Method for rapidly detecting patulin in fruit and vegetable products

Abstract

The invention relates to a method for rapidly detecting patulin in fruit and vegetable products, and belongs to the technical field of patulin detection. The patulin detection method mainly comprises the following steps: extracting a to-be-detected product by using an extraction reagent consisting of thiosalicylic acid, acetic acid, ethanol and ethyl acetate to obtain a to-be-detected sample, and detecting by using a high performance liquid chromatography. The method disclosed by the invention is simple to operate, capable of realizing accurate detection of patulin, free of interference and relatively high in accuracy and sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of patulin detection technology and relates to a method for rapid detection of patulin in fruit and vegetable products. Background Technology

[0002] Fruits and vegetables are important sources of nutrition for humans, providing essential vitamins, minerals, and dietary fiber. After harvesting, due to their high water content and rich nutrients, fruits and vegetables are susceptible to infection and spoilage by pathogenic fungi during storage, transportation, and processing. Diseased fruits and vegetables result in a loss of 35% to 55% of total yield. These infected fruits and vegetables accumulate large amounts of mycotoxins. Mycotoxins are secondary metabolites produced by fungi, and diseased fruits and vegetables mainly contain mycotoxins such as Alternaria toxin, ochratoxin, and patulin. Among these, patulin primarily contaminates fruits and their processed products. Patulin (PAT) can be produced by 60 different fungi, such as *Penicillium expansum*, *Penicillium albopictus*, *Penicillium patellae*, *Penicillium patellae*, and *Aspergillus*, with *Penicillium expansum* being the main fungus causing patulin contamination in food. Penicillium expansum primarily infects ripe fruits through wounds and other routes. Its host range includes common and processed fruits such as apples, peaches, pears, grapes, kiwifruit, and hawthorn, making it one of the most important postharvest pathogens. During infection, Penicillium expansum produces large amounts of patulin, which accumulates in rotten parts of the fruit and spreads to unaffected areas. It enters the food chain during fresh fruit distribution and processing, endangering consumer health. Processed products such as fruit juice, jam, and puree are the main food categories contaminated with patulin.

[0003] Fungal toxins not only cause spoilage and quality reduction in fruits and vegetables, resulting in losses for the fruit and vegetable industry, but also, after entering the human body through the food chain, can cause toxic effects such as metabolic disorders, nerve paralysis, organ failure, carcinogenesis, and immune system damage. Therefore, developing sensitive and reliable detection methods for major fungal toxins in fruits and vegetables, monitoring their content, and reducing their levels are of significant practical importance for the development of the fruit and vegetable industry and human health.

[0004] Traditional quantitative analysis methods for patulin in food mainly include: high-performance liquid chromatography (HPLC), HPLC-tandem mass spectrometry (HPLC-MS / MS), thin-layer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), and capillary electrophoresis. These traditional methods often encounter interference from impurities, requiring sample pretreatment to obtain more accurate qualitative and quantitative information. Pretreatment methods typically include liquid-liquid extraction, solid-phase extraction, and dispersive solid-phase extraction; however, sample purification is a key challenge in the determination of patulin. Summary of the Invention

[0005] The main objective of this invention is to provide a method for rapid detection of patulin in fruit and vegetable products. The method is simple to operate, can achieve rapid detection of patulin in fruit and vegetable products, and has high accuracy and sensitivity.

[0006] The present invention employs the following technical solutions to achieve the above objectives: A rapid method for detecting patulin in fruit and vegetable products, the method mainly involves pre-treating the product to be tested and then detecting it using high performance liquid chromatography.

[0007] The method described above mainly includes the following steps: Step 1, Sample pretreatment: Extraction reagent I is added to the product to be tested and stirred for extraction. The mixture is allowed to stand and separate into layers. The upper liquid I is reserved. The lower liquid is taken out and extraction reagent II is added and stirred for extraction. The mixture is allowed to stand and separate into layers to obtain the upper liquid II. The upper liquid I and the upper liquid II are combined and Na2CO3 solution is added. After shaking, the mixture is allowed to stand and separate into layers. The upper solution is taken out, dried, and redissolved in acetic acid aqueous solution to obtain the sample to be tested. Step 2, Preparation of standard solution: Accurately weigh 10 mg of patulin standard into a 10 mL volumetric flask, dissolve in acetonitrile and dilute to volume to prepare a 1.0 mg / mL standard stock solution; accurately measure the standard stock solution and dilute with 0.5% acetic acid aqueous solution to prepare a 1 μg / mL standard solution. Step 3, determination by high performance liquid chromatography: The sample to be tested and the standard solution were injected into the high performance liquid chromatograph, and the determination was carried out by gradient elution. The gradient elution procedure is as follows: 0-5 min: acetonitrile 5%, water 95%; 5-15 min: acetonitrile 5%~80%, water 95%~20%; 15-20 min: acetonitrile 80%~95%, water 20%~5%; 20-25 min: acetonitrile 95%-5%, water 5%-95%; 25-30 min: acetonitrile 5%, water 95%.

[0008] Specifically, the extraction reagent I used in step 1 is composed of thiosalicylic acid, acetic acid, ethanol, and ethyl acetate; A further preferred embodiment is that each 100 mL of extraction reagent I contains 1-1.3 g of thiosalicylic acid, 3-4 g of acetic acid, 6-8 mL of ethanol, and 92-94 mL of ethyl acetate.

[0009] Specifically, the extraction reagent II used in step 1 is composed of thiosalicylic acid, acetic acid, ethanol, and ethyl acetate; Further preferred, each 100 mL of extraction reagent II contains 2-2.2 g of thiosalicylic acid, 2-2.5 g of acetic acid, 10-12 mL of ethanol, and 88-90 mL of ethyl acetate.

[0010] Specifically, the extraction temperature in step 1 is 30-35℃.

[0011] Specifically, the mass fraction of the Na2CO3 solution in step 1 is 0.5%.

[0012] Specifically, the mass fraction of acetic acid in the acetic acid aqueous solution in step 1 is 0.5%.

[0013] The present invention has the following beneficial effects: In this invention, an extraction solvent prepared from thiosalicylic acid, acetic acid, ethanol, and ethyl acetate is first used to extract the fruit and vegetable products to be tested. This solvent can effectively enrich patulin in the fruit and vegetable products without enriching other components and without interfering with the detection of patulin. At the same time, the high-performance liquid chromatography conditions are optimized, and a gradient elution method is used to detect patulin in the sample to be tested, which has high accuracy and no interference from impurity peaks.

[0014] The product pretreatment process used in this invention is simple, requiring no complex column purification steps, and can be obtained simply through conventional reagent extraction. It is easy to operate and enables rapid sample processing and detection. Attached Figure Description

[0015] Figure 1 Comparison of the detection effects of different detection methods on patulin, where A is the chromatogram of the standard solution in Example 1, B is the chromatogram of the sample to be tested in Example 1, C is the chromatogram of the spiked sample to be tested in Example 2, and D is the chromatogram of the spiked sample to be tested in Comparative Example 1. Figure 2 Comparison of the detection effects of different detection methods on patulin, where A is the chromatogram of the standard solution in Comparative Example 2, and B is the chromatogram of the spiked sample in Comparative Example 2. Detailed Implementation

[0016] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.

[0017] Example 1 Step 1, Sample pretreatment: Take 20g of a commercially available brand of apple and hawthorn juice, add 20mL of water, and mix well. Add 100mL of extraction reagent I (containing 1.3g of thiosalicylic acid, 4g of acetic acid, 8mL of ethanol, and 92mL of ethyl acetate), and extract by stirring in a 35℃ water bath for 10min. Allow to stand and separate the layers, and store the upper liquid I in a separate container. Take out the lower liquid and add 100mL of extraction reagent II (containing 2.2g of thiosalicylic acid, 2.5g of acetic acid, 10mL of ethanol, and 90mL of ethyl acetate), and extract by stirring in a 35℃ water bath for 10min. Allow to stand and separate the layers, and take out the upper liquid II. Combine the upper liquid II with the upper liquid I, add 10mL of 1.0% Na2CO3 solution, shake, and allow to stand and separate the layers. Take out the upper solution, evaporate to dryness under reduced pressure at 35℃, and redissolve in 5mL of 0.5% acetic acid aqueous solution to obtain the sample to be tested. Step 2, Preparation of standard solution: Accurately weigh 10 mg of patulin standard into a 10 mL volumetric flask, dissolve and dilute to volume with acetonitrile to prepare a 1.0 mg / mL standard stock solution; accurately measure the standard stock solution and dilute it with 0.5% acetic acid aqueous solution to prepare a 1 μg / mL standard solution.

[0018] Step 3, determination by high performance liquid chromatography: Inject the sample to be tested and the standard solution into the high-performance liquid chromatograph and perform the determination under the following conditions.

[0019] The chromatographic conditions are as follows: Instrument: Waters e2695 high performance liquid chromatograph; Column: Agilent Eclipse Plus C 18 4.6×250mm, 5μm; Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 275 nm; Injection volume: 10 μL; Mobile phase: Acetonitrile (phase A) - 0.1% acetic acid aqueous solution (phase B); Washing procedure:

[0020] Record and analyze the chromatogram: No peak was observed in the beverage chromatogram at the corresponding position of the reference standard, indicating that patulin was not detected in the beverage and that there were no other impurities interfering with the peak position.

[0021] Example 2 Step 1, Sample pretreatment: Take 20g of the same apple and hawthorn juice beverage as in Example 1, add 20mL of water, and mix well; add 100mL of extraction reagent I (containing 1g of thiosalicylic acid, 3g of acetic acid, 6mL of ethanol, and 94mL of ethyl acetate), stir in a 30℃ water bath for 10min to extract, let stand to separate the layers, and store the upper liquid I in a separate container; take out the lower liquid and add 100mL of extraction reagent II (containing 2g of thiosalicylic acid, 2g of acetic acid, 12mL of ethanol, and 88mL of ethyl acetate), stir in a 30℃ water bath for 10min to extract, let stand to separate the layers, and take out the upper liquid II; combine the upper liquid II with the upper liquid I, add 8-10mL of 1.0% Na2CO3 solution, shake, let stand to separate the layers, take out the upper solution, evaporate under reduced pressure to dryness at 30℃, redissolve in 5mL of 0.5% acetic acid aqueous solution, and then add the following 1.0mg / mL standard stock solution to prepare a spiked test sample containing 0.3μg / mL of patulin; Step 2, Preparation of standard solution: Accurately weigh 10 mg of patulin standard into a 10 mL volumetric flask, dissolve and dilute to volume with acetonitrile to prepare a 1.0 mg / mL standard stock solution; accurately measure the standard stock solution and dilute it with 0.5% acetic acid aqueous solution to prepare a 1 μg / mL standard solution.

[0022] Step 3, determination by high performance liquid chromatography: Inject the spiked sample or standard solution into the high-performance liquid chromatograph and perform the determination under the following conditions.

[0023] The chromatographic conditions are as follows: Instrument: Waters e2695 high performance liquid chromatograph; Column: Agilent Eclipse Plus C 18 4.6×250mm, 5μm; Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 275 nm; Injection volume: 10 μL; Mobile phase: Acetonitrile (phase A) - 0.1% acetic acid aqueous solution (phase B); Washing procedure:

[0024] Record and analyze the chromatogram: At the corresponding position of the reference standard, a chromatographic peak appears in the spiked sample, and there is no interference from other impurities at this peak position.

[0025] Example 3 Step 1, Sample pretreatment: Take 10g of a commercially available brand of carrot and apple puree, add 20mL of water, and mix well. Add 100mL of extraction reagent I (containing 1g of thiosalicylic acid, 3.5g of acetic acid, 7mL of ethanol, and 93mL of ethyl acetate), and extract by stirring in a water bath at 30-35℃ for 10min. Allow to stand and separate the layers, and store the upper liquid I in a separate container. Take out the lower liquid and add 100mL of extraction reagent II (containing 2.2g of thiosalicylic acid, 2.3g of acetic acid, 11mL of ethanol, and 89mL of ethyl acetate), and extract by stirring in a water bath at 30-35℃ for 10min. Allow to stand and separate the layers, and take out the upper liquid II. Combine the upper liquid II with the upper liquid I, add 8-10mL of 1.0% Na2CO3 solution, shake, and allow to stand and separate the layers. Take out the upper solution, evaporate to dryness under reduced pressure at 30-35℃, and redissolve in 5mL of 0.5% acetic acid aqueous solution to obtain the sample to be tested. Step 2, Preparation of standard solution: Accurately weigh 10 mg of patulin standard into a 10 mL volumetric flask, dissolve and dilute to volume with acetonitrile to prepare a 1.0 mg / mL standard stock solution; accurately measure the standard stock solution and dilute it with 0.5% acetic acid aqueous solution to prepare a 1 μg / mL standard solution.

[0026] Step 3, determination by high performance liquid chromatography: Inject the sample to be tested and the standard solution into the high-performance liquid chromatograph and perform the determination under the following conditions.

[0027] The chromatographic conditions are as follows: Instrument: Waters e2695 high performance liquid chromatograph; Column: Agilent Eclipse Plus C 18 4.6×250mm, 5μm; Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 275 nm; Injection volume: 10 μL; Mobile phase: Acetonitrile (phase A) - 0.1% acetic acid aqueous solution (phase B); Washing procedure:

[0028] Record and analyze the chromatograms: No peak was observed in the chromatogram of the fruit pulp sample at the corresponding position of the reference standard, indicating that patulin was not detected in the fruit pulp and that there were no other impurities interfering at the peak position.

[0029] Comparative Example 1 Step 1, Sample pretreatment: Take 20g of the same apple and hawthorn juice beverage as in Example 1, add 20mL of water, and mix well; add 100mL of ethyl acetate, and extract by stirring in a 30℃ water bath for 30min, allowing it to stand and separate into layers, and store the upper liquid I in a separate container; take out the lower liquid, add another 100mL of ethyl acetate, and extract by stirring in a 30℃ water bath for 30min, allowing it to stand and separate into layers, and take out the upper liquid II; combine the upper liquid II with the upper liquid I, add 5mL of 1.5% Na2CO3 solution, shake, and allow it to stand and separate into layers, take out the upper solution, add 5mL of pH3.0 H3PO4 solution, shake, separate into layers, take the upper solution, evaporate to dryness under reduced pressure at 30℃, redissolve in 5mL of 0.5% acetic acid aqueous solution, and then add the following 1.0mg / mL standard stock solution to prepare a spiked test sample containing 0.3μg / mL of patulin; Step 2, Preparation of standard solution: Accurately weigh 10 mg of patulin standard into a 10 mL volumetric flask, dissolve and dilute to volume with acetonitrile to prepare a 1.0 mg / mL standard stock solution; accurately measure the standard stock solution and dilute it with 0.5% acetic acid aqueous solution to prepare a 1 μg / mL standard solution.

[0030] Step 3, determination by high performance liquid chromatography: Inject the spiked sample or standard solution into the high-performance liquid chromatograph and perform the determination under the following conditions.

[0031] The chromatographic conditions are as follows: Instrument: Waters e2695 high performance liquid chromatograph; Column: Agilent Eclipse Plus C 18 4.6×250mm, 5μm; Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 275 nm; Injection volume: 10 μL; Mobile phase: Acetonitrile (phase A) - 0.1% acetic acid aqueous solution (phase B); Washing procedure:

[0032] Record and analyze the chromatograms: If cross-peaks appear in the chromatogram of the spiked sample at the corresponding position of the reference standard, it indicates that there are other impurities interfering with the elution site of patulin, which will affect the detection effect.

[0033] Comparative Example 2 Step 1, Sample pretreatment: Take 20g of the same apple and hawthorn juice beverage as in Example 1, add 20mL of water, and mix well; add 100mL of extraction reagent I (containing 1.3g of thiosalicylic acid, 4g of acetic acid, 8mL of ethanol, and 92mL of ethyl acetate), stir in a 35℃ water bath for 10min to extract, let stand to separate the layers, and store the upper liquid I in a separate container; take out the lower liquid and add 100mL of extraction reagent II (containing 2.2g of thiosalicylic acid, 2.5g of acetic acid, 10mL of ethanol, and 90mL of ethyl acetate), stir in a 35℃ water bath for 10min to extract, let stand to separate the layers, and take out the upper liquid II; combine the upper liquid II with the upper liquid I, add 10mL of 1.0% Na2CO3 solution, shake, let stand to separate the layers, take out the upper solution, evaporate under reduced pressure to dryness at 35℃, redissolve in 5mL of 0.5% acetic acid aqueous solution, and then add the following 1.0mg / mL standard stock solution to prepare a spiked test sample containing 0.3μg / mL of patulin; Step 2, Preparation of standard solution: Accurately weigh 10 mg of patulin standard into a 10 mL volumetric flask, dissolve and dilute to volume with acetonitrile to prepare a 1.0 mg / mL standard stock solution; accurately measure the standard stock solution and dilute it with 0.5% acetic acid aqueous solution to prepare a 1 μg / mL standard solution.

[0034] Step 3, determination by high performance liquid chromatography: Inject the spiked sample or standard solution into the high-performance liquid chromatograph and perform the determination under the following conditions.

[0035] The chromatographic conditions are as follows: Instrument: Waters e2695 high performance liquid chromatograph; Column: Agilent Eclipse Plus C 18 4.6×250mm, 5μm; Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 275 nm; Injection volume: 10 μL; Mobile phase: Acetonitrile (phase A) - 0.1% acetic acid aqueous solution (phase B); Washing procedure:

[0036] Record and analyze the chromatograms: At the corresponding positions of the reference standard, the chromatogram of the spiked test sample shows multiple cross-chromatographic peaks, which cannot be effectively separated. Under these chromatographic conditions, effective detection of patulin is not possible.

Claims

1. A method for rapid detection of patulin in fruit and vegetable products, characterized in that, The method involves pre-treating the product to be tested and then detecting it using high-performance liquid chromatography (HPLC). The method includes the following steps: Step 1, Sample pretreatment: Extraction reagent I is added to the product to be tested and stirred for extraction. The mixture is allowed to stand and separate into layers. The upper liquid I is reserved. The lower liquid is taken out and extraction reagent II is added and stirred for extraction. The mixture is allowed to stand and separate into layers to obtain the upper liquid II. The upper liquid I and the upper liquid II are combined and Na2CO3 solution is added. After shaking, the mixture is allowed to stand and separate into layers. The upper solution is taken out, dried, and redissolved in acetic acid aqueous solution to obtain the sample to be tested. Step 2, Preparation of standard solution: Accurately weigh 10 mg of patulin standard into a 10 mL volumetric flask, dissolve in acetonitrile and dilute to volume to prepare a 1.0 mg / mL standard stock solution; accurately measure the standard stock solution and dilute with 0.5% acetic acid aqueous solution to prepare a 1 μg / mL standard solution. Step 3, determination by high performance liquid chromatography: The sample to be tested and the standard solution were injected into the high performance liquid chromatograph, and the determination was carried out by gradient elution. The gradient elution procedure is as follows: 0-5 min: acetonitrile 5%, water 95%; 5-15 min: acetonitrile 5%~80%, water 95%~20%; 15-20 min: acetonitrile 80%~95%, water 20%~5%; 20-25 min: acetonitrile 95%-5%, water 5%-95%; 25-30 min: acetonitrile 5%, water 95%.

2. The method as described in claim 1, characterized in that, The extraction reagent I used in step 1 consists of thiosalicylic acid, acetic acid, ethanol, and ethyl acetate.

3. The method as described in claim 2, characterized in that, Each 100 mL of extraction reagent I contains 1-1.3 g of thiosalicylic acid, 3-4 g of acetic acid, 6-8 mL of ethanol, and 92-94 mL of ethyl acetate.

4. The method as described in claim 1, characterized in that, The extraction reagent II used in step 1 consists of thiosalicylic acid, acetic acid, ethanol, and ethyl acetate.

5. The method as described in claim 4, characterized in that, Each 100 mL of Extraction Reagent II contains 2-2.2 g of thiosalicylic acid, 2-2.5 g of acetic acid, 10-12 mL of ethanol, and 88-90 mL of ethyl acetate.

6. The method as described in claim 1, characterized in that, The extraction temperature in step 1 is 30-35℃.

7. The method as described in claim 1, characterized in that, The mass fraction of the Na2CO3 solution in step 1 is 0.5%.

8. The method as described in claim 1, characterized in that, The mass fraction of acetic acid in the acetic acid aqueous solution in step 1 is 0.5%.

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