A method for detecting multi-component content of a cold heat-clearing preparation

Abstract

The application discloses a multi-component content detection method for a cold heat-clearing preparation, and comprises the following steps: setting chromatographic conditions, detecting a wavelength of 250 nm, setting a column temperature to 30 DEG C, taking C18 as a chromatographic column, taking a methanol (A)-water (B) system as a mobile phase system, gradient elution, and setting a program as follows: 0-10 min, 5%-20% A; 10-40 min, 20%-30% A; 40-50 min, 30%-80% A; 50-65 min, 80%-95% A; respectively sucking test sample solutions and mixed control samples for testing, and calculating 3'-hydroxymethyl puerarin, puerarin, 3'-methoxymethyl puerarin and apiosyl puerarin glycoside contents of the cold heat-clearing granules according to a chromatogram. The cold heat-clearing preparation component content is simultaneously detected by the application, the result is accurate, comprehensive and stable, and the application is beneficial to comprehensively and rapidly checking the product quality.

Description

Technical fields:

[0001] This invention relates to a method for detecting the content of multiple components in a cold and fever-relieving preparation. Background technology:

[0002] Cold and Fever Relief Granules are composed of eleven traditional Chinese medicines, including Schizonepeta tenuifolia, Mentha haplocalyx, Saposhnikovia divaricata, Bupleurum chinense, Perilla frutescens, Pueraria lobata, Platycodon grandiflorus, Prunus armeniaca, Angelica dahurica, Viola yedoensis, and Phragmites communis. They have the effects of dispelling wind and cold, relieving exterior symptoms, and clearing heat. The 2015 edition of the Chinese Pharmacopoeia only uses puerarin as an indicator component for content determination. However, this product contains many herbs and has complex components. The combined effect of these multiple components reflects the overall efficacy of the compound preparation. Therefore, the determination of the content of multiple components and their comprehensive evaluation are of great significance for the quality control of Cold and Fever Relief Granules. Summary of the Invention:

[0003] The purpose of this invention is to provide a method for detecting the content of multiple components in a cold and fever-relieving preparation.

[0004] This invention is achieved through the following technical solutions:

[0005] A method for detecting the content of multiple components in a cold and fever-relieving preparation includes the following steps:

[0006] (1) Preparation of mixed reference standard: Take appropriate amounts of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin and apigenin puerarin glycoside, accurately weigh them, add 50wt% methanol to prepare a mixed reference standard solution containing 1.06 mg / ml of 3'-hydroxypuerarin, 1.00 mg / ml of puerarin, 0.52 mg / ml of 3'-methoxypuerarin and 0.51 mg / ml of apigenin puerarin glycoside, and filter through a microporous membrane to obtain the solution;

[0007] (2) Preparation of test solution: Take 1g of cold and fever clearing granules, weigh accurately, place in a stoppered conical flask, add 50ml of distilled water accurately, stopper tightly, weigh, sonicate, cool, weigh again, add methanol to make up the weight, shake well, filter through a microporous membrane to obtain the solution.

[0008] (3) Chromatographic conditions: The detection wavelength was 250 nm, the column temperature was 30 °C, a C18 column was used, the mobile phase was methanol (A)-water (B), and gradient elution was performed. The program was as follows: 0-10 min, 5%-20% A; 10-40 min, 20%-30% A; 40-50 min, 30%-80% A; 50-65 min, 80%-95% A.

[0009] (4) Take the test solution and the mixed reference standard separately for testing, and calculate the contents of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin and apigenin puerarin glycoside in cold and fever relief granules according to the chromatogram.

[0010] The beneficial effects of this invention are as follows:

[0011] 1) This invention simultaneously measures the content of multiple indicators in cold and fever-reducing preparations, resulting in accurate, comprehensive, and stable results, which is beneficial for comprehensive and rapid quality control of products. Attached image description:

[0012] Figure 1 This is a comparison chart of chromatographic results of test sample solutions prepared with different solvents;

[0013] In this composition, A is water, B is 50 wt% methanol, and C is pure methanol.

[0014] Figure 2 This is a comparison of chromatographic results eluted with different mobile phases;

[0015] Where A is methanol-water and B is acetonitrile-water.

[0016] Figure 3 This is a comparison of chromatographic results for different elution gradients using methanol-water as the mobile phase;

[0017] Where A is gradient 1, B is gradient 2, C is gradient 3, and D is gradient 4.

[0018] Figure 4 This is the HPLC chromatogram of the mixed reference standard (A) and the cold and fever relief granules test sample (B).

[0019] Wherein 1 is 3'-hydroxypuerarin, 2 is puerarin, 3 is 3'-methoxypuerarin, and 4 is apigenin puerarin glycoside. Detailed implementation method:

[0020] The following is a further description of the invention, but not a limitation thereof.

[0021] Example 1: A method for detecting the content of multiple components in a cold and fever-reducing preparation.

[0022] Experimental materials:

[0023] Waters 2695 high-performance liquid chromatograph, Waters 2996 detector, and Empower 3 workstation (Waters Corporation, USA); CP225D 0.0001 g electronic analytical balance (Sartorius Scientific Instruments Co., Ltd.); KQ-600VDB dual-frequency digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.). Cold and fever-reducing granules (commercially available; sample information is shown in Table 1, where S1-S10 are samples from different batches of the same manufacturer, and the rest are samples from different manufacturers). 3'-Hydroxypuerarin (batch number 19082021), 3'-methoxypuerarin (batch number 19081921), and apigenin puerarin glycoside (batch number 19061221) were all purchased from Shanghai Tongtian Biotechnology Co., Ltd.; puerarin (batch number 110752-201816) was purchased from the China National Institutes for Food and Drug Control. Methanol (chromatographic grade, Merck, Germany), distilled water, and other reagents were of analytical grade.

[0024] Table 1. Source Information of 29 Batches of Cold and Fever Relief Granules

[0025]

[0026]

[0027] Includes the following steps:

[0028] 1) Preparation of mixed reference standard: Weigh 1 mg each of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin and apigenin puerarin glycoside, add 50 wt% methanol to prepare a mixed reference standard solution containing 1.06 mg / ml of 3'-hydroxypuerarin, 1.00 mg / ml of puerarin, 0.52 mg / ml of 3'-methoxypuerarin and 0.51 mg / ml of apigenin puerarin glycoside, filter through a 0.22 μm microporous membrane, and store at 4℃ for later use;

[0029] 2) Preparation of the test solution: Take 1g of cold and fever clearing granules, accurately weigh them, place them in a stoppered conical flask, accurately add 50ml of distilled water, seal tightly, weigh, sonicate, cool, weigh again, make up the weight with methanol, shake well, filter through a microporous membrane, and the solution is ready.

[0030] For the preparation of the test solution, distilled water, 50 wt% methanol, and pure methanol were compared as solvents. The results showed that distilled water as the solvent extracted more chromatographic information and had better peak shapes (see...). Figure 1 Therefore, distilled water was chosen as the extraction solvent.

[0031] 3) Chromatographic conditions: The detection wavelength was 250 nm, the column temperature was 30 °C, a Waters Bridge C18 (4.6 mm × 150 mm, 5 μm) column was used, the flow rate was 1.0 ml / min, the injection volume was 10 μl, and the mobile phase system was methanol (A)-water (B) with gradient elution. The program was as follows: 0–10 min, 5%–20% A; 10–40 min, 20%–30% A; 40–50 min, 30%–80% A; 50–65 min, 80%–95% A.

[0032] Regarding the choice of mobile phase, the effects of methanol-water and acetonitrile-water systems on the results were investigated, and it was found that the methanol-water system performed better as a mobile phase (see...). Figure 2 The experiment used methanol (A)-water (B) as the mobile phase and compared the effects of different gradient conditions on the detection results. Gradient 1: 0-10 min, 20%-40% A; 10-15 min, 40%-75% A; 15-20 min, 75%-65% A; 20-30 min, 65%-20% A. Gradient 2: 0-15 min, 5%-25% A; 15-55 min, 25%-70% A; 55-75 min, 70%-90% A. Gradient 3: 0-10 min, 5%-25% A; 10-25 min, 25%-40% A; 25-30 min, 40%-70% A; 30-40 min, 70%-95% A; 40-45 min, 95%-95% A. Gradient 4 was set at 5%-20% A for 0-10 min; 20%-30% A for 10-40 min; 30%-80% A for 40-50 min; and 80%-95% A for 50-65 min. Results (see...) Figure 3 The chromatogram obtained by gradient 4 shows good separation and suitable retention time, so gradient 4 was selected as the chromatographic condition for the detection of cold and fever relieving granules.

[0033] 4) Test the sample solution and the mixed reference standard separately. The chromatogram is shown in Figure 4. The theoretical plate number, calculated based on 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, and apigenin puerarin glycoside, is greater than 6000. Calculate the content of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, and apigenin puerarin glycoside in the cold and fever-reducing granules according to the chromatogram. The results are shown in Table 1.

[0034] Table 1. Content determination results (mg / g)

[0035]

[0036]

[0037] Example 2: Methodological Investigation

[0038] Linearity assessment: Referring to Example 1, the mixed reference solution was accurately pipetted and diluted with 50 wt% methanol to prepare a series of mixed reference solutions with varying concentration gradients (3'-hydroxypuerarin 1.06 × 10³ μg / ml, 530.00 μg / ml, 106.00 μg / ml, 53.00 μg / ml, 10.60 μg / ml, 2.65 μg / ml; puerarin 1.00 × 10³ μg / ml, 500.00 μg / ml, 100.00 μg / ml, 50.00 μg / ml, 10.00 μg / ml, 2.50 μg / ml; 3'-methoxypuerarin 515). The concentrations of apigenin (510.00 μg / ml, 257.50 μg / ml, 51.50 μg / ml, 25.75 μg / ml, 5.15 μg / ml, 1.29 μg / ml) were determined according to the chromatographic conditions set in this invention. A standard curve was plotted with peak area (Y) as the ordinate and injection volume (X) as the abscissa, and the regression equation was calculated. The results showed that each component exhibited a good linear relationship within the corresponding concentration range, as detailed in Table 2.

[0039] Table 2. Linear relationships among the four components of Cold and Fever Relief Granules

[0040]

[0041]

[0042] Precision test: Referring to Example 1, the mixed reference solution was precisely pipetted and injected six times consecutively under the chromatographic conditions of this invention. The peak areas of each component were recorded, and the RSD of the peak areas was calculated. The results (see Table 3) show that the RSDs of the peak areas of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, and apigenin puerarin glycoside were 0.86%, 1.21%, 1.45%, and 1.57%, respectively, indicating that the instrument has good precision.

[0043] Table 3 Precision test results

[0044]

[0045] Repeatability test: Approximately 1 g of sample (numbered S3) was accurately weighed, and six test solutions were prepared in parallel according to Example 1. The results showed that the RSDs of the peak areas of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, and apigenin were 2.63%, 0.92%, 1.36%, and 1.33%, respectively, indicating good repeatability of the method.

[0046] Table 4 Results of Repeatability Tests

[0047]

[0048]

[0049] Stability test: Approximately 1 g of sample (number S3) was accurately weighed and the test solution was prepared according to Example 1. The solution was injected and measured at 0, 3, 6, 9, 12, and 24 hours. The results showed that the peak area RSDs of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, and apigenin puerarin glycoside were 2.76%, 1.99%, 2.28%, and 2.74%, respectively, indicating that the test solution had good stability within 24 hours.

[0050] Table 5. Results of stability tests

[0051]

[0052] Recovery test: Take about 0.5 g of sample with known content (number S3), weigh accurately, make 6 parallel samples, add a certain amount of reference standard to each sample accurately, prepare the test solution according to Example 1, inject and determine according to the chromatographic conditions under section "2.1", calculate the recovery rate, and the results are shown in Table 6.

[0053] Table 6 Results of the recovery test (n=6)

[0054]

[0055]

Claims

1. A method for detecting the content of multiple components in a cold and fever-relieving preparation, characterized in that, The procedure includes the following steps: Test solutions and mixed reference standards are separately sampled and tested under the following chromatographic conditions: detection wavelength 250 nm, column temperature 30℃, C18 column, and mobile phase system of methanol-water (methanol as phase A, water as phase B). Gradient elution is used with the following program: 0–10 min, 5%–20% A; 10–40 min, 20%–30% A; 40–50 min, 30%–80% A; 50–65 min, 80%–95% A. The contents of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, and apigenin puerarin glycoside in the cold and fever-reducing granules are calculated based on the chromatogram. The preparation of the mixed reference standard includes the following steps: Appropriate amounts of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, and apigenin puerarin glycoside are accurately weighed and added to 50 wt% methanol to prepare a solution containing 1.06 mg / ml of 3'-hydroxypuerarin and 1.00 mg / ml of puerarin. A mixed reference solution of 0.52 mg / ml 3'-methoxypuerarin and 0.51 mg / ml apigenin was obtained by filtration through a microporous membrane. The preparation of the test solution included the following steps: 1 g of cold and fever-reducing granules was accurately weighed, placed in a stoppered conical flask, 50 ml of distilled water was accurately added, the flask was sealed, the weight was measured, the flask was sonicated, cooled, and weighed again. The weight was adjusted with methanol, the flask was shaken well, and the solution was filtered through a microporous membrane.

2. The method for detecting the content of multiple components in a cold and fever-relieving preparation according to claim 1, characterized in that, Flow rate 1.0 ml / min; injection volume 10 μl.

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