Method for determining content of fructus forsythiae component in children lung heat cough and asthma granules

Abstract

The invention discloses a method for determining the content of fructus forsythiae in children lung heat cough and asthma granules, which comprises the following steps: step 1, precisely weighing forsythiaside A, forsythiaside H and forsythiaside I, and adding 50v / v% methanol solution for dissolving to obtain a mixed reference solution; 2, precisely weighing the ground sample to be detected, adding a 50v / v% methanol solution, carrying out ultrasonic treatment, cooling, weighing, complementing the reduced weight with the 50v / v% methanol solution, filtering, and taking a subsequent filtrate to obtain a test solution; and step 3, adopting high performance liquid chromatography, and carrying out sample injection detection. According to the method, the content of the fructus forsythiae related components in the infantile lung-heat cough and asthma granules is simultaneously determined by adopting the high performance liquid chromatography, the method is simple, reliable, good in durability, advanced and feasible, and the purpose of effectively controlling the quality of the fructus forsythiae related components in the infantile lung-heat cough and asthma granules is achieved; and a technical basis can be provided for internal quality control of pharmaceutical production enterprises and quality standard improvement of supervision departments.

Description

Technical Field

[0001] The present invention relates to the technical field of forsythia component detection, and in particular to a method for determining the content of forsythia components in Xiaoer Feire Kechuan granules. Background Art

[0002] Xiaoer Feire Kechuan Granules are composed of 11 Chinese herbs: Ephedra, Honeysuckle, Bitter Apricot, Anemarrhena, Forsythia, Scutellaria, Licorice, Radix Isatidis, Radix Ophiopogonis, Herba Houttuyniae, Ophiopogon, and Gypsum. They have the effects of clearing heat and detoxifying, promoting lung function and resolving phlegm. This preparation has many medicinal ingredients, a complicated process, and the various ingredients interact with each other. However, the standards implemented only include properties, identification, and inspection items, with few inspection items. This standard cannot reflect the quality of the medicinal materials used and the implementation of the production process, and cannot effectively meet the overall drug quality control requirements. Currently, most reports on the quality research of Xiaoer Feire Kechuan Granules use thin-layer chromatography to identify the medicinal materials of Ephedra and Scutellaria and high-performance liquid chromatography to determine the content of ephedrine hydrochloride, forsythin, and baicalin. However, the control index for the content of Forsythia is single, which cannot effectively control the product quality.

[0003] A Chinese invention patent (CN105675751B) provides a method for detecting forsythiaside A, H, and I in an antiviral oral solution using high-performance liquid chromatography. The method discloses a liquid sample preparation method that primarily involves extraction, evaporation to dryness, and reconstitution. Furthermore, the method utilizes a three-phase mobile phase system, making the experimental operation complex, time-consuming, and costly. A Chinese invention patent (CN110632232B) discloses a method for establishing a fingerprint of Forsythia suspensa. This method utilizes a 0.1% formic acid solution-acetonitrile solution as the mobile phase, with a measurement time of 200 minutes. The experimental operation is time-consuming, and the cycle for multi-batch measurements is long. Furthermore, the evaluation metric used is simply similarity, which is relatively simple and cannot comprehensively control the quality of Forsythia suspensa.

[0004] Therefore, it is urgent to establish a simple, accurate and practical method for determining the content of Forsythia suspensa-related components in solid samples. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for determining the content of the forsythia component in Xiaoer Feire Kechuan granules in order to address the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] Provided is a method for determining the content of the forsythia component in Xiaoer Feire Kechuan granules, comprising the following steps:

[0008] Step 1, preparation of reference solution: accurately weigh forsythiaside A, forsythiaside H, and forsythiaside I, and dissolve them in 50 v / v% methanol solution to obtain a mixed reference solution;

[0009] Step 2: Preparation of test solution:

[0010] Accurately weigh the ground sample, add 50 v / v% methanol solution, ultrasonically treat, let cool, weigh again, make up the lost weight with 50 v / v% methanol solution, filter, and take the filtrate to obtain the test solution;

[0011] Step 3, using high performance liquid chromatography for sample injection detection;

[0012] The chromatographic conditions are:

[0013] C18 chromatographic column; detection wavelength of 203 nm; column temperature of 30-40°C; injection volume of 5-10 μL; flow rate of 0.8-1.5 mL / min; mobile phase A is methanol, mobile phase B is an aqueous solution containing 0.05-0.1 v / v% formic acid; gradient elution.

[0014] Furthermore, the chromatographic conditions were as follows: C18 chromatographic column, column length 10 cm, inner diameter 4.6 mm, particle size 2.7 μm; detection wavelength 203 nm; column temperature 35°C; injection volume 5 μL; flow rate 0.8 mL / min; mobile phase A was methanol, mobile phase B was an aqueous solution containing 0.05 v / v% formic acid; gradient elution.

[0015] Furthermore, the gradient elution procedure is: 0-10 min, 23% A; 10-15 min, 23%-26% A; 15-29 min, 26%-30% A; 29-33 min, 30%-70% A; 33-34 min, 70%-23% A; 34-38 min, 23% A.

[0016] Furthermore, each 1 ml of the mixed reference solution contains 0.05 mg of forsythiaside A, 0.05 mg of forsythiaside H, and 0.05 mg of forsythiaside I.

[0017] Furthermore, the ultrasonic treatment conditions are 37KHz, 380W; and the treatment time is 30min.

[0018] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0019] The present invention adopts high performance liquid chromatography to simultaneously determine the content of forsythia-related components (forsythiaside A, forsythiaside H, and forsythiaside I) in the pediatric lung heat cough and asthma granules. The method is simple, reliable, durable, advanced and feasible, and can achieve the purpose of effectively controlling the quality of the forsythia-related components in the pediatric lung heat cough and asthma granules. It can provide a technical basis for the internal quality control of pharmaceutical manufacturers and the improvement of quality standards of regulatory departments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The figure is a typical chromatogram in one embodiment of the present invention; wherein, A-blank solvent; B-negative preparation lacking Forsythia suspensa; C-mixed reference solution; D-test solution; 1-forsythoside I; 2-forsythoside H; 3-forsythoside A. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0022] Example 1

[0023] This embodiment provides a method for determining the content of the Forsythia component in Xiaoer Feire Kechuan granules, which specifically comprises the following steps:

[0024] Step 1, preparation of reference solution:

[0025] Accurately weigh appropriate amounts of forsythiaside A, forsythiaside H, and forsythiaside I, and add 50 v / v% methanol to prepare a mixed reference solution containing 0.05 mg each of forsythiaside A, forsythiaside H, and forsythiaside I per 1 mL.

[0026] Step 2: Preparation of test solution:

[0027] Take the sample to be tested, grind it into powder, take 1 g, weigh it accurately, put it into a stoppered conical flask, accurately add 25 mL of 50 v / v% methanol, weigh it, and treat it ultrasonically (37 kHz, 380w) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 v / v% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0028] Step 3, using high performance liquid chromatography for sample injection detection;

[0029] The chromatographic conditions are:

[0030] C18 chromatographic column, column length, 10 cm, inner diameter, 4.6 mm, particle size, 2.7 μm; detection wavelength, 203 nm; column temperature, 35°C; injection volume, 5 μL; flow rate, 0.8 mL / min; theoretical plate number, calculated based on the forsythiaside I peak, should be no less than 10,000; mobile phase A was methanol, mobile phase B was an aqueous solution containing 0.05 v / v% formic acid; gradient elution;

[0031] The gradient elution procedure is: 0-10 min, 23% A; 10-15 min, 23%-26% A; 15-29 min, 26%-30% A; 29-33 min, 30%-70% A; 33-34 min, 70%-23% A; 34-38 min, 23% A.

[0032] Verify the above method:

[0033] 1.1 Linear

[0034] Five mixed reference solutions of varying concentrations were prepared, and 5 μL of each solution was injected into the liquid chromatograph. Linear regression was performed using the reference solution concentration as the x-axis and the peak area as the y-axis. The results showed that forsythiaside I, forsythiaside H, and forsythiaside A exhibited good linearity within each linear range (Table 1).

[0035] Table 1

[0036]

[0037] 1.2 Precision

[0038] The test solution was injected six times continuously to determine the peak areas of forsythiaside I, forsythiaside H, and forsythiaside A, and their relative standard deviations (RSD%) were calculated. The results showed that the RSDs of the peak areas of forsythiaside I, forsythiaside H, and forsythiaside A were all less than 1%, indicating good precision of the method.

[0039] 1.3 Repeatability

[0040] Six aliquots of the test solution from the same batch were prepared according to the method described in "Test Solution Preparation," injected, and assayed. The RSDs for forsythiaside I, forsythiaside H, and forsythiaside A were all less than 1%, indicating good reproducibility of the method.

[0041] 1.4 Accuracy

[0042] Weigh 9 portions of a test sample solution of known content and add each reference substance at concentrations equivalent to 50%, 100%, and 150% of the forsythoside I, forsythoside H, and forsythoside A content in the test sample, with triplicate replicates for each concentration. Prepare the test sample solution according to the method under "Test Solution Preparation," inject the sample, and measure the peak area. Calculate the content of each component using the external standard method, and calculate the recovery. The results are shown in Table 2.

[0043] Table 2

[0044]

[0045] 1.5 Intermediate precision

[0046] Different analysts, on different testing dates, prepared six aliquots of the same batch of test solution using the method described in "Test Solution Preparation." The samples were injected and assayed. The RSDs for forsythiaside I, forsythiaside H, and forsythiaside A were all less than 4%, indicating good intermediate precision.

[0047] 1.6 Stability

[0048] Prepare one aliquot of the test solution from the same batch according to the method described in "Test Solution Preparation." Inject the solution at 0, 4, 8, 12, 16, and 24 hours for determination. The RSDs for forsythiaside I, forsythiaside H, and forsythiaside A were all less than 1%, indicating good sample stability.

[0049] 1.710 batches of samples were measured

[0050] Ten batches of Xiaoer Feire Kechuan Granules (produced by Heilongjiang Sunflower Pharmaceutical Co., Ltd., batch numbers: 24070006, 2407001, 2311024, 2310091, 2311025, 2311037, 23091373, 2310080, 2304103, 22121233) were used to prepare test solutions according to the method under "Preparation of Test Solution." The total content of forsythiaside I, forsythiaside H, and forsythiaside A in all ten batches of Xiaoer Feire Kechuan Granules was greater than 2.89 mg as determined by the above method.

[0051] Example 2

[0052] This example provides a robustness analysis of the method for determining the content of the Forsythia component in the Xiaoer Feire Kechuan granules, specifically including:

[0053] 2.1 Flow rate is 0.75 mL / min

[0054] 1) Preparation of reference solution: same as in Example 1

[0055] 2) Preparation of test solution: same as in Example 1

[0056] 3) Preparation of mobile phase: same as in Example 1

[0057] 4) Chromatographic conditions: flow rate 0.75 mL / min; other conditions are the same as in Example 1

[0058] 5) Sample determination: Accurately aspirate 5 μL of the reference solution and the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram. 29 H 36 O 15 ), forsythiaside H(C 29 H 36 O 15 ), forsythiaside A (C 29 H 36 O 15 ) content is 4.57 mg in total.

[0059] 2.2 Flow rate is 0.85 mL / min

[0060] 1) Preparation of reference solution: same as in Example 1

[0061] 2) Preparation of test solution: same as in Example 1

[0062] 3) Preparation of mobile phase: same as in Example 1

[0063] 4) Chromatographic conditions: flow rate 0.85 mL / min; other conditions are the same as in Example 1

[0064] 5) Sample determination: Accurately aspirate 5 μL of the reference solution and the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram. 29 H 36 O 15 ), forsythiaside H(C 29 H 36 O 15 ), forsythiaside A (C 29 H 36 O 15 ) content is 4.21 mg in total.

[0065] 2.3 Column temperature is 33°C

[0066] 1) Preparation of reference solution: same as in Example 1

[0067] 2) Preparation of test solution: same as in Example 1

[0068] 3) Preparation of mobile phase: same as in Example 1

[0069] 4) Chromatographic conditions: column temperature was 33°C; other conditions were the same as in Example 1

[0070] 5) Sample determination: Accurately aspirate 5 μL of the reference solution and the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram. 29 H 36 O 15 ), forsythiaside H(C 29 H 36 O 15 ), forsythiaside A (C 29 H 36 O 15 ) content is calculated on a total basis and is 4.40 mg.

[0071] 2.4 Column temperature is 37°C

[0072] 1) Preparation of reference solution: same as in Example 1

[0073] 2) Preparation of test solution: same as in Example 1

[0074] 3) Preparation of mobile phase: same as in Example 1

[0075] 4) Chromatographic conditions: column temperature was 37°C; other conditions were the same as in Example 1

[0076] 5) Sample determination: Accurately aspirate 5 μL of the reference solution and the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram. 29 H 36 O 15 ), forsythiaside H(C 29 H 36 O 15 ), forsythiaside A (C 29 H 36 O 15 ) content is 4.34 mg in total.

[0077] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the content of the forsythia component in Xiaoer Feire Kechuan granules, characterized in that: The steps include: Step 1, preparation of reference solution: accurately weigh forsythiaside A, forsythiaside H, and forsythiaside I, and dissolve them in 50 v / v% methanol solution to obtain a mixed reference solution; Step 2: Preparation of test solution: Accurately weigh the ground sample, add 50 v / v% methanol solution, ultrasonically treat, let cool, weigh again, make up the lost weight with 50 v / v% methanol solution, filter, and take the filtrate to obtain the test solution; Step 3, using high performance liquid chromatography for sample injection detection; The chromatographic conditions are: C18 chromatographic column; detection wavelength of 203 nm; column temperature of 30-40°C; injection volume of 5-10 μL; flow rate of 0.8-1.5 mL / min; mobile phase A is methanol, mobile phase B is an aqueous solution containing 0.05-0.1 v / v% formic acid; gradient elution.

2. The method for determining the content of the forsythia component in the Xiaoer Feire Kechuan granules according to claim 1, characterized in that: The chromatographic conditions are: C18 chromatographic column, column length, 10 cm, inner diameter, 4.6 mm, particle size, 2.7 μm; detection wavelength, 203 nm; column temperature, 35°C; injection volume, 5 μL; flow rate, 0.8 mL / min; mobile phase A, methanol, mobile phase B, aqueous solution containing 0.05 v / v% formic acid; gradient elution.

3. The method for determining the content of the forsythia component in the Xiaoer Feire Kechuan granules according to claim 2, characterized in that: The gradient elution procedure is: 0-10 min, 23% A; 10-15 min, 23%-26% A; 15-29 min, 26%-30% A; 29-33 min, 30%-70% A; 33-34 min, 70%-23% A; 34-38 min, 23% A.

4. The method for determining the content of the forsythia component in the Xiaoer Feire Kechuan granules according to claim 1, characterized in that: Each 1 ml of the mixed reference solution contains 0.05 mg each of forsythiaside A, forsythiaside H, and forsythiaside I.

5. The method for determining the content of the forsythia component in the Xiaoer Feire Kechuan granules according to claim 1, characterized in that: The ultrasonic treatment conditions are 37KHz, 380W; and the treatment time is 30min.

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