Pesticide mixed standard substance as well as preparation method and application thereof

Abstract

The invention discloses a pesticide mixed standard substance as well as a preparation method and application thereof, 54 pesticide monomer purity standard substances are selected as raw materials, the molecular connectivity index of each pesticide monomer and related solvent is calculated by adopting QSPR (Quantitative Surface Plasmon Resonance), methodological verification is performed on a gas chromatograph-mass spectrometer and a liquid chromatograph-mass spectrometer valuing method, and the molecular connectivity index of each pesticide monomer and related solvent is calculated by adopting QSPR (Quantitative Surface Plasmon Resonance). The method comprises the following steps: carrying out valuing on 33 pesticide mixed standard substances, and preparing standard curves adopted in the valuing process by adopting primary or secondary national standard substances of each pesticide monomer solution, so that a valuing result has traceability; the uniformity and stability of the prepared pesticide mixed standard substance are evaluated, the relative expansion uncertainty of the pesticide mixed standard substance is evaluated through the uncertainty propagation rate, the relative expansion uncertainty Urel of each pesticide monomer is smaller than or equal to 5%, and k is equal to 2; the method can be used for quality control operations such as standard recovery and reference substance verification in pesticide residue detection of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of standard substances, and in particular to a pesticide mixed standard substance and a preparation method and application thereof. Background Art

[0002] Pesticides are agents used to prevent and control pests and diseases, regulate plant growth, etc., in order to increase product yield and quality. Due to climate, soil, and the composition of the crops themselves, pests and diseases are common, and the use of pesticides is essential. Many surveys have shown that in the process of planting some Chinese medicinal materials, because growers often lack basic knowledge of plant protection, the preferred standard for pesticides is high efficiency and low cost. In addition, unscientific pesticide use habits can easily cause pesticide residues in medicinal materials.

[0003] Regarding promoting the inheritance and innovative development of traditional Chinese medicine, it is proposed to "strictly manage the use of pesticides, fertilizers, plant growth regulators, etc., and improve the pesticide residues and heavy metal limit standards for Chinese medicinal materials by region and variety" in strengthening the quality control of Chinese medicinal materials. The new 2020 edition of the "Chinese Pharmacopoeia" General Chapter 2341 Pesticide Residue Determination Method added a fifth method for detecting the residues of 33 banned pesticides in Chinese medicinal materials, marking a new height in the quality supervision of Chinese medicinal materials.

[0004] However, according to the standard method of the 2020 edition of the Chinese Pharmacopoeia, the determination of banned pesticide residues in traditional Chinese medicines requires the use of corresponding standard substances for the establishment of calibration curves, verification of spiked recovery rates, etc. At present, there are no directly usable pesticide mixed standard substances with measurement uncertainty on the market. Therefore, it is of great significance to develop a 33-item pesticide mixed standard substance with measurement uncertainty. Summary of the invention

[0005] Technical issues solved:

[0006] The purpose of the present invention is to overcome the technical problems existing in the prior art, such as the lack of standard substances in the fifth method of the determination of pesticide residues in 2341 of the general rules of the fourth volume of the 2020 edition of the "Chinese Pharmacopoeia". The present application provides a mixed pesticide standard substance and its preparation method and application. The standard substance has an accurate quantity value, a reliable and accurate uncertainty reference range, a traceable value setting process and a stable and uniform packaging unit. The use of the standard substance to establish a standard curve, spike recovery, and comparison verification can improve the accuracy of the determination of pesticide residues in traditional Chinese medicines and ensure the safety and controllability of the quality of traditional Chinese medicines.

[0007] Technical solution:

[0008] To achieve the above objectives, this application is implemented through the following technical solutions:

[0009] A method for preparing a pesticide mixed standard substance comprises the following steps:

[0010] Step 1: Select 54 kinds of pesticide monomer purity standards as raw materials. The purity of each pesticide monomer purity standard is ≥95%. Use nuclear magnetic resonance and mass spectrometry to characterize the structure of the pesticide monomer purity standard and verify the purity:

[0011] Step 2: Calculate the molecular connectivity index of each pesticide monomer and the relevant solvent using the Quantitative Structure-Property Relationship model QSPR, where the solvent is acetone, acetonitrile, methanol, n-hexane, toluene, chloroform or cyclohexane;

[0012] Step 3: Methodological validation of the gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry determination methods, and relevant validation of linearity, repeatability, reproducibility, limit of quantification, limit of detection, and recovery parameters;

[0013] Step 4: The 33 pesticide mixed standard substances were calibrated. The standard curves used in the calibration process were prepared with the first-level or second-level national standard substances of each pesticide monomer solution, with a concentration of 100 mg / L, so that the calibration results were traceable;

[0014] Step 5: Evaluate the uniformity and stability of the pesticide mixed standard substances, and use the uncertainty propagation rate to assess its relative expanded uncertainty. The relative expanded uncertainty U of each pesticide monomer is rel Both ≤5%, k=2.

[0015] Preferably, the method for characterizing the structures of the 54 kinds of pesticide monomer purity standard products in the first step is two-dimensional nuclear magnetic resonance, the method for verifying the purity of the 54 kinds of pesticide monomer purity standard products is nuclear magnetic hydrogen spectrum and high-resolution mass spectrometry, and the 54 kinds of pesticide monomer purity standard products are methyl parathion, methyl parathion, parathion, monocrotophos, phosphamidon, α-BHC, β-BHC, lindane, δ-BHC, p,p'-DDT, o,p'-DDT, p,p'-DDD, p,p'-DDD, chlordimeform, herbicide ether, aldrin, dieldrin, fenamiphos, fenamiphos sulfone, One or more of fenamiphos sulfoxide, fenthion, cadusafos, coumaphos, terbufos, terbufos sulfone, terbufos sulfoxide, chlorsulfuron, ethametsulfuron, metsulfuron-methyl, phorate, phorate sulfone, phorate sulfoxide, methyl isofenphos, demeton, carbofuran, trihydroxycarbofuran, aldicarb, aldicarb sulfoxide, ethoxyprolin, chlorfenapyr, isocarb, endosulfan sulfate, α-endosulfan, β-endosulfan, fipronil, flunitrile, fipronil sulfone, fipronil sulfoxide, 2,4'-dicofol, dicofol, thiocyanate, and methyl thiocyanate.

[0016] Preferably, the pesticide monomer in the first step is ethoprophos.

[0017] Preferably, in the second step, QSPR is used to calculate the molecular connectivity index of each pesticide monomer and the solvent, thereby determining that the solvent is acetonitrile.

[0018] Preferably, the methodological validation qualification standard for the fixed value method in the third step is: R 2 ≥99.0%, RSD of repeatability <10.0%, RSD of reproducibility <10.0%, spiked recovery rates were all between 60% and 130%, detection limit <5ng / ml and quantification limit <10ng / ml.

[0019] Preferably, the fourth step of preparing the mixed pesticide standard substance is as follows: weigh 0.200-0.500 mg of 54 kinds of pesticide monomer purity standards according to the mass-to-volume ratio, dissolve them in a 1L volumetric flask containing 500 mL of glacial acetonitrile, and after complete dissolution, let the acetonitrile stand until it returns to room temperature, and make up the volume with acetonitrile to obtain the mixed pesticide standard substance, in which the concentration of the 54 kinds of pesticide monomer purity standards is 200-500 ng / mL. After the preparation is completed, it is packaged into 1000 3mL ampoules, each with 1mL, and 3 bottles of samples are randomly selected for dilution 10 times, followed by fixed value detection.

[0020] Preferably, when evaluating the relative expanded uncertainty in the fifth step, full consideration is given to one or more uncertainty influencing factors such as uniformity, stability, preparation process, value determination method, and instrumentation, and finally the measurement uncertainty of the pesticide mixed standard substance is evaluated; in the fifth step, 25 packaging units of the pesticide mixed standard substance are randomly selected for uniformity assessment, and the Shapiro-Wilk method is used to test the normality of the data, and the Dixon's and Grubbs's tests are used to eliminate suspicious values, and finally the obtained data is subjected to an F test to verify whether there is a significant difference, and finally the standard uncertainty component introduced by the uniformity is calculated.

[0021] Preferably, the minimum time span for evaluating the stability of the mixed pesticide standard substance in the fifth step is 6 months, and 3 bottles of mixed pesticide standard substance are selected for stability evaluation, and the stability of the standard substance at 4°C is evaluated over a time span of half a year. The obtained data are subjected to linear regression analysis to calculate the regression parameters.

[0022] A pesticide mixed standard substance prepared by any of the above preparation methods.

[0023] The present application also discloses the use of the mixed pesticide standard substance prepared by any of the above-mentioned preparation methods in the determination of pesticide residues in traditional Chinese medicines.

[0024] The technical principle of the present invention is: pesticide monomer purity standard substances are used to obtain pesticide mixed standard substances after preparation, and a corresponding standard curve is established through the standard substances of each pesticide monomer, and the prepared pesticide mixed standard substances are quantified to achieve traceability of the value, and the relative standard uncertainty of each pesticide monomer is calculated, further increasing the reliability of pesticide detection.

[0025] Beneficial effects:

[0026] The present application provides a pesticide mixed standard substance and its preparation method and application, which have the following beneficial effects:

[0027] 1. This application provides a mixed pesticide standard substance, which can be used to establish a standard curve in the fifth method of the determination of pesticide residues in General Rules 2341 of Part IV of the 2020 edition of the Chinese Pharmacopoeia;

[0028] 2. This application provides a pesticide mixed standard substance, which can be used for quality control operations such as spike recovery and reference substance verification in the detection of pesticide residues in traditional Chinese medicines;

[0029] 3. This application provides a pesticide mixed standard substance, which can realize the traceability of the value of the determination of pesticide residues in traditional Chinese medicines and strengthen the quality supervision of traditional Chinese medicines;

[0030] 4. Pesticide mixed standard substances are obtained after preparation through pesticide monomer purity standard substances, and the corresponding standard curve is established through the standard substances of each pesticide monomer. The prepared pesticide mixed standard substances are quantified to achieve the traceability of the value, and the relative standard uncertainty of each pesticide monomer is calculated, which further increases the reliability of pesticide detection;

[0031] 5. Compared with existing products on the market, the sample in this application has an additional uncertainty, which can be used as a reference. The rest of the items are consistent with existing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Gas chromatography-mass spectrometry of the pesticide mixed standard substance for this application;

[0033] Figure 2 Liquid chromatography-mass spectrometry of the pesticide mixed standard substance for this application;

[0034] Figure 3 The following are the nuclear magnetic resonance hydrogen spectrum and mass spectrum of chloranthiocarb in the examples of the present application, wherein the upper figure is the nuclear magnetic resonance spectrum and the lower figure is the mass spectrum. DETAILED DESCRIPTION

[0035] The following is a further description of a pesticide mixed standard substance involved in the present invention by combining examples. The standard substance covers 54 pesticide monomers. The following examples only take ethoprophos monomer as an example, and the remaining pesticide monomers are prepared by reference.

[0036] Embodiment 1: A method for preparing a pesticide mixed standard substance comprises the following steps:

[0037] Step 1: Select the purity standard of thiopromide as raw material. The purity of the purity standard of thiopromide is 99%. The method for characterizing the structure of the purity standard of thiopromide is two-dimensional nuclear magnetic resonance. The method for verifying the purity of the purity standard of thiopromide is nuclear magnetic hydrogen spectrum and high-resolution mass spectrum. The obtained spectrum is shown in the attached Figure 3 , the data obtained is: 1 H NMR (600MHz, Acetone-d6) δ4.19 (dq, J=9.8, 7.1Hz, 2H), 2.88 (dt, J=15.3, 7.2Hz, 4H), 1.75 (h, J=7.3Hz, 4H), 1.34 (t, J=7.1Hz,

[0038] 3H), 1.00 (t, J = 7.4Hz, 6H).; M = 242M / z:

[0039] Step 2: Quantitative Structure-Property Relationship (QSPR) model was used to calculate the molecular connectivity index of anthocyanidin and related solvents. The solvents were acetone, acetonitrile, methanol, n-hexane, toluene, chloroform or cyclohexane. The calculation results are as follows:

[0040]

[0041]

[0042] According to the QSPR calculation results, it can be seen that the 1 x V The / f value is closest to acetonitrile, so acetonitrile is selected as the solvent for the pesticide mixed standard substance;

[0043] Step 3: The determination method used is the determination method recommended in the determination method of pesticide residues in Part IV of the 2020 edition of the Chinese Pharmacopoeia. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are used for joint determination. The determination methods of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are methodologically verified. The linearity, repeatability, reproducibility, limit of quantification, limit of detection, and recovery rate parameters are verified. The linear correlation coefficient R 2=99.935%; repeatability RSD of ethoxyproline = 1.7%; reproducibility RSD of ethoxyproline = 1.2%; recovery rate of ethoxyproline = 109.3%; detection limit of ethoxyproline = 0.22ng / mL; quantitative limit of ethoxyproline = 0.72ng / ml;

[0044] Step 4: Weigh 0.200 mg of the purity standard of ethoxyprofen and other pesticide purity standards of corresponding mass, dissolve in a 1L volumetric flask containing 500 mL of glacial acetonitrile, and after complete dissolution, let the acetonitrile stand until it returns to room temperature, and dilute to volume with acetonitrile to obtain a mixed pesticide standard substance, in which the concentration of ethoxyprofen is 200 ng / mL. After preparation, dispense into 1000 3mL ampoules, randomly select 3 bottles of samples, dilute 10 times, and then perform fixed value detection. The test results are as follows:

[0045]

[0046] The standard curves used in the determination process are prepared with the first-level or second-level national standard substances of each pesticide monomer solution, with a concentration of 100 mg / L, so that the determination results are traceable;

[0047] Step 5: Randomly select 25 bottles of the prepared 1000 bottles of pesticide mixed standard substances for uniformity assessment, and use the Shapiro-Wilk method to test the normality of the data. Then use Dixon's and Grubbs's tests to eliminate suspicious values. Finally, perform an F test on the obtained data to verify whether there is a significant difference, and finally calculate the standard uncertainty component introduced by uniformity. The test results are as follows:

[0048]

[0049] After F test, there was no significant difference in the mixed pesticide standard substances, the units were mixed evenly, and the standard uncertainty introduced by uniformity was 0.09 ng / mL.

[0050] Three bottles of pesticide mixed standard substances were selected for stability assessment. The minimum stability time span was 6 months. The stability of the standard substances at 4°C was assessed over a time span of half a year. The obtained data were subjected to linear regression analysis, the regression parameters were calculated, and the slope β was subjected to a t test to test the significance of β. The test results are as follows:

[0051] Compound Name 0 months 2 months 4 months 6 months <![CDATA[β1]]> <![CDATA[s(β1)]]> n <![CDATA[s(β1)*t 0.95,2 ]]> in conclusion Ethoprophos 22.38 21.37 21.30 21.33 0.3202 0.3090 4 1.3287 Stablize

[0052] At 4℃, |β1|<t 0.95，n-2 ·s(β1), that is, there is no significant difference. The results show that the developed pesticide mixed standard is stable; stability has a certain linear relationship with time, and the mathematical formula between the two is u s=s(β1)·X, where X is the validity period (time) of the standard substance and s(β1) is the standard deviation of the straight line. The results show that the standard substance is stable for 6 months at 4°C. The standard uncertainty component introduced by the stability calculation of ethoxychlor is 1.85 ng / mL.

[0053] The uncertainty propagation rate is used to evaluate the relative expanded uncertainty, the expanded uncertainty of the invented pesticide mixed standard substance is evaluated, and the corresponding mathematical model is established:

[0054]

[0055] Where: c—the amount of banned pesticides in the standard substance, in ng / mL;

[0056] ρ—mass concentration of banned pesticides in standard working solution, in ng / mL;

[0057] A—Mass spectrum peak area of ​​banned pesticides in the developed standard substances;

[0058] A S —Mass spectrum peak area of ​​national certified pesticide solution standard substance;

[0059] V1—total volume of solution used for testing after dilution, in mL, V1=1mL;

[0060] V2: The volume of the original solution taken out for the standard test, in mL, V2 = 0.1 mL.

[0061] The sources of uncertainty are mainly the following:

[0062] Ⅰ. Uncertainty u1 introduced by constant value repeatability;

[0063] II. Uncertainty introduced by the resolution of LC / MS and GC / MS;

[0064] III. Uncertainty u2 introduced by the electronic balance and volumetric flask during the configuration process;

[0065] IV. Uncertainty u introduced by the liquid chromatography-mass spectrometry used in the determination process L ;

[0066] Ⅴ. Uncertainty introduced by the gas chromatography-mass spectrometry used in the determination process G ;

[0067] VI. Uncertainty u introduced by homogeneity of reference material bb ;

[0068] VII. Uncertainty u introduced by the stability of reference materials ss .

[0069] After evaluating each uncertainty component separately, the uncertainty propagation rate formula is used to synthesize them to obtain the synthetic standard uncertainty.

[0070]

[0071] Compound Name <![CDATA[u1(ng / mL)]]> <![CDATA[u2(ng / mL)]]> <![CDATA[u M (ng / mL)]]> <![CDATA[u bb (ng / mL)]]> <![CDATA[u ss (ng / mL)]]> <![CDATA[u c (ng / mL)]]> Ethoprophos 0.12 29 1.52 0.09 1.85 3.8

[0072] The input and output of the standard measurement are both in accordance with the normal distribution, so k=2; the expanded uncertainty U of the standard can be obtained by the following formula:

[0073] U=k×u c

[0074] Relative expanded uncertainty of the standard product U rel (k=2) can be obtained by the following formula:

[0075]

[0076] Compound Name <![CDATA[u c (ng / mL)]]> U (ng / mL) (k=2) <![CDATA[U rel (k=2)]]> Ethoprophos 3.8 8 3

[0077] Therefore, the final value of cyproconazole in the pesticide mixed standard substance is y = 217 ng / mL, U rel =3%, k=2, fully considering uniformity, stability, preparation process, determination method, one or several uncertainty influencing factors in instruments and equipment, and finally evaluating the measurement uncertainty of pesticide mixed standard substances.

[0078] The final amount table of the pesticide mixed standard substance in this embodiment is:

[0079]

[0080]

[0081] Embodiment 2: A method for preparing a pesticide mixed standard substance comprises the following steps:

[0082] Step 1: Select the purity standard of thiopromide as raw material. The purity of the purity standard of thiopromide is 99%. The method for characterizing the structure of the purity standard of thiopromide is two-dimensional nuclear magnetic resonance. The method for verifying the purity of the purity standard of thiopromide is nuclear magnetic hydrogen spectrum and high-resolution mass spectrum. The obtained spectrum is shown in the attached Figure 3 , the data obtained is: 1 H NMR (600MHz, Acetone-d6) δ4.19 (dq, J=9.8, 7.1Hz, 2H), 2.88 (dt, J=15.3, 7.2Hz, 4 H), 1.75 (h, J = 7.3Hz, 4H), 1.34 (t, J = 7.1Hz, 3H), 1.00 (t, J = 7.4Hz, 6H).; M = 242M / z:

[0083] Step 2: Quantitative Structure-Property Relationship (QSPR) model was used to calculate the molecular connectivity index of anthocyanidin and related solvents. The solvents were acetone, acetonitrile, methanol, n-hexane, toluene, chloroform or cyclohexane. The calculation results are as follows:

[0084] Solvent Name <![CDATA[ 1 x V / f]]> acetone 1.204 Acetonitrile 1.332 Methanol 0.447 n-Hexane 2.914 Toluene 2.411 Chloroform 0.743 Cyclohexane 1.455 Pesticide monomer name <![CDATA[ 1 x V / f]]> Ethoprophos 1.322

[0085] According to the QSPR calculation results, it can be seen that the 1 x V The / f value is closest to acetonitrile, so acetonitrile is selected as the solvent for the pesticide mixed standard substance;

[0086] Step 3: The determination method used is the determination method recommended in the determination method of pesticide residues in Part IV of the 2020 edition of the Chinese Pharmacopoeia. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are used for joint determination. The determination methods of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are methodologically verified. The linearity, repeatability, reproducibility, limit of quantification, limit of detection, and recovery rate parameters are verified. The linear correlation coefficient R 2 =99.935%; repeatability RSD of ethoxyproline = 1.7%; reproducibility RSD of ethoxyproline = 1.2%; recovery rate of ethoxyproline = 109.3%; detection limit of ethoxyproline = 0.22ng / mL; quantitative limit of ethoxyproline = 0.72ng / ml;

[0087] Step 4: Weigh 0.500 mg of the purity standard of ethoxyprofen and other pesticide purity standards of corresponding mass, dissolve in a 1L volumetric flask containing 500 mL of glacial acetonitrile, and after complete dissolution, let the acetonitrile stand until it returns to room temperature, and dilute to volume with acetonitrile to obtain a mixed pesticide standard substance, in which the concentration of ethoxyprofen is 500 ng / mL. After preparation, dispense into 1000 3 mL ampoules, randomly select 3 bottles of samples, dilute 10 times, and then perform fixed value detection. The test results are as follows:

[0088]

[0089] The standard curves used in the determination process are prepared with the first-level or second-level national standard substances of each pesticide monomer solution, with a concentration of 100 mg / L, so that the determination results are traceable;

[0090] Step 5: Randomly select 25 bottles of the prepared 1000 bottles of pesticide mixed standard substances for uniformity assessment, and use the Shapiro-Wilk method to test the normality of the data. Then use Dixon's and Grubbs's tests to eliminate suspicious values. Finally, perform an F test on the obtained data to verify whether there is a significant difference, and finally calculate the standard uncertainty component introduced by uniformity. The test results are as follows:

[0091]

[0092] After F test, there was no significant difference in the mixed pesticide standard substances, the units were mixed evenly, and the standard uncertainty introduced by uniformity was 0.98ng / mL.

[0093] Three bottles of pesticide mixed standard substances were selected for stability assessment. The minimum stability time span was 6 months. The stability of the standard substances at 4°C was assessed over a time span of half a year. The obtained data were subjected to linear regression analysis, the regression parameters were calculated, and the slope β was subjected to a t test to test the significance of β. The test results are as follows:

[0094]

[0095] At 4℃, |β1|<t 0.95，n-2 ·s(β1), that is, there is no significant difference. The results show that the developed pesticide mixed standard is stable; stability has a certain linear relationship with time, and the mathematical formula between the two is u s =s(β1)·X, where X is the validity period (time) of the standard substance and s(β1) is the standard deviation of the straight line. The results show that the standard substance is stable for 6 months at 4°C. The standard uncertainty component introduced by the stability calculation of ethoxychlor is 2.61 ng / mL.

[0096] The uncertainty propagation rate is used to evaluate the relative expanded uncertainty, the expanded uncertainty of the invented pesticide mixed standard substance is evaluated, and the corresponding mathematical model is established:

[0097]

[0098] Where: c—the amount of banned pesticides in the standard substance, in ng / mL;

[0099] ρ—mass concentration of banned pesticides in standard working solution, in ng / mL;

[0100] A—Mass spectrum peak area of ​​banned pesticides in the developed standard substances;

[0101] A S —Mass spectrum peak area of ​​national certified pesticide solution standard substance;

[0102] V1—total volume of solution used for testing after dilution, in mL, V1=1mL;

[0103] V2: The volume of the original solution taken out for the standard test, in mL, V2 = 0.1 mL.

[0104] The sources of uncertainty are mainly the following:

[0105] Ⅰ. Uncertainty u1 introduced by constant value repeatability;

[0106] II. Uncertainty introduced by the resolution of LC / MS and GC / MS;

[0107] III. Uncertainty u2 introduced by the electronic balance and volumetric flask during the configuration process;

[0108] IV. Uncertainty u introduced by the liquid chromatography-mass spectrometry used in the determination process L ;

[0109] Ⅴ. Uncertainty introduced by the gas chromatography-mass spectrometry used in the determination process G ;

[0110] VI. Uncertainty u introduced by homogeneity of reference material bb ;

[0111] VII. Uncertainty u introduced by the stability of reference materials ss .

[0112] After evaluating each uncertainty component separately, the uncertainty propagation rate formula is used to synthesize them to obtain the synthetic standard uncertainty.

[0113]

[0114] Compound Name <![CDATA[u1(ng / mL)]]> <![CDATA[u2(ng / mL)]]> <![CDATA[u M (ng / mL)]]> <![CDATA[u bb (ng / mL)]]> <![CDATA[u ss (ng / mL)]]> <![CDATA[u c (ng / mL)]]> Ethoprophos 0.14 29 3.60 0.98 2.61 5.4

[0115] The input and output of the standard measurement are both in accordance with the normal distribution, so k=2; the expanded uncertainty U of the standard can be obtained by the following formula:

[0116] U=k×u c

[0117] Relative expanded uncertainty of the standard product U rel (k=2) can be obtained by the following formula:

[0118]

[0119] Compound Name <![CDATA[u c (ng / mL)]]> U (ng / mL) (k=2) <![CDATA[U rel (k=2)]]> Ethoprophos 5.4 11 2

[0120] Therefore, the final value of y = 515 ng / mL in the pesticide mixed standard substance, U rel=2%, k=2, fully considering one or several uncertainty influencing factors such as uniformity, stability, preparation process, setting method, and instruments and equipment, and finally evaluating the measurement uncertainty of the pesticide mixed standard substance.

[0121] The final amount table of the pesticide mixed standard substance in this embodiment is:

[0122]

[0123]

[0124]

[0125] The above description of the examples is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a mixed pesticide standard substance, characterized in that: The following steps are involved: Step 1: Select 54 kinds of pesticide monomer purity standards as raw materials. The purity of each pesticide monomer purity standard is ≥95%. Use nuclear magnetic resonance and mass spectrometry to characterize the structure of the pesticide monomer purity standard and verify the purity: Step 2: Calculate the molecular connectivity index of each pesticide monomer and the relevant solvent using the Quantitative Structure-Property Relationship model QSPR, where the solvent is acetone, acetonitrile, methanol, n-hexane, toluene, chloroform or cyclohexane; Step 3: Methodological validation of the gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry determination methods, and relevant validation of linearity, repeatability, reproducibility, limit of quantification, limit of detection, and recovery parameters; Step 4: The 33 pesticide mixed standard substances were calibrated. The standard curves used in the calibration process were prepared with the first-level or second-level national standard substances of each pesticide monomer solution, with a concentration of 100 mg / L, so that the calibration results were traceable; Step 5: Evaluate the uniformity and stability of the pesticide mixed standard substances, and use the uncertainty propagation rate to assess its relative expanded uncertainty. The relative expanded uncertainty U of each pesticide monomer is rel Both ≤5%, k=2.

2. The method for preparing a mixed pesticide standard substance according to claim 1, characterized in that: The method for characterizing the structures of the 54 pesticide monomer purity standards in the first step is two-dimensional nuclear magnetic resonance, the method for verifying the purity of the 54 pesticide monomer purity standards is nuclear magnetic hydrogen spectrum and high-resolution mass spectrometry, and the 54 pesticide monomer purity standards are methyl parathion, methyl parathion, parathion, monocrotophos, phosphamidon, α-BHC, β-BHC, lindane, δ-BHC, p,p'-DDT, o,p'-DDT, p,p'-DDD, p,p'-DDD, chlordimeform, herbicide ether, aldrin, dieldrin, fenamiphos, fenamiphos sulfone ... One or more of phosphine sulfoxide, chlorpyrifos, cadusin, coumaphos, terbufos, terbufos sulfone, terbufos sulfoxide, chlorsulfuron, ethametsulfuron, metsulfuron-methyl, phorate, phorate sulfone, phorate sulfoxide, methyl isofenphos, demeton, carbofuran, trihydroxycarbofuran, aldicarb, aldicarb sulfoxide, ethoxyprolin, chlorfenapyr, isocarb, endosulfan sulfate, α-endosulfan, β-endosulfan, fipronil, flunitrile, fipronil sulfone, fipronil sulfoxide, 2,4'-dicofol, dicofol, thiocyanate, and methyl thiocyanate.

3. The method for preparing a mixed pesticide standard substance according to claim 2, characterized in that: In the first step, the pesticide monomer is ethoprophos.

4. The method for preparing a pesticide mixed standard substance according to claim 1, characterized in that: In the second step, QSPR was used to calculate the molecular connectivity index of each pesticide monomer and the solvent, thereby determining that the solvent was acetonitrile.

5. The method for preparing a pesticide mixed standard substance according to claim 1, characterized in that: The qualification criteria for the methodological validation of the value determination method in the third step are: R 2 ≥99.0%, RSD of repeatability <10.0%, RSD of reproducibility <10.0%, spiked recovery rates were all between 60% and 130%, detection limit <5ng / ml and quantification limit <10ng / ml.

6. The method for preparing a pesticide mixed standard substance according to claim 1, characterized in that: The fourth step of preparing the pesticide mixed standard substance is as follows: weigh 0.200-0.500 mg of 54 kinds of pesticide monomer purity standards according to the mass-to-volume ratio, dissolve them in a 1L volumetric flask containing 500 mL of glacial acetonitrile, and after complete dissolution, let the acetonitrile stand until it returns to room temperature, and use acetonitrile to make up the volume to obtain the pesticide mixed standard substance, in which the concentration of the 54 kinds of pesticide monomer purity standards is 200-500 ng / mL. After the preparation is completed, it is divided into 1000 3mL ampoules, each with 1mL, and 3 bottles of samples are randomly selected for dilution 10 times, followed by fixed value detection.

7. The method for preparing a pesticide mixed standard substance according to claim 1, characterized in that: When evaluating the relative expanded uncertainty in the fifth step, full consideration is given to one or more uncertainty influencing factors such as uniformity, stability, preparation process, determination method, and instruments and equipment, and finally the measurement uncertainty of the pesticide mixed standard substance is evaluated; in the fifth step, 25 packaging units of the pesticide mixed standard substance are randomly selected for uniformity assessment, and the Shapiro-Wilk method is used to test the normality of the data, and the Dixon's and Grubbs's tests are used to eliminate suspicious values, and finally the F test is performed on the obtained data to verify whether there is a significant difference, and finally the standard uncertainty component introduced by uniformity is calculated.

8. The method for preparing a mixed pesticide standard substance according to claim 1, characterized in that: The minimum time span for evaluating the stability of the pesticide mixed standard substance in the fifth step is 6 months. Three bottles of pesticide mixed standard substance are selected for stability evaluation. The stability of the standard substance at 4°C is evaluated over a time span of half a year. The obtained data is subjected to linear regression analysis to calculate the regression parameters.

9. A pesticide mixed standard substance prepared by the preparation method according to any one of claims 1 to 8.

10. Use of a mixed pesticide standard substance prepared by the preparation method according to any one of claims 1 to 8 in the determination of pesticide residues in traditional Chinese medicines.

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