Method for detecting matrine in tea leaves

Abstract

The invention relates to a method for detecting sophocarpidine in tea leaves. The method sequentially comprises the following steps: step 1, taking a proper amount of tea leaves, putting the tea leaves into a homogenizer, and uniformly crushing the tea leaves; step 2, weighing 2.0 g of the sample in the step 1, placing the sample in a 50 mL polypropylene centrifuge tube, adding 0.1 mL of a 1 mg / L internal standard working solution, and adding 10 mL of water and 10 mL of acetonitrile; step 3, shaking the sample in the step 2 for 30 minutes, and centrifuging for 10 minutes under the condition of 4000 rpm (revolutions per minute); step 4, taking 1mL of supernate in the step 3, putting the supernate into a 2ml plastic centrifugal tube, performing vortex oscillation for 1min, and centrifuging for 5min under the condition of 13000rpm; and step 5, taking the supernate in the step 4, passing through a 0.22 mu m filter membrane, and analyzing by using a liquid chromatograph-mass spectrometer. The method does not use a large amount of acetonitrile-toluene solution, and is environment-friendly. According to the method, an SPE column is not used for purification, so that the pretreatment time is greatly shortened, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field related to chemical analysis and detection, and in particular to a method for detecting matrine in tea leaves. Background Art

[0002] Matrine, molecular formula is C 15 H 24 N2O is an alkaloid extracted from the dried roots, plants and fruits of the leguminous plant Sophora flavescens by organic reagents such as ethanol. In agricultural production, matrine, as a natural botanical pesticide, is low-toxic to humans and animals, and has obvious control effects on armyworms, cabbage worms, aphids and red spiders on various crops. In addition, it also has good control effects on vegetable downy mildew, blight and anthrax. Therefore, matrine has gradually become an active ingredient in many insecticides and fungicides and is widely used. In recent years, matrine, as a pesticide not approved by the European Union in food, implements its default limit standard, i.e. 0.01 mg / Kg, and has been notified many times for exceeding the limit standard. Tea is a major export product of my country, and the detection technology of matrine in tea is increasingly receiving attention from all parties. There is no detection method for matrine in tea in the current effective national food safety standards and published academic papers.

[0003] Currently, there are two types of detection methods for pesticide residues in tea: gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Based on the properties of different pesticides, both methods use mass spectrometry to perform qualitative and quantitative analysis of target pesticides after appropriate pretreatment.

[0004] 1. GB 23200.13-2016-National Food Safety Standard-Determination of 448 Pesticides and Related Chemicals Residues in Tea-Liquid Chromatography-Mass Spectrometry is applicable to the qualitative identification of 448 pesticides and related chemicals residues in green tea, black tea, Pu'er tea, and oolong tea, and is also applicable to the quantitative determination of 418 pesticides and related chemicals residues. The sample is extracted with acetonitrile homogenate, purified by solid phase extraction column, eluted with acetonitrile-toluene solution (3+1), detected by liquid chromatography-tandem mass spectrometry, and quantified by external standard method.

[0005] 2. GB / T 23204-2008-Determination of 519 pesticides and related chemicals residues in tea-gas chromatography-mass spectrometry, applicable to the qualitative identification of 490 pesticides and related chemicals residues in green tea, black tea, Pu'er tea, and oolong tea, and can also quantitatively determine 453 pesticides and related chemicals. The sample is extracted with acetonitrile homogenate, purified by solid phase extraction column, eluted with acetonitrile-toluene solution (3+1), detected by gas chromatography-mass spectrometry, and quantified by internal standard method.

[0006] 3. GB 23200.121-2021-National Food Safety Standard-Determination of 331 Pesticides and Their Metabolites in Plant-derived Foods-Liquid Chromatography-Mass Spectrometry, applicable to the determination of 331 pesticides and their metabolites in plant-derived foods. The sample is extracted with acetonitrile, the extract is purified by dispersed solid phase extraction, detected by liquid chromatography-mass spectrometry, and quantified by external standard method.

[0007] 4. GB 23200.113-2018-National Food Safety Standard-Determination of 208 Pesticides and Their Metabolites in Plant-derived Foods-Gas Chromatography-Mass Spectrometry, applicable to the determination of 208 pesticides and their metabolites in plant-derived foods. Tea samples are extracted with acetonitrile, the extract is purified by solid phase extraction or dispersed solid phase extraction, and detected by gas chromatography-mass spectrometry, and quantified by internal standard method or external standard method.

[0008] The disadvantages of the prior art are:

[0009] The current detection methods for pesticide residues in tea cause the loss of matrine during the pre-treatment process and are therefore unable to detect matrine;

[0010] And there is no specific matrine detection method for tea samples.

[0011] In view of the above-mentioned defects, the designers actively carried out research and innovation in order to create a method for detecting matrine in tea to make it more valuable for industrial use. Summary of the invention

[0012] In order to solve the above technical problems, the object of the present invention is to provide a method for detecting matrine in tea leaves.

[0013] To achieve the above object, the present invention adopts the following technical solution:

[0014] A method for detecting matrine in tea leaves comprises the following steps in sequence:

[0015] Step 1: Take an appropriate amount of tea leaves and grind them evenly in a homogenizer;

[0016] Step 2, weigh 2.0 g of the sample in step 1 above into a 50 mL polypropylene centrifuge tube, add 0.1 mL of 1 mg / L internal standard working solution, and then add 10 mL of water and 10 mL of acetonitrile;

[0017] Step 3, shaking the sample in step 2 for 30 min, and centrifuging at 4000 rpm for 10 min;

[0018] Step 4: Take 1 mL of the supernatant in step 3 above and place it in a 2 mL plastic centrifuge tube. Vortex for 1 min and centrifuge at 13,000 rpm for 5 min.

[0019] Step 5: Take the supernatant in step 4 above, filter it through a 0.22 μm filter membrane, and analyze it using a liquid chromatography-mass spectrometer.

[0020] As a further improvement of the present invention, the 2 ml plastic centrifuge tube in step 4 is filled with 10 mg of ethylenediamine-N-propylsilane.

[0021] As a further improvement of the present invention, the chromatographic conditions in the LC-MS analysis in step 5 are:

[0022] Column: BEH Amide, 2.1×100 mm, 1.7 μm;

[0023] Column temperature: 40°C;

[0024] Injection volume: 2 μL;

[0025] Flow rate: 0.40ml / min;

[0026] Mobile phase: Mobile phase A: 50 mM ammonium formate aqueous solution, mobile phase B: 100% acetonitrile.

[0027] As a further improvement of the present invention, the liquid phase gradient in the LC-MS analysis in step 5 is:

[0028] Initial: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40 mL / min;

[0029] 3min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min;

[0030] 6min: mobile phase A: 30%, mobile phase B: 70%, flow rate: 0.40mL / min;

[0031] 7min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min;

[0032] 8min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min;

[0033] 8.2min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min;

[0034] 12min: Mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min.

[0035] As a further improvement of the present invention, the mass spectrometry conditions in the LC-MS analysis in step 5 are:

[0036] Ionization mode:ESIPositive;

[0037] Sheath gas pressure: 35psi;

[0038] Auxiliary gas flow: 9arb;

[0039] Ion spray voltage: 5500V;

[0040] Capillary temperature: 550℃;

[0041] Auxiliary gas heater temperature 1: 60℃;

[0042] Auxiliary gas heater temperature 2: 60℃.

[0043] As a further improvement of the present invention, the ion pair combination under the mass spectrometry condition in the LC-MS analysis in step 5 is:

[0044] Matrine quantitative ion pair: 249->148;

[0045] Matrine qualitative ion pair: 249->98;

[0046] Tritiated matrine quantitative ion pair: 252->148;

[0047] Tritiated matrine qualitative ion pair: 252->179.

[0048] By means of the above scheme, the present invention has at least the following advantages:

[0049] The invention does not use a large amount of acetonitrile-toluene solution and is environmentally friendly.

[0050] The present invention does not use SPE column purification, which greatly reduces the time of pre-treatment and improves the detection efficiency.

[0051] The present invention uses LC-MS / MS detection, and the quantitative limit can be 0.01 mg / Kg, which is lower than the requirements of various countries and regions on the limit of matrine in tea.

[0052] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0054] Figure 1 It is a flow chart of a method for detecting matrine in tea leaves of the present invention;

[0055] Figure 2 It is a quantitative ion diagram of matrine in a matrine chromatogram obtained by applying a method for detecting matrine in tea leaves of the present invention;

[0056] Figure 3 It is a qualitative ion diagram of matrine in a matrine chromatogram obtained by applying a method for detecting matrine in tea leaves of the present invention;

[0057] Figure 4 It is a quantitative ion diagram of tritiated matrine in a matrine chromatogram obtained by using a method for detecting matrine in tea of ​​the present invention;

[0058] Figure 5 The invention discloses a qualitative ion diagram of tritiated matrine in a matrine chromatogram obtained by applying a method for detecting matrine in tea leaves of the invention. DETAILED DESCRIPTION

[0059] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0060] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. The components of the embodiment of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiment of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiment of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0061] Example

[0062] like Figure 1 to Figure 5 As shown,

[0063] A method for detecting matrine in tea leaves comprises the following steps in sequence:

[0064] Step 1: Take an appropriate amount of tea leaves and grind them evenly in a homogenizer;

[0065] Step 2, weigh 2.0 g of the sample in step 1 above into a 50 mL polypropylene centrifuge tube, add 0.1 mL of 1 mg / L internal standard working solution, and then add 10 mL of water and 10 mL of acetonitrile;

[0066] Step 3, shaking the sample in step 2 for 30 min, and centrifuging at 4000 rpm for 10 min;

[0067] Step 4: Take 1 mL of the supernatant in step 3 above and place it in a 2 mL plastic centrifuge tube. Vortex for 1 min and centrifuge at 13,000 rpm for 5 min.

[0068] Step 5: Take the supernatant in step 4 above, filter it through a 0.22 μm filter membrane, and analyze it using a liquid chromatography-mass spectrometer.

[0069] Preferably, the 2 ml plastic centrifuge tube in step 4 contains 10 mg of ethylenediamine-N-propylsilane.

[0070] Preferably, the chromatographic conditions in the LC-MS analysis in step 5 are:

[0071] Column: BEH Amide, 2.1×100 mm, 1.7 μm;

[0072] Column temperature: 40°C;

[0073] Injection volume: 2 μL;

[0074] Flow rate: 0.40ml / min;

[0075] Mobile phase: Mobile phase A: 50 mM ammonium formate aqueous solution, mobile phase B: 100% acetonitrile.

[0076] Preferably, the liquid phase gradient in the LC-MS analysis in step 5 is:

[0077] Initial: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40 mL / min;

[0078] 3min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min;

[0079] 6min: mobile phase A: 30%, mobile phase B: 70%, flow rate: 0.40mL / min;

[0080] 7min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min;

[0081] 8min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min;

[0082] 8.2min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min;

[0083] 12min: Mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min.

[0084] Preferably, the mass spectrometry conditions in the LC-MS analysis in step 5 are:

[0085] Ionization mode:ESIPositive;

[0086] Sheath gas pressure: 35psi;

[0087] Auxiliary gas flow: 9arb;

[0088] Ion spray voltage: 5500V;

[0089] Capillary temperature: 550℃;

[0090] Auxiliary gas heater temperature 1: 60℃;

[0091] Auxiliary gas heater temperature 2: 60℃.

[0092] Preferably, the ion pair combination under mass spectrometry conditions in the LC-MS analysis in step 5 is:

[0093] Matrine quantitative ion pair: 249->148;

[0094] Matrine qualitative ion pair: 249->98;

[0095] Tritiated matrine quantitative ion pair: 252->148;

[0096] Tritiated matrine qualitative ion pair: 252->179.

[0097] The first embodiment of the present invention:

[0098] The method for detecting matrine in tea leaves of the present invention is applied to solve the following problems:

[0099] 1) Explore the extraction method of tea samples so that matrine in tea can be fully extracted and reduce the occurrence of false negative results.

[0100] 2) Explore the purification method of the sample solution after tea sample extraction, so that the interfering impurities generated in the tea during the detection of matrine can be fully removed to ensure the accuracy of qualitative detection.

[0101] 3) Explore the instrumental conditions for detecting matrine by mass spectrometry so that the quantitative limit of the detection method is no higher than 0.01 mg / Kg.

[0102] This method aims to establish a detection method for matrine in tea.

[0103] The sample was extracted with water and acetonitrile, the extract was purified by dispersive solid phase extraction, and determined by liquid chromatography-mass spectrometry. The internal standard method was used for quantification, and the limit of quantification was 0.01 mg / Kg.

[0104] Reagents:

[0105] Deionized water, PSA (ethylenediamine-N-propylsilane), formic acid, ammonium formate, acetonitrile, matrine, tritiated matrine.

[0106] Instruments and equipment:

[0107] Balance, liquid chromatography-mass spectrometry / mass spectrometer (AB Sciex 5500+), high-speed centrifuge, oscillator, vortexer, homogenizer.

[0108] Sample preparation:

[0109] Take an appropriate amount of tea leaves and grind them evenly in a homogenizer, then weigh 2.0 g of the sample into a 50 mL polypropylene centrifuge tube, add 0.1 mL of 1 mg / L internal standard working solution, and then add 10 mL of water and 10 mL of acetonitrile.

[0110] Shake for 30 minutes, centrifuge at 4000 rpm for 10 minutes. Take 1 mL of the supernatant into a 2 ml plastic centrifuge tube (containing 10 mg PSA), vortex for 1 minute, and centrifuge at 13000 rpm for 5 minutes.

[0111] The supernatant was filtered through a 0.22 μm filter membrane and analyzed by liquid chromatography-mass spectrometry.

[0112] Instrument settings:

[0113] Column: BEH Amide, 2.1×100mm, 1.7μm

[0114] Column temperature: 40°C

[0115] Injection volume: 2 μL

[0116] Mobile phase A: 50 mM ammonium formate in water

[0117] Mobile phase B: 100% acetonitrile

[0118] gradient:

[0119] Initial: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40 mL / min;

[0120] 3min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min;

[0121] 6min: mobile phase A: 30%, mobile phase B: 70%, flow rate: 0.40mL / min;

[0122] 7min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min;

[0123] 8min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min;

[0124] 8.2min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min;

[0125] 12min: Mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min.

[0126] Mass spectrometry parameters:

[0127] Ionization mode:ESIPositive;

[0128] Sheath gas pressure: 35psi;

[0129] Auxiliary gas flow: 9arb;

[0130] Ion spray voltage: 5500V;

[0131] Capillary temperature: 550℃;

[0132] Auxiliary gas heater temperature 1: 60℃;

[0133] Auxiliary gas heater temperature 2: 60℃.

[0134] Ion Pair:

[0135] Matrine quantitative ion pair: 249->148;

[0136] Matrine qualitative ion pair: 249->98;

[0137] Tritiated matrine quantitative ion pair: 252->148;

[0138] Tritiated matrine qualitative ion pair: 252->179.

[0139] This method was applied to tea samples for spike verification at the 0.010 mg / Kg level, and the recovery rate was 70-130%, with RSD <20%. More than 500 batches of tea samples have been tested, covering black tea, green tea, scented tea and other tea samples. In addition, for the same batch of tea samples, the data tested by this method were compared with the Eurofins Dr. Specht Express laboratory, one of the official laboratories designated by the European Union, and there was no significant difference in the results. Under this method, the chromatogram of matrine obtained is as follows Figures 2 to 5 shown.

[0140] The invention does not use a large amount of acetonitrile-toluene solution (3+1), and is environmentally friendly.

[0141] The present invention does not use SPE column purification, which greatly reduces the time of pre-treatment and improves the detection efficiency.

[0142] The present invention uses LC-MS / MS detection, and the quantitative limit can be 0.01 mg / Kg, which is lower than the requirements of various countries and regions on the limit of matrine in tea.

[0143] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly referring to the number of technical features indicated. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0144] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installation", "connection" and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be the internal connection of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood through specific circumstances.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting matrine in tea, characterized in that: The following steps are included in sequence: Step 1: Take an appropriate amount of tea leaves and grind them evenly in a homogenizer; Step 2, weigh 2.0 g of the sample in step 1 above into a 50 mL polypropylene centrifuge tube, add 0.1 mL of 1 mg / L internal standard working solution, and then add 10 mL of water and 10 mL of acetonitrile; Step 3, shaking the sample in step 2 for 30 min, and centrifuging at 4000 rpm for 10 min; Step 4: Take 1 mL of the supernatant in step 3 above and place it in a 2 mL plastic centrifuge tube. Vortex for 1 min and centrifuge at 13,000 rpm for 5 min. Step 5: Take the supernatant in step 4 above, filter it through a 0.22 μm filter membrane, and analyze it using a liquid chromatography-mass spectrometer.

2. The method for detecting matrine in tea leaves according to claim 1, characterized in that: The 2 ml plastic centrifuge tube in step 4 is filled with 10 mg of ethylenediamine-N-propylsilane.

3. The method for detecting matrine in tea leaves according to claim 1, characterized in that: The chromatographic conditions in the LC-MS analysis in step 5 are: Column: BEH Amide, 2.1×100 mm, 1.7 μm; Column temperature: 40°C; Injection volume: 2 μL; Flow rate: 0.40ml / min; Mobile phase: Mobile phase A: 50 mM ammonium formate aqueous solution, mobile phase B: 100% acetonitrile.

4. The method for detecting matrine in tea leaves according to claim 3, characterized in that: The liquid phase gradient in the LC-MS analysis in step 5 is: Initial: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40 mL / min; 3min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min; 6min: mobile phase A: 30%, mobile phase B: 70%, flow rate: 0.40mL / min; 7min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min; 8min: mobile phase A: 60%, mobile phase B: 40%, flow rate: 0.40mL / min; 8.2min: mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min; 12min: Mobile phase A: 10%, mobile phase B: 90%, flow rate: 0.40mL / min.

5. The method for detecting matrine in tea leaves according to claim 1, characterized in that: The mass spectrometry conditions in the LC-MS analysis in step 5 are: Ionization mode:ESIPositive; Sheath gas pressure: 35psi; Auxiliary gas flow: 9arb; Ion spray voltage: 5500V; Capillary temperature: 550℃; Auxiliary gas heater temperature 1: 60℃; Auxiliary gas heater temperature 2: 60℃.

6. The method for detecting matrine in tea leaves according to claim 5, characterized in that: The ion pair combination under mass spectrometry conditions in the LC-MS analysis in step 5 is: Matrine quantitative ion pair: 249->148; Matrine qualitative ion pair: 249->98; Tritiated matrine quantitative ion pair: 252->148; Tritiated matrine qualitative ion pair: 252->179.

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