Method for detecting seven alternaria toxins in edible vegetable oil

Abstract

The invention relates to a method for detecting seven alternaria toxins in edible vegetable oil, and belongs to the technical field of food safety detection. The method comprises the following steps: S1, sample extraction: weighing a uniformly mixed edible oil sample, dissolving with an ethyl acetate / cyclohexane solution in a volume ratio of 1: 1, fixing the volume, and transferring into a GPC (Gel Permeation Chromatography) sample injection bottle; s2, sample purification: purifying the sample by using gel permeation chromatography (GPC), taking ethyl acetate / cyclohexane with a volume ratio of 1: 1 as a mobile phase, and collecting leacheate in a specific time period; s3, sample concentration treatment: transferring the collected leacheate into a rotary evaporation flask, carrying out rotary evaporation until the leacheate is nearly dry, and dissolving residues with an acetonitrile-water solution; s4, sample centrifugation: centrifuging the dissolved sample under specific conditions, and taking supernate; and S5, instrument detection: detecting the seven kinds of alternaria toxins in the supernate by using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

Description

Technical Field

[0001] The present invention relates to a food safety detection method, specifically to a method for detecting 7 alternaria toxins in edible vegetable oil, belonging to the technical field of food safety detection. Background Art

[0002] Alternaria toxins are a class of secondary metabolites produced by fungi of the genus Alternaria, mainly including tenuazonic acid (TeA), altenusin (ALS), alternariol (ALT), alternariol monomethyl ether (AME), alternariol (AOH), alternatoxin I (ATX-I), tentoxin (TEN), etc. These toxins have biological activities such as mutagenicity, teratogenicity and cytotoxicity, and long-term ingestion may cause harm to human health.

[0003] Oilseeds such as peanuts, corn, sunflower seeds, olives, sesame seeds, soybeans, rapeseeds, etc. are extremely vulnerable to contamination by alternaria toxins. Vegetable oil occupies an important component in the diet of residents, and its hygienic quality directly affects people's health and safety. Therefore, the monitoring of alternaria toxin contamination in vegetable oil is of great significance. At present, the detection of alternaria toxins mainly focuses on matrices such as fruits, vegetables and their products, and grain processed products. There are few reports on the detection of edible oils, and the number of toxin types determined simultaneously is not very large.

[0004] In the prior art, et al. used a methanol / water mixed solution extraction to determine 5 alternaria toxins in sunflower oil, Honglin Lin et al. used cold-induced liquid-liquid microextraction to determine 4 alternaria toxins in olive oil, and Li Lei et al. used solid-phase extraction purification to determine 4 alternaria toxins in rapeseed oil. The above methods all use isotope internal standards to correct the matrix effects generated during the determination process. However, isotope internal standards are expensive, and there is a lack of isotope standards for some alternaria toxins in the current market. Moreover, at most only 5 alternaria toxins are determined, which limits the application of related detection technologies.

[0005] In addition, edible oils contain a large amount of interfering substances such as lipids and pigments, which are likely to interfere with instrument detection. Therefore, selecting a suitable purification method is particularly crucial for the establishment of the method. The existing purification methods mainly include liquid-liquid extraction and solid-phase extraction methods. However, these methods have problems such as incomplete sample purification and easy generation of matrix effects during the determination process. Therefore, isotope internal standards are needed to correct the matrix effects, which not only increases the detection cost, but also there is no commercially available isotope internal standard for some alternaria toxins (such as ALS) currently.

[0006] Therefore, there is an urgent need to develop a method for simultaneously detecting multiple alternaria toxins in edible oils, which does not rely on isotope internal standards and has good purification effects, so as to meet the needs of food safety monitoring. Summary of the Invention

[0007] Based on the above background, the object of the present invention is to provide a method for detecting 7 alternaria mycotoxins in edible vegetable oil, so as to solve the problems described in the background art.

[0008] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0009] A method for detecting 7 alternaria mycotoxins in edible vegetable oil, comprising the following steps:

[0010] S1: Sample extraction: Weigh a well-mixed edible oil sample, dissolve it with an ethyl acetate / cyclohexane solution with a volume ratio of 1:1 and make up the volume, and transfer it to a GPC injection vial.

[0011] S2: Sample purification: Use gel permeation chromatography (GPC) to purify the sample, with the mobile phase being ethyl acetate / cyclohexane with a volume ratio of 1:1, and collect the eluent in a specific time period.

[0012] S3: Sample concentration treatment: Transfer the collected eluent to a rotary evaporation flask, rotary evaporate until nearly dry, and dissolve the residue with an acetonitrile - aqueous solution.

[0013] S4: Sample centrifugation: Centrifuge the dissolved sample under specific conditions and take the supernatant.

[0014] S5: Instrument detection: Use ultra-high performance liquid chromatography - tandem mass spectrometry (UPLC-MS / MS) to detect 7 alternaria mycotoxins in the supernatant, including tenuazonic acid (TeA), altenusin (ALS), alternariol (ALT), alternariol methyl ether (AME), alternariol (AOH), alternatoxin I (ATX-I) and tentoxin (TEN).

[0015] Gel permeation chromatography (GPC) is a sample pretreatment technology developed in recent years, also known as size exclusion chromatography, which is a kind of liquid phase distribution chromatography. It uses a porous gel as the stationary phase and utilizes the space size effect of the gel pores to separate molecules of different sizes. As the mobile phase moves, components with large molecular weights mainly move along the pores between the gel particles, with a shorter migration path and eluting out of the chromatographic column first; components with small molecular weights diffuse into the interior of the gel particles due to diffusion, with a longer migration path and eluting out of the chromatographic column later. In the present invention, GPC is used to purify the edible oil sample, and macromolecular oils, pigments, alkaloids, polymers, etc. are eluted first, and the alternaria mycotoxins have relatively small molecular weights and are eluted later. By collecting the eluate in a specific time period, most of the lipid, pigment, etc. interference in the edible vegetable oil can be effectively removed.

[0016] Preferably, in step S1, 1 g of the edible oil sample (accurate to 0.01 g) is weighed into a 10.0 mL brown volumetric flask, dissolved with ethyl acetate / cyclohexane with a volume ratio of 1:1, and made up to 10.0 mL.

[0017] Preferably, in step S2, 5.0 mL of the sample solution is aspirated by GPC for purification, the flow rate is 5 mL / min, the packing of the gel chromatography column (25 mm × 500 mm) is Bio-Beads SX3, the eluate in the first 1200 s is discarded, the eluate in the next 600 s is collected, and finally the GPC column is eluted with 240 s.

[0018] Preferably, in step S3, it is rotary evaporated to near dryness at 40 °C, and the residue is dissolved with 1.00 mL of 50% acetonitrile-aqueous solution.

[0019] Preferably, in step S4, it is centrifuged at 12000 r / min at 4 °C for 10 min.

[0020] Preferably, in step S5, the chromatographic conditions are as follows: the chromatographic column is an Intersil ODS-3 C18 chromatographic column (100 mm × 2.1 mm, 1.7 μm); mobile phase A is 0.002% ammonia water, B is acetonitrile; the injection volume is 5.0 μL; the flow rate is 0.25 mL / min; the column temperature is 30 °C; the gradient elution program is as follows: at 0 - 1.0 min, B is 5%; at 1.0 - 6.0 min, B is 5% - 80.0%; at 6.0 - 6.5 min, B is 80.0% - 90.0%; at 6.5 - 7.5 min, B is 90.0%; at 7.5 - 8.0 min, B is 90.0% - 5.0%; at 8.0 - 10.0 min, B is 5%.

[0021] Preferably, in step S5, the mass spectrometry conditions are as follows: electrospray ionization (ESI) source, negative ion (ESI-) scan mode; spray voltage 4500 V; ion source temperature 500 °C; curtain gas pressure 35 psi; nebulizer gas 1 pressure 50 psi; nebulizer gas 2 pressure 50 psi.

[0022] Preferably, the present invention further includes the step of preparing a matrix-matched standard working curve: weighing a negative matrix sample, treating it according to steps S1 to S4 to obtain a blank matrix extraction and concentration solution, and preparing standard curve solutions with the blank matrix extraction and concentration solution. The mass concentrations of the standard curve solutions are 1.0, 5.0, 10.0, 50.0, 100.0, and 200.0 μg / L respectively.

[0023] Preferably, step S5 is directly carried out after step S4, and the sample is not filtered with filters such as NYL and PTFE, because the filter membrane will adsorb components such as AME and AOH, resulting in a low determination result.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The detection method for 7 alternaria toxins in an edible vegetable oil of the present invention realizes the simultaneous detection of 7 alternaria toxins in the edible oil for the first time. Compared with the prior art which can detect at most 5 alternaria toxins, the detection range is wider, and the safety of the edible oil can be evaluated more comprehensively.

[0026] 2. The present invention adopts gel permeation chromatography (GPC) purification technology, which separates according to the steric exclusion effect of different-sized molecules in the gel pores. Macromolecular oils, pigments, alkaloids, polymers, etc. are eluted first, and the alternaria toxins with smaller molecular weights are eluted later. By collecting the eluate in a specific time period, most of the lipid, pigment and other interfering substances in the edible vegetable oil can be effectively removed, significantly reducing the matrix effect. There is no need to use expensive isotope internal standards to correct the matrix effect, reducing the detection cost.

[0027] 3. The method of the present invention avoids using a filter membrane for filtration during the sample treatment process, preventing components such as AME and AOH from being adsorbed by the filter membrane, resulting in a low determination result, and improving the accuracy of the detection result.

[0028] 4. The detection limit and quantification limit of the method of the present invention are relatively low, and the spike recovery rate and precision both meet the requirements of the analysis method verification, and it is applicable to the detection of 7 alternaria toxins in various edible vegetable oils. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the MRM diagram of 7 alternaria toxins. DETAILED DESCRIPTION OF THE INVENTION

[0030] The technical solutions of the present invention will be further specifically described below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0031] In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0032] The reagents used in the following embodiments can be obtained from a conventional biochemical reagent store without special instructions.

[0033] Instruments and Reagents

[0034] SCIEX 5500 +Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer; GPC Vario automatic gel permeation chromatography purifier; JY500 electronic balance; XS-205DU electronic balance; X-30 high-speed refrigerated centrifuge; Auto EVA 30Plus automatic parallel concentrator; Mili-Q Reference ultrapure water instrument.

[0035] Single standard stock solutions: tenuazonic acid (TeA), altenusin (ALS), alternene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), alternatoxin I (ATX-I), and tentoxin (TEN) standard solutions, all with a concentration of 100 μg / mL.

[0036] The examples of the present invention disclose a method for detecting 7 alternaria mycotoxins in edible vegetable oil. This method uses gel permeation chromatography purification technology and ultra-high performance liquid chromatography-tandem mass spectrometry detection technology, and can simultaneously detect the contents of 7 alternaria mycotoxins in edible oil, including tenuazonic acid (TeA), altenusin (ALS), alternene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), alternatoxin I (ATX-I), and tentoxin (TEN).

[0037] Examples

[0038] A method for detecting 7 alternaria mycotoxins in edible vegetable oil, the specific steps are as follows:

[0039] S1. Sample extraction

[0040] Weigh 1 g (accurate to 0.01 g) of the well-mixed edible oil sample into a 10.0 mL brown volumetric flask, dissolve and make up the volume with ethyl acetate / cyclohexane (1:1), and transfer all the solution to a GPC injection vial.

[0041] S2. Sample purification

[0042] The GPC aspirates 5.0 mL of the sample solution for purification at a flow rate of 5 mL / min. The mobile phase is ethyl acetate / cyclohexane (1:1). The packing material of the gel chromatography column (25 mm × 500 mm) is Bio-Beads SX3. Discard the eluate in the first 1200 s, collect the eluate in the next 600 s, and finally wash the GPC column with 240 s of eluent.

[0043] S3. Sample concentration treatment

[0044] Transfer the collected solution to a rotary evaporation flask, rotary evaporate to nearly dry at 40 °C, and dissolve the residue with 1.00 mL of 50% acetonitrile-aqueous solution.

[0045] S4. Sample centrifugation

[0046] The sample after the previous step of dissolution was centrifuged at 12,000 r / min for 10 min at 4 °C, and the supernatant was taken.

[0047] It should be noted that the sample dried by nitrogen blowing was dissolved with 1.00 mL of 50% acetonitrile-aqueous solution and could not be filtered with filters such as NYL and PTFE, because the filter membrane would adsorb components such as AME and AOH, resulting in a low determination result. Therefore, it was necessary to centrifuge at 12,000 r / min for 10 min at 4 °C and then directly measure it on the machine.

[0048] S5. Instrument detection

[0049] The supernatant in S4 was detected using an ultra-high performance liquid chromatography-tandem mass spectrometer, and the detection conditions were as follows: 1. Chromatographic conditions

[0050] Chromatographic column: Intersil ODS-3 C18 chromatographic column (100 mm × 2.1 mm, 1.7 μm); mobile phase A was 0.002% ammonia water, B was acetonitrile; injection volume was 5.0 μL; flow rate was 0.25 mL / min; column temperature was 30 °C; gradient elution program: from 0 to 1.0 min, B was 5%; from 1.0 to 6.0 min, B was 5% - 80.0%; from 6.0 to 6.5 min, B was 80.0% - 90.0%; from 6.5 to 7.5 min, B was 90.0%; from 7.5 to 8.0 min, B was 90.0% - 5.0%; from 8.0 to 10.0 min, B was 5%.

[0051] 2. Mass spectrometry conditions

[0052] Electrospray ionization (ESI) source, negative ion (ESI - ) scan mode; spray voltage: 4500 V; ion source temperature 500 °C; curtain gas pressure 35 psi; nebulizing gas 1 pressure 50 psi; nebulizing gas 2 pressure 50 psi; multiple reaction monitoring (MRM) mode; the retention time, characteristic ion pairs, declustering voltage and collision energy parameters of the compound are shown in Table 1, where "*" represents the quantitative ion.

[0053] Table 1 Mass spectrometry parameters

[0054]

[0055] Preparation of matrix-matched standard working curve:

[0056] Preparation of blank matrix extraction concentrate: Weigh 1 g of negative matrix sample (edible oil sample that has been tested and confirmed to contain no 7 alternaria toxins), and process it according to the above steps S1 to S4 to obtain the blank matrix extraction concentrate, which is used to prepare the standard working curve.

[0057] Matrix-matched standard working curve: Use the above-mentioned blank matrix extraction concentrate to calculate the usage amount of the standard curve solution to be prepared, and prepare 7 kinds of Alternaria toxin mixed standard series working solutions with mass concentrations of 1.0, 5.0, 10.0, 50.0, 100.0, and 200.0 μg / L respectively.

[0058] Establishment of the standard curve: Measure the prepared standard series working solutions according to step S5. Take the peak area of each component as the ordinate (y) and the corresponding mass concentration as the abscissa (x) to plot the standard working curve and perform linear regression analysis.

[0059] Linear range, detection limit, and quantification limit: According to the established standard working curve, measure the linear equation, correlation coefficient, detection limit, and quantification limit of 7 kinds of Alternaria toxins. The MRM diagrams of 7 kinds of Alternaria toxins are as Figure 1 shown. The linear range of the standard curve and the detection limit of 7 kinds of Alternaria toxins are shown in Table 2.

[0060] Table 2

[0061]

[0062] The results show that the standard curve established by this method has a good linear relationship, the correlation coefficients are all greater than 0.998, the detection limits and quantification limits are both low, meeting the requirements of food safety detection.

[0063] Results of the spiked recovery determination: Add a mixed standard solution of 7 kinds of Alternaria toxins at 3 different concentrations (2.0, 10.0, and 50.0 μg / kg) to the blank edible vegetable oil sample, perform spiked recovery according to the above-mentioned pretreatment method and instrument determination conditions, conduct 6 parallel experiments for each addition level, and the determination results of the added concentration, average recovery rate, and relative standard deviation (RSD) are shown in Table 3.

[0064] Table 3

[0065]

[0066] The results show that the average recovery rates of 7 kinds of Alternaria toxins by this method at different spiked levels are 81.0% - 97.5%, and the relative standard deviations (RSD) are 2.59% - 8.12%, all meeting the requirements of the analysis method verification, indicating that this method has good accuracy and precision.

[0067] Application example: Detection of actual samples

[0068] The method of the present invention was used to detect the contents of 7 Alternaria mycotoxins in 20 different types of edible vegetable oil samples collected from the market, including camellia oil, rice bran oil, flaxseed oil, olive oil, soybean oil, rapeseed oil, sunflower seed oil, edible blended oil, corn oil, peanut oil, etc. The actual measured results of 7 Alternaria mycotoxins in edible vegetable oils are shown in Table 4.

[0069] Table 4

[0070]

[0071] In summary, the method for detecting 7 Alternaria mycotoxins in edible vegetable oils provided by the present invention has the following significant advantages compared with the prior art: it realizes the simultaneous detection of 7 Alternaria mycotoxins in edible oils for the first time, with a wider detection range; it adopts GPC purification technology to effectively remove interfering substances such as lipids and pigments in edible oils, with a small matrix effect; it does not need to use expensive isotope internal standards to correct the matrix effect, reducing the detection cost; it avoids using filter membranes for filtration to prevent the adsorption of components such as AME and AOH, improving the accuracy of the detection results; the spike recovery rate and precision of the method meet the requirements of analytical method validation, and it is applicable to the detection of 7 Alternaria mycotoxins in various edible vegetable oils.

[0072] Specific examples are used in this article to elaborate on the principle and implementation mode of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. For example, the GPC purification parameters can be adjusted according to the characteristics of different types of edible oils, different brands of liquid chromatography - mass spectrometry instruments can be used, the chromatographic conditions and mass spectrometry conditions can be optimized, etc. As long as it does not deviate from the technical solution and technical idea of the present invention, it belongs to the protection scope of the present invention.

Claims

1. A method for detecting seven types of Alternaria toxins in edible vegetable oils, characterized in that: The method comprises the following steps: S1: Sample extraction: Weigh the mixed edible oil sample, dissolve it in ethyl acetate / cyclohexane solution with a volume ratio of 1:1 and make up to volume, and transfer it to the GPC injection bottle; S2: Sample cleanup: The samples were cleaned up using gel permeation chromatography (GPC) with a mobile phase of ethyl acetate / cyclohexane in a volume ratio of 1:1, and the eluent was collected for a specific period of time; S3: Sample concentration treatment: transfer the collected eluent to a rotary evaporation bottle, evaporate it to near dryness, and dissolve the residue with acetonitrile-water solution; S4: Sample centrifugation: centrifuge the dissolved sample under specific conditions and take the supernatant; S5: Instrumental detection: Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used to detect seven types of Alternaria toxins in the supernatant, including tenuisporic acid (TeA), altricialosporin (ALS), alternariae (ALT), alternariae methyl ether (AME), alternariae ol (AOH), alternariae toxin I (ATX-I) and tengdotoxin (TEN).

2. The detection method according to claim 1, characterized in that: In step S1, 1 g (accurate to 0.01 g) of edible oil sample was weighed into a 10.0 mL brown volumetric flask, and dissolved in ethyl acetate / cyclohexane with a volume ratio of 1:1 and the volume was adjusted to 10.0 mL.

3. The detection method according to claim 1, characterized in that: In step S2, GPC absorbs 5.0 mL of sample solution for purification, the flow rate is 5 mL / min, the gel chromatography column (25 mm × 500 mm) is filled with Bio-BeadsSX3, the first 1200 s of eluent is discarded, the next 600 s of eluent is collected, and finally the GPC column is eluted for 240 s.

4. The detection method according to claim 1, characterized in that: In step S3, the mixture was rotary evaporated to near dryness at 40°C, and the residue was dissolved with 1.00 mL of 50% acetonitrile-water solution.

5. The detection method according to claim 1, characterized in that: In step S4, centrifugation was performed at 12000 r / min at 4°C for 10 min.

6. The detection method according to claim 1, characterized in that: In step S5, the chromatographic conditions are as follows: the chromatographic column is an Intersil ODS-3C18 chromatographic column (100 mm×2.1 mm, 1.7 μm); the mobile phase A is 0.002% ammonia water, and B is acetonitrile; the injection volume is 5.0 μL; the flow rate is 0.25 mL / min; the column temperature is 30° C.; the gradient elution program is: from 0 to 1.0 min, B is 5%; From 1.0 to 6.0 min, B is 5% to 80.0%; from 6.0 to 6.5 min, B is 80.0% to 90.0%; from 6.5 to 7.5 min, B is 90.0%; from 7.5 to 8.0 min, B is 90.0% to 5.0%; from 8.0 to 10.0 min, B is 5%.

7. The detection method according to claim 1, characterized in that: In step S5, the mass spectrometry conditions are: electrospray ionization (ESI) source, negative ion (ESI-) scanning mode; spray voltage 4500V; ion source temperature 500°C; curtain gas pressure 35psi; nebulizer gas 1 pressure 50psi; nebulizer gas 2 pressure 50psi; multiple reaction monitoring (MRM) mode.

8. The detection method according to claim 1, characterized in that: The method also includes the step of preparing a matrix matching standard working curve: weighing a negative matrix sample, processing according to steps S1 to S4 to obtain a blank matrix extraction concentrate, and using the blank matrix extraction concentrate to prepare a standard curve solution.

9. The detection method according to claim 8, characterized in that: The mass concentrations of the standard curve solutions are 1.0, 5.0, 10.0, 50.0, 100.0, and 200.0 μg / L, respectively.

10. The detection method according to claim 1, characterized in that: After step S4, step S5 is directly performed without using a filter membrane such as NYL or PTFE to filter the sample.

Images