CN115524434A - Universal purification method for measuring various mycotoxins in edible oil - Google Patents

Universal purification method for measuring various mycotoxins in edible oil Download PDF

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CN115524434A
CN115524434A CN202211209332.1A CN202211209332A CN115524434A CN 115524434 A CN115524434 A CN 115524434A CN 202211209332 A CN202211209332 A CN 202211209332A CN 115524434 A CN115524434 A CN 115524434A
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purification
oil
edible oil
mycotoxins
acetonitrile
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CN115524434B (en
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周健
金米聪
陈晓红
王立
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Ningbo Municipal Center For Disease Control & Prevention
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N30/02—Column chromatography
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    • G01N30/02—Column chromatography
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Abstract

The invention discloses a universal purification method for determining various mycotoxins in edible oil, belonging to the field of food detection. The method comprises the following steps: freezing and purifying acetonitrile extract of edible oil, collecting supernatant, and transferring to container C 18 And an extraction column of EMR mixed filler, purifying in a passing mode, leaching and collecting purified extracting solution. The invention uses the purification column to combine with the freezing low-temperature auxiliary purification, and can realize the monitoring of 51 mycotoxins in the edible oil sample at lower cost and higher speed. After purification, the oil and pigment components in the extract are obviously reduced, and the fungus toxicity is reducedThe matrix inhibition effect of the toxin, especially the weak polar toxin, is obviously improved, and the sensitivity of the method is greatly improved; the coverage range of the target object and the applicability of the substrate are all beyond the previous research, the method has obvious practical significance for investigating the mycotoxin pollution condition in the edible oil, and meanwhile, the method has few experimental steps, strong operability and low professional requirement of personnel, and has popularization and application values.

Description

Universal purification method for determining various mycotoxins in edible oil
Technical Field
The invention relates to the field of food detection, in particular to a universal purification method for measuring various mycotoxins in edible oil.
Background
Mycotoxins (mycotoxins) are secondary metabolites produced by toxigenic fungi including Penicillium (Penicillium spp.), aspergillus (Aspergillus spp.), fusarium spp.), and Alternaria (Alternaria app) under suitable environmental conditions. According to the food and agriculture organization statistics of the united nations, about 25% of crops are contaminated with mycotoxins every year worldwide. The probability of acute toxicity caused by mycotoxins is extremely low, but long-term, low-concentration chronic exposure is a truly non-negligible threat to human health, since even trace concentrations of toxins have triphasic effects, genetic and immunotoxicity.
In crops, oil crops are serious disaster areas infected and bred by fungi due to rich nutrient substances, meanwhile, the oil crops are planted, grown, harvested, transported to be stored, the whole production link is long in period, and toxic fungi can be greatly proliferated as long as the temperature and humidity conditions are proper. At present, more than 400 toxins with known chemical structural formulas are known, but the dietary exposure evaluation and pollutant monitoring work carried out on mycotoxins in food at home and abroad mainly focuses on aflatoxin, zearalenone, ochratoxin, deoxynivalenol and other conventionally monitored toxins, but for monitoring the edible oil substrate, the toxins are only focused on aflatoxin in peanut oil and zearalenone in corn oil, and other toxins, particularly weak-polarity toxins such as alternariol monomethyl ether, tenuton, beauvericin, variegated aspergillus toxin and four enrofloxacin and other related works are hardly carried out, and the pollution condition information is very little. The development and establishment of the method suitable for the multiple mycotoxins in the edible oil have important practical significance for investigating the distribution of the mycotoxins in different edible oil matrixes and the pollution condition of specific toxins and ensuring the health of people. However, the variety of edible oil products is various, the matrix components are complex, the oil content is extremely high, the difference of physicochemical properties of different mycotoxins is huge, and the requirement of multi-component analysis on the professional performance of operators is high, which is the main reason for limiting the development of the multi-mycotoxin method. At present, the mainstream purification technology (such as a general solid phase extraction technology, a multifunctional toxin purification column and a dispersive solid phase extraction method) is difficult to separate weak polar toxin from a large amount of fatty acid components, so that the oil content of a sample solution is high, the matrix inhibition effect is strong, and the sensitivity of the method and the accuracy of a trace measurement result are seriously influenced.
Disclosure of Invention
The invention aims to provide a universal purification method for determining various mycotoxins in edible oil, which solves the problems in the prior art, obviously improves the inhibition effect of an oil substrate on weak polar mycotoxins (beauvericin, enrofloxacin, alternariol monomethyl ether, tenutoxin and the like), reduces the possibility of false negative results, and greatly improves the detection sensitivity.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a universal purification method for measuring various mycotoxins in edible oil, which comprises the following steps:
freezing and purifying acetonitrile extract of edible oil, collecting supernatant, and transferring to container C 18 And an extraction column of EMR mixed filler, purifying in a passing mode, leaching and collecting purified extracting solution.
Preferably, the method for obtaining the acetonitrile extracting solution comprises the following steps: mixing and extracting the edible oil and the formic acid acetonitrile solution according to the mass volume ratio of 1g.
Preferably, the formic acid acetonitrile solution is an acetonitrile solution containing 0.5 mass% of formic acid.
Preferably, the freeze purification is: freezing at the temperature of minus 20 ℃ to minus 40 ℃ for 1 to 12 hours.
Preferably, the freeze purification is: freezing at-20 deg.C for 8h.
Preferably, the supernatant, C 18 The dosage ratio of the filling material to the EMR filling material is 5mL:76mg:32mg.
Preferably, the extraction column is rinsed with acetonitrile containing 0.5% formic acid, wherein the volume ratio of the acetonitrile containing 0.5% formic acid to the supernatant is 2.
The invention also provides application of the method in monitoring edible oil mycotoxin.
Preferably, the mycotoxins comprise less polar toxins.
The invention discloses the following technical effects:
(1) The invention discloses a through type purification column method suitable for 51 mycotoxins in edible oil. Aiming at the characteristics of components of edible oil, a large amount of fatty acid interference components in an extracting solution are quickly and efficiently reduced based on the combination of low-temperature freezing and purification and a pass-type impurity removal and extraction technology, the inhibition effect of an oil matrix on weak-polarity mycotoxins (beauvericin, enrofloxacin, alternariol monomethyl ether, tenutoxin and the like) can be obviously improved, the possibility of false negative results is reduced, and the detection sensitivity is greatly improved. The method can meet the purification requirement required by detection while keeping the high-flux characteristic of the multi-mycotoxin, has light pollution degree to an analysis system, and simultaneously, the mycotoxin coverage type and the substrate applicability are far superior to those of the previous similar researches, and particularly covers five new toxins which are low in polarity and attention but cannot be ignored in actual pollution in edible oils such as new toxins and alternariol monomethyl ether.
(2) The purifying agent used in the invention is C 18 And EMR, the dosage and proportion of which are determined after optimization based on a response surface-center combined design method, can control the loss of the weak-polarity toxin within an acceptable range (the loss of recovery rate is within 25%) on the premise of ensuring the purification effect of the weak-polarity impurities such as fatty acid in the extracting solution, and meanwhile, the substrate inhibition effect can be controlled within 50% by more than sixty percent of the purified mycotoxins. The verification proves that the recovery rate of all target toxins can be controlled between 75.8 and 109.5 percent, the RSD is less than 7.9 percent, the detection limit of the method is between 0.05 mu g/kg and 20.0 mu g/kg, and the accuracy, the precision and the sensitivity are high.
(3) The purification method disclosed by the invention only comprises five steps of sample weighing, extraction, freezing purification, column passing and sample loading, and leaching in the whole treatment process, and does not need the steps of activation, elution and the like. Although freeze purification (recommended time of 8h and more) and pass-through column purification seem to require much time, the above process really takes little time for experimenters, hardly prolongs the experimental flow, and can be expected to save more than half of the time compared with the conventional SPE technology. Meanwhile, the pretreatment step only needs basic operations such as sample weighing, liquid transferring and the like, the requirement on the specialty of experimenters is low, the operability is strong, and the result reproducibility is high. On the other hand, the single-pass purifying column uses less filler, can save the consumption of organic solvent, and the actual cost is less than half of the price of the traditional general solid phase extraction column.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a target mycotoxin multi-response monitoring profile;
FIG. 2 shows the freezing and purifying effect of acetonitrile extract of edible oil; 1: rapeseed oil, 2: camellia oil, 3: sesame oil, 4: blend oil, 5: sunflower oil, 6: soybean oil, 7: corn oil, 8: rice bran oil, 9: olive oil, 10: the oil is separated out from the sediment at the bottom of the peanut oil;
FIG. 3 is a flow chart of the edible oil purification treatment;
FIG. 4 shows the comparison of sample extraction solution purification and the purification effect of the purification column; 1-3: respectively extracting olive oil, rapeseed oil and sesame oil, and mixing the following raw materials in parts by weight: purifying the above extractive solution, and purifying with pure white filler (7-9): the effects of the three extracting solutions after purification are respectively achieved;
FIG. 5 is a comparison of the purification effect of sample solutions; 1-3: respectively injecting sample solutions of olive oil, rapeseed oil and sesame oil which are purified by a passing purification column, wherein the sample solutions are as follows, and the sample injection ratio is 4-6: respectively injecting sample solution into unpurified olive oil, rapeseed oil and sesame oil;
FIG. 6 is a comparison of the inhibitory effect of a typical less polar toxin purified versus an unpurified substrate in an olive oil sample solution;
FIG. 7 is a matrix effect of mycotoxins in ten typical edible oil matrices;
fig. 8 is a secondary fragment pattern of a high resolution orbitrap for a suspected positive sample.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1 Mass Spectrometry Condition optimization of mycotoxin Single Standard solutions
The UHPLC-MS/MS system consists of an ExionLC UHPLC and a Sciex Q-Trap 6500plus mass spectrum, is mainly used for quantitative analysis, and can perform qualitative analysis on part of low-content suspicious results based on retention time and fragment ion ratio. The hardware conditions and operating parameters for the instrument process are shown in table 1 below.
TABLE 1 UHPLC-MS/MS operating conditions
Figure BDA0003873948970000041
Tables 2-3 show the gradient elution conditions for the two polarity detection modes; instrument control and data acquisition and processing were performed by software analysis 1.7.1 (Sciex), experimental Design and statistical analysis using Design-expert 8.0.6.0 (Stat-Ease inc., minneapolis, USA), minitab 17.1.0 (Minitab inc., USA) and IBM SPSS stattics 19 (SPSS inc., chicago, USA), respectively.
TABLE 2 Positive ESI detection mode gradient elution procedure
Figure BDA0003873948970000051
TABLE 3 gradient elution procedure for negative ESI detection mode
Figure BDA0003873948970000052
Specific optimized MRM parameters such as parent/daughter ion selection, target retention time, cone aperture voltage, collision energy are detailed in table 4 (see fig. 1 for a typical MRM map).
TABLE 4 mycotoxin multireaction monitoring parameters
Figure BDA0003873948970000053
Figure BDA0003873948970000061
Figure BDA0003873948970000071
Example 2 Low temperature refrigeration purification treatment of edible oil
In this embodiment, the acetonitrile extract is first subjected to a low-temperature freezing purification treatment technique (-20 ℃ to-40 ℃), so as to reduce the solubility of fatty acid in an organic solvent, promote the fatty acid to coagulate and precipitate, and have little influence on trace amount of mycotoxin.
Based on the acetonitrile freezing point (-48 ℃), the test is carried out at three temperature levels of-20 ℃, 30 ℃ and 40 ℃ with the freezing time of 1h, 2h, 3h, 4h, 6h, 8h and 12h.
The results show that when the mixture is frozen at-20 ℃ for 8h, at-30 ℃ for 6h and at-40 ℃ for 4h, the fatty acid is separated out and settled down obviously, and the fatty acid separation amount is not increased obviously when the time is increased continuously. Considering that-20 ℃ is the temperature achievable in a domestic refrigerator, conditions are recommended for freezing at-20 ℃ for 8h or overnight. Finally, the effect of the freeze purification treatment is shown in fig. 2 (5 mL of extract, equivalent to 1.0g of oil sample), and it can be found that although part of fatty acid is precipitated after the freeze treatment, the color of the sample extract is still dark, and meanwhile, more oil and pigment components still exist in the sample extract, and further purification is required subsequently.
Example 3 method evaluation, optimization and validation for commonly used mycotoxin treatment methods
The applicability of the mycotoxins in this study was investigated against three common pretreatment methods for multi-mycotoxins, including solid phase extraction column (SPE) methods (HLB, HLB PRiME, MAX, MCX PRiME), multifunctional purification column (MFC) methods (MycoSep 226, 227) and dispersed solid phase extraction technology (d-SPE).
For both the SPE and MFC methods, evaluation tests were carried out using standard solutions according to the respective conditions recommended in example 1; the conditions for the d-SPE method are as follows: after transferring 3mL of the standard solution into a dispersion tube containing 200mg of each test adsorbent, shaking and centrifuging, 2.0mL of the resulting supernatant was concentrated to 1.0mL for analysis, and the results are shown in tables 5 to 6.
TABLE 5 recovery results for various SPE, MFC for multiple mycotoxins
Figure BDA0003873948970000081
Figure BDA0003873948970000091
TABLE 5 recovery results for various SPE, MFC for multiple mycotoxins
Figure BDA0003873948970000092
Figure BDA0003873948970000101
Figure BDA0003873948970000111
From the results in tables 5-6, it can be seen that, for the SPE column, HLB and HLB PRiME show good affinity for lipophilic and hydrophilic analytes (excluding toxoflavin, penitrem a, etc.), despite the large difference in physicochemical properties of most toxins. The strong polar toxin such as nivalenol, deoxynivalenol and its derivatives, patulin, etc. are difficult to retain in C 18 And (4) carrying out column extraction. In addition, both multifunctional columns have been shown to be suitable for only one type of toxin, e.g., mycosep 226 is suitable for aflatoxins and zearalenone, while Mycosep 227 is suitable for trichothecenes. Although it is a mixture ofHowever, the d-SPE technique has been shown to be suitable for multi-component assays, but there are still situations where a portion of the adsorbent can cause significant loss of analyte in multi-toxin applications. According to the results, C 18 While both EMR and EMR fillers show good applicability to most mycotoxins in sample solutions, it is also noted that while EMR has good lipid removal, excessive use can result in significant loss of some of the lipid-soluble toxins (e.g., beauvericin, alternaria tetrastigrins, aflatoxins, penicilltremorine a, etc.).
Besides the recovery rate, researchers also consider the purification effect of various methods on actual oil samples and the matrix effect of blank matrix after adding the standard, extract 5.0g of oil samples, purify the oil samples by using the method, add the standard and concentrate the oil samples to 1.0mL. The observation of the redissolution can find that only C is contained in the d-SPE purificant 18 The oil removal effect of EMR and GCB is good, no obvious oil drop can be seen by naked eyes, but the GCB purifying agent has good oil removal and pigment removal effects, but the GCB purifying agent cannot be eluted due to strong adsorption capacity to most mycotoxins (the recovery rate is close to 0%). And SPE aspect, C 18 The degreasing effect is good, and the HLB column effect is second (the HLB PRIME has oil drops visible to naked eyes).
In summary, the HLB column in the SPE technique and the C in the d-SPE method were selected 18 Purifiers, EMR purifiers were investigated as candidate methods for matrix effect. The result shows that after the purification is completed by using the HLB column, most of weak polar toxins are remarkably inhibited by the matrix, especially beauvericin and four enrofloxacin can inhibit more than 95 percent of signal intensity, and the sensitivity of the method is seriously influenced, which indicates that the purification effect of the method on actual samples is very unsatisfactory; the inhibitory effect of the less polar toxins in the EMR-purified matrix solution is greatly improved, but this may be due to the greater adsorption of EMR to less polar impurities, including toxins; c 18 The toxin with either polarity or weak polarity has good matrix effect control effect, most of the fungaltoxin inhibition effect can be controlled within 50%, but the matrix effect of the weak polarity toxin is still obvious.
From the above embodimentsConsidering the results of (A), although the existing SPE and d-SPE technical methods are mature, the purification of the edible oil matrix is still difficult to deal with by only adopting the single method. Considering detection cost, analysis speed and step number, C is to be adopted in the research 18 And further verifying the purification of the mycotoxin in the edible oil extraction liquid by an EMR mixed purifying agent in a through type purification column mode so as to realize the rapid and efficient purification of the mycotoxin in the edible oil extraction liquid.
Example 4 optimization of C in response to the surface method 18 Purifying edible oil extractive solution with EMR purifying agent
For C 18 And EMR, using a Central Composite Design (CCD) test, the specific test matrix is shown in Table 7. Wherein, each factor sets up 5 investigation levels altogether, and five groups of experiments are parallelly connected to the central point, add a virtual variable in order to monitor data model accuracy. After the experiment is carried out according to the experiment matrix, the obtained data is subjected to quadratic multiple regression fitting, and a regression equation for evaluating the primary term, the interactive term and the secondary term of the experiment factors is obtained through arrangement.
TABLE 7 CCD experiment design matrix and each factor value
Figure BDA0003873948970000121
Analysis of variance was performed on the data (see Table 8 for results) and showed that all fitted equation model terms were extremely significant (p)<0.0001 None of the vectorial terms is significant (p)>0.05 In the model coefficient term, C) 18 The amount used had a significant effect on 35 mycotoxins, while EMR had a significant effect on 46 mycotoxins, while none of the virtual variables had a significant effect on any of the toxins. The degree of fit and model quality of the polynomial model equation are determined by a coefficient (R) 2 ) Indicating that good model fit generally requires at least R 2 At least greater than 0.8, adjusting the determination factor (Adj-R) 2 ) And a prediction determination coefficient (Pred-R) 2 ) The difference between them is less than 0.2. The results show that R is in this experiment 2 0.939 to 0.999,adj-R 2 Is 0.884 to 0.999 percent,Pred-R 2 is 0.702-0.999 (and Adj-R) 2 The difference is 0.001-0.196, and all are less than 0.2), which shows that the generated equation has at least 93.9% of conformity with experimental data, can explain at least 88.4% of change effect, and has excellent prediction capability and higher reliability on response values.
TABLE 8 analysis of variance of CCD experimental design
Figure BDA0003873948970000131
Figure BDA0003873948970000141
In order to obtain the optimal condition considering all the recovery rates of the target mycotoxins, the maximum value of a response value Y in a fitting equation is solved by utilizing the Numerical Solutions function of software, the first order partial derivative is respectively solved for each variable, and the optimal experimental condition is obtained through calculation. The result shows that when C 18 And EMR at 76mg and 32mg respectively, the expected value for the experimental combination was highest, while the experimental predicted recovery under these conditions ranged from 79.4% to 104.4%. After determining the optimal conditions, supplementary experiments (n = 3) were carried out to verify the prediction results. The prediction accuracy of the established model was considered satisfactory in most cases when the deviation between the actual recovery and the theoretical prediction was less than 15%, except for ENB1 (-20%) in rapeseed oil matrix and β -ZEL (-18%) in corn oil.
Example 5 establishing optimized edible oil purification method
Weighing 5.0g of oil sample into a 50mL centrifuge tube, adding 25.0mL of 0.5% formic acid acetonitrile, performing vortex oscillation extraction for 20min, and centrifuging at 8500rpm for 3min after the end. The supernatant was taken out into a 15mL centrifuge tube and frozen overnight at-20 ℃. The next day, 5.0mL of the supernatant was taken, and 100. Mu.L of a mixed internal standard working solution (the types of internal standards are shown in Table 4) was added thereto, followed by transferring it to a medium previously charged with 76mg of C 18 And 32mg of EMR mixed packing, and purification was accomplished by passing (scheme shown in FIG. 3), after which 2.0mL of 0.5%And leaching the extraction column by acetonitrile formate. Collecting purified extractive solution (i.e. collecting the first liquid after the sample extractive solution passes through the column, and continuously eluting the collected second liquid with acidic acetonitrile, and mixing the two liquids) in a glass test tube at 45 deg.C, blowing nitrogen to near dryness, adding 150 μ L acetonitrile for redissolving, adding 0.85mL pure water, centrifuging at 16000 × g for 15min, and collecting sufficient supernatant for sample analysis.
Example 6 application of edible oil purification method
The edible oil purification method of example 5 was established according to the above-described exploration of various purification conditions of examples 1-4. This example will verify the different edible oil purification methods based on the purification method of example 5.
(1) Methodological parameter validation
Three typical complex edible oils (olive oil, rapeseed oil and sesame oil) are selected for extraction, the operation is completed according to a pretreatment method, and compared with the extraction solution before and after purification (see figure 4), a passing purification column can be found to adsorb a large amount of fatty acids and pigments, and the color of the purified extraction solution is obviously lightened.
Meanwhile, the inventor also compares the purification effect with the non-purification effect, namely directly blowing nitrogen to concentrate and redissolve the extracting solution after the extracting solution is not purified, as shown in figure 5. Although the sample solution is still slightly turbid after purification, the purification effect can meet the detection requirement in practice, and meanwhile, the inventor bases the same BEH C 18 The chromatographic column completes more than 350 times of actual sample analysis, the front pressure and the rear pressure are compared, and the result shows that the pressure of the chromatographic column is not obviously improved under the condition of the same mobile phase and proportion (<3 MPa), which indicates that the sample solution contains less low-polarity impurities and does not significantly pollute chromatography consumables. On the other hand, the unpurified sample solution is observed to be in an emulsion state, the pigment components are more, oil drops visible to naked eyes float on the upper layer, and the oil inevitably causes serious pollution to an instrument sample injector, a pipeline, a chromatographic column and an ion source after multiple sample injections. After matrix labeling is carried out on the sample solution, the sample finds that most of weak polar toxins (penicillium tremorine A, beauvericin, enrofloxacin, variegated aspergillotoxin and the like) are remarkably inhibited and the matrix effect is improved on the premise of not being purifiedMore than 90% of the total concentration of the sample can be obtained, and the sensitivity of the method and the accuracy of a low-concentration measurement result are seriously influenced. The signal intensity of five typical weak polar toxins of the same olive oil sample solution after purification and unpurified is compared (see fig. 6), and it can be found that under the same concentration, the target toxin signal in the unpurified sample solution is completely inhibited by the matrix, so that a false negative result is generated subsequently, and serious misjudgment can be generated on the pollution condition of the mycotoxin in the oil matrix. In conclusion, the purifying column can be proved to have great help for analyzing the mycotoxins in the edible oil sample, especially the low-polarity mycotoxin, the detection sensitivity can be improved, and the data reliability is ensured.
Subsequently, MRM spectra of the standard solution, the blank sample solution and the blank matrix-added standard solution were compared, and the results showed that no interference peak was found in 10 typical edible oil matrices around the retention time of each target toxin, indicating that the established experimental conditions were able to distinguish between analyte and matrix interfering components, and the method selectivity was good. Preparing a matrix matching curve in a blank matrix, preparing a standard working curve with the same concentration by using the initial flow phase, and comparing the slopes of the two curves to obtain the matrix effect. The results of matrix effect are shown in fig. 7, in which the inhibitory effect of mycotoxins is controlled within 50% in most cases, indicating that the purification effect of the method is acceptable and that the matrix effect is not sufficient to have a significant effect on the trace results. Preparing a substrate standard curve according to the concentration designed by the experiment, and finally obtaining the linear correlation coefficient R of all target compounds 2 Are all larger than 0.9991, which indicates that the substrate matching curve has good linearity and can meet the requirement of quantitative analysis. The accuracy and precision of the established method was determined by means of a standard test in ten typical edible oil bases, the final results are shown in table 9 (i.e. three parts on pages 17-19 of the description are the results of table 9), it was found that all the mycotoxin recoveries of interest were between 75.8% and 109.5%, the RSD was less than 7.9%, and the results of the method accuracy and precision were satisfactory. Method sensitivity was determined using various, low concentration level spiking experiments with signal to noise ratio 3.
Figure BDA0003873948970000171
Figure BDA0003873948970000181
Figure BDA0003873948970000191
(2) Quantitative and qualitative analysis of actual sample
The results of analysis of the contamination (including concentration range, positive rate, and average contamination concentration) of 221 samples of 10 types of edible oils collected from the above experiment are shown in tables 10 to 19. It should be noted that, the collected samples are all single grease components (if the olive oil only contains the olive oil, but no other oil components) except the blend oil, only one part of the samples in the same batch is collected, the oil of the same brand and different specifications is not repeatedly collected, and multiple brands of oil are selected as much as possible for collection. The results show that various edible oils are seriously polluted by mycotoxin, and particularly 26 toxins are detected in the olive oil, the corn oil, the peanut oil and the sesame oil. The detection rates of five emerging toxins, namely beauvericin and four enrofloxacin, are highest, especially in olive oil, while the aflatoxin with the highest concern is detected only in peanut oil. Therefore, the difference of different edible oils in the dominant bacterial and infectious virus production process can be found. Most previous studies have not further confirmed the nature of unconventionally monitored toxins after their detection. In this study, qualitative analysis based on retention time and precursor-product ion ratio by means of a triple quadrupole analyzer was still required, since the concentrations of some toxins (such as alternariol monomethyl ether, destruxins a/B, snakesins and mycophenolic acid) were too low to excite sufficient response intensity on a high resolution orbitrap mass spectrometer. The result shows that the difference between the retention time of the standard peak and the suspicious peak and the peak area ratio of the product ion is within 5 percent, and the result of the triple quadrupole mass spectrometer has good reliability. For beauvericin, enrofloxacin and tenuton, the exact mass numbers of parent and daughter ions (same retention time and target ion error less than 5ppm, see table 20 for results) and ion fragmentation characteristics were used as important criteria on high resolution MS. More specifically, precursor ions of the target compound are compared with theoretical molecular masses calculated from the chemical formula, and fragment ions of suspected results are compared with ions of a standard solution.
According to the results of Table 20, it was revealed that the errors between the exact mass number and the theoretical mass number of the parent ion were each between-1.44 ppm and 1.74 ppm. The secondary mass spectral signals were then acquired via the data-dependent mode, with the results shown in FIG. 8. The deviation of the suspected positive result from the standard solution is at an acceptable level (-0.34 ppm to 1.74 ppm), and meanwhile, all the fragment ion peaks of the standard substance can be found in the spectrum of the suspected analyte, which shows that the two ion fragmentation modes are consistent, and the deviation can be used as the exact evidence for the unconventional monitoring of the existence of the toxin, and the results of the pretreatment method are proved to be high in reliability from the side, and the false positive possibility is extremely low.
TABLE 10 detection results and contamination status of mycotoxins in olive oil
Figure BDA0003873948970000201
TABLE 11 detection results and contamination status of multiple mycotoxins in corn oil
Figure BDA0003873948970000202
Figure BDA0003873948970000211
TABLE 12 detection results and contamination status of multiple mycotoxins in peanut oil
Figure BDA0003873948970000212
TABLE 13 detection results and contamination status of mycotoxins in rapeseed oil
Figure BDA0003873948970000213
TABLE 14 detection results and contamination status of multiple mycotoxins in camellia oil
Figure BDA0003873948970000214
TABLE 15 detection results and contamination status of mycotoxins in blend oils
Figure BDA0003873948970000215
Figure BDA0003873948970000221
TABLE 16 detection results and contamination status of mycotoxins in rice bran oil
Figure BDA0003873948970000222
TABLE 17 detection results and contamination status of multiple mycotoxins in sunflower oil
Figure BDA0003873948970000223
TABLE 18 detection results and contamination status of mycotoxins in soybean oil
Figure BDA0003873948970000224
TABLE 19 detection results and contamination status of mycotoxins in sesame oil
Figure BDA0003873948970000225
Figure BDA0003873948970000231
TABLE 20 qualitative analysis results of high resolution orbitrap ion trap for suspected positive samples
Figure BDA0003873948970000232
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (9)

1. A universal purification method for measuring various mycotoxins in edible oil is characterized by comprising the following steps:
freezing and purifying acetonitrile extract of edible oil, collecting supernatant, and transferring to container C 18 And an extraction column of EMR mixed filler, purifying in a passing mode, leaching and collecting purified extracting solution.
2. The method of claim 1, wherein the acetonitrile extract is obtained by a method comprising: mixing and extracting the edible oil and the formic acid acetonitrile solution according to the mass volume ratio of 1g.
3. The method of claim 1, wherein the formic acid acetonitrile solution is an acetonitrile solution containing 0.5% formic acid.
4. The method of claim 1, wherein the cryo-purification is: freezing at-20 deg.c to-40 deg.c for 1-12 hr.
5. The method of claim 4, wherein the freeze purification is: freezing at-20 deg.C for 8h.
6. The method of claim 1, wherein the supernatant, C, is 18 The dosage ratio of the filling material to the EMR filling material is 5mL:76mg:32mg.
7. The method of claim 1, wherein the extraction column is rinsed with acetonitrile containing 0.5% formic acid, and the volume ratio of the acetonitrile containing 0.5% formic acid to the supernatant is 2.
8. Use of the method of claim 1 for monitoring mycotoxins in edible oils.
9. The use of claim 6, wherein said mycotoxins comprise less polar toxins.
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