Method for detecting 7 kinds of alternaria toxins in edible vegetable oil

Abstract

The present application relates to a kind of detection methods of 7 kinds of alternaria toxins in edible vegetable oil, belong to food safety detection technical field.The method includes the following steps: S1: sample extraction: the mixed uniform edible oil sample is weighed, with the volume ratio of 1:1 ethyl acetate / cyclohexane solution is dissolved and constant volume, transfer to GPC sample bottle;S2: sample purification: using gel permeation chromatography (GPC) to purify sample, mobile phase is the volume ratio of 1:1 ethyl acetate / cyclohexane, the eluate of specific time period is collected;S3: sample concentration treatment: the collected eluate is transferred to rotary evaporation bottle, rotary evaporation is nearly dry, with acetonitrile-water solution is dissolved residue;S4: sample centrifugation: after dissolving, the sample is centrifuged under specific conditions, and the supernatant is taken;S5: instrument detection: using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to detect 7 kinds of alternaria toxins in supernatant.

Description

Technical Field

[0001] This invention relates to a food safety testing method, specifically a method for detecting seven types of Alternaria mycotoxins in edible vegetable oils, belonging to the field of food safety testing technology. Background Technology

[0002] Alternaria toxins are a class of secondary metabolites produced by fungi of the genus *Alternaria*, mainly including TeA, alginate (ALS), alginate (ALT), AME, AOH, ATX-I, and TEN. These toxins possess mutagenic, teratogenic, and cytotoxic biological activities, and long-term intake may pose health risks.

[0003] Oilseeds such as peanuts, corn, sunflower seeds, olives, sesame seeds, soybeans, and rapeseed are highly susceptible to contamination by Alternaria alternata toxins. Since vegetable oils constitute a significant portion of the diet, their hygiene quality directly impacts public health and safety. Therefore, monitoring Alternaria alternata toxin contamination in vegetable oils is of great importance. Currently, the detection of Alternaria alternata toxins mainly focuses on matrices such as fruits, vegetables and their products, and processed grain products. Reports on the detection of Alternaria alternata toxins in edible oils are relatively few, and the number of toxins tested simultaneously is also limited.

[0004] In the existing technology, Five Alternaria toxins in sunflower seed oil were determined using methanol / water mixed solution extraction; four Alternaria toxins in olive oil were determined using cold-induced liquid-liquid microextraction; and four Alternaria toxins in rapeseed oil were determined using solid-phase extraction purification. All of these methods used isotope internal standards to correct for matrix effects during the determination process. However, isotope internal standards are expensive, and there is a current lack of isotope standards for some Alternaria toxins on the market. Furthermore, these methods have only determined a maximum of five Alternaria toxins, limiting the application of these detection technologies.

[0005] Furthermore, edible oils contain a large amount of lipids and pigments, which can easily interfere with instrument detection. Therefore, choosing a suitable purification method is crucial for establishing the method. Existing purification methods mainly include liquid-liquid extraction and solid-phase extraction, but these methods suffer from incomplete sample purification and matrix effects during the measurement process. Therefore, isotope internal standards are needed to correct for matrix effects, which not only increases the detection cost, but also, for some Alternaria mycotoxins (such as ALS), there are currently no commercially available isotope internal standards available.

[0006] Therefore, there is an urgent need to develop a method for simultaneously detecting multiple alginate toxins in edible oils, which is independent of isotope internal standards and has good purification effects, in order to meet the needs of food safety monitoring. Summary of the Invention

[0007] Based on the above background, the purpose of this invention is to provide a method for detecting seven types of Alternaria mycotoxins in edible vegetable oils, thereby solving the problems described in the background art.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A method for detecting seven alginate toxins in edible vegetable oils, comprising the following steps:

[0010] S1: Sample extraction: Weigh the well-mixed edible oil sample, dissolve it in a 1:1 volume ratio ethyl acetate / cyclohexane solution and make up to volume, then transfer it to a GPC injection bottle;

[0011] S2: Sample purification: The sample was purified using gel permeation chromatography (GPC) with ethyl acetate / cyclohexane in a 1:1 volume ratio as the mobile phase, and the eluent was collected at specific time intervals.

[0012] S3: Sample concentration treatment: Transfer the collected eluent to a rotary evaporation flask, evaporate to near dryness, and dissolve the residue with acetonitrile-water solution;

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

[0014] S5: Instrument detection: Seven algal toxins in the supernatant were detected using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), including algal ketoacid (TeA), algalin (ALS), algal spore (ALT), algal methyl ether (AME), algal phenol (AOH), algal toxin I (ATX-I), and tengin (TEN).

[0015] Gel permeation chromatography (GPC) is a sample pretreatment technique developed in recent years, also known as size exclusion chromatography. It is a type of liquid partition chromatography that uses a porous gel as the stationary phase and leverages the spatial size effect of the gel pores to separate molecules of different sizes. As the mobile phase moves, components with larger molecular weights mainly move along the pores between gel particles, having a shorter migration path and eluting from the column first. Components with smaller molecular weights diffuse into the interior of the gel particles, having a longer migration path and eluting from the column later. In this invention, GPC is used to purify edible oil samples. Large molecules such as lipids, pigments, alkaloids, and polymers are eluted first, while Alternaria alternata toxins, with their relatively small molecular weight, are eluted later. By collecting the eluent over a specific time period, most of the interference from lipids and pigments in edible vegetable oils can be effectively removed.

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

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

[0018] Preferably, in step S3, the residue is dissolved in 1.00 mL of 50% acetonitrile-water solution after rotary evaporation at 40°C until nearly dry.

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

[0020] Preferably, in step S5, the chromatographic conditions are as follows: the column is an Intersil ODS-3C18 column (100 mm × 2.1 mm, 1.7 μm); mobile phase A is 0.002% ammonia, and mobile phase B is acetonitrile; the injection volume is 5.0 μL; the flow rate is 0.25 mL / min; the column temperature is 30 °C; and the gradient elution program is as follows: 0–1.0 min, B is 5%; 1.0–6.0 min, B is 5%–80.0%; 6.0–6.5 min, B is 80.0%–90.0%; 6.5–7.5 min, B is 90.0%; 7.5–8.0 min, B is 90.0%–5.0%; and 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-) scanning mode; spray voltage 4500V; ion source temperature 500℃; curtain gas pressure 35psi; nebulizer gas 1 pressure 50psi; nebulizer gas 2 pressure 50psi.

[0022] Preferably, the present invention further includes the step of preparing a matrix-matched standard working curve: weighing a negative matrix sample, processing it 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, wherein the mass concentrations of the standard curve solutions are 1.0, 5.0, 10.0, 50.0, 100.0, and 200.0 μg / L.

[0023] As a preferred option, step S5 is performed directly after step S4, without using NYL, PTFE or other filter membranes to filter the sample, because filter membranes will adsorb components such as AME and AOH, resulting in lower measurement results.

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

[0025] 1. The present invention provides a method for detecting seven types of alginate toxins in edible vegetable oils, which for the first time achieves the simultaneous detection of seven types of alginate toxins in edible oils. Compared with the prior art, which can only detect a maximum of five types of alginate toxins, the detection range is wider and can more comprehensively assess the safety of edible oils.

[0026] 2. This invention employs gel permeation chromatography (GPC) purification technology, which separates molecules of different sizes based on the spatial exclusion effect in the gel pores. Large molecules such as oils, pigments, alkaloids, and polymers are eluted first, while smaller molecules such as Alternaria mycotoxins are eluted later. By collecting the eluent over a specific time period, most of the interfering substances such as lipids and pigments in edible vegetable oils can be effectively removed, significantly reducing the matrix effect. This eliminates the need for expensive isotope internal standards to correct for the matrix effect, thus reducing detection costs.

[0027] 3. The method of the present invention avoids the use of filter membranes during sample processing, preventing components such as AME and AOH from being adsorbed by the filter membrane, which would lead to lower measurement results and improve the accuracy of the detection results.

[0028] 4. The detection limit and quantitation limit of the method of the present invention are low, and the spiked recovery rate and precision meet the requirements of analytical method validation. It is applicable to the detection of seven alginate toxins in various edible vegetable oils. Attached Figure Description

[0029] Figure 1 This is the MRM diagram of 7 Alternaria toxins. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0031] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0032] Unless otherwise specified, the reagents used in the following examples can be purchased from a regular biochemical reagent store.

[0033] Instruments and reagents

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

[0035] Single standard stock solutions: standard solutions of Alternaria ketoacid (TeA), Alternaria spp. (ALS), Alternaria spp. (ALT), Alternaria spp. methyl ether (AME), Alternaria spp. (AOH), Alternaria spp. I (ATX-I), and Alternaria spp. EN, all with a concentration of 100 μg / mL.

[0036] This invention discloses a method for detecting seven alginate toxins in edible vegetable oils. The method employs gel permeation chromatography purification technology and ultra-high performance liquid chromatography-tandem mass spectrometry detection technology, which can simultaneously detect the content of seven alginate toxins in edible oils, including alginate ketone acid (TeA), alginate succinate (ALS), alginate thiocyanate (ALT), alginate methyl ether (AME), alginate phenol (AOH), alginate toxin I (ATX-I), and tengin (TEN).

[0037] Example

[0038] A method for detecting seven alginate toxins in edible vegetable oils, the specific steps of which are as follows:

[0039] S1, Sample Extraction

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

[0041] S2, Sample Cleaning

[0042] GPC was purified by taking 5.0 mL of sample solution, with a flow rate of 5 mL / min, using ethyl acetate / cyclohexane (1:1) as the mobile phase, and a Bio-Beads SX3 gel chromatography column (25 mm × 500 mm) as the packing material. The first 1200 s of eluent was discarded, the next 600 s of eluent was collected, and finally the GPC column was eluted for 240 s.

[0043] S3, Sample Concentration Processing

[0044] The collected liquid was transferred to a rotary evaporation flask and evaporated at 40°C until nearly dry. The residue was dissolved in 1.00 mL of 50% acetonitrile-water solution.

[0045] S4, Sample centrifugation

[0046] After dissolving the sample in the previous step, centrifuge it at 12000 r / min at 4℃ for 10 min and collect the supernatant.

[0047] It should be noted that the sample dried by nitrogen blowing should be dissolved in 1.00 mL of 50% acetonitrile-water solution. It should not be filtered with NYL, PTFE or other filter membranes, because the filter membrane will adsorb components such as AME and AOH, which will lead to lower test results. Therefore, it is necessary to centrifuge at 12000 r / min at 4℃ for 10 min and then directly test it.

[0048] S5, Instrument Testing

[0049] The supernatant in S4 was analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) under the following conditions: 1. Chromatographic conditions

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

[0051] 2. Mass spectrometry conditions

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

[0053] Table 1 Mass Spectrometry Parameters

[0054]

[0055] Preparation of matrix matching standard working curves:

[0056] Preparation of blank matrix extract concentrate: Weigh 1g of negative matrix sample (edible oil sample confirmed by testing to be free of 7 types of Alternaria toxins), process it according to steps S1 to S4 above, and obtain blank matrix extract concentrate. The obtained blank matrix extract concentrate is used to prepare standard working curve.

[0057] Matrix-matched standard working curve: Using the above blank matrix to extract the concentrate, calculate the amount of standard curve solution to be prepared, and prepare a series of mixed standard working solutions of seven Alternaria toxins with mass concentrations of 1.0, 5.0, 10.0, 50.0, 100.0 and 200.0 μg / L.

[0058] Establishment of standard curve: The prepared standard series working solutions were measured according to step S5. The peak area of ​​each component was plotted as the ordinate (y) and the corresponding mass concentration was plotted as the abscissa (x). Linear regression analysis was then performed.

[0059] Linear range, limit of detection, and limit of quantitation: Based on the established standard working curves, the linear equations, correlation coefficients, limits of detection, and limits of quantitation for the seven alginate toxins were determined. The linear range, correlation coefficients, limits of detection, and limits of quantitation for the seven alginate toxins are shown in the figure below. Figure 1 As shown in Table 2, the linear range and detection limits of the standard curves for the seven Alternaria toxins are presented.

[0060] Table 2

[0061]

[0062] The results showed that the standard curve established by this method had a good linear relationship, with correlation coefficients all greater than 0.998, and low limits of detection and quantitation, which met the requirements for food safety testing.

[0063] Spiked recovery results: Mixed standard solutions of 7 alginate toxins at three different concentrations (2.0, 10.0 and 50.0 μg / kg) were added to blank edible vegetable oil samples. Spiked recovery was performed according to the above pretreatment method and instrument measurement conditions. Six parallel experiments were performed for each spiking level. The spiking concentration, average recovery rate and relative standard deviation (RSD) results are shown in Table 3.

[0064] Table 3

[0065]

[0066] The results showed that the average recoveries of the seven Alternaria toxins under different spiking levels were 81.0%–97.5%, and the relative standard deviations (RSDs) were 2.59%–8.12%, all of which met the requirements for analytical method validation, indicating that the method has good accuracy and precision.

[0067] Application example: Actual sample testing

[0068] The method of this invention was used to detect the content of seven alginate 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, blended edible oil, corn oil, and peanut oil. The measured results of the seven alginate mycotoxins in edible vegetable oils are shown in Table 4.

[0069] Table 4

[0070]

[0071] In summary, the detection method for seven alginate toxins in edible vegetable oils provided by this invention has the following significant advantages compared to existing technologies: it achieves simultaneous detection of seven alginate toxins in edible oils for the first time, with a wider detection range; it employs GPC purification technology to effectively remove interfering substances such as lipids and pigments in edible oils, resulting in a small matrix effect; it eliminates the need for expensive isotope internal standards to correct for matrix effects, reducing detection costs; it avoids the use of filter membranes, preventing the adsorption of components such as AME and AOH, thus improving the accuracy of detection results; the spiked recovery rate and precision of the method meet the requirements for analytical method validation, and it is applicable to the detection of seven alginate toxins in various edible vegetable oils.

[0072] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of 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, and chromatographic and mass spectrometric conditions can be optimized, etc. As long as they do not deviate from the technical solution and technical ideas of the present invention, they all fall within the protection scope of the present invention.

Claims

1. A method for detecting seven alginate toxins in edible vegetable oils, characterized in that, The method includes the following steps: S1: Sample extraction: Weigh the well-mixed edible oil sample, dissolve it in a 1:1 volume ratio ethyl acetate / cyclohexane solution and make up to volume, then transfer it to a GPC injection bottle; S2: Sample purification: The sample was purified using gel permeation chromatography (GPC) with ethyl acetate / cyclohexane in a 1:1 volume ratio as the mobile phase, and the eluent was collected over a specific time period. S3: Sample concentration treatment: Transfer the collected eluent to a rotary evaporation flask, evaporate to near dryness, and dissolve the residue with acetonitrile-water solution; S4: Sample centrifugation: Centrifuge the dissolved sample under specific conditions and collect the supernatant; S5: Instrument detection: Seven Alternaria toxins in the supernatant were detected using ultra-high performance liquid chromatography-tandem mass spectrometry, including Alternaria ketoacid, Alternaria spp., Alternaria spp., Alternaria spp. methyl ether, Alternaria spp., Alternaria spp. I and Alternaria spp. In step S2, 5.0 mL of sample solution was taken from the GPC for purification at a flow rate of 5 mL / min. The gel chromatography column packing material was Bio-BeadsSX3 with a column size of 25 mm × 500 mm. The first 1200 s of eluent was discarded, the next 600 s of eluent was collected, and finally the GPC column was eluted for 240 s. In step S5, the chromatographic conditions are as follows: the column is an Intersil ODS-3 C18 column with dimensions of 100 mm × 2.1 mm and a diameter of 1.7 µm; mobile phase A is 0.002% ammonia, and mobile phase B is acetonitrile; the injection volume is 5.0 µL; the flow rate is 0.25 mL / min; the column temperature is 30 °C; and the gradient elution program is as follows: 0–1.0 min, B is 5%; 1.0–6.0 min, B is 5%–80.0%; 6.0–6.5 min, B is 80.0%–90.0%; 6.5–7.5 min, B is 90.0%; 7.5–8.0 min, B is 90.0%–5.0%; and 8.0–10.0 min, B is 5%. In step S5, the mass spectrometry conditions are as follows: electrospray ionization source, negative ion scanning mode; spray voltage 4500V; ion source temperature 500℃; curtain gas pressure 35psi; nebulizer gas 1 pressure 50psi; nebulizer gas 2 pressure 50psi; multiple reaction monitoring mode.

2. The detection method according to claim 1, characterized in that, In step S1, 1 g of edible oil sample was weighed into a 10.0 mL brown volumetric flask, 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 S3, the mixture is rotary evaporated to near dryness at 40°C, and the residue is dissolved in 1.00 mL of 50% acetonitrile-water solution.

4. The detection method according to claim 1, characterized in that, In step S4, centrifuge at 12000 r / min at 4℃ for 10 min.

5. The detection method according to claim 1, characterized in that, It also includes the step of preparing a matrix-matched standard working curve: weigh the negative matrix sample, process it according to steps S1 to S4 to obtain a blank matrix extraction concentrate, and use the blank matrix extraction concentrate to prepare a standard curve solution.

6. The detection method according to claim 5, characterized in that, The mass concentrations of the standard curve solutions were 1.0, 5.0, 10.0, 50.0, 100.0, and 200.0 μg / L, respectively.

7. The detection method according to claim 1, characterized in that, After step S4, proceed directly to step S5 without using NYL or PTFE filter membranes to filter the sample.

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