Method for determining volatile components in human placenta

Abstract

The application relates to a determination method of volatile components in human dan, wherein the human dan comprises a core and a coating wrapped on the core; the material of the core comprises menthol, cassia, liquorice, catechu, costus, borneol, platycodon, camphor, cumin, cardamom and clove basil oil; the material of the coating is a non-aluminum water-soluble material; the determination method comprises the following steps: taking a human dan sample to be determined, adding an organic solvent and water to extract, and preparing a test sample solution; taking standard samples of menthol, borneol and camphor, adding a solvent to dissolve, and preparing a reference solution; taking the test sample solution and the reference solution, and performing gas chromatography analysis, and determining by using an external standard method. The determination method has the advantages of few interfering substances in the test sample solution, complete effective components, simplicity, feasibility, accuracy, rapidness and good stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine drug quality detection and analytical chemistry, and particularly relates to a method for determining volatile components in Rendan. BACKGROUND

[0002] Rendan is originally derived from ancient prescription "Zhugeliujunshansan". The prescription has evolved into a traditional Chinese medicine prescription mainly composed of menthol, cassia, licorice, catechu, safflower, borneol, platycodon, camphor, cumin, cardamom and clove basil oil through years of application and practice. The function and indication of Rendan are to dispel wind and invigorate the stomach, and it is used for indigestion, nausea and vomiting, seasickness, mild heatstroke and drunkenness. Rendan is currently collected in Traditional Chinese Medicine Prescriptions of the Ministry of Public Health of the People's Republic of China, Volume 19 (standard number WS3-B-3528-98). Since Rendan contains a large amount of volatile components, the Rendan products on the market are generally coated with aluminum foil to prevent volatilization and prolong the shelf life.

[0003] However, some studies have shown that long-term intake of aluminum foil by the human body may pose a health risk. At present, researchers have developed a non-aluminum water-soluble coated Rendan product, and there is no report on the quality detection method for the Rendan product. SUMMARY

[0004] Therefore, the technical problems to be solved by the present application include providing a method for determining volatile components in Rendan. The Rendan of the present application includes a core and a coating coated on the core. The material of the core includes menthol, cassia, licorice, catechu, safflower, borneol, platycodon, camphor, cumin, cardamom and clove basil oil. The material of the coating is a non-aluminum water-soluble material. The determination method of the present application is stable, reliable and accurate, and can accurately determine the volatile components in Rendan.

[0005] Specifically, the determination method comprises the following steps:

[0006] Taking the Rendan sample to be tested, adding an organic solvent and water for extraction to prepare a test solution;

[0007] Taking the standard samples of menthol, borneol and camphor, dissolving them in a solvent to prepare a reference solution;

[0008] Taking the test solution and the reference solution, performing gas chromatography analysis, and determining by external standard method.

[0009] In some embodiments of the present application, in the determination method, the conditions of the gas chromatography analysis include: detector: hydrogen flame ionization detector;

[0010] chromatographic column: capillary column with cross-linked bonded polyethylene glycol as stationary phase;

[0011] Temperature rising program: 90-110℃, holding for 3-6min, then rising to 150-180℃ at 4-8℃ / min, holding for 3-8min;

[0012] Injection port temperature: 200-250℃;

[0013] Detector temperature: 200-250℃;

[0014] Split ratio (10-20): 1.

[0015] In some embodiments of the present application, the organic solvent in the determination method is ethyl acetate.

[0016] In some embodiments of the present application, the mass-volume ratio of the human semen sample and the organic solvent in the determination method is 1g:100mL.

[0017] The mass-volume ratio of the human semen sample and the water is 1g:(5-7)mL.

[0018] In some embodiments of the present application, the coating material in the determination method comprises talcum powder, carmine and red iron oxide.

[0019] In some embodiments of the present application, the mass ratio of the core and the coating in the determination method is 1:(0.02-0.05).

[0020] In some embodiments of the present application, the material of the core in the determination method comprises 40.6±m parts of menthol, 20.3±m parts of cassia, 476±m parts of licorice, 40.6±m parts of catechu, 10.2±m parts of saussurea, 20.3±m parts of borneol, 325±m parts of platycodon, 20.3±m parts of camphor, 20.3±m parts of cumin, 10.2±m parts of cardamom and clove basil oil, in weight fraction;

[0021] wherein all m are the same, and m is 1, 0.5, 0.2, 0.1 or 0;

[0022] The mass-volume ratio of the core and the clove basil oil is (900-1000)g:5mL.

[0023] In some embodiments of the present application, the preparation of the reference solution in the determination method comprises the following steps for preparing a single standard control solution: taking standard samples of camphor, menthol and borneol, respectively adding the solvent to dissolve, to prepare camphor control solution, menthol control solution and borneol control solution;

[0024] The following steps are taken to prepare the mixed control solution: mixing camphor standard, borneol standard and menthol standard, and dissolving them with the solvent.

[0025] In some embodiments of the present application, in the determination method, the mass-volume concentration of menthol in the mixed control solution is 54-66 μg / mL, the mass-volume concentration of borneol is 54-66 μg / mL, and the mass-volume concentration of camphor is 180-220 μg / mL.

[0026] In some embodiments of the present application, in the determination method, the determination is performed by using the external standard method, and the retention time of the menthol, the borneol and the camphor in the determination result is as follows: camphor: 11.048±1 min, menthol: 13.437±1 min, and borneol: 14.810±1 min.

[0027] The present application is based on the water-soluble outer coating material of Ren Dan and the characteristics of the core traditional Chinese medicinal material, and uses gas chromatography as the detection means, adopts the external standard method to scientifically and accurately determine the content of volatile components in Ren Dan based on suitable extraction methods and chromatographic conditions. The chromatographic parameters, reagent types and reagent amounts in the above determination, especially the preparation method of the test solution, are obtained through long-term complex experiments, and are the synergistic effect of the above factors to support the beneficial effects of the present application.

[0028] The determination method provided by the present application has the advantages of simple and feasible, accurate and fast, and good stability. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0030] Figure 1 The chromatogram of the blank solution prepared in 2.3.2;

[0031] Figure 2 The chromatogram of the control solution prepared in 2.3.2;

[0032] Figure 3 The chromatogram of the negative test solution without camphor, menthol and borneol prepared in 2.3.2;

[0033] Figure 4chromatogram of the camphor-free test solution prepared in 2.3.2;

[0034] Figure 5 chromatogram of the menthol-free test solution prepared in 2.3.2;

[0035] Figure 6 chromatogram of the borneol-free test solution prepared in 2.3.2;

[0036] Figure 7 linear plot of camphor prepared in 2.4.2 linear test;

[0037] Figure 8 linear plot of menthol prepared in 2.4.2 linear test;

[0038] Figure 9 linear plot of borneol prepared in 2.4.2 linear test. DETAILED DESCRIPTION

[0039] The application will be further described below in conjunction with the embodiments, examples and drawings. It should be understood that these examples are only used to illustrate the application and not intended to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content taught in the application, and these equivalent forms also fall within the protection scope of the appended claims of the application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0041] Terminology

[0042] Unless otherwise indicated, or unless the context clearly indicates otherwise, the terms or phrases used in this application have the following meanings:

[0043] In this application, "preferably", "more preferably", "even more preferably" and the like are used to describe the embodiments and / or examples of the application and are not used to limit the scope of the application.

[0044] In this application, "further", "furthermore", "in addition" and the like are used to describe additional features or embodiments and are not used to limit the scope of the application.

[0045] In this application, the technical features described in an open form include both the closed technical solution consisting of the listed features and the open technical solution containing the listed features.

[0046] In the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. the minimum and maximum values) of the numerical range, as well as every numerical value between the two numerical endpoints. When a numerical interval refers only to integers within the numerical interval, unless otherwise specified, both numerical endpoint integers, as well as every integer between the two numerical endpoints, are included. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein.

[0047] In the present application, unless otherwise specified, the temperature parameter allows for both constant temperature processing, as well as fluctuations within a certain temperature interval. It should be understood that the constant temperature processing allows for fluctuations within the accuracy range of the instrument control. Fluctuations within a range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0048] In the present application, weight can be μg, mg, g, kg, and other mass units commonly known in the chemical industry.

[0049] In a first aspect of the present application, a method for determining volatile components in human dan is provided. The method is stable, reliable, and accurate, and can precisely determine the volatile components in human dan.

[0050] The human dan of the present application includes a core and a coating covering the core.

[0051] The material of the core is a traditional Chinese medicine active ingredient, including menthol, cassia, licorice, catechu, safflower, borneol, platycodon, camphor, cumin, cardamom, and clove basil oil.

[0052] In some embodiments, the material of the core includes: 40.6±m parts of menthol, 20.3±m parts of cassia, 476±m parts of licorice, 40.6±m parts of catechu, 10.2±m parts of safflower, 20.3±m parts of borneol, 325±m parts of platycodon, 20.3±m parts of camphor, 20.3±m parts of cumin, 10.2±m parts of cardamom, and clove basil oil, in terms of weight fraction;

[0053] wherein all m are the same, and m is 1, 0.5, 0.2, 0.1, or 0;

[0054] The mass-to-volume ratio of the core material and the clove basil oil is (900-1000) g:5 mL.

[0055] The material of the coating is a non-aluminum water-soluble material, which has high dissolution in the body, and can well replace aluminum foil to achieve the effect of drug dissolution.

[0056] In some embodiments, the coating material comprises talc, carmine and red iron oxide. Further, the mass ratio of talc, carmine and red iron oxide is (250-300):(10-20):50.

[0057] Preferably, the mass ratio of talc, carmine and red iron oxide is (270-280):(12-16):50. The loss of volatile ingredients in the core can be effectively preserved and moisture can be prevented.

[0058] In some embodiments, the mass ratio of the core and the coating is 1:(0.02-0.05), which can further be 1:(0.03-0.04), for example, the mass ratio can be 1:0.02, 1:0.03, 1:0.04, 1:0.05, etc.

[0059] The current determination method of the effective ingredients in the human dan has the problem of inaccurate determination results. Based on a large number of experimental explorations, the researchers found that this is mainly due to the fact that the effective ingredients in the human dan contain a variety of volatile substances, such as menthol, borneol and camphor, which are prone to volatilize or sublimate during storage and analysis, which can easily lead to analysis. Therefore, it is of great significance to develop a method for accurately determining the content of volatile ingredients in human dan. In this paper, menthol (menthol, Menthol) refers to the compound with CAS number 89-78-1; borneol (natural borneol, Borneol) refers to the compound with CAS number 507-70-0; camphor (Camphor) refers to the compound with CAS number 464-49-3.

[0060] The determination method of the human dan in the present application takes into account the specific coating material of the sample to be tested and the characteristics of the core traditional Chinese medicinal material, uses gas chromatography as the detection means, and scientifically and accurately determines the content of volatile ingredients in the human dan based on suitable extraction methods and chromatographic conditions.

[0061] In some embodiments of the present application, the determination method comprises the following steps:

[0062] S100: Taking the human dan sample to be tested, adding an organic solvent and water for extraction to prepare a test solution;

[0063] S200: Taking standard samples of menthol, borneol and camphor, dissolving them in a solvent to prepare a reference solution;

[0064] S300: Taking the test solution and the reference solution, performing gas chromatography analysis, and determining by external standard method.

[0065] S100 Preparation of sample solution

[0066] Applicant found that the best extraction effect can be obtained by directly dissolving and extracting the sample of Renshen without crushing through long-term experience accumulation and a large number of experimental explorations. In the traditional preparation, the extraction after crushing can make the core material fully infiltrate and improve the extraction efficiency, but the crushing process can cause loss of a large number of volatile components in Renshen, resulting in inaccurate test results.

[0067] In the present application, in step S100, the sample of Renshen is extracted with a suitable amount of water and an organic solvent, which can separate the coating layer from the core material of traditional Chinese medicine well, effectively extract the effective components of the core material of traditional Chinese medicine, and avoid loss of volatile substances, thereby improving the accuracy.

[0068] In some embodiments, in step S100, the organic solvent is ethyl acetate. Further, the mass-volume ratio of the sample of Renshen and the organic solvent is 1 g:100 mL, and the mass-volume ratio of the sample of Renshen and water is 1 g:(5-7) mL.

[0069] In some embodiments, in step S100, the extraction is by stirring, which can be further magnetic stirring. In some embodiments, the stirring speed is 200 r / min-400 r / min. It can be further 250 r / min-350 r / min. As an example, the stirring speed can be 200 r / min, 250 r / min, 300 r / min, 350 r / min, 400 r / min, etc. In some embodiments, the stirring time can be 10 min-20 min, which can be further 10 min-15 min. As an example, the stirring time can be 10 min, 12 min, 15 min, 18 min, 20 min, etc.

[0070] In some embodiments, the step of preparing the test sample solution comprises: accurately weighing 1 g of the sample of Renshen, placing it in a conical flask with a plug, adding 60 mL of ethyl acetate, adding 5 mL of water, tightly plugging, and stirring at 200 r / min-400 r / min.

[0071] After stirring, the liquid in the conical flask with a plug is transferred to a 100 mL volumetric flask, the conical flask is rinsed with ethyl acetate, and then the volume is adjusted with ethyl acetate and shaken well.

[0072] S200 Preparation of reference solution

[0073] In the present application, in step S200, the standard samples of menthol, borneol and camphor are dissolved in a solvent to prepare a reference solution. Preferably, the solvent in step S200 is the same as the organic solvent in step S100.

[0074] In some embodiments, the preparation of the reference solution in step S200 comprises the following sub-steps:

[0075] S210 Preparation of single standard control solution: Take camphor, menthol and borneol standards, respectively, add solvent to dissolve, prepare camphor control solution, menthol control solution and borneol control solution;

[0076] S220 Preparation of mixed control solution: Take camphor, borneol and menthol standards and mix, dissolve with solvent to prepare mixed control solution.

[0077] In some embodiments, in step S220, take camphor, borneol and menthol standards, respectively, add solvent to dissolve, prepare camphor stock solution, borneol stock solution and menthol stock solution, take camphor stock solution, borneol stock solution and menthol stock solution, mix, dilute with ethyl acetate.

[0078] In some embodiments, in step S220, the mass concentration of menthol in the mixed control solution is 54-66 μg / mL.

[0079] In some embodiments, in step S220, the mass concentration of borneol in the mixed control solution is 54-66 μg / mL.

[0080] In some embodiments, in step S220, the mass concentration of camphor in the mixed control solution is 180-220 μg / mL.

[0081] In some specific embodiments, the step of preparing the reference solution comprises:

[0082] Precisely weigh 60 mg of menthol standard, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and prepare a menthol control stock solution;

[0083] Precisely weigh 40 mg of borneol standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and prepare a borneol control stock solution;

[0084] Precisely weigh 100 mg of camphor standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and prepare a camphor control stock solution;

[0085] Precisely take the menthol control stock solution, the borneol control stock solution and the camphor control stock solution, place them in the same volumetric flask, dilute to the mark with solvent, shake well, and obtain the mixed control solution.

[0086] S300 Determination method

[0087] In this application, in step S300, take the test solution and the reference solution, perform gas chromatography analysis, and determine by external standard method.

[0088] In some embodiments of the present application, the conditions of gas chromatography analysis include:

[0089] Detector: hydrogen flame ionization detector;

[0090] Chromatographic column: capillary column with cross-linked bonded polyethylene glycol as stationary phase;

[0091] Temperature program: initial temperature 90-110℃, holding for 3-6min, then rising to 150-180℃ at 4-8℃ / min, holding for 3-8min;

[0092] Injection port temperature: 200-250℃;

[0093] Detector temperature: 200-250℃;

[0094] Split ratio (10-20): 1.

[0095] The separation degree between any two components of the active ingredients determined by the determination method of the present application is higher than 2, which can effectively avoid interference and has high precision. In some embodiments, the separation degree between any two of camphor, menthol and borneol is ≥2, and further can be 2-3.

[0096] In some embodiments of the present application, the determination is performed by external standard method, and the retention time of menthol, borneol and camphor in the obtained determination result is as follows: camphor: 11.048±1min; menthol: 13.437±1min; borneol: 14.810±1min.

[0097] The following are some specific examples.

[0098] In the following specific examples, the experimental parameters not written are preferably referred to the guidance given in the present application document, and can also be referred to the experimental manual in the art or other experimental methods known in the art, or the experimental conditions recommended by the manufacturer; the raw materials and reagents involved can be obtained by market, or can be prepared by the skilled person in the art according to known means.

[0099] The products of human dan used in the following examples are from Guangzhou Wanglaoji Pharmaceutical Co., Ltd., and the batch numbers are as follows: 2006013, 2006014, 2006015.

[0100] 1. Development of extraction method of test sample

[0101] 1.1. First extraction method and selection of extraction solvent

[0102] By comparing the same solvent and different extraction methods, the same extraction method and different solvents, the method with less impurities and high extraction content is selected as the pretreatment method. The specific method is shown in Table 1-1-1, the initial chromatographic column is shown in Table 1-1-2, and the experimental results are shown in Table 1-1-3.

[0103] Table 1-1-1 Preparation of sample solution

[0104]

[0105] Table 1-1-2 Initial chromatographic conditions

[0106] Carrier gas nitrogen (N2) Chromatographic column DB-WAX (30 m*0.32 mm*0.5 um) Flow rate 1 mL / min Injection volume 1 μL Injection mode Split Split ratio 1：1 Detector Flame ionization detector (FID) Injection port temperature 220℃ Detector temperature 220℃ Column temperature Initial temperature 100 °C, hold for 4 min, increase to 160 °C at 5 °C per minute, hold for 8 min. Analysis time About 24 minutes

[0107] Table 1-1-3 Sample content results

[0108]

[0109] Result analysis: The above two reagents are compared, and in the same extraction method, the target content extracted from the human dan product by using ethyl acetate as the extraction reagent is higher; by comprehensive comparison, the target content extracted from the human dan product by using ethyl acetate ultrasonic extraction for 30 min is the highest.

[0110] During the experiment, it was found that since the target substances camphor, menthol and borneol are easy to sublimate and volatile substances, which can easily lead to poor reproducibility, therefore, the sample processing should not be ground and crushed, and the sample pretreatment should be re-processed.

[0111] 1.2. Second extraction method and selection of extraction solvent

[0112] The organic reagent cannot dissolve the sample coating, and a proper amount of water is added to dissolve the sample coating, and then the organic reagent is used to extract the target substance. The solubility of water in organic reagent is different, and the extraction effect will also change. The addition of water extraction needs to be re-determined the organic solvent and the extraction method.

[0113] The sample has more impurities, and the sample injection split ratio is changed from 1:1 to 10:1. The extraction method is shown in Table 1-2-1, the chromatographic column is shown in Table 1-2-2, and the experimental results are shown in Table 1-2-3.

[0114] Table 1-2-1 Sample extraction method

[0115]

[0116] Table 1-2-2 Chromatographic conditions

[0117] Carrier gas nitrogen (N2) Chromatographic column DB-WAX (30 m*0.32 mm*0.5 um) Flow rate 1 mL / min Injection volume 1 μL Injection mode Split Split ratio 10：1 Detector Flame ionization detector (FID) Injection port temperature 220℃ Detector temperature 220℃ Column temperature Initial temperature 100 °C, hold for 4 min, increase to 160 °C at 5 °C per minute, hold for 4 min. Analysis time About 20 minutes

[0118] Table 1-2-3 Sample content

[0119]

[0120]

[0121] Results analysis: compared with two kinds of solvents, ethyl acetate extraction effect is more ideal; with ethyl acetate as solvent, compared with two kinds of extraction methods, magnetic stirring for 10 min can completely extract the target.

[0122] Through the above extraction method and extraction solvent, it is determined that the extraction solvent is ethyl acetate, and the extraction method is magnetic stirring for 10 min. Combined with the feasibility of batch operation, under the premise of not changing the sample amount and sample concentration, the sample preparation method is adjusted, and the sample preparation method is preliminarily determined as follows: take 1g of human dan sample, accurately weigh, put it in a conical flask with a plug, add 60mL of ethyl acetate and 5mL of water, tightly plug, stir with a magnetic stirrer for 10min(300r / min), collect the extraction liquid in a 100mL volumetric flask, rinse the conical flask with ethyl acetate, collect the rinse liquid in a 100mL volumetric flask, and dilute with ethyl acetate, shake well.

[0123] 2. Determination method verification process

[0124] 2.1. Chromatographic conditions

[0125] Table 2-1-1 Chromatographic conditions

[0126] Carrier gas nitrogen (N2) Chromatographic column DB-WAX (30 m*0.32 mm*0.5 um) Flow rate 1 mL / min Injection volume 1 μL Injection mode Split Split ratio 10:1 Detector Flame ionization detector (FID) Injection port temperature 220℃ Detector temperature 220℃ Column temperature Initial temperature 100 °C, hold for 4 min, increase to 160 °C at 5 °C per minute, hold for 4 min. Analysis time About 20 minutes

[0127] 2.2. Preparation of solution

[0128] 2.2.1. Preparation of control stock solution

[0129] Take camphor 60mg, accurately weigh, put it in a 100mL volumetric flask, dissolve and dilute to the mark with ethyl acetate, shake well, and prepare camphor control stock solution.(The actual concentration needs to be corrected by the purity of the control sample, and the concentration is about 0.6mg / mL)

[0130] Take borneol 40mg, accurately weigh, put it in a 50mL volumetric flask, dissolve and dilute to the mark with ethyl acetate, shake well, and prepare borneol control stock solution.(The actual concentration needs to be corrected by the purity of the control sample, and the concentration is about 0.8mg / mL)

[0131] Take menthol 100mg, accurately weigh, put it in a 50mL volumetric flask, dissolve and dilute to the mark with ethyl acetate, shake well, and prepare menthol control stock solution.(The actual concentration needs to be corrected by the purity of the control sample, and the concentration is about 2.0mg / mL)

[0132] 2.2.2. Preparation of blank solution

[0133] Take the appropriate amount of ethyl acetate into a 10 mL volumetric flask, and dilute to the calibration line with ethyl acetate to prepare a blank solution.

[0134] 2.2.3. Preparation of the reference solution

[0135] Take 1 mL of camphor stock solution, 0.75 mL of borneol reference stock solution, and 1 mL of menthol stock solution respectively into the same 10 mL volumetric flask, dilute to the calibration line with ethyl acetate, and shake well to obtain the solution. (The concentrations of camphor and borneol are about 60 μg / mL, and the concentration of menthol is about 200 μg / mL)

[0136] 2.2.4. Preparation of the test solution

[0137] Take 1 g of human dan sample, accurately weigh, and place into a conical flask with a stopper. Add 60 mL of ethyl acetate and 5 mL of water, tightly stopper, and stir for 10 min (300 r / min) with a magnetic stirrer. Collect the extract into a 100 mL volumetric flask, rinse the conical flask with ethyl acetate three times, collect the rinse liquid into a 100 mL volumetric flask, dilute with ethyl acetate, shake well, and obtain the solution.

[0138] 2.2.5. Preparation of the negative test solution

[0139] Take 1 g of each of the negative sample without camphor, the negative sample without menthol, the negative sample without borneol, and the negative sample without camphor, menthol, and borneol, accurately weigh, add 60 mL of ethyl acetate and 5 mL of water, and stir for 10 min (300 r / min) with a magnetic stirrer. Collect the extract into a 100 mL volumetric flask, rinse the conical flask with ethyl acetate three times, collect the rinse liquid into a 100 mL volumetric flask, dilute with ethyl acetate, shake well, and obtain the negative test solution.

[0140] 2.3. Specificity

[0141] 2.3.1. Test method

[0142] According to the chromatographic conditions in 2.1, detect the blank solution, the reference solution, and the negative test solution.

[0143] 2.3.2. Test results

[0144] There is no interference at the retention time of the target peak in the blank solution. In the reference solution, the separation degree between the peaks of each component is greater than 2.0. In the negative test solution, there is no interference at the retention time of the target peak corresponding to each negative test solution. The specificity of the method meets the requirements (≥2.0).

[0145] The chromatograms of the blank solution, the control solution, the camphor-free, menthol-free, borneol-free negative sample solutions, the camphor-free negative sample solution, the menthol-free negative sample solution, and the borneol-free negative sample solution are shown in the attached Figures 1 to 6 .

[0146] 2.4. Linearity

[0147] 2.4.1. Preparation of the linear solution

[0148] The control stock solution was taken according to Table 2-4-1 and placed in a 2 mL volumetric flask, and then diluted to the mark with ethyl acetate.

[0149] Table 2-4-1 Preparation of the linear solution

[0150]

[0151] 2.4.2. Test results

[0152] The linear solution prepared in 2.4.1 was injected for testing at different concentrations, and the linear results are shown in Tables 2-4-2 to 2-4-4. The standard curve (camphor linear curve, menthol linear curve, and borneol linear curve, in turn, as shown in the attached Figures 7 to 9 ) was plotted with the concentration (μg / mL) as the abscissa and the peak area as the ordinate, and the linear regression correlation coefficients of each component met the requirements.

[0153] Table 2-4-2 Camphor linear results table

[0154]

[0155] Table 2-4-3 Menthol linear results table

[0156]

[0157]

[0158] Table 2-4-4 Borneol linear results table

[0159]

[0160] 2.5. Accuracy

[0161] 2.5.1. Preparation of the accuracy solution

[0162] The sample was taken according to Table 2-5-1, accurately weighed, placed in a conical flask with a stopper, the control stock solution was added according to Table 2-5-1, an appropriate amount of ethyl acetate and 5 mL of water were added, and the mixture was stirred with a magnetic stirrer for 10 min (300 r / min); the extract was taken into a 100 mL volumetric flask, the conical flask was rinsed with ethyl acetate three times, the rinsing liquid was placed in the same volumetric flask, and the volume was adjusted with ethyl acetate, and then the mixture was shaken well to obtain the solution.

[0163] Prepare 3 concentrations of accuracy solutions, 3 solutions for each concentration. Calculate the recovery rate by deducting the amount in the accuracy sample blank solution.

[0164] Table 2-5-1 Preparation of accuracy solutions

[0165]

[0166] 2.5.2 Test results

[0167] Each sample of accuracy A0-A3 solutions was injected once, the amount of target in the sample was converted by A0, and the RSD value of the recovery rate calculated by deducting the amount of target in the sample met the requirements. The accuracy results are shown in Tables 2-5-2 to 2-5-4.

[0168] Table 2-5-2 Camphor accuracy results table

[0169]

[0170] Table 2-5-3 Menthol accuracy results table

[0171]

[0172] Table 2-5-4 Borneol accuracy results table

[0173]

[0174]

[0175] 2.6. Precision

[0176] 2.6.1. Repeatability

[0177] Weigh 1 g of human dan sample accurately, place it in a conical flask with a stopper, add 60 mL of ethyl acetate and 5 mL of water, tightly seal it, and stir it with a magnetic stirrer for 10 min (300 r / min); collect the extract in a 100 mL volumetric flask, rinse the conical flask with ethyl acetate three times, collect the rinse liquid in a 100 mL volumetric flask, and dilute it with ethyl acetate, shake it well, and test 6 parallel test samples to calculate the relative standard deviation of the content of the 6 samples.

[0178] 2.6.2. Intermediate precision

[0179] Prepare 6 parallel test samples by different analysts on different dates, test them, and calculate the relative standard deviation of the content of the 12 samples.

[0180] 2.6.3. Precision results

[0181] 6 Method repeatability and intermediate precision of test solution The RSD values of each control content in 12 solutions met the precision requirements. The repeatability results are shown in Table 2-6-1, and the precision results are shown in Table 2-6-2.

[0182] Table 2-6-1 Repeatability results table

[0183]

[0184] Table 2-6-2 Precision results table

[0185]

[0186]

[0187] 2.7. Solution stability

[0188] 2.7.1. Stability of control solution and test solution

[0189] Accurately pipette 1 mL of camphor stock solution, 0.75 mL of borneol control stock solution, and 1 mL of menthol stock solution into the same 10 mL volumetric flask, dilute to the calibration mark with ethyl acetate, shake well, and analyze at 0, 2, 4, 8, 12, and 24 hours. Calculate the RSD% value of each control peak area to investigate the stability.

[0190] Take 1 g of human dan sample, accurately weigh, and place in a conical flask with a stopper. Add 60 mL of ethyl acetate and 5 mL of water, tightly stopper, and stir with a magnetic stirrer for 10 min (300 r / min). Collect the extract in a 100 mL volumetric flask, rinse the conical flask with ethyl acetate three times, collect the rinse liquid in a 100 mL volumetric flask, and dilute with ethyl acetate. Shake well, and analyze at 0, 2, 4, 8, 12, and 24 hours. Calculate the RSD% value of each control peak area to investigate the stability.

[0191] The RSD values of each control peak area of 6 control solutions and 6 test solutions at 0, 2, 4, 8, 12, and 24 hours met the requirements. The solutions were stable at room temperature in the dark for 24 h. The stability results are shown in Table 2-7-1.

[0192] Table 2-7-1 Solution stability results table

[0193]

[0194] 2.7.2. Stability of control stock solution

[0195] The prepared control stock solution was stored at 2-8 °C in the dark for 5 days, and then was restored to room temperature. 1 mL of camphor stock solution, 0.75 mL of borneol control stock solution, and 1 mL of menthol stock solution were precisely measured and placed in the same 10 mL volumetric flask, which was diluted to the calibration mark with ethyl acetate, and then was shaken uniformly. The stability was investigated by comparing the prepared control stock solution with the newly prepared control stock solution. The stability results are shown in Table 2-7-2.

[0196] The recovery rates of camphor, menthol, and borneol were in the range of 90-108%, and the stock solution was stable when stored at 2-8 °C in the dark for 5 days.

[0197] Table 2-7-2 Stability Results of Solution

[0198]

[0199] 2.8. Robustness

[0200] 2.8.1. Changing the detector temperature

[0201] The detector temperature was changed from 220 °C to 210 °C and 230 °C, and the blank and test sample solutions were tested under the condition that other parameters were unchanged. The results are shown in Table 2-8-1.

[0202] Table 2-8-1 Robustness Results of Changing the Detector Temperature

[0203]

[0204] 2.8.2. Changing the chromatographic column

[0205] The chromatographic column DB-wax (30 m x 320 mm x 0.50 μm) was changed to the chromatographic column DB-waxetr (30 m x 320 mm x 0.25 μm), and the blank and test sample solutions were tested under the condition that other parameters were unchanged. The results are shown in Table 2-8-2.

[0206] Table 2-8-2 Robustness Results of Changing the Chromatographic Column

[0207]

[0208] 3. Confirmation of the Content Determination Method

[0209] The content of 4 batches of samples was determined according to the content determination method developed above, and the specific test parameters are as follows:

[0210] 3.1. Chromatographic Conditions and System Suitability Test

[0211] Capillary column with cross-linked polyethylene glycol as stationary phase; column temperature: programmed temperature, initial temperature 100℃, hold for 4 min, increase to 160℃ at the rate of 5℃ per min, hold for 4 min; injection port temperature 220℃; detector temperature 220℃; split injection, split ratio 10:1. Theoretical plate number should not be less than 5000 calculated by menthol peak.

[0212] 3.2. Preparation of control solution

[0213] Take camphor, borneol and menthol control solution respectively, accurately weigh, dissolve in ethyl acetate solution to prepare camphor, borneol control solution containing about 60 μg per 1 mL and menthol control solution containing about 0.2 mg per 1 mL as control solution.

[0214] 3.3. Preparation of test solution

[0215] Take 1 g of the product, accurately weigh, put into a conical flask with plug, add 60 mL of ethyl acetate and 5 mL of water, tightly plug, stir for 10 minutes (300 r / min) with magnetic stirrer, collect the extract in a 100 mL volumetric flask, rinse the conical flask with ethyl acetate, collect the rinse liquid in the volumetric flask, and then constant volume with ethyl acetate, shake well, filter, and then collect the filtrate, which is the test solution.

[0216] 3.4. Determination method

[0217] Accurately take 1 μL of control solution and test solution, inject into gas chromatograph, and then determine, which is the determination result.

[0218] 3.5. Determination result (Table 3)

[0219] Table 3: Determination result of human dan sample

[0220]

[0221] According to Table 3, the above-mentioned determination method is feasible and accurate.

[0222] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. Unless and to the extent that the documents cited in the present application conflict with the application purpose and / or technical scheme of the present application, the cited documents are cited in the present application in their entirety and in their entirety purpose. When the present application refers to the cited documents, the definition of the related technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present application refers to the cited documents, the examples and preferred modes of the cited related technical features can also be cited as references in the present application, but to the extent that the present application can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application is correct or is modified according to the description in the present application.

[0223] Each technical feature in the above-described embodiments and examples can be combined in any suitable manner, and the present application can be applied to some technical features. Therefore, it is contemplated that the technical features of the above-described embodiments and examples can be combined in any suitable manner, and the present application can be applied to some technical features. It is contemplated that all possible combinations have been disclosed in the specification, unless the combinations are contradictory or the combinations do not make sense.

[0224] The above-described embodiments are merely illustrative for the present application and should not be understood as limiting the scope of the present application. It should be noted that, for those skilled in the art, some modifications and improvements can be made to the present application without departing from the concept of the present application, and these modifications and improvements should also be considered within the scope of the present application. In addition, it should be understood that, after reading the above description of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms thus obtained should also fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, and these technical solutions should also fall within the scope of the claims of the present application. Therefore, the scope of the patent protection of the present application should be subject to the claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for measuring the volatile components of human semen, characterized by, The human dan comprises a core and a coating covering the core; the material of the core comprises menthol, cassia, licorice, catechu, costus, borneol, platycodon, camphor, cumin, cardamom and clove basil oil; the material of the coating is a non-aluminum water-soluble material; the material of the coating comprises talc, carmine and red iron oxide; The determination method comprises the following steps: Take 1g of the human dan sample to be tested, add 60mL of organic solvent and 5mL of water, mix, and then extract with a magnetic stirrer at 300r / min for 10min; collect the extract, and then use ethyl acetate to make up to 100mL to obtain a test sample solution; the organic solvent is ethyl acetate; Take standard samples of menthol, borneol and camphor, add a solvent to dissolve, and then prepare a reference solution; Take the test sample solution and the reference solution, and then perform gas chromatography analysis and determination by using an external standard method; The gas chromatography analysis conditions comprise: a detector: hydrogen flame ionization detector; A chromatographic column: a capillary column with cross-linked bonded polyethylene glycol as a stationary phase; A temperature rising program: an initial temperature of 90-110℃, maintained for 3-6min, then increased to 150-180℃ at a rate of 4-8℃ / min, maintained for 3-8min; An injection port temperature: 200-250℃; A detector temperature: 200-250℃; A split ratio (10-20): 1; The determination by using an external standard method, the retention times of the menthol, the borneol and the camphor in the determination results are as follows: camphor: 11.048±1min, menthol: 13.437±1min, and borneol: 14.810±1min.

2. The assay method according to claim 1, characterized by The mass ratio of the core to the coating is 1:(0.02-0.05).

3. The assay method according to claim 1, characterized by, The material of the core comprises 40.6±m parts of menthol, 20.3±m parts of cassia, 476±m parts of licorice, 40.6±m parts of catechu, 10.2±m parts of costus, 20.3±m parts of borneol, 325±m parts of platycodon, 20.3±m parts of camphor, 20.3±m parts of cumin, 10.2±m parts of cardamom and clove basil oil, in terms of weight fraction; All the m are the same, and m is 1, 0.5, 0.2, 0.1 or 0; The mass-volume ratio of the core to the clove basil oil is (900-1000)g:5mL.

4. The assay method according to claim 1, characterized by, The preparation of the reference solution comprises the following steps of preparing a single standard control solution: taking standard samples of camphor, menthol and borneol, respectively adding a solvent to dissolve, and then preparing camphor control solution, menthol control solution and borneol control solution; Preparing a mixed control solution by mixing camphor standard sample, borneol standard sample and menthol standard sample, and then dissolving with the solvent.

5. The assay method according to claim 4, characterized in that, In the mixed control solution, the mass-volume concentration of menthol is 54-66μg / mL, the mass-volume concentration of borneol is 54-66μg / mL, and the mass-volume concentration of camphor is 180-220μg / mL.

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