CN111289630A - Method for measuring content of menthol in traditional Chinese medicine composition by gas chromatography - Google Patents
Method for measuring content of menthol in traditional Chinese medicine composition by gas chromatography Download PDFInfo
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- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 title claims abstract description 61
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 title claims abstract description 58
- 229940041616 menthol Drugs 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 49
- 239000003814 drug Substances 0.000 title claims abstract description 34
- 239000000203 mixture Substances 0.000 title claims abstract description 32
- 238000004817 gas chromatography Methods 0.000 title claims abstract description 23
- 239000012088 reference solution Substances 0.000 claims abstract description 28
- 238000002360 preparation method Methods 0.000 claims abstract description 26
- 239000012085 test solution Substances 0.000 claims abstract description 16
- 239000000523 sample Substances 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 239000007921 spray Substances 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 239000007789 gas Substances 0.000 claims description 12
- 238000005259 measurement Methods 0.000 claims description 12
- 239000012159 carrier gas Substances 0.000 claims description 9
- 230000005526 G1 to G0 transition Effects 0.000 claims description 8
- 239000002202 Polyethylene glycol Substances 0.000 claims description 8
- 239000002737 fuel gas Substances 0.000 claims description 8
- 239000001257 hydrogen Substances 0.000 claims description 8
- 229910052739 hydrogen Inorganic materials 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 229920001223 polyethylene glycol Polymers 0.000 claims description 8
- 238000004132 cross linking Methods 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 5
- 239000006187 pill Substances 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 4
- 239000002775 capsule Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000006188 syrup Substances 0.000 claims description 2
- 235000020357 syrup Nutrition 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 230000007547 defect Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 13
- 201000007100 Pharyngitis Diseases 0.000 description 10
- 241000219109 Citrullus Species 0.000 description 8
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 206010068319 Oropharyngeal pain Diseases 0.000 description 7
- 239000013078 crystal Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000001525 mentha piperita l. herb oil Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 235000019477 peppermint oil Nutrition 0.000 description 4
- 210000003800 pharynx Anatomy 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
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- 208000016150 acute pharyngitis Diseases 0.000 description 2
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- 238000011835 investigation Methods 0.000 description 2
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- 238000003908 quality control method Methods 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 208000019505 Deglutition disease Diseases 0.000 description 1
- 244000303040 Glycyrrhiza glabra Species 0.000 description 1
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000100289 Lonicera confusa Species 0.000 description 1
- 235000014435 Mentha Nutrition 0.000 description 1
- 241001072983 Mentha Species 0.000 description 1
- 244000245214 Mentha canadensis Species 0.000 description 1
- 235000001878 Mentha haplocalyx Nutrition 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 240000004534 Scutellaria baicalensis Species 0.000 description 1
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- 229910021538 borax Inorganic materials 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000011477 liquorice Nutrition 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
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- 235000010339 sodium tetraborate Nutrition 0.000 description 1
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- 238000013112 stability test Methods 0.000 description 1
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- 208000011580 syndromic disease Diseases 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Immunology (AREA)
- Pathology (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
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Abstract
The invention belongs to the field of detection of traditional Chinese medicine preparations, and particularly relates to a method for determining the content of menthol in a traditional Chinese medicine composition by using gas chromatography. The method comprises the steps of preparation of a test solution, preparation of a reference solution, chromatographic conditions and a determination method, wherein the column temperature under the chromatographic conditions is constant temperature operation. The constant temperature operation is that the column temperature is controlled at 120 ℃. The method for measuring the content of the menthol in the traditional Chinese medicine composition by using the gas chromatography provided by the invention does not need to adopt a temperature programming method, and overcomes the defects of long measuring time, complex program and the like in the prior art which needs to adopt the temperature programming method.
Description
Technical Field
The invention belongs to the field of detection of traditional Chinese medicine preparations, and particularly relates to a method for determining the content of menthol in a traditional Chinese medicine composition by using gas chromatography.
Background
The watermelon frost sore throat spray and the Shuyanqing spray are prepared from watermelon frost, scutellaria baicalensis, subprostrate sophora, lonicera confusa, menthol, borax, liquorice and other traditional Chinese medicines, are two traditional Chinese medicine preparations which are produced by Guilin Sanjin pharmaceutical industry GmbH and can be used for treating acute pharyngitis caused by wind heat, and the symptoms are as follows: sore throat, dry and burning throat, red and swollen throat, or dysphagia; acute pharyngitis or acute attack of chronic pharyngitis with the above syndrome.
The menthol is saturated cyclic alcohol obtained by steam distilling, freezing and recrystallizing fresh stem and leaf of Mentha haplocytic x Briq, and has white crystal, special fragrance of Mentha haplocalyx, and cool and fresh taste. It can be used as irritant in medicine, and has effects in refreshing and relieving itching; the oral preparation can be taken as a wind-expelling medicine for treating headache, inflammation of nose, pharynx, larynx and the like, is a common medicine for treating oral inflammation, is also one of main components in watermelon crystal sore throat spray and Shuyanqing spray, has larger influence on the efficacy due to the content of the menthol in the two preparations, and needs to be accurately measured and controlled. Patent CN03137148.5 discloses a Chinese medicinal composition for treating oral and throat diseases and a preparation method thereof, and the patent technology also discloses a quality control method of the Chinese medicinal composition spray, the method mainly comprises the identification of some specific components, the content determination of volatile components in the volatile components is not related, and no method for determining the content of menthol components in the Chinese medicinal composition is found in other existing documents. The gas chromatography technology has been developed into a mature and widely-used separation and analysis technology for complex mixtures, has the advantages of high separation speed, small sample consumption, high sensitivity and the like, and is widely applied to drug analysis, particularly analysis of volatile components.
CN108020613A discloses a method for measuring the content of menthol in a Chinese medicinal composition, which adopts gas chromatography to measure the content of menthol in a formula and can detect the content of menthol in the formula.
CN104914194A discloses a method for detecting the content of menthol in a peppermint oil drop pill by using a gas chromatograph. The method comprises preparing Mentholum reference solution, preparing test solution, sucking Mentholum reference solution, injecting into gas chromatograph, measuring peak area, and drawing standard curve to obtain regression equation of standard curve; then absorbing the test solution, injecting the test solution into a gas chromatograph for determination, recording a chromatogram, and calculating the content of the menthol by using an external standard method; or calculating the content of menthol by using the regression equation of the obtained standard curve; wherein the gas chromatograph is a capillary column taking polyethylene glycol 20000 as a stationary phase; the split ratio is 10-20: 1; the initial temperature of the column temperature is 50-115 ℃, the column temperature is kept for 4-5 minutes, the temperature is raised to 200 ℃ at the speed of 1.5-4 ℃ per minute, and the column temperature is kept for 2-3 minutes; the temperature of a sample inlet is 230 ℃; the temperature of the detector is 230 ℃; the carrier gas flow is 1-2 ml/min.
CN02131128.5 discloses a method for detecting the quality of peppermint oil, which takes the identification and content measurement of menthol as the quality control standard of peppermint oil, and can strictly control the quality of peppermint oil.
However, the above-mentioned methods have disadvantages such as requiring a temperature-programmed method, long measurement time, and complicated procedure.
The present invention has been made in view of the above.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for measuring the content of menthol in a traditional Chinese medicine composition by using a gas chromatography, which does not need a temperature programming method and overcomes the defects.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for measuring the content of menthol in a Chinese medicinal composition by gas chromatography comprises the preparation of a test solution, the preparation of a reference solution, chromatographic conditions and a measuring method, wherein the column temperature is constant under the chromatographic conditions.
Further, the constant temperature operation is that the column temperature is controlled at 120 ℃.
Further, the chromatographic conditions are as follows: a chromatographic column: a capillary column with a cross-linking bond and polyethylene glycol as stationary phases; a detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15-25: 1; the fuel gas proportion is as follows: air-hydrogen 450: 45; the sample size was 1. mu.L.
Further, the capillary column is long: 30m, inner diameter: 0.53mm, film thickness: 1.0 μm.
Further, the method for measuring the content of the menthol in the traditional Chinese medicine composition by using the gas chromatography comprises the following steps:
1) preparation of control solutions: taking a proper amount of menthol control, precisely weighing, and adding organic solvent to prepare a solution containing 0.2mg per 1 ml;
2) preparation of a test solution: taking 1ml of the product, placing the product in a 100ml measuring flask, adding an organic solvent to dilute the product to a scale, and shaking up the product to obtain the product;
3) chromatographic conditions are as follows: a chromatographic column: a capillary column taking a cross-linking bond and polyethylene glycol as a stationary phase, wherein the column length is as follows: 30m, inner diameter: 0.53mm, film thickness: 1.0 μm; a detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15-25: 1; the fuel gas proportion is as follows: air-hydrogen 450: 45; the sample volume is 1 mu L;
4) the determination method comprises the following steps: precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a gas chromatograph, and measuring.
Wherein, the organic solvent in the step 1) and the step 2) is absolute ethyl alcohol.
In the invention, the traditional Chinese medicine composition is prepared from the following raw material medicines:
or, the traditional Chinese medicine composition is prepared from the following raw material medicines:
furthermore, the traditional Chinese medicine composition is added with auxiliary materials to be prepared into pharmaceutically acceptable dosage forms, preferably spray, tablets, capsules, syrup, granules, dripping pills, pills and patches, and preferably spray.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for measuring the content of menthol in a traditional Chinese medicine composition by using a gas chromatography, which does not need to adopt a temperature programming method and overcomes the defects of long measuring time, complex procedure and the like in the prior art which needs to adopt the temperature programming method.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a gas chromatogram of watermelon frost laryngalgia treating spray of the invention;
FIG. 2 is a standard graph of the linear relationship examination of the present invention.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
A method for measuring the content of menthol in a traditional Chinese medicine composition by gas chromatography is characterized in that the method for measuring the content of the menthol is as follows:
1) preparation of control solutions: taking a proper amount of menthol control, precisely weighing, and adding absolute ethanol to prepare a solution containing 0.2mg per 1 ml;
2) preparation of a test solution: taking 1ml of watermelon frost sore throat spray, placing into a 100ml measuring flask, adding absolute ethyl alcohol to dilute to scale, and shaking up to obtain the product;
3) chromatographic conditions are as follows: a chromatographic column: a capillary column (HP-FFAP, manufacturer: Agilent, column length: 30m, internal diameter: 0.53mm, film thickness: 1.0 μm) using a cross-linking bond and polyethylene glycol as a stationary phase. A detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15: 1; the fuel gas proportion is as follows: air-hydrogen 450: 45; the sample volume is 1 mu L;
4) the determination method comprises the following steps: precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a gas chromatograph, and measuring.
By adopting the method, 3 batches of watermelon frost sore throat spray samples (U17031, U17032 and U17033 respectively) are measured, peak areas are recorded, and the measurement results are respectively as follows: 18.98mg/ml, 18.74mg/ml, 18.71 mg/ml.
Example 2
A method for measuring the content of menthol in a traditional Chinese medicine composition by gas chromatography is characterized in that the method for measuring the content of the menthol is as follows:
1) preparation of control solutions: taking a proper amount of menthol control, precisely weighing, and adding absolute ethanol to prepare a solution containing 0.2mg per 1 ml;
2) preparation of a test solution: taking 1ml of Shuyanqing spray, placing into a 100ml measuring flask, adding absolute ethyl alcohol to dilute to a scale, and shaking up to obtain the spray;
3) chromatographic conditions are as follows: a chromatographic column: a capillary column (HP-FFAP, manufacturer: Agilent, column length: 30m, internal diameter: 0.53mm, film thickness: 1.0 μm) using a cross-linking bond and polyethylene glycol as a stationary phase. A detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15: 1; the fuel gas proportion is as follows: air-hydrogen 450: 45; the sample volume is 1 mu L;
4) the determination method comprises the following steps: precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a gas chromatograph, and measuring.
By adopting the method, 3 batches of spray samples (170111, 170302 and 170506 respectively) of the Shuyanqing are measured, peak areas are recorded, and the measurement results are respectively as follows: 2.23mg/ml, 2.19mg/ml, 2.20 mg/ml.
Test example 1
1. Instrument and reagent
1.1 instruments
7890A gas chromatograph (manufacturer: Agilent technologies), Mettler-Torredo XP-205 electronic balance, Brand Transferpette pipette (100. mu.l-1000. mu.l).
1.2 reagent
Menthol control (batch number: 200506; source: China food and drug testing research institute). Absolute ethyl alcohol (analytical purity, manufacturer: national pharmaceutical group chemical reagents limited; batch number: 20170111). All other reagents used were analytical grade.
2. Method and results
2.1 preparation of test and control solutions
2.1.1 selection of solvent and amount
The experiment researches that the sample is prepared by using methanol, absolute ethyl alcohol and ethyl acetate as solvents, and through comprehensive analysis, the absolute ethyl alcohol is found to be used as the solvent, so that the sample is well separated and has good repeatability. In addition, the menthol is easy to dissolve in the absolute ethyl alcohol, and has no influence on a capillary chromatographic column, so the absolute ethyl alcohol is selected as a solvent.
The sample to be detected is a liquid preparation, so the sample can be directly measured and diluted. We tested the samples diluted 10, 25, 50 and 100 times respectively and the results are shown in table 1. The result shows that the separation degree, the theoretical plate number and the symmetry factor of the menthol crystal detection peak are better when the menthol crystal is diluted by 100 times, so the menthol crystal is diluted by 100 times.
TABLE 1 table of solvent and dosage selection test results in the preparation of test solutions
| Numbering | Dilution factor | Peak area | Number of theoretical plates | Symmetry factor |
| 1 | 10 | 27540 | 27540 | 1.943 |
| 2 | 25 | 11472 | 25119 | 1.295 |
| 3 | 50 | 5662 | 25746 | 1.123 |
| 4 | 100 | 2916 | 25670 | 1.014 |
2.1.2 preparation of control solutions
Taking appropriate amount of Mentholum reference substance, precisely weighing, and adding anhydrous ethanol to obtain solution containing 0.2mg per 1 ml.
2.1.3 preparation of test solutions
Precisely measuring 1ml of the product, placing into a 100ml measuring flask, adding anhydrous ethanol to dilute to scale, and shaking.
2.1.4 negative control solution preparation
Taking a negative control sample of menthol (prepared according to the proportion in the prescription and the sample preparation method), and preparing the negative control solution according to the preparation method of the test solution under the item '2.1.3'.
2.2 chromatographic conditions
The test of the invention selects three different brands of chromatographic columns HP-FFAP (Agilent, 30m multiplied by 0.53mm multiplied by 1.0 μm), TR-WAXMS (Thermo, 30m multiplied by 0.32mm multiplied by 0.25 μm), EC-1000(Alltech, 30m multiplied by 0.53mm multiplied by 1.2 μm) to be tested under the same condition, namely a detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15: 1; the fuel gas proportion is as follows: air-hydrogen 450:45, a first step of; the amount of the sample was 1. mu.L, and the measurement results are shown in Table 2. The measurement results showed that HP-FFAP was separated well and the theoretical plate number was the highest, so HP-FFAP column was used.
TABLE 2 number of theoretical plates
Therefore, the chromatographic conditions selected by the invention are as follows: a chromatographic column: a capillary column (HP-FFAP, manufacturer: Agilent, column length: 30m, internal diameter: 0.53mm, film thickness: 1.0 μm) using a cross-linking bond and polyethylene glycol as a stationary phase. A detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15: 1; the fuel gas proportion is as follows: air-hydrogen 450:45, a first step of; the sample volume is 1 mu L;
2.3 specificity test
Precisely sucking 1 μ l of each of the reference solution, the sample solution, the negative reference solution and the solvent blank (anhydrous ethanol), injecting into a gas chromatograph, and measuring according to the chromatographic condition under the item of 2.2, wherein the sample chromatogram (shown in figure 1) shows a chromatographic peak with the same retention time as the reference chromatographic peak, and the solvent blank and the negative reference solution of menthol do not show a chromatographic peak with the same retention time as the reference chromatographic peak. Indicating that the solvent blank and negative control solution were not interfering with the assay results.
2.4 Linear relationship investigation
Precisely weighing 96.33mg of menthol control, placing the menthol control in a 100ml measuring flask, adding absolute ethanol to dilute the menthol control to scale, shaking the menthol control to obtain reference stock solution with the menthol content of 0.9633mg/ml, taking 2.5ml of the solution, placing the solution in a 5ml measuring flask, adding absolute ethanol to dilute the solution to scale, shaking the solution to obtain ① reference solution, taking 4ml of ① reference solution, placing the ① reference solution in a 5ml measuring flask, adding absolute ethanol to dilute the solution to scale, shaking the solution to obtain ② reference solution, taking 3.75ml of ② reference solution, placing the solution in a 5ml measuring flask, adding absolute ethanol to dilute the solution to scale, shaking the reference solution to obtain ③ reference solution, taking 3.333ml of ③ reference solution, placing the solution in a 5ml measuring flask, adding absolute ethanol to dilute the solution to scale, shaking the reference solution to obtain ④ reference solution, taking 2.5ml of ④ reference solution, placing the reference solution in a 365 ml measuring flask, adding absolute ethanol to dilute the reference solution to scale, adding absolute ethanol to obtain 3.23 ml of the reference solution, shaking the reference solution, and measuring the absolute ethanol to obtain the reference solution under the condition of 365 ml, measuring the absolute ethanol, and measuring the measuring result of 365 ml.
TABLE 3 Linear relationship investigation experiment
| Reference substance | Peak area of control (A) | Sample size C (μ g) |
| Number 1 | 7773 | 0.48165 |
| Number 2 | 6479 | 0.38532 |
| No. 3 | 4803 | 0.28899 |
| Number 4 | 3324 | 0.19264 |
| |
1714 | 0.09632 |
| Number 6 | 894 | 0.048160 |
And drawing a standard curve by taking the sample volume (mug) as a vertical coordinate and the peak area of the reference sample as a horizontal coordinate, wherein the standard curve is shown in figure 2, and the regression equation is obtained as follows: the result shows that the sampling amount of the menthol is in a range of 0.048160-0.48165 mu g, and the peak area and the sampling amount have a good linear relation.
2.5 methodological considerations
2.5.1 precision test
Preparing a watermelon frost sore throat spray sample (batch number: U170301) into a test solution, precisely sucking 1 mul, and carrying out continuous sample injection for 6 times according to the chromatographic condition under the item of 2.2, wherein the test result is shown in Table 4, and the RSD of the menthol content in 6 times is 0.40%.
TABLE 4 Instrument precision test
2.5.2 stability Studies
Taking the sample solution under the item "2.1.2", precisely sucking 1 μ l, injecting into gas chromatograph at 0, 2, 4, 8, 16, 24, 33 hours respectively, and determining menthol content according to the chromatographic conditions under the item "2.2", the determination results are shown in Table 5. The measurement result shows that the RSD of the menthol content is 1.05%, and the stability of the menthol is good within 33 hours.
TABLE 5 stability test
2.5.3 repeatability test
6 parts of the test solution are prepared by the same method as the preparation method of the test solution under the item 2.1.2, and the measurement results are shown in the table 6 according to the chromatographic conditions under the item 2.2. The measurement result shows that the RSD of the menthol content in 6 test sample solutions is 0.79 percent, and the repeatability meets the regulation.
TABLE 6 repeatability tests
2.5.4 sample recovery test
Precisely measuring 9 parts of 0.5ml of watermelon crystal sore throat spray with known content, placing into a 100ml measuring flask, precisely adding 5ml, 10ml and 15ml of menthol control stock solutions under item 2.1.1 into each of the three parts, diluting with anhydrous ethanol to scale, and shaking. The above 9 parts of the solution were taken, and the recovery rate was calculated by measuring under the chromatographic conditions under the item "2.2", and the measurement and calculation results are shown in Table 7. The measurement and calculation results show that the average sample recovery rate of the menthol is 100.62 percent, the RSD is 2.68 percent, and the recovery rate meets the specification.
TABLE 7 sample recovery test
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A method for measuring the content of menthol in a traditional Chinese medicine composition by gas chromatography is characterized by comprising the steps of preparing a test solution, preparing a reference solution, and performing chromatographic condition and measurement, wherein the column temperature is constant-temperature operation under the chromatographic condition.
2. The method for measuring the content of menthol in a Chinese medicinal composition by gas chromatography as claimed in claim 1, wherein the constant temperature is controlled to 120 ℃.
3. The method for measuring the content of menthol in a traditional Chinese medicine composition by using gas chromatography as claimed in claim 1 or 2, wherein the chromatographic conditions are as follows: a chromatographic column: a capillary column with a cross-linking bond and polyethylene glycol as stationary phases; a detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15-25: 1; the fuel gas proportion is as follows: air-hydrogen 450: 45; the sample size was 1. mu.L.
4. The method for measuring the content of menthol in a Chinese medicinal composition by gas chromatography as claimed in claim 3, wherein the capillary column has a length: 30m, inner diameter: 0.53mm, film thickness: 1.0 μm.
5. The method for measuring the content of the menthol in the traditional Chinese medicine composition by using the gas chromatography as claimed in any one of claims 1 to 4, which is characterized in that the method for measuring the content of the menthol in the traditional Chinese medicine composition by using the gas chromatography comprises the following steps:
1) preparation of control solutions: taking a proper amount of menthol control, precisely weighing, and adding organic solvent to prepare a solution containing 0.2mg per 1 ml;
2) preparation of a test solution: taking 1ml of the product, placing the product in a 100ml measuring flask, adding an organic solvent to dilute the product to a scale, and shaking up the product to obtain the product;
3) chromatographic conditions are as follows: a chromatographic column: a capillary column taking a cross-linking bond and polyethylene glycol as a stationary phase, wherein the column length is as follows: 30m, inner diameter: 0.53mm, film thickness: 1.0 μm; a detector: FID, column temperature 120 ℃; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 230 ℃; the carrier gas is nitrogen, and the flow rate is as follows: 1 mL/min; split-flow sample introduction, split-flow ratio: 15-25: 1; the fuel gas proportion is as follows: air-hydrogen 450: 45; the sample volume is 1 mu L;
4) the determination method comprises the following steps: precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a gas chromatograph, and measuring.
6. The method for measuring the content of menthol in a Chinese medicinal composition by using the gas chromatography as claimed in claim 5, wherein the organic solvent used in the steps 1) and 2) is absolute ethyl alcohol.
7. The method for measuring the content of menthol in the traditional Chinese medicine composition by using the gas chromatography as claimed in any one of claims 1 to 6, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines:
8. the method for measuring the content of menthol in the traditional Chinese medicine composition by using the gas chromatography as claimed in any one of claims 1 to 6, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines:
9. the method for measuring the content of menthol in the traditional Chinese medicine composition by using the gas chromatography as claimed in claim 7 or 8, wherein the traditional Chinese medicine composition is added with auxiliary materials to be prepared into pharmaceutically acceptable dosage forms, preferably sprays, tablets, capsules, syrups, granules, dripping pills, pills and patches, preferably sprays.
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