass spectrometry parameters of DON are as follows:

[0021] The fragment ion pair (m / z) of DON was 297.1 / 202.9, the quantitative ion pair (m / z) was 297.1 / 202.9, the cone voltage was 20 V, and the collision energy was 16 V;

[0022] The fragment ion pair (m / z) of vomitoxin was 297.1 / 249.1, the quantitative ion pair (m / z) was 297.1 / 202.9, the cone voltage was 20V, and the collision energy was 10V.

[0023] According to another aspect of the present invention, a method for rapidly detecting the content of vomitoxin in pig liver is provided for use in detecting the content of vomitoxin in pig liver.

[0024] Beneficial effects of the present invention: The present invention establishes a method for determining the content of vomitoxin in pig liver by high performance liquid chromatography-mass spectrometry, which not only removes most of the matrix interference in the sample, but also improves the efficiency of the overall experimental pre-treatment. The standard curve of vomitoxin is obtained when the concentration range of vomitoxin is 1.00-80.0 ng / mL, wherein the linear correlation coefficient R 2 =0.999479, the detection limit is 0.147μg / kg; the spiked recovery rate of vomitoxin is 82.8% to 99.2% in the concentration range of 1.00-80.0ng / mL, and the precision is 2.8% to 5.3%. The present invention fills the gap in the detection of vomitoxin in pig liver matrix, has a low detection limit, high precision, is economical and practical, and has good sample extraction stability, and is suitable for rapid and accurate qualitative and quantitative detection of vomitoxin in pig liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The standard curve diagram of the vomitoxin in the spiked concentration range of 1.00-80.0 ng / mL in the method for rapidly detecting the vomitoxin content in pig liver of the present invention.

[0026] Figure 2This is the LC-MS / MS spectrum of vomitoxin when the Kinetex 1.7μXB-C18-HILIC-DIOL chromatographic column series system is used in Example 7 of the present invention.

[0027] Figure 3 This is the LC-MS / MS spectrum of vomitoxin when the Acquity UPLC BEH C18-HILIC-DIOL chromatographic column series system is used in Example 7 of the present invention.

[0028] Figure 4 This is the LC-MS / MS spectrum of vomitoxin when the Yuexu AQ-C18-HILIC-DIOL chromatographic column series system is used in Example 7 of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below in conjunction with the embodiments.

[0030] Example 1 A method for rapidly detecting the content of vomitoxin in pig liver comprises the following steps:

[0031] S1. Grind the pig liver sample to be tested with a grinder and mix it evenly. Weigh 2-6 g of the sample to an accuracy of 0.01 g, add 5-10 mL of 0.3% formic acid aqueous solution, vortex for 5-10 min, ultrasonicate for 10-20 min at 4 ° C, and centrifuge at 8000-10000 r / min for 5 min to obtain the supernatant and precipitate.

[0032] S2. Transfer all the supernatant to the IPS-RT-Y53 automatic column analyzer for sample loading. The IPS-RT-Y53 automatic column analyzer uses a DON-IPS special column. The model of the DON-IPS special column is Prebon IAC-030-3, the specification is 25T, the column capacity is 3mL, the column pressure is 0.2MPa, the flow rate is 2mL / min, the activation liquid is methanol + water (volume ratio is 1:1), the eluent is water, and the eluent is methanol + 25% ammonium hydroxide solution + water (volume ratio is 7:0.5:2.5). Collect 2mL of the eluate in a 15mL centrifuge tube;

[0033] S3. The collected eluate was vortexed and mixed for 1 min, and filtered through a 0.22 μm microporous filter membrane to obtain a test solution;

[0034] S4. The test liquid is subjected to high performance liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​vomitoxin, and the content of vomitoxin in the test pig liver is calculated based on the standard curve of vomitoxin.

[0035] (1) Liquid chromatography conditions

[0036] Chromatographic columns: The series chromatographic column system is: Acquity UPLC BEH C18- HILIC-DIOL, the specifications of the Acquity UPLCBEH C18 column are: 2.1 m×100 mm, 1.7μm; the specifications of the HILIC-DIOL column are: 4.6m×250mm, 5μm; injection volume: 5μL; column temperature: 30℃; flow rate: 0.20mL / min; mobile phases A and B are used for gradient elution, mobile phase A is 0.1% formic acid methanol solution, and mobile phase B is 0.1% formic acid pure water; the gradient elution conditions are shown in Table 1.

[0037] Table 1 Liquid phase gradient elution program

[0038]

[0039] (2) Mass spectrometry conditions

[0040] Ion source: electrospray ionization source (ESI); scanning mode: multiple reaction monitoring (MRM); positive and negative ion mode: positive ion mode; mode temperature: 550℃; time: 6min; capillary voltage: 1.00 kV; ion source temperature (TEM): 550℃; desolvation gas flow: nitrogen, 1000 L / h; collision gas: argon; the remaining nebulizer gas, curtain gas, and auxiliary gas used in mass spectrometry detection are all high-purity nitrogen. The mass spectrometry parameters of vomitoxin are shown in Table 2 below.

[0041]

[0042] (3) Establishment of standard curve

[0043] To the negative pig liver sample without vomitoxin, vomitoxin was added at the concentrations of 1.00 ng / mL, 2.00 ng / mL, 10.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, and 80.0 ng / mL, respectively, to obtain gradient spiked samples with vomitoxin concentrations of 1.00 ng / mL, 2.00 ng / mL, 10.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, and 80.0 ng / mL. The spiked sample solution to be tested was prepared according to steps S1-S3, and the LC-MS analysis was performed with reference to the above-mentioned liquid phase and mass spectrometry conditions. The standard working curves of vomitoxin were drawn with the concentration of vomitoxin added as the horizontal coordinate and the peak area as the vertical coordinate, and the linear regression equation and correlation coefficient were obtained. The results are shown in Tables 3 and Figure 1 .

[0044] Table 3 Standard curve regression equation and correlation coefficient table

[0045]

[0046] As shown in Table 3 and Figure 1 As shown, the detection method of this embodiment has a good linear relationship within the linear range of 1.00-80.0 ng / mL spiked concentration of vomitoxin, and the correlation coefficient R 2 >0.999479.

[0047] Example 2 Determination of the detection limit of a method for rapid detection of vomitoxin content in pig liver

[0048] According to the detection method of Example 1, seven parallel addition experiments were performed with crushed pig liver as the matrix, and the concentration of vomitoxin added was 1.00 μg / kg. At the same time, a sample blank experiment was performed, and pretreatment was performed according to steps S1-S3 in Example 1. The concentration of vomitoxin in each sample was measured on the machine using the external standard method, and with reference to the following calculation formula, it was calculated that the detection limit of the detection method of the present invention for vomitoxin in pig liver was 0.147 μg / kg.

[0049] The calculation formula is as follows:

[0050] Limit of detection (LOD) = K × Sb × C / X (_)

[0051] Where: K is the confidence factor, which is 3; Sb is the standard deviation of the content of parallel test samples; C is the spike concentration; X(_) is the average value of the content of parallel samples.

[0052] Example 3 Recovery and precision determination of a method for rapid detection of vomitoxin content in pig liver

[0053] The crushed pig liver samples were spiked with low, medium and high concentrations of DON (1.00 μg / kg, 2.00 μg / kg and 10.0 μg / kg, respectively), and a sample blank experiment was performed. Six parallel tests were performed for each concentration level according to the detection method of Example 1, and the precision (RSD, %) and recovery (%) of each group were calculated at the same time. The results are shown in Table 4.

[0054] Table 4 Recovery and precision of vomitoxin spike

[0055]

[0056] As can be seen from Table 4, when samples with different added concentrations were detected by liquid chromatography-mass spectrometry, the recovery rate of the detection method of the present invention was 82.8% to 99.2%, and the precision was 2.8% to 5.3%, indicating that the detection method of the present invention has high precision and recovery rate.

[0057] Example 4 Effect of different extraction agents on the recovery rate of vomitoxin

[0058] Deoxynivalenol (deoxynivalenol) has a large solubility in aqueous solution, and the extraction process of aqueous solution is relatively simple. Considering the use of aqueous solution as the extractant to complete the corresponding extraction, however, since the structure of deoxynivalenol itself in solvent water is relatively easy to decompose, its stability under different pH conditions is different. When the pH value is high or low, its solubility in the extract will be affected. Therefore, it is necessary to add an appropriate amount of formic acid solution as a buffer to help adjust the pH value of the extractant. Since deoxynivalenol is a polar compound with a strong affinity for water, adding appropriate formic acid to the extractant can increase the interaction between the extractant and deoxynivalenol and improve the extraction efficiency.

[0059] 10mL of water, 0.1% formic acid aqueous solution, 0.3% formic acid aqueous solution, 0.5% formic acid aqueous solution, methanol, 0.1% formic acid methanol solution, 0.3% formic acid methanol solution, 0.5% formic acid methanol solution, acetonitrile, 0.1% formic acid acetonitrile solution, 0.3% formic acid acetonitrile solution, 0.5% formic acid acetonitrile solution, a total of 12 different ratio systems were used as different treatments of the extractant, and 3 replicates were set for each treatment. Referring to the method of Example 1, vomitoxin was detected, and the extraction efficiency of the above-mentioned treatments was compared and analyzed, and the most suitable extractant was finally determined. The specific results are shown in Table 5.

[0060] Table 5 Comparison of extraction efficiency of different solvents

[0061]

[0062] Among the above 12 extractants, 10mL of methanol solution and acetonitrile solution have average extraction effects, and adding formic acid cannot improve the recovery rate. After adding formic acid to the aqueous solution, the recovery rate ranges from 60% to 96%, showing a relatively excellent extraction effect, especially the 0.3% formic acid aqueous solution has a relatively excellent recovery rate, ranging from 85% to 96%, with high precision and good stability, and can have a good extraction effect on vomitoxin in pig liver samples. Therefore, considering the above factors, the present invention uses 0.3% formic acid aqueous solution as the extraction agent.

[0063] Example 5 Effects of different column flow methods on the column flow efficiency, controllability, sample saving, and repeatability of DON

[0064] In this example, conventional (manual) column flow and IPS-RT-Y53 automatic column flow were used as the objects of investigation, and the "time cost", "number of single column flow", "column flow rate and pressure per unit time", "remaining sample volume before and after column flow", "recovery rate" and "precision" were used as indicators to compare the effects of conventional (manual) column flow and IPS-RT-Y53 automatic column flow on the detection of vomitoxin. In this example, the detection of 40 pig liver samples using conventional (manual) column flow and IPS-RT-Y53 automatic column flow were compared. The column flow used 5.00 mL of activation solution, which was methanol + water (volume ratio of 1:1), 10.0 mL of extraction sample solution, 2.00 mL of eluent, which was water, and 2.00 mL of eluent, which was methanol + 25% ammonium hydroxide solution + water (volume ratio of 7:0.5:2.5). The specific results are shown in Table 6.

[0065] Table 6 Comparison of two column passing methods

[0066]

[0067] Conventional (manual) column passing requires manual transfer of the reserve solution to the column one by one, but the present invention uses the IPS-RT-Y53 automatic column passing instrument, which can process 40 sample solutions at the same time, greatly saving the time for transferring the sample solutions. Taking 40 samples as an example, the labor time cost saved is 3.5 hours, which greatly shortens the sample purification time.

[0068] During the conventional (manual) column flow, solution dripping and liquid residue in the column are prone to occur, which increases the sample loss in the pre-treatment process. The IPS-RT-Y53 automatic column flow meter uses automated operation to control the column flow pressure, which effectively ensures the sample loss before and after the column flow. As shown in Table 6, the average residual volume of 10mL sample extract after manual column flow is 8.4mL, while the average residual volume of the sample after the IPS-RT-Y53 automatic column flow meter is 9.8mL, which effectively saves samples.

[0069] During conventional (manual) column passing, the flow rate per unit time will change. In the case of more liquid in the early stage, the pressure in the column is higher and the flow rate per unit time is faster. However, as the amount of liquid decreases, the pressure in the column decreases, and the flow rate will slow down. The overall unit flow rate range is between 1mL / min and 3mL / min, and bubble blockage is prone to occur. After the column is completed, the solvent needs to be cleaned to flow out of the column, which greatly increases the time of the column. The IPS-RT-Y53 automatic column passing instrument can regulate the column pressure, stabilize the column pressure at 0.2MPa, and ensure the flow rate per unit time at 2mL / min, ensuring the controllability of the column process. At the same time, the column can be automatically cleaned after the column is completed, saving a lot of time.

[0070] From the results in Table 6, it can be seen that the use of IPS-RT-Y53 automatic column flowmeter can greatly shorten the column flow time for extracting vomitoxin from pig liver. At the same time, the pressure and flow rate can be controlled during the column flow, which effectively saves the sample volume. According to the subsequent test results, it can be found that the recovery rate and precision of vomitoxin are good, with a recovery rate between 94% and 101%, which is about 20 percentage points higher than manual column flow. The precision is also greatly improved, with excellent reproducibility.

[0071] Example 6 Effects of different column conditions on the recovery rate and precision of vomitoxin

[0072] After selecting the IPS-RT-Y53 automatic column analyzer, the specific parameters of "different affinity column types", "column pressure" and "flow rate per unit time" were compared and explored. The specific results are shown in Table 7.

[0073] Table 7 Comparison of recovery and precision of DON under different column conditions

[0074]

[0075] As shown in Table 7, the IPS-RT-Y53 automatic column analyzer has the best recovery and precision for vomitoxin when using a DON-IPS column (Purebon IAC-030-3, 25T, 3mL), a column pressure of 0.2Mpa, and a flow rate per unit time of 2.00 mL / min. The DON-IPS column selectively captures vomitoxin by using its specificity in the mixed solution, which can better distinguish vomitoxin from other impurities and obtain vomitoxin with higher purity. At the same time, the optimal flow rate and column pressure can minimize the residence time of the target in the column while maintaining the recovery rate and precision, avoiding sample loss and improving column efficiency.

[0076] The IPS-RT-Y53 automatic column flow meter used in this embodiment can achieve the following results: ① The 32-bit high-speed embedded distributed control system can complete the processing of 40 extracts at the same time, and the time used is only 20% of that of manual column flow. This system can realize multi-method editing, can be directly called when used, and can be controlled in sections, and can preset column flow parameters for different batches; ② The operating instrument is driven by a contactless touch panel, equipped with a 10-inch full-color wide-angle touch screen, guided operation, and provides humanized interaction. It can monitor in real time with closed-loop control, dynamically detect the flow rate through the column, and automatically adjust the pressure through the column according to the actual situation of the column to ensure the stability of the flow rate through the column. At the same time, real-time detection can prevent the escape of toxins, effectively avoid the leakage of vomitoxin, and ensure the safety of laboratories and testing personnel; ③ It contains a flow path pre-filling function, which can realize instrument cleaning and reagent filling, and the waste liquid is automatically extracted to the waste liquid port to avoid volatile pollution; ④ It can realize unattended operation, and the whole process of loading, enrichment, elution, elution, and collection is automatically completed, which effectively saves labor costs; ⑤ Using a DON-IPS special column, the specific degassed snow mold enol (DON) monoclonal antibody is cross-linked on agarose gel microspheres, based on antigen-antibody specific binding, toxins are effectively captured, impurities are separated, and the sample is purified, and the loss of vomitoxin can be minimized; ⑥ According to previous literature reports, there is no precedent for using the automatic column mode for the treatment of toxin substances. Considering the toxicity of toxin substances and their structural particularity, the present invention creatively adopts an automatic column instrument to optimize the experimental process. In summary, the IPS-RT-Y53 automatic column instrument of this embodiment is used for the purification of vomitoxin in pig liver, which has the advantages of high efficiency, sensitivity, simplicity, economy and safety.

[0077] Example 7 Effects of different chromatographic column systems on the separation effect and signal intensity of vomitoxin

[0078] In this example, the chromatographic peak separation effect and signal intensity obtained by using different chromatographic columns in series were compared, and three chromatographic column series systems were investigated, namely, ①Kinetex 1.7μXB-C18 (100A 100×2.1 mm)-HILIC-DIOL (4.6m×250 mm, 5μm), ②Acquity UPLC BEH C18 (2.1 m×100 mm, 1.7μm)-HILIC-DIOL (4.6m×250 mm, 5μm), and ③Yuexu AQ-C18 (3.0×100mm, 1.7μm)-HILIC-DIOL (4.6m×250 mm, 5μm).

[0079] Standard solutions were prepared, and standard solutions of vomitoxin with concentrations of 1.00, 2.00, 5.00, 8.00, and 10.0 ng / ml were tested on the machine to examine the column efficiency of the above three chromatographic column combination systems. The results are shown in Table 8 and Figure 2-Figure 4 .

[0080] Table 8 Linear regression equation, separation and correlation coefficient of three chromatographic column series systems

[0081]

[0082] As shown in Table 8, among the three chromatographic column series systems, Kinetex 1.7μXB-C18- HILIC-DIOL has the worst column efficiency, with a linear correlation coefficient of 0.98432, which is difficult to meet the requirement of 0.99, and the separation is low. Compared with Yuexu AQ-C18- HILIC-DIOL, Acquity UPLC BEHC18- HILIC-DIOL can obtain higher precision, and the separation is 1.9 higher than the latter, which can achieve better results. The recovery rate and precision are also consistent with the performance of related parameters. The Acquity UPLC BEH C18- HILIC-DIOL column series system can obtain higher recovery rate and better precision. At the same time, Figure 2-Figure 4 It can be seen that the separation effect of the target peak and the impurity peak in the chromatographic column series system ① and ③ is poor, and the target peak of vomitoxin has a tailing phenomenon, and the peak area response of the chromatographic peak is low. The target peak of system ② has a sharp peak shape, high response, and good separation from other chromatographic peaks. Therefore, the present invention selects the chromatographic column series system ②Acquity UPLCBEH C18 (2.1 m×100 mm, 1.7μm)-HILIC-DIOL (4.6m×250 mm, 5μm) to separate vomitoxin.

[0083] The present invention creatively applies the dual-column series mode for the separation of vomitoxin for the first time. Since vomitoxin is a secondary product of polar fungal toxins, it causes great interference in the separation process of the target. When the sample matrix is ​​complex and the target content is high, the column efficiency of the chromatographic column will be greatly reduced, and it will cause great health risks to the experimental environment and the experimenters themselves. The non-polar C18 column and the hydrophilic column HILIC are used in series, which can not only greatly reduce the interference of the sample matrix itself (weakly polar amino acids and fatty acids, etc.) at the front end of the separation, but also reduce the interference of impurities on vomitoxin, improve the column efficiency of the chromatographic column of the overall separation system, and effectively separate the vomitoxin and impurity interference components in the sample, greatly improving the accuracy of the detection system and the sample recovery rate. At the same time, the C18 column at the front end of the dual-series system is equivalent to the step of pre-treatment solid phase extraction, which allows more impurities to remain at the front end, which is good for the environment and personnel health, further reduces the workload of detection and reduces the residual toxin impurities, and creatively provides a safe and effective way for the detection of vomitoxin.

[0084] Example 8 Effects of different mobile phases on the results of DON assay

[0085] When the mobile phase A of the present invention is 0.1% formic acid methanol solution and the mobile phase B is formic acid aqueous solution, the vomitoxin obtained in the above extraction process has better peak appearance, higher recovery rate and accuracy. Therefore, this embodiment mainly discusses the effect of mobile phase B formic acid aqueous solution on the efficiency of vomitoxin extraction in pig liver at different ratios.

[0086] The spiked sample was prepared with a concentration of 10 ng / mL, and the detection method of Example 1 was used to determine the content of vomitoxin in pig liver. In the mobile phase conditions, mobile phase A was 0.1% formic acid methanol solution, and mobile phase B was formic acid aqueous solution with volume ratios of 0%, 0.01%, 0.05%, 0.1%, 0.2%, and 0.5%, respectively. The spiked recoveries and retention times of the samples were determined with the six different proportions of mobile phase B. The specific results are shown in Table 9.

[0087] Table 9 Recovery and precision of spiked vomitoxin with different mobile phases B

[0088]

[0089] As shown in Table 9, when formic acid is added to mobile phase B, the recovery rate and precision are improved, and when 0.1% formic acid aqueous solution is selected, the recovery rate and precision are optimal. This is because formic acid has good ion pairing ability, can combine with charged vomitoxin to form ion pairs, increase its polarity and solubility in the liquid phase, thereby accelerating its migration speed in the chromatographic column, and increasing its Coulomb integral area, improving the response signal. In addition, the formic acid aqueous solution can also play a buffering role in separation, maintaining the stability and constant pH of the mobile phase. However, as the content of formic acid increases, the recovery rate and precision decrease instead. This may be due to the increase in the formic acid concentration, resulting in an excessive number of ion pairs formed by vomitoxin and formic acid, resulting in a low signal-to-noise ratio, making the test results unstable and interfering. In addition, formic acid is highly acidic, and excessive concentration can easily cause the chromatographic column to fail, affecting the accuracy of the test. Therefore, the present invention uses 0.1% formic acid aqueous solution as mobile phase B, which can promote the precipitation of vomitoxin and improve the sensitivity and accuracy of the test results.

[0090] Example 9 A comparative test of a method for rapid detection of vomitoxin content in pig liver

[0091] The detection method of Example 1 was used to perform liquid chromatography-mass spectrometry on 10 pig livers randomly purchased from the farmers' market. The 10 samples were all subjected to double parallel determination, and the average concentration was taken as the final determination concentration of vomitoxin in the sample. The specific determination results are shown in Table 10.

[0092] Table 10 Measured results of vomitoxin content in pig liver samples

[0093]

[0094] Note: “ND” in the table means not detected.

[0095] As shown in the test results in Table 10, vomitoxin was detected in pig liver (samples 1, 3, 5, 6, 7, 8, and 10), and vomitoxin was not detected in pig liver (samples 2, 4, and 9). The detection rate of vomitoxin in pig liver was as high as 14%.

[0096] The present invention compares different types of extractants, the effects of manual column flow and IPS-RT-Y53 automatic column flow, the peak effects of different chromatographic columns, the comparison of different mobile phase systems and the method conditions of high performance liquid chromatography-mass spectrometry detection. Based on this, a method for determining the content of vomitoxin in pig liver by high performance liquid chromatography-mass spectrometry is established, which not only removes most of the matrix interference in the sample, but also improves the efficiency of the overall experimental pretreatment. The standard curve of vomitoxin is obtained when the concentration range of vomitoxin is 1.00-80.0 ng / mL, wherein the linear correlation coefficient R 2=0.999479, the detection limit is 0.147μg / kg; the spiked recovery rate of vomitoxin is 82.8% to 99.2% and the precision is 2.8% to 5.3% in the concentration range of 1.00-80.0ng / mL; the present invention avoids the shortcomings of cumbersome operation, long time consumption, poor sensitivity and specificity of thin layer chromatography, enzyme-linked immunosorbent assay and liquid chromatography in the current common standard for vomitoxin detection methods, and fills the gap in the detection of vomitoxin in pig liver matrix. The present invention has a low detection limit, high precision, economical and practical, and good sample extraction stability, so that the detection method of the present invention is suitable for rapid and accurate qualitative and quantitative detection of vomitoxin in pig liver.

[0097] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for rapidly detecting the content of vomitoxin in pig liver, comprising the following steps: S1. The pig liver sample to be tested was crushed with a grinder and mixed, the sample was weighed, an aqueous formic acid solution was added, vortexed, and centrifuged after ultrasonication to obtain a supernatant and a precipitate, wherein the aqueous formic acid solution is an aqueous formic acid solution with a concentration of 0.3%; S2. All the supernatant was transferred to the IPS-RT-Y53 automatic column analyzer for sample loading, and the eluent was collected in a centrifuge tube. The IPS-RT-Y53 automatic column analyzer used a DON-IPS special column. The model of the DON-IPS special column was Prebon IAC-030-3, the specification was 25T, the column capacity was 3mL, the column pressure was 0.2MPa, the flow rate was 2mL / min, the activation solution was methanol + water, the volume ratio was 1:1, the eluent was water, and the eluent was methanol + 25% ammonium hydroxide solution + water, the volume ratio was 7:0.5:2.5; S3. Vortex the collected eluate and filter it through a microporous membrane to obtain a test solution; S4. The test liquid is subjected to high performance liquid chromatography-mass spectrometry to obtain the peak area of ​​vomitoxin, and the content of vomitoxin in the pig liver to be tested is calculated based on the standard curve of vomitoxin. The chromatographic conditions of the liquid phase are as follows: chromatographic column: Acquity UPLC BEH C18-HILIC-DIOL tandem chromatographic column system, the specifications of the Acquity UPLC BEH C18 chromatographic column are: 2.1 m×100 mm, 1.7μm; the specifications of the HILIC-DIOL chromatographic column are: 4.6m×250 mm, 5μm; injection volume: 5μL; mobile phases A and B are used for gradient elution, mobile phase A is 0.1% formic acid methanol solution, and mobile phase B is 0.1% formic acid pure water.

2. The method for rapidly detecting the content of vomitoxin in pig liver according to claim 1, characterized in that: The temperature condition of the ultrasonic treatment in step S1 is 4° C., and the speed of the centrifugation in step S1 is 8000-10000 r / min.

3. The method for rapidly detecting the content of vomitoxin in pig liver according to claim 1, characterized in that: The pore size of the microporous filter membrane in step S3 is 0.22 μm.

4. The method for rapidly detecting the content of vomitoxin in pig liver according to claim 1, characterized in that: The liquid chromatography conditions in step S4 are: column temperature: 30°C; flow rate: 0.20 mL / min; and the gradient elution procedure is as follows: 0~0.5min, mobile phase A: 95%, mobile phase B: 5%; 0.5-1.5min, mobile phase A: decreased from 95% to 35%, mobile phase B: increased from 5% to 65%; 1.5-4.5 min, mobile phase A: 35%, mobile phase B: 65%; 4.5-4.6 min, mobile phase A: increased from 35% to 95%, mobile phase B: decreased from 65% to 5%; 4.6~6.0min, mobile phase A: 95%, mobile phase B: 5%.

5. The method for rapidly detecting the content of vomitoxin in pig liver according to claim 1, characterized in that: The mass spectrometry conditions in step S4 are: ion source: electrospray ionization source; scanning mode: multiple reaction monitoring; positive and negative ion mode: positive ion mode; mode temperature: 550°C; time: 6 min; capillary voltage: 1.00 kV; ion source temperature: 550°C; desolvation gas flow rate: nitrogen, 1000 L / h; collision gas: argon; the remaining nebulizing gas, curtain gas, and auxiliary gas used in mass spectrometry detection are all high-purity nitrogen.

6. The method for rapidly detecting the content of vomitoxin in pig liver according to claim 1, characterized in that: During mass spectrometry detection in step S4, the mass spectrometry parameters of vomitoxin are as follows: The fragment ion pair of DON was 297.1 / 202.9, the quantitative ion pair was 297.1 / 202.9, the cone voltage was 20 V, and the collision energy was 16 V; The fragment ion pair of vomitoxin was 297.1 / 249.1, the quantitative ion pair was 297.1 / 202.9, the cone voltage was 20V, and the collision energy was 10V.

7. Use of the method for rapidly detecting the content of vomitoxin in pig liver according to any one of claims 1 to 6 in detecting the content of vomitoxin in pig liver.

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