A method for determining fumonisin and ochratoxin in cereals

Abstract

The present invention discloses a method for determining fumonisins and ochratoxins in cereals, comprising: adding cereal powder to a mixed isotope internal standard, then adding an extracting solution and vortexing and stirring for extraction, subjecting the extracting solution to low-temperature centrifugation, and after phase separation, removing a lower layer solution to obtain the extracting solution; the extracting solution is an acetonitrile-water solution containing aqueous ammonia. The method of the present invention adopts a low-temperature centrifugation induced phase separation integrated method to achieve a one-step operation process integrating target extraction, enrichment, purification and centrifugation, and by optimizing the extracting solution, fumonisins and ochratoxins of different polarity properties can be simultaneously transferred to the same small volume collection layer without the need for additional tedious manual operations, greatly improving the extraction, enrichment and purification efficiency, having the advantages of simple operation, short time consumption and low cost, and providing a new determination method for sample preparation and detection of fumonisins and ochratoxins in cereals.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and in particular to a method for determining fumonisin and ochratoxin in cereals. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced by filamentous fungi, primarily including aflatoxins, ochratoxins, zearalenone, vomitoxin, and fumonisins. These mycotoxins can contaminate crops and their by-products, such as peanuts, corn, and wheat. Mycotoxins-contaminated foods can cause acute or chronic poisoning, further damaging the liver, kidneys, and nervous system. Fumonisins (FBs) and ochratoxins (OTs) are common mycotoxins in my country. They are Class 2B carcinogens and are listed as priority pollutants by the Global Environment Monitoring System / Food (GEMS / Food). FBs and OTs contamination in cereals poses a threat to public health. Therefore, the detection of fumonisins and ochratoxins in cereals is crucial.

[0003] The methods currently used in the existing mycotoxin detection standards include thin layer chromatography, fluorescence spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry, etc. The determination and analysis of mycotoxins in cereal samples rich in starch and protein has always been challenging. The extraction efficiency of mycotoxins is adversely affected by the co-extraction of interfering substances in the matrix, and the presence of co-extracted interfering substances will adversely affect the recovery, repeatability and sensitivity of subsequent determination methods. To overcome this problem, sample preparation is considered an important step to eliminate analytical interference. In addition, due to the low concentration levels of mycotoxins, enrichment is required to obtain sufficient detection sensitivity. Therefore, developing an effective sample preparation procedure to extract and enrich mycotoxins, as well as to remove co-extracted interfering substances, remains a major challenge and research focus for the accurate determination of mycotoxins in cereal samples.

[0004] Common sample preparation methods include QuEChERS and solid-phase extraction (SPE) using reversed-phase, anion-exchange, and immunoaffinity columns. Fumonisin toxins contain multiple carboxylic acid groups, making it difficult to extract the target compounds into the acetonitrile organic phase via salt-induced phase separation during QuEChERS extraction. Furthermore, excessive use of the purification adsorbent PSA can lead to adsorption of fumonisin toxins during subsequent cleanup. For acidic mycotoxins, anion-exchange columns are often used for SPE cleanup during routine sample preparation, effectively enriching and removing acidic mycotoxins. However, for target components of varying acidity, it is often necessary to select an appropriate anion-exchange column, such as a strong or weak anion-exchange column. Furthermore, extraction results can vary between different brands of anion-exchange SPE columns. When analyzing large numbers of samples, the cost and time associated with using solid-phase extraction (SPE) using anion-exchange and immunoaffinity columns are high. Therefore, developing simpler, more convenient, time-saving, and cost-effective high-throughput analytical methods is crucial. Summary of the Invention

[0005] To solve the above technical problems, the purpose of the present invention is to develop a method for the rapid extraction, enrichment and purification of fumonisins and ochratoxins in cereals, and to widely apply it to the high-throughput detection of fumonisins and ochratoxins in food safety risk monitoring.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for extracting fumonisin and ochratoxin from cereals comprises the following steps:

[0008] The grain powder is added with a mixed isotope internal standard, and then the extract is added and vortexed. The extract is subjected to low-temperature centrifugation. After phase separation, the lower layer solution is taken to obtain the extract; the extract is an acetonitrile-water solution containing ammonia water.

[0009] The present invention provides a centrifugation-integrated low-temperature induced phase separation method, which realizes a one-step operation process of target extraction, enrichment, purification and centrifugation. The core idea of ​​this study is to use the solubility difference of acetonitrile and water at different temperatures to achieve effective phase separation, and combine pH adjustment to change the molecular form of the analyte, thereby improving the extraction efficiency of the target, while also smoothly removing low-polarity interfering components from the extraction solution. This achieves the goal of solving the problems of extraction, enrichment and purification in the same process. In addition, the centrifugation operation can significantly improve the performance of low-temperature induced phase separation. Therefore, the integration of a low-temperature centrifuge allows the target substance to be directly transferred to a microliter volume collection layer without the need for additional manual operation.

[0010] The mixed isotope internal standard of the present invention is 13 C-FB1 and 13C-OTA.

[0011] Preferably, in the extract, the volume concentration of the acetonitrile-water solution is 50-80%, and the volume concentration of the ammonia water is 1-10%.

[0012] Preferably, the pH value of the extract is ≥9.

[0013] Preferably, the mass volume ratio of the cereal powder to the extract is 1:10-1:30 g / mL.

[0014] Preferably, the vortex extraction time is 10-30 min.

[0015] Preferably, the temperature of the low-temperature centrifugation is -15 to -10°C, the time is 15 to 30 minutes, and the rotation speed is 10,000 to 13,000 rpm.

[0016] The present invention also provides a method for detecting fumonisins and ochratoxins in cereals, comprising the following steps:

[0017] (1) extracting the cereal powder using the above extraction method to obtain a sample to be tested;

[0018] (2) Liquid chromatography-mass spectrometry was used to detect the samples.

[0019] Preferably, in step (2), the liquid chromatography conditions are:

[0020] An HSS T3 chromatographic column was used, with 0.1% formic acid aqueous solution as mobile phase A and methanol / acetonitrile (1 / 1, v / v) solution as mobile phase B. The gradient elution process was set as follows: 0-1 min, 30% B, 1-8 min, 30-70% B, 8-10 min, 70-99% B, 10-12 min 99% B, 12-12.1 min 99%-30% B, 12-15 min 30% B. The flow rate of the column was 0.3 mL and the column temperature was 40°C.

[0021] Preferably, in step (2), the mass spectrometry conditions are:

[0022] Data were collected using a Q Exactive™ quadrupole-Orbitrap HRMS in positive ion mode with the following ion source parameters: spray voltage 3.8 kV; sheath gas 40 arb; auxiliary gas 10 arb; capillary temperature 320°C; heater temperature 400°C;

[0023] Targeted single ion monitoring (tSIM) / data-dependent MS / MS (ddMS 2) acquisition mode for qualitative and quantitative analysis of fumonisins and ochratoxins; tSIM parameters: mass resolution, 70,000 FWHM; AGC, 1×10 5 ; Separation window, 4Da; MSX, 4; ddMS 2 Parameters: mass resolution, 17,500 FWHM; AGC, 2×10 4 ; TopN, 5; dynamic exclusion, 6s; NCE is 15%, 35% and 55%.

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

[0025] The present invention provides a method for determining fumonisins and ochratoxins in cereals. The method adopts a small-scale centrifugation integrated low-temperature induced phase separation method to realize a one-step operation process of target extraction, enrichment, purification and centrifugation. Through the optimization of the extract, fumonisins and ochratoxins can be directly transferred to a microliter collection layer at the same time without the need for additional manual operation, greatly improving the extraction, enrichment and purification efficiency. The method has the advantages of simple operation, short time consumption, low cost, etc., and provides a new determination method for sample preparation and detection of fumonisins and ochratoxins in cereals. DETAILED DESCRIPTION

[0026] The present invention will be further described below by way of specific embodiments. The following examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples.

[0027] Example 1:

[0028] A method for extracting fumonisin and ochratoxin from cereals comprises the following steps:

[0029] Add 0.1 g of cereal powder sample to a 2 mL centrifuge tube, and then add 40 μL of mixed isotope internal standard ( 13 C-FB1 and 13 C-OTA) and 1.5 mL of extraction solution (acetonitrile / water / concentrated ammonia solution; 75 / 24 / 1, v / v / v), vortex extraction for 10 min, and then centrifuge at 10000 rpm in a -10 ° C centrifuge for 20 min. After phase separation, the lower layer solution was removed to obtain the sample to be tested.

[0030] Example 2: Optimization of extraction method

[0031] (1) Effect of pH value of the extract

[0032] This method investigates the extraction efficiency of fumonisin and ochratoxin in the lower aqueous phase after adding different concentrations (0%-10%) of ammonia solution to a 75% acetonitrile-water solution to adjust the pH value.

[0033] As shown in Table 1, when no ammonia water is added to the extract, ochratoxin is mainly enriched in the upper solution, and fumonisin is mainly enriched in the lower solution, and ochratoxin and fumonisin cannot be transferred to the lower solution at the same time. When the pH value of the extract is adjusted to alkaline conditions (≥9), the distribution of fumonisin and ochratoxin in the lower solution is significantly improved. Among them, when the concentration of ammonia water reaches or exceeds 1%, it is ensured that the absolute extraction recovery rate of all fumonisin and ochratoxin is high. Therefore, the preferred volume concentration of ammonia water is 1-10%.

[0034] Table 1 Effect of ammonia concentration on the recovery of fumonisin and ochratoxin / %

[0035]

[0036]

[0037] Note: FB1 is fumonisin 1, FB2 is fumonisin 2, FB3 is fumonisin 3, OTA is ochratoxin A, OTB is ochratoxin B, the same as in the table below.

[0038] (2) Effect of the concentration of acetonitrile-water solution in the extract

[0039] The extraction efficiency and enrichment factors of fumonisins and ochratoxins were evaluated using acetonitrile-water solutions of varying concentrations (50%, 60%, 70%, 75%, and 80%), supplemented with 1% ammonia water (Table 2). The results showed that the majority of ochratoxins and fumonisins were concentrated in the lower aqueous phase. The enrichment factor increased with increasing acetonitrile-water concentration from 50% to 75%, but peak areas did not change significantly when the acetonitrile-water concentration exceeded 75%.

[0040] Table 2 Effect of acetonitrile-water solution concentration on fumonisin and ochratoxin

[0041]

[0042] (3) Influence of rotation speed and temperature under centrifugal action

[0043] To enhance operational convenience and high throughput, this method combines centrifugation with low-temperature-induced phase separation. Under the optimal conditions described above, the extract was placed in a centrifuge to evaluate the effectiveness of low-temperature-induced phase separation. Results showed that, compared with traditional low-temperature-induced phase separation, phase separation could be stably achieved at higher temperatures under centrifugal force, which also helped shorten the time it took for phase separation to reach equilibrium. Table 3 tests the effects of different temperatures (-5, -10, and -15°C) and rotation speeds (0, 7,000, 10,000, and 13,000 rpm) on phase separation. Results showed that, at -5°C, no stable phase separation could be achieved within 30 minutes. At -10°C, phase separation occurred under centrifugal force, while no phase separation occurred in the control group without centrifugation. At -15°C, phase separation occurred in just 15 minutes under centrifugal force, while the control group without centrifugation required 30 minutes, shortening the phase separation time. In addition, at a rotation speed of 13,000 rpm, the centrifuge requires a longer equilibrium time to reach the preset temperature (-10 and -15°C). Therefore, the preferred temperature and rotation speed conditions of the present invention are a rotation speed of 10,000 rpm at -10°C.

[0044] Table 3 Effect of rotation speed and temperature under centrifugal force

[0045]

[0046] Note: “-” indicates homogeneous phase without phase separation; “+” indicates phase separation.

[0047] (4) Effect of cold induction time under centrifugal force

[0048] Under the above optimal conditions, different low-temperature centrifugation times (10, 15, 20, 25, and 30 minutes) were evaluated to determine the optimal low-temperature centrifugation time. Table 4 shows that the optimal extraction effect was achieved after a time exceeding 15 minutes. Therefore, the present invention preferably uses a low-temperature centrifugation time of 15-30 minutes.

[0049] Table 4 Effect of centrifugation time on the recovery of fumonisin and ochratoxin / %

[0050]

[0051] Example 3:

[0052] A method for detecting fumonisins and ochratoxins in cereals comprises the following steps:

[0053] The sample obtained in Example 1 was tested using liquid chromatography-mass spectrometry; wherein the liquid chromatography conditions were:

[0054] An HSS T3 chromatographic column was used, with 0.1% formic acid aqueous solution as mobile phase A and methanol / acetonitrile (1 / 1, v / v) solution as mobile phase B. The gradient elution process was set as follows: 0-1 min, 30% B, 1-8 min, 30-70% B, 8-10 min, 70-99% B, 10-12 min 99% B, 12-12.1 min 99%-30% B, 12-15 min 30% B. The flow rate of the column was 0.3 mL and the column temperature was 40°C.

[0055] Mass spectrometry conditions are:

[0056] Q Exactive TM Data were collected in positive ion mode using a quadrupole Orbitrap HRMS with the following ion source parameters: spray voltage 3.8 kV; sheath gas 40 arb; auxiliary gas 10 arb; capillary temperature 320 °C; heater temperature 400 °C.

[0057] Targeted single ion monitoring (tSIM) / data-dependent MS / MS (ddMS 2 ) acquisition mode for quantitative analysis of fumonisins and ochratoxins; tSIM parameters: mass resolution, 70,000 FWHM; AGC, 1×10 5 ; Separation window, 4Da; MSX, 4; ddMS 2 Parameters: mass resolution, 17,500 FWHM; AGC, 2×10 4 ; TopN, 5; dynamic exclusion, 6s; NCE were 15%, 35% and 55%. The results of the methodological investigation are shown in Table 5. The results show that all fumonisins and ochratoxins in the cereal matrix have a good linear relationship (R 2 >0.998). The limits of detection and quantification for fumonisins and ochratoxins were 0.3-0.36 μg / kg and 0.06-0.1 μg / kg, respectively. Matrix effects were 1.05-1.13 and 0.98-1.03, respectively. Spiked recoveries were 83.1-106.5% and 82.9-95.4%, respectively, demonstrating excellent performance.

[0058] Table 5 Methodological investigation

[0059]

Claims

1. A method for extracting fumonisin and ochratoxin from cereals, characterized in that: The steps include: The cereal powder is added with a mixed isotope internal standard, and then the extract is added and vortexed. The extract is subjected to low-temperature centrifugation. After phase separation, the lower layer solution is taken to obtain the extract; the extract is an acetonitrile-water solution containing aqueous ammonia; In the extract, the volume concentration of the acetonitrile-water solution is 50-80%, and the volume concentration of the ammonia solution is 1-10%; The pH value of the extract is ≥9; The low-temperature centrifugation temperature is -15 ~ -10 ° C, the time is 15-30 min, and the speed is 10000-13000 rpm; The fumonisin toxin is FB1, FB2 or FB3; the ochratoxin is OTB.

2. The method for extracting fumonisin and ochratoxin from cereals according to claim 1, characterized in that: The mass volume ratio of the cereal powder to the extract is 1:10-1:30 g / mL.

3. The method for extracting fumonisin and ochratoxin from cereals according to claim 1, characterized in that: The vortex extraction time is 10-30 min.

4. A method for detecting fumonisin and ochratoxin in cereals, characterized in that: The steps include: (1) Extracting cereal powder using the extraction method according to any one of claims 1 to 3 to obtain a sample to be tested; (2) Liquid chromatography-mass spectrometry was used to detect the samples.

5. The detection method according to claim 4, characterized in that In step (2), the liquid chromatography conditions are as follows: using an HSS T3 column, 0.1% formic acid aqueous solution as mobile phase A, methanol / acetonitrile 1 / 1, v / v solution as mobile phase B, the gradient elution process is set as follows: 0-1 min, 30% B, 1-8 min, 30-70% B, 8-10 min, 70-99% B, 10-12 min 99% B, 12-12.1 min 99%-30% B, 12-15 min 30% B, the flow rate of the column is 0.3 mL, and the column temperature is 40 °C.

6. The detection method according to claim 4, characterized in that In step (2), the mass spectrometry conditions are: Data were collected using a Q Exactive™ quadrupole-Orbitrap HRMS in positive ion mode with the following ion source parameters: spray voltage 3.8 kV; sheath gas 40 arb; auxiliary gas 10 arb; capillary temperature 320 °C; heater temperature 400 °C; targeted single ion monitoring (tSIM) / data-dependent MS / MS (ddMS) 2 ) acquisition mode for qualitative and quantitative analysis of fumonisins and ochratoxins; tSIM parameters: mass resolution, 70,000 FWHM; AGC, 1×10 5 ; Separation window, 4 Da; MSX, 4; ddMS 2 Parameters: mass resolution, 17,500 FWHM; AGC, 2×10 4 ; TopN, 5; dynamic exclusion, 6 s; NCE were 15%, 35% and 55%.

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