A method for extracting and detecting matrine drugs in tea leaves

Abstract

The application belongs to the technical field of analysis and detection, and particularly relates to a method for extracting and detecting matrine alkaloids in tea. First, magnetic nanomaterial (Fe3O4-PDA) modified with polydopamine on the surface is prepared as an adsorbent, then the material is mixed with a tea sample to be detected extracted by an acidic aqueous solution, the magnetic nanomaterial selectively adsorbs matrine alkaloids in the sample under neutral and slightly acidic conditions with pH 6.5, then the magnetic material is quickly separated by an external magnetic field, and impurities such as uncombined pigments and polyphenols are removed; after alkaline methanol solution is used to elute matrine alkaloid targets combined with the material, the eluate is filtered. Based on the electrostatic interaction and pi-pi conjugation between polydopamine and matrine alkaloids, the application realizes rapid and selective extraction, enrichment and analysis of matrine alkaloids from a complex matrix of tea, and has the advantages of simple operation, short time consumption, high sensitivity, batch preparation of the material and the like.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for extracting and detecting matrine-like drugs in tea. Background Technology

[0002] Matrine-based pesticides, as natural plant-derived biological pesticides, mainly include matrine (MA) and oxymatrine (OMA), and are characterized by low toxicity, easy degradation, and environmental friendliness. They are widely used for pest and disease control in economic crops such as tea and fruits and vegetables. my country has established temporary limits for matrine residues in some fruits and vegetables, while the European Union explicitly lists it as an unauthorized pesticide, stipulating that the residue limit in tea must not exceed 0.01 mg·kg⁻¹, placing extremely high demands on detection sensitivity.

[0003] Currently, the pretreatment methods for detecting matrine-like substances in tea mainly employ liquid-liquid extraction, solid-phase extraction (SPE), and QuEChERS, while instrumental detection primarily uses high-performance liquid chromatography (HPLC) or liquid chromatography-tandem mass spectrometry (LC-MS / MS). Existing technologies have significant shortcomings: First, the pretreatment process suffers from low selectivity and poor purification. The tea matrix is ​​rich in interfering substances such as pigments, polyphenols, and polysaccharides, leading to matrix effects that interfere with subsequent detection. Conventional pretreatment purification methods are ineffective, severely reducing detection sensitivity and accuracy. Second, traditional solid-phase extraction is cumbersome, time-consuming, and requires large amounts of organic solvents. It also has limited adsorbent selectivity and low sensitivity due to insufficient enrichment efficiency of the target analyte.

[0004] Therefore, developing a highly selective, effective, rapid, simple, and sensitive method for the extraction and detection of matrine-like drugs in tea is of great practical significance for ensuring the quality and safety of tea and meeting export testing requirements. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies of the existing technology and provide a method for the extraction and detection of matrine-like drugs in tea. This invention first prepares polydopamine-modified magnetic nanomaterials (Fe3O4-PDA) as a magnetic solid-phase extraction adsorbent. After activation with methanol, the adsorbent is mixed with tea extract adjusted to a suitable pH. Under mild conditions, matrine-like drugs in the sample are selectively adsorbed through electrostatic and π-π conjugation interactions. Subsequently, the magnetic material is rapidly separated by an external magnetic field to remove unbound impurities such as pigments and polyphenols. The target analytes are eluted with an alkaline methanol solution. After filtration through a filter membrane, the eluent is qualitatively and quantitatively detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS), thus achieving efficient extraction and accurate detection of matrine-like drugs in tea.

[0006] This invention utilizes the specific adsorption of matrine-like drugs by Fe3O4-PDA material, which can efficiently eliminate interference from the complex matrix of tea, significantly reduce the matrix effect, and achieve rapid enrichment and highly selective extraction of trace matrine-like drugs. Simultaneously, the magnetic separation operation is simple and rapid, eliminating the need for centrifugation and column chromatography, greatly shortening the pretreatment time. This invention achieves simultaneous extraction, efficient purification, and sensitive detection of matrine-like drugs in tea, offering advantages such as good purification effect, simple operation, rapid and efficient operation, high sensitivity, and low organic solvent consumption, meeting the domestic and international requirements for the limit detection of matrine-like pesticide residues in tea.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A method for extracting and detecting matrine-like drugs in tea, comprising the following steps:

[0009] (1) Fe3O4 magnetic nanospheres were obtained by purchasing or synthesizing them in-house. Fe3O4 magnetic nanospheres were dispersed in a mixture of anhydrous ethanol and Tris buffer solution, and dopamine hydrochloride aqueous solution was added. The mixture was stirred at room temperature to carry out a self-polymerization reaction. After magnetic separation, washing with water and ethanol alternately, and vacuum drying, Fe3O4-PDA material, i.e., magnetic nanomaterials coated with polydopamine, was obtained.

[0010] (2) Grind the tea sample into powder, add an aqueous solution of acetic acid with pH 3.5, vortex mix, then perform ultrasonic extraction, centrifuge, take the supernatant and adjust the pH value to obtain the tea extract to be tested;

[0011] (3) Activate the Fe3O4-PDA material obtained in step (1) with methanol, vortex and magnetically separate to discard the supernatant, add the tea extract obtained in step (2) to be tested, vortex and oscillate to make matrine drugs specifically bind to Fe3O4-PDA material; apply an external magnetic field to separate and remove the supernatant containing impurities, wash the material with deionized water to remove residual matrix interferences;

[0012] (4) Add alkaline methanol eluent to the washed material, vortex eluent to remove the target substance, magnetically separate the eluent, filter it through an organic phase filter membrane, and perform qualitative and quantitative detection by liquid chromatography-tandem mass spectrometry to complete the extraction and detection of matrine drugs in tea.

[0013] Further, in step (1), the concentration of the Tris buffer in the synthesis of Fe3O4-PDA magnetic nanomaterials is 5~15mM, the pH is 6.0~10.0, the reaction temperature is 5~30 ℃, the stirring speed is 100~500 rpm, and the reaction time is 6~20 h.

[0014] Further, in step (2), the ratio of tea powder to acetic acid aqueous solution is 2 g: 15 mL; the ultrasonic extraction time is 10~60 min; the centrifugation speed is 2000~12000 rpm and the centrifugation time is 1~10 min; the pH of the supernatant after adjustment is 5.0~7.0.

[0015] Further, in step (3), the amount of Fe3O4-PDA material used is 10~30 mg / mL of the sample solution to be tested; the activation solvent is methanol, and the activation vortex time is 0.5~5.0 min; the adsorption vortex rotation speed is 1000~3000 rpm, and the adsorption time is 2~5 min; the washing solvent is deionized water, and the washing is performed twice. Each vortex lasts for 5~30 s.

[0016] Further, in step (4), the eluent is a methanol-ammonia mixture with a volume ratio of 85:15; the elution vortex speed is 1000~3000 rpm, the elution time is 0.5~5.0 min; and the filtration uses a 0.22 μm organic phase needle filter.

[0017] Further, in step (4), the liquid chromatography conditions are as follows: the chromatographic column is a C18 column; the mobile phase A is 10 mM ammonium acetate aqueous solution, the mobile phase B is methanol, A:B=65:35 (v:v); the flow rate is 0.4 mL / min; the column temperature is 20~60 ℃; and the injection volume is 2~10 μL.

[0018] Furthermore, in step (4), the applicable objects of this method include, but are not limited to, teas such as green tea, black tea, Pu'er tea, and oolong tea, and the analytes include, but are not limited to, matrine, oxymatrine, and other matrine-like drugs.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) High selectivity: This invention utilizes the electrostatic interaction and π-π conjugation between matrine-like drugs and polydopamine. Through the specific adsorption of matrine and oxymatrine in tea by Fe3O4-PDA magnetic nanomaterials, it can efficiently remove matrix interference substances such as pigments, polyphenols, and polysaccharides. This solves the drawbacks of poor purification effect and serious non-specific adsorption of traditional pretreatment methods, significantly reduces matrix effect, and improves the accuracy and reliability of target analyte detection.

[0021] (2) Simple and efficient operation: The magnetic solid phase extraction technology is adopted, combined with the rapid magnetic response characteristics of magnetic nanomaterials. The material and sample liquid can be separated instantaneously by an external magnetic field. There is no need for cumbersome steps such as centrifugation, column chromatography, and concentration. The pretreatment time is greatly shortened. The processing time for a single sample is only about 5 minutes. The operation is simple and the process is fast. It is easy to standardize and conduct high-throughput detection, which is suitable for the rapid detection needs of tea export.

[0022] (3) Green, economical and environmentally friendly: The Fe3O4-PDA material used has a mild synthesis process, high single-batch yield, can be prepared in batches and has good stability; the amount of organic solvent used is significantly less than liquid-liquid extraction, traditional solid-phase extraction and other methods, which is more green and low in toxicity; no expensive extraction columns and special equipment are required, the detection cost is low, and it is suitable for daily laboratory testing and large-scale sample screening.

[0023] (4) Excellent sensitivity and precision: The method efficiently enriches trace matrines by magnetic solid phase extraction and combines them with high-sensitivity detection by LC-MS / MS. The detection limit meets the strict limit requirement of 0.01 mg·kg⁻¹ in the European Union (for example, the detection limit of matrine and oxymatrine is as low as 1.88 μg·kg⁻¹). The method has good recovery and precision, and the data is stable and reliable, filling the gap in the lack of national standard sensitive detection method for matrine pesticide residues in tea.

[0024] (5) Wide applicability: It is applicable to various tea substrates such as green tea, black tea, oolong tea, and Pu'er tea. The pretreatment and detection conditions are mild and do not degrade or damage the target. It can simultaneously extract and quantitatively detect multiple matrine drugs such as matrine and oxymatrine, and has broad application prospects. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the preparation process of the Fe3O4-PDA nanomaterials in Example 1.

[0026] Figure 2 The images are TEM images of the materials at each stage of the preparation process in Example 1, including: (a1, a2) Fe3O4 magnetic spheres and (b1, b2) Fe3O4-PDA nanomaterials.

[0027] Figure 3 Infrared spectra of Fe3O4 magnetic spheres and Fe3O4-PDA nanomaterials in Example 1

[0028] Figure 4 The hysteresis curves are those of the Fe3O4 magnetic spheres and Fe3O4-PDA nanomaterials in Example 1.

[0029] Figure 5 The thermogravimetric curves of Fe3O4 magnetic spheres and Fe3O4-PDA nanomaterials in Example 1 are shown.

[0030] Figure 6 The representative LC-MS / MS spectrum of Example 1 Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0032] Unless otherwise specified, all raw materials used in this invention are commercially available products.

[0033] Magnetic solid-phase extraction (MSPE), as a novel sample pretreatment technique, combines the advantages of magnetic separation and solid-phase extraction. It is characterized by its simplicity, speed, efficiency, low organic solvent consumption, and environmental friendliness, and has shown promising application prospects in the field of food pesticide residue detection. Matrine is a polar alkaloid that can form stable electrostatic and π-π conjugation interactions with polydopamine. However, interfering substances such as pigments, polyphenols, and polysaccharides in the tea matrix are difficult to bind efficiently to polydopamine modification sites. This characteristic provides an important basis for achieving highly selective extraction and enrichment of matrine. Based on this, this invention discloses a rapid and sensitive analytical method for the efficient extraction and detection of matrine in tea. The core of this method is the preparation of surface-modified polydopamine magnetic nanomaterials (Fe3O4-PDA) as adsorbents. Utilizing the specific interaction between matrine-like drugs and polydopamine, and combining the advantages of magnetic solid-phase extraction and liquid chromatography-tandem mass spectrometry, the method achieves selective enrichment, purification, and precise quantification of the target analytes. The method mainly includes three core steps: the preparation of Fe3O4-PDA magnetic nanomaterials, the construction of a magnetic solid-phase extraction system, and the extraction and detection of matrine-like drugs in tea using this adsorbent. Details are as follows:

[0034] (I) Preparation of polydopamine-modified magnetic nano-adsorbent (Fe3O4-PDA)

[0035] The preparation of the magnetic nano-adsorbent sequentially includes the solvothermal synthesis of Fe3O4 magnetic nanospheres and in-situ coating with a polydopamine (PDA) layer, ultimately yielding a Fe3O4-PDA core-shell structured material, which serves as a specific enrichment and adsorption carrier for matrine (MA) and oxymatrine (OMA), as detailed below:

[0036] 1. Acquisition of magnetic nanomaterials: Purchase or prepare monodisperse Fe3O4 magnetic nanospheres using a solvothermal method. The types include, but are not limited to, pure Fe3O4 microspheres and composite nanospheres modified with Fe3O4 on the surface or inside. This material serves as the magnetic response core of the adsorbent and can achieve rapid solid-liquid separation within 1 minute by applying an external magnetic field, providing a basis for the simplicity of subsequent extraction operations.

[0037] 2. Coating the surface of magnetic nanomaterials with a polydopamine layer: Dry Fe3O4 magnetic nanospheres were dispersed in anhydrous ethanol, and Tris buffer was added to the system to polymerize dopamine hydrochloride on the material surface under alkaline conditions to form polydopamine (PDA). The PDA coating significantly enhances the surface activity and adsorption selectivity of the material. It specifically recognizes matrine alkaloids through electrostatic interactions and π-π conjugation, providing stable binding sites for the target analyte, while repelling matrix interferences such as pigments and polyphenols.

[0038] 3. Post-treatment and activation of Fe3O4-PDA material: The dried Fe3O4-PDA material was activated with methanol before use. The activation solution was then discarded after magnetic separation. This material possesses a clear core-shell structure, strong magnetic response, large specific surface area, and high adsorption capacity, enabling highly selective enrichment of trace amounts of matrine and oxymatrine in complex tea matrices.

[0039] (II) Selective extraction and purification of matrine-like drugs in tea

[0040] Using the Fe3O4-PDA magnetic nanomaterials prepared above, matrine and oxymatrine were selectively extracted and purified from tea sample extracts. The specific steps are as follows:

[0041] After activating the Fe3O4-PDA material with methanol, the adsorbent was weighed at a ratio of 10-30 mg of Fe3O4-PDA material per 1 mL of tea sample solution. An external magnetic field was applied, the activation supernatant was discarded, and the pH-adjusted tea extract solution was added. Vortexing was used to ensure the matrine-like drugs in the sample fully combined with the Fe3O4-PDA material. After vortexing, the sample supernatant containing impurities was removed by magnetic separation, and the material was washed with deionized water. A methanol-ammonia solution was added as the eluent to efficiently elute the target analyte from the material. An external magnetic field was applied to collect the eluent, which was then filtered through a 0.22 μm organic phase filter membrane. The resulting filtrate was analyzed by LC-MS / MS.

[0042] In this invention, the samples to be processed include, but are not limited to, various tea substrates such as green tea, black tea, oolong tea, and pu-erh tea, which can be widely applied to routine detection, export screening, and quality control of matrine pesticide residues in tea.

[0043] Example 1

[0044] Matrine and oxymatrine were extracted, purified, and detected from green tea samples using Fe3O4-PDA magnetic nanomaterials. In this example, Fe3O4-PDA is a polydopamine-modified core-shell magnetic nanomaterial, and its preparation process is illustrated in the schematic diagram below. Figure 1 As shown, the specific steps are as follows:

[0045] (a) Synthesis of polydopamine-modified magnetic nano-adsorbent (Fe3O4-PDA)

[0046] (1) Synthesis of Fe3O4 nanospheres by solvothermal method: 1.35 g of ferric chloride hexahydrate and 75 mL of ethylene glycol were added to a beaker and stirred until completely dissolved to obtain an orange transparent solution; then 3.60 g of anhydrous sodium acetate was added and stirred for another 30 minutes. The reaction solution was transferred to a 200 mL high-pressure reactor and reacted at 200 °C for 16 hours to obtain Fe3O4 magnetic beads. The product was collected using an external magnetic field and washed three times each with deionized water and anhydrous ethanol, and then dried under vacuum at 50 °C for 12 hours to obtain Fe3O4 nanospheres with uniform particle size and monodisperse structure.

[0047] (2) Synthesis of polydopamine-coated magnetic nanomaterials (Fe3O4-PDA): 200 mg of dried Fe3O4 magnetic beads were dispersed in 400 mL of anhydrous ethanol, and then 200 mL of 10 mM Tris buffer (pH=8.5) was added to the system. The mixture was sonicated for 30 minutes until uniformly dispersed. 300 mL of freshly prepared 2.7 mg / mL dopamine hydrochloride aqueous solution was slowly added dropwise under stirring. The entire reaction system was placed at room temperature (20℃) and reacted for 12 hours under mechanical stirring at 300 rpm. Fe3O4-PDA was collected by applying an external magnetic field and washed three times each with deionized water and anhydrous ethanol, and then vacuum dried at 50℃ for 12 hours to obtain a magnetic nano-adsorbent with a uniformly coated polydopamine surface.

[0048] (II) Pretreatment of tea samples and preparation of test solutions

[0049] (1) Tea sample extraction: Take commercially available green tea samples, grind them with a grinder and sieve them; accurately weigh 2.0 g of tea powder and place it in a 50 mL centrifuge tube, add 15 mL of acetic acid aqueous solution with pH=3.5, vortex for 30 seconds, and sonicate for 30 min; centrifuge at 8000 rpm for 5 minutes, take the supernatant, adjust the pH to 6.5 with dilute ammonia water, and use it as the tea extract solution to be tested.

[0050] (2) Preparation of blank matrix and spiked sample: Select green tea samples in which matrine and oxymatrine were not detected, and prepare blank extract according to the above method; accurately transfer 980 μL of blank matrix, add 10 μL of matrine / oxymatrine standard working solution of different concentrations and 10 μL of 1 μg / mL aminopyrine internal standard working solution respectively, mix well, and prepare a series of matrix-matched standard solutions and quality control samples.

[0051] (II) Characterization of the prepared Fe3O4 magnetic nanospheres and Fe3O4-PDA nanomaterials

[0052] The structural morphology of the products from each synthesis step was characterized using transmission electron microscopy (TEM). For sample preparation, small amounts of Fe3O4 magnetic nanospheres and Fe3O4-PDA nanomaterials were first dispersed in 1.5 mL of anhydrous ethanol and ultrasonicated to ensure uniform dispersion. After standing for a short time, a small amount of the supernatant was taken and dropped onto a copper grid to form a droplet. This droplet was then transferred to an infrared lamp for drying and covered with another copper grid. After sample preparation, the samples were placed in a TEM, and the morphology of each nanoparticle was observed and recorded at 200 kV. Figure 2 As shown, Fe3O4 microspheres are nearly spherical with a particle size of approximately 150 nm. After being coated with PDA, a uniform organic layer with a thickness of approximately 50 nm appears on their outer surface, confirming that the Fe3O4 microspheres are completely coated with PDA with a smooth surface and no exposed particles. In the TEM image of the Fe3O4@PDA material, the core-shell structure is clear, the polydopamine layer is continuous and uniform, the particles are well dispersed, and there is no obvious agglomeration, further demonstrating that the magnetic nanomaterial modification can meet the application requirements of magnetic solid-phase extraction.

[0053] The surface functional groups of the material were characterized using Fourier transform infrared spectroscopy (FT-IR), such as... Figure 3 As shown, Fe3O4 exhibits a characteristic Fe-O absorption peak near 580 cm⁻¹. After being coated with PDA, characteristic peaks of benzene ring C-C stretching vibration and CO stretching vibration appear at 1509 cm⁻¹ and 1284 cm⁻¹, respectively, which correspond to the polydopamine structure, confirming that a polydopamine layer was successfully loaded on the Fe3O4 surface.

[0054] The magnetic properties of the material were characterized using a vibrating sample magnetometer, and the hysteresis loop of the material was recorded at room temperature. The magnetic response properties of the material were characterized, and the hysteresis loop is shown below. Figure 4 As shown, all materials exhibit superparamagnetism at room temperature. The saturation magnetization of the Fe3O4 microspheres and the Fe3O4@PDA material was measured to be 70.6 emu / g and 46.77 emu / g, respectively. Although the saturation magnetization decreased with increasing coating thickness of the Fe3O4 microspheres, the final Fe3O4@PDA material still exhibited good magnetic response properties. This material could be uniformly dispersed in tea matrix extract and rapidly separated from the matrix solution within 10 seconds after the application of an external magnetic field.

[0055] The thermal stability of the material was characterized, and the thermogravimetric curve of the material is shown in the figure. Figure 5As shown, the Fe3O4 magnetic spheres lost only about 7.3% of their weight during temperature changes, and the weight loss process was relatively stable, indicating good thermal stability. In contrast, the Fe3O4-PDA nanomaterials experienced a total weight loss of approximately 52.7%, with significant weight loss occurring at around 680℃. This is because the polydopamine outer shell rapidly decomposes at this temperature, and the remaining magnetic spheres exhibit good thermal stability, allowing the thermogravimetric curve to return to a stable state. This further demonstrates the successful synthesis of the polydopamine layer on the surface of the magnetic spheres and indicates that the material maintains good thermal stability within its normal application temperature range.

[0056] (3) Methodological validation of the established and optimized methods for the detection of matrine and oxymatrine pesticide residues, in accordance with the requirements of the EU SANTE / 11312 / 2021 guidelines.

[0057] The extraction and detection performance of matrine and oxymatrine in tea matrix was analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). For sample preparation, appropriate amounts of blank tea matrix solution, matrine standard working solution, and spiked tea extract were taken and filtered through a 0.22 μm organic filter membrane to remove fine tea debris and insoluble impurities. After a short period of degassing, a small amount of the test solution was extracted and placed into a sample vial for automatic sample injection. The samples were then placed into the LC-MS / MS detection system after preparation.

[0058] The chromatography-mass spectrometry conditions are as follows:

[0059] (1) Liquid chromatography conditions

[0060] Chromatographic separation was performed using a Welch Xtimate C18 column (2.1 × 150 mm) from Yuexu Company. Mobile phase A: 10 mM ammonium acetate aqueous solution; mobile phase B: methanol; A:B = 65:35 (v:v); flow rate: 0.4 mL / min. -1 Column oven temperature: 40℃; injection volume: 5μL.

[0061] (2) Mass spectrometry conditions

[0062] Electrospray ionization (ESI) source, positive ion mode scanning; mass spectrometry scanning mode: multiple reaction monitoring (MRM); GS1: 50 psi; GS2: 50 psi; ion source temperature: 500℃; ion spray voltage: 5500V; other relevant mass spectrometry parameters are shown in Table 1.

[0063] Table 1 Mass Spectrometry Conditions

[0064]

[0065] The peak shape, resolution, and interference from impurities of the target analyte were analyzed under optimized chromatographic and mass spectrometric parameters, such as... Figure 6 As shown, the retention times of matrine, oxymatrine, and aminopyrine were 4.69, 1.61, and 5.63 min, respectively. Both matrine and oxymatrine exhibited independent chromatographic peaks with symmetrical shapes and uniform widths, indicating good separation of the two target alkaloids without tailing or overlap. In the optimized detection system, the target compounds showed stable peak positions and low baseline noise, confirming that the tea pretreatment process effectively removes matrix interference, enabling accurate separation and detection of the target compounds. This meets the application requirements for qualitative and quantitative analysis of matrine residues in tea.

[0066] The linearity, detection limit, and quantitation limit of the detection system were characterized by method validation experiments. A series of mixed standard working solutions of matrine and oxymatrine with gradient concentrations were prepared for instrument detection, and a standard curve of target concentration versus mass spectrometry response intensity was established. The linear fitting results are shown in Table 2.

[0067] Table 2 Linear Fitting Results

[0068]

[0069] Using the target compound concentration at a signal-to-noise ratio of 3 as the limit of detection, a limit of detection of 0.25 ng·mL⁻¹ was obtained. Matrine and oxymatrine showed good linearity in the range of 0.5–25 ng·mL⁻¹ (R²). 2 >0.999), and their detection limits are all 0.25 ng·mL. -1 Matrine and oxymatrine showed excellent linearity within the set concentration range, with correlation coefficients greater than 0.999, indicating a good linear correspondence between the concentration and response value of the two target alkaloids within the detection interval, enabling accurate quantitative detection. The limit of detection and limit of quantitation were determined using the signal-to-noise ratio method. The optimized detection system exhibited excellent sensitivity, accurately capturing trace matrine residues in the tea matrix. Even at extremely low concentrations, the target analytes showed stable mass spectrometry response signals, making it suitable for detecting trace pesticide residues in tea.

[0070] The reliability and repeatability of the detection method were verified by precision and accuracy tests. Spiked recovery tests at three concentration levels (high, low, and medium) were conducted to determine the recoveries and relative standard deviations of matrine and oxymatrine in tea samples, thus verifying the method's detection stability. The results are shown in Table 3. The experimental results indicate that the accuracy and precision of the method for both matrine and oxymatrine are within acceptable ranges, suggesting that the method's accuracy and precision are good and meet the requirements.

[0071] Table 3 Accuracy and Precision Results

[0072]

[0073] Based on the tea sample extraction process used in this method, the limit of detection (LOD) for matrine and oxymatrine pesticide residues in tea samples is 1.88 μg·kg⁻¹. -1 The linear range is 3.75-187.50 μg·kg⁻¹. -1 It fully meets the EU's limits for matrine and oxymatrine in tea (10 μg·kg). -1 ) testing needs.

[0074] The matrix effect of the method was evaluated by examining the ratio of the peak area of ​​the spiked solution after magnetic solid-phase extraction of green tea matrix to the peak area of ​​the spiked solution without matrix (magnetic solid-phase extraction eluent), using 2 ng / mL. -1 and 20 ng·mL -1 The matrix effect was investigated using two concentrations of quality control samples, with each concentration measured in triplicate. The results are shown in Table 4.

[0075] Table 4 Results of matrix effect investigation

[0076]

[0077] The established magnetic solid-phase extraction-liquid chromatography-tandem mass spectrometry (MS / MS) method was applied to the extraction and detection of matrine-like drugs in commercially available tea samples. The method is simple, rapid, requires minimal organic solvent, and the pretreatment time for a single sample is less than 5 minutes. Actual sample detection results demonstrate that this method is highly selective and sensitive, accurately determining trace amounts of matrine and oxymatrine in tea. It is suitable for routine monitoring and high-throughput screening of matrine-like pesticide residues in tea.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting and detecting matrine-like drugs in tea, characterized in that, Includes the following steps: (1) Polydopamine-coated iron oxide nanospheres (Fe3O4-PDA) were synthesized by solvothermal method. Iron oxide nanospheres (Fe3O4) with a particle size in the range of 50-300 nm were obtained by purchasing or preparing them by themselves. The Fe3O4 nanospheres were dispersed in a mixture of Tris buffer and anhydrous ethanol. Dopamine hydrochloride aqueous solution was added and stirred at room temperature to carry out self-polymerization reaction. After magnetic separation, the nanospheres were washed with water and anhydrous ethanol alternately and dried under vacuum to obtain Fe3O4-PDA magnetic nanomaterials. (2) Grind the tea sample into powder, add acidic aqueous solution for ultrasonic extraction, centrifuge and take the supernatant, adjust the pH value of the supernatant to obtain the sample solution to be tested; (3) The Fe3O4-PDA magnetic nanomaterials obtained in step (1) are activated with methanol, and the sample solution to be tested obtained in step (2) is added. The matrine-like drugs are selectively bound to the material by vortexing and the supernatant is separated by an external magnetic field. After washing the material with water, alkaline methanol eluent is added and vortexed. The eluent is separated by a magnetic field and filtered through a filter membrane. The qualitative and quantitative detection is performed by liquid chromatography-tandem mass spectrometry.

2. The method for extraction and detection of matrine-like drugs in tea according to claim 1, characterized in that, In step (1), the Tris buffer used in the synthesis of Fe3O4-PDA magnetic nanomaterials is 5~15 mM, pH is 6.0~10.0; the reaction temperature is 5~30 ℃, the stirring speed is 100~500 rpm, and the reaction time is 6~20 h.

3. The method for extraction and detection of matrine-like drugs in tea according to claim 1, characterized in that, In step (2), the acidic aqueous solution is an acetic acid aqueous solution with a pH of 2.0~5.0; the material-to-liquid ratio is 2 g:15 mL; the ultrasonic extraction time is 10~60 min; the centrifugation speed is 2000~12000 rpm and the centrifugation time is 1~10 min; the pH of the supernatant after adjustment is 5.0~7.

0.

4. The method for extraction and detection of matrine-like drugs in tea according to claim 1, characterized in that, In step (3), the amount of Fe3O4-PDA magnetic nanomaterial used is 10~30 mg of Fe3O4-PDA magnetic nanomaterial per milliliter of the sample solution to be tested; the activation solvent is methanol, the activation vortex time is 0.5~5.0 min; the adsorption vortex speed is 1000~3000 rpm, the adsorption time is 2~5 min; the washing solvent is deionized water, and the washing is performed twice.

5. The method for extraction and detection of matrine-like drugs in tea according to claim 1, characterized in that, In step (3), the eluent is a mixed solution of methanol and ammonia in a volume ratio of 85:15; the elution vortex rotation speed is 1000~3000 rpm, the elution time is 0.5~5.0 min; and a 0.22 μm organic phase needle filter is used for filtration.

6. The method for extraction and detection of matrine-like drugs in tea according to claim 1, characterized in that, In step (3), the liquid chromatography conditions are as follows: the column is a C18 column; the mobile phase A is 10 mM ammonium acetate aqueous solution, the mobile phase B is methanol, A:B=65:35 (v:v); the flow rate is 0.4 mL / min; the column temperature is 20~60 ℃; and the injection volume is 2~10 μL.

7. The method for extraction and detection of matrine-like drugs in tea according to claim 1, characterized in that, This method is applicable to the simultaneous extraction and detection of matrine-like drugs (such as matrine and oxymatrine) in tea (such as green tea, black tea, and Pu-erh tea) matrices.

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