Method for determining content of camphor in external therapeutic arthralgia-relieving pharmaceutical preparation

Abstract

The application provides a method for determining camphor content in external treatment arthralgia medicine preparation, and belongs to the technical field of detection methods. The method comprises the following steps: (1) preparing a test sample solution; (2) preparing an internal standard solution; (3) preparing a control sample solution; (4) preparing a correction solution; and (5) sample injection and detection. The method for determining camphor in external treatment arthralgia medicine preparation is simple, accurate and durable, has linear correlation within a certain concentration range, can effectively control the quality of products, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection method, in particular to a method for determining the content of camphor in a drug preparation for treating arthralgia. BACKGROUND

[0002] The arthralgia syndrome in traditional Chinese medicine refers to the pain of meridians, skin, joints, tendons and bones, numbness, heaviness, difficulty in flexing and extending or joint swelling, stiffness, deformity, muscle atrophy and movement disorder caused by the invasion of wind, cold, damp and heat on the body qi and blood, meridians and collaterals, and the obstruction of pathogenic factors. This kind of disease is a common and frequently-occurring disease, and is difficult to cure. It is one of the key projects in the medical field. This disease is similar to the rheumatic disease in modern medicine.

[0003] The arthralgia syndrome is similar to the rheumatic disease in western medicine. At present, western medicine adopts surgery, oral administration or local injection of anti-inflammatory agents or anesthetics, which has certain curative effect. However, the maintenance time is limited, the patient cannot operate by himself, the treatment is not convenient, and the side effect is strong.

[0004] Traditional Chinese medicine has achieved satisfactory curative effect in the treatment of this kind of disease, and the treatment methods are various, such as oral administration, external application, fumigation and washing, acupoint injection, hot compress and medicine embedding. At present, although there are various external Chinese medicine preparations, they are mainly used for treating rheumatic arthralgia syndrome. One typical external drug preparation for treating arthralgia syndrome (Chinese invention patent CN 102293977B) has the following raw material components: salvia miltiorrhiza, piper longum, cyathula officinalis, piper nigrum, zanthoxylum bungeanum, ligusticum sinense, smilax riparia, kaempferia, alpinia officinarum, camphor and borneol. Camphor is one of the traditional Chinese medicine components, which is easy to volatilize and sublimate. Therefore, it is difficult to detect the content of camphor. The usual method is often difficult to obtain the accurate content of camphor. Therefore, a new and more accurate method is needed to detect the content of camphor in the external drug preparation for treating arthralgia syndrome. SUMMARY

[0005] The purpose of the present application is to provide a method for determining the content of camphor in an external drug preparation for treating arthralgia syndrome. The detection method is simple, accurate and durable, has linear correlation in a certain concentration range, can effectively control the quality of the product, and has a wide application prospect.

[0006] The technical scheme of the present application is as follows:

[0007] The present application provides a method for determining the content of camphor in an external drug preparation for treating arthralgia syndrome, which comprises the following steps:

[0008] (1) Preparation of test solution: take the sample, remove the cover liner, cut into small pieces, put into a flask with a stopper, add dichloromethane, add adsorbent, stir and adsorb, filter, add ethyl acetate to the adsorbent, desorb by ultrasonic, filter, wash the solid, dry, reuse, dilute the solution to volume, and prepare the test solution;

[0009] (2) Preparation of internal standard solution: accurately weigh naphthalene, add ethyl acetate to prepare the internal standard solution;

[0010] (3) Preparation of reference solution: accurately weigh camphor reference, add ethyl acetate to prepare the reference solution;

[0011] (4) Preparation of calibration solution: accurately measure the internal standard solution and the reference solution, dilute to volume to prepare the calibration solution;

[0012] (5) Sample injection detection: analyze the content of camphor in the test solution by gas chromatography.

[0013] As a further improvement of the present application, the preparation method of the adsorbent is as follows:

[0014] S1. Preparation of UiO-66-OH: add zirconium tetrachloride and 2-hydroxyterephthalic acid into a solvent, ultrasonic dispersion, hydrothermal reaction, centrifugation, washing, drying, and preparing UiO-66-OH;

[0015] S2. Preparation of alkenylated UiO-66-OH material: add UiO-66-OH into dichloromethane, add chloroethyl acrylate and alkali, heat reflux stirring reaction, centrifugation, washing, drying, and preparing alkenylated UiO-66-OH material;

[0016] S3. Ion liquid loading: add 1-allyl-3-methyl imidazolium chloride and alkenylated UiO-66-OH material into dichloromethane, add alkali, heat reflux stirring reaction, centrifugation, washing, drying, and preparing alkenylated UiO-66-OH material loaded with ion liquid;

[0017] S4. Preparation of molecularly imprinted polymer: mix alkenylated UiO-66-OH material loaded with ion liquid, camphor, monomer, crosslinking agent, and emulsifier into acetonitrile, mix uniformly, drop into water, emulsify, add initiator under inert gas protection, heat stirring reaction, filter the product, wash, dry, and prepare molecularly imprinted polymer;

[0018] S5. Preparation of adsorbent: put the molecularly imprinted polymer into a Soxhlet extractor, extract with mixed solvents until no camphor is detected, wash, dry, and prepare the adsorbent.

[0019] As a further improvement of the present application, the molar ratio of the zirconium tetrachloride and 2-hydroxyterephthalic acid in step S1 is 1:1, the solvent is a mixed solvent of N,N-dimethylformamide and acetic acid, the volume ratio is 38-39:1-2, the temperature of the hydrothermal reaction is 110-130℃, and the time is 20-28h.

[0020] As a further improvement of the present application, the mass ratio of the UiO-66-OH, chloroethyl acrylate and base in step S2 is 10:3-5:4-6, the base is selected from at least one of triethylamine, diethylamine, NaOH, KOH, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate, and the time of the heating reflux stirring reaction is 2-4h.

[0021] As a further improvement of the present application, the mass ratio of the 1-allyl-3-chloromethyl imidazole, allylated UiO-66-OH material and base in step S3 is 2-4:10:3-5, the time of the heating reflux stirring reaction is 3-5h, and the base is selected from at least one of triethylamine, diethylamine, NaOH, KOH, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate.

[0022] As a further improvement of the present application, the mass ratio of the supported ionic liquid allylated UiO-66-OH material, camphor, monomer, crosslinking agent, emulsifier and initiator in step S4 is 4-6:3-4:12-15:0.3-0.7:0.3-0.5:0.05-0.1, the temperature of the heating stirring reaction is 55-65℃, the time is 3-5h, the crosslinking agent is ethylene glycol dimethacrylate, the emulsifier is selected from at least one of Span-20, Tween-20, Span-40, Tween-40, Span-60, Tween-60, Span-80, Tween-80, Span-85, and Tween-85, the monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide and styrene, the mass ratio is 3-5:2-4:3-5:1-3, and the initiator is selected from at least one of sodium persulfate, potassium persulfate and ammonium persulfate.

[0023] As a further improvement of the present application, the mixed solvent in step S5 is a mixed solvent of ethyl acetate and methanol, and the volume ratio is 5-8:2-3.

[0024] As a further improvement of the present application, the mass ratio of the sample to the adsorbent in step (1) is 1:0.3-0.5, the stirring adsorption time is 1-2h, the ultrasonic desorption power is 1000-2000W, and the time is 20-40min; in step (2), the concentration of naphthalene in the internal standard solution is 20-30mg / mL; in step (3), the concentration of camphor in the control solution is 20-30mg / mL; in step (4), the volume ratio of the internal standard solution to the control solution is 1:1.

[0025] As a further improvement of the present application, in step (5), the gas chromatography analysis conditions are as follows: polyethylene glycol capillary column, injection port temperature is 200-240℃, detector temperature is 240-260℃, split ratio is 8-12:1, column temperature is programmed to rise, initial temperature is 70-80℃, and the temperature is raised by 5-10℃ per minute to 120-130℃, and maintained for 15-25 minutes.

[0026] As a further improvement of the present application, the ratio of camphor peak area to internal standard peak area is Y, the concentration is X, the regression equation is Y=1.4681X+0.005, the linear correlation coefficient r=1, and the camphor peak area to internal standard peak area ratio in the concentration range of 0.04mg / mL-1.00mg / mL has a good linear relationship.

[0027] The present application has the following beneficial effects:

[0028] The external treatment of arthralgia medicine preparation, such as Dansheng Tongluo paste, contains a variety of functional components, and after being soaked and extracted by an organic solvent, a large number of loaded active molecules are dissolved and extracted out, and in the subsequent gas chromatography detection process, a large number of impurity peaks will appear, resulting in a large error in the detection of camphor content and low accuracy, therefore, in order to efficiently and accurately detect the camphor content, the present application prepares an adsorbent with high specificity.

[0029] The present application uses molecularly imprinted polymers to adsorb and fix camphor in the sample solution of the external treatment of arthralgia medicine preparation, and after separating the adsorbent, ultrasonic and organic solvents with stronger affinity are used for desorption, so that most of the camphor can be extracted and separated, the purity of camphor in the test sample solution is high, the content of other impurities is extremely low during detection, and the accuracy of camphor detection is greatly improved.

[0030] The molecularly imprinted polymer adsorbent prepared by the template method is convenient to store, realizes specific adsorption of camphor, has high selectivity, can be repeatedly used, reduces cost, and has low consumption, the UiO-66-OH material with a hydroxyl group on the surface is prepared first, the specific surface area is large, and the adsorption amount of camphor is greatly improved, the surface of the prepared UiO-66-OH material is provided with a double bond through the reaction of the hydroxyl group and chloroethyl acrylate, and the further surface unreacted hydroxyl group is reacted with 1-allyl-3-chloromethyl imidazole, so that the UiO-66-OH material surface is further provided with ionic liquid material, the ionic liquid material has high solubility and high adsorption to camphor, so that the adsorption and separation of camphor are rapidly realized, and the subsequent double bond can be crosslinked with monomers to form a polymer in a polymerization reaction process, wherein, the ionic liquid loaded allylated UiO-66-OH material can be a cross point of the polymer, and the mechanical strength of the adsorbent is improved.

[0031] The detection method of camphor in the external use drug preparation for treating arthralgia has simple detection, high accuracy, good durability, linear correlation in a certain concentration range, can effectively control the quality of the product, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0033] Figure 1 The linear regression graph in embodiment 3 of the present application. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0035] Preparation example 1: preparation of adsorbent

[0036] The method is as follows:

[0037] S1. Preparation of UiO-66-OH: 0.01 mol of zirconium tetrachloride and 0.01 mol of 2-hydroxyterephthalic acid were added to 100 mL of solvent, ultrasonic dispersion was performed at 800 W for 20 min, hydrothermal reaction was performed at 110°C for 20 h, centrifugation, washing, and drying were performed, and UiO-66-OH was prepared;

[0038] The solvent is a mixed solvent of N,N-dimethylformamide and acetic acid, and the volume ratio is 38:2;

[0039] S2. Preparation of an alkenylated UiO-66-OH material: 10 g of UiO-66-OH was added to 200 mL of dichloromethane, 3 g of chloroethyl acrylate and 4 g of NaOH were added, and the reaction was stirred under heating reflux for 2 h, centrifugation, washing, and drying were performed, and the alkenylated UiO-66-OH material was prepared;

[0040] S3. Ion liquid loading: 2 g of 1-allyl-3-methyl imidazole chloride and 10 g of the alkenylated UiO-66-OH material were added to 200 mL of dichloromethane, 3 g of NaOH was added, and the reaction was stirred under heating reflux for 3 h, centrifugation, washing, and drying were performed, and the ion liquid-loaded alkenylated UiO-66-OH material was prepared;

[0041] S4. Preparation of a molecularly imprinted polymer: 4 g of the ion liquid-loaded alkenylated UiO-66-OH material, 3 g of camphor, 12 g of monomers, 0.3 g of ethylene glycol dimethacrylate, 0.15 g of Span-60, and 0.15 g of Tween-60 were mixed and added to 150 mL of acetonitrile, mixed uniformly, added dropwise to 300 mL of water, emulsified at 8000 r / min for 15 min, 0.05 g of sodium persulfate was added under nitrogen protection, heated to 55°C, and stirred for 3 h, the product was filtered, washed, and dried, and the molecularly imprinted polymer was prepared;

[0042] The monomers are a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene, and the mass ratio is 3:2:3:1;

[0043] S5. Preparation of an adsorbent: the molecularly imprinted polymer was added to a Soxhlet extractor, and mixed solvents were extracted until no camphor was detected, washed, and dried, and the adsorbent was prepared;

[0044] The mixed solvents are a mixed solvent of ethyl acetate and methanol, and the volume ratio is 5:2.

[0045] Preparation of an adsorbent

[0046] The method is as follows:

[0047] S1. Preparation of UiO-66-OH: 0.01 mol of zirconium tetrachloride and 0.01 mol of 2-hydroxyterephthalic acid were added to 100 mL of solvent, ultrasonic dispersion was performed at 800 W for 20 min, hydrothermal reaction was performed at 130 DEG C for 28 h, centrifugation, washing, and drying were performed, and UiO-66-OH was prepared;

[0048] The solvent is a mixed solvent of N,N-dimethylformamide and acetic acid, and the volume ratio is 39:1;

[0049] S2. Preparation of alkenylated UiO-66-OH material: 10 g of UiO-66-OH was added to 200 mL of dichloromethane, 5 g of chloroethyl acrylate and 6 g of diethylamine were added, heating reflux stirring reaction was performed for 4 h, centrifugation, washing, and drying were performed, and the alkenylated UiO-66-OH material was prepared;

[0050] S3. Ion liquid loading: 4 g of 1-allyl-3-methyl imidazole chloride and 10 g of alkenylated UiO-66-OH material were added to 200 mL of dichloromethane, 5 g of diethylamine was added, heating reflux stirring reaction was performed for 5 h, centrifugation, washing, and drying were performed, and the alkenylated UiO-66-OH material loaded with ion liquid was prepared;

[0051] S4. Preparation of molecularly imprinted polymer: 6 g of alkenylated UiO-66-OH material loaded with ion liquid, 4 g of camphor, 15 g of monomer, 0.7 g of ethylene glycol dimethacrylate, 0.25 g of Span-20, and 0.25 g of Tween-20 were mixed and added to 150 mL of acetonitrile, mixed uniformly, added dropwise to 300 mL of water, emulsified at 8000 r / min for 15 min, 0.1 g of potassium persulfate was added under nitrogen protection, heated to 65 DEG C, stirring reaction was performed for 5 h, the product was filtered, washed, and dried, and the molecularly imprinted polymer was prepared;

[0052] The monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene, and the mass ratio is 5:4:5:3;

[0053] S5. Preparation of adsorbent: the molecularly imprinted polymer was added to a Soxhlet extractor, mixed solvent extraction was performed until no camphor was detected, washing, and drying were performed, and the adsorbent was prepared;

[0054] The mixed solvent is a mixed solvent of ethyl acetate and methanol, and the volume ratio is 8:3.

[0055] Preparation of adsorbent

[0056] The method is as follows:

[0057] S1. Preparation of UiO-66-OH: 0.01 mol of zirconium tetrachloride and 0.01 mol of 2-hydroxyterephthalic acid were added to 100 mL of solvent, ultrasonic dispersion was performed at 800 W for 20 min, hydrothermal reaction was performed at 120 DEG C for 24 h, centrifugation, washing, and drying were performed, and UiO-66-OH was prepared;

[0058] The solvent is a mixed solvent of N,N-dimethylformamide and acetic acid, and the volume ratio is 38:2;

[0059] S2. Preparation of allylated UiO-66-OH material: 10 g of UiO-66-OH was added to 200 mL of dichloromethane, 4 g of chloroethyl acrylate and 5 g of triethylamine were added, heating reflux stirring reaction was performed for 3 h, centrifugation, washing, and drying were performed, and the allylated UiO-66-OH material was prepared;

[0060] S3. Ion liquid loading: 3 g of 1-allyl-3-methyl imidazole chloride and 10 g of allylated UiO-66-OH material were added to 200 mL of dichloromethane, 4 g of triethylamine was added, heating reflux stirring reaction was performed for 4 h, centrifugation, washing, and drying were performed, and the ion liquid loaded allylated UiO-66-OH material was prepared;

[0061] S4. Preparation of molecularly imprinted polymer: 5 g of ion liquid loaded allylated UiO-66-OH material, 3.5 g of camphor, 13 g of monomer, 0.5 g of ethylene glycol dimethacrylate, 0.2 g of Span-85, and 0.2 g of Tween-85 were mixed and added to 150 mL of acetonitrile, mixed uniformly, added dropwise to 300 mL of water, emulsified at 8000 r / min for 15 min, 0.07 g of ammonium persulfate was added under nitrogen protection, heated to 60 DEG C, stirring reaction was performed for 4 h, the product was filtered, washed, and dried, and the molecularly imprinted polymer was prepared;

[0062] The monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene, and the mass ratio is 4:3:4:2;

[0063] S5. Preparation of adsorbent: the molecularly imprinted polymer was added to a Soxhlet extractor, mixed solvent extraction was performed until no camphor was detected, washing, and drying were performed, and the adsorbent was prepared;

[0064] The mixed solvent is a mixed solvent of ethyl acetate and methanol, and the volume ratio is 6:2.

[0065] Comparative Preparation Example 1

[0066] Compared with Preparation Example 3, the difference is that step S3 is not performed.

[0067] The specific process is as follows:

[0068] S1. Preparation of UiO-66-OH: 0.01 mol of zirconium tetrachloride and 0.01 mol of 2-hydroxyterephthalic acid were added to 100 mL of solvent, ultrasonic dispersion was performed at 800 W for 20 min, hydrothermal reaction was performed at 120 DEG C for 24 h, centrifugation, washing, and drying were performed, and UiO-66-OH was prepared;

[0069] The solvent is a mixed solvent of N,N-dimethylformamide and acetic acid, and the volume ratio is 38:2;

[0070] S2. Preparation of alkenylated UiO-66-OH material: 10 g of UiO-66-OH was added to 200 mL of dichloromethane, 4 g of chloroethyl acrylate and 5 g of triethylamine were added, and the reaction was stirred under reflux for 3 h, centrifugation, washing, and drying were performed, and the alkenylated UiO-66-OH material was prepared;

[0071] S3. Preparation of molecularly imprinted polymer: 5 g of alkenylated UiO-66-OH material, 3.5 g of camphor, 13 g of monomer, 0.5 g of ethylene glycol dimethacrylate, 0.2 g of Span-85, and 0.2 g of Tween-85 were mixed and added to 150 mL of acetonitrile, mixed uniformly, and added dropwise to 300 mL of water, emulsified at 8000 r / min for 15 min, 0.07 g of ammonium persulfate was added under nitrogen protection, heated to 60 DEG C, and stirred for 4 h, the product was filtered, washed, and dried, and the molecularly imprinted polymer was prepared;

[0072] The monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene, and the mass ratio is 4:3:4:2;

[0073] S4. Preparation of adsorbent: the molecularly imprinted polymer was added to a Soxhlet extractor, and mixed solvent extraction was performed until no camphor was detected, washing, and drying were performed, and the adsorbent was prepared;

[0074] The mixed solvent is a mixed solvent of ethyl acetate and methanol, and the volume ratio is 6:2.

[0075] Comparative Preparation Example 2

[0076] Compared with Preparation Example 3, the difference is that step S2 is not performed.

[0077] Specifically as follows:

[0078] S1. Preparation of UiO-66-OH: 0.01 mol of zirconium tetrachloride and 0.01 mol of 2-hydroxyterephthalic acid were added to 100 mL of solvent, ultrasonic dispersion was performed at 800 W for 20 min, hydrothermal reaction was performed at 120 DEG C for 24 h, centrifugation, washing, and drying were performed, and UiO-66-OH was prepared;

[0079] The mixed solvent is a mixture of N,N-dimethylformamide and acetic acid, with a volume ratio of 38:2;

[0080] S2. Loading of the ionic liquid: 3g of 1-allyl-3-methyl imidazolium chloride and 10g of UiO-66-OH were added to 200mL of dichloromethane, 4g of triethylamine was added, and the reaction was stirred under reflux heating for 4h, centrifuged, washed, and dried to obtain the ionic liquid-loaded allylated UiO-66-OH material;

[0081] S4. Preparation of the molecularly imprinted polymer: 5g of the ionic liquid-loaded allylated UiO-66-OH material, 3.5g of camphor, 13g of monomer, 0.5g of ethylene glycol dimethacrylate, 0.2g of Span-85, and 0.2g of Tween-85 were mixed in 150mL of acetonitrile, mixed uniformly, and added dropwise into 300mL of water, emulsified at 8000r / min for 15min, 0.07g of ammonium persulfate was added under nitrogen protection, heated to 60℃, and stirred for 4h, and the product was filtered, washed, and dried to obtain the molecularly imprinted polymer;

[0082] The monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene, with a mass ratio of 4:3:4:2;

[0083] S5. Preparation of the adsorbent: the molecularly imprinted polymer was added to a Soxhlet extractor, and extracted with the mixed solvent until no camphor was detected, washed, and dried to obtain the adsorbent;

[0084] The mixed solvent is a mixture of ethyl acetate and methanol, with a volume ratio of 6:2.

[0085] Comparative Preparation Example 3

[0086] Compared with Preparation Example 3, the difference is that steps S2 and S3 are not performed.

[0087] The details are as follows:

[0088] S1. Preparation of UiO-66-OH: 0.01mol of zirconium tetrachloride and 0.01mol of 2-hydroxyterephthalic acid were added to 100mL of solvent, ultrasonically dispersed at 800W for 20min, hydrothermally reacted at 120℃ for 24h, centrifuged, washed, and dried to obtain UiO-66-OH;

[0089] The mixed solvent is a mixture of N,N-dimethylformamide and acetic acid, with a volume ratio of 38:2;

[0090] S2. Preparation of the molecularly imprinted polymer: 5 g of UiO-66-OH, 3.5 g of camphor, 13 g of monomer, 0.5 g of ethylene glycol dimethacrylate, 0.2 g of Span-85, and 0.2 g of Tween-85 were mixed in 150 mL of acetonitrile, mixed uniformly, added dropwise into 300 mL of water, emulsified at 8000 r / min for 15 min, 0.07 g of ammonium persulfate was added under nitrogen protection, heated to 60 DEG C, stirred for 4 h, the product was filtered, washed, and dried to obtain the molecularly imprinted polymer;

[0091] The monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene, and the mass ratio is 4:3:4:2.

[0092] S3. Preparation of the adsorbent: the molecularly imprinted polymer was added to a Soxhlet extractor, extracted with a mixed solvent until no camphor was detected, washed, and dried to obtain the adsorbent.

[0093] The mixed solvent is a mixed solvent of ethyl acetate and methanol, and the volume ratio is 6:2.

[0094] Comparative Preparation Example 4

[0095] Compared with Preparation Example 3, the difference lies in that the allylated UiO-66-OH material loaded with ionic liquid is replaced by equal mass of 1-allyl-3-methylimidazole chloride salt.

[0096] Comparative Preparation Example 5

[0097] Compared with Preparation Example 3, the difference lies in that no allylated UiO-66-OH material loaded with ionic liquid is added in step S4.

[0098] Specifically as follows:

[0099] S4. Preparation of the molecularly imprinted polymer: 3.5 g of camphor, 18 g of monomer, 0.5 g of ethylene glycol dimethacrylate, 0.2 g of Span-85, and 0.2 g of Tween-85 were mixed in 150 mL of acetonitrile, mixed uniformly, added dropwise into 300 mL of water, emulsified at 8000 r / min for 15 min, 0.07 g of ammonium persulfate was added under nitrogen protection, heated to 60 DEG C, stirred for 4 h, the product was filtered, washed, and dried to obtain the molecularly imprinted polymer.

[0100] The monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene, and the mass ratio is 4:3:4:2. Example 1

[0101] The present example provides a method for determining the content of camphor in an external use drug preparation for treating arthralgia, comprising the following steps:

[0102] (1) Preparation of test solution: 1 g of the sample of Danshen Tongluo Ointment was taken, the cover liner was removed, and the sample was cut into small pieces, which were placed in a stoppered triangular flask, 50 mL of dichloromethane was added, 0.4 g of the adsorbent prepared in Preparation Example 1 was added, and stirring adsorption was performed for 1.5 h, filtration was performed, the adsorbent was added to 30 mL of ethyl acetate, desorption was performed by ultrasonic treatment at 1500 W for 30 min, filtration was performed, the solid was washed, and drying was performed, the solution was diluted to 50 mL, and the test solution was prepared;

[0103] (2) Preparation of internal standard solution: naphthalene was accurately weighed and added to ethyl acetate to prepare an internal standard solution with a concentration of 25 mg / mL;

[0104] (3) Preparation of control solution: camphor was accurately weighed, and added to ethyl acetate to prepare a control solution with a concentration of 25 mg / mL;

[0105] (4) Preparation of calibration solution: 1 mL of the internal standard solution and 1 mL of the control solution were accurately measured, and diluted to 50 mL to prepare a calibration solution;

[0106] (5) Sample injection and detection: the content of camphor in the test solution was analyzed by gas chromatography;

[0107] The conditions for gas chromatography analysis were as follows: polyethylene glycol capillary column, injection port temperature of 220 °C, detector temperature of 250 °C, split ratio of 10:1, column temperature of programmed temperature, initial temperature of 80 °C, temperature increase of 5 °C per minute to 130 °C, and maintenance for 20 minutes. Example 2

[0108] According to the preparation of the control solution in Example 1, 6 consecutive injections were performed, the peak area was recorded, and the correction factor (f) and relative standard deviation (RSD) of camphor and the internal standard were calculated, and the results are shown in Table 1.

[0109] Table 1 Results of instrument precision test

[0110] ;

[0111] Conclusion: The RSD of the correction factor is 0.28%, indicating that the instrument precision is good.

[0112] Example 3

[0113] The control solution with concentration ranging from 10% to 200% was prepared respectively, 50 mg of camphor control sample was weighed into a 25 mL volumetric flask, and dissolved by shaking with ethyl acetate, and then diluted to volume, as a control stock solution; 125 mg of internal standard naphthalene was weighed into a 25 mL volumetric flask, and dissolved by shaking with ethyl acetate, and then diluted to volume, as an internal standard stock solution; 0.2 mL, 0.5 mL, 1 mL, 2 mL, 2.5 mL, 3 mL, 4 mL, 5 mL of the control stock solution and 1.0 mL of the internal standard stock solution were precisely taken into a 10 mL volumetric flask, and diluted to volume with ethyl acetate as a control sample solution. The sample was injected, and the peak area was recorded. The results are shown in Table 2. The regression equation and linear correlation coefficient Y = 1.4681X + 0.005, r = 1.0 were obtained by linear regression according to the ratio of camphor peak area to internal standard peak area as the ordinate (Y) and the concentration as the abscissa (X). The results show that the ratio of camphor peak area to internal standard peak area is in good linear relationship within the concentration range of 0.04 mg / mL to 1.00 mg / mL.

[0114] Table 2 Linear test results

[0115] ;

[0116] Example 4 Stability test

[0117] According to the chromatographic conditions under the content determination, the control sample solution and the test sample solution were respectively injected at 0 h, 3 h, 6 h, 9 h, 14 h, 19 h, and 24 h, and the peak area of camphor and the peak area of the internal standard were recorded. The results are shown in Tables 3 and 4.

[0118] Table 3 Stability test results of the control sample solution (peak area)

[0119] ;

[0120] Table 4 Stability test results of the sample solution (peak area)

[0121] ;

[0122] Conclusion: The control sample solution and the test sample solution are stable within 24 h.

[0123] Example 5 Reproducibility test

[0124] The control sample and the sample were prepared according to the above-mentioned “control sample solution preparation method” and “test sample solution preparation method”. Six samples of the same batch were prepared in parallel by the same method. The sample was injected, and the content was calculated by the internal standard method according to the peak area. The results are shown in Table 5.

[0125] Table 5 Reproducibility test results

[0126] ;

[0127] Conclusion: The repeatability is good, and the relative standard deviation is less than 1.5%.

[0128] Intermediate precision test in Example 6

[0129] Different personnel prepared the same batch of test sample, and determined the content with different gas chromatographs. The determination results are shown in Table 6.

[0130] Table 6: Results of intermediate precision test

[0131]

[0132] Conclusion: The RSD value determined by different personnel and different instruments is 1.62%, and the intermediate precision is good.

[0133] Durability test in Example 7

[0134] The split ratio in the chromatographic condition was changed to 5:1 and 15:1, respectively; the injection port temperature was 210°C and 230°C; the detector (FID) temperature was 240°C and 260°C, respectively; the test sample solution and the control sample solution were injected, and the determination results are shown in Table 7.

[0135] Table 7: Results of durability test

[0136]

[0137] Conclusion: With small changes in the split ratio, the injection port temperature, and the detector temperature, the separation degree of the main peak of camphor and the adjacent peaks of the internal standard substance is greater than 2.0, and the method is feasible.

[0138] Accuracy test in Example 8

[0139] Preparation of internal standard stock solution: 300 mg of internal standard naphthalene was accurately weighed into a 50 mL volumetric flask, and then an appropriate amount of ethyl acetate was added to dissolve and dilute to the mark, serving as the internal standard stock solution.

[0140] Preparation method of control sample solution: 75 mg of camphor control sample was weighed into a 50 mL volumetric flask, and then an appropriate amount of ethyl acetate was added to dissolve and dilute to the mark, serving as the control stock solution.

[0141] Take 1 / 4 piece of Danshen Tongluo Paste, and process it according to the method in Example 1 to obtain the test sample solution. Add 2 mL of the control stock solution and 2 mL of the internal standard stock solution to each sample, and then add 10 mL of ethyl acetate to each sample, and ultrasonically treat for 10 min. Filter, and then take the filtrate into a 25 mL volumetric flask. Wash the flask with an appropriate amount of ethyl acetate, and then add the washing liquid into the volumetric flask. Dilute to the mark with ethyl acetate, and then serve as the accuracy test sample solution. Inject each sample, and the results are shown in Table 8. The adsorbent in samples 1-3 is prepared according to Preparation Examples 2-3, and the adsorbent in samples 4-8 is prepared according to Comparative Preparation Examples 1-5. A total of 8 samples are prepared. ​​

[0142] Table 8 accuracy test results

[0143]

[0144] From the above table, the recovery rate in samples 1-3 is 98-100%, and the accuracy is good. In samples 4-8, the adsorbent prepared by comparative preparation example 1-5 is used for treatment, and the recovery rate is significantly reduced.

[0145] Content determination of three batches of samples

[0146] According to the method in Example 1, the content of camphor in three batches of Danshen Tongluo Paste preparation samples (batch number: 20200523002, 20200817003, 20201202004) was determined, and the determination results are shown in Table 9.

[0147] Table 9 Camphor content determination results in samples

[0148]

[0149] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.​​

Claims

1. A method for determining the content of camphor in an external therapeutic preparation for arthralgia, characterized by, The method comprises the following steps: (1) Preparation of test sample solution: take the sample, remove the cover liner, cut into small pieces, put into a flask with a stopper, add dichloromethane, add adsorbent, stir and adsorb, filter, add ethyl acetate to the adsorbent, ultrasonic desorption, filter, wash the solid, dry, reuse, dilute the solution to a certain volume, and prepare the test sample solution; (2) Preparation of internal standard solution: accurately weigh naphthalene, add ethyl acetate, and prepare the internal standard solution; (3) Preparation of reference substance solution: accurately weigh camphor reference substance, add ethyl acetate, and prepare the reference substance solution; (4) Preparation of calibration solution: accurately measure the internal standard solution and the reference substance solution, respectively, dilute to a certain volume, and prepare the calibration solution; (5) Sample injection detection: analyze the content of camphor in the test sample solution by gas chromatography; The preparation method of the adsorbent is as follows: S1. Preparation of UiO-66-OH: add zirconium tetrachloride and 2-hydroxyterephthalic acid into a solvent, ultrasonically disperse uniformly, hydrothermally react, centrifuge, wash, and dry to prepare UiO-66-OH; the solvent is a mixed solvent of N,N-dimethylformamide and acetic acid; S2. Preparation of alkenylated UiO-66-OH material: add UiO-66-OH into dichloromethane, add chloroethyl acrylate and alkali, heat and reflux, stir and react, centrifuge, wash, and dry to prepare the alkenylated UiO-66-OH material; S3. Ion liquid loading: add 1-allyl-3-methyl imidazole chloride and the alkenylated UiO-66-OH material into dichloromethane, add alkali, heat and reflux, stir and react, centrifuge, wash, and dry to prepare the alkenylated UiO-66-OH material loaded with ion liquid; S4. Preparation of molecularly imprinted polymer: mix the alkenylated UiO-66-OH material loaded with ion liquid, camphor, monomer, crosslinking agent, and emulsifier into acetonitrile, mix uniformly, drop into water, emulsify, add initiator under inert gas protection, heat and stir to react, filter the product, wash, and dry to prepare the molecularly imprinted polymer; the crosslinking agent is ethylene glycol dimethacrylate, the monomer is a mixture of methyl methacrylate, butyl acrylate, acrylamide, and styrene with a mass ratio of 3-5:2-4:3-5:1-3, and the initiator is at least one selected from sodium persulfate, potassium persulfate, and ammonium persulfate; S5. Preparation of adsorbent: put the molecularly imprinted polymer into a Soxhlet extractor, extract with a mixed solvent until no camphor is detected, wash, and dry to prepare the adsorbent.

2. The method for determining the content of camphor in the external therapeutic arthromyodynia medicated preparation according to claim 1, characterized in that, In step S1, the molar ratio of zirconium tetrachloride to 2-hydroxyterephthalic acid is 1:1, the volume ratio of N,N-dimethylformamide to acetic acid in the solvent is 38-39:1-2, the temperature of the hydrothermal reaction is 110-130℃, and the time is 20-28h.

3. The method for determining the content of camphor in the external therapeutic arthromyodynia medicated preparation according to claim 1, characterized in that, The mass ratio of the UiO-66-OH, chloroethyl acrylate and the base in step S2 is 10:3-5:4-6, the base is at least one selected from triethylamine, diethylamine, NaOH, KOH, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, the heating reflux stirring reaction time is 2-4h.

4. The method for determining the content of camphor in the external therapeutic arthromyodynia medicated preparation according to claim 1, characterized in that, The mass ratio of the 1-allyl-3-chloromethyl imidazole, allylated UiO-66-OH material and the base in step S3 is 2-4:10:3-5, the heating reflux stirring reaction time is 3-5h, the base is at least one selected from triethylamine, diethylamine, NaOH, KOH, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate.

5. The method for determining the camphor content of a therapeutic external preparation for arthralgia according to claim 1, characterized by, The mass ratio of the supported ionic liquid allylated UiO-66-OH material, camphor, monomer, crosslinking agent, emulsifier and initiator in step S4 is 4-6:3-4:12-15:0.3-0.7:0.3-0.5:0.05-0.1, the heating stirring reaction temperature is 55-65℃, the time is 3-5h, the emulsifier is at least one selected from Span-20, Tween-20, Span-40, Tween-40, Span-60, Tween-60, Span-80, Tween-80, Span-85, Tween-85.

6. The method for determining the camphor content of a therapeutic external preparation for arthralgia according to claim 1, characterized by, The mixed solvent in step S5 is a mixed solvent of ethyl acetate and methanol, the volume ratio is 5-8:2-3.

7. The method for determining the camphor content of a therapeutic external preparation for arthralgia according to claim 1, wherein The mass ratio of the sample and the adsorbent in step (1) is 1:0.3-0.5, the stirring adsorption time is 1-2h, the ultrasonic desorption power is 1000-2000W, the time is 20-40min; the concentration of naphthalene in the internal standard solution in step (2) is 20-30mg / mL; the concentration of camphor in the control sample solution in step (3) is 20-30mg / mL; the volume ratio of the internal standard solution and the control sample solution in step (4) is 1:

1.

8. The method for determining the camphor content of a therapeutic external preparation for arthralgia according to claim 1, characterized by, The gas chromatography analysis conditions in step (5) are: polyethylene glycol capillary column, the injection port temperature is 200-240℃, the detector temperature is 240-260℃, the split ratio is 8-12:1, the column temperature is programmed, the initial temperature is 70-80℃, the temperature is increased by 5-10℃ per minute to 120-130℃, and maintained for 15-25 minutes.

9. The method for determining the camphor content of a therapeutic external preparation for arthralgia according to claim 1, wherein The ratio of camphor peak area to internal standard peak area is Y, the concentration is X, the regression equation is: Y=1.4681X+0.005, the linear correlation coefficient r=1, the camphor peak area and the internal standard area ratio show a good linear relationship in the concentration range of 0.04mg / mL-1.00mg / mL.

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