Pretreatment method for determining zearalenone and alternaria toxin in grain or feed

Abstract

The invention discloses a pretreatment method for determining zearalenone and alternaria toxin in grain or feed, which comprises the following steps: adding an isotope internal standard into a grain or feed sample, adding an acetonitrile aqueous solution for vortex extraction, carrying out freezing centrifugation on the extracting solution for two-phase layering, and collecting an upper organic phase; adding an ammonia water solution into an upper-layer organic phase, uniformly swirling, freezing and centrifuging again, layering two phases, implementing back extraction of pH regulation and control, and collecting a lower-layer water phase as a to-be-detected solution. According to the method, two-phase layering is induced through freezing centrifugation of the acetonitrile aqueous solution, freezing centrifugation of pH regulation and control of the ammonia aqueous solution is combined for further purification, the pretreatment purification effect of the sample is greatly improved, matrix interference of grains or feed can be effectively reduced, the detection accuracy of zearalenone and alternaria toxin is obviously improved, and the method is suitable for large-scale popularization and application. The method has the advantages of simple operation, low solvent consumption, low cost and the like.

Description

Technical Field

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

[0002] Cereals are the most important food source for humans and are particularly susceptible to mycotoxin contamination. Therefore, all countries are strengthening the monitoring of mold in cereals to avoid harm to human health. At present, mass spectrometry analysis methods for mycotoxins in various cereals have been established, but the complexity of cereal food and feed matrices seriously affects the accuracy of mass spectrometry quantification. Traditional QuEChERS and SPE methods are widely used for the enrichment and purification of mycotoxins. This method has many operating steps, high costs and reagent consumption.

[0003] In recent years, the two-phase aqueous phase separation system based on liquid-liquid extraction technology, especially the low-temperature induced two-phase aqueous phase separation system without the use of additional phase inducers, has attracted extensive attention in the field of food contaminant analysis due to its advantages such as good biocompatibility, low interfacial tension and low cost. However, this method still has certain defects. For example, interfering substances with similar polarity to the target analyte will also be extracted into the analysis solution, which will bring obvious matrix effect problems to the subsequent mass spectrometry detection and 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 feeds comprises the following steps:

[0007] (1) Add an isotope internal standard to a grain or feed sample, then add an acetonitrile aqueous solution and perform vortex extraction. The extract is subjected to refrigerated centrifugation to separate the two phases, and the upper organic phase is collected;

[0008] (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.

[0009] Temperature has a certain effect 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 reduced to 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 makes full use of the low temperature induced phase separation of acetonitrile aqueous 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 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 then are extracted into the upper organic phase in the centrifugal-assisted freezing-induced two-phase separation, so as to remove 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 analysis target is ionized by adding an ammonia solution, thereby enhancing the water solubility of the target, and then reversely extracting into the aqueous phase solution in the centrifugal-assisted freezing-induced two-phase separation, so as to remove low-polarity interferences, thereby achieving a double purification effect, effectively reducing the interference of 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 aqueous ammonia solution is 5-15%.

[0013] Preferably, in step (2), the ratio of the grain or feed sample to the aqueous 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 10000-13000 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: using the above-mentioned pretreatment method to treat the grain or feed sample to obtain a test solution; and using liquid chromatography-mass spectrometry to detect the test solution.

[0016] Among them, the 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 (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; scanning 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; scanning 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.

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

[0019] The present invention adopts acetonitrile aqueous solution centrifugal assisted freezing induced two-phase stratification and combines ammonia aqueous solution pH control centrifugal assisted freezing induced two-phase stratification 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, and 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 is further described below by means of specific implementation methods. The following examples are preferred implementation methods of the present invention, but the implementation methods 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, put it into a 1.5 mL centrifuge tube, and add [ 13 C 18]-Zerallarenone isotope internal standard, add 1mL of 80% acetonitrile aqueous solution (v / v), vortex extract for 5min, place the extract in a -12℃ centrifuge, centrifuge at 10000r / m for 15min, collect the upper organic phase, add 200μL of 10% ammonia solution, vortex for 2min, and place again in -12℃

[0025] Centrifuge at 10000r / m for 15min 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 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.

[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 volume concentration of acetonitrile aqueous solution on the extraction of zearalenone and alternaria toxin

[0031] Weigh 0.1 g of grain sample, put it into a 1.5 mL centrifuge tube, and add [ 13 C 18 ]-Zeralenone isotope internal standard, add 1mL of 50%-80% acetonitrile aqueous solution (v / v), vortex extract for 5min, place the extract in a -12℃ centrifuge, and centrifuge at 10000r / m for 15min. Collect the upper organic phase, add 200μL of 10% ammonia solution, vortex for 2min, place in a -12℃ centrifuge again, and centrifuge at 10000r / m for 15min. The lower phase centrifuge was subjected to mass spectrometry detection. 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] As can be seen from the above results, after freezing centrifugation and two-phase separation with 50% to 80% acetonitrile aqueous solution, the analyte enters the upper acetonitrile organic layer. As the acetonitrile content increases, the extraction efficiency shows an upward trend. When the concentration of the acetonitrile aqueous solution reaches 80%, the extraction rate of zearalenone-4-O-β-D-glucoside reaches 91%, and the extraction rates of the remaining 9 analytes reach more than 96%. Therefore, the preferred volume concentration of the acetonitrile aqueous solution is 70-80%.

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

[0036] Weigh 0.1 g of sample, put it into a 1.5 mL centrifuge tube, and add [ 13 C 18 ]-Zerallarenone isotope internal standard, add 1mL, 80% acetonitrile aqueous solution (v / v), vortex extraction for 5min. The extract was placed in a -12℃ centrifuge and centrifuged at 10000r / m for 15min. The upper organic phase was collected, 200μL of 1-15% ammonia solution was added, vortexed for 2min, and placed in a -12℃ centrifuge again, and centrifuged at 10000r / m for 15min. The lower phase centrifuge was subjected to mass spectrometry detection, 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 the ammonia solution is fixed at 200 μL, the stripping efficiency of the analyte increases with the increase of the concentration of the ammonia solution. When the concentration of the ammonia solution reaches 10%, the stripping efficiency reaches equilibrium. Therefore, it is preferred to add a 5-15% ammonia solution as the stripping solvent.

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

[0041] Weigh 0.1 g of sample, put it into a 1.5 mL centrifuge tube, and add [ 13 C 18]-Zeralenone isotope internal standard, add 1mL, 80% acetonitrile aqueous solution (v / v), vortex for 5min. Place in a -12℃ centrifuge, centrifuge at 10000r / m for 15min. Collect the upper phase centrifuge, add 0.1-0.3mL of 10% ammonia solution, vortex for 2min, place in a -12℃ centrifuge again, centrifuge at 10000r / m for 15min. The lower phase centrifuge was subjected to mass spectrometry detection. 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 gradually increases, the concentration of the analyte in the lower aqueous phase also gradually increases. However, when the volume is >200 μL, the concentration of the analyte decreases with the increase in volume. This is mainly due to the decrease in the proportion of acetonitrile aqueous solution in the extraction system, which ultimately leads to an increase in the volume of the lower aqueous phase during centrifugation. Therefore, the preferred 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, put it into a 1.5 mL centrifuge tube, and add [ 13 C 18 ]-Zerallarenone isotope internal standard, add 1mL of 80% acetonitrile aqueous solution (v / v), vortex extract for 5min to obtain solution ①. Place solution ① in a -12℃ centrifuge, centrifuge at 10000r / m for 15min, take the upper solution to obtain solution ②, add 0.2mL of 10% ammonia solution to solution ②, vortex for 2min, place in a -12℃ centrifuge again, centrifuge at 10000r / m for 15min, and obtain the lower aqueous phase centrifuge ③.

[0048] Take zearalenone and alternaria toxin standards and use the solutions in ①, ②, ③ and pure acetonitrile solvent to prepare standard curve working solutions with a concentration of 0.01-10 μg / mL, perform HPLC-MS analysis, and fit the linear standard curve. Divide the difference between the slope of the linear matrix standard curve obtained in ①, ②, ③ and the slope of the acetonitrile solution standard curve by the slope of the acetonitrile solution standard curve to obtain the matrix effect (ME). The matrix effect (ME) value between -20% and 20% is considered to have no obvious matrix effect, and the effect on the result can be ignored. When ME is greater than 20%, it is considered to be a matrix enhancement effect. When ME is less than -20%, it is considered to be a matrix inhibition 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 freezing-induced two-phase demixing 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 shows that when the sample is treated using this method, the matrix effect is significantly reduced 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 grains 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 verified by analyzing six independent replicates, with an emphasis on discerning 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 was required to meet 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 toxins in cereal or feed matrices showed good linear relationships within the given analytical range (R 2 >0.9987). The detection limit and quantification limit were 0.03-0.18 μg / kg and 0.09-0.54 μg / kg, respectively; the matrix effect was -18--2%, respectively, with little matrix interference; the average recovery rate at high, medium and low levels was 68.5%-119.1%, proving the accuracy of the method. The RSD values ​​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 grains or feed, characterized in that: The steps include: (1) Add isotope internal standard to grain or feed samples, then add acetonitrile aqueous solution for vortex extraction, separate the two phases by frozen centrifugation, and collect the upper organic phase; (2) Take the upper organic phase, add ammonia solution, vortex evenly, freeze centrifuge again to separate the two phases, perform pH-controlled back extraction, and collect the lower aqueous phase enriched with zearalenone and alternaria toxin as the test solution.

2. The pretreatment method for determining zearalenone and alternaria toxin in grains or feed according to claim 1, characterized in that: In step (1), the volume concentration of the acetonitrile aqueous solution is 70-80%.

3. The pretreatment method for determining zearalenone and alternaria toxin in grains or feed according to claim 1, characterized in that: 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.

4. The pretreatment method for determining zearalenone and alternaria toxin in grains or feed according to claim 1, characterized in that: In step (2), the volume concentration of the ammonia solution is 5-15%.

5. The pretreatment method for determining zearalenone and alternaria toxin in grains or feed according to claim 4, characterized in that: In step (2), the ratio of the grain or feed sample to the ammonia solution is 0.1 g: 0.1-0.2 ml.

6. The pretreatment method for determining zearalenone and alternaria toxin in grains or feed according to any one of claims 1 to 5, characterized in that: The temperature of the refrigerated centrifugation is -12 to -15°C, the centrifugal speed is 10000-13000r / m, and the centrifugal time is 15-20min.

7. A method for determining zearalenone and alternaria toxin in grains or feed, characterized in that: The method comprises the following steps: using the pretreatment method according to any one of claims 1 to 6 to treat a grain or feed sample to obtain a test solution; and using liquid chromatography-mass spectrometry to detect the test solution.

8. The method for determining zearalenone and alternaria toxin in cereals or feed according to claim 7, characterized in that: The liquid chromatography conditions were: 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.

9. The method for determining zearalenone and alternaria toxin in cereals or feed according to claim 7, characterized in that: The mass spectrometry conditions were: Ion source: electrospray ion source; Scan mode: negative ion mode; Spray voltage: 3.0Kv; Capillary temperature: 320℃; Heater temperature: 400℃; Sheath gas: 40arb; Auxiliary gas: 10arb; 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.

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