Detection method for pyrrolizidine alkaloids in foods

Abstract

The present invention relates to a method for detecting pyrrolizidine alkaloids in food, and belongs to the field of detection technology. The technical problem to be solved by the present invention is to provide a new method for detecting pyrrolizidine alkaloids in food. The detection method comprises the following steps: A. food processing, including the processing of solid food or the processing of liquid food, the processing of solid food is crushing, extraction twice with 95% ethanol solution containing 0.1% formic acid, centrifugation, and purification by PXC-SPE column after activation to obtain supernatant 4; the processing of liquid food is extraction with H2SO4 solution, centrifugation, and purification by PXC-SPE column after activation to obtain supernatant 4; B. determination of pyrrolizidine alkaloids in supernatant 4 by liquid chromatography-tandem mass spectrometry. The present invention effectively solves the bottleneck problems such as lack of index coverage and complex matrix interference, establishes a liquid chromatography-tandem mass spectrometry accurate detection method for pyrrolizidine alkaloid contamination in a variety of foods, and detects more comprehensive types of pyrrolizidine alkaloids.

Description

Technical Field

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

[0002] As typical plant-derived secondary metabolite toxins, pyrrolizidine alkaloids migrate across media within ecosystems, exhibiting a three-stage contamination pathway: plant-soil / water-crop. They are then transferred to animals through feed, causing contamination of animal-derived food. Furthermore, long-term consumption of foods containing pyrrolizidine alkaloids can cause chronic damage to human health, leading to hepatotoxicity, pulmonary toxicity, carcinogenicity, and teratogenicity. Therefore, the regulation and 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. The current pyrrolizidine alkaloid pollution control faces several challenges: the stereoisomer diversity of pyrrolizidine alkaloids and the complexity of food matrices lead to technical barriers to detection; the detection methods lack the accuracy to resolve trace isomers; the traditional "end-of-pipe treatment" model is difficult to deal with this type of pollutants with biomagnification effects, and a full-chain control system from weed control to processing and degradation needs to be established; in addition, the detection limit and quantification limit of the method need to be further improved.

[0004] Yao Leijun, Chen Yanqiu, et al. Determination of 27 pyrrolizidine alkaloids in tea by integrated QuEChERS cleanup-ultra-performance liquid chromatography-tandem mass spectrometry [J]. Tea Science, 2024(5). A method for the detection of 27 pyrrolizidine alkaloids in tea was disclosed. However, it can only be used to detect specific food teas and cannot simultaneously cover the detection of other foods that are susceptible to pyrrolizidine alkaloid contamination (such as honey, herbs, and grains). In addition, its detection limit is high and the quantification limit range is narrow. 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 improved.

[0005] Chen Yankai. Rapid determination 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). A method for rapid determination of 26 pyrrolizidine alkaloids in milk powder and liquid milk by solid phase extraction combined with ultra-high performance liquid chromatography-tandem mass spectrometry is disclosed. However, it can only be used for the detection of specific food milk powder and liquid milk, and cannot simultaneously cover the detection of other foods that are susceptible to pyrrolizidine alkaloid contamination. It has not verified the complexity of the matrix, such as whether defatting will affect the test results. At the same time, its quantitative limit is narrow, and the types of pyrrolizidine alkaloids detected need to be further improved. Summary of the Invention

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

[0007] To achieve the above-mentioned object of the present invention, the method for detecting pyrrolizidine alkaloids in food comprises the following steps:

[0008] A. Food processing: including solid food processing or liquid food processing:

[0009] Solid food processing is:

[0010] a. After the solid food is crushed, pass through a 60-mesh sieve to obtain the undersize and oversize;

[0011] b. The sieve underfill from step a was 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 a supernatant 1 and a residue 1. The mass volume ratio of the sieve underfill to the 95% ethanol solution containing 0.1% formic acid was 1 g:10 mL.

[0012] c. The residue 1 was then 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;

[0013] d. Combine supernatant 1 and supernatant 2, blow down to less than 5 mL with nitrogen, then dilute to 10 mL with 0.1% formic acid aqueous solution, and centrifuge to obtain supernatant 3;

[0014] e. Take 3 to 5 mL of supernatant 3 and load it onto the activated PXC-SPE cartridge. After loading, rinse with 3 mL of water, then with 3 mL of methanol. After rinsing, blow dry the eluent in the PXC-SPE cartridge and elute with 5 mL of 5% ammonia methanol solution. Collect the eluate, blow dry the eluate with nitrogen at 40°C, dilute to 1 mL with a resolvent, and filter through a 0.22 μm nylon filter to obtain supernatant 4. The PXC-SPE cartridge is activated by sequentially passing through the PXC-SPE cartridge with 3 mL of methanol, 3 mL of water, and 3 mL of 0.1% formic acid aqueous solution. The resolvent is a 50% methanol solution containing 0.1% formic acid.

[0015] Liquid food processing is:

[0016] The liquid food was mixed with the H2SO4 solution, subjected to ultrasonic extraction at 40 Hz for 30 minutes, cooled to room temperature, and then centrifuged at 8500 rpm for 5 minutes to obtain supernatant 3. Supernatant 3 was 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 was 1 g:10 mL; the concentration of the H2SO4 solution was 0.05 mol / L.

[0017] B. Determination of pyrrolizidine alkaloids in supernatant 4 by liquid chromatography-tandem mass spectrometry;

[0018] The percentages 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.

[0019] The experiment showed that with the increase of the sample amount on the PXC-SPE column, the recovery rate of the target pyrrolizidine alkaloid compound generally showed a downward trend, and the highest recovery rate was achieved when the sample amount was 3-5 mL.

[0020] The experimental results showed that the elution conditions were 3 mL of pure water first and then 3 mL of methanol, and the target pyrrolizidine alkaloid compounds in the sample would not be lost by the eluent.

[0021] Experiments showed that selecting a 5% ammonia-methanol solution as the elution solvent had the best elution ability for the pyrrolizidine alkaloids. The elution ability of 5 mL of a 5% ammonia-methanol solution could meet the recovery rate requirements, and adding 1 mL of elution solvent did not result in significant difference.

[0022] In step e, the eluent is the purified sample solution. To improve detection efficiency and the accuracy of the test results, the eluent is concentrated by nitrogen blowing. A large number of experiments have shown that the response of each target compound in 50% methanol containing 0.1% formic acid is the highest, while the response of each target compound in other solvents, such as 20% methanol, 50% methanol, and 50% methanol containing 0.1% ammonia water, is poor.

[0023] Experiments have shown that filtering the sample through a 0.22 μm filter membrane after reconstitution has no effect on the results and will not cause clogging of the instrument.

[0024] In a specific embodiment, the solid food is tea, chrysanthemum, corn or rice; and the liquid food is honey.

[0025] In a specific embodiment, the pyrrolizidine alkaloids include: melanocyte-stimulating hormone, melanocyte-stimulating hormone nitrogen oxide, lycopodium, lycopodium nitrogen oxide, sinapine, cyanidin, cyanidin nitrogen oxide, pilocarpine, pilocarpine nitrogen oxide, monocrotaline, sinapine nitrogen oxide, heliotropin, heliotropin nitrogen oxide, 7-acetyl melanocyte-stimulating hormone, 7-acetyl melanocyte-stimulating hormone nitrogen oxide, monocrotaline nitrogen oxide, sennaine, sennaine, sennaine nitrogen oxide ...

[0026] In a specific embodiment, when the food is tea, the liquid chromatography-tandem mass spectrometry determination adopts the matrix matching standard curve method; when the food is chrysanthemum, corn, rice or honey, the liquid chromatography-tandem mass spectrometry determination adopts the solvent standard curve method; the preparation method of the matrix mixed standard working solution of the matrix matching standard curve method is: select a blank sample with the same properties as the food to be tested, prepare 7 blank matrix solutions according to step A, and then accurately add the mixed standard stock solution respectively, blow nitrogen until 10 to 20 μL of liquid remains, add 1.0 mL of 50% methanol resolvent containing 0.1% formic acid to dissolve the residue, pass through a 0.22 μm nylon filter membrane, and prepare a series of matrix mixed standard working solutions with a mass concentration of each pyrrolizidine alkaloid 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, which are prepared and used immediately.

[0027] Numerous experiments have shown that matrix effects are not significant in honey, corn, rice, and chrysanthemum. However, in the tea matrix, more than 90% of the target compounds exhibit significant matrix inhibition effects, especially Senecio nitrososide, Senecio arugula, and Senecio roxburghii, all of which exhibit inhibition levels above -60%. Therefore, a matrix-matched standard curve method was used to reduce the impact of the tea matrix on the quantitative analysis of the 31 target compounds, thereby ensuring optimal accuracy of the results.

[0028] 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.

[0029] In a specific embodiment, the liquid chromatography column is: Agilent Eclipse PlusC 18RRHD chromatographic column; the liquid chromatography mobile phase includes three solutions A, B, and C; A is a 0.1% volume concentration formic acid aqueous solution containing 5 mmol / L ammonium formate, B is methanol, and C is acetonitrile; the mobile phase is used for gradient elution, and the gradient elution program is as follows: 0-2 min, 95% to 90% volume of phase A, 5% to 10% volume of phase B; 2-5 min, 90% to 80% volume of phase A, 10% to 20% volume of phase B; 5-10 min, 80% volume of phase A, 20% volume of phase B; 10-16 min, 80% to 60% volume of phase A, 20% to 40% volume of phase B; 16-18 min, 60% to 50% volume of phase A, 40% to 50% volume of phase B; 18-22 min, 50% volume of phase A, 50% volume of phase B; 22-25 min, 50% to 5% volume of phase A, 50% to 0% volume of phase B, 0% to 95% volume of phase C; 25-28 min, phase A volume 5%, phase B volume 0%, phase C volume 95%; 28-28.1 min, phase A volume 5% to 95%, phase B volume 0% to 5%, phase C volume 95% to 0%; 28.1-35 min, phase A volume 95%, phase B volume 5%; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 2 μL.

[0030] The experimental results showed that 31 pyrrolizidine alkaloids could obtain better responses in the positive ion ionization mode of ESI source.

[0031] The experiment showed that 31 kinds of target objects were detected in the AgilentEclipse Plus C 18 The peak shape on the column is good, the retention time range is 3.0 min to 25.0 min, and several pairs of isomers can be effectively separated. Other columns, such as the Waters HSST3 column with a size of 3.0 mm × 100 mm and a 2.5 μm column, and the Agilent RRHD SB-C column with a size of 2.1 mm × 100 mm and a 1.8 μm column, are also available. 18 The separation effect of the Agilent SB-Phenly RRHD column with a size of 2.1×100 mm and a 1.8 μm column was poor. Figure 1-4 .

[0032] The experiment also showed that when a 0.1% volume concentration of formic acid aqueous solution containing 5 mmol / L ammonium formate and methanol were used as the mobile phase and the above-mentioned gradient elution procedure was adopted, all 31 target compounds had good separation, chromatographic peak shape and mass spectrometric response.

[0033] In a specific embodiment, the Agilent Eclipse Plus C 18 The specifications of the RRHD column are 3.0 mm × 150 mm, 1.8 μm.

[0034] In a specific embodiment, the mass spectrometry conditions of the tandem mass spectrometry include:

[0035] Ion source: electrospray ion source; scan mode: positive ion mode; monitoring mode: multiple reaction monitoring; drying gas N2 temperature: 350℃; nebulizing gas N2 pressure: 275.8 kPa; drying gas flow rate: 12.0 L / min; sheath gas N2 temperature: 350℃; sheath gas flow rate: 12.0 L / min; capillary voltage: 3500 V in negative ion mode, 3500 V in positive ion mode.

[0036] A large number of experiments showed that two monitoring ion pairs were finally selected for each target compound. The specific MRM parameters are shown in Table 1. The MRM diagram of melanocyte-stimulating hormone is shown in Table 1. Figure 5 At the same time, the experiment also studied the plasma source parameters such as drying gas temperature, drying gas flow, nebulizer pressure, capillary voltage, sheath gas temperature, and sheath gas flow. Using the mass spectrometry conditions of the present invention, the ionization efficiency of each target to be measured was optimized.

[0037] In a specific embodiment, the reference retention time, quantitative ion pair, qualitative ion pair, in-source fragmentation voltage, collision energy and other parameters of each pyrrolizidine alkaloid are shown in Table 1:

[0038] Table 1 Reference retention times and mass spectrometry parameters for 31 pyrrolizidine alkaloids

[0039]

[0040] In all tables of the present invention, compounds 1 to 31 represent, in order, monocrotaline, arugula senecioline, monocrotaline N oxide, arugula senecioline N oxide, senecine, eurynein, melanocyte-stimulating hormone, lycopodiamine, eurynein N oxide, eurynein N oxide, rutinine, rutinine N oxide, rutinine N oxide, rutinine, tricholoma pine, eurynein, tricholoma pine N oxide, rutinine, 7-acetyl melanocyte-stimulating hormone, tricholoma pine N oxide, 7-acetyl melanocyte-stimulating hormone, tricholoma pine N oxide, rutinine, rutinine, rutinine N oxide, rutinine N oxide, cynomolgus, cynomolgus N oxide, clematis pine, pilocarpine, pilocarpine N oxide.

[0041] In a specific embodiment, the method has an average spiked recovery of 66.6% to 101.6%, an average relative standard deviation of 0.5% to 6.0%, a detection limit of 0.06 μg / kg to 0.66 μg / kg, and a quantification limit of 0.2 μg / kg to 2 μg / kg.

[0042] Beneficial effects: The present invention effectively solves bottleneck problems such as insufficient indicator coverage and complex matrix interference, establishes a liquid chromatography-tandem mass spectrometry precise detection method for pyrrolizidine alkaloid contamination in a variety of foods, and detects a more comprehensive range of pyrrolizidine alkaloid types, providing decision-making basis and reference for the formulation of relevant limit standards, detection standards, risk prevention and control measures, etc., providing a scientific basis for product quality grading and certification, promoting high-quality development of the local economy, and providing continuous technical guarantee for food import and export trade and food safety.

[0043] The method of the present invention has an average spiked recovery rate of 66.6% to 101.6% at three concentration levels of 2 μg / kg, 5 μg / kg, and 10 μg / kg, an average relative standard deviation of 0.1% to 6.0%, a detection limit of 0.06 μg / kg to 0.66 μg / kg, and a quantification limit of 0.2 μg / kg to 2 μg / kg. It has the characteristics of simplicity, efficiency, accuracy, sensitivity, and economy, and can well meet the needs of trace and even ultratrace simultaneous accurate determination of 31 pyrrolizidine alkaloids in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the chromatogram of the target compound separated using a 3.0 mm × 100 mm, 2.5 μm Waters HSS T3 column.

[0045] Figure 2 This is the chromatogram of the target compound separated using an Agilent RRHDSB-C18 column with dimensions of 2.1 mm × 50 mm and 1.8 μm.

[0046] Figure 3 This is the chromatogram of the target compound separated using an Agilent EclipsePlus C18 column with a size of 3.0 mm × 150 mm and a 1.8 μm column.

[0047] Figure 4 This is the chromatogram of the target compound separated using an Agilent SB-Phenly RRHD column with dimensions of 2.1 mm × 100 mm and 1.8 μm.

[0048] Figure 5 This is the MRM diagram of melanocyte-stimulating hormone. DETAILED DESCRIPTION

[0049] To achieve the above-mentioned object of the present invention, the method for detecting pyrrolizidine alkaloids in food comprises the following steps:

[0050] A. Food processing: including solid food processing or liquid food processing:

[0051] Solid food processing is:

[0052] a. After the solid food is crushed, pass through a 60-mesh sieve to obtain the undersize and oversize;

[0053] b. The sieve underfill from step a was 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 a supernatant 1 and a residue 1. The mass volume ratio of the sieve underfill to the 95% ethanol solution containing 0.1% formic acid was 1 g:10 mL.

[0054] c. Residue 1 was then 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;

[0055] d. Combine supernatant 1 and supernatant 2, blow down to less than 5 mL with nitrogen, then dilute to 10 mL with 0.1% formic acid aqueous solution, and centrifuge to obtain supernatant 3;

[0056] e. Take 3 to 5 mL of supernatant 3 and load it onto the activated PXC-SPE cartridge. After loading, rinse with 3 mL of water, then with 3 mL of methanol. After rinsing, blow dry the eluent in the PXC-SPE cartridge, then elute with 5 mL of 5% ammonia methanol solution. Collect the eluate, blow dry the eluate with nitrogen at 40°C, dilute to 1 mL with a resolvent, and filter through a 0.22 μm nylon filter membrane to obtain supernatant 4. The PXC-SPE cartridge is activated by sequentially passing through the PXC-SPE cartridge with 3 mL of methanol, 3 mL of water, and 3 mL of 0.1% formic acid aqueous solution. The resolvent is a 50% methanol solution containing 0.1% formic acid.

[0057] Liquid food processing is:

[0058] The liquid food was mixed with the H2SO4 solution, subjected to ultrasonic extraction at 40 Hz for 30 minutes, cooled to room temperature, and then centrifuged at 8500 rpm for 5 minutes to obtain supernatant 3. Supernatant 3 was 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 was 1 g:10 mL; the concentration of the H2SO4 solution was 0.05 mol / L.

[0059] B. Determination of pyrrolizidine alkaloids in supernatant 4 by liquid chromatography-tandem mass spectrometry;

[0060] The percentages 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.

[0061] In a specific embodiment, the solid food is tea, chrysanthemum, corn or rice; and the liquid food is honey.

[0062] In a specific embodiment, the pyrrolizidine alkaloids include: melanocyte-stimulating hormone, melanocyte-stimulating hormone nitrogen oxide, lycopodium, lycopodium nitrogen oxide, sinapine, cyanidin, cyanidin nitrogen oxide, pilocarpine, pilocarpine nitrogen oxide, monocrotaline, sinapine nitrogen oxide, heliotropin, heliotropin nitrogen oxide, 7-acetyl melanocyte-stimulating hormone, 7-acetyl melanocyte-stimulating hormone nitrogen oxide, monocrotaline nitrogen oxide, sennaine, sennaine, sennaine nitrogen oxide ...

[0063] In a specific embodiment, when the food is tea, the liquid chromatography-tandem mass spectrometry determination adopts the matrix matching standard curve method; when the food is chrysanthemum, corn, rice or honey, the liquid chromatography-tandem mass spectrometry determination adopts the solvent standard curve method; the preparation method of the matrix mixed standard working solution of the matrix matching standard curve method is: select a blank sample with the same properties as the food to be tested, prepare 7 blank matrix solutions according to step A, and then accurately add the mixed standard stock solution respectively, blow nitrogen until 10 to 20 μL of liquid remains, add 1.0 mL of 50% methanol resolvent containing 0.1% formic acid to dissolve the residue, pass through a 0.22 μm nylon filter membrane, and prepare a series of matrix mixed standard working solutions with a mass concentration of each pyrrolizidine alkaloid 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, which are prepared and used immediately.

[0064] 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.

[0065] In a specific embodiment, the liquid chromatography column is: Agilent Eclipse PlusC 18 RRHD chromatographic column; the liquid chromatography mobile phase includes three solutions A, B, and C; A is a 0.1% volume concentration formic acid aqueous solution containing 5 mmol / L ammonium formate, B is methanol, and C is acetonitrile; the mobile phase is used for gradient elution, and the gradient elution program is as follows: 0-2 min, 95% to 90% volume of phase A, 5% to 10% volume of phase B; 2-5 min, 90% to 80% volume of phase A, 10% to 20% volume of phase B; 5-10 min, 80% volume of phase A, 20% volume of phase B; 10-16 min, 80% to 60% volume of phase A, 20% to 40% volume of phase B; 16-18 min, 60% to 50% volume of phase A, 40% to 50% volume of phase B; 18-22 min, 50% volume of phase A, 50% volume of phase B; 22-25 min, 50% to 5% volume of phase A, 50% to 0% volume of phase B, 0% to 95% volume of phase C; 25-28 min, phase A volume 5%, phase B volume 0%, phase C volume 95%; 28-28.1 min, phase A volume 5% to 95%, phase B volume 0% to 5%, phase C volume 95% to 0%; 28.1-35 min, phase A volume 95%, phase B volume 5%; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 2 μL.

[0066] In a specific embodiment, the Agilent Eclipse Plus C 18 The specifications of the RRHD column are 3.0 mm × 150 mm, 1.8 μm.

[0067] In a specific embodiment, the mass spectrometry conditions of the tandem mass spectrometry include:

[0068] Ion source: electrospray ion source; scan mode: positive ion mode; monitoring mode: multiple reaction monitoring; drying gas N2 temperature: 350℃; nebulizing gas N2 pressure: 275.8 kPa; drying gas flow rate: 12.0 L / min; sheath gas N2 temperature: 350℃; sheath gas flow rate: 12.0 L / min; capillary voltage: 3500 V in negative ion mode, 3500 V in positive ion mode.

[0069] In a specific embodiment, the parameters of the pyrrolizidine alkaloids, such as retention time, quantitative ion pair, qualitative ion pair, in-source fragmentation voltage, collision energy, etc., are shown in Table 1 above.

[0070] In a specific embodiment, the method has an average spiked recovery of 66.6% to 101.6%, an average relative standard deviation of 0.5% to 6.0%, a detection limit of 0.06 μg / kg to 0.66 μg / kg, and a quantification limit of 0.2 μg / kg to 2 μg / kg.

[0071] The specific embodiments of the present invention are further described below in conjunction with examples, but the present invention is not limited to the scope of the examples.

[0072] Example 1

[0073] Instruments and Equipment

[0074] Agilent 1290 InfinityⅡ / G 6470 C high performance liquid chromatography-tandem mass spectrometry: equipped with an electrospray ionization source (ESI source) and Version 1.1 data processing system.

[0075] Materials and reagents

[0076] Materials: 0.22 μm nylon filter membrane; 50 mL centrifuge tubes, 15 mL centrifuge tubes, 10 mL volumetric flasks; PXC-SPE cartridges.

[0077] Chromatographic grade reagents: methanol, formic acid and acetonitrile.

[0078] Analytical grade reagents: anhydrous ethanol, methanol, ammonia.

[0079] Pyrrolizidine alkaloid standards: melanocyte-stimulating hormone, melanocyte-stimulating hormone N oxide, lycopodium, lycopodium N oxide, heliotrope, cynomolgus acid, heliotrope N oxide, heliotropeine, heliotrope N oxide, heliotropeine, heliotrope N oxide, 7-acetyl-melanotropin, 7-acetyl-melanotropin N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide, senecine, senecine N oxide,

[0080] Solution preparation:

[0081] 0.1% formic acid aqueous solution: Take 1000 mL of water, add 1 mL of formic acid, and mix well.

[0082] 5% ammonia methanol solution: Take 25 mL of ammonia, dilute to 500 mL with methanol and mix thoroughly.

[0083] Containing 5 mmol / L ammonium formate and 0.1% formic acid aqueous solution: Measure 1000 mL of water, add 1 mL of 5 mol / L ammonium formate and 1 mL of formic acid, and mix well.

[0084] Reconstitution of the solution in 50% methanol containing 0.1% formic acid: Measure 25 mL of water, add 25 mL of methanol and 50 μL of formic acid, and mix well.

[0085] Mixed standard stock solution: Accurately pipette each single standard solution separately, dilute with methanol, and prepare a mixed standard stock solution with a mass concentration of each pyrrolizidine alkaloid of 10.0 μg / L. Store below -18°C. The shelf life is 1 month.

[0086] Solvent mixed standard working solution: Accurately add the mixed standard stock solution respectively, and dilute it stepwise with the resolvent 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, and 10.0 μg / L, which should be prepared for use immediately.

[0087] Matrix mixed standard working solution: Select a blank sample with the same properties as the sample to be tested, and prepare 7 blank matrix solutions according to the sample pretreatment method of the present invention. Then, accurately add the mixed standard stock solution to each blank solution, blow with nitrogen until 10-20 μL of liquid remains, add 1.0 mL of resolvent to dissolve the residue, and filter through a 0.22 μm nylon filter membrane to 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, and 10.0 μg / L, which are prepared and used immediately.

[0088] Sample preparation

[0089] The samples of tea, corn, and rice were crushed and passed through a 60-mesh sieve for later use. 1 g of each sample (accurate to 0.01 g) was accurately weighed into a 50 mL centrifuge tube, and 10 mL of 95% ethanol solution containing 0.1% formic acid was added. The tube was vortexed for 1 min, ultrasonically extracted at 40 Hz for 30 min, and centrifuged at 8500 rpm for 5 min. The supernatant was removed, and 10 mL of 95% ethanol solution containing 0.1% formic acid was added to the residue and extracted again. The two supernatants were combined, nitrogen was blown to below 5 mL, and the volume was made up to 10 mL with a 0.1% formic acid aqueous solution. After vortex mixing, centrifugation was performed, and 5 mL was accurately transferred as the extract to be purified. Use 3 mL of methanol, 3 mL of water, and 3 mL of 0.1% formic acid aqueous solution to activate the PXC-SPE column in sequence. After activation, the extract to be purified is loaded onto the SPE column. After loading, it is first rinsed with 3 mL of water, then with 3 mL. After rinsing, the eluent in the SPE column is blown dry, and then eluted with 5 mL of 5% ammonia methanol. The eluate is collected in a 10 mL glass test tube, and the collected eluate is blown dry with nitrogen at 40°C, diluted to 1 mL with a resolvent, filtered through a 0.22 μm nylon filter membrane, and stored in an injection vial for liquid chromatography-tandem mass spectrometry determination.

[0090] After the honey sample is homogenized, 1 g (accurate to 0.01 g) is weighed into a 10 mL volumetric flask, 0.05 mol / L H2SO4 solution is added close to the scale line, vortex mixed, and ultrasonic extraction is performed at 40 Hz for 30 min. After cooling to room temperature, the volume is adjusted to the scale line with the extract solution. After shaking, the sample is transferred to a centrifuge tube and centrifuged at 8500 rpm for 5 min. 5 mL is accurately transferred as the extract to be purified. The subsequent purification process of the extract to be purified is the same as that of tea, corn, and rice samples.

[0091] Chromatographic conditions

[0092] Chromatographic column: Agilent Eclipse Plus C, 3.0 mm × 150 mm, 1.8 μm 18RRHD; mobile phase: A: 0.1% formic acid in water containing 5 mmol / L ammonium formate, B: methanol; gradient elution procedure as described above; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 2 μL.

[0093] Mass spectrometry conditions

[0094] Ion source: electrospray ion source; scan mode: positive ion mode; monitoring mode: 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, 3500 V in positive ion mode; parameters such as retention time, quantitative ion pair, qualitative ion pair, in-source fragmentation voltage, collision energy, etc. of each analyte are shown in Table 1 above.

[0095] Example 2

[0096] Refer to Example 1 for other examples and prepare five 5 μg / L solvent mixed standard solutions. In a 40°C water bath, the effects of five concentration methods on the recovery of each target compound were investigated: drying with nitrogen, blowing with nitrogen until nearly dry, blowing with nitrogen for an additional hour after drying, and adding 10% glycerol and 0.1% vitamin C followed by nitrogen drying. The results are shown in Table 2. Blowing with nitrogen until nearly dry means leaving 10 to 20 μL of liquid. As shown in Table 2, the longer the nitrogen blowing time, the greater the loss of the compound. The recoveries of nitrogen drying and nitrogen drying after adding 10% glycerol were generally higher than those of the other three methods. Nitrogen drying offers strong operability and lower detection costs. Nitrogen drying is the optimal method for concentration in the present invention.

[0097] Table 2 Recovery rates of various target substances under different concentration conditions in Example 2

[0098]

[0099] Example 3

[0100] Extraction recovery tests were conducted using a prepared 10 μg / kg standard solvent mixture solution, using six extraction solvents: 0.1% formic acid in water, 0.05 mol / L H₂SO₄ solution, 50% ethanol, 95% ethanol, 0.05 mol / L sulfuric acid in 95% ethanol, and 0.1% formic acid in 95% ethanol, to determine the optimal extraction solvent for pretreatment of each target compound. All other extraction solvents were the same as in Example 1.

[0101] The experimental results showed that the recovery rate of 0.1% formic acid aqueous solution ranged from 65% to 136%, the recovery rate of 0.05 mol / L H2SO4 solution ranged from 70.1% to 126.3%, the recovery rate of 50% ethanol ranged from 71.2% to 134.6%, the recovery rate of 95% ethanol solution containing 0.1% formic acid ranged from 71% to 124.1%, and the recovery rate of 95% ethanol solution containing 0.05 mol / L H2SO4 ranged from 60.1% to 114.5%. Compared with the two acidic aqueous solutions, the recovery rate of 0.05 mol / L H2SO4 solution was slightly better than that of 0.1% formic acid aqueous solution. The recovery rates of the four organic solvents showed little difference.

[0102] Example 4

[0103] The experiments were similar to Example 3, except that blank tea and honey were used as food samples, with the average recovery of each target substance at a spike level of 10 μg / kg as the indicator. The samples were extracted using three different extraction solvents: 0.05 mol / L H₂SO₄ solution, 0.05 mol / L H₂SO₄ in 95% ethanol, and 0.1% formic acid in 95% ethanol. Table 3 shows the effects of the three extraction solvents on the average recovery of pyrrolizidine alkaloids in tea.

[0104] Table 3 Effects of three extraction solvents on the average recovery of pyrrolizidine alkaloids in tea

[0105]

[0106] As shown in Table 3, the recoveries of 0.05 mol / L H₂SO₄ solutions ranged from 43.8% to 134%, with the recoveries of 7-acetyl-MSH N₂O₀, senecine, senecinoline, senecinoline N₂O₀, senegiline, senegiline N₂O₀, senegiline N₂O₀, senecinoline N₂O₀, senecinoline, and senegiline less than 70%. The recoveries of 95% ethanol solutions containing 0.05 mol / L H₂SO₄ ranged from 56.1% to 103.8%, with the recoveries of monocrotaline, 7-acetyl-MSH N₂O₀, senecine, senecinoline, senecinoline, senegiline N₂O₀, senegiline, senegiline N₂O₀, senegiline, senegiline N₂O₀, senegiline N₂O₀, and senegiline less than 70%. The recovery rates of 95% ethanol solutions containing 0.1% formic acid ranged from 63.7% to 105.5%. Only echinoidine, 7-acetyl-MSH N-oxide, senecine, rutinine, rutinine, and rutinine N-oxide had recoveries below 70%. 95% ethanol solutions containing 0.1% formic acid were the optimal solvent for extracting solid foods.

[0107] Honey can be dissolved in acidic aqueous solutions, and the spiked recovery rate in the preliminary experiment fully meets the requirements for spiked recovery rate in relevant national standards such as GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for National Food Safety Standards". 0.05 mol / L H2SO4 solution is the best extraction solvent for honey.

[0108] Example 5

[0109] Using blank (negative) tea leaves as the matrix and the average recovery rate of each target compound at a spike level of 5 μg / kg as an indicator, sample pretreatment was performed using an MCX cartridge with a specification of 60 mg / 3 mL, a PXC cartridge with a specification of 60 mg / 3 mL, and an HLB cartridge with a specification of 60 mg / 3 mL, respectively, to purify the matrix interfering substances in the tea leaves. Other procedures were the same as in Example 1. The results are shown in Table 4.

[0110] Table 4 Effects of three different purification columns on tea purification recovery

[0111]

[0112]

[0113] Results showed that the HLB cartridge failed to retain nearly 10 pyrrolizidine alkaloids, with only a few compounds achieving recoveries that met standard requirements. Cleanup results using both the MCX and PXC cartridges demonstrated that the recoveries of the target compounds met the additive recovery requirements of relevant national standards, such as GB5009.295-2023, "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standard," and GB / T 27404-2008, "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods." However, the PXC cartridge's faster flow rate compared to the MCX cartridge significantly improved the experimental speed. Furthermore, the PXC cartridge used in the experiment was less expensive than the MCX cartridge, making the PXC-SPE cartridge the optimal sample cleanup cartridge.

[0114] Example 6

[0115] A series of solvent-matrix-matched mixed standard working solutions with mass concentrations of each target substance to be measured 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 were prepared using blank solvent and blank tea matrix solution, respectively. The measurements were performed according to the optimal instrument parameter conditions determined by the present invention. The solvent-matrix-matched standard working curve was plotted with the mass concentration of each target substance (x) as the abscissa and the quantitative ion peak area as the ordinate. The detection limit (LOD) of the corresponding target substance was determined with a signal-to-noise ratio (S / N) of 3, and the quantification limit (LOQ) of the corresponding target substance was determined with an average signal-to-noise ratio (S / N) of 6 times as 10. The results for the blank solvent are shown in Table 5, and the results for the blank tea matrix are shown in Table 6. As shown in Tables 5 and 6, the 31 target compounds showed good linear relationships in the mass concentration range of 0.5 μg / L to 10.0 μg / L, with correlation coefficients r>0.99. The detection limits ranged from 0.06 μg / kg to 0.66 μg / kg, and the quantification limits ranged from 0.2 μg / kg to 2 μg / kg. This indicates that this method can well meet the needs of trace and even ultratrace determination of 31 pyrrolizidine alkaloids, such as melanocyte-stimulating hormone, lycopodipine, and senecioin, in foods.

[0116] Table 5 Regression equation, correlation coefficient, detection limit and quantification limit of each target compound in the blank solvent standard curve

[0117]

[0118] Table 6 Regression equation, correlation coefficient, detection limit and quantification limit of each target compound in the blank tea matrix standard curve

[0119]

[0120] Example 7

[0121] A solvent mixed standard solution with a mass concentration of 100 μg / L for each pyrrolizidine alkaloid substance was prepared. Blank honey, tea, rice, and corn were used as sample matrices. Different amounts of the 100 μg / L solvent mixed standard solution were added to each sample matrix, so that the mass concentration of each target substance in the spiked samples was 2 μg / kg, 5 μg / kg, and 10 μg / kg. Four replicates were prepared for each spiked concentration level. Pretreatment and determination analysis were performed according to Example 1 to investigate the accuracy and precision of this method. The determination results are shown in Tables 7 and 8. As can be seen from the table, the average recoveries of 31 pyrrolizidine alkaloids in honey at three spiked levels of 2 μg / kg, 5 μg / kg, and 10 μg / kg were 68.6%-121.1%, with relative standard deviations of 0.3%-5.0%; the average recoveries of 31 pyrrolizidine alkaloids in tea at three spiked levels were 66.6%-101.6%, with relative standard deviations of 0.5%-6.0%; the average recoveries of 31 pyrrolizidine alkaloids in rice at three spiked levels were 74.8%-111.2%, with relative standard deviations of 0.4%-6.3%; the average recoveries of 31 pyrrolizidine alkaloids in corn at three spiked levels were 70.6%-116.2%, with relative standard deviations of 0.2%-9.7%, all meeting the requirements of GB The requirements for method recovery and precision in relevant standards such as GB / T 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for National Food Safety Standard", GB / T 27404-2008 "Laboratory Quality Control Specification for Physical and Chemical Testing of Foods", GB / T 32465-2015 "Requirements for Validation and Confirmation of Chemical Analysis Methods and Internal Quality Control", and GB / T 27417-2017 "Guidelines for Validation and Verification of Chemical Analysis Methods for Conformity Assessment" indicate that the method established in the present invention can well meet the needs of simultaneous and accurate determination of 31 pyrrolizidine alkaloids in food.

[0122] Table 7 Recovery and precision of each target compound in honey and tea matrices

[0123]

[0124] Table 8 Recovery and precision of the target compounds in rice and corn matrices

[0125]

Claims

1. A method for detecting pyrrolizidine alkaloids in food, characterized in that: The following steps are involved: A. Food processing: including solid food processing or liquid food processing: Solid food processing is: a. After the solid food is crushed, pass through a 60-mesh sieve to obtain the undersize and oversize; b. The sieve residue from step a was 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 a supernatant 1 and a residue 1. The mass volume ratio of the sieve residue to the 95% ethanol solution containing 0.1% formic acid was 1 g:10 mL. c. Residue 1 was then 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. Combine supernatant 1 and supernatant 2, blow down to less than 5 mL with nitrogen, then dilute to 10 mL with 0.1% formic acid aqueous solution, and centrifuge to obtain supernatant 3; e. Take 3 to 5 mL of supernatant 3 and load it onto the activated PXC-SPE cartridge. After loading, rinse with 3 mL of water, then with 3 mL of methanol. After rinsing, blow dry the eluent in the PXC-SPE cartridge and elute with 5 mL of 5% ammonia methanol solution. Collect the eluate, blow dry it with nitrogen at 40°C, dilute it to 1 mL with a resolvent, and filter it through a 0.22 μm nylon filter membrane to obtain supernatant 4. The PXC-SPE cartridge is activated by sequentially passing 3 mL of methanol, 3 mL of water, and 3 mL of 0.1% formic acid aqueous solution through the PXC-SPE cartridge. The resolvent is a 50% methanol solution containing 0.1% formic acid. Liquid food processing is: The liquid food was mixed with the H2SO4 solution, subjected to ultrasonic extraction at 40 Hz for 30 minutes, cooled to room temperature, and then centrifuged at 8500 rpm for 5 minutes to obtain supernatant 3. Supernatant 3 was 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 was 1 g:10 mL; the concentration of the H2SO4 solution was 0.05 mol / L. B. Determination of pyrrolizidine alkaloids in supernatant 4 by liquid chromatography-tandem mass spectrometry; The percentages of 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 by volume. The liquid chromatography column is an Agilent Eclipse Plus C18 RRHD column; the liquid chromatography mobile phase includes three solutions A, B, and C; A is a 0.1% volume concentration formic acid aqueous solution containing 5 mmol / L ammonium formate, B is methanol, and C is acetonitrile; the mobile phase is used for gradient elution, and the gradient elution program is as follows: 0-2 min, 95% to 90% volume of phase A, 5% to 10% volume of phase B; 2-5 min, 90% to 80% volume of phase A, 10% to 20% volume of phase B; 5-10 min, 80% volume of phase A, 20% volume of phase B; 10-16 min, 80% to 60% volume of phase A, 20% to 40% volume of phase B; 16-18 min, 60% to 50% volume of phase A, 40% to 50% volume of phase B; 18-22 min, 50% volume of phase A, 50% volume of phase B; 22-25 min, phase A volume 50% to 5%, phase B volume 50% to 0%, phase C volume 0% to 95%; 25-28 min, phase A volume 5%, phase B volume 0%, phase C volume 95%; 28-28.1 min, phase A volume 5% to 95%, phase B volume 0% to 5%, phase C volume 95% to 0%; 28.1-35 min, phase A volume 95%, phase B volume 5%; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 2 μL; The mass spectrometry conditions of the tandem mass spectrometry include: Ion source: electrospray ion source; scan mode: positive ion mode; monitoring mode: multiple reaction monitoring; drying gas N2 temperature: 350℃; nebulizing gas N2 pressure: 275.8 kPa; drying gas flow rate: 12.0 L / min; sheath gas N2 temperature: 350℃; sheath gas flow rate: 12.0 L / min; capillary voltage: 3500 V in negative ion mode, 3500 V in positive ion mode.

2. The method for detecting pyrrolizidine alkaloids in food according to claim 1, wherein The solid food is tea, chrysanthemum, corn or rice; and the liquid food is honey.

3. The method for detecting pyrrolizidine alkaloids in food according to claim 1 or 2, wherein: The pyrrolizidine alkaloids include: melanocyte-stimulating hormone, melanocyte-stimulating hormone nitrogen oxide, lycopodium, lycopodium nitrogen oxide, sinapine, cyanidin, cyanidin nitrogen oxide, pilocarpine, pilocarpine nitrogen oxide, monocrotaline, sinapine nitrogen oxide, heliotropin, heliotropin nitrogen oxide, 7-acetyl melanocyte-stimulating hormone, 7-acetyl melanocyte-stimulating hormone nitrogen oxide, monocrotaline nitrogen oxide, seneciopine, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, seneciopine, seneciopine nitrogen oxide, 4. The method for detecting pyrrolizidine alkaloids in food according to claim 2, wherein When the food is tea, the liquid chromatography-tandem mass spectrometry determination adopts the matrix matching standard curve method; when the food is chrysanthemum, corn, rice or honey, the liquid chromatography-tandem mass spectrometry determination adopts the solvent standard curve method; the preparation method of the matrix mixed standard working solution of the matrix matching 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, and then accurately add the mixed standard stock solution respectively, blow nitrogen until 10 to 20 μL of liquid remains, add 1.0 mL of 50% methanol resolvent containing 0.1% formic acid to dissolve the residue, pass through a 0.22 μm nylon filter membrane, and prepare a series of matrix mixed standard working solutions with a mass concentration of each pyrrolizidine alkaloid 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, which are prepared and used immediately.

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, wherein The specifications of the Agilent Eclipse Plus C18 RRHD chromatographic column are 3.0 mm×150 mm, 1.8 μm.

7. The method for detecting pyrrolizidine alkaloids in food according to claim 1 or 2, characterized in that: The average spiked recoveries of the method were 66.6%-101.6%, the average relative standard deviations were 0.5%-6.0%, the limits of detection were 0.06 μg / kg-0.66 μg / kg, and the limits of quantification were 0.2 μg / kg-2 μg / kg.

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