CN112505178A - Method for detecting dissolution rate of Xihuang pills - Google Patents

Abstract

The invention discloses a method for detecting dissolution rate of Xihuang pills. The detection method comprises the steps of preparation of a dissolution medium, preparation of a reference solution, preparation of a test solution, determination of preparation process parameters, formulation of chromatographic conditions, determination of the content of the test solution and the like, wherein bilirubin is exactly related to clinical curative effect, so that bilirubin is selected as an evaluation index to perform dissolution research on the Xihuang pill, the quality and dissolution behavior of a preparation can be objectively and truly reflected, and scientific data are provided for clinical application of the preparation. The invention determines proper dissolution method, rotating speed, volume ratio and the like through the dissolution characteristics of the Xihuang pill, performs methodology verification, has high precision, good reproducibility, good stability and high recovery rate, and provides theoretical basis for further research on pharmacological tests of the Xihuang pill by conjecturing dissolution position and time of the Xihuang pill in vivo through in vitro tests.

Description

Method for detecting dissolution rate of Xihuang pills

Technical Field

The invention relates to the field of medicines, and discloses a method for detecting dissolution rate of Xihuang pills.

Background

Xihuang Wan, also called Xihuang Wan, is originated from Qing, Wang hong Shu (Collection of medical diseases from the whole life) and Juan Si, is a paste pill prepared by crushing 4 traditional Chinese medicines of musk, bezoar, frankincense and myrrh which are processed with vinegar and then adding sticky rice, and has the efficacies of clearing away heat and toxic materials, regulating ying and relieving swelling, and activating blood and dissolving stasis. Can be used for treating carbuncle, cellulitis, furunculosis, scrofula, multiple abscess, and cancer. It is the famous prescription for treating "mammary rock", "scrofula", "phlegmonous nodule" and "pulmonary abscess". The detection contents of the Xihuang pill, which is executed in the 'Chinese pharmacopoeia' 2015 edition at present, mainly comprise characters, identification, dissolution time limit, content measurement and the like. In order to better evaluate the quality of the Xihuang pill, a dissolution rate determination method of the Xihuang pill needs to be established by simulating the dissolution condition of a medicament in a human body in vitro, and data support and theoretical basis are provided for quality control of the Xihuang pill.

Disclosure of Invention

The invention provides a detection method capable of simulating dissolution of a Xihuang pill in a human body in vitro, which solves the problems in the prior art.

The technical scheme adopted by the invention for solving the technical problems is as follows: a method for detecting dissolution rate of Xihuang pills,

a method for detecting dissolution rate of Xihuang pills is characterized by comprising the following steps:

(1) preparing a dissolution medium, namely simulating artificial gastric juice with pH of 1.2, taking 16.4ml of dilute hydrochloric acid, adding about 800ml of water, stirring, adding water and fixing the volume to 1000 ml;

(2) accurately weighing bilirubin reference substance about 10mg, placing in a 50ml measuring flask, adding dichloromethane to dissolve and dilute to scale, and shaking;

(3) taking 0.6-6g of Xihuang pills by a small cup method or a paddle method, placing the Xihuang pills in a dissolution instrument at the rotating speed of 75-125r/min, adding 100ml of hydrochloric acid solution with the pH value of 1.2, namely the volume of the medicine and the solvent is 0.6-6g/100ml, adding 0.1-1ml of Tween-80, namely 0.1-1% of the volume of the solvent, sampling 5ml in 180min, filtering by adopting a 0.45-micron microporous membrane, and simultaneously supplementing the dissolution medium with the same volume of the same temperature;

(4) the measuring device used in the step (3) is a small cup method or a paddle method, preferably a small cup method;

(5) the volume of the medicine and the solvent in the step (3) is 0.6-6g/100ml, and preferably 3g/100 ml;

(6) the dissolving-out speed in the step (3) is 75-125r/min, preferably 100 r/min;

(7) adding 0.1-1% of tween-80 into the dissolution medium in the step (3), preferably 1%;

(8) the dissolution time of the step (3) is 5, 15, 30, 45, 60, 90, 120, 180 and 240min, preferably 180min reaches the maximum dissolution amount;

(9) establishing chromatographic conditions: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-1% acetic acid water (95: 5) is used as a mobile phase; the sample amount is 5-20 mu L; the column temperature is 25-30 ℃; the flow rate is 0.8-1.2 mL/min; the detection wavelength was 450 nm. The number of theoretical plates is not less than 3000 calculated according to bilirubin peak;

(10) the sample injection amount in the step (9) is 5-20 mu L, and preferably 20 mu L;

(11) the column temperature in the step (9) is 25-30 ℃, and 25 ℃ is preferred;

(12) the flow rate in step (9) is 0.8-1.2 mL/min, preferably 1 mL/min.

By adopting the method, the bioavailability of the Xihuang pill in vivo can be predicted through an in vitro simulation experiment, the in vitro dissolution rate and the in vivo bioavailability show positive correlation, and the dissolution rate is high, so that the medicine can be fully absorbed by a human body. Bilirubin is the main effective component of bezoar in the prescription of Xihuang pill, and is exactly related to the clinical curative effect thereof. The dissolution rate research of the Xihuang pill is carried out by selecting bilirubin as an evaluation index, the quality and the dissolution behavior of the preparation can be objectively and truly reflected, and scientific data is provided for the clinical application of the preparation. And the dissolution position and time of the Xihuang pill in vivo are presumed through in vitro tests, so that a theoretical basis can be provided for further researching the pharmacological test of the Xihuang pill.

Drawings

FIG. 1 is a bilirubin profile;

FIG. 2 is a high performance liquid chromatogram of XIHUANG pill;

FIG. 3 is a bilirubin HPLC chromatogram of a sample prepared by the paddle method;

FIG. 4 is a bilirubin HPLC chromatogram of a sample prepared from 0.6g of the test substance;

FIG. 5 is a bilirubin HPLC chromatogram of a sample prepared from 3 g;

FIG. 6 is a bilirubin HPLC chromatogram of a sample prepared from 6 g;

FIG. 7 is a bilirubin HPLC chromatogram of a sample prepared at a rotation speed of 75 r/min;

FIG. 8 is a bilirubin HPLC chromatogram of a sample prepared at a rotation speed of 100 r/min;

FIG. 9 is a bilirubin HPLC chromatogram of a sample prepared at a rotation speed of 125 r/min;

FIG. 10 is a bilirubin HPLC chromatogram of a test sample prepared from 0.5ml of Tween-80;

FIG. 11 is a bilirubin HPLC chromatogram of a sample prepared from 1ml of Tween-80;

FIG. 12 is a graph showing the dissolution profiles of XIHUANG pill in simulated artificial gastric fluid at various time points (bilirubin measured by HPLC is used as an evaluation index);

FIG. 13 is a bilirubin HPLC chromatogram of 5ul sample size;

FIG. 14 is a bilirubin HPLC chromatogram of a sample loading of 10 ul;

FIG. 15 is a bilirubin HPLC chromatogram of a sample loading of 20 ul;

FIG. 16 is a bilirubin HPLC chromatogram at a flow rate of 0.8 mL/min;

FIG. 17 is a bilirubin HPLC chromatogram at a flow rate of 1 mL/min;

FIG. 18 is a bilirubin HPLC chromatogram at a flow rate of 1.2 mL/min;

FIG. 19 shows the precision, reproducibility and stability of bilirubin of Table 1;

FIG. 20 shows the sample recovery of bilirubin of Table 2;

figure 21 is the dissolution data for the ten samples of table 3.

Detailed Description

Embodiment 1

In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings.

Instruments and reagents:

comparison products: bilirubin (batch number: 100077-201808, purity 98.9%) purchased from China institute for testing and testing food and drug;

reagent: acetonitrile is chromatographically pure from OMNI, acetic acid is analytically pure from Tianjin Kemiou chemical reagent, Inc., hydrochloric acid is from refining plant of Laiyang economic development area, dichloromethane and Tween-80 are from Fuyu Fine chemical Co., Ltd, and water is ultrapure water;

and (3) testing the sample: 16 samples of Xihuang pill;

the instrument comprises the following steps: agilent 1260 high performance liquid system.

The method and the result are as follows:

2.1 preparation of control solutions: precisely weighing about 10mg of bilirubin reference substance, placing in a 50ml measuring flask, adding dichloromethane to dissolve and dilute to scale, and shaking.

Preparation of a test solution: taking 3g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 100r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling for 180 minutes for 5ml, and filtering with a 0.45um microporous membrane.

Establishing chromatographic conditions:

a chromatographic column: agilent ZORBAX SB C18 (250 mm. times.4.6 mm, 5 μm);

mobile phase: acetonitrile-1% acetic acid water (95: 5) as mobile phase; the sample amount is 20 mu L, the column temperature is 25 ℃, the flow rate is 1.0 mL/min, and the detection wavelength is 450 nm.

Linear range investigation of controls:

precisely weighing a proper amount of bilirubin, adding dichloromethane to prepare mother liquor, precisely diluting the mother liquor into 5 solutions containing 0.626, 1.252, 2.53, 5.06 and 10.12ug/ml bilirubin in each 1ml, respectively, measuring according to 2.3 chromatographic conditions, and drawing a standard curve by taking the concentration as an abscissa and the corresponding peak area as an ordinate to obtain a regression equation y =41.994x-2.6275 (R2 = 0.9994). It was shown that bilirubin showed a good linear relationship with peak area in the range of 0.0626-10.012ug/ml, as shown in FIG. 1.

And (3) special investigation:

preparation of negative sample: grinding artificial Moschus, sieving with 100 mesh sieve, steaming appropriate amount of semen Panici Miliacei, oven drying, pulverizing with vinegar Olibanum and vinegar Myrrha into fine powder, sieving with 80 mesh sieve, mixing with artificial Moschus powder, grinding, sieving, mixing, removing embryo, making pill, and drying in the shade to obtain test solution according to 2.2.

Taking appropriate amount of the sample solution, bilirubin reference solution, blank solution and negative sample solution under item 2.2, detecting according to the method under 2.3 chromatographic conditions, and recording chromatogram, as shown in figure 2. The result shows that under the chromatographic condition, only one peak of the sample corresponds to the retention time of the peak of the reference substance, and the negative sample has no peak at the retention time position corresponding to the absorption peak of the bilirubin reference substance, which indicates that other raw materials and auxiliary materials have no influence on the measurement of the sample.

Precision test

Taking a sample solution, continuously injecting a sample for 6 needles according to a method under the 2.3 chromatographic condition, recording the peak area, and showing the result in table 1 that the RSD of the bilirubin peak area is 1.17% (n = 6), which indicates that the precision of the method is good.

Repeatability test

6 test samples were prepared in parallel according to the method of 2.2, and the peak areas were measured, and the results are shown in Table 1.

Stability test

The same sample solution was taken for 0h, 2h, 4h, 8h and 12h for analysis, and the peak areas were recorded, with the results shown in Table 1.

2.9 sample recovery

Accurately weighing 6 parts of the Xihuang pill sample, respectively adding a certain amount of reference substances, preparing the sample according to the method for treating the test solution, extracting a proper amount of dichloromethane, measuring, and calculating the sample adding recovery rate. The results are shown in Table 2.

Dissolution rate measurement of samples of different batches

Taking 10 batches of Xihuang pill samples, taking a PH1.2 solution as a dissolution medium, carrying out a dissolution test at a concentration of 3g/100ml by a small cup method and a rotating speed of 100r/min, sampling at a time point of 180min, and measuring according to a method of 2.3. The average dissolution rates of the ten batches are shown in table 3.

Conclusion

The hydrochloric acid solution with the pH value of 1.2 selected in the experiment simulates the environment of artificial gastric juice, the dissolution quality of the Xihuang pill is high under the condition, the dissolution amount at each time point is shown in figure 3, and the research of methodology shows that the precision, the repeatability, the stability, the recovery rate test and the negative sample test all accord with the related requirements of the known dissolution rate principle of Chinese pharmacopoeia 2015 edition and oral solid preparations; and ten batches of samples are tested to show that the method is simple, accurate and feasible. The method can be used for quality control of the Xihuang pill preparation, and also provides data support for establishment of dissolution rate methodologies of other pills.

Example II

Preparing a test sample: taking 6g of the product by a paddle method, placing the product in a dissolution instrument at the rotating speed of 100r/min, adding 500 ml of hydrochloric acid solution with the pH value of 1.2 and 5ml of Tween-80, sampling 5ml in 180 minutes, and filtering by using a 0.45um microporous filter membrane to obtain the product. The measurement was carried out in accordance with example one, and the results are shown in FIG. 3. Compared with the paddle method, the small cup method is more suitable in the aspects of cost, accurate result of measured data and the like.

Example three

Preparing a test sample: taking 0.6g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 100r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling 5ml in 180 minutes, and filtering with a 0.45um microporous membrane to obtain the final product. The measurement was carried out in accordance with example one, and the results are shown in FIG. 4.

Preparing a test sample: taking 3g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 100r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling for 180 minutes for 5ml, and filtering with a 0.45um microporous membrane. The measurement was carried out in accordance with example one, and the results are shown in FIG. 5.

Preparing a test sample: taking 6g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 100r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling 5ml in 180 minutes, and filtering with a 0.45um microporous membrane to obtain the final product. The measurement was carried out in accordance with example one, and the results are shown in FIG. 6.

Compared with three different medicine quantities, 3g is optimal in terms of operation difficulty and data accuracy.

Example four

Preparing a test sample: taking 3g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 75r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling for 180 minutes for 5ml, and filtering with a 0.45um microporous membrane. The measurement was carried out in accordance with example one, and the results are shown in FIG. 7.

Preparing a test sample: taking 3g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 100r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling for 180 minutes for 5ml, and filtering with a 0.45um microporous membrane. The measurement was carried out in accordance with example one, and the results are shown in FIG. 8.

Preparing a test sample: taking 3g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 125r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling for 180 minutes for 5ml, and filtering with a 0.45um microporous membrane. The measurement was carried out in accordance with example one, and the results are shown in FIG. 9.

Compared with the three rotating speeds, the dissolving effect of 75r/min is slightly poor, the difference between 100r/min and 125r/min is almost zero, and the 100r/min is considered to be optimal comprehensively.

Example five

Preparing a test sample: taking 3g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 100r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 0.5ml of Tween-80, sampling 5ml in 180 minutes, and filtering with a 0.45um microporous membrane to obtain the product. The measurement was carried out in accordance with example one, and the results are shown in FIG. 10.

Preparing a test sample: taking 3g of the product by a small cup method, placing in a dissolution instrument at a rotation speed of 100r/min, adding 100ml of hydrochloric acid solution with pH of 1.2 and 1ml of Tween-80, sampling for 180 minutes for 5ml, and filtering with a 0.45um microporous membrane. The measurement was carried out in accordance with example one, and the results are shown in FIG. 11.

Compared with the sample loading amounts of the two kinds of Tween-80, the sample loading amounts are not very different from each other in terms of results, and the effect is better when the ratio of Tween to the solvent is 1% in terms of the dissolution state in the experimental process.

Example six

Preparing a test sample: taking 3g of the product by a small cup method, placing the product in a dissolution instrument at the rotating speed of 100r/min, adding 100ml of hydrochloric acid solution with the pH value of 1.2 and 1ml of Tween-80, respectively sampling 5ml in 5, 15, 30, 45, 60, 90, 120, 180 and 240 minutes, and filtering by adopting a 0.45-micrometer microporous filter membrane to obtain the product. The measurement was carried out in the same manner as in the first embodiment, and the peak areas measured were plotted, and the results are shown in FIG. 12.

When the time reaches 180min, the dissolution rate of the Xihuang pills reaches the maximum, and then the platform period is entered. Therefore, 180min was selected as the maximum amount of dissolution.

Example seven

Preparation of test sample according to embodiment one, the measurement method changes the sample amount to 5, 10, 20 μ L, and records chromatogram respectively, as shown in FIGS. 13, 14, 15.

When the sample amount is 5, 10 and 20 mu L, the peak area of the sample can be measured, but when the sample amount is 20 mu L, the response value is higher, the signal-to-noise ratio is higher, and the data is more accurate.

Eight implementation cases

According to the first embodiment, the column oven is changed to 25 deg.C and 30 deg.C, and the chromatogram is recorded. As a result, the difference between the two column chambers is small, and 25 ℃ is preferable from the viewpoint of safety.

Example nine

According to the first embodiment, the flow rate of the test sample is changed to 0.8, 1 and 1.2 mL/min by the determination method, and chromatograms are recorded respectively. See fig. 16, 17, 18. As a result: 1.2 mL/min is the earliest peak, 0.8 mL/min is the latest peak, 1 mL/min is in the middle, and 1 mL/min is preferred in the aspects of comprehensive cost and system applicability.

The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.

The present invention is not described in detail, but is known to those skilled in the art.

Claims (9)

1. A method for detecting dissolution rate of Xihuang pills is characterized by comprising the following steps:

(1) taking 0.6-6g of Xihuang pills by a small cup method or a paddle method, placing the Xihuang pills in a dissolution instrument at the rotating speed of 75-125r/min, adding 100ml of hydrochloric acid solution with the pH value of 1.2, namely the volume of the medicine and the solvent is 0.6-6g/100ml, adding 0.1-1ml of Tween-80, namely 0.1-1% of the volume of the solvent, sampling 5ml in 180min, filtering by adopting a 0.45-micron microporous membrane, and simultaneously supplementing the dissolution medium with the same volume of the same temperature;

(2) establishing chromatographic conditions: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-1% acetic acid water (95: 5) is used as a mobile phase; the sample amount is 5-20 mu L; the column temperature is 25-30 ℃; the flow rate is 0.8-1.2 mL/min; the detection wavelength is 450 nm; the number of theoretical plates is not less than 3000 calculated according to bilirubin peak.

2. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: the measuring device used in the step (1) is a small cup method or a paddle method, and preferably a small cup method.

3. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: the volume of the medicament and the solvent in the step (1) is 0.6-6g/100ml, and 3g/100ml is preferred.

4. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: the rotation speed of the dissolution in the step (1) is 75-125r/min, preferably 100 r/min.

5. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: adding 0.1-1% of Tween-80 into the dissolution medium in the step (1), and preferably 1%.

6. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: the dissolution time of the step (1) is 5, 15, 30, 45, 60, 90, 120, 180 and 240min, preferably 180min to reach the maximum dissolution amount.

7. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: the sample injection amount in the step (2) is 5-20 mu L, and preferably 20 mu L.

8. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: the column temperature in the step (2) is 25-30 ℃, and preferably 25 ℃.

9. The method for detecting dissolution rate of XIHUANG pill according to claim 1, wherein: the flow rate in step (2) is 0.8-1.2 mL/min, preferably 1 mL/min.

Images