Method for detecting pesticide residues in fungus traditional Chinese medicinal materials

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for detecting pesticide residues in fungus traditional Chinese medicinal materials. The method for detecting the pesticide residues of the fungus traditional Chinese medicinal materials comprises the following steps: preparing a pesticide mixed reference substance solution; preparing a to-be-detected sample solution; detecting the pesticide mixed reference substance solution and the to-be-detected sample solution by adopting a gas chromatography-tandem mass spectrometry method, and qualitatively detecting the pesticide residue condition in the to-be-detected sample solution; a chromatographic column heating procedure of gas chromatography of the gas chromatography tandem mass spectrometry comprises the following steps: keeping the initial temperature at 90-110 DEG C for 40-80 seconds, heating to 110-130 DEG C at the rate of 25-35 DEG C / min, heating to 150-170 DEG C at the rate of 5-15 DEG C / min, heating to 220-240 DEG C at the rate of 2-6 DEG C / min, heating to 310-330 DEG C at the rate of 10-20 DEG C / min, and keeping for 1-3 minutes. In the application, on the premise of ensuring the sensitivity and the resolution, the sample flux can be improved, and the analysis efficiency is high.

Description

Technical Field

[0001] This application relates to the field of analytical detection technology, and particularly relates to a method for detecting pesticide residues in fungal Chinese medicinal materials. Background Art

[0002] Fungal medicinal materials such as Ganoderma lucidum and Poria cocos are commonly used traditional Chinese medicines, mainly from artificial cultivation. During the cultivation process, in order to prevent and control pests and diseases, most Chinese medicinal material growers will use various pesticides to reduce insect pests. In recent years, due to the extensive use of pesticides, fungal medicinal materials such as Ganoderma lucidum and Poria cocos are also often affected by pesticide residues during the planting process. The residues of prohibited pesticides in Chinese medicinal materials will not only affect the quality of Chinese medicinal materials, but also pose safety problems and damage human health. Therefore, the detection of pesticide residues in fungal medicinal materials such as Ganoderma lucidum and Poria cocos is particularly important.

[0003] The pesticide residues in fungal medicinal materials such as Ganoderma lucidum and Poria cocos are at trace levels. With the development of mass spectrometry technology, gas chromatography-mass spectrometry has high separation efficiency, low detection limit, and also has the characteristics of high selectivity and strong anti-interference ability. Therefore, they are the most effective tools for detecting pesticide residues. However, the matrix of pesticide residues in fungal medicinal materials such as Ganoderma lucidum and Poria cocos is complex. The traditional gas chromatography-tandem mass spectrometry detection method has a too long running time. Under the current regulatory detection requirements, the detection items of prohibited pesticide residues in medicinal materials will increase significantly. Under a large number of inspection items, how to improve the detection efficiency is an urgent problem to be solved. Summary of the Invention

[0004] Based on this, this application provides a method for detecting pesticide residues in fungal Chinese medicinal materials, and its technical solution is as follows:

[0005] A method for detecting pesticide residues in fungal Chinese medicinal materials includes the following steps:

[0006] Prepare a mixed pesticide reference solution, wherein the mixed pesticide reference solution includes one or more of ethoprophos, phorate, α-BHC, β-BHC, γ-BHC, δ-BHC, fipronil, flonicamid, fipronil sulfoxide, fipronil sulfone, aldrin, dieldrin, α-endosulfan, β-endosulfan, endosulfan sulfate, p,p'-DDD, o,p'-DDT, p,p'-DDE, p,p'-DDT, demeton (O+S), terbufos, methyl parathion, parathion, dicofol, isofenphos-methyl, isocarbophos, nitrofen, fenamiphos, and sulprofos;

[0007] Mix the fungal Chinese medicinal materials to be tested, a salting-out agent and a solvent, collect the extract, purify the extract, and prepare a sample solution to be tested;

[0008] The pesticide mixed reference solution and the sample solution to be tested are detected by gas chromatography - tandem mass spectrometry to qualitatively detect the pesticide residues in the sample solution to be tested;

[0009] The chromatographic column temperature - rising program of the gas chromatography in the gas chromatography - tandem mass spectrometry includes: an initial temperature of 90°C to 110°C, held for 40 s to 80 s, heated at a rate of 25°C / min to 35°C / min to 110°C to 130°C, then heated at a rate of 5°C / min to 15°C / min to 150°C to 170°C, then heated at a rate of 2°C / min to 6°C / min to 220°C to 240°C, and then heated at a rate of 10°C / min to 20°C / min to 310°C to 330°C, held for 1 min to 3 min.

[0010] In some embodiments, the pesticide mixed reference solution includes ethoprophos, phorate, α - hexachlorocyclohexane, β - hexachlorocyclohexane, γ - hexachlorocyclohexane, δ - hexachlorocyclohexane, fipronil, flonicamid, fipronil sulfoxide, fipronil sulfone, aldrin, dieldrin, α - endosulfan, β - endosulfan, endosulfan sulfate, p,p’ - DDE, o,p’ - DDT, p,p’ - DDD, p,p’ - DDT, demeton (O + S), terbufos, parathion - methyl, parathion, dicofol, isofenphos - methyl, isocarbophos, nitrofen, fenamiphos, and sulprofos. On the premise of ensuring sensitivity and resolution, the above - mentioned method can simultaneously detect 30 kinds of banned pesticides such as ethoprophos, and the detected pesticide types are numerous and comprehensive.

[0011] In some embodiments, the chromatographic column of the gas chromatography in the gas chromatography - tandem mass spectrometry is a fused - silica capillary column with (50% phenyl) - methyl polysiloxane as the stationary liquid.

[0012] In some embodiments, the column length of the chromatographic column is 30 m, the column inner diameter is 0.25 mm, and the film thickness is 0.25 μm.

[0013] In some embodiments, the gas chromatography conditions of the gas chromatography - tandem mass spectrometry include at least one of the following conditions:

[0014] (1) The inlet temperature is 240°C to 260°C; (2) Splitless injection; (3) The carrier gas is helium; (4) The inlet is in a constant - pressure mode; (5) The column - front pressure is 135 kPa to 155 kPa.

[0015] In some embodiments, the mass spectrometry conditions of the gas chromatography - tandem mass spectrometry include at least one of the following conditions:

[0016] (1) Detection by a triple quadrupole tandem mass spectrometer; (2) The ion source is an electron impact source; (3) The ion source temperature is 240°C to 260°C; (4) The collision gas is nitrogen or argon; (5) The mass spectrometry transfer interface temperature is 240°C to 260°C; (6) The mass spectrometry monitoring mode is multiple reaction monitoring.

[0017] In some embodiments, the salting-out agent includes sodium chloride;

[0018] In some embodiments, the solvent includes acetonitrile.

[0019] In some embodiments, the mass ratio of the Chinese medicinal mushroom to be tested to the salting-out agent is (4 - 6):1.

[0020] In some embodiments, purifying the extract includes the following steps: passing the extract through a solid-phase extraction column filled with a hydrophilic lipophilic balance material.

[0021] In some embodiments, the solvent in the pesticide mixed reference solution includes acetonitrile.

[0022] In some embodiments, it further includes the following steps: quantitatively detecting the pesticide residue in the Chinese medicinal mushroom by the internal standard method, and the internal standard substance of the internal standard method is triphenyl phosphate.

[0023] In some embodiments, before detecting the pesticide mixed reference solution and the sample solution to be tested by gas chromatography-tandem mass spectrometry, it includes the following steps: adding an internal standard solution to the pesticide mixed reference solution and the sample solution to be tested respectively, the internal standard solution includes the internal standard substance and an internal standard solvent, and the internal standard solvent includes acetonitrile.

[0024] In some embodiments, the Chinese medicinal mushroom is Ganoderma lucidum medicinal material, Ganoderma lucidum cut pieces, Poria cocos medicinal material or Poria cocos cut pieces.

[0025] Compared with the traditional solution, the present application has the following beneficial effects:

[0026] The detection method of the present application can quickly and accurately detect trace pesticide residues in Chinese medicinal mushrooms. The traditional method of using a gas chromatography-tandem mass spectrometer to determine 25 kinds of prohibited pesticides such as ethoprophos takes more than 55 minutes to analyze a sample each time. In the present application, on the premise of ensuring sensitivity and resolution, the sample throughput can be increased, the analysis time of each time can be compressed to 33 minutes, reducing the analysis time by 40%, and the analysis efficiency is high. Description of the Drawings

[0027] To more clearly illustrate the technical solutions in the embodiments of the present application and to more comprehensively understand the present application and its beneficial effects, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those skilled in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0028] Figure 1 It is the linear relationship diagram of the concentration and peak area of ethoprophos and sulprofos;

[0029] Figure 2 It is the linear relationship diagram of the concentration and peak area of phorate and α - hexachlorocyclohexane;

[0030] Figure 3 It is the linear relationship diagram of the concentration and peak area of terbufos and demeton (O + S);

[0031] Figure 4 It is the linear relationship diagram of the concentration and peak area of γ - hexachlorocyclohexane and β - hexachlorocyclohexane;

[0032] Figure 5 It is the linear relationship diagram of the concentration and peak area of flonicamid and δ - hexachlorocyclohexane;

[0033] Figure 6 It is the linear relationship diagram of the concentration and peak area of aldrin and methyl parathion;

[0034] Figure 7 It is the linear relationship diagram of the concentration and peak area of fipronil sulfoxide and fipronil;

[0035] Figure 8 It is the linear relationship diagram of the concentration and peak area of parathion and dicofol;

[0036] Figure 9 It is the linear relationship diagram of the concentration and peak area of phosmet and isocarbophos;

[0037] Figure 10 It is the linear relationship diagram of the concentration and peak area of α - endosulfan and fipronil sulfone;

[0038] Figure 11 It is the linear relationship diagram of the concentration and peak area of p,p’ - DDE and dieldrin;

[0039] Figure 12 It is the linear relationship diagram of the concentration and peak area of fenamiphos and phosfolan - methyl;

[0040] Figure 13 It is the linear relationship diagram of the concentration and peak area of nitrofen and o,p’ - DDT;

[0041] Figure 14It is the linear relationship diagram of the concentrations and peak areas of p,p’-DDD and β-endosulfan;

[0042] Figure 15 It is the linear relationship diagram of the concentrations and peak areas of p,p’-DDT and endosulfan sulfate;

[0043] Figure 16 It is the TIC diagram of the blank solvent and the blank poria cocos matrix solution;

[0044] Figure 17 It is the TIC diagram of the multi-pesticide residue reference solution and the mixed reference solution of poria cocos matrix;

[0045] Figure 18 It is the LC-MS spectrum of ethoprophos in the blank poria cocos matrix solution;

[0046] Figure 19 It is the LC-MS spectrum of ethoprophos in the multi-pesticide residue reference solution;

[0047] Figure 20 It is the LC-MS spectrum of ethoprophos in the mixed reference solution of poria cocos matrix. Detailed implementation manners

[0048] The following further describes the present application in detail with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present application more thorough and comprehensive.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0050] Example 1

[0051] 1 Instruments, reagents and test drugs

[0052] Instruments: Agilent gas chromatography-tandem mass spectrometry (Agilent 8890-7000D, Agilent Technologies Co., Ltd.), Agilent J&W VF-17ms (30m×0.25mm, 0.25μm) chromatographic column; ten-thousandth electronic analytical balance (ME204E, Mettler-Toledo Co., Ltd.), Anting centrifuge (LXJ-II C, Shanghai Anting Scientific Instrument Factory), ultrapure water system (Milli-Q Direct, Merck KGaA), nitrogen evaporator (EVA 30, Ruker Instruments (Xiamen) Co., Ltd.).

[0053] Reagents: Methanol (Tianjin Fuyu Fine Chemical Co., Ltd.), analytical reagent grade; formic acid (Cominbio Co., Ltd.), acetonitrile (Merck KGaA), methanol (Merck KGaA), chromatographic grade; water, ultrapure water (prepared in the laboratory).

[0054] Test drugs: Multiresidue reference substance solution (batch number: 380 - 03381, solvent: acetonitrile; source: Shimadzu (Shanghai) Experimental Equipment Co., Ltd.). Specific information is shown in Table 1.

[0055] Table 1

[0056]

[0057] Poria cocos medicinal materials (batch numbers: 20200824, 20201103, 20201104), Poria cocos blank matrix (batch number: FLKB2006)

[0058] Ganoderma lucidum medicinal materials (batch number: 20200763), Ganoderma lucidum blank matrix (batch number: LZKB2003)

[0059] 2 Chromatographic conditions

[0060] Gas chromatography - tandem mass spectrometry chromatographic conditions: An elastic quartz capillary column with (50% phenyl)-methyl polysiloxane as the stationary liquid (column length: 30 m, column inner diameter: 0.25 mm, film thickness: 0.25 μm). Injection port temperature: 250 °C, splitless injection. Carrier gas: high - purity helium (He). The injection port is in constant - pressure mode, and the column - front pressure is 146 kPa. Programmed temperature rise: Initial temperature 100 °C, hold for 1 minute, increase at a rate of 30 °C / min to 120 °C, then increase at a rate of 10 °C / min to 160 °C, then increase at a rate of 4 °C / min to 230 °C, and finally increase at a rate of 15 °C / min to 300 °C, hold for 2 minutes.

[0061] Mass spectrometry conditions: Detected by a triple - quadrupole tandem mass spectrometer; the ion source is an electron impact source (EI), ion source temperature 250 °C. Collision gas: nitrogen or argon. Mass spectrometry transfer interface temperature 250 °C. The mass spectrometry monitoring mode is multiple reaction monitoring (MRM). The reference retention time, monitored ion pairs, and collision energy (CE) information of each compound are shown in Table 2. To improve the detection sensitivity, each pesticide can be detected in segments according to the retention time.

[0062] Table 2

[0063]

[0064]

[0065] 3 Preparation of reference solution

[0066] Precisely measure 1 mL of the multi-pesticide residue reference solution (batch number: 380 - 03381), transfer it to a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and mix well.

[0067] 4 Preparation of the internal standard solution

[0068] Weigh an appropriate amount of triphenyl phosphate reference substance accurately, dissolve it in acetonitrile and prepare a solution containing 0.1 mg per 1 mL; precisely measure an appropriate amount and dilute and make up the volume, add acetonitrile to make a solution containing 0.1 μg per 1 mL, and that is the internal standard solution.

[0069] 5 Preparation of the blank matrix solution

[0070] Take the blank matrix sample, and process it in the same way as the preparation method of the test sample solution to make the blank matrix solution.

[0071] 6 Preparation of the matrix mixed reference solution

[0072] Precisely measure 1.0 mL of the blank matrix solution, place it on a nitrogen evaporator, concentrate it to about 0.6 mL in a 40 °C water bath, add 50 μL of the multi-pesticide residue reference solution (batch number: 380 - 03381), dilute it to 1 mL with acetonitrile, and vortex to mix well, then you get it. The concentrations of the matrix mixed reference solution are as follows:

[0073] C(ethoprophos) = 0.01025 μg / ml, C(sulfotep) = 0.01025 μg / ml, C(phorate) = 0.01025 μg / ml, C(BHC-alpha) = 0.02475 μg / ml, C(BHC-gamma) = 0.0235 μg / ml, C(BHC-beta) = 0.0245 μg / ml, C(BHC-delta) = 0.02375 μg / ml, C(terbufos) = 0.0105 μg / ml, C(demeton) = 0.0105 μg / ml, C(aldrin) = 0.02375 μg / ml, C(dieldrin) = 0.02575 μg / ml, C(flonicamid) = 0.01075 μg / ml, C(fipronil) = 0.0105 μg / ml, C(fipronil sulfoxide) = 0.01 μg / ml, C(fipronil sulfone) = 0.00975 μg / ml, C(o,p-DDT) = 0.02525 μg / ml, C(p,p-DDT) = 0.02525 μg / ml, C(methyl parathion) = 0.0095 μg / ml, C(parathion) = 0.01μg / ml, C(isofenphos-methyl) = 0.0105 μg / ml, C(isocarbophos) = 0.02525 μg / ml, C(phosfolan-methyl) = 0.01575 μg / ml, C(p-p'-DDE) = 0.0235 μg / ml, C(o-p'-DDT) = 0.0245 μg / ml, C(p-p'-DDD) = 0.02375 μg / ml, C(p-p'-DDT) = 0.02375 μg / ml, C(fenamiphos) = 0.01 μg / ml, C(nitrofen) = 0.0255 μg / ml, C(alpha-endosulfan) = 0.026 μg / ml, C(beta-endosulfan) = 0.0245 μg / ml, C(endosulfan sulfate) = 0.02425 μg / ml, C(triphenyl phosphate) = 0.1 μg / ml.

[0074] 7 Preparation of the test sample solution

[0075] Take 5 g of Poria cocos powder (passed through No. 3 sieve), accurately weigh, add 1 g of sodium chloride, immediately disperse by shaking, then add 50 mL of acetonitrile, homogenize for 2 minutes (rotation speed not less than 12,000 revolutions per minute), centrifuge (4,000 revolutions per minute), separate the supernatant, add another 50 mL of acetonitrile to the precipitate, homogenize for 1 minute, centrifuge, combine the supernatants extracted twice, concentrate under reduced pressure to about 3 mL - 5 mL, cool, dilute to 10 mL with acetonitrile, and shake well. Measure 3 mL - 5 mL of the above solution, purify it through a hydrophilic-lipophilic balance material solid-phase extraction column (HLB SPE) (200 mg, 6 mL), collect all the purified solutions, mix well, and you will get it.

[0076] 8 Determination method

[0077] Accurately pipette 1 mL each of the above matrix mixed control solution and the test sample solution respectively, accurately add 0.3 mL of the internal standard solution, mix well, filter, and take the subsequent filtrate. Accurately pipette 1 μL each of the above two solutions respectively, carry out the determination according to the chromatographic conditions under item "2", and calculate according to the internal standard method to obtain the result.

[0078] 9 Methodological verification

[0079] (1) Linear investigation

[0080] Take six portions of 5 g of Poria cocos medicinal powder (sieved through No. 3 sieve, batch number: FLKB2006), accurately weigh them, prepare the blank matrix solution of Poria cocos according to the method determined under item "7", accurately measure 1.0 mL respectively, place them on a nitrogen evaporator, concentrate them to about 0.6 mL in a 40 °C water bath, and add 50 μL, 100 μL, 250 μL, 500 μL, 750 μL, and 1000 μL of the multi-pesticide residue reference substance solution (batch number: 380-03381) respectively; dilute them to 1 mL with acetonitrile respectively, and vortex and mix to obtain matrix mixed reference solutions with different concentrations.

[0081] Accurately pipette 1 μL of each of the above matrix mixed reference solutions respectively, inject them into a gas chromatography-tandem mass spectrometer, and perform the determination under the conditions of item "2" to draw a linear relationship graph between the concentration of each prohibited pesticide residue compound and the peak area. The results are shown in Figures 1 - 15 。

[0082] The results show that the correlation coefficients r of 30 compounds are ≥ 0.990, indicating that the linear relationship between the concentration of each prohibited pesticide residue compound and the peak area is good.

[0083] (2) Repeatability investigation

[0084] Take six portions of 5 g of Poria cocos medicinal powder (sieved through No. 3 sieve, batch number: FLKB2006), accurately weigh them, prepare the blank matrix solution of Poria cocos according to the method determined under item "7", accurately measure 1.0 mL respectively, place them on a nitrogen evaporator, concentrate them to about 0.6 mL in a 40 °C water bath, add 50 μl of the multi-pesticide residue reference substance solution (batch number: 380-03381) respectively, dilute them to 1 mL with acetonitrile respectively, and vortex and mix to obtain matrix mixed reference solutions.

[0085] Take six portions of 5 g of Poria cocos medicinal powder (sieved through No. 3 sieve, batch number: 20201104), accurately weigh them, and prepare the test sample solution according to the method determined under item "7".

[0086] Perform the determination according to the determination method under item "8", calculate the amount of each prohibited pesticide residue compound by the internal standard method, and calculate the repeatability of six samples. The results are shown in Table 3.

[0087] Table 3

[0088]

[0089]

[0090] The results show that the RSDs of the contents of prohibited pesticide residue compounds in 6 samples are all less than 10%, indicating good repeatability.

[0091] (3) Recovery investigation

[0092] Take six portions of 5 g of Poria cocos powder (passed through No. 3 sieve, batch number: FLKB2006), accurately weigh them, and prepare Poria cocos blank matrix solution according to the method determined under item "7". Accurately measure 1.0 mL of each solution, place it on a nitrogen blower, and concentrate it to about 0.6 mL in a 40°C water bath. Add 50 μL of multi-pesticide residue reference solution (batch number: 380-03381) to each solution, dilute it to 1 mL with acetonitrile, and vortex mix to obtain a matrix mixed control solution.

[0093] Take six portions of 5 g of Poria cocos powder (passed through No. 3 sieve, batch number: 20201104), accurately weigh them, prepare the sample solution to be tested according to the method determined under item "7", accurately measure 1.0 mL of each solution, place it on a nitrogen blower, concentrate it in a 40°C water bath to about 0.6 mL, add 50 μL of multi-pesticide residue reference solution (batch number: 380-03381) respectively, dilute it to 1 mL with acetonitrile, vortex mix, and obtain the spiked and recovered sample solution to be tested.

[0094] The matrix mixed control solution and the spiked recovered sample solution were determined according to the determination method under item "8". The amount of each banned pesticide residue was calculated by the internal standard method, and the recovery rate was calculated. The results are shown in Table 4.

[0095] Table 4

[0096]

[0097]

[0098] The results showed that the average recoveries of the banned pesticide residues ranged from 76.61% to 104.91%, and the RSDs were all less than 10%, indicating a good recovery rate.

[0099] (3) Durability study at different initial column temperatures

[0100] The effects of different initial column temperatures of 98℃, 100℃ and 102℃ on the determination of multiple pesticide residues in the mixed reference solution of Poria cocos medicinal material matrix were compared.

[0101] Take 5g of Poria cocos powder (passed through No. 3 sieve, batch number: FLKB2006), accurately weigh, and prepare Poria cocos blank matrix solution according to the method determined under "7". Take multiple pesticide residue reference solution (batch number: 380-03381) and prepare matrix mixed reference solution according to the method determined under "6". Take 5g of Poria cocos powder (passed through No. 3 sieve, batch number: 20201104) in six portions, accurately weigh, and prepare the sample solution to be tested according to the method determined under "7". Except that the initial column temperature was 98℃, 100℃ and 102℃ respectively, the other chromatographic conditions were determined according to the determination method under "8". The amount of each banned pesticide residue compound was calculated by the internal standard method. The experimental results are shown in Table 4.

[0102] Table 4

[0103]

[0104] The results showed that the RSD values of the contents of each pesticide residue compound in the mixed reference solution of Poria cocos matrix with different initial column temperatures were all less than 15%, indicating that the analytical method had good durability at different initial column temperatures.

[0105] (4) Investigation on durability at different pre-column pressures

[0106] Compare the effects of different pre-column pressures of 139 kPa, 146 kPa, and 153 kPa on the determination of multi-pesticide residues in the mixed reference solution of Poria cocos medicinal material matrix.

[0107] Take 5 g of Poria cocos medicinal material powder (passed through No. 3 sieve, batch number: FLKB2006), accurately weighed, prepare the blank matrix solution of Poria cocos according to the method determined under item "7". Additionally, take the multi-pesticide residue reference solution (batch number: 380-03381) and prepare the mixed reference solution of the matrix according to the method determined under item "6". Take six portions of 5 g of Poria cocos medicinal material powder (passed through No. 3 sieve, batch number: 20201104), accurately weighed, and prepare the test sample solution according to the method determined under item "7". Except for the pre-column pressures of 139 kPa, 146 kPa, and 153 kPa, other chromatographic conditions were determined according to the method under item "8" for determination. The amounts of each prohibited pesticide residue compound were calculated by the internal standard method, and the experimental results are shown in Table 5.

[0108] Table 5

[0109]

[0110] The results showed that the RSD values of the contents of each pesticide residue compound in the mixed reference solution of Poria cocos matrix with different pre-column pressures were all less than 15%, indicating that the analytical method had good durability at different pre-column pressures.

[0111] Determination of 10 blank solvent, blank matrix solution, multi-pesticide residue reference solution, and mixed reference solution of the matrix

[0112] Take acetonitrile as the blank solvent.

[0113] Take Poria cocos medicinal material powder (passed through No. 3 sieve, batch number: FLKB2006) and prepare the blank matrix solution of Poria cocos according to the method determined under item "7".

[0114] Take the multi-pesticide residue reference solution (batch number: 380-03381).

[0115] Take Poria cocos powder (passed through No. 3 sieve, batch number: FLKB2006) and prepare Poria cocos blank matrix solution according to the method determined under item "7". Accurately measure 1.0 mL, place it on a nitrogen blower, and concentrate it to about 0.6 mL in a 40°C water bath. Add 50 μL of multi-pesticide residue reference solution (batch number: 380-03381) respectively, dilute it to 1 mL with acetonitrile, and vortex mix to obtain a Poria cocos matrix mixed control solution.

[0116] According to the chromatographic conditions under item "2", 1 mL of blank solvent, blank Poria matrix solution, multi-pesticide residue reference solution and mixed Poria matrix reference solution were accurately aspirated for determination. The total ion current chromatogram (TIC) is as follows: Figures 16 - 17 Among them, the LC-MS spectrum of ethoprophos in the blank matrix solution of Poria cocos is shown in Figure 18 As shown in the figure, the LC / MS spectrum of the multi-pesticide residue reference solution is as follows: Figure 19 As shown in Figure 2, the LC-MS spectrum of ethoprophos in the Poria matrix mixed control solution is as follows: Figure 20 shown.

[0117] 11 Sample determination

[0118] Take 5 g each of Poria cocos powder (passed through No. 3 sieve, batch numbers: 20200824, 20201103, 20201104) and Ganoderma lucidum powder (passed through No. 3 sieve, batch number: 20200763), accurately weigh them, and prepare Poria cocos sample solution and Ganoderma lucidum sample solution according to the method determined under item "7", respectively.

[0119] Take 5 g of Poria cocos powder (passed through No. 3 sieve, batch number: FLKB2006) and Ganoderma lucidum powder (passed through No. 3 sieve, batch number: LZKB2003), prepare Poria cocos and Ganoderma lucidum blank matrix solutions according to the method determined under item "7", accurately measure 1.0 mL of each, place on a nitrogen blowdown instrument, and concentrate in a 40°C water bath to about 0.6 mL, add 50 μL of multi-pesticide residue reference solution (batch number: 380-03381) respectively, dilute with acetonitrile to 1 mL, vortex mix, and obtain a matrix mixed reference solution.

[0120] The determination was carried out according to the determination method under item "8" and the amount of each banned pesticide residue was calculated by the internal standard method. The results are shown in Table 6.

[0121] Table 6

[0122]

[0123] The results showed that no banned pesticide residues were detected in three batches of Poria cocos and one batch of Ganoderma lucidum, and the test results all met the requirements of pesticide residue standards.

[0124] Comparative Example 1

[0125] 1. Instruments, reagents and test drugs

[0126] Same as Example 1

[0127] 2 Chromatographic conditions

[0128] Gas chromatography - tandem mass spectrometry chromatographic conditions: An elastic quartz capillary column with (50% phenyl)-methyl polysiloxane as the stationary liquid (column length 30 m, column inner diameter 0.25 mm, film thickness 0.25 μm). The injection port temperature is 250 °C, and splitless injection is used. The carrier gas is high-purity helium (He). The injection port is in constant pressure mode, and the column inlet pressure is 146 kPa. Programmed temperature rise: The initial temperature is 60 °C, held for 1 minute, then raised to 120 °C at a rate of 30 °C / min, then raised to 160 °C at a rate of 10 °C / min, then raised to 230 °C at a rate of 2 °C / min, and finally raised to 300 °C at a rate of 15 °C / min and held for 6 minutes.

[0129] Mass spectrometry conditions: Detected with a triple quadrupole tandem mass spectrometer; the ion source is an electron impact source (EI), and the ion source temperature is 250 °C. The collision gas is nitrogen or argon. The mass spectrometry transfer interface temperature is 250 °C. The mass spectrometry monitoring mode is multiple reaction monitoring (MRM). The reference retention time, monitored ion pairs, and collision energy (CE) information for each compound are shown in Table 7. To improve the detection sensitivity, each pesticide can be detected in segments according to the retention time.

[0130] Table 7

[0131]

[0132] 3 Preparation of control solution

[0133] Same as Example 1

[0134] 4 Preparation of internal standard solution

[0135] Same as Example 1

[0136] 5 Preparation of blank matrix solution

[0137] Take 5 g of Poria cocos medicinal material powder (passed through No. 3 sieve, batch number: FLKB2006), accurately weighed, and prepare the Poria cocos blank matrix solution according to the method determined in item "7" of Example 1.

[0138] 6 Preparation of matrix mixed control solution

[0139] Precisely measure 1.0 mL of the Poria cocos blank matrix solution, place it on a nitrogen blower, concentrate it to about 0.6 mL in a 40 °C water bath, add 50 μL of the multi-pesticide residue reference solution (batch number: 380 - 03381), dilute it to 1 mL with acetonitrile, and vortex to mix evenly to obtain the matrix mixed control solution.

[0140] 7 Preparation of test sample solution

[0141] Take Poria cocos medicinal material powder (passed through No. 3 sieve, batch number: 20200824), and prepare the Poria cocos sample solution to be measured according to the method determined in item "7" of Example 1.

[0142] Precisely pipette 1 μl of each of the above two Poria cocos sample solutions to be measured and the matrix mixed control solution respectively, and perform the determination according to the chromatographic conditions in item "2" of Example 1 and the chromatographic conditions in item "2" of Comparative Example 1. The retention time results are shown in Table 8.

[0143] Table 8

[0144]

[0145] The results show that compared with Example 1, the retention time of Comparative Example 1 increased by about 40%. That is, the detection method of Example 1 can reduce the analysis time by 40% and has high analysis efficiency.

[0146] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0147] The above-described embodiments only represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A method for detecting pesticide residues in fungal Chinese medicinal materials, characterized in that, Comprising the following steps: Preparing a pesticide mixed reference solution, wherein the pesticide mixed reference solution includes one or more of ethoprophos, phorate, α-HCH, β-HCH, γ-HCH, δ-HCH, fipronil, flonicamid, fipronil sulfoxide, fipronil sulfone, aldrin, dieldrin, α-endosulfan, β-endosulfan, endosulfan sulfate, p,p'-DDE, o,p'-DDT, p,p'-DDD, p,p'-DDT, demeton (O+S), terbufos, methyl parathion, parathion, dicofol, isofenphos-methyl, isocarbophos, nitrofen, fenamiphos, and cyanophos; Mixing the Chinese medicinal materials of the fungi to be tested, a salting-out agent and a solvent, collecting the extract, purifying the extract, and preparing a sample solution to be tested; Detecting the pesticide mixed reference solution and the sample solution to be tested by gas chromatography-tandem mass spectrometry, and qualitatively detecting the pesticide residue situation in the sample solution to be tested; The chromatographic column temperature programming of the gas chromatography in the gas chromatography-tandem mass spectrometry includes: an initial temperature of 90°C to 110°C, holding for 40 s to 80 s, heating at a rate of 25°C / min to 35°C / min to 110°C to 130°C, then heating at a rate of 5°C / min to 15°C / min to 150°C to 170°C, then heating at a rate of 2°C / min to 6°C / min to 220°C to 240°C, and then heating at a rate of 10°C / min to 20°C / min to 310°C to 330°C, holding for 1 min to 3 min.

2. The detection method of pesticide residues in fungal Chinese medicinal materials according to claim 1, characterized in that, The pesticide mixed reference solution includes ethoprophos, phorate, α-HCH, β-HCH, γ-HCH, δ-HCH, fipronil, flonicamid, fipronil sulfoxide, fipronil sulfone, aldrin, dieldrin, α-endosulfan, β-endosulfan, endosulfan sulfate, p,p'-DDE, o,p'-DDT, p,p'-DDD, p,p'-DDT, demeton (O+S), terbufos, methyl parathion, parathion, dicofol, isofenphos-methyl, isocarbophos, nitrofen, fenamiphos, and cyanophos.

3. The detection method for pesticide residues in fungal Chinese medicinal materials according to claim 1, characterized in that, The chromatographic column of the gas chromatography in the gas chromatography-tandem mass spectrometry is a fused silica capillary column with (50% phenyl)-methyl polysiloxane as the stationary liquid.

4. The detection method for pesticide residues in fungal Chinese medicinal materials according to claim 1, characterized in that, The gas chromatography conditions of the gas chromatography-tandem mass spectrometry include at least one of the following conditions: (1) The inlet temperature is 240°C to 260°C; (2) Splitless injection; (3) The carrier gas is helium; (4) The inlet is in a constant pressure mode; (5) The column head pressure is 135 kPa to 155 kPa.

5. The detection method for pesticide residues in fungal Chinese medicinal materials according to claim 1, characterized in that, The mass spectrometry conditions of the gas chromatography-tandem mass spectrometry include at least one of the following conditions: (1) Detecting with a triple quadrupole tandem mass spectrometer; (2) The ion source is an electron impact source; (3) The ion source temperature is 240°C to 260°C; (4) The collision gas is nitrogen or argon; (5) The mass spectrometry transfer interface temperature is 240°C to 260°C; (6) The mass spectrometry monitoring mode is multiple reaction monitoring.

6. The detection method for pesticide residues in fungal Chinese medicinal materials according to any one of claims 1 to 5, characterized in that, Including at least one of the following features: (1) The salting-out agent includes sodium chloride; (2) The solvent includes acetonitrile; (3) The mass ratio of the Chinese medicinal material of the fungus to be tested to the salting-out agent is (4-6):

1.

7. The detection method for pesticide residues in fungal Chinese medicinal materials according to any one of claims 1 to 5, characterized in that, Purifying the extract includes the following steps: flowing the extract through a solid-phase extraction column filled with a hydrophilic-lipophilic balance material.

8. The detection method for pesticide residues in fungal Chinese medicinal materials according to any one of claims 1 to 5, characterized in that, The solvent in the pesticide mixed reference solution includes acetonitrile.

9. The detection method for pesticide residues in fungal Chinese medicinal materials according to any one of claims 1 to 5, characterized in that, It also includes the following steps: Quantitatively detecting the pesticide residue in the Chinese medicinal material of the fungus by using the internal standard method, and the internal standard substance of the internal standard method is triphenyl phosphate.

10. The detection method for pesticide residues in fungal Chinese medicinal materials according to any one of claims 1 to 5, characterized in that, The Chinese medicinal material of the fungus is Ganoderma lucidum medicinal material, Ganoderma lucidum cut crude drug, Poria cocos medicinal material or Poria cocos cut crude drug.

Images