A pretreatment method for determining zearalenone and alternaria toxin in grains or feed

Abstract

The present invention discloses a pretreatment method for determining zearalenone and alternaria toxin in grains or feeds, comprising the steps of: adding an isotope internal standard to a grain or feed sample, then adding an acetonitrile aqueous solution for vortex extraction, subjecting the extract to refrigerated centrifugation for separation of the two phases, and collecting the upper organic phase; adding an ammonia aqueous solution to the upper organic phase, vortexing evenly, refrigerating centrifuging the two phases again for separation, performing pH-controlled back extraction, and collecting the lower aqueous phase as a test solution. The present invention uses refrigerated centrifugation of an acetonitrile aqueous solution to induce two-phase separation and combines it with refrigerated centrifugation with pH-controlled ammonia aqueous solution for further purification, thereby greatly improving the pretreatment purification effect of the sample. The method can effectively reduce matrix interference in the grain or feed, significantly improve the accuracy of zearalenone and alternaria toxin detection, and has the advantages of simple operation, low solvent consumption, and low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and particularly relates to a pretreatment method for determining zearalenone and alternaria toxin in grains or feed. Background Art

[0002] Cereals are a vital food source for humans and are particularly susceptible to mycotoxin contamination. Consequently, countries around the world are strengthening their monitoring of mold in cereals to prevent harm to human health. Mass spectrometry methods for the analysis of mycotoxins in various cereals have been established, but the complexity of cereal food and feed matrices significantly impacts the accuracy of mass spectrometry quantification. Traditional QuEChERS and SPE methods are widely used for the enrichment and purification of mycotoxins, but these methods involve numerous steps, resulting in high costs and reagent consumption.

[0003] In recent years, aqueous two-phase systems based on liquid-liquid extraction (LILE), particularly those that require no additional phase inducers and require low-temperature induced LILE, have garnered widespread attention in the analysis of food contaminants due to their advantages, including good biocompatibility, low interfacial tension, and low cost. However, this method still has certain drawbacks. For example, interfering substances with similar polarity to the target analyte may also be extracted into the analytical solution, leading to significant matrix effects in subsequent mass spectrometry analysis. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to develop a pretreatment method for determining zearalenone and alternaria toxin in grains or feed. When the sample is treated with the pretreatment method of the present invention, the matrix effect is significantly reduced and the accuracy of sample determination is significantly improved.

[0005] The present invention is achieved through the following technical solutions:

[0006] A pretreatment method for determining zearalenone and alternaria toxin in grains or feed comprises the following steps:

[0007] (1) Grain or feed samples were spiked with an isotope internal standard and then vortex-extracted with an acetonitrile-water solution. The extract was subjected to refrigerated centrifugation and the two phases were separated, and the upper organic phase was collected;

[0008] (2) The upper organic phase was added with aqueous ammonia solution, vortexed evenly, and the two phases were separated again by refrigerated centrifugation. pH-controlled back extraction was performed, and the lower aqueous phase enriched with zearalenone and alternaria toxin was collected as the test solution.

[0009] Temperature has a certain impact on the solubility of acetonitrile in water. At room temperature, acetonitrile is completely dissolved in water and exists as a homogeneous solution, but once the temperature is dropped below -10°C, the solubility will drop significantly, causing acetonitrile and water to form two phases, namely an upper organic phase and a lower aqueous phase. The present invention fully utilizes the low-temperature induced phase separation property of acetonitrile-water solution and innovatively introduces a low-temperature centrifuge into this operation process to improve the high-throughput characteristics of sample processing. The low-temperature centrifuge can integrate phase separation and centrifugation into a one-step process, which not only speeds up the procedure but also reduces the potential human errors associated with multi-step operations. The present invention utilizes in step (1) that the target analytes zearalenone and alternaria toxin exist in the solution in the form of molecular free state in the acetonitrile aqueous solution, and are then extracted into the upper organic phase in the centrifugation-assisted freeze-induced two-phase decomposition, thereby removing medium-polarity and high-polarity matrix interferences; in the operation of step (2), based on the characteristics of zearalenone and alternaria toxin containing acidic phenolic hydroxyl groups, the pH of the upper organic phase is controlled, and the target analysis object is ionized by adding an ammonia solution, thereby enhancing the water solubility of the target object, and then reversely extracting it into the aqueous phase solution in the centrifugation-assisted freeze-induced two-phase decomposition, thereby removing low-polarity interferences, achieving a double purification effect, effectively reducing the interference of the grain or feed matrix, and improving the accuracy of detection.

[0010] Preferably, in step (1), the volume concentration of the acetonitrile aqueous solution is 70-80%.

[0011] Preferably, in step (1), the ratio of the grain or feed sample to the acetonitrile aqueous solution is 0.1 g: 0.8-1.2 ml.

[0012] Preferably, in step (2), the volume concentration of the ammonia solution is 5-15%.

[0013] Preferably, in step (2), the ratio of the grain or feed sample to the ammonia solution is 0.1 g: 0.1-0.2 ml.

[0014] Preferably, the temperature of the refrigerated centrifugation is -12 to -15°C, the centrifugal speed is 10,000-13,000 r / m, and the centrifugal time is 15-20 min.

[0015] The present invention also provides a method for determining zearalenone and alternaria toxin in grains or feed, comprising the following steps: treating the grain or feed sample using the above-mentioned pretreatment method to obtain a test solution; and detecting the test solution using liquid chromatography-mass spectrometry.

[0016] Among them, the liquid chromatography conditions are as follows: chromatographic column: C18 column; mobile phase: phase A is a mixture of methanol and acetonitrile in a ratio of 1:1 (v / v); phase B is water; gradient elution conditions are: 0-1 min, 10% A; 1-18 min, 10-100% A; 18-20 min, 100% A; column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 10 μL.

[0017] The mass spectrometry conditions were as follows: ion source: electrospray ion source; scan mode: negative ion mode; spray voltage: 3.0 kV; capillary temperature: 320°C; heater temperature: 400°C; sheath gas: 40 arb; auxiliary gas: 10 arb; scan mode: tSIM / data-dependent MS / MS mode; tSIM parameters: mass resolution, 70,000 FWHM; AGC, 1×10 5 ; 4 m / z isolation window; ddMS2 parameters: mass resolution, 17,500 FWHM; AGC, 2 × 10 4 ; TopN 5; dynamic repulsion, 8 seconds; collision energy NCE is 15, 35, 55.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention adopts centrifugal-assisted freezing-induced two-phase decomposition of acetonitrile aqueous solution and combines it with ammonia aqueous solution pH control centrifugal-assisted freezing-induced two-phase decomposition back extraction for further purification, which greatly improves the pretreatment effect of the sample. The method can effectively reduce the matrix interference of grains or feeds, significantly improves the accuracy of zearalenone and alternaria toxin detection, and has the advantages of simple operation, low solvent consumption, low cost, etc. DETAILED DESCRIPTION

[0020] 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.

[0021] The reagents and samples used in the present invention are all commercially available.

[0022]

[0023] 1. Sample pretreatment method:

[0024] Weigh 0.1 g of grain sample, place it in a 1.5 mL centrifuge tube, and add [ 13 C 18]-Zerallarenone isotope internal standard, add 1 mL of 80% acetonitrile aqueous solution (v / v), vortex extract for 5 minutes, place the extract in a -12 ° C centrifuge, centrifuge at 10000 r / m for 15 minutes, collect the upper organic phase, add 200 μL of 10% ammonia solution, vortex for 2 minutes, and place in a -12 ° C centrifuge again.

[0025] Centrifuge at 10,000 rpm for 15 min and take the lower aqueous phase as the test solution.

[0026] 2. Determination method:

[0027] Liquid chromatography conditions: chromatographic column: C18 column; mobile phase: phase A is methanol / acetonitrile (1:1, v / v); phase B is water; gradient elution conditions: 0-1 min, 10% A; 1-18 min, 10-100% A; 18-20 min, 100% A; column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 10 μL.

[0028] Mass spectrometry conditions: ion source: electrospray ion source; scan mode: negative ion mode; spray voltage: 3.0 KV; capillary temperature: 320°C; heater temperature: 400°C; sheath gas: 40 arb; auxiliary gas: 10 arb; scan mode: tSIM / data-dependent MS / MS mode; tSIM parameters: mass resolution, 70000 FWHM; AGC, 1×10 5 ; 4 m / z isolation window; ddMS2 parameters: mass resolution, 17,500 FWHM; AGC, 2×10 4 ; TopN 5; dynamic repulsion, 8 seconds; collision energy NCE is 15, 35, 55.

[0029] 3. Result analysis:

[0030] 3.1 Effect of acetonitrile aqueous solution volume concentration on the extraction of zearalenone and alternaria toxin

[0031] Weigh 0.1 g of grain sample, place it in a 1.5 mL centrifuge tube, and add [ 13 C 18 ]-Zeralenone isotope internal standard was added to 1 mL of 50%-80% acetonitrile aqueous solution (v / v), and the extract was vortexed for 5 minutes. The extract was centrifuged at 10,000 rpm in a -12°C centrifuge for 15 minutes. The upper organic phase was collected, 200 μL of 10% ammonia solution was added, vortexed for 2 minutes, and centrifuged again at 10,000 rpm in a -12°C centrifuge for 15 minutes. The lower centrifuge was subjected to mass spectrometry. The results are shown in Table 1.

[0032] Table 1 Effect of acetonitrile aqueous solution concentration on the extraction of zearalenone and alternaria toxin (%)

[0033]

[0034] The above results indicate that after refrigerated centrifugation with a 50% to 80% acetonitrile-water solution, the analytes enter the upper acetonitrile organic layer. Extraction efficiency increases with increasing acetonitrile content. When the acetonitrile-water concentration reaches 80%, the extraction rate of zearalenone-4-O-β-D-glucoside reaches 91%, and the extraction rates of the remaining nine analytes exceed 96%. Therefore, a volume concentration of 70-80% acetonitrile-water solution is preferred.

[0035] 3.2 Effect of ammonia concentration on the extraction of zearalenone and alternaria toxin (peak area)

[0036] Weigh 0.1g of sample, place in a 1.5mL centrifuge tube, add [ 13 C 18 ]-Zerallarenone isotope internal standard, 1 mL of 80% acetonitrile aqueous solution (v / v) was added and vortexed for 5 minutes. The extract was placed in a -12°C centrifuge and centrifuged at 10,000 rpm for 15 minutes. The upper organic phase was collected, 200 μL of 1-15% ammonia solution was added, vortexed for 2 minutes, and placed in a -12°C centrifuge again and centrifuged at 10,000 rpm for 15 minutes. The lower phase centrifuge was subjected to mass spectrometry analysis, and the results are shown in Table 2.

[0037] Table 2 Effect of ammonia concentration on the extraction of zearalenone and alternaria toxin (peak area)

[0038]

[0039] As shown in Table 2, when the volume of ammonia solution is fixed at 200 μL, the stripping efficiency of the analytes increases continuously with increasing ammonia concentration. When the ammonia concentration reaches 10%, the stripping efficiency reaches equilibrium. Therefore, it is preferred to add ammonia solution with a concentration of 5-15% as the stripping solvent.

[0040] 3.3 Effect of ammonia volume on the extraction of zearalenone and alternaria toxin (peak area)

[0041] Weigh 0.1g of sample, place in a 1.5mL centrifuge tube, add [ 13 C 18Add 1 mL of 80% acetonitrile in water (v / v) to the zearalenone isotope internal standard and vortex for 5 minutes. Centrifuge at 10,000 rpm at -12°C for 15 minutes. Collect the upper phase centrifuge, add 0.1-0.3 mL of 10% ammonia solution, vortex for 2 minutes, and centrifuge again at 10,000 rpm at -12°C for 15 minutes. The lower phase centrifuge was analyzed by mass spectrometry. The results are shown in Table 3.

[0042] Table 3 Effect of ammonia volume on the extraction of zearalenone and alternaria toxin (peak area)

[0043]

[0044]

[0045] The results in Table 3 show that as the volume of ammonia solution increases, the analyte concentration in the lower aqueous phase also increases. However, when the volume exceeds 200 μL, the analyte concentration decreases with increasing volume. This is primarily due to the reduction in the acetonitrile-water ratio in the extraction system, which ultimately increases the volume of the lower aqueous phase during centrifugation. Therefore, the optimal ratio of grain or feed sample to ammonia solution is 0.1 g: 0.1-0.2 ml.

[0046] 4. Investigation of Matrix Effects

[0047] Weigh 0.1 g of grain sample, place it in a 1.5 mL centrifuge tube, and add [ 13 C 18 ]-zearalenone isotope internal standard, add 1 mL of 80% acetonitrile aqueous solution (v / v), and vortex extract for 5 minutes to obtain solution ①. Place solution ① in a -12℃ centrifuge and centrifuge at 10,000 rpm for 15 minutes. Take the upper layer to obtain solution ②. Add 0.2 mL of 10% ammonia aqueous solution to solution ②, vortex for 2 minutes, and place in a -12℃ centrifuge again and centrifuge at 10,000 rpm for 15 minutes to obtain the lower aqueous centrifuge ③.

[0048] Zearalenone and Alternaria toxin standards were prepared using the solutions in steps ①, ②, and ③, respectively, and pure acetonitrile solvent to create standard curve working solutions ranging from 0.01 to 10 μg / mL. High-performance liquid chromatography-mass spectrometry analysis was performed, and linear standard curves were fitted. The matrix effect (ME) was calculated by dividing the difference between the slopes of the linear matrix standard curves obtained in steps ①, ②, and ③ and the slope of the acetonitrile standard curve by the slope of the acetonitrile standard curve. A matrix effect (ME) value between -20% and 20% was considered to be nonsignificant and negligible, with a negligible impact on the results. An ME greater than 20% was considered a matrix-enhancing effect. An ME less than -20% was considered a matrix-inhibiting effect. The results are shown in Table 4.

[0049] As shown in Table 4, the ME of the unpurified matrix extract (① solution) ranged from -24% to -58%. The ME of some analytes in the matrix extract of ② solution was reduced to a negligible level, but some analytes still showed significant ME (-20% to -35%). This indicates that the single centrifugation-assisted freeze-induced two-phase decomposition method (② solution) can reduce the ME of some analytes in the matrix extract to a negligible level, but some analytes still showed significant ME (-21% to -27%). After purification using the method of the present invention (③ solution), the ME of all analytes was reduced to a negligible level (-2% to -18%). This indicates that the matrix effect is significantly reduced when the sample is treated with this method, and the accuracy of sample measurement is significantly improved.

[0050] Table 4 Effect of different treatments on matrix effect (%)

[0051]

[0052]

[0053] 5. Methodological Investigation

[0054] According to the Eurachem laboratory analytical method validation guidelines, the present invention has been rigorously validated for cereals and feeds. The method validation included method selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision (intra-day variability RSD r and inter-day variability RSD R ). The selectivity of the proposed method was tested by analyzing six independent replicates, with an emphasis on identifying any potential interferences present in the complex sample matrix. The target analytes were identified by retention time and accurate mass of the quasi-molecular ion, where the mass error should be within 5 ppm. Calibration was performed using solvent and matrix-matched standards within the given concentration range. The LOD and LOQ of each analyte were determined using the lowest matrix-matched standard solution. The LOD was determined as three times the standard deviation, where the standard deviation was obtained by measuring ten replicates of the lowest matrix-matched standard solution. The limit of quantification was evaluated as the lowest spike concentration of the sample that required good accuracy and precision. Accuracy and precision were calculated by spiking three different concentrations of analyte into the blank sample. Recovery (%) was used to evaluate accuracy, which was calculated from six replicates at three spiking levels. Eighteen replicates of three batches were analyzed on one day and on three consecutive days to evaluate the RSD. r and RSD R .

[0055] The results of the methodological investigation are shown in Table 5. All 10 zearalenone and alternaria mycotoxins in cereal or feed matrices showed good linearity within the given analytical range (R 2 >0.9987). The limits of detection and quantification were 0.03-0.18 μg / kg and 0.09-0.54 μg / kg, respectively. Matrix effects ranged from -18% to -2%, respectively, indicating minimal matrix interference. Average recoveries at high, medium, and low levels ranged from 68.5% to 119.1%, demonstrating the accuracy of the method. RSDs were <10.8% and <14.2%, respectively, confirming the reliability of the method.

[0056] Table 5 Methodological investigation results

[0057]

[0058]

Claims

1. A pretreatment method for determining zearalenone and alternaria toxin in cereals, characterized in that: The steps include: (1) Add an isotope internal standard to a grain sample, then add an acetonitrile-water solution and perform vortex extraction. The extract is separated into two phases by refrigerated centrifugation, and the upper organic phase is collected. The volume concentration of the acetonitrile-water solution is 70-80%. The dosage ratio of the grain sample to the acetonitrile-water solution is 0.1 g: 0.8-1.2 ml. (2) The upper organic phase was added with an ammonia solution, vortexed evenly, and the two phases were separated by refrigerated centrifugation again. The pH-controlled back extraction was performed, and the lower aqueous phase enriched with zearalenone and alternaria toxin was collected as the test solution; the volume concentration of the ammonia solution was 5-15%; and the dosage ratio of the grain sample to the ammonia solution was 0.1 g: 0.1-0.2 ml.

2. The pretreatment method for determining zearalenone and alternaria toxin in cereals according to claim 1, characterized in that: The temperature of the refrigerated centrifugation is -12 to -15°C, the centrifugal speed is 10,000-13,000 rpm, and the centrifugal time is 15-20 minutes.

3. A method for determining zearalenone and alternaria toxin in cereals, characterized in that: The method comprises the following steps: treating a grain sample by the pretreatment method according to claim 1 or 2 to obtain a solution to be tested; and detecting the solution to be tested by liquid chromatography-mass spectrometry.

4. The method for determining zearalenone and alternaria toxin in cereals according to claim 3, characterized in that: Liquid chromatography conditions are: Chromatographic column: C18 column; mobile phase: phase A is a mixture of methanol and acetonitrile in a ratio of 1:1; phase B is water; gradient elution conditions are: 0-1 min, 10% A; 1-18 min, 10-100% A; 18-20 min, 100% A; column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 10 μL.

5. The method for determining zearalenone and alternaria toxin in cereals according to claim 3, characterized in that: Mass spectrometry conditions are: Ion source: electrospray ion source; scan mode: negative ion mode; spray voltage: 3.0 kV; capillary temperature: 320°C; heater temperature: 400°C; sheath gas: 40 arb; auxiliary gas: 10 arb; scan mode: tSIM / data-dependent MS / MS mode; tSIM parameters: mass resolution, 70,000 FWHM; AGC, 1×105; 4 m / z isolation window; ddMS2 parameters: mass resolution, 17,500 FWHM; AGC, 2×10 4 ; TopN 5; dynamic repulsion, 8 seconds; collision energy NCE is 15, 35, 55.

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