阅读说明：本技术 一种消炎癣湿药膏的检测方法 (Detection method of anti-inflammatory tinea-dampness ointment ) 是由 贾瑞巧 何井亮 戴丽萍 吕国强 于 2019-12-04 设计创作，主要内容包括：本发明公开了一种消炎癣湿药膏的检测方法,该检测方法包括：性状：本品为黄褐色软膏,具有特异臭气和清凉感；鉴别：通过薄层色谱法对蛇床子和冰片进行成分鉴别,通过高效气相色谱法对樟脑进行成分鉴别；该检测方法还包括苯酚和升华硫的含量测定,含量测定均采用高效液相色谱法进行。该方法可以对消炎癣湿药膏的主要药物成分进行更有效控制,可使产品的有效性和安全性得到有力的保障,弥补目前质量控制过程的不足,提高产品的质量监控水平,也有利于管理部门对产品的监测。(The invention discloses a detection method of an anti-inflammatory tinea-dampness ointment, which comprises the following steps: the characteristics are as follows: the product is brown ointment, and has special odor and refreshing feeling; and (3) identification: identifying fructus Cnidii and Borneolum Syntheticum by thin layer chromatography, and identifying Camphora by high performance gas chromatography; the detection method also comprises the content measurement of phenol and sublimed sulfur, and the content measurement is carried out by adopting a high performance liquid chromatography. The method can effectively control the main medicinal components of the ointment for treating inflammation, tinea and dampness, effectively ensure the effectiveness and safety of the product, make up the defects of the existing quality control process, improve the quality monitoring level of the product and facilitate the monitoring of the product by management departments.)

1. The detection method of the anti-inflammatory tinea-dampness ointment is characterized by comprising the following steps:

the characteristics are as follows: the product is brown ointment, and has special odor and refreshing feeling;

and (3) identification: (1) taking 3.0g of finished ointment, adding 20mL of absolute ethanol, heating and melting in a water bath at 50 ℃, carrying out ultrasonic treatment for 30min, and filtering to obtain filtrate as a test solution; adding anhydrous ethanol into a certain amount of reference osthole to obtain a solution of 1mg/mL as reference solution; performing thin-layer chromatography test, sucking the two solutions, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-ethyl acetate-n-hexane at a ratio of 3:2 as developing agent, taking out, air drying, placing under 365nm ultraviolet lamp for inspection, and displaying fluorescent spots of the same color in the chromatogram of the test sample at the position corresponding to the chromatogram of the reference sample;

(2) taking 3.0g of finished ointment, adding 20mL of absolute ethanol, heating and melting in a water bath at 50 ℃, carrying out ultrasonic treatment for 30min, and filtering to obtain filtrate as a test solution; adding anhydrous ethanol into a certain amount of Borneolum Syntheticum as reference substance to obtain 2mg/mL solution as reference substance solution; performing thin layer chromatography test, sucking the two solutions 4 μ L, respectively dropping on the same silica gel G thin layer plate, developing with 17:4 n-hexane-ethyl acetate as developing agent, taking out, air drying, spraying 1% vanillin-sulfuric acid solution, heating at 105 deg.C until the spots are clear, and displaying the spots with the same color in the sample chromatogram at the position corresponding to the control chromatogram;

(3) the camphor is identified by adopting high performance gas chromatography

Chromatographic conditions are as follows: a chromatographic column: the HP-50+ capillary column adopts a 50% -phenyl-methyl polysiloxane stationary phase, and the size of the capillary column is 30m multiplied by 0.25mm and is 0.25 mu m; temperature programming: the initial temperature is 85 ℃, the temperature is increased to 95 ℃ at the speed of 2 ℃/min, then the temperature is increased to 110 ℃ at the speed of 1 ℃/min, then the temperature is increased to 200 ℃ at the speed of 8 ℃/min, and the temperature is kept for 2 min; the temperature of a sample inlet is 200 ℃; FID detection; the temperature of the detector is 200 ℃; the carrier gas is nitrogen, the flow rate is 0.7mL/min, and the split flow is injected, wherein the split flow ratio is 5: 1; the hydrogen flow rate is 50 mL/min; the air flow rate is 400 mL/min;

solution preparation: taking 0.5g of finished ointment, adding 40mL of absolute ethanol, heating and melting in a water bath at 50 ℃, ultrasonically treating for 20min, cooling, refrigerating at 4 ℃ overnight, and filtering to obtain filtrate as a test solution; adding anhydrous ethanol into a certain amount of reference camphor to obtain a solution of 0.5mg/mL as a reference solution;

the method comprises the following operation steps: and (4) performing gas chromatography, sucking 1 μ L of the two solutions under the chromatographic conditions, injecting the two solutions into a gas chromatograph, and allowing the sample solution to have consistent chromatographic peaks at the same retention time positions as the chromatographic peaks of the reference substance in the chromatogram.

2. The method as claimed in claim 1, further comprising the steps of:

the examination should comply with the regulations under ointment:

(1) measuring the content of phenol by high performance liquid chromatography:

chromatographic conditions are as follows: a chromatographic column: agilent TC-C ₁₈250X 4.6mm, 5 μm; mobile phase: 62: 38 methanol-water; flow rate: 1.0 mL/min; detection wavelength: 271 nm; column temperature: 45 ℃;

solution preparation: accurately weighing 0.2g of finished ointment, placing into a conical flask with a plug, accurately adding 50mL of absolute ethanol solution, weighing, heating in a water bath at 50 ℃ for 30min, carrying out ultrasonic treatment in a hot water bath for 30min, cooling, weighing again, supplementing the lost weight with the absolute ethanol solution, shaking, refrigerating at 4 ℃ for 2h, and filtering to obtain a filtrate as a test solution; accurately weighing a certain amount of reference substance phenol, and adding mobile phase to obtain 36 μ g/mL solution as reference substance solution;

the determination method comprises the following steps: precisely absorbing 5 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, respectively recording chromatograms, and calculating the content of phenol in the sample solution by peak area according to an external standard method; the product contains 30.29-37.02 mg/g of phenol;

(2) measuring the content of sublimed sulfur by high performance liquid chromatography:

chromatographic conditions are as follows: a chromatographic column: agilent TC-C ₁₈250X 4.6mm, 5 μm; mobile phase: methanol; flow rate: 1.0 mL/min; detection wavelength: 240 nm; column temperature: 30 ℃;

solution preparation: accurately weighing 0.8g of finished ointment, placing into a conical flask with a plug, adding 50mL of ethyl acetate, heating in water bath at 60 ℃ for 50min, cooling, transferring into a 100mL volumetric flask, adding ethyl acetate to dilute to constant volume, shaking up, and refrigerating at 4 ℃ for 2 h; precisely weighing 2mL of diluted solution from a 100mL volumetric flask into a 10mL volumetric flask, adding ethyl acetate for diluting to a constant volume, shaking up, and filtering to obtain a filtrate as a test solution; accurately weighing a certain amount of sublimed sulfur as a reference substance, and adding ethyl acetate to prepare a solution of 40 mug/mL as the reference substance solution;

the determination method comprises the following steps: precisely absorbing 5 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, respectively recording chromatograms, and calculating the content of sublimed sulfur in the sample solution according to an external standard method by peak area; the product contains 35.35-43.21 mg/g of sublimed sulfur.

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to a detection method of ointment, in particular to a detection method of anti-inflammatory tinea-dampness ointment.

Background

The ointment for diminishing inflammation, tinea and dampness has the effects of sterilizing, astringing dampness and relieving itching, is clinically suitable for treating tinea capitis, tinea corporis, tinea pedis, chronic eczema, water-nourishing pruritus, scabies and the like, and is a better Chinese patent medicine for treating skin diseases clinically.

Fructus cnidii, borneol and camphor are characteristic index components of the anti-inflammatory tinea-dampness ointment respectively. In the existing production process of the anti-inflammatory tinea-dampness ointment, due to the lack of an effective identification method, the cnidium fruit, the borneol and the camphor in the finished anti-inflammatory tinea-dampness ointment are not separately identified, so that the main medicinal components of the finished anti-inflammatory tinea-dampness ointment are uncertain, the product quality difference is large, and the instability of the curative effect is certainly caused.

In order to further ensure the quality of the anti-inflammatory tinea-dampness ointment and be more beneficial to supervision and management of the quality, the effective components of the anti-inflammatory tinea-dampness ointment, namely the fructus cnidii, the borneol and the camphor, are subjected to identification detection and content measurement and detection of phenol and sublimed sulfur, so that the quality and the curative effect of the anti-inflammatory tinea-dampness ointment are further ensured, adverse reactions are reduced to the minimum, and the medication safety of patients is ensured.

Disclosure of Invention

The invention aims to provide a method for detecting an anti-inflammatory tinea-dampness ointment, which can more effectively control the main medicinal components of the anti-inflammatory tinea-dampness ointment, can powerfully guarantee the effectiveness and safety of a product, makes up the defects of the existing quality control process, improves the quality monitoring level of the product, and is also beneficial to monitoring the product by management departments.

In order to achieve the aim, the invention provides a detection method of an anti-inflammatory tinea-dampness ointment, which comprises the following steps:

the characteristics are as follows: the product is brown ointment, and has special odor and refreshing feeling;

and (3) identification: (1) taking 3.0g of finished ointment, adding 20mL of absolute ethanol, heating and melting in a water bath at 50 ℃, carrying out ultrasonic treatment for 30min, and filtering to obtain filtrate as a test solution; adding anhydrous ethanol into a certain amount of reference osthole to obtain a solution of 1mg/mL as reference solution; performing thin-layer chromatography test, sucking the two solutions, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-ethyl acetate-n-hexane at a ratio of 3:2 as developing agent, taking out, air drying, placing under 365nm ultraviolet lamp for inspection, and displaying fluorescent spots of the same color in the chromatogram of the test sample at the position corresponding to the chromatogram of the reference sample;

(2) taking 3.0g of finished ointment, adding 20mL of absolute ethanol, heating and melting in a water bath at 50 ℃, carrying out ultrasonic treatment for 30min, and filtering to obtain filtrate as a test solution; adding anhydrous ethanol into a certain amount of Borneolum Syntheticum as reference substance to obtain 2mg/mL solution as reference substance solution; performing thin layer chromatography test, sucking the two solutions 4 μ L, respectively dropping on the same silica gel G thin layer plate, developing with 17:4 n-hexane-ethyl acetate as developing agent, taking out, air drying, spraying 1% vanillin-sulfuric acid solution, heating at 105 deg.C until the spots are clear, and displaying the spots with the same color in the sample chromatogram at the position corresponding to the control chromatogram;

(3) the camphor is identified by adopting high performance gas chromatography

Chromatographic conditions are as follows: a chromatographic column: the HP-50+ capillary column adopts a 50% -phenyl-methyl polysiloxane stationary phase, and the size of the capillary column is 30m multiplied by 0.25mm and is 0.25 mu m; temperature programming: the initial temperature is 85 ℃, the temperature is increased to 95 ℃ at the speed of 2 ℃/min, then the temperature is increased to 110 ℃ at the speed of 1 ℃/min, then the temperature is increased to 200 ℃ at the speed of 8 ℃/min, and the temperature is kept for 2 min; the temperature of a sample inlet is 200 ℃; FID detection; the temperature of the detector is 200 ℃; the carrier gas is nitrogen, the flow rate is 0.7mL/min, and the split flow is injected, wherein the split flow ratio is 5: 1; the hydrogen flow rate is 50 mL/min; the air flow rate is 400 mL/min;

solution preparation: taking 0.5g of finished ointment, adding 40mL of absolute ethanol, heating and melting in a water bath at 50 ℃, ultrasonically treating for 20min, cooling, refrigerating at 4 ℃ overnight, and filtering to obtain filtrate as a test solution; adding anhydrous ethanol into a certain amount of reference camphor to obtain a solution of 0.5mg/mL as a reference solution;

the method comprises the following operation steps: and (4) performing gas chromatography, sucking 1 μ L of the two solutions under the chromatographic conditions, injecting the two solutions into a gas chromatograph, and allowing the sample solution to have consistent chromatographic peaks at the same retention time positions as the chromatographic peaks of the reference substance in the chromatogram.

The detection method of the anti-inflammatory tinea-dampness ointment further comprises the following steps:

the examination should comply with the regulations under ointment:

(1) measuring the content of phenol by high performance liquid chromatography:

chromatographic conditions are as follows: a chromatographic column: agilent TC-C ₁₈250X 4.6mm, 5 μm; mobile phase: 62: 38 methanol-water; flow rate: 1.0 mL/min; detection wavelength: 271 nm; column temperature: 45 ℃;

solution preparation: accurately weighing 0.2g of finished ointment, placing into a conical flask with a plug, accurately adding 50mL of absolute ethanol solution, weighing, heating in a water bath at 50 ℃ for 30min, carrying out ultrasonic treatment in a hot water bath for 30min, cooling, weighing again, supplementing the lost weight with the absolute ethanol solution, shaking, refrigerating at 4 ℃ for 2h, and filtering to obtain a filtrate as a test solution; accurately weighing a certain amount of reference substance phenol, and adding mobile phase to obtain 36 μ g/mL solution as reference substance solution;

the determination method comprises the following steps: precisely absorbing 5 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, respectively recording chromatograms, and calculating the content of phenol in the sample solution by peak area according to an external standard method; the product contains 30.29-37.02 mg/g of phenol;

(2) measuring the content of sublimed sulfur by high performance liquid chromatography:

chromatographic conditions are as follows: a chromatographic column: agilent TC-C ₁₈250X 4.6mm, 5 μm; mobile phase: methanol; flow rate: 1.0 mL/min; detection wavelength: 240 nm; column temperature: 30 ℃;

solution preparation: accurately weighing 0.8g of finished ointment, placing into a conical flask with a plug, adding 50mL of ethyl acetate, heating in water bath at 60 ℃ for 50min, cooling, transferring into a 100mL volumetric flask, adding ethyl acetate to dilute to constant volume, shaking up, and refrigerating at 4 ℃ for 2 h; precisely weighing 2mL of diluted solution from a 100mL volumetric flask into a 10mL volumetric flask, adding ethyl acetate for diluting to a constant volume, shaking up, and filtering to obtain a filtrate as a test solution; accurately weighing a certain amount of sublimed sulfur as a reference substance, and adding ethyl acetate to prepare a solution of 40 mug/mL as the reference substance solution;

the determination method comprises the following steps: precisely absorbing 5 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, respectively recording chromatograms, and calculating the content of sublimed sulfur in the sample solution according to an external standard method by peak area; the product contains 35.35-43.21 mg/g of sublimed sulfur.

Compared with the prior art, the invention adds the identification of the thin-layer chromatography of the common cnidium fruit and the borneol in the production process of the existing product, adds the identification of the high-efficiency gas chromatography of camphor and adds the content measurement of the high-efficiency liquid chromatography of phenol and sublimed sulfur, thereby realizing more effective detection of the main medicinal components of the xiayan xian shi ointment, ensuring that the main medicinal components in the finished product of the xiao xian shi ointment are determined relatively, and greatly improving the monitoring level of the product quality; the content of phenol and sublimed sulfur is quantitatively controlled, so that the method plays a positive role in reducing adverse reactions of products, further defines the accuracy of the administration dosage and effectively guarantees the effectiveness and safety of the products. The application of the invention is not only beneficial to the detection of manufacturers and management departments on products, but also provides better guarantee for the treatment of medical departments and patients.

Drawings

FIG. 1 is a TLC chromatogram for identifying fructus Cnidii of the present invention; in the figure, 1 and 2 are test solution, 3 is control solution, and 4 is negative control solution;

FIG. 2 is a TLC chromatogram for identifying borneol in the example of the present invention; in the figure, 1 and 2 are test solution, 3 is control solution, and 4 is negative control solution;

FIG. 3 is a GC identification chromatogram of camphor in an example of the invention; in the figure, a is a test solution, b is a control solution, and c is a negative control solution;

FIG. 4 is an HPLC detection chromatogram of phenol in an example of the present invention; in the figure, a is a test solution, b is a control solution, and c is a negative control solution;

FIG. 5 is a graph of HPLC detection linearity of phenol in an example of the present invention;

FIG. 6 is a graph of HPLC detection linearity of sublimed sulfur in an example of the present invention;

FIG. 7 is an HPLC detection chromatogram of sublimed sulfur in an example of the present invention; in the figure, a is a test solution, b is a control solution, and c is a negative control solution.

Detailed Description

The present invention will be described in further detail with reference to examples.

The detection method of the antiphlogistic tinea-dampness ointment comprises the following steps of preparing 50g of medicine-raising bottom, 50g of common cnidium fruit, 50g of sublimed sulfur, 50g of camphor, 30g of borneol and 40ml of phenol into the ointment according to the parts by weight; grinding the five medicinal materials of the Shengyao, the fructus cnidii, the sublimed sulfur, the camphor and the borneol respectively according to the proportion, sieving the medicinal materials for later use, taking 1000g of vaseline for heating and melting, filtering the mixture, adding the sieved Shengyao, the fructus cnidii, the sublimed sulfur and the phenol, stirring the mixture evenly, adding the camphor and the borneol after cooling, and stirring the mixture evenly to obtain the anti-inflammatory tinea-eczema ointment; the detection method comprises the following steps:

the characteristics are as follows: the product is brown ointment, and has special odor and refreshing feeling;

and (3) identification: (1) taking 3.0g of finished ointment, adding 20mL of absolute ethanol, heating and melting in a water bath at 50 ℃, carrying out ultrasonic treatment for 30min, and filtering to obtain filtrate as a test solution; taking a certain amount of reference substance osthole (batch No. 110822-201609, the mass fraction is 99.0%, which is provided by the verification of Chinese medicine biological products), and adding absolute ethyl alcohol to prepare a solution of 1mg/mL, which is used as a reference substance solution; taking 3.0g of negative preparation without fructus Cnidii, and treating according to the preparation method of the test solution to obtain negative control solution; testing by thin layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), sucking 4 μ L of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-n-hexane at a ratio of 3:2 as developing agent, taking out, air drying, placing under 365nm ultraviolet lamp for inspection, displaying fluorescent spots of the same color in the chromatogram of the test sample at the position corresponding to the chromatogram of the reference sample, wherein the negative reference is free of interference, and the thin layer chromatogram is shown in FIG. 1;

in the experiment, the extraction mode of the test solution is inspected, and two modes of ultrasonic treatment and direct ultrasonic treatment after water bath heating are inspected, so that the ultrasonic decomposition effect is better after the ointment is melted by the water bath heating; the proportion and the sample amount of the developing agent are adjusted, the developing agent toluene-ethyl acetate-n-hexane (3:3:2, 3:2:1, 3:2:2) with different proportions and different sample amounts (2 muL, 4 muL, 6 muL) are considered, and finally, the spots are determined to be clear and round when the proportion of the developing agent toluene-ethyl acetate-n-hexane is 3:2:2 and the sample amount is 4 muL.

(2) Taking 3.0g of finished ointment, adding 20mL of absolute ethanol, heating and melting in a water bath at 50 ℃, carrying out ultrasonic treatment for 30min, and filtering to obtain filtrate as a test solution; taking a certain amount of reference borneol (batch No. 110881-201508, the mass fraction is 99.0 percent and is provided by China pharmaceutical and biological product assay), and adding absolute ethyl alcohol to prepare a solution of 2mg/mL as a reference solution; taking 3.0g of negative preparation without borneol, and treating according to the preparation method of the test solution to serve as a negative control solution; testing by thin layer chromatography (0502 of the general rules of the four parts of the book of Chinese pharmacopoeia 2015), sucking 4 μ L of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with 17:4 n-hexane-ethyl acetate as developing agent, taking out, air drying, spraying 1% vanillin-sulfuric acid solution, heating at 105 deg.C until the spots are clear, and displaying the spots of the same color in the chromatogram of the test sample at the positions corresponding to those of the chromatogram of the reference sample; negative control without interference, and thin layer chromatogram is shown in FIG. 2;

in the experiment, the identification mode of borneol (synthetic borneol) is inspected, physical and chemical identification and thin-layer identification are sequentially inspected, and the thin-layer identification spots are clear and the result is better. Then, the selection of the thin layer identification developing agent n-hexane-ethyl acetate ratio (17:3, 17:4), the spotting amount (2 μ L, 4 μ L) and the color developing agent (1% vanillin sulfuric acid solution, 10% phosphomolybdic acid ethanol solution) is considered, and the result shows that spots are clearer when the developing agent n-hexane-ethyl acetate ratio is 17:4, the spotting amount is 4 μ L, and the 1% vanillin sulfuric acid solution is used as the color developing agent.

(3) The camphor is identified by adopting high performance gas chromatography

Chromatographic conditions are as follows: agilent 7890B gas chromatograph, column: the HP-50+ capillary column adopts a 50% -phenyl-methyl polysiloxane stationary phase, and the size of the capillary column is 30m multiplied by 0.25mm and is 0.25 mu m; temperature programming: the initial temperature is 85 ℃, the temperature is increased to 95 ℃ at the speed of 2 ℃/min, the temperature is increased to 110 ℃ at the speed of 1 ℃/min, the temperature is increased to 200 ℃ at the speed of 8 ℃/min, and the temperature is kept for 2 min; the temperature of a sample inlet is 200 ℃; FID detection; the temperature of the detector is 200 ℃; the carrier gas is nitrogen, the flow rate is 0.7mL/min, and the split flow is injected, wherein the split flow ratio is 5: 1; the hydrogen flow rate is 50 mL/min; the air flow rate is 400 mL/min;

solution preparation: taking 0.5g of finished ointment, adding 40mL of absolute ethanol, heating and melting in a water bath at 50 ℃, ultrasonically treating for 20min, cooling, refrigerating at 4 ℃ overnight, and filtering to obtain filtrate as a test solution; taking a certain amount of reference substance camphor (batch No. 110747-170502S with the mass fraction of 99.0 percent, provided by Nanjing Dow Biotechnology Limited), and adding absolute ethyl alcohol to prepare 0.5mg/mL solution as a reference substance solution; taking 0.5g of negative preparation without camphor, and treating according to the preparation method of the test solution to serve as a negative control solution;

the method comprises the following operation steps: performing gas chromatography (0521 of general rules of the four parts of the edition of Chinese pharmacopoeia 2015), respectively sucking 1 μ L of the three solutions under the chromatographic conditions, injecting into a gas chromatograph, and allowing the sample solution to have consistent chromatographic peaks at the same retention time positions as those of the reference substance in the chromatogram; the negative preparation has no interference, and the gas chromatogram map is shown in figure 3;

in the experimental process, chromatographic conditions are optimized, different temperature programming modes are examined in sequence and compared, and finally the temperature programming modes are determined, so that the chromatographic peak separation degree is good, and the camphor can be identified.

The detection method of the anti-inflammatory tinea-dampness ointment further comprises the following steps:

the examination should comply with the regulations under ointment:

(1) the phenol content was measured by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition):

chromatographic conditions are as follows: agilent1260 high performance liquid chromatograph, quaternary pump, DAD detector; a chromatographic column: AgilentTC-C ₁₈250X 4.6mm, 5 μm; mobile phase: 62: 38 methanol-water; flow rate: 1.0 mL/min; detection wavelength: 271 nm; column temperature: 45 ℃;

solution preparation: accurately weighing 0.2g of finished ointment, placing into a conical flask with a plug, accurately adding 50mL of absolute ethanol solution, weighing, heating in a water bath at 50 ℃ for 30min, carrying out ultrasonic treatment in a hot water bath for 30min, cooling, weighing again, supplementing the lost weight with the absolute ethanol solution, shaking, refrigerating at 4 ℃ for 2h, and filtering to obtain a filtrate as a test solution; precisely weighing a certain amount of reference phenol (batch No. 100509-170610D, 99.0% by mass, provided by Nanjing Dow Biotechnology Ltd.), and adding mobile phase to obtain 36 μ g/mL solution as reference solution; taking 0.2g of negative preparation containing no phenol, and treating according to the preparation method of the test solution to serve as a negative control solution;

the determination method comprises the following steps: precisely absorbing 5 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, respectively recording chromatograms, and calculating the content of phenol in the sample solution by peak area according to an external standard method; the product contains 30.29-37.02 mg/g of phenol;

in the experiment, the extraction solvent prepared by the sample is examined, methanol, a mobile phase and absolute ethyl alcohol are successively examined as the extraction solvent, and the result shows that the dispersibility is poor when the methanol and the mobile phase are used as the extraction solvent, and the dispersion effect of the absolute ethyl alcohol is good, and finally the absolute ethyl alcohol is determined to be used as the extraction solvent.

In the determination of the phenol content, the following methodological investigations were carried out:

(1-1) specificity investigation: under the chromatographic conditions, phenol and other peaks in the HPLC chromatogram of the sample are well separated, the negative control is not interfered, and the gas chromatogram is shown in figure 4;

(1-2) examination of the Linear relationship: taking a proper amount of phenol reference substance, precisely weighing, adding fluidity to obtain a mixture of 4.51. mu.g/mL, 9.02. mu.g/mL, 18.03. mu.g/mL, 36.06. mu.g/mL, 72.13. mu.g/mL,180.32 μ g/mL and 360.64 μ g/mL, and 5 μ L of each of the phenol control solutions having different concentrations was precisely pipetted to measure the peak area under the above chromatographic conditions. Calculating to obtain a linear regression equation by taking the peak area A as an ordinate and the concentration C of the phenol reference substance as an abscissa: 4.4586X +3.5377, R ²Linear range 0.9990: 4.51-360.64 mu g/mL, the experimental results are shown in the following table 1, and the linear relation curve is shown in fig. 5.

TABLE 1 phenol Linear relationship examination

The experimental result shows that the peak area and the sample injection amount of phenol have good linear relation in the concentration range of 4.51-360.64 mu g/mL.

(1-3) precision test: the same phenol control solution was sampled and measured for 6 times, respectively, and the peak area integral value RSD was 0.16%, and the experimental results are shown in Table 2 below. The result shows that the precision of the detection instrument is good.

Table 2 precision test results (n ═ 6)

(1-4) repeatability test: the same batch of samples were precisely weighed, treated and measured 6 times in the same way, and the RSD of the phenol content was 2.07%, and the experimental results are shown in table 3 below. The results show that the process is very reproducible.

Table 3 repeatability test results (n ═ 6)

(1-5) stability test: the same sample solution is taken and subjected to sample injection measurement for 0h, 2h, 4h, 8h, 12h, 24h and 48h respectively, the peak area integral value RSD of phenol is 0.37%, and the experimental results are shown in the following table 4. The result shows that the test solution is stable within 48h, and the peak area integral value is not obviously changed before and after.

TABLE 4 stability test results

(1-6) recovery test: precisely weighing about 0.1g (the content of phenol is 32.050mg/g) of the same batch of samples with known content, adding 1mL of 3.538mg/mL of phenol reference solution into each batch of samples, preparing according to the preparation method of the test solution, filtering, and taking the subsequent filtrate to obtain the test solution; the sample solutions were each precisely aspirated by 5. mu.L each, and the phenol content was measured under the above-mentioned chromatographic conditions, and the recovery rates were calculated, as shown in Table 5, with an average recovery rate of phenol of 101.00% and an RSD of 0.64%. The results show that the method has good recovery rate.

TABLE 5 recovery test results

(1-7) sample measurement: the 6 batches of samples produced were tested according to the above test methods and conditions, and the results are shown in Table 6 below.

Table 6 measurement of phenol content (n ═ 6)

According to the results of the six samples in Table 6, the product contains phenol within the range of 30.29-37.02 mg/g, which is the qualified standard.

(2) Measuring the content of sublimed sulfur according to high performance liquid chromatography (China pharmacopoeia 2015 edition general rules 0512):

chromatographic conditions are as follows: agilent1260 high performance liquid chromatograph, quaternary pump, DAD detector; a chromatographic column: AgilentTC-C ₁₈250X 4.6mm, 5 μm; mobile phase: methanol; flow rate: 1.0 mL/min; detection wavelength: 240 nm; column temperature: 30 ℃;

solution preparation: accurately weighing 0.8g of finished ointment, placing into a conical flask with a plug, adding 50mL of ethyl acetate, heating in water bath at 60 ℃ for 50min, cooling, transferring into a 100mL volumetric flask, adding ethyl acetate to dilute to constant volume, shaking up, and refrigerating at 4 ℃ for 2 h; precisely weighing 2mL of diluted solution from a 100mL volumetric flask into a 10mL volumetric flask, adding ethyl acetate for diluting to a constant volume, shaking up, and filtering to obtain a filtrate as a test solution; precisely weighing a certain amount of sublimation sulfur (batch No. 7704349-L1609054, mass fraction 99.95%, provided by Shanghai Aladdin Biotechnology Co., Ltd.), and adding ethyl acetate to obtain 40 μ g/mL solution as reference solution; taking 0.8g of negative preparation without sublimed sulfur, and treating according to the preparation method of the test solution to serve as a negative control solution;

the determination method comprises the following steps: precisely absorbing 5 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, respectively recording chromatograms, and calculating the content of sublimed sulfur in the sample solution according to an external standard method by peak area; the product contains 35.35-43.21 mg/g of sublimed sulfur.

In the experimental process, the extraction solvent for preparing the test sample is inspected, chloroform, sodium hydroxide-diluted ethanol and ethyl acetate are sequentially inspected to be used as the extraction solvent, and the result shows that the extraction effect is good and the peak shape is excellent when the ethyl acetate is used as the extraction solvent. The examination of the selection of the mobile phase under the chromatographic condition successively examines isopropanol and methanol, and as a result, the separation effect is better when the methanol is the mobile phase, so the methanol is selected as the mobile phase.

In the determination of the sublimed sulphur content, the following methodological investigations can be carried out:

(2-1) specificity investigation: under the chromatographic conditions, the sublimed sulfur in the HPLC chromatogram of the sample is well separated from other peaks, the negative control is not interfered, and the gas chromatogram is shown in figure 7;

(2-2) examination of the Linear relationship: taking a proper amount of sublimed sulfur reference substances, accurately weighing, adding ethyl acetate to prepare series of sublimed sulfur reference substance solutions of 10.23 mu g/mL, 20.46 mu g/mL, 40.92 mu g/mL, 81.85 mu g/mL, 163.70 mu g/mL and 327.40 mu g/mL, accurately sucking 5 mu L of each sublimed sulfur reference solution with different concentrations, and measuring the peak area according to the chromatographic conditions. Calculating to obtain a regression equation by taking the peak area A as a vertical coordinate and the concentration C of the sublimed sulfur reference substance as a horizontal coordinate: 8.4237X +1.4652, R ²Linear range 0.9999: 10.23-327.40 mu g/mL, the experimental results are shown in the following table 7, and the linear relation curve is shown in fig. 6.

TABLE 7 sublimation sulfur Linear relationship examination

The experimental result shows that the peak area and the sample injection amount show good linear relation when the concentration of the sublimed sulfur is within the range of 10.23-327.40 mu g/mL.

(3-3) precision test: the same sublimed sulfur reference substance solution is taken and subjected to sample injection measurement for 6 times respectively, the peak area integral value RSD is 0.20%, and the experimental results are shown in the following table 8. The result shows that the precision of the detection instrument is good.

Table 8 precision test results (n ═ 6)

(2-4) repeatability test: the same batch of samples were precisely weighed, treated and measured 6 times in the same way, and the RSD of the sublimed sulfur content was 1.01%, and the experimental results are shown in table 9 below. The results show that the process is very reproducible.

Table 9 repeatability test results (n ═ 6)

(2-5) stability test: the same sample solution is taken and subjected to sample injection measurement for 0h, 2h, 4h, 8h, 12h, 24h and 48h respectively, the peak area integral value RSD of the sublimed sulfur is 0.70%, and the experimental results are shown in the following table 10. The result shows that the test solution is stable within 48h, and the peak area integral value is not obviously changed before and after.

TABLE 10 stability test results

(2-6) recovery test: precisely weighing about 0.4g (the content of sublimed sulfur is 39.015mg/g) of the same batch of samples with known content, and 6 parts in total, respectively adding 15mL of sublimed sulfur reference solution with the content of 1.040mg/mL into each batch of samples, preparing according to the preparation method of the test solution, filtering, and taking the subsequent filtrate to obtain the test solution; the sample solutions were each precisely aspirated by 5. mu.L each, the sublimed sulfur content was measured under the above chromatographic conditions, and the recovery rates were calculated, as shown in Table 11, the average recovery rate of sublimed sulfur was 100.37%, and the RSD was 2.63%. The results show that the method has good recovery rate.

TABLE 11 results of recovery test

(2-7) sample measurement: the 6 batches of samples produced were tested according to the above test methods and conditions, and the results are shown in Table 12 below.

TABLE 12 measurement of sublimation sulfur content (n ═ 6)

According to the results of the six samples in Table 6, the product contains sublimed sulfur within the range of 35.35-43.21 mg/g, which is the qualified standard.

To summarize: after the research of analysis methodology verification (specificity, linear relation research, repeatability test, precision test, stability test, recovery rate test and sample measurement) is carried out on the revised method for measuring the content of the phenol and the sublimed sulfur, the method is simple, convenient, sensitive and good in precision, and can be used as a method for measuring the phenol and the sublimed sulfur in the Xiaoyanxuening ointment.