Method for detecting residual solvent in paeonol raw material

Abstract

The invention relates to a method for detecting a residual solvent in a paeonol raw material. The method comprises the following steps: (1) preparing a reference solution; (2) preparing a test solution; and (3) respectively injecting the reference solution and the test solution into a gas chromatograph in a headspace sampling manner, and calculating the content of the residual solvent by peak area according to an external standard method. According to the method, the contents of seven residual solvents including methanol, ethanol, acetone, acetonitrile, dichloromethane, ethyl acetate and trichloromethane in the paeonol raw material or preparation can be detected at the same time, and the detection time and cost are greatly saved. Moreover, the method can be used for detecting residual solvents in paeonol extracted by different processes, and has the advantages of high practicability, simplicity in operation, high specificity, high accuracy, high precision and the like. Reference and basis are provided for revision of residual solvent inspection items in paeonol quality standards, the quality of raw material medicines is effectively improved, and clinical medication risks are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection, and particularly relates to a method for detecting residual solvents in a paeonol raw material. Background Art

[0002] Paeonol is a small molecule compound isolated from the dried root bark of the Ranunculaceae plants peony and peony and the root or whole herb of the Apocynaceae plant Cynanchum wilfordii. It has the main effects of analgesia, antipyretic, antibacterial and anti-inflammatory, anti-atherosclerosis, inhibition of platelet aggregation, enhancement of cellular immune function, and antioxidant. It has strong pharmacological activity and low toxic and side effects, and is widely used in clinical practice.

[0003] Paeonol can be extracted from natural products or prepared by chemical methods. Its chemical structure is shown below:

[0004]

[0005] Common extraction methods for paeonol include alcohol extraction, steam distillation, supercritical CO2 extraction, etc. Its synthesis process is also relatively mature. It is generally obtained by acetylmethylation with resorcinol as the raw material, with a high yield. However, residual solvents such as methanol, ethanol, acetonitrile, and dichloromethane may be introduced during the synthesis process. The extraction process of paeonol is different, and the types of solvents used in the extraction process are also different. If the process parameters are not strictly controlled, there is a possibility of residual organic solvents in the finished product. When the residual solvent level in the drug is higher than the safety value, it will cause harm to the human body or the environment. However, the Chinese Pharmacopoeia currently does not include a detection method for residual solvents in paeonol.

[0006] Lin Maoquan et al. used headspace gas chromatography to determine the residual amounts of 13 solvents, including methanol, ethanol, ether, 500 mg of acetone, isopropanol, acetonitrile, dichloromethane, n-hexane, n-propanol, ethyl acetate, tetrahydrofuran, cyclohexane and toluene, in stanozolol raw materials (Lin Maoquan et al., 2021); Lu Shihua et al. used headspace gas chromatography to establish a method for determining the residual amounts of 12 organic solvents, including methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, n-propanol, ethyl acetate, tetrahydrofuran, cyclohexane, n-butanol and toluene, in metronidazole raw materials (Lu Shihua et al., 2020); Chinese patent CN 111855837 A discloses a method for determining the residual solvents of methanol, ethanol, acetone, ethyl acetate and dichloromethane in ceftriaxone sodium by headspace gas chromatography. However, since the gas chromatography method for detecting organic solvent residues is specific and different substances interact differently with residual solvents, the detection method described in the above literature can only detect residual solvents mentioned in specific raw materials, and cannot simultaneously detect other residual solvents in the same raw material, let alone other residual solvents in other raw materials. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting residual solvents in paeonol raw materials, so as to overcome the defect that the existing organic solvent detection method cannot meet the demand.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for simultaneously detecting multiple residual solvents in paeonol raw materials, wherein the multiple residual solvents are methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane and chloroform, and detecting them by gas chromatography using headspace injection

[0010] The chromatographic conditions are:

[0011] Chromatographic column: capillary gas chromatography column with 50% phenyl-50% dimethylpolysiloxane or 6% cyanopropylbenzene-94% dimethylpolysiloxane as stationary liquid;

[0012] Heating program: starting temperature is 30-40℃, rising to 45℃ at 1℃ / min; then rising to 60℃ at 3℃ / min; finally rising to 180℃ at 30℃ / min, and holding for 15min;

[0013] Split ratio: 10:1;

[0014] Headspace equilibrium temperature: 80~110℃;

[0015] Headspace equilibrium time: 20-40 min;

[0016] The following steps are involved:

[0017] (1) Preparation of reference solution: Accurately weigh appropriate amounts of methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane, and chloroform, and add N,N-dimethylformamide to prepare a solution containing 3000 μg of methanol, 5000 μg of ethanol, 5000 μg of acetone, 410 μg of acetonitrile, 600 μg of dichloromethane, 5000 μg of ethyl acetate, and 60 μg of chloroform per 1 mL of N,N-dimethylformamide; accurately measure 2 mL, place in a 20 mL headspace bottle, and seal to serve as the reference solution;

[0018] (2) Preparation of test solution: Take the paeonol raw material to be tested, accurately weigh 0.2 g and place it in a 20 mL headspace bottle, add 2 mL of N,N-dimethylformamide, and seal it to serve as the test solution;

[0019] (3) Determination: Inject the reference solution and the test solution into a gas chromatograph by headspace injection, and calculate the residual solvent content by peak area using the external standard method.

[0020] In the preferred technical solution of the present invention, the temperature of the headspace injection port is 245°C to 255°C.

[0021] In the preferred technical solution of the present invention, the starting temperature of the gas chromatography column is 35°C.

[0022] In the preferred technical solution of the present invention, the carrier gas of the gas chromatograph is nitrogen, and the carrier gas flow rate is 0.9 mL / min to 1.1 mL / min.

[0023] In the preferred technical solution of the present invention, the gas chromatograph detector is a FID detector, and the temperature of the FID detector is 275°C to 285°C.

[0024] In the preferred technical solution of the present invention, the hydrogen flow rate of the FID detector is 40 mL / min, the air flow rate is 400 mL / min, and the compensation gas flow rate is 40 mL / min.

[0025] In the preferred technical solution of the present invention, the headspace equilibrium temperature is 100° C. and the headspace equilibrium time is 30 min.

[0026] In the preferred technical solution of the present invention, the chromatographic column of the gas chromatograph uses 6% cyanopropylphenyl and 94% dimethylpolysiloxane as the stationary liquid.

[0027] In the preferred technical solution of the present invention, the specific calculation formula for calculating the residual solvent content by peak area according to the external standard method is as follows:

[0028]

[0029] Where: A spl A is the peak area of ​​the residual solvent to be tested in the test solution; std V is the peak area of ​​the residual solvent to be tested in the reference solution; std V is the dilution volume of the reference solution, mL; spl W is the dilution volume of the test solution, mL; std is the sample weight of the reference substance, mg; W spl is the sample weight of the test sample, mg; P is the purity of the reference sample.

[0030] The present invention also provides application of the method in detecting the content of residual solvent in paeonol raw material.

[0031] The beneficial effects of the present invention are:

[0032] The invention provides a method for detecting residual solvents in a paeonol raw material. By adopting specific chromatographic conditions such as a heating program, an equilibrium temperature and an equilibrium time, not only can the contents of seven residual solvents of methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane and chloroform in the paeonol raw material or preparation be detected simultaneously, but also the residual solvents in the paeonol obtained by different extraction processes can be detected, thereby greatly saving detection time and cost. The method has good specificity, a low detection limit and high accuracy, can detect the residual solvents in the paeonol obtained by different extraction processes, and has the advantages of strong practicability, simple operation, strong specificity, high accuracy, good precision and the like.

[0033] The method of the present invention has high sensitivity, good repeatability and is simple and easy to operate, and will provide a reference and basis for the revision of the residual solvent inspection item in the quality standard of paeonol, effectively improve the quality of raw materials, reduce the risk of clinical drug use, and ensure drug safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the blank solution chromatogram in Example 4.

[0035] Figure 2 It is the chromatogram of the reference substance solution in Example 4.

[0036] Figure 3 This is the chromatogram of the methanol positioning solution in Example 4.

[0037] Figure 4 It is the chromatogram of the ethanol positioning solution in Example 4.

[0038] Figure 5 This is the chromatogram of the acetone positioning solution in Example 4.

[0039] Figure 6 This is the chromatogram of the acetonitrile positioning solution in Example 4.

[0040] Figure 7 This is the chromatogram of the dichloromethane-based solution in Example 4.

[0041] Figure 8 This is the chromatogram of the ethyl acetate positioning solution in Example 4.

[0042] Fig. 9 This is the chromatogram of the chloroform positioning solution in Example 4.

[0043] Fig.10 It is a linear correlation diagram between the concentration of methanol and the peak area in Example 4.

[0044] Fig.11 It is a linear correlation diagram between the concentration of ethanol and the peak area in Example 4.

[0045] Fig.12It is a linear correlation diagram between the concentration of acetone and the peak area in Example 4.

[0046] Fig.13 It is a linear correlation diagram of the concentration of acetonitrile and the peak area in Example 4.

[0047] Fig.14 It is a linear correlation diagram between the concentration of dichloromethane and the peak area in Example 4.

[0048] Fig.15 It is a linear correlation diagram between the concentration of ethyl acetate and the peak area in Example 4.

[0049] Fig.16 It is a linear correlation diagram between the concentration of chloroform and the peak area in Example 4. DETAILED DESCRIPTION

[0050] In order to make the technical problems and technical solutions solved by the present invention clearer, the present invention is described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0051] Example 1

[0052] A method for detecting residual solvents in paeonol, wherein the instruments and reagents used in the method are shown in Table 1.

[0053] Table 1 Instruments and reagents used in this detection method

[0054] Instrument / reagent name Model / Batch No. Source / Manufacturer Electronic balance BV25S Beijing Sartorius Scientific Instruments Co., Ltd. Gas Chromatograph GC-2014C Shimadzu Enterprise Management (China) Co., Ltd. Headspace Sampler HS-20 Opule Instrument Co., Ltd. Chromatographic columns DB-624 Agilent Technologies China Co., Ltd. N,N-Dimethylformamide Chromatographic pure Tianjin Komiou Chemical Reagent Co., Ltd. Methanol Chromatographic pure Tianjin Concord Technology Co., Ltd. Ethanol Chromatographic pure Tianjin Komiou Chemical Reagent Co., Ltd. acetone Chromatographic pure Tianjin Komiou Chemical Reagent Co., Ltd. Acetonitrile Chromatographic pure Tianjin Komiou Chemical Reagent Co., Ltd. Dichloromethane Chromatographic pure Tianjin Komiou Chemical Reagent Co., Ltd. Ethyl acetate Chromatographic pure Tianjin Komiou Chemical Reagent Co., Ltd. Chloroform Chromatographic pure Tianjin Komiou Chemical Reagent Co., Ltd. Paeonol 230711100088 Shanghai Myril Chemical Technology Co., Ltd.

[0055] At the same time, the detection method uses headspace sampling to perform gas chromatography detection, wherein the chromatographic conditions are:

[0056] (1) The injection port temperature of the gas chromatograph is 245°C to 255°C;

[0057] (2) The chromatographic column of the gas chromatograph uses 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase;

[0058] (3) Temperature program of the gas chromatography column: the initial column temperature was 35 °C, which was increased to 45 °C at 1 °C / min, then to 60 °C at 3 °C / min, and finally to 180 °C at 30 °C / min and maintained for 15 min;

[0059] (4) The carrier gas of the gas chromatograph is nitrogen, and the carrier gas flow rate is 1.0 mL / min;

[0060] (5) The detector of the gas chromatograph is a flame ionization detector (FID), the temperature is 280°C, the detector hydrogen flow rate is 40 mL / min, the air flow rate is 400 mL / min, and the makeup gas flow rate is 40 mL / min;

[0061] (6) The split ratio of the gas chromatograph is 10:1;

[0062] (7) The volume of the test solution in the headspace bottle is 2 mL;

[0063] (8) The headspace equilibrium temperature is 100 °C and the headspace equilibrium time is 30 min;

[0064] (9) The diluent is N,N-dimethylformamide (DMF).

[0065] The specific detection process includes the following steps:

[0066] (1) Preparation of reference solution: Accurately weigh appropriate amounts of methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane, and chloroform, and add N,N-dimethylformamide to prepare a solution containing approximately 3000 μg of methanol, 5000 μg of ethanol, 5000 μg of acetone, 410 μg of acetonitrile, 600 μg of dichloromethane, 5000 μg of ethyl acetate, and 60 μg of chloroform per 1 mL of N,N-dimethylformamide. Accurately measure 2 mL, place in a 20 mL headspace bottle, and seal.

[0067] (2) Preparation of test solution: Accurately weigh 2 mL of N,N-dimethylformamide and place it in a 20 mL headspace bottle. Accurately weigh 0.2 g of paeonol and place it in the same headspace bottle and seal it.

[0068] (3) Determination: Inject the reference solution and the test solution into a gas chromatograph by headspace injection, and calculate the residual solvent content by peak area using the external standard method.

[0069] Example 2 Determination of headspace equilibrium temperature and equilibrium time

[0070] 1. Headspace equilibrium temperature investigation

[0071] When the headspace equilibrium time was 20 min, the headspace equilibrium temperatures were investigated respectively: 60°C, 80°C, 100°C, and 120°C. The detection was carried out according to the detection process described in Example 1. The results are shown in Table 2.

[0072] Table 2 Test results at different headspace equilibrium temperatures

[0073]

[0074] The results show that as the headspace equilibrium temperature increases, the response of each residual solvent increases. Among the residual solvents to be tested, acetonitrile has the highest boiling point, which is 81.6℃. In order to increase the response of acetonitrile, the headspace equilibrium temperature is selected as 100℃.

[0075] 2. Headspace equilibration time

[0076] When the headspace equilibrium temperature was 100°C, the headspace equilibrium times were investigated at 10 min, 20 min, 30 min, and 60 min, respectively. The detection was carried out according to the detection process described in Example 1. The results are shown in Table 3.

[0077] Table 3 Detection results at different headspace equilibrium times

[0078]

[0079] The results show that with the increase of headspace equilibration time, the responses of each solvent changed significantly. Among all solvents, ethanol had the largest change in peak area (RSD = 1.06%). Referring to General Chapter 0861 of the 2020 edition of the "Chinese Pharmacopoeia", an equilibration time of 30 min was selected.

[0080] Experimental Example 3: Durability Study on Equilibrium Temperature and Equilibrium Time

[0081] Referring to the chromatographic conditions in Example 1, the durability of the equilibrium temperature and equilibrium time was investigated. The results are shown in Table 4:

[0082] Table 4 Durability test results of equilibrium temperature and equilibrium time

[0083]

[0084]

[0085] The results in Table 4 show that at different equilibrium temperatures and different equilibrium times, the RSD values ​​of the measured amounts of the residual solvents to be tested are all less than 10%, and the durability is good.

[0086] Example 4 Verification of the detection method of Example 1

[0087] 1. Specificity and system suitability test

[0088] Blank solution: Accurately measure 2 mL of diluent (N,N-dimethylformamide), place it in a 20 mL headspace bottle, and tighten the bottle cap.

[0089] Reference solution: Accurately weigh appropriate amounts of methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane, and chloroform; add N,N-dimethylformamide to prepare a solution containing approximately 3000 μg of methanol, 5000 μg of ethanol, 5000 μg of acetone, 410 μg of acetonitrile, 600 μg of dichloromethane, 5000 μg of ethyl acetate, and 60 μg of chloroform per 1 mL of N,N-dimethylformamide; accurately measure 2 mL, place in a 20 mL headspace bottle, and seal.

[0090] Positioning solutions for each residual solvent to be tested: prepare positioning solutions of methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane, and chloroform according to the limits (refer to the limits and requirements for common residual solvents in the 2020 edition of the "Chinese Pharmacopoeia").

[0091] Take blank solution, reference solution and each residual solvent location solution to be tested respectively, inject them into gas chromatograph and record the chromatogram.

[0092] Acceptable standards: In the blank solution chromatogram, there should be no interfering peaks at the retention time of each residual solvent to be tested. If there is, its peak area should not exceed 2% of the peak area of ​​each residual solvent to be tested in the reference solution; in the reference solution chromatogram, the separation between each residual solvent to be tested should be no less than 1.5, and the RSD of the peak area of ​​each residual solvent to be tested in the reference solution for 6 consecutive times should be no more than 10%. In the blank solution chromatogram, there are no interfering peaks at the retention time of methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane, and chloroform; the detection method has strong specificity. Typical chromatograms of blank solution, reference solution, and methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane, and chloroform solutions are shown below. Figures 1 to 9 shown.

[0093] As shown in Table 5, the minimum separation between the residual solvents to be tested is 5.62; the peak areas of the residual solvents to be tested in the reference solution for 6 consecutive times are shown in Table 6: RSD of methanol peak area = 0.630% (n = 6), RSD of ethanol peak area = 0.743% (n = 6), RSD of acetone peak area = 0.837% (n = 6), RSD of acetonitrile peak area = 0.680% (n = 6), RSD of dichloromethane peak area = 0.797% (n = 6), RSD of ethyl acetate peak area = 0.825% (n = 6), RSD of chloroform peak area = 0.923% (n = 6), and the system suitability meets the requirements.

[0094] Table 5 System suitability results

[0095]

[0096]

[0097] Note: “ / ” indicates methanol is the first chromatographic peak and has no separation value.

[0098] Table 6 System suitability results

[0099] Serial number Methanol Ethanol acetone Acetonitrile Dichloromethane Ethyl acetate Chloroform 1 811675 1425898 3999104 1141665 1169273 2481329 32273 2 808901 1414805 3984410 1141260 1157134 2475646 32024 3 806037 1413505 3962320 1141643 1146927 2474249 32044 4 812784 1424894 4010347 1141524 1174126 2486369 32270 5 812464 1424137 4000179 1141852 1170266 2484510 32279 6 811207 1421413 3990922 1141849 1164093 2481423 32012 average value 810511 1420775 3991214 1141632 1163636 2480588 32150 RSD(%) 0.630 0.743 0.837 0.680 0.797 0.825 0.923

[0100] 2. Detection limit and quantification limit verification

[0101] Quantitative limit stock solution: accurately weigh 300 mg of methanol reference substance, 500 mg of ethanol reference substance, 500 mg of acetone reference substance, 41 mg of acetonitrile reference substance, 60 mg of dichloromethane reference substance, 500 mg of ethyl acetate reference substance, and 6 mg of chloroform reference substance, and place them in a 100 mL volumetric flask, dilute to the mark with a diluent (N, N-dimethylformamide), shake well, and use the quantitative limit stock solution. Accurately measure 1 mL of the quantitative limit stock solution, place it in a 100 mL volumetric flask, add a diluent (N, N-dimethylformamide) to dilute to the mark, shake well, and use the quantitative limit solution. Accurately measure 2 mL of the quantitative limit solution, place it in a 20 mL headspace bottle, and press the bottle cap tightly to obtain the product.

[0102] Take 3 mL of the quantitation limit solution and place it in a 10 mL volumetric flask. Add diluent (N,N-dimethylformamide) to dilute to the scale and shake well. This is used as the detection limit solution. Accurately measure 2 mL of the detection limit solution and place it in a 20 mL headspace bottle. Press the bottle cap tightly to obtain the solution.

[0103] Inject blank solution, reference solution, detection limit solution and quantitation limit solution respectively and record the chromatograms.

[0104] Acceptance criteria: The signal-to-noise ratio of each residual solvent peak to be tested in the chromatogram of the quantitative limit solution is not less than 10, and the RSD value of the peak area does not exceed 15%. The signal-to-noise ratio of each residual solvent peak to be tested in the chromatogram of the detection limit solution is not less than 3.

[0105] Table 7 Results of quantitative limit solution and detection limit solution

[0106]

[0107]

[0108] As shown in Table 7, the quantification limit of methanol is 0.090 μg·mL -1 When the concentration is 0.003%, methanol can be accurately detected; the quantitative limit of ethanol is 0.150μg·mL -1 When the concentration is 0.003%, the ethanol concentration can be accurately detected; the quantification limit of acetone is 0.040μg·mL -1 When the concentration is 0.001%, acetone can be accurately detected; the quantification limit of acetonitrile is 0.008 μg·mL-1 When the concentration is 0.002%, acetonitrile can be accurately detected; the limit of quantification of dichloromethane is 0.006μg·mL -1 When the concentration of dichloromethane is 0.001%, it can be accurately detected; the quantitative limit of ethyl acetate is 0.040μg·mL -1 When the concentration is 0.001%, ethyl acetate can be accurately detected; the quantitative limit of chloroform is 0.003 μg·mL -1 When the concentration of chloroform is equal to the limit concentration of 0.005%, it can be accurately detected.

[0109] The detection limit of methanol is 0.300 μg·mL -1 When the concentration of methanol is 0.010%, the detection limit of ethanol is 0.500 μg·mL. -1 When the concentration of ethanol is 0.010%, the detection limit of acetone is 0.180μg·mL -1 When the concentration is 0.004%, acetone can be accurately detected; the detection limit of acetonitrile is 0.025μg·mL -1 When the detection limit concentration is 0.006%, acetonitrile can be accurately detected; the detection limit concentration of dichloromethane is

[0110] 0.020μg·mL -1 When the concentration of dichloromethane is 0.003%, the detection limit of ethyl acetate is 0.170 μg·mL -1 When the concentration of ethyl acetate is 0.003%, the detection limit of chloroform is 0.010 μg·mL. -1 When the concentration of chloroform is equal to the limit of 0.017%, it can be accurately detected.

[0111] 3. Linear relationship analysis

[0112] Linear stock solution: accurately weigh 300 mg of methanol reference substance, 500 mg of ethanol reference substance, 500 mg of acetone reference substance, 41 mg of acetonitrile reference substance, 60 mg of dichloromethane reference substance, 500 mg of ethyl acetate reference substance, and 6 mg of chloroform reference substance, place them in a 100 mL volumetric flask, add diluent (N,N-dimethylformamide) to dilute to the scale, shake well, and use as linear stock solution. Accurately measure an appropriate amount of linear stock solution to prepare 50, 75, 100, 125, 150, and 250 μg mL -1 A series of linear solutions.

[0113] Take blank solution, reference solution and series linear solution respectively, inject them into gas chromatograph and record chromatogram. Under the above optimized chromatographic conditions, inject samples and measure them respectively, and take the mass concentration of each component to be tested (x, μg·mL -1 ) as the horizontal axis and the peak area (y) as the vertical axis for linear regression. Calculate and count: linear equation, correlation coefficient r, linear correlation graph. Figures 10 to 16 The linear correlation graphs of concentration and peak area are respectively for methanol, ethanol, acetone, acetonitrile, dichloromethane, ethyl acetate and chloroform.

[0114] Acceptance criteria: Within the measured concentration range, the response value of each residual solvent to be tested should show good linearity with the concentration, and the linear correlation coefficient r should be no less than 0.990.

[0115] Table 8 Linearity test results

[0116] name Regression equation Correlation coefficient (r) Methanol y=330.3949x+988.5281 0.9997 Ethanol y=842.5486x-1336.1035 0.9996 acetone y=868.6623x-1541.0684 0.9993 Acetonitrile y=691.1518x-640.2877 0.9992 Dichloromethane y=300.9648x+449.0842 0.9992 Ethyl acetate y=712.0947x-464.5263 0.9994 Chloroform y=192.5714x+438.1035 0.9995

[0117] The linear results show (Table 8): in the range of 50 to 250 μg mL -1 In the linear range, the linear equation of methanol is y=330.3949x+988.5281, and the linear correlation coefficient is r=0.9997; the linear equation of ethanol is y=842.5486x-1336.1035, and the linear correlation coefficient is r=0.9996; the linear equation of acetone is y=868.6623x-1541.0684, and the linear correlation coefficient is r=0.9993; the linear equation of acetonitrile is y=691.1518x-640.2877, The linear correlation coefficient is r=0.9992; the linear equation of dichloromethane is y=300.9648x+449.0842, and the linear correlation coefficient is r=0.9992; the linear equation of ethyl acetate is y=712.0947x-464.5263, and the linear correlation coefficient is r=0.9994; the linear equation of chloroform is y=192.5714x+438.1035, and the linear correlation coefficient is r=0.9995. The linear relationship of each residual solvent to be tested is good.

[0118] 4. Accuracy Verification

[0119] Accuracy stock solution ①: Take 50% linear solution under the "Linearity" item as accuracy stock solution ①.

[0120] Accuracy stock solution ②: Take the 100% linear solution under the "Linearity" item as accuracy stock solution ②.

[0121] Accuracy stock solution ③: Take 150% linear solution under the "Linearity" item as accuracy stock solution ③.

[0122] Take blank solution and reference solution respectively, inject into gas chromatograph, record chromatogram. Calculate the recovery rate of each residual solvent to be tested by peak area according to external standard method, and calculate the recovery rate RSD (%).

[0123] Original amount: Measure the result using the test sample solution.

[0124] Acceptance criteria: At the 50% concentration level, 100% concentration level and 150% concentration level, the recovery rate of each residual solvent to be tested should be between 80% and 120%, and RSD≤10% (n=3).

[0125] Table 9 Recovery test results

[0126]

[0127]

[0128] The recovery test results show (Table 9): the recovery rates of each component at 50%, 100% and 150% concentration levels are all between 80% and 120%, with RSD ≤ 10% (n = 3). This shows that the accuracy of each residual solvent to be tested is good.

[0129] 5. Repeatability Verification

[0130] 100% spiked test solution: Prepare 6 100% spiked test solutions in parallel according to the 100% accuracy solution preparation method under "Accuracy". Calculate the content of each residual solvent to be tested in the 100% spiked test solution and its RSD value (n=6) according to the prescribed method.

[0131] Acceptable standard: RSD of the measured amount of each residual solvent to be tested in 6 100% spiked test solution is ≤10%.

[0132] Table 10 Repeatability test results

[0133] Serial number Methanol Ethanol acetone Acetonitrile Dichloromethane Ethyl acetate Chloroform 1 811675 1425898 3999104 1141665 1169273 2481329 32273 2 808901 1414805 3984410 1141260 1157134 2475646 32024 3 806037 1413505 3962320 1141643 1146927 2474249 32044 4 812784 1424894 4010347 1141524 1174126 2486369 32270 5 812464 1424137 4000179 1141852 1170266 2484510 32279 6 811207 1421413 3990922 1141849 1164093 2481423 32012 average value 810511 1420775 3991214 1141632 1163636 2480588 32150 RSD(%) 1.58 2.84 2.07 2.62 2.17 2.51 2.95

[0134] The results of the repeatability experiment show (Table 10): in 6 100% spiked test solutions: the RSD of the measured amount of methanol is 1.58% (n=6); the RSD of the measured amount of ethanol is 2.84% (n=6); the RSD of the measured amount of acetone is 2.07% (n=6); the RSD of the measured amount of acetonitrile is 2.62% (n=6); the RSD of the measured amount of dichloromethane is 2.17% (n=6); the RSD of the measured amount of ethyl acetate is 2.51% (n=6); the RSD of the measured amount of chloroform is 2.95% (n=6); it can be seen that the detection method has good repeatability.

[0135] Through the analysis of Example 2, the detection method of residual solvents in paeonol proposed in Example 1 was finally determined. As can be seen from Example 4, the specificity and system applicability, detection limit and quantification limit, linearity and range, accuracy, precision, durability and other aspects of the method of the present invention have been verified, and the experimental results of each verification item meet the acceptable standards specified in the pharmacopoeia. Therefore, the method provided in Example 1 can be used for the detection of residual solvents in paeonol raw materials or related preparations, and the detection results are accurate and reliable.

[0136] The present invention is described in detail above through specific and preferred embodiments, but those skilled in the art should understand that the present invention is not limited to the embodiments described above, and any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting multiple residual solvents in paeonol raw materials, characterized in that: The multiple residual solvents are methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane and chloroform, and the gas chromatography detection is performed by headspace injection, wherein the chromatographic conditions are: Chromatographic column: capillary gas chromatography column with 50% phenyl-50% dimethylpolysiloxane or 6% cyanopropylbenzene-94% dimethylpolysiloxane as stationary liquid; Heating program: starting temperature is 30-40℃, rising to 45℃ at 1℃ / min; then rising to 60℃ at 3℃ / min; finally rising to 180℃ at 30℃ / min and holding for 15 min; Split ratio: 10:1; Headspace equilibrium temperature: 80~110℃; Headspace equilibration time: 20-40 min; The following steps are involved: (1) Preparation of reference solution: Accurately weigh appropriate amounts of methanol, ethanol, acetone, acetonitrile, ethyl acetate, dichloromethane, and chloroform, and add N,N-dimethylformamide to prepare a mixed solution containing 3000 μg of methanol, 5000 μg of ethanol, 5000 μg of acetone, 410 μg of acetonitrile, 600 μg of dichloromethane, 5000 μg of ethyl acetate, and 60 μg of chloroform per 1 mL of N,N-dimethylformamide; accurately measure 2 mL of the mixed solution, place it in a 20 mL headspace bottle, and seal it as the reference solution; (2) Preparation of the test solution: Take the sample paeonol raw material to be tested, accurately weigh 0.2 g and place it in a 20 mL headspace bottle, add 2 mL of N,N-dimethylformamide, and seal it to serve as the test solution; (3) Determination: Inject the reference solution and the test solution into the gas chromatograph by headspace injection, and calculate the residual solvent content by peak area using the external standard method.

2. The method according to claim 1, characterized in that The headspace injection port temperature is 245℃~255℃.

3. The method according to claim 1, characterized in that The initial temperature of the gas chromatography column was 35°C.

4. The method according to claim 1, characterized in that The carrier gas of the gas chromatograph is nitrogen, and the carrier gas flow rate is 0.9 mL / min~1.1 mL / min.

5. The method for detecting residual solvent in paeonol raw material according to claim 1, characterized in that: The gas chromatograph detector is a FID detector, and the FID detector temperature is 275°C~285°C.

6. The method according to claim 5, characterized in that The hydrogen flow rate of the FID detector was 40 mL / min, the air flow rate was 400 mL / min, and the makeup gas flow rate was 40 mL / min.

7. The method according to claim 1, characterized in that The headspace equilibrium temperature was 100°C and the headspace equilibrium time was 30 min.

8. The method according to claim 1, characterized in that The specific calculation formula for calculating the residual solvent content by peak area according to the external standard method is as follows: ； Where: A spl A is the peak area of ​​the residual solvent to be tested in the test solution; std V is the peak area of ​​the residual solvent to be tested in the reference solution; std V is the dilution volume of the reference solution, mL; spl W is the dilution volume of the test solution, mL; std The sample weight of the reference substance, mg; W spl is the sample weight of the test sample, mg; P is the purity of the reference sample.

9. Use of the method for simultaneously detecting multiple residual solvents in a paeonol raw material as claimed in any one of claims 1 to 8 in detecting the content of residual solvents in the paeonol raw material.

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