CN112067718A - Establishment method of muskiness rheumatalgia-relieving plaster volatile component fingerprint, standard fingerprint and application thereof - Google Patents
Establishment method of muskiness rheumatalgia-relieving plaster volatile component fingerprint, standard fingerprint and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicine preparation identification, in particular to a method for establishing a volatile component fingerprint of a musk rheumatalgia-relieving pain paste, and a standard fingerprint and application thereof. The method comprises a preparation step of a reference substance solution, a preparation step of a test solution and a detection step, wherein in the preparation step of the test solution, N-dimethylformamide is used as a solvent to extract volatile components in a musk rheumatalgia-relieving plaster sample, and a liquid phase part is taken to obtain the test solution. The technical problem that a quality control method for volatile components of the musk rheumatalgia-relieving pain-relieving ointment is lacked can be solved. The standard fingerprint spectrum and the detection method established by the scheme can be applied to identifying the volatile components of rubber plasters, gel plasters and powdery premixed plasters of the musk rheumatalgia-relieving pain paste.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine preparation identification, in particular to a method for establishing a volatile component fingerprint of a musk rheumatalgia-relieving pain paste, and a standard fingerprint and application thereof.
Background
A Chinese medicinal composition, SHEXIANGZHUIFENGZHITONG plaster, is prepared from herba Geranii, cortex Periplocae Radicis, Camphora, Mentholum, Borneolum Syntheticum, methyl salicylate, herba Ruta graveolentis extract (Ruta graveolens L.) and belladonna extract (belladonna), and has good antiinflammatory and analgesic effects and can be used for treating arthralgia, tenderness, difficulty in flexion and extension, and morning stiffness caused by rheumatism. The applicant of the invention applies for the process patent of the musk wind-dispelling pain-relieving plaster in the project establishment research process, application number CN200610079120.0, and then the applicant further performs improved research on the process of the musk wind-dispelling pain-relieving plaster, and applies for the invention patent, application number: CN201810344858.8, both of the two patents are patents of technical invention, and in order to control the quality of the product, it is urgently needed to develop a quality control method for identifying and controlling multiple volatile components in the musk plaster for dispelling wind and relieving pain. Because the product contains more volatile components (camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol), the solubility of the substances and the separation characteristics in a chromatographic column are different, and a stable and reliable method for detecting the volatile components of the relevant product of the musk rheumatalgia-relieving fluid extract is difficult to find.
Disclosure of Invention
The invention aims to provide a method for establishing a volatile component fingerprint spectrum of a musk rheumatalgia-relieving pain-relieving plaster, which is used for solving the technical problem that a quality control method for simultaneously detecting multiple volatile components of the musk rheumatalgia-relieving pain-relieving plaster is lacked.
In order to achieve the purpose, the invention adopts the following technical scheme:
the establishment method of the volatile component fingerprint spectrum of the musk rheumatalgia-relieving pain ointment comprises the steps of preparing a reference substance solution, preparing a test solution and detecting, wherein in the step of preparing the test solution, N-dimethylformamide is used as a solvent to extract volatile components in a musk rheumatalgia-relieving pain ointment sample, and a liquid phase part is taken to obtain the test solution.
The principle and the beneficial effects of the technical scheme are as follows: the N, N-dimethylformamide is used as a solvent to extract volatile components in the musk rheumatalgia-relieving pain paste sample, so that functional components in the musk rheumatalgia-relieving pain paste can be fully extracted and used in the subsequent gas chromatography detection process. The musk rheumatalgia-relieving pain-relieving ointment has more volatile components (camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol), the solubility of all the substances is different, and a solvent which can dissolve and extract all volatile substances is difficult to find. The inventor tries various solvents, and finally finds that only N, N-dimethylformamide can fully extract 10 volatile components in the musk rheumatalgia-relieving plaster, and the fingerprint with clear characteristic peak separation and meeting the requirement can be obtained in the subsequent gas chromatography detection. The N, N-dimethylformamide has good adaptability to fingerprint identification of the musk rheumatalgia-relieving pain-relieving plaster, and when other solvents are used, volatile components in the musk rheumatalgia-relieving pain-relieving plaster cannot be simultaneously and fully extracted. The use of N, N-dimethylformamide has led to unexpected technical results, and the experimental data are detailed in Experimental example 1 and the accompanying FIGS. 3-5 of the description.
The beneficial effects of the technical scheme are summarized as follows:
(1) according to the gas chromatography fingerprint identification spectrum measuring method provided by the invention, the obtained volatile component identification fingerprint spectrum of the musk rheumatalgia-relieving pain paste composition can reflect volatile components in a medicine, wherein the volatile components are respectively selected from camphor, borneol (isoborneol and borneol), menthol, schizonepeta (pulegone), frankincense (octyl acetate), wintergreen oil (methyl salicylate), kaempferia galangal (ethyl p-methylcinnamate) and clove (eugenol).
(2) The gas chromatography fingerprint identification spectrum measuring method provided by the invention has good specificity, precision, repeatability, solution stability and durability.
(3) The gas chromatography fingerprint identification spectrum measuring method provided by the invention can control the internal quality of the product, improve the quality control level and better ensure the quality of the product.
Further, the preparation method of the test solution comprises the following steps: adding N, N-dimethylformamide into a musk rheumatalgia-relieving plaster sample, carrying out ultrasonic treatment under an ice bath condition, standing, taking a supernatant, filtering the supernatant, and taking a liquid phase to obtain the test sample solution.
By adopting the technical scheme, when N, N-dimethylformamide is used as an extraction solvent, the ultrasonic extraction method is simultaneously used, so that the target components can be fully dissolved out of the musk rheumatalgia-relieving pain-relieving ointment.
Further, the preparation method of the test solution comprises the following steps: cutting 1 piece of the musk rheumatalgia-relieving pain paste sample into strips, placing the strips into a conical flask, adding 50ml of N, N-dimethylformamide, carrying out ultrasonic treatment for 15min under the ice bath condition, standing, taking supernatant, filtering the supernatant, taking liquid phase, and obtaining the test solution. By adopting the method, the volatile components in the musk rheumatalgia-relieving pain-relieving plaster sample can be fully extracted for subsequent detection.
Further, in the step of preparing the control solution, a control solution containing camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, octyl acetate, methyl salicylate, ethyl p-methylcinnamate, and eugenol was prepared using N, N-dimethylformamide as a solvent.
By adopting the technical scheme, the position of the characteristic peak of the sample to be detected is determined by using the reference substance solution.
Furthermore, each 1ml of the reference solution contains 0.1-0.8mg of camphor, 0.05-0.3mg of isoborneol, 0.05-0.3mg of borneol, 0.1-0.5mg of menthol, 0.8-2mg of pulegone, 0.05-0.3mg of cinnamaldehyde, 0.05-0.3mg of octyl acetate, 0.1-1.0mg of methyl salicylate, 0.5-2.0mg of ethyl p-methylcinnamate and 0.1-1.0mg of eugenol. By adopting the dosage of the reference substance, the fingerprint of the standard substance with moderate peak area can be obtained.
Further, in the detecting step, volatile components of the test solution and the reference solution are detected using a gas chromatography under a chromatography condition including a column using macrogol nitrobenzene as a stationary phase.
By adopting the technical scheme, the polyethylene glycol nitrobenzene is used as the chromatographic column filler for the meteorological chromatography detection after the volatile components are extracted, 10 target components can be well separated, the characteristic peak type is good, and the theoretical plate number is high.
The applicant has carried out a large number of studies on the identification of gas chromatography, in particular on the selection of chromatography columns, which have been screened from non-polar capillary columns (packing 100% dimethylpolysiloxane), weakly polar capillary columns (packing 5% phenyl 95% dimethylpolysiloxane), weakly medium polar (packing 6% hydroxypropyl-phenyl 94% dimethylpolysiloxane), medium polar capillary columns (packing 35% phenyl 65% dimethylpolysiloxane 50% phenyl 50% dimethylpolysiloxane), polar capillary columns (packing 35% trifluoropropyl 65% dimethylpolysiloxane), and strongly polar capillary columns (packing 50% trifluoropropyl 50% dimethylpolysiloxane, 50% cyanopropyl-phenyl 50% dimethylpolysiloxane, polyethylene glycol-20M, etc.) with poor resolution in application, the target components can not be detected completely, partial peak-peak type is poor, and the theoretical plate number is low (the comparison experiment result is detailed in Table 2). Therefore, the choice of the column is very critical to the process. In the research, according to the characteristics of polyethylene glycol, the inventor combines a chromatographic column manufacturer to perform packing modification on a polyethylene glycol-20M capillary column, and the capillary packing is changed into polyethylene glycol nitrobenzene. The inventors analyzed that the reason why the ideal fingerprint pattern can be obtained by using the polyethylene glycol nitrobenzene as the filler is that: the filler is modified into the polyethylene glycol nitrobenzene, so that the polarity and the ageing resistance of the filler can be enhanced, the more sufficient separation of substances is ensured, and the service life of the column is prolonged. In conclusion, the scheme uses N, N-dimethylformamide as an extraction solvent and uses polyethylene glycol nitrobenzene as chromatographic column filler, and a gas chromatography standard fingerprint and a detection method which are successfully established are used for identifying and controlling the quality of volatile components of the musk rheumatalgia-relieving pain-relieving plaster composition, so that a better effect is obtained.
Further, in the detecting step, the chromatographic conditions of the gas chromatography further include: the chromatographic column is a capillary column with the column length of 30m-60m, the inner diameter of 0.25-0.53mm and the film thickness of 0.25-1.8 um; the carrier gas is N2; the column temperature is 130 ℃ at the initial temperature, the temperature is kept for 20min, the temperature is increased to 205 ℃ at the rate of 6.5-8.5 ℃ per minute, and then the temperature is kept for 20 min; the temperature of a sample inlet is 250 ℃; the detector temperature was 250 ℃. By adopting the detection method, the standard fingerprint spectrum, the fingerprint spectrum of the sample to be detected and the fingerprint spectrum of the reference substance with sharp characteristic peak type and good separation degree can be obtained.
Furthermore, in the detection step, the sampling amount of the reference solution and the sample solution is 2-5 mul.
Further, the standard fingerprint spectrum established by the scheme comprises characteristic chromatographic peaks respectively representing camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, borneol, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol; the retention time of the characteristic chromatographic peak of the camphor is 4.7-4.9 min; the retention time of the characteristic chromatographic peak of the cinnamaldehyde is 5.4-5.6 min; the average retention time of characteristic chromatographic peaks of the pulegone is 5.8-6.0 min; the retention time of the characteristic chromatographic peak of the menthol is 6.3-6.5 min; the retention time of the characteristic chromatographic peak of isoborneol is 7.1-7.3 min; the retention time of the characteristic chromatographic peak of borneol is 8.0-8.3 min; the retention time of the characteristic chromatographic peak of the methyl salicylate is 10.0-10.3 min; the retention time of the characteristic chromatographic peak of the octyl acetate is 11.1-11.3 min; the retention time of the characteristic chromatographic peak of the ethyl methyl cinnamate is 14.9-15.1 min; the retention time of the characteristic chromatographic peak of the eugenol is 23.2-23.4 min. The characteristic peak of each volatile component in the fingerprint is sharp and the separation degree is good.
Further, the standard fingerprint spectrum established by the scheme is applied to identifying rubber plaster of the musk rheumatalgia-relieving pain-relieving plaster, gel plaster of the musk rheumatalgia-relieving pain-relieving plaster or powder premixed plaster of the musk rheumatalgia-relieving pain-relieving plaster. Muskiness rheumatalgia-relieving plaster comprises various dosage forms, such as: rubber plaster, gel plaster and powder premix plaster. The fingerprint spectrum and the identification method established by the scheme can be applied to the identification of the rubber plaster, the gel plaster and the powdery premixed plaster.
Drawings
FIG. 1 is a control profile of example 1 of the present invention.
Fig. 2 is a standard fingerprint established in example 1 of the present invention.
FIG. 3 is a fingerprint of a sample obtained by using methanol as an extraction solvent according to Experimental example 1 of the present invention.
FIG. 4 is a fingerprint of a sample obtained using ethyl acetate as an extraction solvent in Experimental example 1 of the present invention.
FIG. 5 is a fingerprint of a sample obtained using N, N-dimethylformamide as an extraction solvent in Experimental example 1 of the present invention.
FIG. 6 is a fingerprint of a sample obtained by detection using a DB-1 column chromatography in Experimental example 2 of the present invention.
FIG. 7 is a fingerprint of a sample obtained by detection using a DB-5 column chromatography in Experimental example 2 of the present invention.
FIG. 8 is a fingerprint of a sample obtained by detection using a DB-624 chromatographic column in Experimental example 2 of the present invention.
FIG. 9 is a fingerprint of a sample obtained by detection using a DB-35 column chromatography in Experimental example 2 of the present invention.
FIG. 10 is a fingerprint of a sample obtained by detection using a DB-200 column chromatography in Experimental example 2 of the present invention.
FIG. 11 is a fingerprint of a sample obtained by detection using a DB-210 column chromatography in Experimental example 2 of the present invention.
FIG. 12 is a fingerprint of a sample obtained by detection using a DB-225 column chromatography in Experimental example 2 of the present invention.
FIG. 13 is a fingerprint of a sample obtained by DB-WAX chromatography in Experimental example 2 of the present invention.
FIG. 14 is a fingerprint of a sample obtained by detection using HP-FFAP chromatography in Experimental example 2 of the present invention.
Detailed Description
The following is further detailed by way of specific embodiments:
reference numerals in the drawings of the specification include: camphor characteristic chromatographic peak 1, cinnamaldehyde characteristic chromatographic peak 2, pulegone characteristic chromatographic peak 3, menthol characteristic chromatographic peak 4, isoborneol characteristic chromatographic peak 5, borneol characteristic chromatographic peak 6, methyl salicylate characteristic chromatographic peak 7, octyl acetate characteristic chromatographic peak 8, ethyl p-methylcinnamate characteristic chromatographic peak 9 and eugenol characteristic chromatographic peak 10.
Example 1: establishment of standard fingerprint
The establishment method of fingerprint and standard fingerprint are explained by taking the composition of musk rheumatalgia-relieving pain-relieving plaster (rubber plaster, approved article: national standard Z20027408, specification: 7cm x 10cm, content of plaster in each tablet is not less than 1.12 g). The musk rheumatalgia-relieving pain paste sample in the scheme comprises three types of rubber paste of musk rheumatalgia-relieving pain paste, gel paste of musk rheumatalgia-relieving pain paste or powder premixed paste of musk rheumatalgia-relieving pain paste. In this example, the sample of the musk rheumatalgia-relieving pain-relieving plaster is a rubber plaster of the musk rheumatalgia-relieving pain-relieving plaster.
1. Instrument and reagent
(1) Shimadin gas chromatograph (GC-2014)
(2) Shimadzu electronic balance (ATY224)
(3) Musk rheumatalgia-relieving plaster (own product, batch 190301)
(4) Carrier gas and combustion gas: nitrogen, hydrogen and air
(5) DMF (N, N-dimethylformamide, AR Chengdu Kelong reagent factory)
(6) Camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol reference (Chinese institute for testing and testing biological products)
2. Method and results
(1) Chromatographic conditions: shimadzu gas chromatograph (GC-2014), column: a capillary column with polyethylene glycol nitrobenzene as a filler (the column length is 30m, the inner diameter is 0.32mm, and the film thickness is 0.25 μm); carrier gas: n is a radical of2(ii) a Column temperature: the initial temperature is 130 ℃, the temperature is kept for 20min, the temperature is increased to 195 ℃ at the speed of 7.5 ℃ per minute, and the temperature is kept for 20 min; sample inlet temperature: 250 ℃; detector temperature: at 250 ℃ to obtain a mixture.
(2) Preparation of control solutions: taking camphor reference substance, isoborneol reference substance, borneol reference substance, menthol reference substance, pulegone reference substance, octyl acetate reference substance, methyl salicylate reference substance, ethyl p-methylcinnamate and eugenol reference substance, and adding DMF to prepare a mixed solution containing 0.5mg of camphor, 0.1mg of isoborneol, 0.1mg of borneol, 0.3mg of menthol, 1mg of pulegone, 0.1mg of cinnamaldehyde, 0.1mg of octyl acetate, 0.5mg of methyl salicylate, 1.0mg of ethyl p-methylcinnamate and 0.5mg of eugenol in each 1ml as reference substance solution.
(3) Preparation of a test solution: taking 1 tablet (bag) of Moschus rheumatalgia-relieving plaster (specification: 7cm x 10cm), removing the cover liner, cutting into strip shape, getting Moschus rheumatalgia-relieving plaster sample, placing into a conical flask with a plug, adding 50ml of DMF (N, N-dimethylformamide), performing ultrasonic treatment in ice bath for 15min (power 200W, frequency 40kHz), standing, collecting supernatant, filtering, and collecting filtrate as sample solution. 15 batches of musk rheumatalgia-relieving pain-relieving plaster (rubber plaster) are taken, and a test solution (test solution 1-15) is prepared according to the test solution preparation method. Comparing the 15 batches of fingerprints with the reference substance to obtain common characteristic peaks as standard fingerprints, wherein the characteristic peaks sequentially comprise camphor, isoborneol, borneol, menthol, pulegone, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol
(4) And (3) determination: separately, 2. mu.l of each of the control solution and the sample solution was injected into a gas chromatograph and measured. The finger prints of 15 batches of the musk rheumatalgia-relieving plaster and the reference substance are obtained by measurement (figure 1). And comparing the 15 batches of fingerprints with the reference substance to obtain a common characteristic peak serving as a standard fingerprint, wherein the characteristic peaks sequentially comprise camphor, isoborneol, borneol, menthol, pulegone, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol. The standard chromatogram is shown in FIG. 2, and the total length of the fingerprint chromatogram is about 30min (column length is 30m, inner diameter is 0.32mm, and film thickness is 0.25 μm); the retention time of the characteristic chromatographic peak (marked as 1 in the figure) of the camphor is between 4.7 and 4.9 min; the retention time of characteristic chromatographic peaks of cinnamaldehyde (marked as 2 in the figure) is 5.4-5.6 min; the average retention time of characteristic chromatographic peaks of the pulegone (marked as 3 in the figure) is about 5.8-6.0 min; the retention time of characteristic chromatographic peaks of the menthol (marked as 4 in the figure) is 6.3-6.5 min; the retention time of characteristic chromatographic peaks of isoborneol (marked as 5 in the figure) is 7.1-7.3 min; the retention time of characteristic chromatographic peaks of borneol (marked as 6 in the figure) is 8.0-8.3 min; the retention time of the characteristic chromatographic peak of methyl salicylate (marked as 7 in the figure) is 10.0-10.3 min; the retention time of characteristic chromatographic peaks of octyl acetate (marked as 8 in the figure) is 11.1-11.3 min; the retention time of characteristic chromatographic peaks of the ethyl methyl cinnamate (marked as 9 in the figure) is 14.9-15.1 min; the retention time of characteristic chromatographic peaks of eugenol (marked as 10 in the figure) is 23.2-23.4 min.
In the quality retrospective analysis, the volatile component gas chromatography identification fingerprint maps of production batches collected for years are consistent with the standard fingerprint maps of the 15 batches of the musk rheumatalgia-relieving plaster, which shows that the method for measuring the volatile component gas chromatography identification fingerprint maps of the product has strong durability.
Example 2: product quality identification by using established standard fingerprint
The test sample of this example was a musk rheumatalgia-relieving pain-relieving plaster (rubber plaster, approved article: national standard Z20027408, specification: 7cm x 10cm, paste content: not less than 1.12g), and its preparation method was in accordance with the Chinese patent publication No. CN 200610079120.0.
(1) The detection method comprises the following steps: chromatographic conditions are as follows: shimadzu gas chromatograph (GC-2014), column: a capillary column (HP-FFAP, column length of 30m, inner diameter of 0.32mm, film thickness of 0.25 μm) with polyethylene glycol nitrobenzene as a filler; carrier gas: n is a radical of2(ii) a Column temperature: the initial temperature is 130 ℃, the temperature is kept for 20min, the temperature is increased to 195 ℃ at the speed of 7.5 ℃ per minute, and the temperature is kept for 20 min; sample inlet temperature:250 ℃; detector temperature: at 250 ℃ to obtain a mixture.
(2) Preparation of control solutions: taking camphor reference substance, isoborneol reference substance, borneol reference substance, menthol reference substance, pulegone reference substance, octyl acetate reference substance, methyl salicylate reference substance, ethyl p-methylcinnamate and eugenol reference substance, and adding DMF to prepare a mixed solution containing 0.5mg of camphor, 0.1mg of isoborneol, 0.1mg of borneol, 0.3mg of menthol, 1mg of pulegone, 0.1mg of cinnamaldehyde, 0.1mg of octyl acetate, 0.5mg of methyl salicylate, 1.0mg of ethyl p-methylcinnamate and 0.5mg of eugenol in each 1ml as reference substance solution.
(3) Preparation of a test solution: collecting 1 tablet (specification: 7cm x 10cm, paste content: not less than 1.12g) of Moschus rheumatalgia-relieving plaster, removing the lining, cutting into strip, collecting Moschus rheumatalgia-relieving plaster sample, placing into conical flask with plug, adding 50ml of DMF, performing ultrasonic treatment in ice bath for 15min, standing, collecting supernatant, filtering, and collecting filtrate as sample solution.
(4) And (3) determination: separately, 2. mu.l of each of the control solution and the sample solution was injected into a gas chromatograph and measured.
(5) As a result: the fingerprint spectrum of the test solution can find 10 main peaks corresponding to the reference, which are camphor, isoborneol, borneol, menthol, pulegone, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol. The fingerprint of the test sample is consistent with the characteristic peak of the standard fingerprint of example 1.
Example 3: product quality identification by using established standard fingerprint
The test sample of this example was a musk rheumatalgia-relieving plaster (powder premix plaster) with the publication number: CN 201810344858.8.
(1) The detection method comprises the following steps: chromatographic conditions are as follows: shimadzu gas chromatograph (GC-2014), column: a capillary column (HP-FFAP, column length of 30m, inner diameter of 0.32mm, film thickness of 0.25 μm) with polyethylene glycol nitrobenzene as a filler; carrier gas: n2; column temperature: the initial temperature is 130 ℃, the temperature is kept for 20min, the temperature is increased to 195 ℃ at the speed of 7.5 ℃ per minute, and the temperature is kept for 20 min; sample inlet temperature: 250 ℃; detector temperature: at 250 ℃ to obtain a mixture.
(2) Preparation of control solutions: taking camphor reference substance, isoborneol reference substance, borneol reference substance, menthol reference substance, pulegone reference substance, octyl acetate reference substance, methyl salicylate reference substance, ethyl p-methylcinnamate and eugenol reference substance, and adding DMF to prepare a mixed solution containing 0.5mg of camphor, 0.1mg of isoborneol, 0.1mg of borneol, 0.3mg of menthol, 1mg of pulegone, 0.1mg of cinnamaldehyde, 0.1mg of octyl acetate, 0.5mg of methyl salicylate, 1.0mg of ethyl p-methylcinnamate and 0.5mg of eugenol in each 1ml as reference substance solution.
(3) Preparation of a test solution: taking 1 bag of powder (powder bag specification: 7cm x 10cm, powder content 1.15 g/bag) of Moschus rheumatalgia-relieving plaster, placing in a conical flask with a stopper, adding DMF50ml, performing ultrasonic treatment in ice bath for 15min, standing, collecting supernatant, filtering, and collecting filtrate as sample solution.
(4) And (3) determination: separately, 2. mu.l of each of the control solution and the sample solution was injected into a gas chromatograph and measured.
(5) As a result: the fingerprint of the test solution can also find 10 main peaks corresponding to the reference, which are camphor, isoborneol, borneol, menthol, pulegone, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol. The fingerprint of the test sample is consistent with the characteristic peak of the standard fingerprint established in the embodiment 1.
Experimental example 1: selection of sample extraction solvent
Taking the same batch of the musk rheumatalgia-relieving plaster (rubber plaster) in the example 1, respectively selecting methanol, ethyl acetate and DMF (N, N-dimethylformamide) as dissolving and extracting solvents, carrying out ultrasonic treatment for 15min under ice bath conditions, standing, taking supernatant, filtering, taking filtrate as sample solution (3 types), and carrying out gas chromatography detection according to the method in the example 1 to obtain a fingerprint. Identifying cinnamaldehyde and pulegone peaks in fingerprint by methanol extractive solution gas chromatography (figure 3); identifying the peak of octyl acetate in the fingerprint by the gas chromatography of the extract of ethyl acetate (figure 4); the DMF extraction effect is very good, and each component can be effectively extracted (figure 5), which shows that characteristic peaks in a fingerprint can be distinguished by using DMF as an extraction solvent (see the separation effect in example 1). The inventor tries a plurality of extraction solvents, and cannot obtain ideal fingerprint (only two solvents, namely methanol and ethyl acetate, are shown in the experimental example), so that the unexpected technical effect is obtained by using DMF as the extraction solvent of the product.
Experimental example 2: selection of chromatography columns
The same lot of the musk rheumatalgia-relieving pain-relieving plaster (rubber plaster) in example 1 was taken for the experiment of chromatographic column selection, and the experimental process is referred to in example 1.
Chromatographic column brand selection
The chromatographic conditions of this experimental example were: shimadzu gas chromatograph (GC-2014), column: a capillary column with polyethylene glycol nitrobenzene as a filler (the column length is 30m, the inner diameter is 0.32mm, and the film thickness is 0.25 μm); carrier gas: n is a radical of2(ii) a Column temperature: the initial temperature is 130 ℃, the temperature is kept for 20min, the temperature is increased to 195 ℃ at the speed of 7.5 ℃ per minute, and the temperature is kept for 20 min; sample inlet temperature: 250 ℃; detector temperature: at 250 ℃ to obtain a mixture.
Selecting a capillary column with polyethylene glycol nitrobenzene as a filler according to the chromatographic conditions, and selecting SP-1000, OV-351 and HP-FFAP with the column length of 30m, the inner diameter of 0.32mm and the film thickness of 0.25 mu m for testing, wherein the results can reach baseline separation, and the number of theoretical plates can reach more than 6000; an HP-FFAP model is additionally selected: the column length is 30m, the inner diameter is 0.25mm, and the film thickness is 0.5 μm; the column length is 30m, the inner diameter is 0.53mm, the film thickness is 1.8 μm, the column length is 60m, the inner diameter is 0.32mm, the film thickness is 0.25 μm, the test result also reaches the baseline separation, the theoretical plate number can reach more than 6000, the longer the column is, the better the separation degree is, but the longer the time is, the test result shows that the column length is 30m, the inner diameter is 0.32mm, the film thickness is 0.25 μm, the column can reach the measurement requirement, the detection time can be saved, and the efficiency is improved.
Chromatographic column type selection
Currently, the chromatographic columns on the market are mainly classified into nonpolar, weak-polar, weak-medium-polar, polar and strong-polar capillary chromatographic columns, which are detailed in table 1:
TABLE 1 classification and application of common gas chromatography columns
According to the information of the chromatographic columns commonly used at present, the company carries out corresponding screening test work, the chromatographic columns are respectively selected from (DB-1, DB-5, DB-624, DB-35, DB-200, DB-210, DB-225 and DB-WAX), capillary columns with the column length of 30mm, the inner diameter of 0.32mm and the membrane thickness of 0.25 mu m are selected, and carrier gas: n2; column temperature: the initial temperature is 130 ℃, the temperature is kept for 20min, the temperature is increased to 195 ℃ at the speed of 7.5 ℃ per minute, and the temperature is kept for 20 min; sample inlet temperature: 250 ℃; detector temperature: at 250 ℃, the control solutions of each component were prepared and tested, the test results are shown in Table 2, and the results of the study show that 100% dimethylpolysiloxane, 5% phenyl 95% dimethylpolysiloxane, 6% hydroxypropyl-phenyl 94% dimethylpolysiloxane, 35% phenyl 65% dimethylpolysiloxane were selected, 35% trifluoropropyl 65% dimethyl polysiloxane, 50% trifluoropropyl 50% dimethyl polysiloxane, polyethylene glycol-20M and so on are used as the non-polar to strong polar capillary columns made of the fixed phase, each chromatographic column shows that the separation effect on cinnamic aldehyde and pulegone is not ideal, partial chromatographic columns influence the separation degree of isoborneol, borneol, methyl salicylate and octanol, partial chromatographic peak type difference and low theoretical pedal number also appear, and the screening result proves that the conventional chromatographic columns are not favorable for the identification requirement of one-test multi-evaluation of the volatile components of the product.
In order to further search for fillers with better separation effect, the inventors tried that various fillers (see table 2 in detail) could not achieve the ideal effect. When the polyethylene glycol nitrobenzene is selected, the result effect is very satisfactory, the peak type of each peak of the target identification substance peak is sharp, the separation degree can reach more than 1.5, the theoretical plate number is more than 10000, and the identification requirement of the product with multiple scores in one test is met. The polyethylene glycol nitrobenzene filler plays a very key role in the preparation of the fingerprint spectrum of the scheme, and an unexpected technical effect is obtained. According to the analysis of the inventor, the reason for the phenomenon is that the nitrobenzene is introduced into the polyethylene glycol nitrobenzene on the basis of the polyethylene glycol, so that the effect of enhancing the polarity can be realized, the aging of a chromatographic column is prevented, and the separation effect of target components is further improved.
TABLE 2 screening results of volatile components of Moschus rheumatalgia-relieving plaster by gas chromatography
Experimental example 3: specificity test
Blank auxiliary material solutions (comprising rubber, lanolin, zinc oxide, vaseline, liquid paraffin, rosin and the like in the prescribed amount), a reference substance solution and a test substance solution (see example 1) are respectively prepared, and the blank auxiliary materials have no chromatographic peak at the peak positions of camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol according to the gas chromatography condition measurement in example 1, thereby indicating that the specificity of the method is good.
Experimental example 4: precision test
The same batch of musk rheumatalgia-relieving plaster (example 1) was taken, the sample solution was prepared according to the preparation method of the sample solution, and the sample injection was performed 6 times continuously for measurement (refer to example 1), and the measurement results are shown in tables 3-4. The result shows that the sample can have chromatographic peak corresponding to the standard reference fingerprint, the RSD of the retention time and peak area of each main peak is less than 0.5 percent, and the precision of the determination is good.
TABLE 3 precision test retention time statistics
TABLE 4 main peak area statistics of precision tests
Experimental example 5: repeatability test
The test solution was prepared according to the method for preparing the test solution, the test solution was prepared for 6 minutes in the same manner, the relative deviation (RSD) of the peak area corresponding to the standard control fingerprint was calculated according to the method (see example 1), the results are shown in table 5, and the study results show that the main peak of each identified substance corresponds to the standard fingerprint, and the relative deviation of the area is less than 1.0%, indicating that the method has good repeatability.
TABLE 5 results of the repeatability tests
Experimental example 6: stability test
Taking musk rheumatalgia-relieving pain ointment (see example 1), preparing a test solution according to a preparation method of the test solution, determining a fingerprint (see example 1) according to a method every 2 hours, determining for 6 times, calculating the relative deviation (RSD) of peak areas corresponding to standard comparison fingerprints, and finding out that the result is shown in table 6, and research results show that main peaks of all identified substances can correspond to the standard fingerprints, the relative deviation of the main peak areas of corresponding substances in the fingerprints at all time points is less than 0.5%, and the test solution is stable within 10 hours.
TABLE 6 results of solution stability studies
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. The establishment method of the musk rheumatalgia-relieving pain-relieving plaster volatile component fingerprint comprises the preparation steps of a reference substance solution, a test substance solution and a detection step, and is characterized in that: in the preparation step of the test solution, N-dimethylformamide is used as a solvent to extract volatile components in the musk rheumatalgia-relieving plaster sample, and a liquid phase part is taken to obtain the test solution.
2. The method for establishing the fingerprint of the volatile component of the musk rheumatalgia-relieving pain-relieving ointment of claim 1, wherein the fingerprint comprises the following steps: the preparation method of the test solution comprises the following steps: adding N, N-dimethylformamide into a musk rheumatalgia-relieving plaster sample, carrying out ultrasonic treatment under an ice bath condition, standing, taking a supernatant, filtering the supernatant, and taking a liquid phase to obtain the test sample solution.
3. The method for establishing the fingerprint of the volatile component of the musk rheumatalgia-relieving pain-relieving ointment of claim 2, wherein the fingerprint comprises the following steps: the preparation method of the test solution comprises the following steps: cutting 1 piece of the musk rheumatalgia-relieving pain paste sample into strips, placing the strips into a conical flask, adding 50ml of N, N-dimethylformamide, carrying out ultrasonic treatment for 15min under the ice bath condition, standing, taking supernatant, filtering the supernatant, taking liquid phase, and obtaining the test solution.
4. The method for establishing the volatile component fingerprint spectrum of the musk rheumatalgia-relieving pain-relieving ointment as claimed in any one of claims 1 to 3, wherein the fingerprint spectrum comprises the following steps: in the preparation step of the control solution, a control solution containing camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol was prepared using N, N-dimethylformamide as a solvent.
5. The method for establishing the fingerprint of the volatile component of the musk rheumatalgia-relieving pain-relieving ointment of claim 4, wherein the fingerprint comprises the following steps: every 1ml of the reference solution contains 0.1-0.8mg of camphor, 0.05-0.3mg of isoborneol, 0.05-0.3mg of borneol, 0.1-0.5mg of menthol, 0.8-2mg of pulegone, 0.05-0.3mg of cinnamaldehyde, 0.05-0.3mg of octyl acetate, 0.1-1.0mg of methyl salicylate, 0.5-2.0mg of ethyl p-methylcinnamate and 0.1-1.0mg of eugenol.
6. The method for establishing the fingerprint of the volatile component of the musk rheumatalgia-relieving pain-relieving ointment of claim 5, wherein the fingerprint comprises the following steps: in the detection step, volatile components of the test solution and the reference solution are detected by using a gas chromatography, wherein the chromatographic conditions of the gas chromatography comprise a chromatographic column using polyethylene glycol nitrobenzene as a stationary phase.
7. The method for establishing the fingerprint of the volatile component of the musk rheumatalgia-relieving pain-relieving ointment of claim 6, wherein the fingerprint comprises the following steps: in the detecting step, the chromatographic conditions of the gas chromatography further include: the chromatographic column is a capillary column with the column length of 30m-60m, the inner diameter of 0.25-0.53mm and the film thickness of 0.25-1.8 um; the carrier gas is N2; the column temperature is 130 ℃ at the initial temperature, the temperature is kept for 20min, the temperature is raised to 205 ℃ at the rate of 6.5-8.5 ℃/min, and then the temperature is kept for 20 min; the temperature of a sample inlet is 250 ℃; the detector temperature was 250 ℃.
8. The method for establishing the fingerprint of the volatile component of the musk rheumatalgia-relieving pain-relieving ointment of claim 7, wherein the fingerprint comprises the following steps: in the detection step, the sample volumes of the reference solution and the test solution are both 2-5 mul.
9. The standard fingerprint established by the establishing method according to claim 8, wherein: comprises characteristic chromatographic peaks respectively representing camphor, isoborneol, borneol, menthol, pulegone, cinnamaldehyde, borneol, octyl acetate, methyl salicylate, ethyl p-methylcinnamate and eugenol; the retention time of the characteristic chromatographic peak of the camphor is 4.7-4.9 min; the retention time of the characteristic chromatographic peak of the cinnamaldehyde is 5.4-5.6 min; the average retention time of characteristic chromatographic peaks of the pulegone is 5.8-6.0 min; the retention time of the characteristic chromatographic peak of the menthol is 6.3-6.5 min; the retention time of the characteristic chromatographic peak of isoborneol is 7.1-7.3 min; the retention time of the characteristic chromatographic peak of borneol is 8.0-8.3 min; the retention time of the characteristic chromatographic peak of the methyl salicylate is 10.0-10.3 min; the retention time of the characteristic chromatographic peak of the octyl acetate is 11.1-11.3 min; the retention time of the characteristic chromatographic peak of the ethyl methyl cinnamate is 14.9-15.1 min; the retention time of the characteristic chromatographic peak of the eugenol is 23.2-23.4 min.
10. The use of the standard fingerprint of claim 9 for identifying a rubber plaster, a gel plaster or a powder premix plaster of a musk rheumatalgia-relieving pain-relieving plaster.
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| CN113588852A (en) * | 2021-08-16 | 2021-11-02 | 浙江鼎泰药业股份有限公司 | Method for measuring content of volatile components in high-activity transdermal plaster |
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