A method for detecting pesticide residues in Tibetan medicinal materials

Abstract

The present invention relates to a method for controlling the quality of medicinal materials, and provides a method for detecting pesticide residues in Tibetan medicinal materials. The present invention comprises the following steps: step 1, preparing a test solution from a Tibetan medicinal material to be tested; step 2, detecting the test solution by high performance liquid chromatography-tandem mass spectrometry to obtain the result of the determination of pesticide residues; wherein the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler, using 0.05-0.2% formic acid solution as a mobile phase A, and using acetonitrile as a mobile phase B. The present invention applies high performance liquid chromatography-tandem mass spectrometry to optimize the detection conditions, and the provided method can simultaneously detect the residues of 25 banned pesticides from a variety of commonly used Tibetan medicinal materials. And experiments show that the method has good specificity, linearity, sensitivity, precision and accuracy. Therefore, the method provided by the present invention can meet the requirements for detecting pesticides in Tibetan medicinal materials.

Description

Technical Field

[0001] The invention relates to a method for controlling the quality of medicinal materials, and in particular to a method for detecting pesticide residues in Tibetan medicinal materials. Background Art

[0002] Pesticide residues in medicinal materials and pieces of medicine have always been a key issue in drug safety. The General Rules for the Verification of Medicinal Materials and Pieces of the Four Parts of the 2020 Edition of the Chinese Pharmacopoeia stipulate that 33 banned pesticides in medicinal materials and pieces of medicine (plants) shall not be detected (shall not exceed the limit of quantification), and GC-MS / MS and LC-MS / MS methods are recommended for determination. During application, the preparation method and detection conditions of the test solution shall be optimized according to actual conditions. Tibetan medicinal materials, as a kind of ethnic medicine, have outstanding efficacy. With the in-depth study of the efficacy of Tibetan medicine, the demand for medicine has increased, and the artificial planting area and planting varieties have increased year by year. Some commonly used Tibetan medicines such as Rhodiola rosea, Hippophae rhamnoides, and Phyllanthus emblica have large-scale planting bases. In order to reduce pests and diseases during their planting process, pesticides are often used. Therefore, it is very necessary to detect pesticide residues in Tibetan medicinal materials and clarify their contamination status.

[0003] At present, there are few reports on the detection of pesticide residues in Tibetan medicinal materials. The recommended determination method is difficult to be directly used in the detection of Tibetan medicine. It is necessary to optimize the preparation method of the test solution and the detection conditions in a targeted manner to achieve the separation and detection of multiple pesticides in different Tibetan medicine matrices. Summary of the invention

[0004] In view of the problems in the prior art, the present invention provides a method for detecting pesticide residues in Tibetan medicinal materials, aiming to optimize the detection conditions and realize simultaneous and accurate LC-MS / MS detection of 25 banned pesticides in a variety of commonly used Tibetan medicinal materials.

[0005] A method for detecting pesticide residues in Tibetan medicinal materials, comprising the following method:

[0006] Step 1, preparing a test solution of the Tibetan medicinal material to be tested;

[0007] Step 2, detecting the test solution by high performance liquid chromatography-tandem mass spectrometry to obtain the result of pesticide residue determination;

[0008] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler, 0.05-0.2% formic acid solution is used as mobile phase A, and acetonitrile is used as mobile phase B.

[0009] Preferably, the chromatographic conditions further include an elution gradient of:

[0010]

[0011] Preferably, the test solution is prepared by the QuEChERS method.

[0012] Preferably, the chromatographic conditions further include: a chromatographic column Thermo Accucore aQ, with a column length of 10 cm, an inner diameter of 2.1 mm, and a particle size of 2.6 μm.

[0013] Preferably, the chromatographic conditions further include: using 0.1% formic acid solution as mobile phase A and using acetonitrile as mobile phase B.

[0014] Preferably, the chromatographic conditions also include: a flow rate of 0.28-0.32 ml / min, a column temperature of 38-42° C., and an injection volume of 1-5 μl.

[0015] Preferably, in step 2, the mass spectrometry conditions are detected by a triple quadrupole tandem mass spectrometer, the ion source is an electrospray ESI ion source, the positive ion scanning mode, the monitoring mode is multiple reaction monitoring (MRM), the nebulizer temperature is 300-325° C., the gas flow rate is 10-12 L / min, the ion source voltage is 4000-4500 V, and the nebulizer pressure is 15-20 psi; the retention time of each compound, the monitoring ion pair, the collision voltage and the collision energy are as follows:

[0016]

[0017]

[0018]

[0019] Preferably, in step 2, the total ion current graph is collected in three time periods, and the time periods are divided according to the retention time 0-4min, 4-10min, and 10-15min.

[0020] Preferably, the Tibetan medicinal material is Rhodiola rosea, Aucklandia lappa, Phyllanthus emblica, Terminalia chebula, Terminalia chebula, Hippophae rhamnoides, Cyperus rotundus, Pterocephalus chinensis or Lamiaceae.

[0021] Preferably, the pesticide includes ethametsulfuron, fenamiphos, fenamiphos sulfone, fenamiphos sulfoxide, fenfosphos, aldicarb sulfone, aldicarb sulfoxide, phorate sulfone, phorate sulfoxide, metsulfuron, monocrotophos, carbofuran, 3-hydroxycarbofuran, phosphamidon, chlorsulfuron, thiocyanate, cadastre, chlorpyrifos, chlorsulfuron, chlorpyrifos ...

[0022] The present invention provides a method for detecting pesticide residues in Tibetan medicinal materials. For the detection of 25 banned pesticides in commonly used Tibetan medicinal materials, the preparation of test solution, chromatographic conditions and mass spectrometry conditions were optimized. Root and rhizome medicinal materials are rich in starch components, fruit and seed medicinal materials are rich in oil and sugar components, easy to stick during the powdering process and difficult to sieve, and whole herb medicinal materials are rich in pigments. Therefore, we selected 9 commonly used Tibetan medicinal materials in these three different medicinal parts as research objects, and optimized the preparation of test solution. The sample powder and reagent can be effectively mixed by vortexing, and acetonitrile after refrigeration is used for extraction to improve the recovery rate. After purification, the supernatant is directly filtered to obtain the test solution, shortening the preparation cycle of the test solution. Finally, the optimized high performance liquid chromatography-tandem mass spectrometry method is used for detection. The present invention can separate 25 banned pesticides from the chemical components of the commonly used Tibetan medicinal material matrix, thereby realizing the simultaneous and accurate detection of these 25 banned pesticides. The method provided by the invention is simple to operate, has high accuracy (recovery rate 60%-130%), precision (RSD < 4.5%) and sensitivity (detection limit 0.0027-5.5671 ng / ml, quantitative limit 0.0080-16.8700 ng / ml), and has good application prospect in the quality detection of Tibetan medicinal materials.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the total ion current chromatogram of the mixed reference solution;

[0026] Figure 2 This is the total ion flow chromatogram obtained by detecting condition 1 in the elution gradient screening in Experimental Example 1;

[0027] Figure 3 This is the total ion flow chromatogram obtained by detecting condition 2 in the elution gradient screening in Experimental Example 1;

[0028] Figure 4 This is the total ion flow chromatogram obtained by detecting condition 3 in the elution gradient screening in Experimental Example 1;

[0029] Figure 5 This is the total ion flow chromatogram obtained by detecting condition 4 in the elution gradient screening in Experimental Example 1;

[0030] Figure 6 This is the total ion flow chromatogram obtained by detecting condition 5 in the elution gradient screening in Experimental Example 1;

[0031] Figure 7 It is the total ion current diagram of the mixed reference substance in the divided time period and the undivided time period, where: A is the undivided time period, and B is the divided time period;

[0032] Figure 8 The total ion current diagram of the reference substance obtained at different time periods;

[0033] Fig. 9 The total ion current graphs of 9 blank Tibetan medicine matrices, blank solvents and mixed reference solutions, including: A: Hippophae rhamnoides; B: Phyllanthus emblica; C: Pterocephalus chinensis; D: Aucklandia lappa; E: Terminalia chebula; F: Rhodiola rosea; G: Lamiophloea foetida; H: Terminalia chebula; I: Tripterygium wilfordii; a: mixed reference solution; b: blank Tibetan medicine matrix; c: blank solvent. DETAILED DESCRIPTION

[0034] In the following examples and experimental examples, the reagents and raw materials used are all commercially available.

[0035] Example 1 Detection method of 25 pesticide residues in Tibetan medicinal materials

[0036] This embodiment includes the following steps:

[0037] Step 1, using the QuEChERS method to prepare the test Tibetan medicinal materials into a test solution, the specific steps are as follows: take 3.0g of the test powder (passed through a No. 3 sieve), accurately weigh, place in a 50ml polystyrene stoppered centrifuge tube, add 15ml of 1% glacial acetic acid solution, vortex to fully infiltrate the powder, let stand for 30 minutes, accurately add 15ml of acetonitrile that has been refrigerated in advance, vortex to mix, add 7.5g of a mixed powder of anhydrous magnesium sulfate and anhydrous sodium acetate (4:1), and immediately The mixture was shaken and cooled in an ice bath for 10 min. The mixture was centrifuged (3300 rpm) for 3 min. 9 ml of the supernatant was taken and placed in a dispersed solid phase extraction purification tube pre-loaded with purification materials [900 mg of anhydrous magnesium sulfate, 300 mg of N-propylethylenediamine, 300 mg of octadecylsilane bonded silica gel (C18), 300 mg of silica gel, 90 mg of graphitized carbon black (GCB)] and vortexed to mix thoroughly. The supernatant was accurately aspirated and filtered to obtain the product.

[0038] Step 2, detecting the test solution by high performance liquid chromatography-tandem mass spectrometry to obtain the result of pesticide residue content;

[0039] The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler, 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B, flow rate of 0.3 ml / min, column temperature of 40°C, injection volume of 2 μl, elution mode of gradient elution, and the gradient elution procedure is as follows.

[0040] The elution gradient is:

[0041]

[0042]

[0043] The mass spectrometry conditions were:

[0044] The detection was carried out by triple quadrupole tandem mass spectrometer with electrospray ionization (ESI) as the ion source, positive ion scanning mode, multiple reaction monitoring (MRM) as the monitoring mode, nebulizer temperature of 300°C, gas flow rate of 11 L / min, ion source voltage of 4000 V, and nebulizer pressure of 15 psi.

[0045] In this embodiment, the total ion current graph is collected in three time periods, and the time periods are divided according to the retention time 0-4min, 4-10min, and 10-15min.

[0046] This embodiment can be used to simultaneously detect the following 25 banned pesticides: ethametsulfuron, fenamiphos, fenamiphos sulfone, fenamiphos sulfoxide, fenfosphos, aldicarb sulfone, aldicarb sulfoxide, phorate sulfone, phorate sulfoxide, metsulfuron, monocrotophos, carbofuran, 3-hydroxycarbofuran, phosphamidon, chlorsulfuron, thiophanate-methyl, cadusafos, chlorpyrifos, chlorpyrifos, chlorsulfuron, chlorpyrifos, chlorpyrifos, chlorpyrifos, chlorpyrifos, chlorpyrifos, chlorpyrifos, chlorpyrifos, chlorpyrifos, chlorpyrifos, terbufos sulfoxide, coumaphos and chlorpyrifos.

[0047] The total ion current chromatogram obtained by testing the mixed reference solution of the above 25 pesticides is as follows: Figure 1 The retention time, parent ion, daughter ion and corresponding collision voltage and collision energy of 25 pesticides are shown in the following table:

[0048]

[0049]

[0050]

[0051] The peak areas of 25 banned pesticides in each medicinal material matrix mixed control solution are obtained respectively, and the contents of 25 banned pesticides in the sample to be tested are calculated using the matrix matching standard curve method. In the present invention, taking the Tibetan wood fragrance matrix mixed control as an example, the matrix matching standard curve method includes the following steps:

[0052] Take a blank matrix sample of Tibetan wood fragrance, prepare a blank matrix solution according to the test solution preparation method of Example 1, add the pesticide mixed reference solution to prepare a matrix mixed control solution with a concentration of 5ng / ml, 10ng / ml, 20ng / ml, 50ng / ml, 100ng / ml, and 200ng / ml, respectively, and measure, and record the peak area of ​​each concentration of the matrix mixed control solution. With the peak area (y) as the ordinate and the concentration (x, ng / ml) as the abscissa, a standard curve is drawn by linear regression to obtain a regression equation.

[0053] In the present invention, the linear equations, linear ranges and correlation coefficients of 25 banned pesticides in the Tibetan wood matrix mixed control are shown in the table.

[0054] Linear equations and correlation coefficients (R) of 25 banned pesticides in mixed control of Tibetan wood matrix 2 ), linear range:

[0055]

[0056]

[0057] Tibetan medicinal materials applicable to the present embodiment include: Rhodiola rosea, Aucklandia lappa, Phyllanthus emblica, Terminalia chebula, Terminalia chebula, Hippophae rhamnoides, Cyperus rotundus, Pterocephalus chinensis or Lamiaceae.

[0058] The technical solution of the present invention is further described below through experimental examples. The detection conditions not specifically described in the following experimental examples are set with reference to Example 1.

[0059] Experimental Example 1 Optimization of gradient elution conditions and segmentation time

[0060] In order to obtain better separation effect, the gradient elution conditions were optimized.

[0061] Condition 1 is shown in the following table:

[0062]

[0063]

[0064] The obtained chromatogram is as follows Figure 2 shown.

[0065] Condition 2 is shown in the following table:

[0066]

[0067] The obtained chromatogram is as follows Figure 3 shown.

[0068] Condition 3 is shown in the following table:

[0069]

[0070] The obtained chromatogram is as follows Figure 4 shown.

[0071] Condition 4 is shown in the following table:

[0072]

[0073] The obtained chromatogram is as follows Figure 5 shown.

[0074] Condition 5 is shown in the following table:

[0075]

[0076] The obtained chromatogram is as follows Figure 6 shown.

[0077] It can be seen that the change of elution gradient has a significant impact on the peak shape, separation degree and peak distance of the chromatographic peak. In order to accurately detect the quantitative detection and divide the time of subsequent segmented detection, condition 5 was selected as the final gradient elution condition.

[0078] Since 25 banned pesticides were analyzed at the same time, they were divided into three time periods according to retention time. Compared with non-segmented analysis, the number of ions collected in each time period was reduced, the sampling cycle was shortened, and the sensitivity of the instrument was improved. The total ion flow chromatograms of divided and undivided time periods are shown in the figure below. Figure 7 As shown, it can be seen that when the data is collected in different time periods, the peak shape and separation of the chromatogram are better.

[0079] Adjust the segment time and fine-tune the mobile phase ratio at the same time, so that the peak time of the compound to be tested is in the corresponding segment class, avoiding the situation where the peak is generated at the segment node and the peak collection information is incomplete. Figure 8 As shown in the figure, the optimization results of the segment time are 0-4min, 4-10min, and 10-15min.

[0080] Experimental Example 2 Methodology Verification

[0081] 1. Exclusivity

[0082] In the experiment, blank matrix solutions, mixed reference solutions and blank solvents of 9 medicinal materials were injected respectively, and the total ion current chromatograms were recorded after HPLC-MS / MS detection. Figure 8 ,It can be seen from the figure that there is no chemical component interference in the blank matrix and blank solvent, indicating that this method has good specificity.

[0083] 2. Linear range, limit of detection (LOD) and limit of quantification (LOQ)

[0084] First, the mixed reference substance solutions of 25 kinds of banned pesticides are prepared into reference substance solutions with concentrations of 5ng / ml, 10ng / ml, 20ng / ml, 50ng / ml, 100ng / ml, and 200ng / ml, and 2 μl of the above solutions are accurately drawn respectively, injected into a liquid chromatography-tandem mass spectrometer, and the peak areas of the mixed reference substance solutions of each concentration are recorded. 3g of Rhodiola rosea, Tibetan woodruff, Phyllanthus emblica, Hairy Terminalia chebula, Terminalia chebula, Hippophae rhamnoides, Iron club hammer, Pterocephalus scabra, and Herba Leptospermi sibiricum are accurately weighed respectively, and pre-processed according to the test solution preparation method of Example 1, and the mixed reference substance solutions of pesticides are added to be respectively prepared into matrix mixed reference solutions of different concentrations, and measured, and the peak areas of the matrix mixed reference solutions of each concentration are recorded. With the peak area value (y) as the ordinate and the concentration (x, ng / ml) as the abscissa, regression calculation is performed to obtain a regression equation. The results showed that within the linear range of 5ng / ml to 200ng / ml, the linear relationships of the 25 banned pesticides were mostly greater than 0.99, indicating a good linear relationship.

[0085] 3. Limit of detection and limit of quantification

[0086] The limits of quantitation and detection were calculated based on the standard deviation of the response and the slope of the standard curve according to the following expressions:

[0087] LOQ=10δ / S (1)

[0088] LOD=3.3δ / S (2)

[0089] δ is the deviation of the response value; S is the slope of the standard curve. The results show that the detection limit of the method used in the present invention is 0.0027-5.5671 ng / ml, and the quantitative limit is 0.0080-16.8700 ng / ml, which meets the requirements for the detection of banned pesticides.

[0090] 4. Precision

[0091] Take the mixed reference solution of 25 banned pesticides, dilute it with acetonitrile to 100ng / ml, inject it into the LC-MS / MS instrument, inject it 6 times in a row, record the peak area, and calculate the RSD of the peak area of ​​each pesticide. The results show that the RSD of the 25 pesticides is between 0.5% and 4.5%, indicating that the instrument has good precision and meets the standard for pesticide multi-residue detection.

[0092] 5. Recovery rate and repeatability

[0093] From many Tibetan medicines, 9 kinds of medicinal materials that can obtain blank matrices, including Rhodiola rosea, Tibetan wood fragrance, Phyllanthus emblica, Terminalia chebula, Terminalia chebula, Hippophae rhamnoides, Iron club hammer, Pterocephalus pterocarpon, and Du Yiwei, were selected for sample recovery experiments. 3g of blank matrix sample powder of each medicinal material was taken, and 25 kinds of pesticide mixed reference substance solutions were accurately added respectively, and the sample recovery rate test at three concentration levels of low concentration (20ng / ml), medium concentration (50ng / ml), and high concentration (100ng / ml) was performed. Six parallel experiments were performed at each level, and the test solution was prepared according to the method in Example 1. The test solution was taken, measured by liquid chromatography-tandem mass spectrometry, the peak area was recorded, and the sample recovery rate and the corresponding RSD value of each reference substance at three concentration levels were calculated. The recovery rate of most of the pesticides at three different addition levels in the 9 medicinal material matrices ranged from 60% to 130%, and the RSD range was between 0.54% and 9.04%, indicating that the repeatability of the method was good and met the basic requirements of pesticide residue detection.

[0094] The method of the present invention was used to detect 25 banned pesticides in 60 batches of Tibetan medicinal materials samples. The results showed that 1 batch of Pterocephalus and 1 batch of Aucklandia lappa respectively contained chlorpyrifos, both exceeding the quantitative limit; 2 batches of Terminalia chebula, 2 batches of Rhodiola rosea and 1 batch of Tripterygium wilfordii contained methyl parathion, all exceeding the quantitative limit.

[0095] Through the above embodiments and experimental examples, it can be seen that the method provided by the present invention can improve the detection efficiency of pesticide residues in Tibetan medicinal materials, and can simultaneously detect the residues of 25 banned pesticides from 9 commonly used Tibetan medicinal materials. And the experiment shows that the specificity, linearity, sensitivity, precision and sample recovery rate of the method are all very good. Therefore, the method provided by the present invention can meet the requirements of accurately detecting pesticide residues in Tibetan medicinal materials.

Claims

1. A method for detecting pesticide residues in Tibetan medicinal materials, characterized in that: The following methods are included: Step 1, preparing a test solution of the Tibetan medicinal material to be tested; Step 2, using high performance liquid chromatography-tandem mass spectrometry to detect the test solution to obtain the result of pesticide residue determination; the detected pesticides include ethametsulfuron, fenamiphos, fenamiphos sulfone, fenamiphos sulfoxide, difenothion, aldicarb sulfone, aldicarb sulfoxide, phorate sulfone, phorate sulfoxide, metsulfuron, monocrotophos, carbofuran, 3-hydroxycarbofuran, phosphamidon, chlorsulfuron, thiocyanate, cadusafos, chlorfenapyr, ethoprophos, demeton, chlordimeform, terbufos sulfone, terbufos sulfoxide, coumaphos and chlorpyrifos; The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler, the column length is 10 cm, the inner diameter is 2.1 mm, and the particle size is 2.6 µm; 0.05-0.2% formic acid solution is used as the mobile phase A, and acetonitrile is used as the mobile phase B; The elution gradient is: The mass spectrometry conditions were as follows: detection by triple quadrupole tandem mass spectrometer, electrospray ion source, positive ion scan mode, multiple reaction monitoring mode, nebulizer temperature 300-325°C, gas flow rate 10-12 L / min, ion source voltage 4000-4500 V, nebulizer pressure 15-20 psi; The retention time, monitoring ion pair, collision voltage and collision energy of each compound are as follows: In step 2, the total ion current graph is collected in three time periods, and the time periods are divided according to the retention time 0-4 min, 4-10 min, and 10-15 min; The preparation method of the test solution is a QuEChERS method, which includes: taking a powder of a Tibetan medicinal material to be tested, adding a glacial acetic acid solution, vortexing to fully infiltrate the powder, placing it, adding acetonitrile, vortexing to mix it, adding a mixed powder of anhydrous magnesium sulfate and anhydrous sodium acetate, shaking it, cooling it, centrifuging it to obtain a supernatant, placing it in a dispersed solid phase extraction purification tube pre-installed with a purification material, vortexing it to fully mix it, absorbing the supernatant, filtering it, and obtaining a subsequent filtrate; the purification material includes anhydrous magnesium sulfate, N-propylethylenediamine, octadecylsilane bonded silica gel, silica gel and graphitized carbon black; The Tibetan medicinal materials are Rhodiola rosea, Aucklandia lappa, Phyllanthus emblica, Terminalia chebula, Terminalia chebula, Hippophae rhamnoides, Cyperus rotundus, Pterocephalus chinensis or Lamiaceae.

2. The detection method according to claim 1, characterized in that: The chromatographic conditions also include: the chromatographic column is Thermo Accucore aQ.

3. The detection method according to claim 1, characterized in that: The chromatographic conditions also include: using 0.1% formic acid solution as mobile phase A and using acetonitrile as mobile phase B.

4. The detection method according to claim 1, characterized in that: The chromatographic conditions also include: a flow rate of 0.28-0.32 ml / min, a column temperature of 38-42° C., and an injection volume of 1-5 μl.

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