Method for detecting pyrrolizidine alkaloids in food

Abstract

The invention relates to a method for detecting pyrrolizidine alkaloids in food, and belongs to the technical field of detection. The technical problem to be solved by the invention is to provide a novel method for detecting pyrrolizidine alkaloids in food. The detection method comprises the following steps: A, food treatment: solid food treatment or liquid food treatment is carried out, the solid food treatment comprises crushing, twice extraction with a 95% ethanol solution containing 0.1% formic acid, centrifugation and activated PXC-SPE small column purification, and a supernatant 4 is obtained; the treatment of the liquid food comprises the following steps: extracting with an H2SO4 solution, centrifuging, and purifying with an activated PXC-SPE small column to obtain supernate 4; and B, determining the pyrrolizidine alkaloids in the supernate 4 by using liquid chromatography-tandem mass spectrometry. According to the method, the bottleneck problems of less index coverage, complex matrix interference and the like are effectively solved, the liquid chromatography-tandem mass spectrometry accurate detection method for pyrrolizidine alkaloid pollution in various foods is established, and the detected pyrrolizidine alkaloid types are more comprehensive.

Description

Technical Field

[0001] The present invention relates to a method for detecting pyrrolizidine alkaloids in foods, belonging to the technical field of detection. Background Art

[0002] As typical plant-derived secondary metabolite toxins, pyrrolizidine alkaloids have the characteristic of cross-media migration in the ecosystem. Their pollution pathways show a three-level transfer feature of "plant-soil / water-crop", and then transfer to animals through feed, causing animal-derived food pollution. In addition, long-term intake of foods containing pyrrolizidine alkaloids may cause chronic damage to human health, leading to hepatotoxicity, pulmonary toxicity, carcinogenicity and teratogenicity. Therefore, the control of pyrrolizidine alkaloids is of great significance.

[0003] However, currently only a few methods are applicable to the detection of pyrrolizidine alkaloids in specific foods. There are several challenges in the current treatment of pyrrolizidine alkaloid pollution: the diversity of stereoisomers of pyrrolizidine alkaloids and the complexity of food matrices lead to technical barriers in detection; the resolution accuracy of the detection method for trace isomers is insufficient; the traditional "end-treatment" mode is difficult to cope with such pollutants with biological magnification effects, and a whole-chain control system from weed prevention and control to processing and degradation needs to be established; in addition, the detection limit and quantification limit ranges of the methods still need to be further improved.

[0004] Yao Leijun, Chen Yanqiu, etc. Determination of 27 pyrrolizidine alkaloids in tea by integrated QuEChERS purification-ultra high performance liquid chromatography-tandem mass spectrometry [J]. Journal of Tea Science, 2024(5). discloses a method for detecting 27 pyrrolizidine alkaloids in tea. However, it can only detect specific food tea and cannot cover other foods vulnerable to pyrrolizidine alkaloid pollution (such as honey, herbs, grains) at the same time. Moreover, its detection limit is high, the quantification limit range is narrow, and the relative standard deviation of some compounds is above 10%, indicating that the stability of the method in complex matrices needs to be improved, and the types of pyrrolizidine alkaloids detected also need to be further increased.

[0005] Chen Yankai. Rapid determination of the content of 26 pyrrolizidine alkaloids in milk powder and liquid milk by solid-phase extraction combined with ultra high performance liquid chromatography-tandem mass spectrometry [J]. Food Science, 2024(24). discloses a method for rapidly determining the content of 26 pyrrolizidine alkaloids in milk powder and liquid milk by solid-phase extraction combined with ultra high performance liquid chromatography-tandem mass spectrometry. However, it can only detect specific foods, milk powder and liquid milk, and cannot cover other foods vulnerable to pyrrolizidine alkaloid pollution at the same time. Moreover, it does not verify and explain the complexity of the matrix, such as whether defatting will affect the detection results. At the same time, its quantification limit range is narrow, and the types of pyrrolizidine alkaloids detected need to be further increased. Summary of the Invention

[0006] The object of the present invention is to provide a new method for detecting pyrrolizidine alkaloids in food.

[0007] To achieve the above object of the present invention, the method for detecting pyrrolizidine alkaloids in food includes the following steps: A. Food treatment: including the treatment of solid food or liquid food: The treatment of solid food is as follows: a. The solid food is crushed and passed through a 60-mesh sieve to obtain the undersize and oversize; b. The undersize obtained in step a is mixed with a 95% ethanol solution containing 0.1% formic acid, vortexed for 1 min, ultrasonically extracted at 40 Hz for 30 min, and centrifuged at 8500 rpm for 5 min to obtain supernatant 1 and residue 1. The mass-volume ratio of the undersize to the 95% ethanol solution containing 0.1% formic acid is 1 g:10 mL; c. The residue 1 is further mixed with a 95% ethanol solution containing 0.1% formic acid, vortexed for 1 min, ultrasonically extracted at 40 Hz for 30 min, and centrifuged at 8500 rpm for 5 min to obtain supernatant 2 and residue 2; d. The supernatant 1 and supernatant 2 are combined, blown to less than 5 mL with nitrogen, and then made up to 10 mL with a 0.1% formic acid aqueous solution, and centrifuged to obtain supernatant 3; e. Then, 3 - 5 mL of supernatant 3 is loaded onto an activated PXC-SPE small column. After loading, first wash with 3 mL of water, then wash with 3 mL of methanol. After washing, the eluent in the PXC-SPE small column is blown dry, and then eluted with 5 mL of a 5% ammonia-methanol solution. The eluate is collected, and the eluate is blown dry with nitrogen at 40°C, made up to 1 mL with a reconstitution solvent, and filtered through a 0.22 μm nylon filter membrane to obtain supernatant 4. The activation method of the PXC-SPE small column is to pass 3 mL of methanol, 3 mL of water, and 3 mL of a 0.1% formic acid aqueous solution through the PXC-SPE small column in sequence. The reconstitution solvent is a 50% methanol solution containing 0.1% formic acid; The treatment of liquid food is as follows: The liquid food is mixed with an H2SO4 solution, ultrasonically extracted at 40 Hz for 30 min, cooled to room temperature, and then centrifuged at 8500 rpm for 5 min to obtain supernatant 3. The supernatant 3 is subjected to subsequent operations according to step e to obtain supernatant 4. The mass-volume ratio of the liquid food to the 0.05 mol / L H2SO4 solution is 1 g:10 mL. The concentration of the H2SO4 solution is 0.05 mol / L; B. Determination of pyrrolizidine alkaloids in supernatant 4 by liquid chromatography - tandem mass spectrometry; Among them, the percentage contents in the 95% ethanol solution containing 0.1% formic acid, the 5% ammonia methanol solution, the 50% methanol solution containing 0.1% formic acid, and the 0.1% formic acid aqueous solution are all volume percentages.

[0008] Experiments show that as the sample loading amount on the PXC - SPE column increases, the recovery rate of the target compounds of pyrrolizidine alkaloids generally shows a downward trend, and the recovery rate is the highest when the sample loading amount is 3 - 5 mL.

[0009] Experimental results show that under the elution conditions of first eluting with 3 mL of pure water and then with 3 mL of methanol, the target compounds of pyrrolizidine alkaloids in the sample will not be lost due to elution by the eluent.

[0010] Experiments show that choosing the 5% ammonia methanol solution as the elution solvent has good elution ability for various pyrrolizidine alkaloids. The elution ability of 5 mL of the 5% ammonia methanol solution can meet the recovery rate requirements, and there is no obvious difference when adding 1 mL more of the elution solvent.

[0011] In step e, the eluate is the purified sample solution. In order to improve the detection efficiency and the accuracy of the detection results, the eluate is concentrated by nitrogen blowing. A large number of experiments show that the response of each target compound is the highest in 50% methanol containing 0.1% formic acid, and the responses of each target compound in other solvents, such as 20% methanol, 50% methanol, and 50% methanol containing 0.1% ammonia, are not good.

[0012] Experiments show that filtering through a 0.22 μm filter membrane after re - dissolution has no effect on the results and will not cause instrument blockage.

[0013] In a specific embodiment, the solid food is tea, chrysanthemum, corn or rice; the liquid food is honey.

[0014] In a specific embodiment, the pyrrolizidine alkaloids include at least one of: melanocyte - stimulating hormone, melanocyte - stimulating hormone N - oxide, lycoclavine, lycoclavine N - oxide, heliotrine, echimidine, echimidine N - oxide, lasiocarpine, lasiocarpine N - oxide, monocrotaline, heliotrine N - oxide, europine, europine N - oxide, 7 - acetyl - melanocyte - stimulating hormone, 7 - acetyl - melanocyte - stimulating hormone N - oxide, monocrotaline N - oxide, senecionine, seneciphylline, senecionine N - oxide, seneciphylline N - oxide, jacobine, jacobine N - oxide, erucifoline, retrorsine, erucifoline N - oxide, retronecine, trichodesmine, seneciphylline N - oxide.

[0015] In a specific embodiment, when the food is tea, matrix-matched standard curve method is used for liquid chromatography-tandem mass spectrometry determination; when the food is chrysanthemum, corn, rice or honey, solvent standard curve method is used for liquid chromatography-tandem mass spectrometry determination; the preparation method of the matrix mixed standard working solution for the matrix-matched standard curve method is as follows: select a blank sample with the same properties as the food to be tested, prepare 7 blank matrix solutions according to step A, then accurately add the mixed standard stock solution respectively, blow with nitrogen until 10-20 μL of liquid remains, add 1.0 mL of 50% methanol re-solvent containing 0.1% formic acid to dissolve the residue, filter through a 0.22 μm nylon filter membrane, and prepare a series of matrix mixed standard working solutions with the mass concentration of each pyrrolizidine alkaloid being 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 2.5 μg / L, 5.0 μg / L, 7.5 μg / L, 10.0 μg / L respectively, and use them freshly prepared.

[0016] A large number of experiments show that the matrix effect is not obvious in honey, corn, rice and chrysanthemum, but there is a relatively obvious matrix suppression effect for more than 90% of the target compounds in the tea matrix. Especially for seneciphylline N-oxide, jacobine and retrorsine, they are all above -60%. Therefore, the matrix-matched standard curve method is used to reduce the influence of the tea matrix on the quantitative analysis of 31 target analytes to ensure the best accuracy of the results.

[0017] In a specific embodiment, the mass concentration of each pyrrolizidine alkaloid in the mixed standard stock solution is 10.0 μg / L, and the solvent of the mixed standard stock solution is methanol.

[0018] In a specific embodiment, the chromatographic column of the liquid chromatography is: Agilent Eclipse PlusC 18RRHD chromatographic column; The mobile phase of liquid chromatography includes three solutions A, B, and C; A is an aqueous formic acid solution with a volume concentration of 0.1% containing 5 mmol / L ammonium formate, B is methanol, and C is acetonitrile; Gradient elution is carried out successively with the mobile phase, and the gradient elution program is as follows: 0 - 2 min, the volume of phase A is from 95% to 90%, and the volume of phase B is from 5% to 10%; 2 - 5 min, the volume of phase A is from 90% to 80%, and the volume of phase B is from 10% to 20%; 5 - 10 min, the volume of phase A is 80%, and the volume of phase B is 20%; 10 - 16 min, the volume of phase A is from 80% to 60%, and the volume of phase B is from 20% to 40%; 16 - 18 min, the volume of phase A is from 60% to 50%, and the volume of phase B is from 40% to 50%; 18 - 22 min, the volume of phase A is 50%, and the volume of phase B is 50%; 22 - 25 min, the volume of phase A is from 50% to 5%, the volume of phase B is from 50% to 0%, and the volume of phase C is from 0% to 95%; 25 - 28 min, the volume of phase A is 5%, the volume of phase B is 0%, and the volume of phase C is 95%; 28 - 28.1 min, the volume of phase A is from 5% to 95%, the volume of phase B is from 0% to 5%, and the volume of phase C is from 95% to 0%; 28.1 - 35 min, the volume of phase A is 95%, and the volume of phase B is 5%; Flow rate: 0.3 mL / min; Chromatographic column temperature: 40 °C; Injection volume: 2 μL.

[0019] The experimental results show that thirty-one pyrrolizidine alkaloids can obtain better responses in the positive ion ionization mode of the ESI source.

[0020] The experiment shows that the thirty-one target analytes have good peak shapes on an Agilent Eclipse Plus C chromatographic column with specifications: 3.0 mm × 150 mm, 1.8 μm, and the retention time range is 3.0 min - 25.0 min, and several pairs of isomers can be effectively separated. For other chromatographic columns, such as the Waters HSST3 chromatographic column with specifications 3.0 mm × 100 mm, 2.5 μm, the Agilent RRHD SB-C chromatographic column with specifications 2.1 mm × 100 mm, 1.8 μm 18 and the Agilent SB-Phenly RRHD chromatographic column with specifications 2.1 × 100 mm, 1.8 μm have poor separation effects, as shown in 18 . Figures 1-4 .

[0021] The experiment also shows that when using an aqueous formic acid solution with a volume concentration of 0.1% containing 5 mmol / L ammonium formate and methanol as the mobile phase composition and adopting the above gradient elution program, the thirty-one target analytes all have good resolution, chromatographic peak shapes, and mass spectrometry responses.

[0022] In a specific embodiment, the Agilent Eclipse Plus C 18 RRHD chromatographic column has a specification of 3.0 mm × 150 mm, 1.8 μm.

[0023] In a specific embodiment, the mass spectrometry conditions of the tandem mass spectrometry include: Ion source: electrospray ionization source; Scanning mode: positive ion mode; Monitoring mode: multiple reaction monitoring; Temperature of drying gas N2: 350 °C; Pressure of nebulizing gas N2: 275.8 kPa; Flow rate of drying gas: 12.0 L / min; Temperature of sheath gas N2: 350 °C; Flow rate of sheath gas: 12.0 L / min; Capillary voltage: 3500 V in negative ion mode and 3500 V in positive ion mode.

[0024] A large number of experiments show that finally 2 monitoring ion pairs are selected for each target analyte. The specific multiple reaction monitoring parameters are shown in Table 1. The MRM diagram of melanocyte-stimulating hormone is shown in detail in Figure 5 . Meanwhile, the experiment also studies the ion source parameters such as the temperature of the drying gas, the flow rate of the drying gas, the pressure of the sprayer, the capillary voltage, the temperature of the sheath gas, and the flow rate of the sheath gas. Using the mass spectrometry conditions of the present invention, the ionization efficiency of each target analyte reaches the best.

[0025] In a specific embodiment, the reference retention times, quantitative ion pairs, qualitative ion pairs, in-source fragmentation voltages, collision energies and other parameters of the respective pyrrolizidine alkaloids are shown in Table 1: Table 1 Reference retention times and mass spectrometry parameter conditions of thirty-one pyrrolizidine alkaloids

[0026] In all the tables of the present invention, Compounds 1 to 31 represent monocrotaline, senecionine, monocrotaline N-oxide, senecionine N-oxide, seneciphylline, heliotrine, melanocyte-stimulating hormone, lycopsamine, seneciphylline N-oxide, retrorsine N-oxide, riebeckine, heliotrine N-oxide, melanocyte-stimulating hormone N-oxide, lycopsamine N-oxide, retrorsine, lasiocarpine, jacobine, heliotridine, 7-acetylmelanocyte-stimulating hormone, jacobine N-oxide, 7-acetylmelanocyte-stimulating hormone N-oxide, heliotridine N-oxide, vernolicine, senecionine N-oxide, vernolicine N-oxide, echinatine, echinatine N-oxide, clivorine, lasiocarpine N-oxide, lasiocarpine N-oxide in sequence.

[0027] In a specific embodiment, the average spiked recovery rate of the method is 66.6% - 101.6%, the average relative standard deviation is 0.5% - 6.0%, the detection limit is 0.06 μg / kg - 0.66 μg / kg, and the quantification limit is 0.2 μg / kg - 2 μg / kg.

[0028] Beneficial effects: The present invention effectively solves bottleneck problems such as few indicator coverages and complex matrix interferences, establishes a precise liquid chromatography - tandem mass spectrometry detection method for the contamination of pyrrolizidine alkaloids in various foods, has a more comprehensive variety of detected pyrrolizidine alkaloids, provides a decision - making basis and reference for formulating relevant limit standards, detection standards, risk prevention and control measures, etc., provides a scientific basis for product quality grading and certification, boosts the high - quality development of the local economy, and provides continuous technical support for food import and export trade and food safety.

[0029] The average spiked recovery rate of the method of the present invention at three concentration levels of 2 μg / kg, 5 μg / kg, and 10 μg / kg is between 66.6% and 101.6%, the average relative standard deviation is 0.1% - 6.0%, the detection limit is 0.06 μg / kg - 0.66 μg / kg, and the quantification limit is 0.2 μg / kg - 2 μg / kg. It has the characteristics of simplicity, high efficiency, accuracy, sensitivity, economy, etc., and can well meet the need for accurate simultaneous determination of trace and even ultra - trace amounts of thirty - one pyrrolizidine alkaloids in foods. Description of the Drawings

[0030] Figure 1 It is the chromatogram of separating the target analyte using a Waters HSS T3 chromatographic column with a specification of 3.0 mm × 100 mm and 2.5 μm. Figure 2 It is the chromatogram of separating the target analyte using an Agilent RRHDSB - C18 chromatographic column with a specification of 2.1 mm × 50 mm and 1.8 μm. Figure 3 It is the chromatogram of separating the target analyte using an Agilent EclipsePlus C18 chromatographic column with a specification of 3.0 mm × 150 mm and 1.8 μm. Figure 4 It is the chromatogram of separating the target analyte using an Agilent SB - Phenly RRHD chromatographic column with a specification of 2.1 mm × 100 mm and 1.8 μm. Figure 5 It is the MRM diagram of melanocyte - stimulating hormone. Specific Embodiments

[0031] To achieve the above object of the present invention, the method for detecting pyrrolizidine alkaloids in food comprises the following steps: A. Food treatment: including the treatment of solid food or liquid food: The treatment of solid food is as follows: a. The solid food is crushed and passed through a 60-mesh sieve to obtain the undersize and oversize; b. The undersize obtained in step a is mixed with a 95% ethanol solution containing 0.1% formic acid, vortexed for 1 min, ultrasonically extracted at 40 Hz for 30 min, and centrifuged at 8500 rpm for 5 min to obtain supernatant 1 and residue 1. The mass-volume ratio of the undersize to the 95% ethanol solution containing 0.1% formic acid is 1 g:10 mL; c. Residue 1 is further mixed with a 95% ethanol solution containing 0.1% formic acid, vortexed for 1 min, ultrasonically extracted at 40 Hz for 30 min, and centrifuged at 8500 rpm for 5 min to obtain supernatant 2 and residue 2; d. Supernatant 1 and supernatant 2 are combined, blown to less than 5 mL with nitrogen, and then made up to 10 mL with a 0.1% formic acid aqueous solution, and centrifuged to obtain supernatant 3; e. Then, 3 - 5 mL of supernatant 3 is loaded onto an activated PXC-SPE small column. After loading, first wash with 3 mL of water, then wash with 3 mL of methanol. After washing, the eluate in the PXC-SPE small column is blown dry, and then eluted with 5 mL of a 5% ammonia-methanol solution. The eluate is collected, and the eluate is blown dry with nitrogen at 40°C, made up to 1 mL with a reconstitution solvent, and filtered through a 0.22 μm nylon filter membrane to obtain supernatant 4. The activation method of the PXC-SPE small column is to pass 3 mL of methanol, 3 mL of water, and 3 mL of a 0.1% formic acid aqueous solution through the PXC-SPE small column in sequence. The reconstitution solvent is a 50% methanol solution containing 0.1% formic acid; The treatment of liquid food is as follows: The liquid food is mixed with an H2SO4 solution, ultrasonically extracted at 40 Hz for 30 min, cooled to room temperature, and then centrifuged at 8500 rpm for 5 min to obtain supernatant 3. Supernatant 3 is subjected to subsequent operations according to step e to obtain supernatant 4. The mass-volume ratio of the liquid food to the 0.05 mol / L H2SO4 solution is 1 g:10 mL. The concentration of the H2SO4 solution is 0.05 mol / L; B. Determine the pyrrolizidine alkaloids in supernatant 4 by liquid chromatography-tandem mass spectrometry; Wherein, the percentage contents in the 95% ethanol solution containing 0.1% formic acid, the 5% ammonia-methanol solution, the 50% methanol solution containing 0.1% formic acid, and the 0.1% formic acid aqueous solution are all volume percentage contents.

[0032] In a specific embodiment, the solid food is tea, chrysanthemum, corn or rice; and the liquid food is honey.

[0033] In a specific embodiment, the pyrrolizidine alkaloids include at least one of the following: melanocyte-stimulating hormone, melanocyte-stimulating hormone N-oxide, lycopsamine, lycopsamine N-oxide, heliotrine, echimidine, echimidine N-oxide, lasiocarpine, lasiocarpine N-oxide, monocrotaline, heliotrine N-oxide, europine, europine N-oxide, 7-acetylmelanocyte-stimulating hormone, 7-acetylmelanocyte-stimulating hormone N-oxide, monocrotaline N-oxide, senecionine, seneciphylline, senecionine N-oxide, seneciphylline N-oxide, jacobine, jacobine N-oxide, erucifoline, retrorsine, erucifoline N-oxide, retronecine, trichodesmine, seneciphylline N-oxide.

[0034] In a specific embodiment, when the food is tea, matrix-matched standard curve method is used for liquid chromatography-tandem mass spectrometry determination; when the food is chrysanthemum, corn, rice or honey, solvent standard curve method is used for liquid chromatography-tandem mass spectrometry determination; the preparation method of the matrix mixed standard working solution for the matrix-matched standard curve method is as follows: select a blank sample with the same properties as the food to be measured, prepare 7 blank matrix solutions according to step A, then accurately add the mixed standard stock solution respectively, blow with nitrogen until 10-20 μL of liquid remains, add 1.0 mL of 50% methanol re-solvent containing 0.1% formic acid to dissolve the residue, filter through a 0.22 μm nylon filter membrane, and prepare a series of matrix mixed standard working solutions with the mass concentration of each pyrrolizidine alkaloid being 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 2.5 μg / L, 5.0 μg / L, 7.5 μg / L, 10.0 μg / L, and use them immediately after preparation.

[0035] In a specific embodiment, the mass concentration of each pyrrolizidine alkaloid substance in the mixed standard stock solution is 10.0 μg / L, and the solvent of the mixed standard stock solution is methanol.

[0036] In a specific embodiment, the chromatographic column for the liquid chromatography is: Agilent Eclipse PlusC 18RRHD chromatographic column; The mobile phase of liquid chromatography includes three solutions A, B, and C; A is an aqueous solution of 0.1% (v / v) formic acid containing 5 mmol / L ammonium formate, B is methanol, and C is acetonitrile; Gradient elution is carried out successively with the mobile phase, and the gradient elution program is as follows: 0 - 2 min, the volume of phase A is from 95% to 90%, and the volume of phase B is from 5% to 10%; 2 - 5 min, the volume of phase A is from 90% to 80%, and the volume of phase B is from 10% to 20%; 5 - 10 min, the volume of phase A is 80%, and the volume of phase B is 20%; 10 - 16 min, the volume of phase A is from 80% to 60%, and the volume of phase B is from 20% to 40%; 16 - 18 min, the volume of phase A is from 60% to 50%, and the volume of phase B is from 40% to 50%; 18 - 22 min, the volume of phase A is 50%, and the volume of phase B is 50%; 22 - 25 min, the volume of phase A is from 50% to 5%, the volume of phase B is from 50% to 0%, and the volume of phase C is from 0% to 95%; 25 - 28 min, the volume of phase A is 5%, the volume of phase B is 0%, and the volume of phase C is 95%; 28 - 28.1 min, the volume of phase A is from 5% to 95%, the volume of phase B is from 0% to 5%, and the volume of phase C is from 95% to 0%; 28.1 - 35 min, the volume of phase A is 95%, and the volume of phase B is 5%; Flow rate: 0.3 mL / min; Column temperature: 40 °C; Injection volume: 2 μL.

[0037] In a specific embodiment, the Agilent Eclipse Plus C 18 The specification of the RRHD chromatographic column is 3.0 mm × 150 mm, 1.8 μm.

[0038] In a specific embodiment, the mass spectrometry conditions of the tandem mass spectrometry include: Ion source: electrospray ionization source; Scanning mode: positive ion mode; Monitoring mode: multiple reaction monitoring; Temperature of drying gas N2: 350 °C; Pressure of nebulizing gas N2: 275.8 kPa; Flow rate of drying gas: 12.0 L / min; Temperature of sheath gas N2: 350 °C; Flow rate of sheath gas: 12.0 L / min; Capillary voltage: 3500 V in negative ion mode and 3500 V in positive ion mode.

[0039] In a specific embodiment, the parameters such as retention time, quantitative ion pair, qualitative ion pair, in-source fragmentation voltage, collision energy, etc. of each pyrrolizidine alkaloid are shown in Table 1 above.

[0040] In a specific embodiment, the average spiked recovery rate of the method is 66.6% - 101.6%, the average relative standard deviation is 0.5% - 6.0%, the detection limit is 0.06 μg / kg - 0.66 μg / kg, and the quantification limit is 0.2 μg / kg - 2 μg / kg.

[0041] The following further describes the specific implementation manners of the present invention in conjunction with embodiments, and the present invention is not limited to the scope of the described embodiments.

[0042] Example 1 Instruments and Equipment Agilent 1290 InfinityⅡ / G 6470 C type high performance liquid chromatography - tandem mass spectrometry: equipped with an electrospray ionization source (ESI source) and a data processing system of Version 1.1.

[0043] Materials and Reagents Materials: 0.22 μm nylon filter membrane; 50 mL centrifuge tubes, 15 mL centrifuge tubes, 10 mL volumetric flasks; PXC - SPE columns.

[0044] Chromatographic pure reagents: methanol, formic acid and acetonitrile.

[0045] Analytical pure reagents: anhydrous ethanol, methanol, ammonia water.

[0046] Pyrrolizidine alkaloid standards: melanocyte - stimulating hormone, melanocyte - stimulating hormone N - oxide, lycopsamine, lycopsamine N - oxide, heliotrine, echimidine, echimidine N - oxide, lasiocarpine, lasiocarpine N - oxide, heliotrine N - oxide, europine, europine N - oxide, 7 - acetylmelanocyte - stimulating hormone, 7 - acetylmelanocyte - stimulating hormone N - oxide, senecionine, senecionine N - oxide, jacobine, jacobine N - oxide, sesquiterpene lactone, sesquiterpene lactone N - oxide, retrorsine, retronecine, retronecine N - oxide, clivorine, lasiocarpine, and seneciphylline N - oxide; monocrotaline, monocrotaline N - oxide, seneciphylline, seneciphylline N - oxide, seneciphylline N - oxide, and seneciphylline N - oxide.

[0047] Solution Preparation: 0.1% formic acid aqueous solution: Take 1000 mL of water, add 1 mL of formic acid, and mix well.

[0048] 5% ammonia water methanol solution: Take 25 mL of ammonia water, make up the volume to 500 mL with methanol and mix well.

[0049] 0.1% (v / v) formic acid aqueous solution containing 5 mmol / L ammonium formate: Measure 1000 mL of water, add 1 mL of 5 mol / L ammonium formate and 1 mL of formic acid, and mix well.

[0050] 50% methanol complex solvent containing 0.1% formic acid: Measure 25 mL of water, add 25 mL of methanol and 50 μL of formic acid, and mix well.

[0051] Mixed standard stock solution: Accurately pipette each single standard solution respectively, dilute with methanol, and prepare a mixed standard stock solution with the mass concentration of each pyrrolizidine alkaloid being 10.0 μg / L. Store it below -18 °C, and the validity period is 1 month.

[0052] Solvent mixed standard working solution: Accurately add the mixed standard stock solution respectively, and gradually dilute with the complex solvent to prepare a series of solvent mixed standard working solutions with the mass concentration of each pyrrolizidine alkaloid being 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 2.5 μg / L, 5.0 μg / L, 7.5 μg / L, 10.0 μg / L. Prepare and use immediately.

[0053] Matrix mixed standard working solution: Select blank samples with the same properties as the samples to be measured, prepare 7 blank matrix solutions according to the sample pretreatment method of the present invention, then accurately add the mixed standard stock solution respectively, blow with nitrogen until 10 - 20 μL of liquid remains, add 1.0 mL of complex solvent to dissolve the residue, filter through a 0.22 μm nylon filter membrane, and prepare a series of matrix mixed standard working solutions with the mass concentration of each pyrrolizidine alkaloid being 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 2.5 μg / L, 5.0 μg / L, 7.5 μg / L, 10.0 μg / L. Prepare and use immediately.

[0054] Sample pretreatment Crush the samples of tea, corn, and rice and sieve them through a 60 - mesh sieve for standby. Accurately weigh 1 g of each sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add 10 mL of 95% ethanol solution containing 0.1% formic acid, vortex for 1 min, extract ultrasonically at 40 Hz for 30 min, centrifuge at 8500 rpm for 5 min, take out the supernatant, add another 10 mL of 95% ethanol solution containing 0.1% formic acid to the residue for re - extraction, combine the two supernatants, blow with nitrogen until less than 5 mL, and volume - fix to 10 mL with 0.1% formic acid aqueous solution, vortex and mix well, then centrifuge. Accurately transfer 5 mL as the extraction solution to be purified. Activate the PXC - SPE column by passing 3 mL of methanol, 3 mL of water, and 3 mL of 0.1% formic acid aqueous solution in sequence. After activation, load the extraction solution to be purified onto the SPE column. After loading, first wash with 3 mL of water, then wash with 3 mL. After washing, blow dry the eluent in the SPE column, and then elute with 5 mL of 5% ammonia - methanol. Collect the eluate with a 10 mL glass test tube, dry the collected eluate with nitrogen at 40 °C, volume - fix to 1 mL with the complex solvent, filter through a 0.22 μm nylon filter membrane, and store in an injection vial for determination by liquid chromatography - tandem mass spectrometry.

[0055] After the honey sample was homogenized, 1 g (accurate to 0.01 g) was weighed and placed in a 10 mL volumetric flask. 0.05 mol / L H2SO4 solution was added until it was close to the calibration line. After vortex mixing, it was ultrasonically extracted at 40 Hz for 30 min. After cooling to room temperature, it was made up to the calibration line with the extract. After shaking well, it was transferred to a centrifuge tube and centrifuged at 8500 rpm for 5 min. Exactly 5 mL was accurately pipetted as the extract to be purified. The subsequent purification process of the extract to be purified was the same as that of tea, corn, and rice samples.

[0056] Chromatographic conditions Chromatographic column: The Agilent Eclipse Plus C chromatographic column with a specification of 3.0 mm × 150 mm and 1.8 μm 18 RRHD; Mobile phase: A is an aqueous solution of 0.1% (v / v) formic acid containing 5 mmol / L ammonium formate, B is methanol; The gradient elution program is as described above; Flow rate: 0.3 mL / min; Chromatographic column temperature: 40 °C; Injection volume: 2 μL.

[0057] Mass spectrometry conditions Ion source: Electrospray ionization source; Scanning mode: Positive ion mode; Monitoring method: Multiple reaction monitoring; N2 drying gas temperature: 350 °C; N2 nebulizing gas pressure: 275.8 kPa; Drying gas flow rate: 12.0 L / min; N2 sheath gas temperature: 350 °C; Sheath gas flow rate: 12.0 L / min; Capillary voltage: 3500 V in negative ion mode and 3500 V in positive ion mode; Parameters such as the retention time, quantitative ion pairs, qualitative ion pairs, in-source fragmentation voltage, and collision energy of each analyte can be seen in Table 1 above.

[0058] Example 2 For the rest, refer to Example 1, and prepare 5 portions of 5 μg / L solvent mixed standard solutions. Under the condition of a 40 °C water bath, the effects of five concentration methods on the recovery rates of each analyte were studied, including drying with nitrogen, blowing to near dry with nitrogen, drying with nitrogen and then blowing for 1 h, and drying with nitrogen after adding 10% glycerol and 0.1% vitamin C respectively. The results are shown in Table 2, where blowing to near dry with nitrogen means leaving 10 - 20 μL of liquid remaining. As can be seen from Table 2, the longer the time of blowing with nitrogen, the greater the loss of the compound; The recovery rates of drying with nitrogen and drying with nitrogen after adding 10% glycerol are generally higher than the other three modes. Drying with nitrogen has strong operability and lower detection costs. The best concentration method of the present invention is drying with nitrogen.

[0059] Table 2 Recovery rates of each analyte under different concentration conditions in Example 2 Example 3 Taking the prepared 10 μg / kg solvent-mixed standard solution as the object, extraction recovery tests were respectively carried out using six extraction solvents: 0.1% formic acid aqueous solution, 0.05 mol / L H2SO4 solution, 50% ethanol, 95% ethanol, 95% ethanol solution containing 0.05 mol / L sulfuric acid, and 95% ethanol solution containing 0.1% formic acid, to study the optimal extraction solvent for the pretreatment of each target compound. Others were the same as in Example 1.

[0060] The experimental results showed that the recovery rates of the 0.1% formic acid aqueous solution were between 65% and 136%, those of the 0.05 mol / L H2SO4 solution were between 70.1% and 126.3%, those of the 50% ethanol were between 71.2% and 134.6%, those of the 95% ethanol were between 65.6% and 134.2%, those of the 95% ethanol solution containing 0.1% formic acid were between 71% and 124.1%, and those of the 95% ethanol solution containing 0.05 mol / L H2SO4 were between 60.1% and 114.5%. Comparing the two acid-containing aqueous solutions, the recovery rate of the 0.05 mol / L H2SO4 solution was slightly better than that of the 0.1% formic acid aqueous solution, and there was little difference in the recovery rates among the four organic solvents.

[0061] Example 4 Others were similar to Example 3, except that blank tea leaves and honey were used as food sample objects respectively, and the average recovery rate of each target analyte at the 10 μg / kg addition level was used as the index. The samples were respectively tested using three extraction solvents: 0.05 mol / L H2SO4 solution, 95% ethanol solution containing 0.05 mol / L H2SO4, and 95% ethanol solution containing 0.1% formic acid. The results of the influence of the three extraction solvents on the average recovery rate of pyrrolizidine alkaloids in tea are shown in Table 3.

[0062] Table 3 Influence of Three Extraction Solvents on the Average Recovery Rate of Pyrrolizidine Alkaloids in Tea As can be seen from Table 3, the recovery rates of the 0.05 mol / L H2SO4 solution are between 43.8% and 134%. Among them, the recovery rates of 7-acetylmelanotropin N-oxide, senecionine, seneciphylline, seneciphyllinine, seneciphyllinine N-oxide, vernalicine, vernalicine N-oxide, jacobine, retrorsine, and lasiocarpine are lower than 70%. The recovery rates of the 95% ethanol solution containing 0.05 mol / L H2SO4 are between 56.1% and 103.8%. Among them, the recovery rates of monocrotaline, 7-acetylmelanotropin N-oxide, senecionine, seneciphylline, seneciphyllinine, vernalicine, jacobine, retrorsine, lasiocarpine, jacobine N-oxide, and retrorsine N-oxide are lower than 70%. The recovery rates of the 95% ethanol solution containing 0.1% formic acid are between 63.7% and 105.5%. Among them, only the recovery rates of echimidine, 7-acetylmelanotropin N-oxide, senecionine, vernalicine, jacobine, and jacobine N-oxide are lower than 70%. The 95% ethanol solution containing 0.1% formic acid is the best extraction solvent for solid foods.

[0063] Honey can be dissolved in acid-containing aqueous solutions, and the spike recovery rates in the preliminary experiments fully meet the requirements of relevant national standards such as GB 5009.295-2023 "National Food Safety Standard General Rules for the Verification of Chemical Analysis Methods" for spike recovery rates. It is best to choose the 0.05 mol / L H2SO4 solution as the extraction solvent for honey.

[0064] Example 5 Using blank (negative) tea as the matrix and taking the average recovery rate of each target analyte at the addition level of 5 μg / kg as the index, the sample pretreatment was carried out using MCX cartridges with a specification of 60 mg / 3 mL, PXC cartridges with a specification of 60 mg / 3 mL, and HLB cartridges with a specification of 60 mg / 3 mL to purify the matrix interference substances in the tea. Other procedures were the same as in Example 1. The results are shown in Table 4.

[0065] Table 4 Effects of Three Different Purification Cartridges on the Purification Recovery Rates of Tea The results showed that the HLB cartridges did not retain nearly 10 pyrrolizidine alkaloids, and the recovery rates of only individual compounds met the standard requirements. The purification results of the MCX cartridges and PXC cartridges showed that the recovery rates of the target compounds could meet the requirements for spike recoveries in relevant national standards such as GB 5009.295-2023 "National Food Safety Standard General Rules for Verification of Chemical Analysis Methods" and GB / T 27404-2008 "Quality Control Code for Laboratories - Physical and Chemical Testing of Foods". However, the column passing speed of the PXC cartridges was faster than that of the MCX cartridges, which could improve the experimental rate to a certain extent, and the cost of the PXC cartridges used in the experiment was lower than that of the MCX cartridges. Therefore, it was best to use the PXC-SPE cartridges as the sample purification cartridges.

[0066] Example 6 Series of solvent and matrix-matched mixed standard working solutions with mass concentrations of 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 2.5 μg / L, 5.0 μg / L, 7.5 μg / L, and 10.0 μg / L for each target analyte were prepared using blank solvent and blank tea matrix solution respectively, and were determined according to the optimal instrument parameters determined by the present invention. With the mass concentration (x) of each target analyte as the abscissa and the peak area of its quantitative ion as the ordinate, solvent and matrix-matched standard working curves were plotted. The detection limits (LOD) of the corresponding target analytes were determined with a signal-to-noise ratio (S / N) of 3, and the quantitative limits (LOQ) of the corresponding target analytes were determined with an average signal-to-noise ratio (S / N) of 10 for 6 times. The results of the blank solvent are shown in Table 5, and the results of the blank tea matrix are shown in Table 6. It can be seen from Table 5 and Table 6 that the linear relationships of the thirty-one target analytes were good in the mass concentration range of 0.5 μg / L to 10.0 μg / L, the correlation coefficients r were all > 0.99, the detection limit ranges were 0.06 μg / kg to 0.66 μg / kg, and the quantitative limit ranges were 0.2 μg / kg to 2 μg / kg, indicating that this method could well meet the trace and even ultra-trace determination requirements of thirty-one pyrrolizidine alkaloids such as melanocyte-stimulating hormone, lycopsamine, and senecionine in foods.

[0067] Table 5 Regression equations, correlation coefficients, detection limits, and quantitative limits of each target analyte in the standard curve of blank solvent Table 6 Regression equations, correlation coefficients, detection limits, and quantitative limits of each target analyte in the standard curve of blank tea matrix Example 7 Prepare a solvent mixed standard solution with a mass concentration of 100 μg / L for each pyrrolizidine alkaloid substance. Using blank honey, tea, rice, and corn as sample matrices, add different amounts of the 100 μg / L solution mixed standard solution to each sample matrix so that the mass concentration of each target analyte in the spiked samples is 2 μg / kg, 5 μg / kg, and 10 μg / kg. Prepare 4 parallel samples for each spiked concentration level, perform pretreatment and determination analysis according to Example 1, and investigate the accuracy and precision of this method. The determination results are shown in Tables 7 and 8. As can be seen from the tables, the average recoveries of 31 pyrrolizidine alkaloid substances in honey at the three spiked levels of 2 μg / kg, 5 μg / kg, and 10 μg / kg are 68.6% - 121.1%, and the relative standard deviations are 0.3% - 5.0%; the average recoveries of 31 pyrrolizidine alkaloid substances in tea at the three spiked levels are 66.6% - 101.6%, and the relative standard deviations are 0.5% - 6.0%; the average recoveries of 31 pyrrolizidine alkaloid substances in rice at the three spiked levels are 74.8% - 111.2%, and the relative standard deviations are 0.4% - 6.3%; the average recoveries of 31 pyrrolizidine alkaloid substances in corn at the three spiked levels are 70.6% - 116.2%, and the relative standard deviations are 0.2% - 9.7%. All meet the requirements of relevant standards such as GB 5009.295-2023 "National Food Safety Standard General Rules for Verification of Chemical Analysis Methods", GB / T 27404-2008 "Laboratory Quality Control Specification for Food Physical and Chemical Testing", GB / T 32465-2015 "Requirements for Verification, Validation, and Internal Quality Control of Chemical Analysis Methods", and GB / T 27417-2017 "Conformity Assessment - Guidelines for the Verification and Validation of Chemical Analysis Methods" for the recovery rate and precision of the method, indicating that the method established in the present invention can well meet the need for simultaneous accurate determination of 31 pyrrolizidine alkaloid substances in food.

[0068] Table 7 Spike recoveries and precisions of each target analyte in honey and tea matrices Table 8 Spike recoveries and precisions of each target analyte in rice and corn matrices

Claims

1. Detection method for pyrrolizidine alkaloids in food, characterized in that, It includes the following steps: A. Food treatment: including the treatment of solid food or liquid food: The treatment of solid food is as follows: a. Crush the solid food and pass it through a 60-mesh sieve to obtain the undersize and oversize; b. Take the undersize from step a and mix it with a 95% ethanol solution containing 0.1% formic acid, vortex for 1 min, extract by ultrasonic wave at 40 Hz for 30 min, and centrifuge at 8500 rpm for 5 min to obtain supernatant 1 and residue 1. The mass-volume ratio of the undersize to the 95% ethanol solution containing 0.1% formic acid is 1 g:10 mL; c. Then mix residue 1 with a 95% ethanol solution containing 0.1% formic acid, vortex for 1 min, extract by ultrasonic wave at 40 Hz for 30 min, and centrifuge at 8500 rpm for 5 min to obtain supernatant 2 and residue 2; d. Combine supernatant 1 and supernatant 2, blow it to less than 5 mL with nitrogen, and then make up the volume to 10 mL with a 0.1% formic acid aqueous solution, and centrifuge to obtain supernatant 3; e. Then take 3 - 5 mL of supernatant 3 and load it onto an activated PXC-SPE small column. After loading, first rinse with 3 mL of water, then rinse with 3 mL of methanol. After rinsing, blow dry the eluent in the PXC-SPE small column, and then elute with 5 mL of a 5% ammonia-methanol solution. Collect the eluate, and blow dry the eluate with nitrogen at 40°C, make up the volume to 1 mL with a complex solvent, and filter through a 0.22 μm nylon filter membrane to obtain supernatant 4; The activation method of the PXC-SPE small column is to pass 3 mL of methanol, 3 mL of water, and 3 mL of a 0.1% formic acid aqueous solution through the PXC-SPE small column in sequence; The complex solvent is a 50% methanol solution containing 0.1% formic acid; The treatment of liquid food is as follows: Mix the liquid food with an H2SO4 solution, extract by ultrasonic wave at 40 Hz for 30 min, cool to room temperature, and then centrifuge at 8500 rpm for 5 min to obtain supernatant 3. Perform the subsequent operations on supernatant 3 according to step e to obtain supernatant 4; The mass-volume ratio of the liquid food to the 0.05 mol / L H2SO4 solution is 1 g:10 mL; The concentration of the H2SO4 solution is 0.05 mol / L; B. Determine the pyrrolizidine alkaloids in supernatant 4 by liquid chromatography-tandem mass spectrometry; Among them, the percentage contents in the 95% ethanol solution containing 0.1% formic acid, the 5% ammonia-methanol solution, the 50% methanol solution containing 0.1% formic acid, and the 0.1% formic acid aqueous solution are all volume percentage contents.

2. The detection method of pyrrolizidine alkaloids in food according to claim 1, characterized in that, The solid food is tea, chrysanthemum, corn or rice; The liquid food is honey.

3. The method for detecting pyrrolizidine alkaloids in food according to claim 1 or 2, characterized in that, The pyrrolizidine alkaloids include at least one of the following: melanocyte-stimulating hormone, melanocyte-stimulating hormone N-oxide, lycopodine, lycopodine N-oxide, heliotrine, echimidine, echimidine N-oxide, lasiocarpine, lasiocarpine N-oxide, monocrotaline, heliotrine N-oxide, europine, europine N-oxide, 7-acetylmelanocyte-stimulating hormone, 7-acetylmelanocyte-stimulating hormone N-oxide, monocrotaline N-oxide, senecionine, seneciphylline, senecionine N-oxide, seneciphylline N-oxide, jacobine, jacobine N-oxide, erucifoline, retrorsine, erucifoline N-oxide, retronecine, retronecine N-oxide, clivorine, lasiocarpinine, seneciphylline N-oxide.

4. The detection method of pyrrolizidine alkaloids in food according to claim 2, wherein When the food is tea, matrix-matched standard curve method is used for liquid chromatography-tandem mass spectrometry determination; when the food is chrysanthemum, corn, rice or honey, solvent standard curve method is used for liquid chromatography-tandem mass spectrometry determination; the preparation method of the matrix-mixed standard working solution for the matrix-matched standard curve method is as follows: select a blank sample with the same properties as the food to be tested, prepare 7 blank matrix solutions according to step A, then accurately add the mixed standard stock solution respectively, blow with nitrogen until 10-20 μL of liquid remains, add 1.0 mL of 50% methanol complex solvent containing 0.1% formic acid to dissolve the residue, filter through a 0.22 μm nylon filter membrane, and prepare a series of matrix-mixed standard working solutions with the mass concentration of each pyrrolizidine alkaloid being 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 2.5 μg / L, 5.0 μg / L, 7.5 μg / L, 10.0 μg / L, and use it immediately after preparation.

5. The method for detecting pyrrolizidine alkaloids in food according to claim 4, wherein, The mass concentration of each pyrrolizidine alkaloid in the mixed standard stock solution is 10.0 μg / L, and the solvent of the mixed standard stock solution is methanol.

6. The method for detecting pyrrolizidine alkaloids in food according to claim 1 or 2, characterized in that, The chromatographic column for the liquid chromatography is: Agilent Eclipse Plus C18 RRHD chromatographic column; the mobile phase of the liquid chromatography includes three solutions A, B, and C; A is an aqueous formic acid solution with a volume concentration of 0.1% containing 5 mmol / L ammonium formate, B is methanol, and C is acetonitrile; gradient elution is carried out successively with the mobile phase, and the gradient elution program is as follows: 0 - 2 min, the volume of phase A is 95% to 90%, and the volume of phase B is 5% to 10%; 2 - 5 min, the volume of phase A is 90% to 80%, and the volume of phase B is 10% to 20%; 5 - 10 min, the volume of phase A is 80%, and the volume of phase B is 20%; 10 - 16 min, the volume of phase A is 80% to 60%, and the volume of phase B is 20% to 40%; 16 - 18 min, the volume of phase A is 60% to 50%, and the volume of phase B is 40% to 50%; 18 - 22 min, the volume of phase A is 50%, and the volume of phase B is 50%; 22 - 25 min, the volume of phase A is 50% to 5%, the volume of phase B is 50% to 0%, and the volume of phase C is 0% to 95%; 25 - 28 min, the volume of phase A is 5%, the volume of phase B is 0%, and the volume of phase C is 95%; 28 - 28.1 min, the volume of phase A is 5% to 95%, the volume of phase B is 0% to 5%, and the volume of phase C is 95% to 0%. 28.1 - 35 min, the volume of phase A is 95%, and the volume of phase B is 5%. Flow rate: 0.3 mL / min; chromatographic column temperature: 40 °C; injection volume: 2 μL.

7. The method for detecting pyrrolizidine alkaloids in food according to claim 6, characterized in that, The specification of the Agilent Eclipse Plus C18 RRHD chromatographic column is 3.0 mm × 150 mm, 1.8 μm.

8. The method for detecting pyrrolizidine alkaloids in food according to claim 1 or 2, characterized in that, The mass spectrometry conditions of the tandem mass spectrometry include: Ion source: electrospray ionization source; scan mode: positive ion mode; monitoring method: multiple reaction monitoring; drying gas N2 temperature: 350 °C; nebulizing gas N2 pressure: 275.8 kPa; drying gas flow rate: 12.0 L / min; sheath gas N2 temperature: 350 °C; sheath gas flow rate: 12.0 L / min; capillary voltage: 3500 V in negative ion mode and 3500 V in positive ion mode.

9. The method for detecting pyrrolizidine alkaloids in food according to claim 1 or 2, characterized in that, The average spiked recovery rate of the method is 66.6% - 101.6%, the average relative standard deviation is 0.5% - 6.0%, the detection limit is 0.06 μg / kg - 0.66 μg / kg, and the quantification limit is 0.2 μg / kg - 2 μg / kg.

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