Efficient extraction and detection method of sugarcane neurotoxin 3-nitropropionic acid

Abstract

The application provides a high-efficiency extraction and detection method of sugarcane neurotoxin 3-nitropropionic acid. Specifically, the application provides a sugarcane sample pretreatment method for detecting sugarcane neurotoxin 3-NPA and a detection technology thereof. In the application, the sugarcane sample can be enriched with 3-NPA in the sugarcane to the maximum extent under specific extraction conditions and magnetic solid-phase extraction conditions, so that quantitative detection of 3-NPA in the sugarcane and products thereof can be realized.

Description

Technical Field

[0001] This invention belongs to the field of chemical pollutant detection, specifically relating to a highly efficient extraction and detection method for the sugarcane neurotoxin 3-nitropropionic acid. Background Technology

[0002] 3-Nitropropanoic acid (3-NPA) is a compound produced by *Fusarium moniliforme* (…). Arthrinium Aspergillus ( ) Aspergillus 3-NPA, a potent neurotoxin produced by fungi such as spores, is widely found in moldy sugarcane, corn, and other foods and related products. Acute poisoning by 3-NPA can cause vomiting, dystonia, coma, and even death. Long-term exposure may induce reproductive toxicity and increase the risk of neurodegenerative diseases.

[0003] With global warming, 3-NPA contamination incidents have been reported in many countries. In Serbia, corn contamination rates can reach 44% in hot, dry years, and cases of poisoning from moldy sugarcane have also occurred frequently in southern China. As a major sugarcane producer, China's monitoring of 3-NPA contamination is of significant practical importance for ensuring food safety and public health. However, there is currently no standard detection method for 3-NPA, nor has a highly efficient and universally applicable detection technology been reported.

[0004] Existing technologies commonly use fluorescence sensing, electrochemical methods, and gas chromatography-tandem mass spectrometry to detect 3-NPA, but these methods have many limitations: For example, although fluorescence sensing and electrochemical methods have high sensitivity and extremely fast analysis speed, they are easily affected by food matrices in practical applications, which can affect the accuracy of the results. Gas chromatography-tandem mass spectrometry can improve the accuracy of 3-NPA detection methods, but it requires 3-NPA derivatization pretreatment, which is cumbersome and can easily cause loss of the target analyte. Liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) can achieve accurate detection without derivatization, and the instruments are already widely used in analytical laboratories, making it a promising standard detection technique. However, 3-NPA, as a small molecule organic acid (pKa=3.66), has weak retention on reversed-phase chromatography columns, and the complex sugarcane matrix produces a severe matrix inhibition effect, both of which make it difficult to accurately quantify 3-NPA by HPLC-MS / MS. Solid-phase extraction has certain technical limitations for the extraction of 3-NPA from sugarcane samples. Although many studies have attempted to use solid-phase extraction columns for extraction, a suitable material for the extraction of 3-NPA from sugarcane samples has not yet been found.

[0005] Furthermore, the extremely low concentration of 3-NPA in sugarcane and the lack of suitable internal standard correction further complicate the analysis.

[0006] Therefore, there is an urgent need in the field to develop a highly efficient and accurate method for the quantitative detection of the sugarcane neurotoxin 3-nitropropionic acid (3-NPA) that is highly resistant to matrix interference and / or easy to operate. Summary of the Invention

[0007] The purpose of this invention is to provide a highly efficient and accurate quantitative detection method for the sugarcane neurotoxin 3-nitropropionic acid (3-NPA) that is highly resistant to matrix interference and / or easy to operate.

[0008] In a first aspect of the invention, a method for pretreatment of sugarcane samples for detecting the sugarcane neurotoxin 3-nitropropionic acid (3-NPA) is provided, the method comprising the steps of: (S1) Provides a liquid crude sugarcane extract containing 3-NPA; (S2) The liquid crude sugarcane extract from step (S1) is subjected to magnetic solid-phase extraction using a magnetic solid-phase extraction material to obtain a magnetic solid-phase extraction material adsorbed with 3-NPA (or a "magnetic solid-phase extraction material-3-NPA" complex). The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads (MCX), or N-propylethylenediamine magnetic beads (PSA), or combinations thereof; and (S3) The “magnetic solid phase extraction material-sugarcane neurotoxin 3-NPA” complex is eluted with an eluent (or eluent) to elute the sugarcane neurotoxin 3-NPA, thereby obtaining an eluent enriched with sugarcane neurotoxin 3-NPA.

[0009] In another preferred embodiment, the crude sugarcane extract is in the form of a concentrated liquid.

[0010] In another preferred embodiment, in step (S2), the magnetic solid-phase extraction process includes: mixing liquid sugarcane crude extract with magnetic solid-phase extraction material to obtain a first mixture.

[0011] In another preferred embodiment, the first mixture contains crude sugarcane extract and magnetic solid-phase extraction material.

[0012] In another preferred embodiment, step (S2) further includes: performing magnetic solid-liquid separation on the first mixture to obtain a magnetic solid-phase extraction material adsorbed with 3-NPA.

[0013] In another preferred embodiment, in step (S3), after elution with an eluent, a second mixture containing 3-NPA and the eluted magnetic solid-phase extraction material is obtained.

[0014] In another preferred embodiment, step (S3) further includes: performing magnetic solid-liquid separation on the second mixture to remove the eluted magnetic solid-phase extraction material, thereby obtaining an eluent enriched with sugarcane neurotoxin 3-NPA.

[0015] In another preferred embodiment, step (S2) includes: mixing and incubating the crude sugarcane extract with a magnetic solid-phase extraction material to perform magnetic solid-phase extraction, so that sugarcane neurotoxin 3-NPA is adsorbed on the surface of the magnetic solid-phase extraction material, and then performing magnetic solid-liquid separation to obtain a magnetic solid-phase extraction material adsorbed with 3-NPA.

[0016] In another preferred embodiment, the incubation time is 0.5 to 10 min, more preferably 0.8 to 8 min, and even more preferably 1 to 5 min.

[0017] In another preferred embodiment, step (S1) includes: crudely extracting the sugarcane sample with acetonitrile, then adding water and concentrating the extract to obtain a concentrated form of crude sugarcane extract.

[0018] In another preferred embodiment, the magnetic solid-phase extraction material has an extraction recovery rate of 90-110% for sugarcane neurotoxin 3-NPA, more preferably 95-105%, and even more preferably 96-100%.

[0019] In another preferred embodiment, the magnetic solid-phase extraction material is a mixed-type strong cation exchange magnetic bead (MCX).

[0020] In another preferred embodiment, the method includes the steps of: (S1) Provides a liquid crude sugarcane extract containing 3-NPA; (S2) The liquid crude sugarcane extract from step (S1) is subjected to magnetic solid-phase extraction using an activated magnetic solid-phase extraction material to obtain a magnetic solid-phase extraction material adsorbed with 3-NPA (or a "magnetic solid-phase extraction material-3-NPA" complex). The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads (MCX), or N-propylethylenediamine magnetic beads (PSA), or combinations thereof; and (S3) The “magnetic solid phase extraction material-sugarcane neurotoxin 3-NPA” complex is eluted with an eluent (or eluent) to elute the sugarcane neurotoxin 3-NPA, thereby obtaining an eluent enriched with sugarcane neurotoxin 3-NPA.

[0021] In another preferred embodiment, the magnetic solid-phase extraction material in step (S2) is an activated magnetic solid-phase extraction material that has undergone activation treatment.

[0022] In another preferred embodiment, the method for preparing the liquid crude sugarcane extract containing 3-NPA in step (S1) includes the following sub-steps: (S1a) The sugarcane sample was crudely extracted with acetonitrile and ultrasonically treated under constant temperature to obtain a crude extract solution. (S1b) After adding sodium chloride to the solution obtained in step (S1a), centrifuge and dilute to obtain a liquid crude sugarcane extract containing 3-NPA; (S1c) Step (S1b) Obtain liquid crude sugarcane extract containing 3-NPA, which is then concentrated by nitrogen blowing to obtain sugarcane sample concentrate.

[0023] In another preferred embodiment, the acetonitrile concentration used in step (S1a) or (S1) is 100% v / v.

[0024] In another preferred embodiment, the mixing ratio of acetonitrile to sugarcane sample used in step (S1a) or (S1) is 1g sugarcane (wet weight): 1-5ml acetonitrile, preferably 1g sugarcane (wet weight): 1-3ml acetonitrile.

[0025] In another preferred embodiment, in step (S1a), the temperature of the ultrasound is room temperature, which is 25 to 37°C.

[0026] In another preferred embodiment, in step (S1a), the duration of the ultrasound is 5 to 30 minutes, more preferably 5 to 25 minutes, and even more preferably 5 to 10 minutes.

[0027] In another preferred embodiment, in step (S1b), the mixing ratio of sodium chloride to liquid sugarcane crude extract is 1g sodium chloride: 1-10ml acetonitrile, more preferably 1g sodium chloride: 1-5ml acetonitrile.

[0028] In another preferred embodiment, the activated magnetic solid-phase extraction material is prepared by activation treatment using the following sub-steps: (S2a) Provides a magnetic solid-phase extraction material; The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads (MCX), or N-propylethylenediamine magnetic beads (PSA), or combinations thereof; (S2b) The magnetic solid-phase extraction material is immersed in methanol. After magnetic solid-liquid separation, the supernatant is discarded to obtain the pre-activated magnetic solid-phase extraction material. (S2c) The pre-activated magnetic solid phase extraction material obtained in step (S2b) is immersed in a 2% formic acid aqueous solution for magnetic solid-liquid separation. The supernatant is discarded to obtain the activated magnetic solid phase extraction material.

[0029] In another preferred embodiment, the soaking time for each of steps (S2b) and (S2c) is independently 0.5 to 10 min, more preferably 0.8 to 8 min, and even more preferably 1 to 5 min.

[0030] In another preferred embodiment, the magnetic solid-phase extraction material is a mixed-type strong cation exchange magnetic bead (MCX).

[0031] In another preferred embodiment, the magnetic solid-phase extraction material has an average diameter of 20-40 µm.

[0032] In another preferred embodiment, the magnetic solid-phase extraction material has an average pore size of 60–100 Å; more preferably, it has an average pore size of 70–90 Å.

[0033] In another preferred embodiment, the mixing ratio of the magnetic solid-phase extraction material to the liquid sugarcane crude extract containing 3-NPA is 5-25 mg: 0.6 mL, more preferably 8-25 mg: 0.6 mL, and even more preferably 10 ± 2 mg: 0.6 mL.

[0034] In another preferred embodiment, the eluent used in step (S3) is selected from the group consisting of acetonitrile, acetonitrile-0.5% ammonia solution, acetonitrile-1% ammonia solution, methanol, methanol-0.5% ammonia solution, or methanol-1% ammonia solution.

[0035] In another preferred embodiment, the eluent used in step (S3) is pure acetonitrile (approximately 100% v / v).

[0036] In another preferred embodiment, the eluent used in step (S3) is pure methanol (approximately 100% v / v).

[0037] In another preferred embodiment, the total volume of the eluent used in step (S3) is 0.5 to 3 mL, more preferably 0.8 to 2 mL, and even more preferably 1 ± 0.2 mL.

[0038] In another preferred embodiment, the sugarcane sample is a peeled and homogenized sugarcane stalk or sugarcane juice obtained by pressing sugarcane stalks after peeling.

[0039] In a second aspect of the invention, a reagent combination for detecting sugarcane neurotoxin 3-NPA is provided, the reagent combination comprising: (a) Magnetic solid-phase extraction materials; (b) Organic solvents; in, The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads (MCX), or N-propylethylenediamine magnetic beads (PSA), or combinations thereof; The organic solvent is acetonitrile.

[0040] In another preferred embodiment, the magnetic solid-phase extraction material has an extraction recovery rate of 90-110% for sugarcane neurotoxin 3-NPA, more preferably 95-105%, and even more preferably 96-100%. In another preferred embodiment, the volume of the organic solvent is 2 to 10 mL, more preferably 5 ± 1 mL.

[0041] In a third aspect of the invention, a method for detecting neurotoxin 3-NPA in sugarcane samples is provided, the method comprising: (S1) Provides a liquid crude sugarcane extract containing 3-NPA; (S2) The liquid crude sugarcane extract from step (S1) is subjected to magnetic solid-phase extraction using a magnetic solid-phase extraction material to obtain a magnetic solid-phase extraction material adsorbed with 3-NPA (or a "magnetic solid-phase extraction material-3-NPA" complex). The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads (MCX), or N-propylethylenediamine magnetic beads (PSA), or combinations thereof; (S3) The “magnetic solid-phase extraction material-sugarcane neurotoxin 3-NPA” complex is eluted with an eluent (or eluent) to elute sugarcane neurotoxin 3-NPA, thereby obtaining an eluent enriched with sugarcane neurotoxin 3-NPA; and (S4) Quantitatively determine the content of sugarcane neurotoxin 3-NPA in the eluent enriched with sugarcane neurotoxin 3-NPA.

[0042] In another preferred embodiment, the magnetic solid-phase extraction material adsorbed with 3-NPA in step (S2) is a magnetic solid-phase extraction material adsorbed with 3-NPA that has undergone washing treatment.

[0043] In another preferred embodiment, the quantitative detection method in step (S4) is selected from the group consisting of: mass spectrometry, gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or combinations thereof.

[0044] In another preferred embodiment, the quantitative detection method in step (S4) is HPLC-MS / MS or GC-APCI-MS / MS.

[0045] In another preferred embodiment, the detection limit of the detection method is ≤0.05 μg / kg, such as 0.0001 to 0.05 μg / kg, more preferably 0.001 to 0.05 μg / kg, and even more preferably 0.01 to 0.05 μg / kg.

[0046] In another preferred embodiment, the limit of quantitation of the detection method is ≤0.5 μg / kg, such as 0.0001 to 0.3 μg / kg, more preferably 0.001 to 0.2 μg / kg, and even more preferably 0.01 to 0.1 μg / kg.

[0047] In a fourth aspect of the invention, a use of a magnetic solid-phase extraction material is provided for preparing a material for enriching neurotoxin 3-NPA in sugarcane samples; The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads (MCX), or N-propylethylenediamine magnetic beads (PSA), or combinations thereof.

[0048] In another preferred embodiment, the extraction recovery rate of the magnetic solid-phase extraction material is 90-110%, more preferably 95-105%, and even more preferably 96-100%.

[0049] In a fifth aspect of the invention, there is provided the use of a reagent combination as described in the second aspect of the invention for preparing a kit for enriching neurotoxin 3-NPA in sugarcane samples. The kit includes: (a) A first container, and a magnetic solid-phase extraction material located in the first container; (b) the second container, and the organic solvent located in the second container; in, The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads (MCX), or N-propylethylenediamine magnetic beads (PSA), or combinations thereof; The organic solvent is acetonitrile.

[0050] In another preferred embodiment, the kit further includes: Optional sugarcane neurotoxin 3-NPA standard and instructions.

[0051] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0052] Figure 1 This paper illustrates the effect of different types of magnetic solid-phase extractants on the adsorption efficiency and recovery rate (ER) of 3-nitropropionic acid (3-NPA) in one embodiment of the present invention.

[0053] Figure 2This paper illustrates the effect of different crude extraction conditions, such as extractant volume, vortex / ultrasonic treatment, ultrasonic time, or sodium chloride dosage, on the crude extraction recovery (ER) of 3-nitropropionic acid (3-NPA) in one embodiment of the invention.

[0054] Figure 3 This paper illustrates, in one embodiment of the invention, the effect of the type and amount of magnetic solid-phase extractant and the type of eluent on the recovery (ER) of 3-nitropropionic acid (3-NPA) in a complex matrix. Detailed Implementation

[0055] Through extensive and in-depth research and screening, the inventors unexpectedly discovered for the first time that a specific class of magnetic solid-phase extractants not only specifically adsorb sugarcane neurotoxin 3-nitropropionic acid but also possess excellent resistance to sugarcane matrix interference. This led to the development of a highly efficient and accurate quantitative detection method for sugarcane neurotoxin 3-nitropropionic acid (3-NPA) that is highly resistant to matrix interference and / or easy to operate. This invention was completed based on this discovery.

[0056] Specifically, in this invention, for liquid sugarcane crude extract containing 3-NPA, magnetic solid-phase extraction is first performed using a specific magnetic solid-phase extractant (i.e., MCX magnetic solid-phase extractant) to adsorb 3-NPA onto the surface of the magnetic solid-phase extraction material, forming a "magnetic solid-phase extraction material adsorbed with 3-NPA". After magnetic solid-liquid separation, the magnetic solid-phase extraction material adsorbed with 3-NPA is eluted with a specific eluent (such as acetonitrile) to elute 3-NPA from the magnetic solid-phase extraction material, thereby obtaining a sample that can be used for subsequent HPLC-MS / MS quantitative detection.

[0057] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0059] Solid phase extraction (SPE) and magnetic solid phase extraction (MSPE) As used herein, the terms “solid phase extraction”, “solid phase extraction method”, “SPE”, and “solid phase extraction” are used interchangeably to refer to a sample pretreatment technique based on liquid-solid chromatography theory. It is mainly used for the separation, purification, and concentration of complex matrix samples to improve the recovery rate of analytes and more effectively separate analytes from interfering impurities.

[0060] Solid-phase extraction has certain technical limitations for the extraction of 3-NPA from sugarcane samples. Although many studies have attempted to use solid-phase extraction columns for extraction, a suitable material for the extraction of 3-NPA from sugarcane samples has not yet been found.

[0061] As used in this article, the terms "magnetic solid-phase extraction," "MSPE," and "Magnetic solid-phase extraction" are used interchangeably. They refer to a method that uses magnetic solid-phase extraction materials for adsorption to achieve separation of target substances. Combined with an external magnetic field, it can achieve rapid solid-liquid separation and has advantages such as simple operation, short extraction time, and low consumption of organic solvents.

[0062] In this invention, magnetic solid-phase extraction is selected as an efficient extraction method for recovering 3-NPA from sugarcane samples or sugarcane juice.

[0063] The pretreatment method of the present invention As used herein, “pretreatment method of the present invention”, “pretreatment method of sugarcane sample for detection of sugarcane neurotoxin 3-nitropropionic acid (3-NPA) of the present invention” and “enrichment method of the present invention” are used interchangeably and refer to the pretreatment method as defined in the first aspect of the present invention.

[0064] Specifically, the pretreatment method of the present invention includes: (a) obtaining a crude extract containing 3-NPA from sugarcane by solvent extraction, and (b) efficiently and specifically extracting an eluent containing 3-NPA from the crude extract using the magnetic solid-phase extraction method of the present invention.

[0065] In this invention, the extraction can be performed once or multiple times, preferably once.

[0066] In addition, the crude extract obtained after extraction with the extractant can be further subjected to ultrasonication or vortexing, salting out, centrifugation, dilution, and nitrogen blowing to obtain a sugarcane sample concentrate with 3-NPA.

[0067] For solvent extraction, there are no particular restrictions on the extractant (or extraction solvent) used. Representative examples include (but are not limited to): acetonitrile, water, ethanol, methanol or a mixture of one or more of their aqueous solutions; Preferably, in this invention, acetonitrile is used as the extractant for solvent extraction.

[0068] For sugarcane, it is preferable to directly mix the sugarcane sample with acetonitrile for crude extraction. There are no particular restrictions on the mixing ratio of sugarcane and acetonitrile, but it is usually 1g sugarcane (wet weight): 1-5ml acetonitrile, and more preferably 1g sugarcane (wet weight): 1-3ml acetonitrile.

[0069] For magnetic solid-phase extraction, there are no particular limitations on the magnetic solid-phase extraction materials. Representative examples include (but are not limited to): one or a combination of several of the following: MAX magnetic beads, PSA magnetic beads, NH2 magnetic beads, HILIC magnetic beads, HLB magnetic beads, and MCX magnetic beads.

[0070] In this invention, for the magnetic solid phase extraction method, MCX magnetic solid phase extraction material or PSA magnetic solid phase extraction material is used for extraction, preferably MCX magnetic solid phase extraction material.

[0071] Specifically, the present invention first activates the magnetic solid-phase extraction material, then mixes it with the liquid crude sugarcane extract (or its sample concentrate) of the present invention and incubates it, then performs magnetic solid-liquid separation, eluent is used for elution, and magnetic solid-liquid separation is performed again to collect the eluent containing sugarcane neurotoxin 3-NPA.

[0072] In the magnetic solid-phase extraction process of the present invention, acetonitrile or methanol can be used as the eluent, preferably acetonitrile.

[0073] In a preferred embodiment of the present invention, the preferred crude extraction method in the pretreatment method of sugarcane neurotoxin 3-NPA is as follows: using sugarcane as raw material and acetonitrile as extraction liquid, the mixture is subjected to ultrasonic extraction and salting out to obtain a liquid crude sugarcane extract containing 3-NPA.

[0074] Compared to other extraction methods, the magnetic solid-phase extraction method of this invention enables high recovery of 3-NPA in sugarcane samples and exhibits excellent resistance to matrix interference in complex food matrices (such as sugarcane matrices), thereby obtaining an eluent with a higher content (or relative content) of sugarcane neurotoxin 3-NPA. At the same time, the operation process is simple, time-saving, and significantly simplifies the sample pretreatment process.

[0075] Preferably, the magnetic solid-phase extraction material has an extraction recovery rate of 90-110% for sugarcane neurotoxin 3-NPA, more preferably 95-105%, and even more preferably 96-100%.

[0076] At this point, the extraction recovery rate is the percentage of the actual amount of the target compound detected after sample treatment to the theoretical amount added, which is the apparent recovery rate.

[0077] The MSPE-HPLC-MS / MS detection method of the present invention The present invention also provides a method for quantitative detection of sugarcane neurotoxin 3-NPA, namely magnetic solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS).

[0078] Typically, the MSPE-HPLC-MS / MS of the present invention includes the following steps: (a) For liquid sugarcane crude extract containing 3-NPA, magnetic solid phase extraction is performed by a specific magnetic solid phase extractant (i.e., MCX magnetic solid phase extractant) to adsorb 3-NPA onto the surface of the magnetic solid phase extraction material, thereby forming a first mixture containing sugarcane crude extract and magnetic solid phase extraction material. (b) After solid-liquid separation by magnetic field, a magnetic solid-phase extraction material adsorbed with 3-NPA is obtained; (c) Optionally, the magnetic solid-phase extraction material adsorbed with 3-NPA is washed. (d) Elute the magnetic solid phase extraction material adsorbed with 3-NPA in step (b) or (c) with a specific eluent (such as acetonitrile) to remove 3-NPA from the magnetic solid phase extraction material and obtain a sample that can be used for subsequent HPLC-MS / MS detection. (e) The content of sugarcane neurotoxin 3-NPA in the sample was quantitatively detected by HPLC-MS / MS.

[0079] Compared with other existing analytical methods (such as GC-APCI-MS / MS), the MSPE-HPLC-MS / MS detection results of the present invention have significant advantages such as high stability, high accuracy and high sensitivity.

[0080] The main advantages of this invention include: (a) The sample pretreatment method of the present invention has high resistance to matrix interference and high recovery rate for 3-NPA in sugarcane samples or sugarcane juice.

[0081] (b) The sample pretreatment method of the present invention greatly reduces the amount of organic solvent used and the processing time. While ensuring that 3-NPA in sugarcane samples or sugarcane juice has high resistance to matrix interference and high recovery rate, it simplifies the operation steps, greatly improves the processing efficiency, and effectively saves costs.

[0082] (c) After extensive screening and experimentation, the inventors selected MCX as a magnetic solid-phase extraction material, which can achieve efficient enrichment in complex sample matrices (preferably sugarcane samples or sugarcane juice) and significantly improve the recovery rate of 3-NPA in complex matrices.

[0083] (d) After extensive screening and experimentation, the inventors selected acetonitrile as the eluent for magnetic solid-phase extraction. Compared with conventional eluents in the prior art (such as alkaline eluents), this method achieves efficient elution while improving the stability of the method.

[0084] (e) The present invention uses HPLC-MS / MS to quantitatively detect 3-NPA in batch sugarcane samples, which has low detection limit and high sensitivity, thereby achieving good accuracy and repeatability.

[0085] (f) The method of the present invention can not only extract 3-NPA from sugarcane samples to the maximum extent, but also achieve quantitative detection of the neurotoxin content in sugarcane and its products with only a small amount of sample (such as about 1 ml or less).

[0086] 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. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0087] Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0088] Materials and Methods 1. Experimental Materials MCX (hybrid strong cation exchange magnetic beads, approximately 20-40 µm; approximately 80 Å, polystyrene matrix, specific surface area 680 m²) 2 / g, Ajaliberty, product name: Bonnacats-MA MCX), PSA (magnetic beads with N-propylethylenediamine surface modified, approximately 20-40 µm; approximately 80 Å, specific surface area approximately 680 m²). 2 / g, Ajabel Corporation), MAX (mixed strong anion exchange magnetic beads, approximately 20-40 µm; approximately 80 Å, polystyrene matrix, specific surface area 680 m²), 2 / g, Ajabel Biosciences), HLB (hydrophilic-lipophilic balanced magnetic beads, approximately 20-40 µm; approximately 80 Å, polystyrene matrix, specific surface area 680 m²), 2 / g, Ajaliberty Corporation). Amino magnetic beads (magnetic beads with amino (-NH2) functional groups on the surface, approximately 20-40 µm; approximately 80 Å, specific surface area approximately 680 m²). 2 / g, Ajaliberty Corporation). HILIC magnetic beads (magnetic beads with surfaces typically modified with hydrophilic groups such as amides, glycols, and zwitterions, silica matrix, specific surface area 320 m²). 2 / g, approximately 20-40 µm; approximately 100 Å, specific surface area approximately 680 m² 2 / g, Ajaliberty Inc.

[0089] 2. Instrument conditions 2.3.1 Chromatographic conditions: Analysis was performed using an ACQUITY UPLC-Xevo TQ-XS ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer (HPLC-MS / MS, Waters, USA). Sample separation was performed using a BEH C18 column (10 mm × 2.1 mm, 1.7 µm). The column temperature was 40 °C. The mobile phase consisted of 5 mM ammonium acetate solution and methanol. The gradient elution program was: 0–3 min, 5% methanol; 3–4 min, 40% methanol; 4–5 min, 5% acetonitrile. The mobile phase flow rate was 0.3 mL min⁻¹. The injection volume was 5 μL.

[0090] 2.3.2 Mass spectrometry conditions: The ion source was an electrospray ionization source in negative ion mode (ESI-), with a capillary voltage of 0.5 kV; ion source temperature: 120 ℃; desolvation gas temperature: 450 ℃; desolvation gas flow rate: 1000 L h⁻¹; collision gas was argon, with a flow rate of 0.15 mL min⁻¹; cone voltage: 20 V; detection mode was multiple reaction monitoring (MRM): precursor ion m / z 117.9, characteristic ion m / z 46.1.

[0091] Example 1: Screening of Magnetic Solid Phase Extraction Materials 1) Experimental methods (1) Screening of magnetic solid phase extraction materials: In this embodiment, six magnetic solid phase extraction materials with different properties were selected.

[0092] Functionalized magnetic solid-phase extraction (SPE) materials include: MAX (mixed strong anion exchange magnetic beads), PSA (N-propylethylenediamine magnetic beads), NH2 (amino magnetic beads), HILIC (hydrophilic interaction chromatographic magnetic beads), HLB (hydrophilic-lipophilic balanced magnetic beads), and MCX (mixed strong cation exchange magnetic beads). The effects of different magnetic SPE materials on the adsorption and elution performance of 3-NPA in aqueous solution were investigated.

[0093] (2) MSPE purification process: Weigh 20 mg of candidate magnetic solid phase extraction material, first use 1 mL of methanol, then use 1 mL of 2% formic acid aqueous solution (used when MCX is selected as magnetic solid phase extraction material) or water (used when other types of magnetic solid phase extraction materials are selected) and vortex for 1 min to activate it, and then discard the supernatant.

[0094] Add 1 mL of 3-NPA aqueous solution containing 100 μg / L to each solution, vortex for 1 min, and after magnetic solid-liquid separation, discard the supernatant. Then add 1 mL of 1% ammonia-methanol eluent to each solution, vortex for 1 min, and after magnetic solid-liquid separation, filter the supernatant through a 0.22 μm nylon filter membrane for analysis.

[0095] (3) Recovery rate determination: The recovery rate of 3-NPA in the above 3-NPA aqueous solution was determined by HPLC-MS / MS.

[0096] 2) Experimental Results The effects of different magnetic solid-phase extraction materials on the adsorption and elution performance of 3-NPA in aqueous solution, such as... Figure 1 As shown in Table 1.

[0097] Table 1. The results showed that, compared with other types of magnetic solid-phase extraction materials, the adsorption efficiency of 3-NPA was significantly improved when using PSA magnetic beads, MCX magnetic beads, or MAX magnetic beads as functionalized magnetic solid-phase extraction materials. Furthermore, since MAX magnetic beads are mixed-type strong anion exchange materials, they achieved the highest adsorption efficiency of 3-NPA through electrostatic adsorption, reaching 99.9%. However, due to the strong adsorption force of MAX magnetic beads on 3-NPA, the eluent in this method could not elute it, resulting in a 3-NPA recovery rate of only 0.03%, which was almost impossible to elute.

[0098] The above results indicate that MCX magnetic beads and PSA magnetic beads are preferred magnetic solid-phase extraction materials for recovering 3-NPA from aqueous solutions.

[0099] Example 2: Effect of optimized extraction conditions on the recovery rate of 3-NPA 2.1 Effect of Acetonitrile Addition on 3-NPA Recovery 1) Experimental methods (1) Sample preparation: Peel the sugarcane, cut it into 3cm sections, grind it with a homogenizer, and store it at -18℃ for a long time. Alternatively, squeeze the ground sugarcane to obtain sugarcane juice samples and store them at -18℃ for a long time.

[0100] In this embodiment, ground sugarcane is used as the sample to be tested.

[0101] (2) Crude extraction: Weigh 2 g of sugarcane sample (wet weight) into each 15 mL centrifuge tube, add different volumes of acetonitrile (100% v / v), and extract each tube by sonication at 25℃ for 20 min. Then add 2 g of NaCl, vortex at 2000 rpm for 1.5 min, centrifuge at 10000 r / min for 5 min, and take 1 mL of supernatant (i.e. sample extract) for instrument testing.

[0102] The groups are as follows: Treatment group 2.1.1: 2 mL acetonitrile, Treatment group 2.1.2: 3 mL acetonitrile, Treatment group 2.1.3: 4 mL acetonitrile, Treatment group 2.1.4: 6 mL acetonitrile, Treatment group 2.1.5: 8 mL acetonitrile.

[0103] (3) Recovery rate determination: The recovery rate of 3-NPA was determined by HPLC-MS / MS for the sample extracts obtained under different extraction conditions.

[0104] 2) Experimental Results The results are as follows Figure 2 As shown in Figure A, the 3-NPA recovery rate of treatment group 2.1.3 was unexpectedly significantly improved compared with other treatment groups, increasing from 35.7% to approximately 67.5%. Furthermore, the relative recovery rate of 3-NPA in treatment group 2.1.3 was approximately 1.89 compared with treatment group 2.1.1. This indicates that, under the condition that other treatment conditions remain unchanged, the optimal recovery efficiency for 3-NPA in sugarcane samples is achieved when the amount of acetonitrile added is 4 mL. Therefore, 4 mL of acetonitrile is the optimal amount to be added when extracting 3-NPA from sugarcane samples.

[0105] 2.2 Detection of the effect of different extraction techniques on the recovery rate of 3-NPA 1) Experimental methods (1) Sample preparation: Samples were prepared using the same experimental methods as in Example 2.1.

[0106] (2) Crude extraction: Weigh 2 g of sugarcane sample (wet weight) into each 15 mL centrifuge tube. Group the samples according to different volumes of acetonitrile and different extraction techniques. After processing each group, add 2 g of NaCl, vortex at 2000 rpm for 1.5 min, centrifuge at 10000 r / min for 5 min, and take 1 mL of supernatant (i.e. sample extract) for instrument testing. The groups are as follows: Treatment group 2.2.1: Extracted by vortexing with 2 mL of acetonitrile at 25°C for 20 min. Treatment group 2.2.2: Extracted by ultrasonication with 2 mL acetonitrile at 25°C for 20 min. Treatment group 2.2.3: Extracted by vortexing with 4 mL of acetonitrile at 25°C for 20 min. Treatment group 2.2.4: Extracted by ultrasonication with 4 mL of acetonitrile at 25°C for 20 min.

[0107] (3) Recovery rate determination: The recovery rate of 3-NPA was determined by HPLC-MS / MS for the sample extracts obtained under different extraction conditions.

[0108] 2) Experimental Results The results are as follows Figure 2 As shown in B, when the amount of acetonitrile added was 2 mL, the recovery rate of 3-NPA in treatment group 2.2.2 was unexpectedly significantly improved compared with treatment group 2.2.1, increasing from 20.7% to about 35.7%, and the relative recovery rate of 3-NPA in treatment group 2.2.2 was about 1.73.

[0109] When the amount of acetonitrile added was 4 mL, the recovery rate of 3-NPA in treatment group 2.2.4 was unexpectedly significantly improved compared with treatment group 2.2.3, increasing from 46.0% to 67.5%, and the relative recovery rate of 3-NPA in treatment group 2.2.4 was approximately 1.47.

[0110] This indicates that, under the condition that other treatment conditions remain unchanged, when the amount of acetonitrile added is 4 mL and ultrasonic extraction is used, there is a high recovery efficiency for 3-NPA in sugarcane samples. Therefore, adding 4 mL of acetonitrile and using ultrasonic extraction is the optimal extraction method for extracting 3-NPA from sugarcane samples.

[0111] 2.3 Effect of sonication time on 3-NPA recovery 1) Experimental methods (1) Sample preparation: Samples were prepared using the same experimental methods as in Example 2.1.

[0112] (2) Crude extraction: Weigh 2 g of sugarcane sample (wet weight) into each 15 ml centrifuge tube, add 4 mL of acetonitrile to each group, and perform isothermal ultrasonic extraction at 25℃ for different extraction times. The groups are as follows: Treatment group 2.3.1: Ultrasound for 5 min; Treatment group 2.3.2: Ultrasound for 10 min; Treatment group 2.3.3: Ultrasound for 15 min; Treatment group 2.3.4: Ultrasound for 20 min; Treatment group 2.3.5: Ultrasound for 25 min; Subsequently, 2 g NaCl was added to each group, vortexed at 2000 rpm for 1.5 min, centrifuged at 10000 r / min for 5 min, and 1 mL of supernatant (i.e. sample extract) was taken for instrumental testing.

[0113] (3) Recovery rate determination: The recovery rate of 3-NPA was determined by HPLC-MS / MS for the sample extracts obtained under different extraction conditions.

[0114] 2) Experimental Results The results are as follows Figure 2 As shown in C, there was no significant difference in the recovery rate of 3-NPA among the groups within the ultrasonic time of 5-25 min. This indicates that the ultrasonic cavitation effect can quickly eliminate mass transfer resistance, and the distribution balance of 3-NPA in the two phases can be achieved in just 5 min. Extending the ultrasonic time not only fails to improve the recovery rate, but may also increase the risk of co-extraction of matrix impurities, leading to matrix inhibition effect.

[0115] Therefore, 5 minutes is selected as the optimized ultrasound time in this disclosure.

[0116] 2.4 Effect of NaCl addition on 3-NPA recovery rate Because sugarcane or sugarcane juice has a high water content and is rich in soluble sugars, acetonitrile is easily miscible with water and does not separate into layers. This not only reduces the extraction efficiency of 3-NPA, but also leads to turbidity of the extract and a large amount of co-soluble impurities.

[0117] Therefore, in this embodiment, by exploring the effect of different NaCl addition amounts on salting out, the efficient separation effect of the organic phase and aqueous phase can be optimized, and the ionic strength of the system can be adjusted to improve the distribution of the target substance in the two phases, thereby synergistically improving the extraction efficiency through a dual-action mechanism.

[0118] 1) Experimental methods (1) Sample preparation: Samples were prepared using the same experimental methods as in Example 2.1.

[0119] (2) Crude extraction: Weigh 2 g of sugarcane sample (wet weight) into each 15 ml centrifuge tube, add 4 mL of acetonitrile to each group, and perform isothermal ultrasonic extraction at 25℃ for 5 min. Then, the groups are divided according to the amount of NaCl added. After vortexing at 2000 rpm for 1.5 min, centrifuge at 10000 r / min for 5 min, and take 1 mL of supernatant (i.e. sample extract) for instrument testing. The groups are as follows: Treatment group 2.4.1: The amount of NaCl added was 0.5g. Treatment group 2.4.2: The amount of NaCl added was 1g. Treatment group 2.4.3: The amount of NaCl added was 2g. Treatment group 2.4.4: The amount of NaCl added was 3g. Treatment group 2.4.5: The amount of NaCl added is 5g.

[0120] (3) Recovery rate determination: The recovery rate of 3-NPA was determined by HPLC-MS / MS for the sample extracts obtained under different extraction conditions.

[0121] 2) Experimental Results The results are as follows Figure 2 As shown in Figure D, compared with other treatment groups, the 3-NPA recovery rate of treatment group 2.4.2 was significantly improved, increasing from 63.6% to 75.1%. Furthermore, compared with treatment group 2.4.1, the relative recovery rate of 3-NPA in treatment group 2.4.2 was 1.18. This indicates that, under the condition that other treatment conditions remain unchanged, when the amount of NaCl added is 1g, it has a high recovery efficiency for 3-NPA in sugarcane samples.

[0122] However, with further increases in the amount of NaCl added, the salting-out effect tends to saturate, and the two-phase distribution equilibrium of 3-NPA in the sugarcane sample stabilizes, with the recovery rate remaining essentially stable. Therefore, 1g of NaCl is the optimal amount of NaCl to be added for recovering 3-NPA from the sugarcane sample.

[0123] The above results indicate that the optimized extraction conditions for sugarcane 3-NPA are: acetonitrile volume 4 mL, ultrasonic extraction, ultrasonic time 5 min, and NaCl addition amount 1 g.

[0124] Example 3: MSPE Condition Optimization Based on Complex Matrix 3.1 Screening of Magnetic Solid Phase Extraction Materials Based on Complex Matrices To investigate the extraction performance of magnetic solid phase extraction materials in complex matrices, based on the screening results of Example 1, MCX magnetic beads and PSA magnetic beads were selected as candidate magnetic solid phase extraction materials for MSPE condition optimization in this example, and their extraction effect in sugarcane juice extract was quantitatively detected.

[0125] 1) Experimental methods MSPE purification was performed using the same method as in Example 1, except that: 3-NPA aqueous solution (1 mL containing 100 μg / L 3-NPA) or sample concentrate was added respectively; 20 mg of candidate magnetic solid phase extraction materials, MCX magnetic beads and PSA magnetic beads, were weighed for the experiment; in, Preparation of sample concentrate: Accurately pipette 2 mL of the sample extract after centrifugation of the treatment group in 2.4.2 into a 15 mL centrifuge tube, add 1 mL of pure water, and blow with nitrogen at 40℃ to about 0.6 mL to obtain sample concentrate (1 mL contains about 20 μg / L of 3-NPA).

[0126] Recovery rate determination: The recovery rate of 3-NPA in the eluent obtained under the above conditions was determined by HPLC-MS / MS.

[0127] 2) Experimental Results like Figure 3 As shown in Figure A, when PSA magnetic beads are used as the magnetic solid-phase extraction material, the recovery rate of 3-NPA in the sugarcane matrix extract is significantly reduced compared with that of the aqueous matrix, decreasing from 62.1% to 9.0%.

[0128] However, compared with PSA magnetic beads, MCX magnetic beads can unexpectedly significantly improve the magnetic solid-phase extraction efficiency of 3-NPA in complex sugarcane matrices, from 9.0% to 71.6%, which is comparable to its magnetic solid-phase extraction efficiency of 3-NPA in aqueous matrices (with a recovery rate of approximately 70.5%), and even more significantly improve the recovery rate of 3-NPA in complex matrices.

[0129] This indicates that MCX magnetic beads, as a magnetic solid-phase extraction material, are almost unaffected by matrix interference. Compared with PSA magnetic beads, MCX magnetic beads have higher selectivity and stronger resistance to matrix interference.

[0130] 3.2 Evaluation of the effect of magnetic solid-phase extraction material dosage on recovery rate 1) Experimental methods MSPE purification was performed using the same method as in Example 3.1, except that different amounts (5 mg, 10 mg, 15 mg, 20 mg, 25 mg) of MCX or PSA magnetic beads were weighed to replace the 20 mg candidate magnetic solid phase extraction material in Example 3.1.

[0131] Recovery rate determination: The recovery rate of 3-NPA in the eluent obtained under the above conditions was determined by HPLC-MS / MS.

[0132] 2) Experimental Results like Figure 3 B and Figure 3 As shown in C, when PSA magnetic beads are used as magnetic solid phase extraction material, although the dosage increases from 5 mg to 25 mg, the 3-NPA recovery rate of sugarcane matrix only fluctuates between 8.7% and 9.9%, without a significant improvement effect. This indicates that when PSA magnetic beads are used as magnetic solid phase extraction material, complex matrix interference affects the extraction performance.

[0133] However, unexpectedly, when MCX magnetic beads were used as the magnetic solid-phase extraction material, the recovery rate of 3-NPA from the sugarcane matrix increased significantly from 52.9% to 72.1% as the amount of MCX magnetic beads increased from 5 mg to 10 mg. When the amount of MCX magnetic beads was further increased to 25 mg, the recovery rate of 3-NPA from the sugarcane matrix tended to stabilize.

[0134] In summary, this invention selects 10mg as the optimal dosage of MCX magnetic beads.

[0135] 3.3 Evaluation of the effect of eluent type on recovery rate 1) Experimental methods MSPE purification was performed using the same method as in Example 3.1, except that the eluent in Example 3.1 was replaced with 1 mL acetonitrile, 1 mL methanol, 1 mL acetonitrile / methanol containing 0.5% ammonia, and 1 mL acetonitrile / methanol containing 1% ammonia, respectively.

[0136] Recovery rate determination: The recovery rate of 3-NPA in the eluent obtained under the above conditions was determined by HPLC-MS / MS.

[0137] 2) Experimental Results like Figure 3 As shown in D, compared with other treatment groups, the recovery rate of 3-NPA in sugarcane was significantly improved when acetonitrile or methanol was used as the eluent alone, increasing from 65.8% to 97.6%. In addition, compared with methanol as the eluent alone, the recovery rate of 3-NPA in sugarcane was significantly improved when acetonitrile was used as the eluent alone. Specifically, the recovery rate of 3-NPA in sugarcane was 97.6% when acetonitrile was used as the eluent alone, and 87.6% when methanol was used as the eluent alone.

[0138] However, unexpectedly, when 0.5% or 1% ammonia was added to acetonitrile or methanol, respectively, the recovery rate of 3-NPA in sugarcane decreased significantly. This indicates that acetonitrile or methanol has a highly efficient elution effect on 3-NPA of MCX magnetic beads, and the elution efficiency of acetonitrile on 3-NPA of MCX magnetic beads is more significantly improved than that of methanol.

[0139] Example 4: Validation of the MCX-MSPE-HPLC-MS / MS method and its application in sugarcane samples 4.1 Validation of the MCX-MSPE-HPLC-MS / MS method 1) Experimental methods (1) Samples were prepared using the same experimental method as in Example 2.1.

[0140] (2) Crude extraction: Weigh 2 g (wet weight) of sugarcane sample (accurate to 0.01 g) into a 15 mL centrifuge tube, add 4 mL of acetonitrile, and extract by sonication at 25 °C for 5 min. Then add 1 g of NaCl, vortex at 2000 rpm for 1.5 min, centrifuge at 10000 r / min for 5 min, accurately pipette 2 mL of supernatant (i.e. sample extract) into a 15 mL centrifuge tube, add 1 mL of pure water, and blow with nitrogen at 40 °C to about 0.6 mL to obtain the sample concentrate.

[0141] (3) MSPE purification process: 10 mg of MCX magnetic solid-phase extraction material was activated by vortexing 1 mL of methanol and 1 mL of 2% formic acid aqueous solution for 1 min, and then the supernatant was discarded. The above sample concentrate was added, vortexed for 1 min, and after magnetic solid-liquid separation, the supernatant was discarded. Then 1 mL of acetonitrile was added, vortexed for 1 min, and after magnetic solid-liquid separation, the supernatant was filtered through a 0.22 μm nylon filter membrane and analyzed.

[0142] (4) Recovery rate determination: The recovery rate of 3-NPA in the eluent obtained under the above conditions was determined by HPLC-MS / MS.

[0143] 2) Experimental Results The method validation results of the sugarcane samples obtained using the above method are shown in Table 2.

[0144] Table 2. Method validation results As shown in Table 2, the R of 3-NPA in sugarcane and sugarcane juice 2 All values ​​were 0.999, indicating that the method of the present invention is effective in the range of 0.5-50 μg / kg. -1 It exhibits a good linear relationship within its linear range; Furthermore, the limit of detection (LOD) for 3-NPA in sugarcane samples using the MCX-MSPE-HPLC-MS / MS method of this invention is 0.02-0.04 μg kg. -1 The limit of quantitation (LOQ) is 0.06-0.12 μg / kg. -1 The recoveries of 3-NPA at the three spiking levels ranged from 96.9% to 105.4%, with inter-day and intra-day RSDs ranging from 0.6% to 5.3% and 1.1% to 6.6%, respectively.

[0145] The above results demonstrate that the method of the present invention has good sensitivity, accuracy, and repeatability.

[0146] Therefore, the MSPE-HPLC-MS / MS method of the present invention can be used to accurately quantify 3-NPA in sugarcane and sugarcane juice.

[0147] Example 5: Comparison of Other Detection Methods In this embodiment, the method of the present invention is mainly compared with conventional methods disclosed in the prior art for performance indicators such as recovery rate, sensitivity, accuracy and repeatability of 3-NPA in sugarcane samples.

[0148] In this study, treatment groups 5.4 and 5.5 used the same experimental methods as in Example 4.1 to perform crude extraction, MSPE purification, and recovery rate determination on sugarcane samples (sugarcane juice or sugarcane) (setting conditions are shown in Table 3), thereby screening out a quantitative detection method for 3-NPA with high recovery rate, high accuracy, and high repeatability.

[0149] The comparative group consists of similar test samples (i.e., sugarcane or sugarcane juice samples) used in the prior art. The 3-NPA data of the test samples were obtained under different pretreatment conditions and different analytical methods (the settings are shown in Table 3) with reference to the conventional methods in the prior art.

[0150] The comparison results between the detection method of the present invention and the methods disclosed in the prior art are shown in Table 3.

[0151] Table 3. The results show that Compared with comparative examples 5.1-5.3, treatment groups 5.4 and 5.5 showed significant advantages in the following aspects: (1) The consumption of organic solvents and time efficiency are significantly optimized, which can save time and economic costs in the sample pretreatment process and improve the sample pretreatment efficiency; (2) The linear range for treatments 5.4 and 5.5 was 0.5–50 μg kg. -1 Compared with other comparative groups (e.g., 5.1, 10-100 μg kg), -1 The concentrations of 3-NPA (e.g., 3-NPA) decreased significantly, indicating that the method of this embodiment can still maintain a linear relationship at lower concentrations, and has better accuracy and reliability when detecting 3-NPA in low concentration sugarcane samples. (3) The recovery rates of treatment group 5.4 were between 96.9% and 105.4%, and those of treatment group 5.5 were between 97.4% and 100.2%. Compared with the control group (e.g., the recovery rate of 5.1 was 80.0%-96.8%), the recovery rates were significantly higher and close to 100%. This indicates that the method in this embodiment has high accuracy and can more realistically reflect the actual content of 3-NPA in sugarcane samples, reduce measurement errors, and improve the reliability of the detection results. (4) The LOD of treatment group 5.4 was 0.02 μg kg. -1 The LOD of treatment group 5.5 was 0.04 μg kg. -1 Compared with most comparative groups (such as 5.1), the LOD was 3.6 μg kg. -1 Compared to other methods, the detection limits of treatment groups 5.4 and 5.5 were significantly lower, indicating that the method of the present invention has higher sensitivity and can effectively detect and quantify 3-NPA even when the content is extremely low.

[0152] The above results indicate that when sugarcane juice or sugarcane is used as the sample, 6 ml of organic solvent is used, and the sample pretreatment time is about 50 min, setting up a magnetic solid phase extraction step and using MCX magnetic beads as the magnetic solid phase extraction material, and using HPLC-MS / MS for quantitative detection of 3-NPA in sugarcane samples, the detection operation is simple, effective and cost-saving. The MCX-MSPE purification method used is not easily affected by matrix interference, thus achieving efficient and accurate detection of 3-NPA.

[0153] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for pretreatment of sugarcane samples for detecting the sugarcane neurotoxin 3-nitropropionic acid, characterized in that, The method includes the following steps: (S1) Provides a liquid crude sugarcane extract containing 3-nitropropionic acid; (S2) The liquid crude sugarcane extract from step (S1) is subjected to magnetic solid-phase extraction using a magnetic solid-phase extraction material to obtain a "magnetic solid-phase extraction material-sugarcane neurotoxin 3-nitropropionic acid" complex. The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads, N-propylethylenediamine magnetic beads, or combinations thereof; and (S3) The "magnetic solid phase extraction material-sugarcane neurotoxin 3-nitropropionic acid" complex is eluted with an eluent to elute the sugarcane neurotoxin 3-nitropropionic acid, thereby obtaining an eluent enriched with sugarcane neurotoxin 3-nitropropionic acid. in, The eluent used in step (S3) is selected from the group consisting of: acetonitrile, acetonitrile-0.5% ammonia solution, acetonitrile-1% ammonia solution, methanol, methanol-0.5% ammonia solution, or methanol-1% ammonia solution; The total volume of the elution solution used in step (S3) is 0.5 to 3 mL.

2. The pretreatment method as described in claim 1, characterized in that, The magnetic solid-phase extraction material has an extraction recovery rate of 90-110% for the sugarcane neurotoxin 3-nitropropionic acid.

3. The pretreatment method as described in claim 1, characterized in that, The magnetic solid-phase extraction material has an extraction recovery rate of 95-105% for the sugarcane neurotoxin 3-nitropropionic acid.

4. The pretreatment method as described in claim 1, characterized in that, The magnetic solid-phase extraction material has an extraction recovery rate of 96-100% for the sugarcane neurotoxin 3-nitropropionic acid.

5. The pretreatment method as described in claim 1, characterized in that, The total volume of the elution solution used in step (S3) is 0.8 to 2 mL.

6. The pretreatment method as described in claim 1 or 5, characterized in that, The mixing ratio of the magnetic solid-phase extraction material to the liquid crude sugarcane extract containing 3-NPA is 5-25 mg: 0.6 mL.

7. The pretreatment method as described in claim 1 or 5, characterized in that, The mixing ratio of the magnetic solid-phase extraction material to the liquid crude sugarcane extract containing 3-NPA is 8-25 mg: 0.6 mL.

8. The pretreatment method as described in claim 1 or 5, characterized in that, The mixing ratio of the magnetic solid-phase extraction material to the liquid crude sugarcane extract containing 3-NPA is 10±2 mg: 0.6 mL.

9. A reagent combination for detecting the sugarcane neurotoxin 3-nitropropionic acid, characterized in that, The reagent combination includes: (a) Magnetic solid-phase extraction materials; (b) Organic solvents; in, The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads, or N-propylethylenediamine magnetic beads, or combinations thereof; The organic solvent is acetonitrile; The volume of the organic solvent is 2 to 10 mL.

10. A method for detecting the neurotoxin 3-nitropropionic acid in sugarcane samples, characterized in that, The method includes: (S1) Provides a liquid crude sugarcane extract containing 3-nitropropionic acid; (S2) The liquid crude sugarcane extract from step (S1) is subjected to magnetic solid-phase extraction using a magnetic solid-phase extraction material to obtain a "magnetic solid-phase extraction material-sugarcane neurotoxin 3-nitropropionic acid" complex. The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads, N-propylethylenediamine magnetic beads, or combinations thereof; (S3) The "magnetic solid phase extraction material-sugarcane neurotoxin 3-nitropropionic acid" complex is eluted with an eluent to elute the sugarcane neurotoxin 3-nitropropionic acid and obtain an eluent enriched with sugarcane neurotoxin 3-nitropropionic acid. in, The eluent used in step (S3) is selected from the group consisting of: acetonitrile, acetonitrile-0.5% ammonia solution, acetonitrile-1% ammonia solution, methanol, methanol-0.5% ammonia solution, or methanol-1% ammonia solution; The total volume of the eluent used in step (S3) is 0.5–3 mL; and (S4) Quantitatively detect the content of sugarcane neurotoxin 3-nitropropionic acid in the eluent enriched with sugarcane neurotoxin 3-nitropropionic acid.

11. The detection method as described in claim 10, characterized in that, The detection limit of the detection method is ≤0.05μg / kg.

12. The detection method as described in claim 10, characterized in that, The detection limit of the detection method is 0.0001 to 0.05 μg / kg.

13. The detection method as described in claim 10, characterized in that, The detection limit of the detection method is 0.001 to 0.05 μg / kg.

14. The detection method as described in claim 10, characterized in that, The detection limit of the detection method is 0.01–0.05 μg / kg.

15. The detection method as described in claim 10, characterized in that, The limit of quantitation for the detection method is ≤0.5μg / kg.

16. The detection method as described in claim 10, characterized in that, The limit of quantitation for the detection method is 0.0001–0.3 μg / kg.

17. The detection method as described in claim 10, characterized in that, The limit of quantitation for the detection method is 0.001–0.2 μg / kg.

18. The detection method as described in claim 10, characterized in that, The limit of quantitation for the detection method is 0.01–0.1 μg / kg.

19. Use of the magnetic solid-phase extraction material in the reagent combination as described in claim 9, characterized in that, Materials used to prepare materials for enriching the neurotoxin 3-nitropropionic acid in sugarcane samples; The reagent combination includes: (a) Magnetic solid-phase extraction materials; (b) Organic solvents; The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads, N-propylethylenediamine magnetic beads, or combinations thereof.

20. Use of the reagent combination as described in claim 9, characterized in that, The reagent combination is used to prepare a kit for enriching the neurotoxin 3-nitropropionic acid in sugarcane samples; The kit includes: (a) A first container, and a magnetic solid-phase extraction material located in the first container; (b) the second container, and the organic solvent located in the second container; The reagent combination includes: (a) Magnetic solid-phase extraction materials; (b) Organic solvents; in, The magnetic solid-phase extraction material is selected from the group consisting of: mixed strong cation exchange magnetic beads, or N-propylethylenediamine magnetic beads, or combinations thereof; The organic solvent is acetonitrile; The volume of the organic solvent is 2 to 10 mL.

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