阅读说明：本技术 小儿清热宣肺贴膏中栀子苷和苦杏仁苷的同时测定方法 (Method for simultaneously measuring geniposide and amygdalin in children heat-clearing lung-ventilating plaster ) 是由 王保安 李兴科 于 2020-12-01 设计创作，主要内容包括：本发明公开了一种同时测定小儿清热宣肺贴膏中栀子苷和苦杏仁苷的方法。所公开的方法包括：将待测小儿清热宣肺贴膏用甲醇超声浸泡、过滤,所得滤液为待测样；以对照样的液相色谱测定结果为参照,采用液相色谱测定所述待测样中栀子苷和苦杏仁苷的含量,对照样为合理浓度的栀子苷和苦杏仁苷甲醇溶液；测定条件为：填充剂为十八烷基硅烷键合硅胶、检测波长为207nm、柱温25℃±1、流动相为流动相A和流动相B的混合液,流动相A为乙腈或甲醇,流动相B为磷酸溶液或甲酸溶液。本发明采用合适条件对不同化合物的吸附能力不同以达到对小儿宣清热肺贴膏中两种有效成分栀子苷和苦杏仁苷的分离,实现一次测定完成两种有效成分的定性和定量分析。(The invention discloses a method for simultaneously measuring geniposide and amygdalin in a plaster for clearing heat and ventilating lung of children. The disclosed method comprises: ultrasonically soaking the heat-clearing lung-ventilating plaster for the child to be tested by using methanol, and filtering to obtain filtrate as a sample to be tested; taking the liquid chromatography measurement result of a reference sample as a reference, and measuring the contents of the geniposide and the amygdalin in the sample to be measured by adopting liquid chromatography, wherein the reference sample is a methanol solution of the geniposide and the amygdalin with reasonable concentrations; the measurement conditions were: the filler is octadecylsilane chemically bonded silica, the detection wavelength is 207nm, the column temperature is 25 +/-1 ℃, the mobile phase is a mixed solution of a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile or methanol, and the mobile phase B is a phosphoric acid solution or a formic acid solution. The invention adopts different adsorption capacities to different compounds under proper conditions to separate the jasminoidin and the amygdalin which are two effective components in the plaster for dispersing and clearing heat in children, and realizes one-time determination to finish qualitative and quantitative analysis of the two effective components.)

1. A method for simultaneously measuring geniposide and amygdalin in a plaster for clearing heat and ventilating the lung of an infant is characterized by comprising the following steps:

sample extraction: ultrasonically soaking the heat-clearing lung-ventilating plaster for the child to be tested by using methanol, and filtering to obtain filtrate as a sample to be tested;

taking the liquid chromatography measurement result of a reference sample as a reference, and measuring the contents of the geniposide and the amygdalin in the sample to be measured by adopting liquid chromatography, wherein the reference sample is a methanol solution of the geniposide and the amygdalin with reasonable concentrations; the determination conditions of the liquid chromatogram are as follows: the filler is octadecylsilane chemically bonded silica, the detection wavelength is 207nm, the column temperature is 25 +/-1 ℃, the mobile phase is a mixed solution of a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile or methanol, and the mobile phase B is a phosphoric acid solution with the mass concentration of 0.1-0.5% or a formic acid solution with the mass concentration of 0.5-1.0%.

2. The method for simultaneously measuring geniposide and amygdalin in the children's heat clearing and lung ventilating plaster according to claim 1, wherein the mixing volume ratio of the mobile phase A to the mobile phase B is (7-15): 85-93.

3. The method for simultaneously measuring geniposide and amygdalin in the children's heat-clearing lung-ventilating plaster according to claim 1, wherein the column temperature is 25 ℃.

4. The method for simultaneously measuring geniposide and amygdalin in the infantile heat-clearing lung-ventilating plaster as claimed in claim 1, wherein the reasonable concentrations of the control sample are as follows: geniposide 16 μ g/ml; amygdalin 20 μ g/ml.

5. The method for simultaneously measuring geniposide and amygdalin in the infantile heat-clearing lung-ventilating plaster as claimed in claim 1, wherein the sample extraction comprises: removing the lining of the medicine, cutting, weighing, and cutting into pieces; then ultrasonically soaking the crushed sample by using quantitative methanol for a reasonable time; weighing, and then supplementing methanol to the weight before soaking; then evenly mixing and filtering, and taking a proper amount of filtrate to dilute with methanol to obtain a sample to be detected.

Technical Field

The invention relates to a method for measuring effective components in a plaster for clearing heat and ventilating the lung, in particular to a method for simultaneously measuring two effective components of geniposide and amygdalin in the plaster for clearing heat and ventilating the lung by adopting an HPLC method.

Background

The infantile heat-clearing lung-ventilating plaster is a commonly used pediatric medicament and is used for relieving cough and expectoration caused by infantile acute bronchitis, or symptoms such as nasal obstruction, watery nasal discharge, low fever and the like. The prescription consists of four medicinal materials of raw gardenia, bitter almond, safflower and peach kernel. A large number of researches show that the geniposide in raw gardenia and the amygdalin in bitter almond are two representative effective components.

At present, the qualitative detection of two effective components is mainly carried out by adopting a thin-layer chromatography; or qualitatively and quantitatively detecting one of the two effective components by HPLC. The two methods can not simultaneously carry out qualitative and quantitative analysis on the two effective components, and do not well control the product quality of the infantile heat-clearing lung-ventilating plaster.

Disclosure of Invention

Aiming at the defects or shortcomings of the prior art, the invention aims to provide a method for simultaneously measuring geniposide and amygdalin in a plaster for clearing heat and ventilating the lung for children.

Therefore, the method for simultaneously measuring the geniposide and the amygdalin in the heat-clearing lung-ventilating plaster for the children, which is provided by the invention, comprises the following steps:

sample extraction: ultrasonically soaking the heat-clearing lung-ventilating plaster for the child to be tested by using methanol, and filtering to obtain filtrate as a sample to be tested;

taking the liquid chromatography measurement result of a reference sample as a reference, and measuring the contents of the geniposide and the amygdalin in the sample to be measured by adopting liquid chromatography, wherein the reference sample is a methanol solution of the geniposide and the amygdalin with reasonable concentrations; the liquid chromatography determination conditions are as follows: the filler is octadecylsilane chemically bonded silica, the detection wavelength is 207nm, the column temperature is 25 +/-1 ℃, the mobile phase is a mixed solution of a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile or methanol, and the mobile phase B is a phosphoric acid solution with the mass concentration of 0.1-0.5% or a formic acid solution with the mass concentration of 0.5-1.0%. Wherein the column temperature is preferably 25 ℃.

Further, the mixing volume ratio of the mobile phase A to the mobile phase B is (7-15): 85-93.

Further, the reasonable concentrations of the control were: geniposide 16 μ g/ml; amygdalin 20 μ g/ml.

Further, the sample extraction comprises: removing the lining of the medicine, cutting, weighing, and cutting into pieces; then ultrasonically soaking the crushed sample by using quantitative methanol for a reasonable time; weighing, and then supplementing methanol to the weight before soaking; then evenly mixing and filtering, and taking a proper amount of filtrate to dilute with methanol to obtain a sample to be detected.

Compared with the prior art, the invention adopts different adsorption capacities to different compounds under proper conditions to separate the two effective components of the geniposide and the amygdalin in the plaster for dispersing and clearing heat in children, and realizes one-time determination to finish qualitative and quantitative analysis of the two effective components. The determination method can well control the quality of the 'infantile heat clearing and lung ventilating plaster' and achieve the maximization of the cost-benefit ratio.

Drawings

Fig. 1 is a liquid chromatogram of the plaster for clearing heat and ventilating the lung for children of example 1, in which the two effective components are completely separated, and the abscissa is time and unit: and (5) min.

Detailed Description

Unless otherwise indicated, the terms herein are to be understood in accordance with the conventional knowledge of those skilled in the art.

Based on the conception and the purpose of the invention, the volume ratio of the mobile phase A to the mobile phase B, the reasonable concentration of the control sample, the initial weighing and quantification of the medicine, the using amount of methanol during soaking, the sampling amount of the filtrate and the dilution concentration in the method can be determined according to the specific medicine by adopting the conventional experimental method in the field.

To ensure that the disclosure is complete, the following detailed description is given in conjunction with the specific drawings and embodiments, the examples described in this specification are intended to illustrate and not limit the invention.

The geniposide and amygdalin used in the following examples were purchased from Dorpus biological technologies, Inc., and the batches were 115810-12-3 and 115810-13-4, respectively, and the purities were all greater than 98%. The plaster (to-be-tested) for clearing heat and ventilating lung of the children to be tested is a product (batch No. 20190501) of Shanxi Momei Qi-blood and pharmacy Co.

Example 1:

in the embodiment, HPLC method is adopted to simultaneously determine the contents of two effective components of geniposide and amygdalin in the plaster for clearing heat and ventilating lung of children:

accurately weighing 16.2mg of jasminoidin reference substance and 20.4mg of amygdalin reference substance, placing in a 100ml measuring flask, dissolving with methanol and diluting to scale, accurately sucking 0.5ml, 0.8ml, 1.0ml, 1.2ml and 1.5ml, respectively placing in a 10ml measuring flask, adding methanol to dilute to scale, and shaking;

treating a test sample: taking the product, removing a cover liner, cutting into 6cm in diameter, precisely weighing, shearing, placing in a conical flask with a plug, precisely adding 100ml of methanol, sealing the plug, weighing, performing ultrasonic treatment (power 250W and frequency 50kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, precisely measuring the subsequent filtrate 5ml, placing in a 50ml measuring flask, adding 50% methanol to dilute to scale, shaking up, filtering, and taking the subsequent filtrate;

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, and injecting into a liquid chromatograph with octadecylsilane chemically bonded silica as filler; acetonitrile is used as a mobile phase A, phosphoric acid solution with the mass concentration of 0.1% is used as a mobile phase B, and the ratio of the mobile phase A to the mobile phase B is 7: 93 mixing and filtering to obtain a mobile phase; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak;

recording chromatogram of the reference substance, and performing linear regression on peak area by concentration, wherein the regression equation of geniposide is 2E +06x +1416.1, R2 is 1.0, the regression equation of amygdalin is 2E +06x +60.9, and R2 is 1.0; the result shows that the geniposide is 0.1021-0.3062 mug, the amygdalin is 0.0811-0.2433 mug, the sampling amount and the peak area value have good linear relation;

the test results are shown in FIG. 1.

Example 2:

this example differs from example 1 in that:

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, and injecting into a liquid chromatograph with octadecylsilane chemically bonded silica as filler; methanol is used as a mobile phase A, phosphoric acid solution with the mass concentration of 0.1% is used as a mobile phase B, and the ratio of the mobile phase A to the mobile phase B is 15: 85 as mobile phase after mixing and filtering; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak.

Example 3:

this example differs from example 1 in that:

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, injecting into liquid chromatograph, and measuring with octadecylsilane chemically bonded silica as filler; acetonitrile is used as a mobile phase A, formic acid solution with the mass concentration of 0.5% is used as a mobile phase B, and the ratio of the mobile phase A to the mobile phase B is as follows: 92 mixing and filtering to obtain a mobile phase; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak.

Example 4:

this example differs from example 1 in that:

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, injecting into a liquid chromatograph, and measuring with octadecylsilane chemically bonded silica as filler; methanol is used as a mobile phase A, a formic acid solution with the mass concentration of 0.5% is used as a mobile phase B, and the mobile phase A and the mobile phase B are mixed according to the weight ratio of 15: 85 as mobile phase after mixing and filtering; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak.

Example 5:

this example differs from example 1 in that:

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, and injecting into a liquid chromatograph with octadecylsilane chemically bonded silica as filler; acetonitrile is used as a mobile phase A, phosphoric acid solution with the mass concentration of 0.3% is used as a mobile phase B, and the ratio of the mobile phase A to the mobile phase B is as follows, wherein the ratio of the mobile phase A to the mobile phase B is 8: 92 mixing and filtering to obtain a mobile phase; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak.

Example 6:

this example differs from example 1 in that:

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, injecting into a liquid chromatograph, and measuring with octadecylsilane chemically bonded silica as filler; methanol is used as a mobile phase A, phosphoric acid solution with the mass concentration of 0.5% is used as a mobile phase B, and the mobile phase A and the mobile phase B are mixed according to the weight ratio of 12: 88 mixing and filtering to obtain a mobile phase; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak.

Example 7:

this example differs from example 1 in that:

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, and injecting into a liquid chromatograph with octadecylsilane chemically bonded silica as filler; acetonitrile is used as a mobile phase A, a formic acid solution with the mass concentration of 0.8% is used as a mobile phase B, and the mobile phase A and the mobile phase B are mixed according to the weight ratio of 9: 91 mixing and filtering to obtain a mobile phase; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak.

Example 8:

this example differs from example 1 in that:

and (3) determination: respectively and precisely sucking 10 μ l of reference solution and sample solution, respectively, injecting into a liquid chromatograph, and measuring with octadecylsilane chemically bonded silica as filler; methanol is used as a mobile phase A, a phosphoric acid solution with the mass concentration of 1.0% is used as a mobile phase B, and the ratio of the mobile phase A to the mobile phase B is 10: 90 mixing and filtering to obtain a mobile phase; the detection wavelength is 207 nm; column temperature: 25 ℃; the number of theoretical plates is not less than 3000 calculated according to geniposide peak and amygdalin peak.

Example 9:

taking appropriate amount of jasminoidin reference substance and amygdalin reference substance, precisely weighing, and adding methanol to obtain a mixed solution containing 16 μ g of jasminoidin and 20 μ g of amygdalin per 1ml as reference substance solution;

taking a drug to be tested, removing a cover liner, cutting the drug to be tested into a diameter of 6cm, precisely weighing, shearing, placing the drug in a conical flask with a plug, precisely adding 100ml of methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 50kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, precisely measuring 5ml of subsequent filtrate, placing the subsequent filtrate in a 50ml measuring flask, adding 50% methanol to dilute to a scale, shaking up, filtering, and taking the subsequent filtrate as a sample solution;

preparing negative samples of the unprocessed gardenia and the bitter almond according to the preparation process of the infant lung-ventilating plaster, and obtaining a negative control solution by a preparation method of 2 pieces of test sample solution;

the control solution, the test solution and the negative control solution were each precisely aspirated by 10. mu.l each, and the solutions were injected into a liquid chromatograph, and the measurement was performed by the method described in example 1.

The result shows that the retention time of the geniposide chromatographic peak in the HPLC chromatogram of the test solution is about 31.213min, the retention time of the amygdalin chromatographic peak is 21.394min (refer to figure 1), and the separation between the geniposide chromatographic peak and the amygdalin chromatographic peak and the adjacent chromatographic peaks is good; the peak shapes of the geniposide chromatographic peak and the amygdalin chromatographic peak are good; in the negative control solution chromatogram, no chromatographic peak is found at the position corresponding to the geniposide chromatographic peak and the amygdalin chromatographic peak, and the negative control solution chromatogram is free of interference. Therefore, the method for simultaneously measuring the two effective components of the geniposide and the amygdalin in the infant lung-ventilating plaster by adopting the HPLC method is feasible.

Example 10:

precisely sucking 10 μ l of the control solution of example 9, injecting into a liquid chromatograph, continuously injecting for 6 times, and recording chromatogram.

TABLE 1 sample introduction precision test data

The results shown in Table 1 show that the chromatographic conditions of the area RSD of the geniposide peak is 0.72 percent and the area RSD of the amygdalin peak is 1.05 percent, and the precision of experimental sample injection meets the specification.

Example 11:

precisely sucking 10 μ l of the sample solution of example 9, injecting into a liquid chromatograph, injecting 10 μ l of the sample solution every 2 hours, measuring 7 times, namely injecting the sample solution for 0, 2, 4, 6, 8, 10 and 12 hours, calculating relative standard deviation by peak area, and inspecting the stability of the sample solution.

Table 2 solution stability test data

The results shown in table 2 illustrate that: the average value of the peak area of 7 times of determination of the geniposide is 402065.1, and the RSD is 0.82%; the average value of the peak area of amygdalin in 7 times of measurement is 384047.0, and the RSD is 0.94%; the test solution showed good stability within 12 hours.

Example 12:

taking a medicine with a known content, removing a cover liner, cutting into 2.42cm in diameter, precisely weighing, shearing, placing in a conical flask with a plug, taking 9 parts in total, precisely adding 100ml of methanol, sealing the plug, weighing, ultrasonically treating (with the power of 250W and the frequency of 50kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, and placing in a 50ml measuring flask;

taking 3 parts as a group, respectively weighing 80%, 100% and 120% of the content of the geniposide and the amygdalin in the sample, precisely adding a mixed reference substance, adding 50% methanol for dilution to scale, shaking up, filtering, taking a subsequent filtrate, testing according to the chromatographic conditions, and calculating the recovery rate according to the following formula, wherein the results are shown in tables 3 and 4. As a result, the average recovery was 99.52% and the RSD was 1.74%.

Recovery (%) — measured amount-content in sample ÷ added amount × 100.

TABLE 3 geniposide recovery test data

TABLE 4 amygdalin recovery test data

The results in tables 3 and 4 show that the drug is processed according to the preparation method and measured according to the chromatographic conditions, the recovery rate is high, and the detection method is accurate and reliable.