High-throughput detection method for 60 mycotoxins in pet food

Abstract

The invention provides a high-throughput detection method for 60 mycotoxins in pet food, which is based on ultra-high performance liquid chromatography-tandem mass spectrometry, and adopts an EMR-lipid adsorbent as a purification material to remove a large amount of grease so as to purify a sample; a universal pretreatment method is established through a dispersive solid-phase extraction technology, high-throughput detection of 60 mycotoxins in the pet food is realized by combining an ultra-high performance liquid chromatography-tandem mass spectrometry technology, and aiming at raw material mixing and high grease content in the pet food, an EMR-lipid adsorbent is used as a purification material to remove a large amount of grease, so that the detection accuracy is high. The purpose of purifying the sample is achieved; the interference of grease on a detection result is reduced; the method is wide in coverage range, capable of meeting high-throughput screening of mycotoxin in pet food with complex components, high in recovery rate, low in cost, suitable for complex matrixes such as pet solid food, wet food and snacks, and capable of meeting the requirement for mycotoxin monitoring in production and storage of the pet food.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pet food safety detection, and particularly relates to a high-throughput detection method for 60 mycotoxins in pet food, which is based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) for simultaneous detection and suitable for complex matrices such as pet dry food, pet wet food and pet snacks. BACKGROUND

[0002] In recent years, the pet market has developed rapidly, and the scale of China's pet economy industry reached 493.6 billion yuan in 2022. The scale of the pet food industry has also increased year by year, and the production scale of pet feed in China reached 1.13 million tons in 2021. Pet food refers to pet food that is directly consumed by pets after industrialized production, including compound feed, additive premixed feed and other foods designed to achieve rewards, stimulate chewing, interaction and other purposes. As a multi-source composite system, the complexity of pet food matrix far exceeds that of human food and livestock and poultry feed, and the main raw materials include grains, animal by-products, plant proteins, oils and fats, and various additives. Therefore, the safety of pet food and the potential harm of contaminants in pet food to pet health have also attracted more attention.

[0003] Mycotoxins are a class of toxic secondary metabolites produced by molds, which usually have acute and chronic toxicity such as anorexia, vomiting and diarrhea, and some toxins have serious reproductive toxicity, teratogenicity, carcinogenicity and mutagenicity. They are common toxic chemicals in pet food, which not only affect the nutritional value of pet food, but also pose a great threat to pet health. In 2006, Diamond Pet Food Company in the United States was sued by pet owners after 76 pets died and 21 dogs were sick due to corn raw material contaminated with aflatoxin (raw material detection was not performed), resulting in the recall of 350,000 bags of products and compensation of $3.1 million. In recent years, there have been numerous reports of mycotoxin contamination in pet food in various countries around the world, and the phenomenon of simultaneous contamination of multiple mycotoxins is prominent. Based on this, China formulated and implemented the "Provisions on the Hygiene of Pet Feed" in 2018, increasing the limit of mycotoxins to 8 kinds (AFB1, DON, ZEN, FB1, FB2, OTA, T-2 and HT-2), with a stringent limit of AFB1 of 10 μg / kg (the European Union standard is 5 μg / kg).

[0004] The commonly used mycotoxin detection methods mainly include high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry, colloidal gold immunochromatography, enzyme-linked immunosorbent assay and the like. According to the different feed hygiene standard restriction degrees, detection technical levels and actual production needs in various countries in the world, different countries and regions adopt different mycotoxin detection technologies. Due to the reasons that the mycotoxin pollution usually presents common pollution of multiple types, the physical and chemical properties of different mycotoxins are greatly different, the pollution concentrations are greatly different and the like, the ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) is widely used at present to sensitively and accurately determine multiple mycotoxins in the same sample. The existing UPLC-MS / MS method mainly aims at the detection of common fungal toxins in ordinary livestock and poultry feed, and lacks a high-throughput, sensitive and accurate detection method for new fungal toxins and metabolites (enramycin, fusariocin, beauvericin, hidden mycotoxin, mycophenolic acid and the like) in pet food. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a detection method based on UPLC-MS / MS, which adopts EMR-lipid adsorbent and dispersive solid phase extraction technology, optimizes the pretreatment and instrument analysis conditions, realizes broad-spectrum, high recovery rate, low matrix effect and high-throughput screening, so as to meet the demand of mycotoxin detection in pet food.

[0006] In order to achieve the above-mentioned purpose, the solution of the present application is:

[0007] A high-throughput detection method for 60 kinds of mycotoxins in pet food, comprising the following steps:

[0008] (1) Sample preparation: the pet food sample is crushed to a particle size of less than 0.5-1 mm, mixed uniformly, divided into 100 g, stored in a sample bottle, and sealed and stored at-18℃;

[0009] (2) Sample pretreatment: weigh the sample, mix uniformly with water, add 8 mL of 0.5% acetic acid-acetonitrile solution, perform ultrasonic extraction, and then perform dispersive solid phase extraction using EMR-lipid adsorbent and sodium chloride and anhydrous magnesium sulfate, and then perform nitrogen blowing and drying, and then redissolve, and then pass through a polytetrafluoroethylene filter membrane;

[0010] (3) Instrument analysis: ultra-high performance liquid chromatography-tandem mass spectrometry is adopted, a Waters XBridge BEH-C18 column is used as a chromatographic column, a methanol / water / acetic acid solution containing 5mM ammonium acetate is used as a mobile phase, gradient elution is performed, and 60 kinds of mycotoxins are quantitatively analyzed in a multiple reaction monitoring mode;

[0011] (4) Quantitative analysis: an appropriate amount of standard solution was accurately pipetted to prepare a mixed standard solution, and a series of standard working solutions were prepared by dilution with a blank matrix reconstitution solution. The matrix matching standard curve method was used for quantitative detection of 60 mycotoxins.

[0012] Preferably, in step (1), the pet food includes dog dry food, dog wet food, cat dry food, cat wet food, and pet snacks.

[0013] Preferably, in step (2), the EMR-lipid adsorbent is used to remove high content of oil and fat in the sample, and reduce the matrix effect, with the matrix effect ranging from 81.5% to 117.1%.

[0014] Sample pretreatment: 2.00 g ± 0.01 g of the sample was accurately weighed in a 50 mL centrifuge tube, 6 mL of water was added and vortexed for 1 min, then 8 mL of 0.5% acetic acid-acetonitrile solution was added and vortexed for 1 min, ultrasonic extraction for 5 min, and placed in a vortex mixer at 150 rpm for 30 min. 7 mL of supernatant was taken to a centrifuge tube containing EMR-lipid adsorbent (1 g), vortexed for 1 min, and centrifuged at 5000 rpm for 5 min. 1 g of sodium chloride and 4 g of anhydrous magnesium sulfate were added to the supernatant, vortexed for 1 min, and centrifuged at 5000 rpm for 5 min. The supernatant was transferred to a 10 mL centrifuge tube, dried with nitrogen, reconstituted with the initial mobile phase, and filtered through a 0.22 μm PTFE filter membrane before UPLC-MS / MS determination.

[0015] Preferably, in step (3), the 60 mycotoxins include aflatoxins, fusarium toxins, penicillin toxins, novel fungal toxins, and hidden mycotoxins.

[0016] Further, the aflatoxins include aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, and versicolor.

[0017] The fusarium toxins include zearalenone, deoxynivalenol, T-2 toxin, fumonisin, and enniatin.

[0018] The penicillin toxins include patulin, penicillin, verruculogen, and penicillin tremor A.

[0019] The novel fungal toxins include enniatin, moniliformin, beauvericin, and mycophenolic acid.

[0020] The hidden mycotoxins include deoxynivalenol-3-glucoside, T-2-3-glucoside, and HT-2-3-glucoside.

[0021] UPLC-MS / MS detection: Waters XBridge BEH-C18 column (3.0 x 100 mm, 2.5 μm) was used; mobile phase A was methanol / water / acetic acid solution (97:2:1, v / v / v) containing 5 mM ammonium acetate, and mobile phase B was methanol / water / acetic acid solution (10:89:1, v / v / v) containing 5 mM ammonium acetate; the gradient elution program was 0-1 min, 2% B; 1-2 min, 2-50% B; 2-3 min, 50% B; 3-5.5 min, 50-75% B; 5.5-9.5 min, 75-100% B; 9.5-11.5 min, 100% B; 11.5-12 min, 100% B-2% B; 12-13 min, 2% B; the flow rate was 0.4 mL / min; the injection volume was 3 μL; the column temperature was 40°C. The positive and negative ionization mode (ESI+ and ESI-) was used for scanning by using an electrospray ion source; high-purity air was used as the atomizing gas and auxiliary gas; high-purity nitrogen was used as the collision gas; the atomizing gas (Ion source gas 1, GS1) was 50 Psi; the auxiliary gas (Ion source gas 2, GS2) was 50 Psi; the atomizing temperature was 500.0°C; the spray voltage was 5500 V / -4500 V; the de-clustering voltage was 80 V; the spray voltage gas curtain gas was 35 Psi; the collision gas was 8 Psi. The target compounds were quantified by using the multiple reaction monitoring (MRM) mode, and the parent ions, daughter ions and collision energies of 60 mycotoxins were set according to specific settings.

[0022] Preferably, in step (4), the standard solution is prepared into a mixed standard solution with concentrations of 0.5 μg / mL, 10 μg / mL and 100 μg / mL respectively, and a series of standard working solutions are prepared by dilution with a blank matrix reconstitution solution.

[0023] Preferably, in step (4), the intra-day precision of the quantitative detection is 4.8-12.1%, and the inter-day precision is 7.2-14.5%.

[0024] Due to the use of the above scheme, the beneficial effects of the present application are:

[0025] 1. The EMR-lipid adsorbent is used as a purification material to remove a large amount of oil and fat in pet food with mixed raw materials and high oil and fat content, so as to achieve the purpose of purifying the sample and reduce the interference of oil and fat on the detection results.

[0026] 2. A high-throughput detection method covering a wide range is established, which can simultaneously detect 60 kinds of mycotoxins, and meets the high-throughput screening of mycotoxins in pet food with complex components and uneven raw material quality.

[0027] 3. Based on the dispersed solid phase extraction technology, a universal pretreatment method is established, which is simple and fast in operation, can realize high-throughput screening of samples, and meets the needs of monitoring of mycotoxins in pet food production and storage. And the cost is low, the cost of each sample detection is less than 100 yuan. According to the characteristics of high oil content in pet food, EMR-lipid adsorbent is used to effectively remove oil and reduce matrix interference; Therefore, the method has the characteristics of wide spectrum (covering 60 kinds of toxins), high sensitivity (detection limit 0.05-11.5 μg / kg), high recovery rate (>60%) and low cost (<100 yuan / sample), low matrix effect (81.5-117.1%), good precision (1.1-10.8% within a day, 2.6-13.6% between days), which can effectively cope with the interference of complex matrix in pet food, and meet the high-throughput screening needs of mycotoxins in production, storage and supervision. DETAILED DESCRIPTION

[0028] The application provides a high-throughput detection method for 60 mycotoxins in pet food.

[0029] The application will be described in detail below in combination with examples. However, the embodiments of the application are not limited thereto, and obviously, the examples described below are only part of the embodiments of the application, and for those skilled in the art, other similar embodiments can be obtained without creative labor, which fall within the protection scope of the application.

[0030] Example 1:

[0031] (I) Sample preparation

[0032] Select dog dry food, dog wet food, cat dry food and cat wet food, 4 kinds of samples each 1 kg, use high-speed pulverizer to crush, pass through sieve, make its particle size less than 0.5-1 mm aperture test sieve, mix uniformly, then subdivide to 100 g, store in sample bottle, seal and store in-18℃ refrigerator for standby.

[0033] (II) Reagent

[0034] 60 kinds of mycotoxin standard solution is purchased from Shanghai Academy of Agricultural Sciences, Romer and Purbang company, stored at-20℃ in dark; Methyl alcohol, acetonitrile (chromatographic pure) are purchased from American Merck company; EMR-lipid adsorbent is purchased from Agilent company; Sodium chloride and anhydrous magnesium sulfate are purchased from Shanghai Aladdin company; Chromatographic pure acetic acid and ammonium acetate are purchased from Shanghai Anpu company; Experimental water is prepared by Millopore Synergy ultrapure water instrument (American Millipore company).

[0035] (III) Instrument conditions

[0036] Chromatographic column: Waters XBridge BEH-C18 column (3.0*100mm, 2.5um); mobile phase: A phase is methanol / water / acetic acid solution (97:2:1, v / v / v) containing 5mM ammonium acetate, B phase is methanol / water / acetic acid solution (10:89:1, v / v / v) containing 5mM ammonium acetate; gradient elution: 0-1min, 2% B; 1-2min, 2-50% B; 2-3min, 50% B; 3-5.5min, 50-75% B; 5.5-9.5min, 75-100% B; 9.5-11.5min, 100% B; 11.5-12min, 100% B-2% B; 12-13min, 2% B; flow rate is 0.4mL / min; injection volume is 3uL; column temperature is 40℃.

[0037] Mass spectrometry conditions:

[0038] The ion source is electrospray, positive and negative ionization mode (ESI+ and ESI-) scanning, both atomizing gas and auxiliary gas are high-purity air; the collision gas is high-purity nitrogen; the atomizing gas (Ion source gas 1, GS1) is 50Psi; the auxiliary gas (Ion source gas 2, GS2) is 50Psi; the atomizing temperature is 500.0℃; the spray voltage is 5500V (positive) / -4500V (negative); the cluster voltage is 80V; the spray voltage gas curtain gas is 35Psi; the collision gas is 8Psi. The target compound is accurately quantified by multiple reaction monitoring (MRM) mode, and the parent ions, daughter ions and collision energies of 60 mycotoxins are shown in Table 1. As can be seen from Table 1, the toxins covered by the present application are relatively wide, including some new and hidden toxins.

[0039] (Four) Preparation of standard solution

[0040] An appropriate amount of A, B and C group standard solutions was accurately taken and mixed to prepare a mixed standard solution with concentrations of 0.5ug / mL, 10ug / mL and 100ug / mL, and then diluted with a blank matrix reconstitution solution (mobile phase B) to prepare a series of standard working solutions for making a calibration curve. The concentration range is 0.01-100ug / mL, which is used to construct a calibration curve.

[0041] (Five) Sample pretreatment

[0042] Accurately weigh 2.00 g ± 0.01 g of sample into a 50 mL centrifuge tube, vortex for 1 min after adding 6 mL of water, vortex for 1 min after adding 8 mL of 0.5% acetic acid-acetonitrile solution, ultrasonic for 5 min, and extract for 30 min at 150 rpm in a vortex mixer. Take 7 mL of supernatant into a centrifuge tube containing EMR-lipid adsorbent (1 g), vortex for 1 min, and centrifuge at 5000 rpm for 5 min. Continue to add 1 g of sodium chloride and 4 g of anhydrous magnesium sulfate to the supernatant, vortex for 1 min, and centrifuge at 5000 rpm for 5 min. Transfer the supernatant to a 10 mL centrifuge tube, dry under nitrogen, re-dissolve with 1 mL of the initial mobile phase, pass through a 0.22 μm PTFE filter membrane, and determine by UPLC-MS / MS. To ensure the consistency of the pretreatment, the same batch of reagents is used for each experiment, and the balance and pipette are calibrated regularly

[0043] (VI) Results

[0044] Linearity of the method: 60 mycotoxin standard series working solutions were prepared with blank matrix extractant, with a concentration range of 0.01-100 μg / mL. The concentration of the matrix-matched standard series solution was used as the abscissa, and the chromatographic peak area (response value) was used as the ordinate to draw the standard curve. Each standard curve showed a good linear relationship, and the correlation coefficient was greater than 0.99.

[0045] Limit of detection and limit of quantification of the method: 60 mycotoxin standard series working solutions were prepared with blank matrix extractant. The limit of detection and the limit of quantification of each toxin were determined at 3 times the signal-to-noise ratio and 10 times the signal-to-noise ratio, respectively, with a range of 0.05-11.5 μg / kg and 0.1-30 μg / kg, respectively.

[0046] Recovery rate verification of the method: The recovery rate of the method was investigated by the matrix spiking method. 2 g of blank samples of dog dry food, dog wet food, cat dry food, and cat wet food were weighed, and 60 mycotoxin standard solutions at low, medium, and high concentration levels (1 times LOQ, 5 times LOQ, and 10 times LOQ) were added, with 6 replicates for each. After purification according to the above steps, the samples were detected, quantified by the matrix-matched standard curve external standard method, and the recovery rates of the 60 mycotoxins were calculated. The results showed that the average recovery rate of the 60 mycotoxins was more than 60%, meeting the requirements of the Laboratory Quality Control Specification for Food Physical and Chemical Testing (GB / T 27404-2008).

[0047] Precision verification of the method: Within the same day, 6 parallel samples were tested, and the intra-day precision was 4.8-12.1% after purification and detection. For 5 consecutive days, 5 parallel samples were tested each day, and the inter-day precision was 7.2-14.5% after detection and calculation.

[0048] Matrix effect of mass spectrometry: prepare standard working solution with blank sample extract of typical pet food such as dog dry food, construct matrix matching calibration curve, and evaluate matrix effect by ratio of slope of matrix matching calibration curve to slope of standard solution prepared with pure solvent (calculate ratio of slope of matrix matching calibration curve to slope of calibration curve prepared with pure solvent). 80-120% is low matrix effect, indicating that the matrix effect is small, <80% matrix effect is inhibition, and >120% matrix effect is enhancement. As can be seen from Table 2, except for 12 kinds of mycotoxins such as dihydroxypropyl dihydroxymellein, T-2 tetrol, and hemiacetal fusarenonic acid, the matrix effect of the rest of the mycotoxins is between 81.5-117.1%, indicating that the matrix effect is low. However, due to the diverse categories of samples to be tested, in order to ensure the accuracy of the value results, matrix matching standard curve is used for quantitative analysis to reduce interference.

[0049] Actual sample analysis: 100 samples were randomly collected from the market and enterprise production line, including more than 20 types of full-value cat food, full-value dog food, and full-value dog bone meat paste, which were detected according to the above pretreatment and analysis method. It was found that 52% of the samples detected mycotoxins, among which fumonisin, enniatin, and beauvericin were the main detected mycotoxins, with the highest content of 246.3 μg / kg, indicating that there is a risk of mycotoxin pollution in pet food. In order to further verify the applicability of the method, 10 high-fat pet snacks (containing more than 20% of oil) were selected for detection, and the recovery rate remained above 60%, and the matrix effect was between 85.2-115.8%, indicating that the method has good adaptability to high-fat matrix.

[0050] As can be seen from the above, the detection method of 60 kinds of mycotoxins in pet food can effectively and accurately quantify the mycotoxins in different pet foods, and the types of mycotoxins contained are significantly higher than those of the existing similar analysis methods, and the recovery rate is high and the precision is good. Compared with the existing purification method, the accuracy and reproducibility are ensured or even better, the cost is greatly saved, the matrix interference is significantly reduced, and high-throughput processing of samples is realized.

[0051] In summary, the detection method of the present application has strong spectrum, high sensitivity and low cost, and is suitable for high-throughput screening of mycotoxins in pet food, which is significantly superior to the prior art. As can be seen from Table 3, the recovery rate is >60%, the intra-day precision is 1.1-10.8%, and the inter-day precision is 2.6-13.6%.

[0052] Table 1 main information and mass spectrometry parameters of 60 kinds of mycotoxins

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] Linearities, limits of detection, and limits of quantitation for 60 mycotoxins in a typical dog dry food

[0059]

[0060]

[0061]

[0062] Recoveries and precision (n = 6) for 60 mycotoxins in a typical dog dry food

[0063]

[0064]

[0065]

[0066] The above examples are only used to illustrate the technical solutions of the present application, and not to limit the same; although the present application has been described in detail with reference to the above examples, those skilled in the art can still modify the technical solutions recorded in the above examples, or make equivalent replacements for part or all of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the patent protection scope of the present application. The following describes the present application in detail in conjunction with the examples. However, the embodiments of the present application are not limited thereto, and obviously, the examples described below are only some of the embodiments of the present application, and for those skilled in the art, other similar embodiments obtained without creative labor fall within the protection scope of the present application.

Claims

1. A high-throughput detection method for 60 mycotoxins in pet food, characterized in that, It includes the following steps: (1) Sample preparation: Crush the pet food sample to a particle size of less than 0.5-1 mm, mix evenly and store at -18℃; (2) Sample pretreatment: Weigh the sample, add water and vortex mix, add 8 mL of 0.5% acetic acid-acetonitrile solution, extract by ultrasonication, and then use EMR-lipid adsorbent and sodium chloride and anhydrous magnesium sulfate for dispersion solid phase extraction. After drying with nitrogen, reconstitute and filter through a polytetrafluoroethylene membrane. (3) Instrumental analysis: Ultra-high performance liquid chromatography-tandem mass spectrometry was used with a WatersXBridge BEH-C18 column as the chromatographic column, a methanol / water / acetic acid solution containing 5 mM ammonium acetate as the mobile phase, gradient elution, and multiple reaction monitoring mode to quantitatively analyze 60 mycotoxins; (4) Quantitative analysis: Accurately transfer an appropriate amount of standard solution to prepare a mixed standard solution, and dilute it with blank matrix reconstitution solution to prepare a series of standard working solutions. The matrix matching standard curve method is used to quantitatively detect 60 mycotoxins. In step (3), the 60 mycotoxins include aflatoxins, fusarium toxins, penicillin toxins, novel mycotoxins, and cryptic mycotoxins.

2. The high-throughput detection method for 60 mycotoxins in pet food according to claim 1, characterized in that, In step (3), the aflatoxins include aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2 and aflatoxin variegata. The Fusarium toxins include zearalenone, deoxynivalenol, T-2 toxin, fumonisin, and enfurcin. The penicillin class includes patulin, penicillin, penicillin var. verrucospora, and penicillin tremor A; The novel mycotoxins include Fusarium oxysporin, Fusarium moniliforme, Beauveria bassiana, and mycophenolic acid. The cryptic mycotoxins include deoxynivalenol-3-glucoside, T-2-3-glucoside, and HT-2-3-glucoside.

3. The high-throughput detection method for 60 mycotoxins in pet food according to claim 1, characterized in that, In step (1), the pet food includes dry dog ​​food, wet dog food, dry cat food, wet cat food, and pet snacks.

4. The high-throughput detection method for 60 mycotoxins in pet food according to claim 1, characterized in that, In step (2), the EMR-lipid adsorbent is used to remove high levels of oil in the sample and reduce the matrix effect, which ranges from 81.5% to 117.1%.

5. The high-throughput detection method for 60 mycotoxins in pet food according to claim 1, characterized in that, In step (4), the standard solutions are prepared into mixed standard solutions with concentrations of 0.5 μg / mL, 10 μg / mL and 100 μg / mL, and a series of standard working solutions are prepared by diluting with blank matrix reconstitution solution.

6. The high-throughput detection method for 60 mycotoxins in pet food according to claim 1, characterized in that, In step (4), the intraday precision of the quantitative detection is 1.1–10.8%, and the interday precision is 2.6–13.6%.

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