Improved QuEChERS-UPLC-MS / MS Method for the Determination of 15 Mycotoxins in Food and Drug Dual-Purpose Substances

Abstract

The present invention discloses an improved method for rapidly determining 15 mycotoxins in food and drug dual-purpose substances by QuEChERS-UPLC-MS / MS, which relates to a method for detecting 15 mycotoxins in malt, coix seed, lotus seed and lily by QuEChERS purification-ultra high performance liquid chromatography tandem mass spectrometry. In the present invention, the sample is extracted. After the sample solution is purified, 15 mycotoxins are simultaneously detected by ultra high performance liquid chromatography tandem mass spectrometry, and quantitative analysis of 15 mycotoxins is carried out by preparing a mixed standard working solution. Among them, the pretreatment adopts a QuEChERS purification package material with an optimized ratio. Compared with the prior art, the present invention greatly improves the purification degree of the sample, and has simple operation and low cost; the ultra high performance liquid chromatography tandem mass spectrometry has good separation effect, fast analysis speed, can simultaneously detect a variety of mycotoxins, saves operation time, has high sensitivity, accurate quantification and good repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of instrument detection, and particularly relates to a method for improving the rapid determination of 15 mycotoxins in food and drug dual-purpose substances by QuEChERS-UPLC-MS / MS. Background Art

[0002] The food and drug dual-purpose substances announced by the National Health Commission of China are not only traditional Chinese medicinal materials but also foods widely consumed by people. In recent years, with the improvement of people's living standards, foods with medicinal effects have become increasingly popular among consumers. Therefore, compared with ordinary Chinese medicinal materials, food and drug dual-purpose substances have a higher usage frequency. However, food and drug dual-purpose substances are susceptible to mold infection during growth, harvesting, storage, and transportation, contaminating various mycotoxins, which seriously affects the safety and effectiveness of food and drug dual-purpose substances. Therefore, it is necessary to pay attention to the mold and mycotoxin contamination in food and drug dual-purpose substances, control their quality, and reduce the poisoning risk of consumers.

[0003] Mycotoxin is a class of secondary metabolites with high toxicity that can easily cause pathological changes and physiological abnormalities in humans and animals, produced by various toxigenic molds under suitable conditions. More than 400 mycotoxins have been discovered. In recent years, there have been endless reports on mycotoxins in food and drug dual-purpose substances. Among these more than 400 mycotoxins, the ones that have been studied more deeply are mainly aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), HT-2 toxin (HT-2), etc., which have a relatively high contamination rate, strong toxicity, and seriously endanger human health. These mycotoxins not only have carcinogenic, teratogenic, and mutagenic effects, but also have hepatotoxicity, toxic renal damage, reproductive toxicity, and immunosuppressive effects, seriously affecting the quality and safety of food and drug dual-purpose substances and posing a great threat to human health.

[0004] Common and frequently used substances with both food and drug uses mainly include malt, coix seed, lily, lotus seed, etc. Due to their complex matrix components, the matrix effect is relatively large during the detection process, resulting in problems such as low recovery rate and difficult detection of target compounds. Therefore, it is particularly important to select a suitable pretreatment method. Currently, the sample pretreatment methods for mycotoxin detection mainly include extraction method, QuEChERS method, immunoaffinity column purification method, etc. However, the extraction method uses a large amount of solvent and has cumbersome operations, while the immunoaffinity column purification method has high costs and long time consumption. The QuEChERS method has the characteristics of being fast, simple, inexpensive, effective, reliable, and safe compared with other methods, and it is the preferred choice for the pretreatment method of mycotoxin detection in substances with both food and drug uses at present. There are a large number of QuEChERS products for pesticide residues on the market currently, and the QuEChERS technology for pesticide residues has been quite mature. However, the QuEChERS technology specifically for mycotoxin detection is not yet mature, and selecting an adsorbent suitable for mycotoxin purification is the key to the development of mycotoxin detection technology. On the other hand, since substances with both food and drug uses are all dried in the sun or by drying, their water content is relatively low. Since the original QuEChERS method mainly targets some substances with relatively high water content (such as fruits and vegetables), and substances with both food and drug uses contain a large amount of components such as pigments, organic acids, polysaccharides, and proteins, the extraction efficiency of the original QuEChERS method is not ideal, and its composition and ratio need to be optimized. In addition, a suitable extraction solvent can not only reduce impurities in the sample such as fat and protein, reduce the matrix effect, but also improve the recovery rate. In mycotoxin detection, organic solvent plus water is generally used as the solvent for sample extraction. The water in the extract can moisten the matrix and enhance the penetration ability of the organic solvent. However, the properties of different mycotoxins are significantly different. When using one extraction solvent to extract multiple mycotoxins simultaneously, the recovery rate of each mycotoxin also varies. Therefore, the selection and optimization of the extraction solvent are very important.

[0005] The main mycotoxin detection methods include thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), liquid chromatography (LC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc. TLC belongs to semi-quantitative technology, and this method has low sensitivity and poor repeatability. ELISA is suitable for the screening of single mycotoxins. Due to the cross-reaction between antibodies and false positive or false negative reactions, it cannot be used for the simultaneous detection and final confirmation of multiple mycotoxins. The LC method is the mainstream method for mycotoxin detection currently, and it is mostly used for the detection of one or the same type of mycotoxins, but it has a long analysis time and complex operations. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) combines the advantages of high separation efficiency, fast separation speed, and simple quantitative analysis of chromatography, and also has the characteristics of high sensitivity and strong qualitative ability of mass spectrometry, and can achieve the detection purposes of both qualitative and quantitative analysis simultaneously. Currently, this method has developed rapidly in the simultaneous detection of multiple mycotoxins. Summary of the Invention

[0006] Object of the Invention: Aiming at some problems existing in the prior art, the present invention provides an improved QuEChERS-UPLC-MS / MS method for rapidly determining 15 mycotoxins in food and drug dual-purpose substances. This method has simple sample pretreatment, does not require the addition of expensive isotope internal standards during the detection process, has low cost and short detection time. It mainly solves the problems of complex and difficult sample pretreatment of food and drug dual-purpose substances, and makes up for the deficiency in the detection of multiple mycotoxins in food and drug dual-purpose substances.

[0007] Technical Solution: In order to achieve the above object, the present invention provides an improved QuEChERS-UPLC-MS / MS method for rapidly determining 15 mycotoxins in food and drug dual-purpose substances, which includes the following steps:

[0008] (1) Sample extraction

[0009] Using acetonitrile-acetic acid aqueous solution as the extraction solution, mixing it with the food and drug dual-purpose substance sample and then performing ultrasonic treatment, and centrifuging to obtain the sample supernatant.

[0010] (2) Sample purification

[0011] Adding QuEChERS purifying agent to the sample supernatant obtained by centrifugation, mixing and then centrifuging to obtain the supernatant, and drying it.

[0012] (3) UPLC-MS / MS detection

[0013] Redissolving with methanol solution, filtering through a membrane and then performing UPLC-MS / MS detection. At the same time, preparing a mixed standard working solution for quantitative analysis of 15 mycotoxins.

[0014] Among them, in step (1), 60% - 90% acetonitrile-acetic acid aqueous solution is used as the extraction solution, and the 15 mycotoxins in the food and drug dual-purpose substance sample are extracted according to a solid-liquid ratio of 1:10 - 1:20 (g / mL). After vortexing the extraction solution and the sample for 1 - 5 min, ultrasonic treatment for 10 - 30 min and then centrifuging, the sample supernatant is obtained.

[0015] Preferably, in step (1), 80% acetonitrile aqueous solution containing 1% acetic acid is used as the extraction solution, the solid-liquid ratio is 1:20 (g / mL), vortex for 1 min, and ultrasonic treatment for 20 min.

[0016] Among them, the food and drug dual-purpose substance sample in step (1) includes any one or more of malt, coix seed, lotus seed and lily.

[0017] Among them, the 15 mycotoxins in the food and drug dual-purpose substance sample in step (1) include aflatoxin B 1 、B 2, G 1 , G 2 , deoxynivalenol (DON), ochratoxin A (OTA), fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2 ), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), β-zearalanol (β-ZAL), any one or more of them.

[0018] Among them, the composition and proportion of the QuEChERS purifying agent in step (2) are: 200 - 300 mg of anhydrous MgSO 4 , 50 - 100 mg of PSA, 50 - 100 mg of C18, 50 - 100 mg of Silica, 20 - 100 mg of GCB.

[0019] Preferably, the composition and proportion of the QuEChERS purifying agent in step (2) are: 240 mg of anhydrous MgSO 4 , 80 mg of PSA, 80 mg of C18, 80 mg of Silica, 25 mg of GCB.

[0020] Among them, the ratio of the QuEChERS purifying agent to the sample supernatant in step (2) is 35:1 - 70:1 (mg / mL); after vortexing for 1 - 3 min and mixing evenly, then centrifuging to take the supernatant, taking 2 - 10 mL, and drying it under nitrogen at 40 - 50 °C.

[0021] Preferably, the ratio of the QuEChERS purifying agent to the sample supernatant in step (2) is 505:10 (mg / mL); after vortexing for 1 min and mixing evenly, then centrifuging to take the supernatant, taking 5 mL, and drying it under nitrogen at 45 °C.

[0022] Among them, in step (3), it is reconstituted with a methanol solution with a volume fraction of 10% - 50%, and after passing through a 0.22 μm filter membrane, it is detected by UPLC-MS / MS.

[0023] Preferably, the concentration of the methanol solution with a volume fraction in step (3) is 35%.

[0024] Among them, the liquid chromatography conditions of UPLC-MS / MS in step (3) are:

[0025] (1) Mobile phase: Phase A is formic acid - ammonium acetate aqueous solution, Phase B is methanol; flow rate: 0.1 - 0.3 mL / min; column temperature: 25 - 40 °C; injection volume: 1 - 5 μL;

[0026] (2) The elution gradient program is as follows:

[0027]

[0028] Preferably, the liquid chromatography conditions for UPLC-MS / MS in step (3) are as follows: mobile phase A is 0.1% formic acid - 1 mM ammonium acetate aqueous solution; flow rate is 0.2 mL / min; column temperature is 40 °C; injection volume is 1 μL. The elution gradient program is 0 - 5 min, 80% - 5% A; 5 - 9 min, 5% A; 9 - 10 min, 5% - 80% A; 10 - 15 min, 80% A.

[0029] Among them, the mass spectrometry conditions for UPLC-MS / MS in step (3) are as follows:

[0030] (1) Ion source: electrospray ionization source (ESI);

[0031] (2) Scanning mode: positive and negative ions simultaneously;

[0032] (3) Monitoring mode: multiple reaction monitoring (MRM);

[0033] (4) Interface voltage: 3.0 - 5.0 kV;

[0034] (5) Ion source temperature: 280 - 320 °C;

[0035] (6) Desolvation temperature: 500 - 550 °C;

[0036] (7) Nebulizing gas flow rate: 2.5 - 3.5 L / min;

[0037] (8) Heating gas flow rate: 8 - 12 L / min.

[0038] Preferably, the mass spectrometry conditions for UPLC-MS / MS in step (3) are as follows:

[0039] (1) Ion source: electrospray ionization source (ESI);

[0040] (2) Scanning mode: positive and negative ions simultaneously;

[0041] (3) Monitoring mode: multiple reaction monitoring (MRM);

[0042] (4) Interface voltage: 4.0 kV;

[0043] (5) Ion source temperature: 300 °C;

[0044] (6) Desolvation temperature: 526 °C;

[0045] (7) Nebulizing gas flow rate: 3.0 L / min;

[0046] (8) Heating gas flow rate: 10 L / min.

[0047] Further, the multiple reaction monitoring ion pairs and mass spectrometry related parameters of the 15 mycotoxins in step (3) are shown in Table 1:

[0048] Table 1 Mass spectrometry conditions for 15 mycotoxins

[0049]

[0050] * is the quantitative ion.

[0051] The focus of the present invention is first to select and optimize the extraction solvent for the problem of low recovery rate of individual toxins, and to develop a pretreatment method with low cost, simple operation and good purification effect for the problem of complex matrix of food and drug dual-purpose substances. In addition, a UPLC-MS / MS method for simultaneous detection of multiple mycotoxins is established. Without using isotope internal standards, good sensitivity, accuracy and precision can still be obtained. Moreover, due to the use of an ultra-high performance liquid system, the separation efficiency is greatly improved and the analysis time is shortened. The analysis of 15 mycotoxins can be completed within 6 minutes. The pretreatment method of the present invention uses a purifying agent in a specific ratio, and the addition amount of the purifying agent needs to be appropriately controlled. Excessive addition will cause the mycotoxins in the sample to be adsorbed, resulting in inaccurate detection results, while too little addition will cause incomplete adsorption of impurities and insignificant purification effect. The matrix of food and drug dual-purpose substances is complex, containing a large amount of plant pigments, acids, sugars, proteins, etc. Therefore, it is necessary to achieve the best purification conditions by specifically adjusting the ratio of the purifying agent. On the basis of removing impurities to the greatest extent, the accuracy of the detection results should also be ensured.

[0052] As a method for simultaneous detection of multiple mycotoxins, especially for samples with complex matrices such as food and drug dual-purpose substances, the method of the present invention does not use expensive immunoaffinity columns for purification in the pretreatment, but uses cheap purifying agents, which not only eliminates the cumbersome steps of activation, washing, elution, etc. of the immunoaffinity column, but also greatly reduces the detection cost, and is more suitable for the detection of large batches of samples. In addition, in the subsequent detection, the present invention does not need to use expensive isotope internal standards for result correction, and can also achieve good recovery rate and precision. Due to the introduction of ultra-high performance liquid chromatography, the separation efficiency is also greatly improved and the detection time is shortened; at the same time, the precision is high and the repeatability is good. The present invention realizes the simultaneous detection of multiple mycotoxins by using a very simplified and cheap pretreatment.

[0053] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0054] The present invention extracts 15 mycotoxins in food and drug dual-purpose substances with acetic acid-acetonitrile-aqueous solution. Aiming at the problem of complex matrix of food and drug dual-purpose substances, a QuEChERS purification package with low cost, simple operation and good purification effect is selected for purification. The present invention adjusts the ratio of the purifying agent to achieve the best purification conditions, and on the basis of removing impurities to the greatest extent, ensures the accuracy of the detection results. Finally, a UPLC-MS / MS method for simultaneous detection of multiple mycotoxins is established, and good sensitivity, accuracy and precision are obtained without using isotope internal standards. Moreover, due to the use of an ultra-high performance liquid system, the separation efficiency is greatly improved and the analysis time is shortened. The present invention effectively improves the purification degree of the sample, improves the detection efficiency, simplifies the operation process, has high sensitivity, good repeatability, high accuracy and low cost. It can be used as a reliable method for detecting the content of mycotoxins in food and drug dual-purpose substances. Description of the Drawings

[0055] Figure 1 It is the total ion current chromatogram of 15 mycotoxins in Example 1 of the present invention;

[0056] Figure 2 It is the multiple reaction monitoring chromatogram of 15 mycotoxins in Example 1 of the present invention;

[0057] Figure 3 In Example 2 of the present invention, when 1 mM ammonium acetate aqueous solution and 0.1% acetic acid-1 mM ammonium acetate aqueous solution are used as the mobile phase A phase, FB 1 and FB 2 chromatogram. A and C are the chromatograms of FB 1 and FB 2 when 1 mM ammonium acetate aqueous solution is used as the mobile phase; B and D are the chromatograms of FB 1 and FB 2 when 0.1% acetic acid-1 mM ammonium acetate aqueous solution is used as the mobile phase;

[0058] Figure 4 In Example 2 of the present invention, when 0.1% acetic acid-1 mM ammonium acetate aqueous solution and 0.1% formic acid-1 mM ammonium acetate aqueous solution are used as the mobile phase A phase, FB 1 and FB 2 chromatogram. A and C are the chromatograms of FB 1 and FB 2 when 0.1% acetic acid-1 mM ammonium acetate aqueous solution is used as the mobile phase; B and D are the chromatograms of FB 1 and FB 2 when 0.1% formic acid-1 mM ammonium acetate aqueous solution is used as the mobile phase;

[0059] Figure 5In Example 3 of the present invention, the effects of different extraction solvents on the recoveries of 15 mycotoxins;

[0060] Figure 6 In Example 5 of the present invention, the matrix effects of malt, coix seed, lotus seed and lily on mycotoxins were investigated. Detailed implementation manners

[0061] In the examples, the experimental methods described are all conventional methods unless otherwise specified; the reagents and materials described can be obtained from commercial sources unless otherwise specified.

[0062] In the following examples, the ultra-high performance liquid chromatography-tandem mass spectrometer used was the Shimadzu LCMS-8050 series (SHIMADZU), and the chromatographic column was Shim-pack GIST C 18 , with a specification of 100 mm × 2.1 mm, 2 μm (SHIMADZU).

[0063] Standard products of 15 mycotoxins (purity 99.9%), Jiangsu Meizheng Bio-Tech Co., Ltd.; methanol (mass spectrometry grade), acetonitrile (chromatography grade), Shanghai Macklin Biochemical Co., Ltd.; formic acid (mass spectrometry grade), Fluka Company, USA; ammonium acetate (mass spectrometry grade), Aladdin Company, USA.

[0064] Coix seed, lotus seed, malt and lily are commercially available.

[0065] Example 1

[0066] Optimization of mass spectrometry conditions

[0067] The detection method of 15 mycotoxins provided in this example includes the following steps:

[0068] Aflatoxin B 1 , B 2 , G 1 , G 2 , deoxynivalenol (DON), ochratoxin A (OTA), fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2) T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), β-zearalanol (β-ZAL) standards and other 15 mycotoxins were dissolved in methanol and diluted to a stock solution with a concentration of 100 ng / mL. According to the molecular weight information of the mycotoxins, the ionization mode of each mycotoxin was optimized, and its precursor ion, product ion and collision energy were optimized. Finally, the mass spectrometry conditions for 15 mycotoxins were obtained, as shown in Table 1. The total ion current chromatogram of 15 mycotoxins is shown in Figure 1 , and the multiple reaction monitoring chromatogram of each mycotoxin is shown in Figure 2 .

[0069] Example 2

[0070] Optimization of Chromatographic Conditions

[0071] The detection method for 15 mycotoxins provided in this example includes the following steps:

[0072] Aflatoxin B 1 , B 2 , G 1 , G 2 , deoxynivalenol (DON), ochratoxin A (OTA), fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2 ), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), β-zearalanol (β-ZAL) standards and other 15 mycotoxins were dissolved in methanol and diluted to a standard stock solution with a concentration of 100 μg / mL. An appropriate amount of the standard stock solution was taken and diluted with a 35% (v / v) methanol solution to a mixed standard solution with a concentration of 100 ng / mL for analysis.

[0073] The ultra-high performance liquid chromatography conditions were as follows: chromatographic column Shim-pack GIST C 18 , 100 mm × 2.1 mm, 2 μm; flow rate 0.2 mL / min; column temperature 40 °C; injection volume 1 μL; mobile phase gradient elution, and the elution program is shown in Table 2:

[0074] Table 2 Mobile Phase Elution Program

[0075]

[0076] When methanol was used as mobile phase B, the peak shapes and separation effects of 15 mycotoxins were investigated when mobile phase A was 1 mM ammonium acetate aqueous solution, 0.1% acetic acid - 1 mM ammonium acetate aqueous solution, and 0.1% formic acid - 1 mM ammonium acetate aqueous solution. It was found that the addition of acid to the mobile phase had a significant impact on the peak emergence effects of FB 1 and FB 2 . As shown in Figure 3 A and C, when mobile phase A was 1 mM ammonium acetate aqueous solution, FB 1 and FB 2 did not show peaks; when mobile phase A changed to 0.1% acetic acid - 1 mM ammonium acetate aqueous solution, FB 1 and FB 2 showed normal peaks and good responses, but the peak shapes were a bit tailing ( Figure 3 B and D). Therefore, the peak shapes of FB 1 and FB 2 were further compared when mobile phase A was 0.1% formic acid - 1 mM ammonium acetate aqueous solution and 0.1% acetic acid - 1 mM ammonium acetate aqueous solution. As shown in Figure 4 A and C, when mobile phase A was 0.1% acetic acid - 1 mM ammonium acetate aqueous solution, the peak shapes of FB 1 and FB 2 were tailing; when changed to 0.1% formic acid - 1 mM ammonium acetate aqueous solution, the peak shapes were improved and the responses became higher ( Figure 4 B and D). Therefore, finally, 0.1% formic acid - 1 mM ammonium acetate aqueous solution was determined as mobile phase A and methanol as mobile phase B, and the above 0.1% acetic acid or 0.1% formic acid were both volume fractions in the system.

[0077] Example 3

[0078] Optimization of extraction solvent

[0079] A suitable extraction solvent can not only reduce impurities in the sample such as fat and protein, etc., reduce matrix effects, but also improve the recovery rate. In the detection of mycotoxins, the sample extraction generally uses an organic solvent plus water as the solvent. The extraction solution containing water can moisten the matrix and enhance the penetration ability of the organic solvent. However, due to the obvious differences in the properties of different mycotoxins, when using one extraction solvent to extract multiple mycotoxins simultaneously, there may be significant differences in the recovery rates of each mycotoxin. In order to obtain satisfactory recovery rates for all 15 mycotoxins, it is necessary to optimize the extraction solvent.

[0080] The optimized extraction solvent provided in this example includes the following steps:

[0081] The effects of acetonitrile - water system or acetonitrile - water plus acid system on the extraction rates of 15 mycotoxins were investigated, including 80% acetonitrile aqueous solutions containing 0%, 1%, 5%, and 10% acetic acid, and the above ratios were all volume fractions in the system.

[0082] Preparation of sample solution: Coix seeds without mycotoxins were used as blank samples, and a mixed standard solution of 15 mycotoxins at a certain concentration was added thereto to obtain spiked samples. The final concentrations of the mycotoxins were as follows: AFB 1 and AFG 1 , 10 μg / kg; AFB 2 and AFG 2 , 5 μg / kg; DON, 750 μg / kg; FB 1 and FB 2 , 500 μg / kg; T-2 and OTA, 20 μg / kg; HT-2, 100 μg / kg; ZEN, ZAN, α-ZEL, β-ZEL and β-ZAL, 200 μg / kg. 80% aqueous acetonitrile solutions containing 0%, 1%, 5% and 10% acetic acid were used as extraction solutions respectively, and 15 mycotoxins in the food and drug dual-purpose substance samples were extracted according to a solid-liquid ratio of 1:20 (g / mL). After vortexing for 1 min, ultrasonic treatment was carried out for 20 min, and the sample supernatant was obtained after centrifugation at 8000 rpm for 5 min; QuEChERS purification agent B was added to the sample supernatant obtained by centrifugation (the ratio of the purification agent to the sample supernatant was 505:10 (mg / mL)), vortexed for 1 min, and then centrifuged at 8000 rpm for 5 min. 5 mL of the supernatant was taken and dried under nitrogen at 45 °C. It was re-dissolved with 1 mL of 35% methanol solution, filtered through a 0.22 μm filter membrane and then detected by UPLC-MS / MS.

[0083] UPLC-MS / MS detection conditions: Mobile phase A was 0.1% formic acid - 1 mM ammonium acetate aqueous solution, mobile phase B was methanol, the flow rate was 0.2 mL / min, the column temperature was 40 °C, the injection volume was 1 μL, and the mobile phase elution gradient program was as shown in Table 2 above.

[0084] Mass spectrometry conditions were as follows: Interface voltage 4.0 kV, ion source temperature 300 °C, desolvation temperature 526 °C, nebulizing gas flow rate 3.0 L / min, heating gas flow rate 10 L / min. The ion source was an electrospray ionization source (ESI); the scanning mode was simultaneous positive and negative ion scanning; the monitoring mode was multiple reaction monitoring (MRM). The multiple reaction monitoring ion pairs and mass spectrometry related parameters of 15 mycotoxins are shown in Table 1.

[0085] From the results of the recovery experiment ( Figure 5 ), it can be seen that the change in acid concentration has a great influence on the extraction rate of mycotoxins, especially FB 1 and FB 2 . After adding acetic acid, FB 1 and FB 2The recovery rates increased from 6.67% and 9.63% to 123.89% and 119.58% respectively, showing a very significant difference. This may be because FB 1 and FB 2 have many carboxyl groups, which have requirements for the acidity of the extraction solvent. Therefore, reducing the pH value of the extraction solvent can improve its stability and extraction efficiency. The recovery rates of ZEN, ZAN, α-ZEL, β-ZEL and β-ZAL were the best when containing 1% acetic acid, but started to decline after the acid concentration increased further. When using 80% acetonitrile containing 1% acetic acid, most mycotoxins showed acceptable recovery rates (61.88% - 123.89%), so it was selected as the optimal extraction solvent.

[0086] Example 4

[0087] Optimization of the ratio of QuEChERS purification agents

[0088] The optimized ratio of QuEChERS purification agents provided in this example includes the following steps:

[0089] In the early stage of the present invention, different samples of food and drug dual-purpose substances were detected, and coix seeds without mycotoxins were screened out as blank samples. A mixed standard solution of 15 mycotoxins at a certain concentration was added to them to obtain spiked samples, and the final concentrations of the mycotoxins were: AFB 1 and AFG 1 , 10 μg / kg; AFB 2 and AFG 2 , 5 μg / kg; DON, 750 μg / kg; FB 1 and FB 2, 500 μg / kg; T-2 and OTA, 20 μg / kg; HT-2, 100 μg / kg; ZEN, ZAN, α-ZEL, β-ZEL and β-ZAL, 200 μg / kg. An 80% acetonitrile aqueous solution containing 1% acetic acid was used as the extraction solution, and 15 mycotoxins in the sample were extracted at a solid-liquid ratio of 1:20 (g / mL). After vortexing for 1 min, ultrasonic treatment was carried out for 20 min, and the sample supernatant was obtained after centrifugation at 8000 rpm for 5 min; Two different ratios of QuEChERS purification agents A and B were added to the centrifuged sample supernatant (the ratio of the purification agent to the sample supernatant was 505:10 (mg / mL)), vortexed for 1 min, and then centrifuged at 8000 rpm for 5 min. 5 mL of the supernatant was taken and dried under nitrogen at 45 °C. It was re-dissolved with 1 mL of 35% methanol solution and filtered through a 0.22 μm filter membrane before being detected by UPLC-MS / MS (under the detection conditions optimized in Examples 1 and 2). By comparing the spiked recoveries, the optimal ratio of the QuEChERS purification agent was determined. Among them, the composition and ratio of QuEChERS purification agent A (commercial composition and ratio) were: 900 mg of anhydrous MgSO 4 , 300 mg of PSA, 300 mg of C18, 300 mg of Silica, 90 mg of GCB. The composition and ratio of QuEChERS purification agent B were: 240 mg of anhydrous MgSO 4 , 80 mg of PSA, 80 mg of C18, 80 mg of Silica, 25 mg of GCB. The present invention found that when the QuEChERS purification agent was added in excess, the toxin would also be adsorbed, resulting in a decrease in the recovery rate. The ratio of QuEChERS purification agent B was obtained after a large number of optimizations and adjustments based on the ratio of QuEChERS purification agent A. Under QuEChERS purification agent B, good recoveries were achieved for 15 mycotoxins simultaneously, meeting the detection requirements.

[0090] As can be seen from Table 3, the spiked recovery rate of QuEChERS purification agent A was not ideal. After using QuEChERS purification agent B for pretreatment, the recovery rate was significantly improved. Therefore, QuEChERS purification agent B was selected as the optimal ratio.

[0091] Table 3 Spiked recovery experiments of QuEChERS purification agents with different ratios

[0092]

[0093]

[0094] Example 5

[0095] Investigation of matrix effect

[0096] Since the present invention uses LCMS for detection and the ESI source is used, the complex matrix in the sample has a great interference on the ESI source. Therefore, it is necessary to investigate the matrix effect of the sample first, otherwise it will affect the recovery rate of the method, that is, the accuracy.

[0097] The detection method for 15 mycotoxins provided in this embodiment includes the following steps:

[0098] Preparation of matrix sample solution: Select coix seed, lotus seed, malt, and lily without 15 mycotoxins as sample matrices. Use an 80% acetonitrile aqueous solution containing 1% acetic acid as the extraction solution. According to the solid-liquid ratio of 1:20 (g / mL), vortex for 1 min, then perform ultrasonic treatment for 20 min, and centrifuge at 8000 rpm for 5 min to obtain the sample supernatant; Add QuEChERS purifying agent to the sample supernatant obtained by centrifugation, vortex for 1 min, and then centrifuge at 8000 rpm for 5 min. Take 5 mL of the supernatant and blow it dry with nitrogen at 45°C. Re-dissolve with 1 mL of 35% methanol solution and filter through a 0.22 μm filter membrane to obtain the matrix solutions of coix seed, lotus seed, malt, and lily respectively.

[0099] Preparation of solvent standard curve: Aflatoxin B 1 , B 2 , G 1 , G 2 , deoxynivalenol (DON), ochratoxin A (OTA), fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2 ), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), β-zearalenol (β-ZAL) standard products and other 15 mycotoxins are dissolved in methanol and diluted into a standard stock solution with a concentration of 100 μg / mL. Absorb an appropriate amount of the standard stock solution and dilute it with 35% methanol into a series of concentration gradient mixed standard curve working solutions, which are prepared and used immediately.

[0100] Preparation of matrix-matched standard curve: After the blank samples of malt, coix seed, lotus seed, and lily are processed according to the sample preparation method, the malt matrix blank solution, coix seed matrix blank solution, lotus seed matrix blank solution, and lily matrix blank solution are obtained respectively. Absorb an appropriate amount of the standard stock solution and dilute the standard stock solution into a series of concentration gradient mixed standard curve working solutions with the matrix blank solution, which are prepared and used immediately.

[0101] Matrix effect investigation: Using the concentration of each mycotoxin as the abscissa and the corresponding peak area as the ordinate, a standard curve was plotted and a linear equation was obtained. According to calculation formula (1), the matrix effect of each mycotoxin in matrices of malt, coix seed, lotus seed, and lily was obtained.

[0102] Matrix effect (%) = 100 × (Ka / Kb - 1) Formula (1)

[0103] In the formula, Ka represents the slope of the matrix-matched standard curve, and Kb represents the slope of the solvent standard curve. A matrix effect within ±20% is considered negligible; otherwise, matrix matching should be performed to eliminate the matrix effect for accurate quantification. As Figure 6 shown, malt, coix seed, lotus seed, and lily all have a strong matrix effect on most mycotoxins, so it cannot be ignored and a matrix-matched standard curve is needed to eliminate the matrix effect.

[0104] The concentrations of the working solutions of the standard curves of each mycotoxin are shown in Table 4.

[0105] Table 4 Concentrations of the working solutions of the mixed standard curves of 15 mycotoxins

[0106]

[0107]

[0108] Example 6

[0109] Methodology investigation

[0110] The detection method for 15 mycotoxins provided in this example includes the following steps and is detected under the optimal conditions of each of the above examples:

[0111] Preparation of the sample solution: Using coix seed without mycotoxins as a blank sample, a mixed standard solution of 15 mycotoxins with a certain concentration was added thereto.

[0112] Using an 80% acetonitrile aqueous solution containing 1% acetic acid as the extraction solution, 15 mycotoxins in the food and drug dual-purpose substance sample were extracted at a solid-liquid ratio of 1:20 (g / mL). After vortexing for 1 min, ultrasonic treatment was performed for 20 min, and after centrifugation at 8000 rpm for 5 min, the sample supernatant was obtained; QuEChERS purifying agent B was added to the centrifuged sample supernatant (the ratio of the purifying agent to the sample supernatant was 505:10 (mg / mL)), vortexed for 1 min, and then centrifuged at 8000 rpm for 5 min. 5 mL of the supernatant was taken and dried under nitrogen at 45°C. It was re-dissolved with 1 mL of 35% methanol solution, filtered through a 0.22 μm filter membrane, and then detected by UPLC-MS / MS.

[0113] UPLC-MS / MS detection conditions: Mobile phase A is 0.1% formic acid - 1 mM ammonium acetate aqueous solution, mobile phase B is methanol, flow rate is 0.2 mL / min, column temperature is 40 °C, injection volume is 1 μL. The mobile phase elution gradient program is as shown in Table 2 above.

[0114] The mass spectrometry conditions are as follows: Interface voltage is 4.0 kV, ion source temperature is 300 °C, desolvation temperature is 526 °C, nebulizing gas flow rate is 3.0 L / min, heating gas flow rate is 10 L / min. The ion source is an electrospray ionization source (ESI); the scanning mode is simultaneous positive and negative ion scanning; the monitoring mode is multiple reaction monitoring (MRM). The multiple reaction monitoring ion pairs and mass spectrometry related parameters of 15 mycotoxins are shown in Table 1.

[0115] Aflatoxin B 1 、B 2 、G 1 、G 2 , deoxynivalenol (DON), ochratoxin A (OTA), fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2 ), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), β-zearalanol (β-ZAL) standard products and other 15 mycotoxins are dissolved in methanol and diluted into a standard stock solution with a concentration of 100 μg / mL. Appropriate amounts of the standard stock solution are taken and diluted with the blank matrix solution of coix seed into a series of concentration gradient mixed standard curve working solutions for analysis.

[0116] Blank coix seed samples are selected and mixed standard solutions with low, medium, and high levels (see Table 5 for the spiking levels, and the toxin standard products are directly added to the matrix according to the mass ratio) are added respectively. Pretreatment is carried out according to the sample preparation method, and 3 parallel experiments are conducted for each spiking level. The results are shown in Table 5. The results show that the recoveries at three different spiking levels are between 61.99% and 113.50%, and the relative standard deviations are between 1.60% and 19.55%, indicating that this method has good accuracy, high precision, and low detection limit.

[0117] Table 5 Spiking recovery experiment of mycotoxins in coix seed

[0118]

[0119]

[0120] Example 7

[0121] According to the method of Example 6, replace the coix seed sample with malt, and the results are shown in Table 6. The recovery rates of 15 mycotoxins in malt range from 65.51% to 118.76%, and the precision ranges from 0.78% to 15.78%, indicating that the detection method established by the present invention has good accuracy, high precision and low detection limit.

[0122] Table 6 Spike recovery experiment of mycotoxins in malt

[0123]

[0124]

[0125] Example 8

[0126] According to the method of Example 6, replace the coix seed sample with lotus seeds, and the results are shown in Table 7. The recovery rates of 15 mycotoxins in lotus seeds range from 61.51% to 116.27%, and the precision ranges from 0.96% to 18.47%, indicating that the detection method established by the present invention has good accuracy, high precision and low detection limit.

[0127] Table 7 Spike recovery experiment of mycotoxins in lotus seeds

[0128]

[0129]

[0130] Example 9

[0131] According to the method of Example 6, replace the coix seed sample with lily, and the results are shown in Table 8. The recovery rates of 15 mycotoxins in lily range from 61.04% to 118.75%, and the precision ranges from 0.68% to 18.26%, indicating that the detection method established by the present invention has good accuracy, high precision and low detection limit.

[0132] Table 8 Spike recovery experiment of mycotoxins in lily

[0133]

[0134]

[0135]

[0136] Obviously, the above examples are only for illustration and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementations here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An improved QuEChERS-UPLC-MS / MS method for the rapid determination of 15 mycotoxins in food and drug dual-purpose substances, characterized in that, it comprises the following steps: (1) Sample extraction Using acetonitrile-acetic acid aqueous solution as the extraction solution, mix it with the food and drug dual-purpose substance sample, then perform ultrasonic treatment, and centrifuge to obtain the sample supernatant; (2) Sample purification Add QuEChERS purifying agent to the sample supernatant obtained by centrifugation, mix well and then centrifuge to obtain the supernatant, and dry it; (3) UPLC-MS / MS detection Re-dissolve and filter through a membrane, then perform UPLC-MS / MS detection. At the same time, prepare a mixed standard working solution for quantitative analysis of 15 mycotoxins; In step (1), using acetonitrile-acetic acid aqueous solution as the extraction solution to extract 15 mycotoxins in the food and drug dual-purpose substance sample according to a solid-liquid ratio of 1:10 - 1:20 (g / mL). The acetonitrile-acetic acid aqueous solution contains 60% - 90% acetonitrile and 0% - 10% acetic acid, and the above ratios are all volume fractions in the system; In step (1), the food and drug dual-purpose substance sample is any one or more of malt, coix seed, lotus seed and lily; The 15 mycotoxins in the food and drug dual-purpose substance sample in step (1) include aflatoxin B1, B2, G1, G2, deoxynivalenol (DON), ochratoxin A (OTA), fumonisin B1 (FB1), fumonisin B2 (FB2), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), β-zearalanol (β-ZAL). The composition and ratio of the QuEChERS purifying agent in step (2) are: 200 - 300 mg of anhydrous MgSO4, 50 - 100 mg of PSA, 50 - 100 mg of C18, 50 - 100 mg of Silica, 20 - 100 mg of GCB; In step (3), the liquid chromatography conditions of UPLC-MS / MS are: (1) Mobile phase: Phase A is formic acid-ammonium acetate aqueous solution, Phase B is methanol; Flow rate: 0.1 - 0.3 mL / min; Column temperature: 25 - 40 °C; Injection volume: 1 - 5 μL; (2) The elution gradient program is as follows: ； In step (3), the mass spectrometry conditions of UPLC-MS / MS are: (1) Ion source: Electrospray ionization source (ESI); (2) Scanning mode: Positive and negative ions simultaneously; (3) Monitoring mode: Multiple reaction monitoring (MRM); (4) Interface voltage: 3.0 - 5.0 kV; (5) Ion source temperature: 280 - 320 °C; (6) Desolvation temperature: 500 - 550 °C; (7) Nebulizing gas flow rate: 2.5 - 3.5 L / min; (8) Heating gas flow rate: 8 - 12 L / min.

2. The improved QuEChERS-UPLC-MS / MS method for the rapid determination of 15 mycotoxins in food and drug dual-purpose substances according to claim 1, characterized in that, After vortexing the extraction solution and the sample for 1 - 5 min in step (1), perform ultrasonic treatment for 10 - 30 min, and then centrifuge to obtain the sample supernatant.

3. The improved QuEChERS - UPLC - MS / MS method for rapid determination of 15 mycotoxins in food and drug dual - use substances according to claim 1, characterized in that, in step (2), the ratio of the QuEChERS purifying agent to the sample supernatant is 35:1 - 70:1 (mg / mL); after vortexing for 1 - 3 min to mix evenly, centrifuge again to take the supernatant, take 2 - 10 mL, and blow dry with nitrogen at 40 - 50 °C.

4. The improved QuEChERS - UPLC - MS / MS method for rapid determination of 15 mycotoxins in food and drug dual - use substances according to claim 1, characterized in that, in step (3), redissolve with 10% - 50% methanol solution, filter through a 0.22 μm filter membrane, and then perform UPLC - MS / MS detection.

5. The improved QuEChERS - UPLC - MS / MS method for rapid determination of 15 mycotoxins in food and drug dual - use substances according to claim 1, characterized in that, in step (3), the multiple reaction monitoring ion pairs and mass spectrometry - related parameters of the 15 mycotoxins are as follows: * is the quantitative ion.

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