Quality detection method and application of dracocephalum moldavica formula granules

Abstract

The invention discloses a quality detection method and application of dracocephalum moldavica formula granules. The quality detection method of the dracocephalum moldavica formula granules comprises the following steps: carrying out gradient elution separation on a to-be-detected solution of a test solution 1 containing the dracocephalum moldavica formula granules by high performance liquid chromatography, in the high performance liquid chromatography, a mobile phase A is methanol, and a mobile phase B is a phosphoric acid aqueous solution; the column temperature of the chromatographic column is 30-40 DEG C. The method fills the blank of quality standard research in the prior art, and the constructed method can be used for rapidly and comprehensively controlling the quality of the dracocephalum moldavica formula granules.

Description

Technical Field

[0001] This invention relates to the field of drug quality control technology, specifically to a detection method and application for comprehensively controlling the quality of Xiangqinglan formula granules. Background Technology

[0002] Dracocephalum moldavica L., a plant in the Lamiaceae family, is a dried aerial part of the plant. It is harvested during its peak flowering season in summer, impurities are removed, and it is sun-dried. Currently, it is cultivated throughout Xinjiang. It is classified as a Class II dry-heat herb. Its functions and indications include benefiting the heart and brain, protecting the liver and stomach, enhancing sensory abilities, replenishing protective energy, boosting intelligence, and clearing blockages in the brain. It is used for palpitations, chest pain, dizziness, slow reaction time, decreased sensation, dull thinking, weak stomach and liver, and decreased bodily vitality.

[0003] Modern pharmaceuticals possess three key characteristics: stability, uniformity, safety, and efficacy. For traditional Chinese medicine (TCM) preparations, multiple testing methods are necessary to ensure the reliability and stability of the results. TCM formula granules are single-herb products refined using modern scientific technology, mimicking the traditional decoction process of TCM. These granules involve the extraction, concentration, and drying of raw medicinal herbs. They retain the properties and efficacy of the raw herbs, offering stable quality. They are suitable for dispensing prescriptions in TCM clinical practice, adapting to the needs of syndrome differentiation and treatment, and adapting to prescription changes. They also offer advantages such as not requiring decoction, convenient administration, rapid absorption, accurate dosage, safety, cleanliness, and portability. Compared to traditional medicinal materials, the morphological characteristics of formula granules are significantly altered, making it impossible to detect their authenticity and quality with the naked eye. Therefore, it is necessary to establish a quality evaluation system for TCM formula granules to comprehensively reflect their intrinsic quality.

[0004] Regarding the quality control methods for *Xiangqinglan* medicinal materials, the draft revision of the standards published by the National Pharmacopoeia Commission adopts methods such as morphological identification, cross-sectional identification, powder microscopic identification, thin-layer chromatography, and content determination. Currently, no manufacturers produce *Xiangqinglan* formula granules on the market, and existing patent literature does not disclose quality standards for *Xiangqinglan* formula granules. Our company previously applied for patent CN202211559765.X, which discloses a preparation method for *Xiangqinglan* formula granules. However, a systematic quality testing method for *Xiangqinglan* formula granules has not yet been established. Using only existing medicinal material quality testing methods to test formula granules is insufficient and cannot reflect the overall intrinsic quality of *Xiangqinglan* formula granules, thus failing to meet the quality control requirements for traditional Chinese medicine formula granules. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the deficiency of the lack of quality testing methods for Xiangqinglan formula granules in the prior art, and to provide a quality testing method and application for Xiangqinglan formula granules. The method of this invention fills the gap in the existing technology regarding its quality standards, and the constructed method can be used for rapid and comprehensive quality control of Xiangqinglan formula granules.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solution.

[0007] This invention provides a method for quality testing of Xiangqinglan formula granules, which includes the following steps: separating the test solution 1 containing Xiangqinglan formula granules by gradient elution using high performance liquid chromatography;

[0008] In the high-performance liquid chromatography, mobile phase A is methanol, and mobile phase B is an aqueous solution of phosphoric acid; the column temperature is 30-40℃.

[0009] In this invention, the fragrant orchid formulation granules preferably include araliain and / or rosmarinic acid.

[0010] In this invention, the preparation method of the test solution 1 containing Xiangqinglan formula particles preferably includes the following steps: extracting and processing the mixture including Xiangqinglan formula particles and solvent, filtering, and using the filtrate as test solution 1.

[0011] The particle size of the fragrant orchid formula granules can be such that they pass through a No. 6 sieve.

[0012] The solvent may be an aqueous solution of methanol and / or ethanol. The volume percentage of ethanol in the aqueous solution may be 40%-80%, for example, 70%.

[0013] The mass-to-volume ratio of the fragrant orchid formulation granules to the solvent can be 0.6g:(10-80)mL, preferably 0.6g:(20-70)mL, for example 0.6g:40mL or 0.6g:50mL.

[0014] The extraction process can be performed by ultrasonic treatment or reflux treatment.

[0015] The extraction process can take 0.2-2 hours, preferably 0.5-1.5 hours, for example 1 hour.

[0016] The process typically includes a step of replenishing the weight loss after extraction and before filtration. This replenishment is usually achieved using an aqueous solution of methanol and / or ethanol.

[0017] In a preferred embodiment, the preparation method of the test solution 1 includes the following steps: ultrasonic extraction and filtration of a mixture including Xiangqinglan formula granules and methanol, replenishing the lost weight with methanol, and using the filtrate as the test solution; the mass-volume ratio of Xiangqinglan formula granules to methanol is 0.6g:(20-70)mL; the ultrasonic extraction time is 0.5-1.5h.

[0018] In a preferred embodiment, the preparation method of the test solution 1 includes the following steps: ultrasonic extraction and filtration of a mixture including Xiangqinglan formula granules and methanol, replenishing the lost weight with methanol, and using the filtrate as the test solution; the mass-volume ratio of Xiangqinglan formula granules to methanol is 0.6g:50mL; the ultrasonic extraction time is 1h.

[0019] In this invention, the high-performance liquid chromatography generally also includes the preparation of reference solution 1 and reference medicinal material reference solution 1.

[0020] The reference solution 1 generally comprises a reference standard 1 and a solvent. The reference standard 1 is preferably arvense glycoside and / or rosmarinic acid. The solvent may be an aqueous solution of methanol and / or ethanol. In the aqueous ethanol solution, the volume percentage of ethanol may be 40%-80%, for example, 70%.

[0021] When reference standard 1 includes citronellol, the mass concentration of citronellol in the reference standard solution 1 can be 40-70 μg / mL, for example 60 μg / mL.

[0022] When reference standard 1 includes rosmarinic acid, the mass concentration of rosmarinic acid in the reference standard solution 1 may be 100-130 μg / mL, for example 120 μg / mL.

[0023] In a preferred embodiment, the reference solution 1 comprises argiol at a mass concentration of 40-70 μg / mL and rosmarinic acid at a mass concentration of 100-130 μg / mL.

[0024] In a preferred embodiment, the reference solution 1 comprises piracetam at a mass concentration of 60 μg / mL and rosmarinic acid at a mass concentration of 120 μg / mL.

[0025] The reference medicinal material refers to a standard medicinal material. In this invention, the reference medicinal material is *Cymbidium ensifolium* reference medicinal material.

[0026] The preparation method of the reference solution 1 preferably includes the following steps: extracting and filtering a mixture of reference medicinal material 1 and solvent, and using the filtrate as reference solution 1.

[0027] In the reference solution 1, the solvent may be an aqueous solution of methanol and / or ethanol. The volume percentage of ethanol in the aqueous solution may be 40%-80%, for example, 70%.

[0028] The mass-to-volume ratio of the reference medicinal material 1 to the solvent can be 0.5g:(10-30)mL, for example, 0.5g:20mL.

[0029] The extraction process can be performed by ultrasonic treatment or reflux treatment.

[0030] The extraction process is preferably performed for 0.5-1.5 hours, for example, 1 hour.

[0031] The process typically includes a step of replenishing the weight loss after extraction and before filtration. This replenishment is usually achieved using an aqueous solution of methanol and / or ethanol.

[0032] In a preferred embodiment, the preparation method of the reference solution 1 includes the following steps: ultrasonic extraction and filtration of a mixture including reference medicinal material 1 and methanol, replenishing the lost weight with methanol, and using the filtrate as reference solution 1; the mass-volume ratio of reference medicinal material 1 to methanol is 0.5g:(10-30)mL; the ultrasonic extraction time is 0.5-1.5h.

[0033] In a preferred embodiment, the preparation method of the reference solution 1 includes the following steps: ultrasonic extraction and filtration of a mixture including reference medicinal material 1 and methanol, replenishing the lost weight with methanol, and using the filtrate as reference solution 1; the mass-volume ratio of reference medicinal material 1 to methanol is 0.5g:20mL; the ultrasonic extraction time is 1h.

[0034] In this invention, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution in the high performance liquid chromatography can be 0.1%-0.5%, for example 0.2%, 0.3% or 0.4%.

[0035] In a preferred embodiment, the mobile phase in the high-performance liquid chromatography is methanol (A) - 0.2% phosphoric acid aqueous solution (B).

[0036] In this invention, the column temperature of the chromatographic column in the high-performance liquid chromatography is preferably 32-38℃, for example 35℃.

[0037] In this invention, the flow rate of the mobile phase in the high-performance liquid chromatography is 0.3-1.0 mL / min, preferably 0.5-0.8 mL / min, for example 0.6 mL / min.

[0038] In this invention, the injection volume in the high-performance liquid chromatography can be 10-20 μL, for example 15 μL.

[0039] In this invention, the detection wavelength in the high-performance liquid chromatography can be conventional in the art, preferably 200-400 nm, for example 328 nm.

[0040] In this invention, the chromatographic column in the high-performance liquid chromatography can be a column packed with octadecylsilane-bonded silica gel, preferably GL InertSustain C18, GL InertSustain AQ-C18 or YMC TriartC18.

[0041] The chromatographic column can have a length of 250 mm and an inner diameter of 4.6 mm. The packing material of the chromatographic column can have a particle size of 5 μm.

[0042] In this invention, the gradient elution in the high-performance liquid chromatography can be as follows:

[0043]

[0044]

[0045]

[0046] or,

[0047]

[0048] The percentages in the table represent the volume percentage of each component in the total volume of mobile phase A and mobile phase B, respectively.

[0049] In this invention, the fingerprint spectrum obtained after gradient elution separation of the test solution 1 of the Xiangqinglan formula granules by high performance liquid chromatography may include two characteristic peaks, with the piracetam glycoside peak as the reference peak. The average relative retention time of each characteristic peak is as follows:

[0050] Peak 4 is rosmarinic acid, with an average relative retention time of 0.558 and an RSD of 0.05%.

[0051] In this invention, preferably, the fingerprint spectrum obtained after gradient elution separation of the test solution 1 of the Xiangqinglan formula granules by high performance liquid chromatography includes 9 characteristic peaks, with the piracetam glycoside peak as the reference peak, and the average relative retention times of each characteristic peak are as follows:

[0052] The mean relative retention time of peak 1 was 0.348, with an RSD of 0.02%.

[0053] The mean relative retention time of peak 2 was 0.383, with an RSD of 0.03%.

[0054] The mean relative retention time of peak 3 was 0.498, with an RSD of 0.03%.

[0055] Peak 4 is rosmarinic acid, with an average relative retention time of 0.558 and an RSD of 0.05%.

[0056] The mean relative retention time of peak 5 was 0.619, with an RSD of 0.04%.

[0057] The mean relative retention time of peak 6 was 0.647, with an RSD of 0.04%.

[0058] The mean relative retention time of peak 7 was 0.739, with an RSD of 0.04%.

[0059] The mean relative retention time of peak 9 was 1.017, with an RSD of 0.02%.

[0060] In this invention, the method for quality testing of the Xiangqinglan formula granules preferably further includes the following (1) and / or (2).

[0061] (1) Thin-layer chromatography was used to detect the test solution 2 containing the Xiangqinglan formula particles;

[0062] (2) The content of piracetam in the test solution 3 of the Xiangqinglan formula granules was determined by high performance liquid chromatography.

[0063] In step (1), the method for preparing the test solution 2 containing Xiangqinglan formula particles preferably includes the following steps: ultrasonically treating the Xiangqinglan formula particle solution, filtering, and drying to obtain residue; the solution of the residue is the test solution 2.

[0064] In step (1), the solvent 2-1 in the Xiangqinglan formulation granule solution can be an aqueous solution of methanol and / or ethanol. The volume percentage of ethanol in the aqueous solution can be 40%-80%, for example, 70%.

[0065] In step (1), the mass-to-volume ratio of the Xiangqinglan formula granules to solvent 2-1 in the solution can be 5g:(40-60)mL, for example 5g:50mL.

[0066] In step (1), the ultrasonic treatment time can be 5-30 minutes, for example 10 minutes.

[0067] In step (1), the solvent 2-2 in the residue solution preferably has the same type as the solvent 2-1. The volume ratio of solvent 2-1 to solvent 2-2 can be (4-6):1, for example, 5:1.

[0068] In a preferred embodiment, the preparation method of the test solution 2 containing Xiangqinglan formulation particles in the thin-layer chromatography method includes the following steps: ultrasonically treating a solution of Xiangqinglan formulation particles and 70% ethanol aqueous solution with a mass-to-volume ratio of 5g:50mL for 10min, filtering, and drying to obtain residue; the solution of residue and 70% ethanol aqueous solution is the test solution; the volume ratio of 70% ethanol aqueous solution in the Xiangqinglan formulation particles and 70% ethanol aqueous solution to the volume ratio of 70% ethanol aqueous solution in the residue and 70% ethanol aqueous solution is 5:1.

[0069] In step (1), the preparation of reference solution 2 and reference medicinal material reference solution 2 is generally also carried out.

[0070] In step (1), the reference solution 2 generally includes a reference standard 2 and a solvent. The reference standard 2 is preferably citronellol. The solvent may be an aqueous solution of methanol and / or ethanol. The volume percentage of ethanol in the aqueous solution may be 40%-80%, for example, 70%.

[0071] In step (1), when the reference standard 2 includes citronellol, the mass concentration of citronellol in the reference standard solution 2 can be 0.1-0.3 mg / mL, for example 0.2 mg / mL.

[0072] In a preferred embodiment, the reference solution 2 comprises piracetam at a mass concentration of 0.2 mg / mL.

[0073] In step (1), the preparation method of the reference solution 2 preferably includes the following steps: ultrasonically treating the solution containing the reference medicinal material 2 and solvent 2-3, filtering and drying to obtain residue; the solution of the residue and solvent 2-4 is the reference solution 2.

[0074] In step (1), the solution containing reference medicinal material 2 and solvent 2-3 may contain methanol and / or an aqueous solution of ethanol. The volume percentage of ethanol in the aqueous solution may be 40%-80%, for example, 70%.

[0075] In step (1), the mass-to-volume ratio of the reference medicinal material 2 and solvent 2-3 in the solution can be 1g:(40-60)mL, for example 1g:50mL.

[0076] In step (1), the ultrasonic treatment time can be 5-30 minutes, for example 10 minutes.

[0077] In step (1), the type of solvent 2-4 in the solution of the residue and solvent 2-4 is preferably the same as that of solvent 2-3. The volume ratio of solvent 2-3 to solvent 2-4 can be (4-6):1, for example, 5:1.

[0078] In a preferred embodiment, the preparation method of the reference solution 2 in the thin-layer chromatography includes the following steps: ultrasonically treating the solution of the reference medicinal material 2 and solvent 2-3, filtering, and drying to obtain a residue; the solution of the residue and solvent 2-4 is the reference solution 2; the type of solvent 2-3 is 70% ethanol aqueous solution, the mass-to-volume ratio of the reference medicinal material to solvent 2-3 is 1g:50mL, the ultrasonic treatment time is 10min, the type of solvent 2-4 is 70% ethanol aqueous solution, and the volume ratio of solvent 2-3 to solvent 2-4 is 5:1.

[0079] In step (1), the operation of the thin-layer chromatography method can be referred to the thin-layer chromatography method in the General Chapter 0502 of Part IV of the Chinese Pharmacopoeia 2020.

[0080] In step (1), the thin-layer chromatography preferably includes the following steps: spotting the test solution 2, the reference medicinal material solution 2 and the reference solution 2 onto the same silica gel thin-layer plate, developing, removing, drying, and examining under ultraviolet light.

[0081] In step (1), the spotting volume of the test solution 2, the reference medicinal material solution 2 and the reference solution 2 in the thin-layer chromatography can each be 1-10 μL, preferably 2-5 μL, for example 3 or 4 μL.

[0082] In a preferred embodiment, the spotting amounts of the test solution 2, the reference medicinal material solution 2, and the reference solution 2 in the thin-layer chromatography are 3 μL, respectively.

[0083] In a preferred embodiment, the spotting volumes of the test solution 2, the reference medicinal material solution 2, and the reference solution 2 in the thin-layer chromatography are 5 μL, respectively.

[0084] In a preferred embodiment, the spotting volume of the test solution 2, the reference medicinal material solution 2, and the reference solution 2 in the thin-layer chromatography is 10 μL, respectively.

[0085] In step (1), the wavelength used for testing in the thin-layer chromatography can be conventional in the art, preferably at an ultraviolet wavelength, such as 254 nm or 365 nm. Those skilled in the art will understand that the ultraviolet wavelength is 10-400 nm.

[0086] In step (1), during the thin-layer chromatography, it is preferable to spray a 5% aluminum trichloride ethanol solution during inspection.

[0087] In a preferred embodiment, the thin-layer chromatography is performed by spraying with a 5% aluminum trichloride ethanol solution and examining under ultraviolet light at 365 nm.

[0088] In step (1), the silica gel thin-layer chromatogram can be of a type commonly used in the art, preferably H plate, GF254 or G plate, such as G plate.

[0089] In step (1), the developing solvent used in the thin-layer chromatography can be an aqueous solution of ethyl acetate and formic acid, or an aqueous solution of ethyl acetate, formic acid and toluene.

[0090] When the developing solvent used is an aqueous solution of ethyl acetate and formic acid, the volume ratio of ethyl acetate, formic acid and water can be (6-10):1:(5-7), for example 8:0.5:3.

[0091] When the developing solvent used is an aqueous solution of ethyl acetate, toluene, and formic acid, the volume ratio of the ethyl acetate, toluene, formic acid, and water can be (14-17):(0.5-2.5):1:(0.5-0.7), for example, 16:2:1:0.6 or 16:0.6:1:0.6.

[0092] In step (1), the developing temperature in the thin-layer chromatography can be 10-40℃, for example 25℃.

[0093] In step (1), the humidity of the thin-layer chromatography process can be 10%-90%, for example, 40%.

[0094] In step (2), the preparation method of the test solution 3 containing Xiangqinglan formula particles preferably includes the following steps: extracting and processing the Xiangqinglan formula particle solution, filtering it, and using the filtrate as the test solution 3.

[0095] In step (2), the particle size of the Xiangqinglan formula granules can be such that they pass through a No. 6 sieve.

[0096] In step (2), the solvent 3-1 in the Xiangqinglan formula granule solution can be methanol, an aqueous ethanol solution, or an aqueous methanol solution. In the aqueous ethanol solution, the volume percentage of ethanol can be 40%-80%, for example, 70%. In the aqueous methanol solution, the volume percentage of methanol can be 40%-80%, for example, 70%.

[0097] In step (2), the mass-to-volume ratio of the Xiangqinglan formulation granules to solvent 3-1 can be 0.3g:(20-110)mL, preferably 0.3g:(25-100)mL, for example 0.3g:50mL or 0.3g:80mL.

[0098] In step (2), the extraction process can be ultrasonic treatment or reflux treatment.

[0099] In step (2), the extraction process can take 10-30 minutes, for example, 20 minutes.

[0100] In step (2), after the extraction process and before the filtration, a step to replenish the weight loss is generally included. This replenishment of weight loss is typically achieved using methanol, an aqueous ethanol solution, or an aqueous methanol solution.

[0101] In step (2), the preparation of reference solution 3 and reference medicinal material reference solution 3 is generally also carried out.

[0102] In step (2), the reference solution 3 generally includes reference standard 3 and solvent 3-2. Reference standard 3 is preferably citronellol. The solvent 3-2 can be methanol, an aqueous ethanol solution, or an aqueous methanol solution. The volume percentage of ethanol in the aqueous ethanol solution can be 40%-80%, for example, 70%. The volume percentage of methanol in the aqueous methanol solution can be 40%-80%, for example, 70%.

[0103] When the reference standard 3 is citronellol, the mass concentration of citronellol in the reference standard solution 3 can be 20-30 μg / mL, for example, 26 μg / mL.

[0104] In a preferred embodiment, the reference solution 3 is a mixture of argentin and 70% aqueous ethanol solution, wherein the mass concentration of argentin in the reference solution 3 is 26 μg / mL.

[0105] In step (2), the mobile phase A in the high-performance liquid chromatography can be acetonitrile.

[0106] In step (2), the mobile phase B in the high-performance liquid chromatography can be an aqueous phosphoric acid solution. The volume percentage of phosphoric acid in the aqueous phosphoric acid solution can be 0.1%-0.5%, for example, 0.2%, 0.3% or 0.4%.

[0107] In step (2), the volume ratio of mobile phase A to mobile phase B in the high performance liquid chromatography can be (20-25):(75-80), for example 22:78, 23:77, 25:75 or 20:80.

[0108] In step (2), the detection wavelength in the high-performance liquid chromatography can be conventional in the art, preferably 200-400nm, for example 333nm.

[0109] In step (2), the column temperature of the chromatographic column in the high performance liquid chromatography is preferably 25-40℃, for example 30℃.

[0110] In step (2), the flow rate of the mobile phase in the high-performance liquid chromatography can be 0.5-2 mL / min, for example, 1.0 mL / min.

[0111] In step (2), the injection volume in the high performance liquid chromatography can be 10-20 μL, for example 15 μL.

[0112] The present invention also provides the application of the quality testing method for the Xiangqinglan formula granules as described above in evaluating and / or controlling their quality.

[0113] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0114] The reagents and raw materials used in this invention are all commercially available.

[0115] The positive and progressive effects of this invention are as follows:

[0116] (1) This invention is the first to comprehensively study the quality standards of Xiangqinglan formula granules and provides a quality detection method for Xiangqinglan granules. The method of this invention fills the gap in the existing technology for its quality standard research, and the constructed method can be used to quickly and comprehensively control the quality of Xiangqinglan formula granules.

[0117] Preferably, the method of the present invention controls the quality of Xiangqinglan formula granules from multiple perspectives, including thin-layer identification, characteristic chromatograms, and content determination, so as to ensure the safety and effectiveness of the formula granules and lay a solid foundation for the quality stability of Xiangqinglan formula granules.

[0118] (2) The method of the present invention is simple to operate, can construct feature maps with better separation and clearer features, has good stability, high precision, good reproducibility, high detection efficiency, and low cost. It is suitable for evaluating the quality consistency of Xiangqinglan formula granules from different manufacturers or different batches from the same manufacturer, and provides a stronger guarantee for its industrialization. Attached Figure Description

[0119] Figure 1 The thin-layer chromatography results are shown for Examples 1-1 and 1-2.

[0120] Figure 2 This is a thin-layer chromatography result of Example 2-1.

[0121] Figure 3 The image shows the thin-layer chromatography results of Example 2-2.

[0122] Figure 4 This is a thin-layer chromatography result of Example 3-1.

[0123] Figure 5 This is a thin-layer chromatography result of Example 4-1.

[0124] Figure 6 This is a thin-layer chromatography result diagram of Example 4-2.

[0125] Figure 7 The results are thin-layer chromatograms of Examples 5-1 to 5-3.

[0126] Figure 8 This is a thin-layer chromatography result of Example 6-1.

[0127] Figure 9 This is a thin-layer chromatography result of Example 6-2.

[0128] Figure 10 The image shows the thin-layer chromatography results of Examples 6-3.

[0129] Figure 11 This is a thin-layer chromatography result of Example 7-1.

[0130] Figure 12 This is a thin-layer chromatography result of Example 7-2.

[0131] Figure 13 The image shows the thin-layer chromatography results of Examples 7-3.

[0132] Figure 14 This is a thin-layer chromatography result of Example 8-1.

[0133] Figure 15 This is a thin-layer chromatography result diagram of Example 8-2.

[0134] Figure 16 The image shows the thin-layer chromatography results of Examples 8-3.

[0135] Figure 17 This is a thin-layer chromatography result of Example 9-1.

[0136] Figure 18 The image shows the thin-layer chromatography results of Example 9-2.

[0137] Figure 19 This is a thin-layer chromatography result of Example 10-1.

[0138] Figure 20 The image shows the thin-layer chromatography results of Example 10-2.

[0139] Figure 21 The image shows the thin-layer chromatography results of Examples 10-3.

[0140] Figure 22 The results are thin-layer chromatograms of Examples 10-4.

[0141] Figure 23 This is a thin-layer chromatography result of Example 11-1.

[0142] Figure 24 This is a thin-layer chromatography result of Example 12-1.

[0143] Figure 25 The image shows the thin-layer chromatography results of Example 12-2.

[0144] Figure 26 This is a thin-layer chromatography result of Example 13-1.

[0145] Figure 27 The image shows the thin-layer chromatography results of Example 13-2.

[0146] Figure 28 This is a column chromatography chromatogram of the test solution from Example 14-1.

[0147] Figure 29 This is a column chromatography chromatogram of the test solution from Example 14-2.

[0148] Figure 30 This is a column chromatography chromatogram of the test solution from Examples 14-3.

[0149] Figure 31 The chromatograms are column chromatography diagrams of the test solutions in Examples 14-4.

[0150] Figure 32 The chromatograms are column chromatography diagrams of the test solutions from Examples 14-5.

[0151] Figure 33 This is a specificity assessment diagram of the test sample solution during the construction of the characteristic spectrum.

[0152] Figure 34 This is a graph showing the results of the precision assessment of the reference standard during the construction of the feature map.

[0153] Figure 35 This is a graph showing the results of the precision assessment of the test sample during the construction of the feature map.

[0154] Figure 36 This is a graph showing the results of repeatability tests during the construction of the feature map.

[0155] Figure 37 This is a graph showing the results of intermediate precision tests during the construction of the feature map.

[0156] Figure 38 This is a graph showing the results of stability tests during the construction of the feature map.

[0157] Figure 39 The HPLC characteristic chromatograms of three batches of Xiangqinglan formula granules are shown.

[0158] Figure 40 The linear graph of piracetamine reference standard is shown.

[0159] Figure 41 The chromatogram for the detection limit of Xiangqinglan formula granules. Figure 41 Part A represents the chromatogram of the detection limit of piracetamine reference standard. Figure 41 Part B represents the chromatogram of the detection limit of the test solution.

[0160] Figure 42 The chromatogram of the limit of quantification for the Xiangqinglan formula granules. Figure 42 Part A represents the limit of quantification chromatogram of piracetamine reference standard. Figure 42 Part B represents the limit of quantitation chromatogram of the test solution. Detailed Implementation

[0161] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0162] 1. Experimental Preparation

[0163] 1.1 Instruments, Reagents and Chemicals

[0164] 1.1.1 Instruments

[0165] DT500B electronic balance (Changshu Jiaheng Balance Instrument Co., Ltd.), MS205DU electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), MS105DU electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), AL204 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), ME204E / 02 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), Waters E2695 high performance liquid chromatograph, GL InertSustain C18 column (4.6×250mm, 5μm), Agilent C18 column (4.6×250mm, 5μm), Waters SunFire C18 column (4.6×250mm, 5μm).

[0166] 1.1.2 Reagents

[0167] Methanol (Thermo Fisher Scientific (China) Co., Ltd.) and phosphoric acid (Tianjin Beilian Fine Chemicals Development Co., Ltd.) were of chromatographic grade, water was ultrapure water, and other reagents were of analytical grade.

[0168] 1.1.3 Reference standards and reference medicinal materials

[0169]

[0170] 1.1.4 Sample Information

[0171]

[0172] 2. Thin-layer chromatography identification:

[0173] Example 1 Investigation of the solvent in the test solution

[0174] Example 1-1

[0175] (1) Preparation of test solution: Take 5g of Xiangqinglan formula granules, add 50ml of 70% ethanol, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, add 10ml of 70% ethanol to dissolve the residue, and use it as test solution.

[0176] (2) Preparation of reference medicinal material solution: Weigh 1g of Xiangqinglan reference medicinal material, add 50ml of 70% ethanol, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, add 10ml of 70% ethanol to dissolve the residue, and prepare the reference medicinal material solution.

[0177] (3) Preparation of reference solution: Take citronellol reference standard, add 70% ethanol to prepare a solution containing 0.2 mg per ml, and obtain the reference solution;

[0178] (4) Thin-layer chromatography analysis: Thin-layer chromatography conditions are as follows: thin-layer plate: silica gel GF254 thin-layer plate; sample volume: 10 μl for reference solution, 5 μl for test solution and reference medicinal material solution; developing solvent: a mixed solution of ethyl acetate-formic acid-water with a volume ratio of 8:0.5:0.3 is used as the developing solvent; inspection: under ultraviolet light (254 nm) at 25℃ and 30% humidity.

[0179] Examples 1-2

[0180] (1) Preparation of test solution: Take 5g of Xiangqinglan formula granules, add 50ml of methanol, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, add 10ml of methanol to dissolve the residue, and use it as test solution.

[0181] (2) Preparation of reference medicinal material solution: Weigh 1g of Xiangqinglan reference medicinal material, add 50ml of methanol, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, add 10ml of methanol to dissolve the residue, and prepare the reference medicinal material solution.

[0182] (3) Preparation of reference solution: Take citronellol reference standard, add 70% ethanol to prepare a solution containing 0.2 mg per ml, and obtain the reference solution;

[0183] (4) Thin-layer chromatography analysis: Thin-layer chromatography conditions are as follows: thin-layer plate: silica gel GF254 thin-layer plate; sample volume: 10 μl for reference solution and reference medicinal material solution, and 5 μl for test solution b; developing solvent: a mixed solution of ethyl acetate-formic acid-water with a volume ratio of 8:0.5:0.3 is used as the developing solvent; inspection: under ultraviolet light (254 nm) at 25℃ and 30% humidity.

[0184] The thin-layer chromatography results of Examples 1-1 and 1-2 are shown below. Figure 1 As shown, 1 is 10 μL of piperazine reference solution, 2 is 10 μL of *Cymbidium faberi* reference medicinal material solution prepared with methanol solvent, 3 is 5 μL of *Cymbidium faberi* formula granule test solution prepared with methanol solvent, 4 is 5 μL of *Cymbidium faberi* reference medicinal material solution prepared with 70% ethanol solution, and 5 is 5 μL of *Cymbidium faberi* formula granule test solution prepared with 70% ethanol solution. Figure 1 It can be seen that under these developing solvent conditions, the color development and separation effects of the 70% ethanol solvent for the reference medicinal material and the test sample are better. Therefore, the optimal preparation solvent for the test sample is set as 70% ethanol solvent.

[0185] Example 2 Investigation of developing solvent conditions

[0186] Example 2-1

[0187] The only difference from Example 1-1 is that the developing solvent in step (4) is ethyl acetate-toluene-formic acid-water (volume ratio of 8:1:0.5:0.3).

[0188] Example 2-2

[0189] The only difference from Example 1-1 is that the developing solvent in step (4) is ethyl acetate-toluene-formic acid-water (volume ratio of 8:0.3:0.5:0.3).

[0190] The thin-layer chromatography results of Example 2-1 are as follows: Figure 2 The thin-layer chromatography results of Example 2-2 are as follows: Figure 3 Among them, 1 is a citronella glycoside reference solution, 2 is a *Cymbidium ensifolium* reference medicinal material solution, and 3 is a *Cymbidium ensifolium* formula granule test solution. This can be found from... Figure 2 and Figure 3 The comparison showed that, under ultraviolet light (254 nm), the chromatograms of the reference medicinal material and the test sample under the developing solvent of ethyl acetate-toluene-formic acid-water (8:1:0.5:0.3) showed better colorimetric correspondence and separation.

[0191] Example 3: Examination of inspection conditions

[0192] Example 3-1

[0193] The only difference from Example 2-1 is that the inspection conditions in step (4) are spraying with 5% aluminum trichloride ethanol solution and inspecting under ultraviolet light (365nm).

[0194] The thin-layer chromatography results of Example 3-1 are as follows: Figure 4 The thin-layer chromatography results of Example 2-1 are as follows: Figure 2 Among them, 1 is the reference solution of citronella glycoside, 2 is the reference medicinal material solution of *Cymbidium faberi*, and 3 is the test solution of *Cymbidium faberi* granules. Figure 2 and Figure 4 The comparison showed that after spraying with 5% aluminum trichloride ethanol solution and examining under ultraviolet light (365nm), the spots of the reference standard chromatogram, the reference medicinal material chromatogram, and the test sample chromatogram were clearly visible, and the separation effect was better.

[0195] Example 4: Investigation of different thin-layer plates

[0196] Example 4-1

[0197] The only difference from Example 3-1 is that the thin-layer plate in step (4) is a thin-layer chromatography silica gel G plate.

[0198] Example 4-2

[0199] The only difference from Example 3-1 is that the thin-layer plate in step (4) is a thin-layer chromatography silica gel H plate.

[0200] The thin-layer chromatography results of Example 3-1 are as follows: Figure 4 The thin-layer chromatography result of Example 4-1 is as follows: Figure 5 The thin-layer chromatography results of Example 4-2 are as follows: Figure 6 Among them, 1 is a citronella glycoside reference solution, 2 is a *Cymbidium ensifolium* reference medicinal material solution, and 3 is a *Cymbidium ensifolium* formula granule test solution. After inspection... Figure 4 , Figure 5 and Figure 6 The comparison showed that among the three thin-layer plates, the silica gel G thin-layer plate had the best spot separation and correspondence in the chromatograms of the reference medicinal material and the test sample. Therefore, this experiment selected the silica gel G thin-layer plate as the best thin-layer plate for examining the Xiangqinglan formula granules.

[0201] Example 5: Investigation of Sample Size

[0202] Example 5-1

[0203] The only difference from Example 4-1 is that in step (4), the sample amount is 3 μl each of the reference solution, the reference medicinal material solution and the test solution, and they are spotted onto the same silica gel G thin-layer plate.

[0204] Example 5-2

[0205] The only difference from Example 4-1 is that in step (4), the sample amount is 5 μl each of the reference solution, the reference medicinal material solution and the test solution, and they are spotted onto the same silica gel G thin-layer plate.

[0206] Example 5-3

[0207] The only difference from Example 4-1 is that in step (4), the sample amount is 10 μl each of the reference solution, the reference medicinal material solution and the test solution, and they are spotted onto the same silica gel G thin-layer plate.

[0208] The thin-layer chromatography results of Examples 5-1 to 5-3 are as follows: Figure 7 Among them, 1-3 are the reference solution of citronella glycoside, 4-6 are the reference medicinal material solution of *Cymbidium faberi*, and 7-9 are the test solution of *Cymbidium faberi* granules. The sample volume for 1, 4, and 7 is 3 μl; for 2, 5, and 8, it is 5 μl; and for 3, 6, and 9, it is 10 μl. After inspection... Figure 7 The results showed that when the reference solution, the reference medicinal material solution, and the test solution were all 5 μl, the color development and separation effects of the test solution and the reference solution were the best. Therefore, 5 μl of the reference solution, the reference medicinal material solution, and the test solution were selected as the spotting amounts for this thin-layer chromatography.

[0209] Example 6: Investigation at different temperatures

[0210] Example 6-1

[0211] The reference solution prepared in Example 1-1 was used. The reference medicinal material solution and the test solution were subjected to (4) thin-layer chromatography analysis: The thin-layer chromatography conditions were as follows: thin-layer plate: silica gel G thin-layer plate; sample volume: 5 μl for the reference solution, the test solution and the reference medicinal material solution; developing solvent: ethyl acetate-toluene-formic acid-water solution with a volume ratio of 8:1:0.5:0.3 was used as the developing solvent and developed at an ambient temperature of 25°C; inspection: sprayed with 5% aluminum trichloride ethanol solution and inspected under ultraviolet light (365 nm).

[0212] Example 6-2

[0213] The only difference from Example 6-1 is that the unfolding temperature in step (4) is 10°C.

[0214] Example 6-3

[0215] The only difference from Example 6-1 is that the unfolding temperature in step (4) is 40°C.

[0216] The thin-layer chromatography result of Example 6-1 is as follows: Figure 8The thin-layer chromatography results of Example 6-2 are as follows: Figure 9 The thin-layer chromatography results of Examples 6-3 are as follows: Figure 10 Among them, 1 is a citronella glycoside reference solution, 2 is a *Cymbidium ensifolium* reference medicinal material solution, and 3 is a *Cymbidium ensifolium* formula granule test solution. After inspection... Figure 8-10 The comparison shows that at low temperatures, the color of the test sample chromatogram is not clear at the corresponding positions as that of the reference herb chromatogram. At high temperatures, some chromatographic spots are affected by the high temperature. Therefore, temperature changes affect the separation of chromatograms. This experiment can be carried out at room temperature (25℃).

[0217] Example 7: Investigation of different humidity levels

[0218] Example 7-1

[0219] The reference solution prepared in Example 1-1 was used. The reference medicinal material solution and the test solution were subjected to (4) thin-layer chromatography analysis: The thin-layer chromatography conditions were as follows: thin-layer plate: silica gel G thin-layer plate; sample volume: 5 μl for the reference solution, the test solution and the reference medicinal material solution; developing solvent: ethyl acetate-toluene-formic acid-water solution with a volume ratio of 8:1:0.5:0.3 was used as the developing solvent, and the development was carried out at 25°C and 40% ambient humidity; inspection: sprayed with 5% aluminum trichloride ethanol solution and inspected under ultraviolet light (365nm).

[0220] Example 7-2

[0221] The only difference from Example 7-1 is that the unfolding humidity in step (4) is 10%.

[0222] Example 7-3

[0223] The only difference from Example 7-1 is that the unfolding humidity in step (4) is 90%.

[0224] The thin-layer chromatography result of Example 7-1 is as follows: Figure 11 The thin-layer chromatography results of Example 7-2 are as follows: Figure 12 The thin-layer chromatography results of Examples 7-3 are as follows: Figure 13 Among them, 1 is a citronella glycoside reference solution, 2 is a *Cymbidium ensifolium* reference medicinal material solution, and 3 is a *Cymbidium ensifolium* formula granule test solution. After inspection... Figure 11-13 The comparison showed that the chromatographic spots in the thin-layer chromatogram of the control medicinal material under high humidity were diffused. Therefore, humidity changes affect the separation of chromatograms. Thus, this experiment can be carried out under low humidity (e.g., less than 90%).

[0225] 3. Construction of Feature Maps

[0226] 3.1 Feature Map Construction Method

[0227] 3.1.1 Chromatographic conditions and system suitability experiment:

[0228] The chromatographic column was octadecylsilane-bonded silica gel as the packing material; methanol was used as mobile phase A and 0.2% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 1; the flow rate was 0.5 ml per minute, the column temperature was 35℃, the detection wavelength was 328 nm, and the column type was GL InertSustain C18.

[0229] Table 1 Gradient elution program

[0230]

[0231] 3.1.2 Preparation of the reference solution:

[0232] Accurately weigh 0.5g of the powdered herb *Xiangqinglan* and place it in a 150ml conical flask. Add 20ml of methanol, weigh the solution, heat under reflux for 1 hour, cool, replenish the lost weight with methanol, shake well, filter, and use the filtrate as the reference solution for the herb.

[0233] Take appropriate amounts of argentin and rosmarinic acid reference standards, add 70% ethanol to prepare a mixed reference solution containing 60 μg of argentin and 120 μg of rosmarinic acid per 1 ml, shake well, and use it as the reference solution.

[0234] 3.1.3 Preparation of the test solution:

[0235] Take the granules of Xiangqinglan formula, grind them into a fine powder (pass through a No. 6 sieve), accurately weigh 0.6g, place them in a 150ml conical flask, add 20ml of methanol, weigh the powder, heat under reflux for 1 hour, cool, replenish the lost weight with methanol, shake well, filter, and take the filtrate as the test solution.

[0236] 3.1.4 Determination method:

[0237] Accurately pipette 10 μl each of the reference solution of the reference medicinal material, the reference solution of the reference substance, and the test solution, inject them into the liquid chromatograph, and determine the result.

[0238] 3.2 Methodological Examination of Feature Map Construction

[0239] The sample pretreatment method for the characteristic chromatogram of Xiangqinglan formulation particles was investigated, mainly focusing on the effects of solvent volume, reflux extraction time, different flow rates and different injection volumes on the characteristic chromatogram of Xiangqinglan formulation particles.

[0240] Example 8: Investigation of different solvent amounts

[0241] Example 8-1

[0242] Preparation of the test solution: Take an appropriate amount of Xiangqinglan formula granules, grind them finely (pass through a No. 6 sieve), take about 0.6g, weigh accurately, place in a stoppered conical flask, accurately add 20ml of methanol, weigh, heat under reflux for 1h, cool, weigh again, add methanol to make up the lost weight, shake well, filter, and take the filtrate as the test solution.

[0243] The determination shall be performed using the method described in section “3.1” above.

[0244] Example 8-2

[0245] The only difference from Example 8-1 is that the amount of methanol added is 50 ml.

[0246] Example 8-3

[0247] The only difference from Example 8-1 is that the amount of methanol added is 70 ml.

[0248] The characteristic chromatographic results of Example 8-1 are shown in the figure. Figure 14 The characteristic chromatographic results of Example 8-2 are as follows: Figure 15 The characteristic chromatographic results of Example 8-3 are as follows: Figure 16 The results are shown in Table 2. As the amount of solvent added increases, the peak areas of rosmarinic acid and argentin in the chromatogram decrease, and the peak shape of argentin in the chromatogram is not good when the solvent volume is 70 ml. Therefore, the optimal solvent volume for this experiment is set at 20 ml.

[0249] Table 2. Investigation of different solvent amounts

[0250]

[0251] Example 9: Investigation of different reflux times

[0252] Example 9-1

[0253] The only difference from Example 8-1 is that the heating reflux time is 0.5h.

[0254] Example 9-2

[0255] The only difference from Example 8-1 is that the heating reflux time is 1.5h.

[0256] The characteristic chromatographic results of Example 8-1 are shown in the figure. Figure 14 The characteristic chromatographic results of Example 9-1 are as follows: Figure 17 The characteristic chromatographic results of Example 9-2 are as follows: Figure 18 .contrast Figure 14 , Figure 17 and Figure 18The differences in chromatograms and changes in the peak area of ​​the target peaks were determined, and the results are shown in Table 3 below. When refluxed for 1 hour, the peak areas of both rosmarinic acid and citronellol were relatively high. Therefore, reflux for 1 hour was selected as the optimal reflux extraction time for this experiment.

[0257] Table 3 Comparison of different reflux times

[0258]

[0259] Example 10: Investigation of different mobile phases

[0260] Example 10-1

[0261] The preparation method of the test solution is the same as in Example 8-1.

[0262] Octadecylsilane-bonded silica gel was used as the filler; methanol was used as mobile phase A, and 0.2% phosphoric acid aqueous solution was used as mobile phase B, with a flow rate of 0.5 ml per minute.

[0263] The elution gradient is shown in the table below. Column temperature: 35℃; injection volume: 10μL; detection wavelength: 328nm.

[0264]

[0265] Example 10-2

[0266] The only difference from Example 10-1 is that:

[0267] The elution gradient is shown in the table below: Column temperature: 30℃; Injection volume: 10μL; Detection wavelength: 277nm.

[0268]

[0269] Example 10-3

[0270] The only difference from Example 10-1 is that:

[0271] The elution gradient is as follows: column temperature: 35℃; injection volume: 10μL; detection wavelength: 328nm.

[0272]

[0273] Example 10-4

[0274] The only difference from Example 10-1 is that:

[0275] The elution gradient is as follows: column temperature: 35℃; injection volume: 10μL; detection wavelength: 328nm.

[0276]

[0277] The chromatogram of the characteristic spectrum results of Example 10-1 is as follows: Figure 19 The chromatogram of the characteristic spectral results of Example 10-2 is as follows: Figure 20 The chromatograms of the characteristic spectral results in Examples 10-3 are as follows: Figure 21 The chromatograms of the characteristic spectral results of Examples 10-4 are as follows: Figure 22 .contrast Figures 19-22 The differences in chromatograms and the changes in the peak shape of the target peaks are observed. From the comparison of the chromatogram results, the gradient elution effect of methanol-0.2% phosphoric acid aqueous solution used in this invention is better, and the separation degree and peak shape of each chromatographic peak are better.

[0278] Example 11 Investigation of different flow velocities

[0279] Example 11-1

[0280] The only difference from Example 10-1 is that the flow rate is 0.8 ml / min.

[0281] The chromatogram of the characteristic spectral results of Example 11-1 is as follows: Figure 23 .contrast Figure 23 and Figure 19 The differences in chromatograms and changes in the peak shape of the target peak were observed. Comparison of the chromatogram results showed that the peak shape of piracetam was not good at a flow rate of 0.8 ml / min. Therefore, the optimal experimental flow rate was set at 0.5 ml / min.

[0282] Table 4 Comparison of different flow velocities

[0283]

[0284] Example 12 Investigation of different injection volumes

[0285] Example 12-1

[0286] The only difference from Example 10-1 is that the injection volume is 15 μl.

[0287] Example 12-2

[0288] The only difference from Example 10-1 is that the injection volume is 20 μl.

[0289] The chromatogram of the characteristic spectral results of Example 12-1 is as follows: Figure 24 The chromatogram of the characteristic spectral results in Example 12-2 is as follows: Figure 25 .contrast Figure 24 , Figure 25 and Figure 19 The differences in chromatograms and changes in the peak shape of the target peaks were observed by comparing the chromatogram results. It was found that as the injection volume increased, the chromatographic peaks shifted and their shapes deteriorated, with the optimal injection volume being 10 μl. Therefore, the optimal injection volume for this experiment was set at 10 μl.

[0290] Example 13 Investigation at different column temperatures

[0291] Example 13-1

[0292] The only difference from Example 10-1 is that the column temperature is 30°C.

[0293] Example 13-2

[0294] The only difference from Example 10-1 is that the column temperature is 40°C.

[0295] The chromatogram of the characteristic spectral results of Example 13-1 is as follows: Figure 26 The chromatogram of the characteristic spectral results in Example 13-2 is as follows: Figure 27 .contrast Figure 26 , Figure 27 and Figure 19 The differences in chromatograms and the variations in target peak shapes are evident from the comparison of chromatographic results: the characteristic chromatograms differ at different column temperatures. Comparison of peak shapes and numbers reveals that 35℃ exhibits better peak shapes and a greater number of peaks; therefore, 35℃ is selected as the optimal detection column temperature.

[0296] Example 14 Investigation of different chromatographic columns

[0297] Example 14-1

[0298] The only difference from Example 10-1 is that the chromatographic column is a GL InertSustain AQ-C18.

[0299] Example 14-2

[0300] The only difference from Example 10-1 is that the chromatographic column is a YMC Triart C18.

[0301] Example 14-3

[0302] The only difference from Example 10-1 is that the chromatographic column is GL InertSustain ODS-3.

[0303] Example 14-4

[0304] The only difference from Example 10-1 is that the chromatographic column is an Agilent 5TC-C18.

[0305] Examples 14-5

[0306] The only difference from Example 10-1 is that the chromatographic column is a Waters Xbridge C18.

[0307] The chromatogram of the characteristic spectral results of Example 14-1 is as follows: Figure 28 The chromatogram of the characteristic spectral results of Example 14-2 is as follows: Figure 29 The chromatograms of the characteristic spectral results in Example 14-3 are as follows: Figure 30 The chromatograms of the characteristic spectral results of Examples 14-4 are as follows: Figure 31 The chromatograms of the characteristic spectral results of Examples 14-5 are as follows: Figure 32 .contrast Figure 28 , Figure 29 , Figure 30 , Figure 31 , Figure 32 and Figure 19 The differences in chromatograms and the variations in target peak shapes were observed through a comparison of the chromatographic results. Significant differences were found in the characteristic chromatograms obtained using different columns. Comparison of peak shapes and numbers revealed that GL InertSustain C18, GL InertSustain AQ-C18, and YMC Triart C18 columns showed the best peak shapes and separation performance for each target peak. Other columns could not completely reproduce the nine characteristic peaks in the chromatograms. Therefore, GL InertSustain, GL InertSustain AQ-C18, or YMC Triart C18 were selected as the optimal columns for this experiment.

[0308] 3.4 Methodological Validation of Feature Map Construction

[0309] 3.4.1 Specificity Examination

[0310] Accurately weigh 0.3g of *Cymbidium faberi* (passed through a 100-mesh sieve) and place it in a 150ml Erlenmeyer flask. Add 20ml of methanol, weigh the flask, heat under reflux for 1 hour, cool, and replenish the lost weight with methanol. Shake well, filter, and use the filtrate as the negative test solution. Perform the determination according to the method described in "Example 8-1" above. The results show that the main characteristic peaks in the chromatogram of the test sample are well separated, and the method has good specificity. See details below. Figure 33 .

[0311] 3.4.2 Precision Test

[0312] Precision test of the reference standard: Following the test method in "Example 8-1", the mixed reference standard solution was injected six times consecutively, and the peak area, retention time, and RSD value were measured and calculated. The results showed that the peak area RSD value of rosmarinic acid was 0.11%, and the retention time RSD value was 0.08%; the peak area RSD value of piracetam was 0.13%, and the retention time RSD value was 0.03%. Using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version A)" of the Chinese Pharmacopoeia Commission, the HPLC chromatogram was imported, and after multi-point calibration and data matching, the chromatogram was generated using the average method. (See attached image). Figure 34 As shown in Table 5, the precision of the reference standard is good.

[0313] Table 5 Precision of Reference Standards

[0314]

[0315] Precision determination of the test sample: A sample of Xiangqinglan formula granules (batch number: 20220303) was taken, and a test solution was prepared according to the preparation method of the test sample in Example 8-1. The solution was injected continuously six times under the chromatographic conditions in Example 8-1, and the peak area, retention time, and RSD value were measured and calculated. The HPLC chromatogram was imported using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version A)" of the Chinese Pharmacopoeia Commission. After multi-point correction and data matching, the chromatogram was generated using the average method. Results are shown below. Figure 35 And Table 6, peak area RSD values: 0.62% (peak 1), 0.29% (peak 2), 0.27% (peak 3), 0.29% (peak 4), 2.05% (peak 5), 0.27% (peak 6), 0.57% (peak 7), 0.33% (peak 8 (S)), 0.45% (peak 9); retention time RSD values: 0.06% (peak 1), 0.04% (peak 2), 0.05% (peak 3), 0.05% (peak 4), 0.04% (peak 5) The percentages of peaks 6, 7, 8, and 9 were 0.04%, 0.03%, 0.02%, and 0.02%, respectively. The average retention times relative to peak S were 0.345 (peak 1), 0.381 (peak 2), 0.493 (peak 3), 0.557 (peak 4), 0.615 (peak 5), 0.642 (peak 6), 0.738 (peak 7), 1.000 (peak 8 (S)), and 1.017 (peak 9). The results indicate that the sample has good precision.

[0316] Table 6 Precision of the test sample

[0317]

[0318] Table 6 (continued)

[0319]

[0320] 3.4.3 Repeatability Test

[0321] A sample of Xiangqinglan formula granules (batch number: 20220303) was taken. A test solution was prepared according to the method described in Example 8-1, and six parallel test solutions were prepared. 10 μl of each test solution was injected into a liquid chromatograph and analyzed under the chromatographic conditions described in Example 8-1. The peak area and retention time RSD were calculated. The HPLC chromatogram was imported using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version A)" issued by the Chinese Pharmacopoeia Commission. After multi-point calibration and data matching, the chromatogram was generated using the average method. The results are shown below. Figure 36And as shown in Table 7, the peak area RSD values ​​are: 8.74% (peak 1), 2.22% (peak 2), 3.47% (peak 3), 1.12% (peak 4), 1.92% (peak 5), 2.77% (peak 6), 3.07% (peak 7), 0.83% (peak 8 (S)), and 2.22% (peak 9); the retention time RSD values ​​are: 0.13% (peak 1), 0.11% (peak 2), 0.11% (peak 3), 0.11% (peak 4), and 0.09% (peak 5). The percentages of peaks 6, 7, 8, and 9 were 0.09%, 0.08%, 0.03%, and 0.03%, respectively. The average retention times relative to peak S were 0.346 (peak 1), 0.381 (peak 2), 0.494 (peak 3), 0.558 (peak 4), 0.615 (peak 5), 0.643 (peak 6), 0.738 (peak 7), 1.000 (peak 8), and 1.017 (peak 9). The results indicate that the test sample has good repeatability.

[0322] Table 7 Repeatability of Test Samples

[0323]

[0324] Table 7 (continued)

[0325]

[0326] 3.4.4 Intermediate Precision Test

[0327] Six parallel batches of the same batch of Xiangqinglan formula granules (batch number: 20220303) were prepared at different times according to the preparation method of the test solution in Example 8-1. 10 μl of each test solution was injected into a liquid chromatograph and analyzed under the chromatographic conditions in Example 8-1. The peak area and retention time RSD were calculated. The HPLC chromatogram was imported using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version A)" of the Chinese Pharmacopoeia Commission. After multi-point correction and data matching, the chromatogram was generated using the average method. The results are shown below. Figure 37And Table 8, peak area RSD values: 1.79% (peak 1), 1.29% (peak 2), 2.00% (peak 3), 0.50% (peak 4), 0.46% (peak 5), 1.69% (peak 6), 2.12% (peak 7), 0.66% (peak 8 (S)), 2.54% (peak 9); retention time RSD values: 0.05% (peak 1), 0.04% (peak 2), 0.04% (peak 3), 0.05% (peak 4), 0.04% (peak 5), 0.04% (peak 6), 0.05% (peak 7), 0.03% (peak 8 (S)), 0.03% (peak 9); average retention time relative to peak S: 0.346 (peak 1), 0.381 (peak 2), 0.494 (peak 3), 0.558 (peak 4), 0.616 (peak 9). 5) 0.643 (peak 6), 0.739 (peak 7), 1.000 (peak 8 (S)), 1.017 (peak 9); Peak area RSD values ​​compared with repeatability data: 7.09% (peak 1), 2.31% (peak 2), 3.39% (peak 3), 1.06% (peak 4), 1.58% (peak 5), 2.77% (peak 6), 3.04% (peak 7), 0.72% (peak 8 (S)), 2.54% (peak 9); Retention time RSD values: 0.12% (peak 1), 0.11% (peak 2), 0.13% (peak 3), 0.13% (peak 4), 0.11% (peak 5), 0.11% (peak 6), 0.10% (peak 7), 0.04% (peak 8 (S)), 0.04% (peak 9). The results indicate good intermediate precision.

[0328] Table 8 Intermediate Precision

[0329]

[0330] Table 8 (Continued)

[0331]

[0332] Table 8 (Continued) - 2

[0333]

[0334] Table 8 (Continued) - 3

[0335]

[0336] 3.4.5 Stability Test

[0337] Approximately 0.6 g of Xiangqinglan formula granules (batch number: 20220303) was taken and analyzed at 0 h, 4 h, 9 h, 14 h, 26 h, 37 h, and 48 h according to the preparation method and chromatographic conditions of the test solution in Example 8-1. The results showed that the peak shape and number of peaks in the characteristic chromatogram of the test sample were basically consistent (see...). Figure 33 The characteristic spectrum contains 9 common peaks. Using the piracetam glycoside peak (peak 8) as the reference peak S, the relative retention times and relative peak areas of peaks 1, 2, 3, 4, 5, 6, 7, and 9 with peak S were calculated, and the RSD values ​​were calculated. The results are shown in […]. Figure 38 See Table 9. The calculation results show the peak area RSD values: 0.47% (peak 1), 1.12% (peak 2), 0.88% (peak 3), 1.11% (peak 4), 1.29% (peak 5), 1.11% (peak 6), 1.56% (peak 7), 1.11% (peak 8 (S)), 0.08% (peak 9); retention time RSD values: 0.17% (peak 1), 0.17% (peak 2), 0.14% (peak 3), 0.11% (peak 4), 0.11% (peak 5). The percentages of peaks 6, 7, 8, and 9 were 0.11%, 0.06%, 0.03%, and 0.03%, respectively. The average retention times relative to peak S were 0.346 (peak 1), 0.382 (peak 2), 0.493 (peak 3), 0.557 (peak 4), 0.615 (peak 5), 0.642 (peak 6), 0.738 (peak 7), 1.000 (peak 8 (S)), and 1.017 (peak 9). The results indicate that the sample has good stability within 48 hours.

[0338] Table 9 Stability test results

[0339]

[0340] Table 9 (Continued)

[0341]

[0342] Table 9 (Continued) - 2

[0343]

[0344] In summary, the specificity, precision, repeatability, intermediate precision, and stability of the proposed characteristic spectral method all met the requirements, indicating that the established method can be well used for the determination of characteristic spectra of Xiangqinglan formula granules.

[0345] 3.5 Characterization Analysis of Three Batches of Xiangqinglan Formula Granules

[0346] 3.5.1 Characteristic Spectrum Determination

[0347] Following the preparation method and chromatographic conditions of the test solution in Example 8-1, the characteristic chromatograms of three batches of *Xiangqinglan* formula granules (9220603; 9220604; 9220605) were determined. Localization was performed using piracetamine. The results showed that the characteristic chromatograms of the *Xiangqinglan* formula granules contained nine common peaks, corresponding to the retention times of nine characteristic peaks in the chromatograms of the reference medicinal material. Peak 8 corresponds to the piracetamine reference standard. For details of the common peak characteristic chromatograms, please refer to [link to relevant documentation]. Figure 39 .

[0348] 3.5.2 Relative retention time of characteristic chromatograms

[0349] According to the proposed characteristic spectrum analysis method, the characteristic spectra of three batches of Xiangqinglan formula granules were determined. The results showed that there were 9 common peaks in the characteristic spectrum. Taking the piracetam glycoside peak (peak 8) as the reference peak S, the relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, and 9 with peak S were calculated, and the RSD values ​​were calculated. See Table 10 for details.

[0350] Table 10. Relative retention times of common peaks in three batches of Xiangqinglan formula granules.

[0351]

[0352] In summary, the method for determining the characteristic chromatograms of Xiangqinglan formula granules established using high-performance liquid chromatography (HPLC) was tested for precision, repeatability, intermediate precision, and stability according to the "Guiding Principles for Analytical Method Validation (General Rule 9101)" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, and it met the requirements. Using the proposed characteristic chromatogram analysis method, the characteristic chromatograms of three batches of Xiangqinglan formula granules were determined. The results identified nine common characteristic peaks. Using the piracetamin peak (peak 8) as the reference peak S, the relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, and 9 with peak S were calculated. The average relative retention times of the peaks in the sample precision chromatogram were set as the specified values: 0.345 (peak 1), 0.381 (peak 2), 0.494 (peak 3), 0.556 (peak 4), 0.616 (peak 5), 0.643 (peak 6), 0.737 (peak 7), and 1.017 (peak 9). Considering the errors caused by various factors such as experimental operation, instruments, and reagents, the allowable range for relative retention time is set at ±10%.

[0353] 4. Content determination:

[0354] 4.1 Selection of analytes

[0355] *Cymbidium faberi* contains various chemical components, including flavonoids, volatile oils, terpenes, amino acids, and trace elements. Literature reports that the ethanol extract of *Cymbidium faberi*, after macroporous resin column chromatography, yielded eight compounds from the 50% ethanol eluate, identified as apigenin, luteolin, kaempferol, isorhamnetin, acetin-7-O-(6-O-malonyl-β-D-glμcopyranoside), agstachoside, and eugenol. Other studies have reported that the flavonoid fraction of *Cymbidium faberi* is its active component for treating angina pectoris in coronary heart disease. Among these, acetin, luteolin, kaempferol, 2'-(p-hydroxycinnamoyloxyastragaloside), and isorhamnetin exhibit significant pharmacological activities such as anti-myocardial ischemia, with acetin being the main active ingredient. Therefore, acetin was used as a control indicator for flavonoids in *Cymbidium faberi*. HPLC was used to determine the content of acetin in *Cymbidium faberi*, and corresponding content limits were established to control the quality of the drug.

[0356] 4.2 Test Methods

[0357] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0358] Chromatographic conditions and system suitability test:

[0359] The column was filled with octadecylsilane-bonded silica gel (4.6 mm × 150 mm, 5 μm); the mobile phase was acetonitrile-0.3% phosphoric acid aqueous solution (volume ratio 22:78); the detection wavelength was 333 nm; the flow rate was 1.0 mL / min; the column temperature was 30 ℃; the injection volume was 10 µl; the theoretical plate number calculated based on the piracetam peak should not be less than 2000.

[0360] Preparation of reference solution: Weigh an appropriate amount of piracetamine reference standard accurately, add 70% ethanol to prepare a solution containing 26 μg per ml, shake well, and the solution is ready.

[0361] Preparation of the test solution: Take an appropriate amount of Xiangqinglan formula granules, grind them finely (pass through a No. 6 sieve), take about 0.3g, weigh accurately, place in a 150ml stoppered conical flask, accurately add 50ml of 70% methanol solution, weigh, sonicate for 20 minutes, take it out, let it stand at room temperature, make up the missing weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0362] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0363] 4.3 Methodological Investigation of Content Determination

[0364] The sample pretreatment method for determining the content of *Xiangqinglan* formula granules was investigated, mainly focusing on the effects of extraction solvent, extraction method, extraction time, and solvent volume on the content of piracetamin in *Xiangqinglan* formula granules.

[0365] 4.3.1 Investigation of different extraction solvents

[0366] Example 15-1

[0367] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder (pass through a No. 6 sieve), take about 0.3g, weigh it accurately, place it in a 150ml stoppered conical flask, accurately add 25ml of 70% methanol solution, weigh it, sonicate for 10 minutes, take it out, let it stand at room temperature, make up the missing weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0368] Test according to the test method in 4.2.

[0369] Example 15-2

[0370] The only difference from Example 15-1 is that the solvent used is 70% ethanol.

[0371] Example 15-3

[0372] The only difference from Example 15-1 is that the solvent used is methanol.

[0373] The chromatographic conditions under section 4.2 were followed. The results showed that the content of araliain extracted by different extraction solvents varied greatly, with 70% methanol showing the best extraction effect. Therefore, 70% methanol was determined to be the optimal solvent, and the results are shown in Table 11.

[0374] Table 11 Investigation of different extraction solvents

[0375]

[0376] 4.3.2 Examination of Different Extraction Methods

[0377] Example 16-1

[0378] The only difference from Example 15-1 is that the test sample is prepared by ultrasonic extraction.

[0379] Example 16-2

[0380] The only difference from Example 16-1 is that the test sample is prepared by reflux extraction.

[0381] The results showed that the content changes little under ultrasound and reflux, with an RSD of 0.72%. Ultrasonic extraction was convenient and had good parallelism, so it was chosen. The results are shown in Table 12.

[0382] Table 12 Examination of Different Extraction Methods

[0383]

[0384] 4.3.3 Investigation of different ultrasound times

[0385] Example 17-1

[0386] The only difference from Example 15-1 is that the preparation and extraction time is 20 minutes.

[0387] Example 17-2

[0388] The only difference from Example 15-1 is that the preparation and extraction time is 30 minutes.

[0389] Example 17-3

[0390] The only difference from Example 15-1 is that the preparation and extraction time is 10 minutes.

[0391] The results showed that the content varied significantly between ultrasound for 10 minutes, 20 minutes, and 30 minutes, with the highest content observed after 20 minutes of ultrasound. Therefore, ultrasound for 20 minutes was selected. The results are shown in Table 13.

[0392] Table 13 Investigation of different ultrasound times

[0393]

[0394] 4.3.4 Solvent Ratio Examination

[0395] Example 18-1

[0396] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder (pass through a No. 6 sieve), accurately weigh about 0.3g, place it in a 150ml stoppered volumetric flask, accurately add 25ml of 70% methanol solution, weigh, sonicate for 20 minutes, remove, let it stand at room temperature, make up the missing weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0397] Example 18-2

[0398] The only difference from Example 18-1 is that the amount of 70% methanol solvent added is 50 ml.

[0399] Example 18-3

[0400] The only difference from Example 18-1 is that the amount of 70% methanol solvent added is 100 ml.

[0401] The results showed that the extraction effect was best when the solvent volume of the test sample was 50 ml. Therefore, the solvent volume was determined to be 50 ml. The results are shown in Table 14.

[0402] Table 14 Solvent Ratio Study

[0403]

[0404] In summary, the main parameters for the preparation method of the test solution are as follows: Take an appropriate amount of this product, grind it finely (passing through a No. 6 sieve), take about 0.3g, weigh it accurately, place it in a stoppered conical flask, accurately add 50ml of 70% methanol, seal tightly, weigh it, sonicate (power 250W, frequency 53kHz) for 20 minutes, remove it, cool it, weigh it again, replenish the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0405] 4.4 Methodological Investigation of Content Determination

[0406] The chromatographic conditions for determining the content of Xiangqinglan formula granules were investigated.

[0407] 4.4.1 Volume ratio of the mobile phase

[0408] Example 19-1

[0409] The preparation of the test solution is the same as in Example 15-1.

[0410] Chromatographic conditions: Column: Agilent C18 (4.6×150mm, 5μm); Detection wavelength: 333nm; Mobile phase: acetonitrile-0.3% phosphoric acid aqueous solution (volume ratio 22:78); Flow rate: 1.0mL / min; Column temperature: 30℃.

[0411] Example 19-2

[0412] The only difference from Example 19-1 is that the mobile phase is acetonitrile-0.3% phosphoric acid aqueous solution (volume ratio 25:75).

[0413] Example 19-3

[0414] The only difference from Example 19-1 is that the mobile phase is acetonitrile-0.3% phosphoric acid aqueous solution (volume ratio 23:77).

[0415] Example 19-4

[0416] The only difference from Example 19-1 is that the mobile phase is acetonitrile-0.3% phosphoric acid aqueous solution (volume ratio 20:80).

[0417] The results of the separation, symmetry and peak area of ​​piracetamine under different mobile phase ratios of acetonitrile-0.3% phosphoric acid aqueous solution were examined and compared. It was found that the separation and symmetry of the test sample met the requirements when the mobile phase ratio was acetonitrile-0.3% phosphoric acid aqueous solution (22:78). Considering all factors, acetonitrile-0.3% phosphoric acid (22:78) was selected as the optimal experimental mobile phase. The results are shown in Table 15.

[0418] Table 15 Results of investigation on different mobile phase ratios

[0419]

[0420] 4.4.2 Investigating different column temperatures

[0421] Example 20-1

[0422] The preparation of the test solution is the same as in Example 15-1.

[0423] Chromatographic conditions: Column: Agilent C18 (4.6×150mm, 5μm); Detection wavelength: 333nm; Mobile phase: acetonitrile-0.3% phosphoric acid (volume ratio 22:78); Flow rate: 1.0mL / min; Column temperature: 30℃.

[0424] Example 20-2

[0425] The only difference from Example 20-1 is that the column temperature is 25°C.

[0426] Example 20-3

[0427] The only difference from Example 20-1 is that the column temperature is 40°C.

[0428] The separation, symmetry, and peak area of ​​piracetam were examined and compared at column temperatures of 25℃, 30℃, and 40℃. It was found that the separation and symmetry of piracetam met the requirements at different column temperatures, but the content decreased at 40℃. After comprehensive consideration, 30℃ was selected as the detection column temperature. The results are shown in Table 16.

[0429] Table 16 Results of investigation at different column temperatures

[0430]

[0431] In the actual development process of the method for detecting the content of Xiangqinglan formula granules, optimization was carried out in batches on a parameter-by-parameter basis. Therefore, situations may arise where the scheme is consistent but the results are different. To further verify the accuracy of the experiment, RSD verification was performed on the results obtained from different batches with the same scheme as Example 15-1. The results are shown in Table 17 below.

[0432] Table 17

[0433]

[0434] As shown in the table, when the experimental conditions are consistent with those in Example 15-1, the RSD is less than 5% in each batch, indicating that when all conditions in the content determination are consistent, there is no significant difference in the test results at different times.

[0435] 4.5 Validation of content determination analytical methods

[0436] 4.5.1 Linearity Test

[0437] Accurately pipette 0.5 ml, 2 ml, 4 ml, 6 ml, 8 ml, and 9 ml of the citronellol reference solution (104.958 μg / ml) into 10 ml volumetric flasks, dilute to the mark with 70% ethanol, and mix well. Accurately pipette 10 μL into each flask and determine the peak area. The results showed that linear regression with citronellol concentration (μg / ml) as the x-axis and peak area as the y-axis yielded the regression equation Y = 0.4846X + 0.0424, R² = 1. The results indicate that citronellol exhibits good linearity in the range of 5.2479 μg / ml to 94.4622 μg / ml. (See attached figure). Figure 40 Table 18.

[0438] Table 18. Experimental data on the linear relationship of citronellol reference standard.

[0439]

[0440] 4.5.2 Precision Test

[0441] The reference solution and test solution prepared according to the method in section 4.2 were accurately pipetted and injected into the liquid chromatograph for determination 6 times. The RSD value of the reference solution was 0.11% and the RSD value of the test solution was 0.12%. The results showed that the precision was good. The results are shown in Tables 19 and 20.

[0442] Table 19 Precision Test of Reference Standard

[0443]

[0444] Table 20 Precision Test of Test Samples

[0445]

[0446] 4.5.3 Repeatability Test

[0447] Six samples from the same batch were accurately weighed and operated according to the preparation and determination method of the test solution under section 4.2. The RSD (%) of the piracetamine content was calculated. The results showed good repeatability with an RSD of 0.55%. The results are shown in Table 21.

[0448] Table 21 Repeatability of Test Samples

[0449]

[0450] 4.5.4 Intermediate Precision Test

[0451] Six samples of the same batch of this product were prepared on the first day and six samples were prepared on the third day. The test solutions were prepared according to the test solution preparation method under section 4.2. 10 μL of the test solution was injected into the liquid chromatograph for determination and the content of piracetamine was calculated. The results are shown in Table 22. The intermediate precision is good.

[0452] Table 22 Intermediate Precision

[0453]

[0454] 4.5.5 Stability

[0455] The same sample solution was measured at 0, 6, 12, 21, 24, 30, 36, 45, and 48 hours after preparation, and the RSD (%) was calculated. The results showed that the RSD of the sample was 0.54% within 48 hours, indicating that the sample was stable within 48 hours. The results are shown in Table 23.

[0456] Table 23 Stability test results

[0457]

[0458] 4.5.6 Accuracy (Spiking and Recovery) Test

[0459] Preparation of reference stock solution: Accurately weigh 0.01071 g of piperazine reference standard (potency 98%), place it in a 100 ml volumetric flask, add 70% ethanol to dissolve and dilute to the mark, shake well, and the solution is ready.

[0460] 50% Spiked Solution: Accurately pipette 5 ml of the reference standard stock solution into a 150 ml Erlenmeyer flask, evaporate to dryness, accurately weigh 0.15 g of the test sample, place it in the same Erlenmeyer flask, accurately add 50 ml of 70% methanol, sonicate for 20 minutes, remove, allow to room temperature, dilute to the mark with 70% methanol, and shake well. Perform three parallel treatments.

[0461] Accurately pipette 6.5 ml of the reference standard stock solution into a 150 ml Erlenmeyer flask and evaporate to dryness. Accurately weigh 0.15 g of the test sample and place it in the same Erlenmeyer flask. Accurately add 50 ml of 70% methanol, sonicate for 20 minutes, remove, and allow to reach room temperature. Dilute to the mark with 70% methanol and shake well. Perform three parallel treatments.

[0462] 150% Spiked Solution: Accurately pipette 8 ml of the reference standard stock solution into a 150 ml Erlenmeyer flask, evaporate to dryness, accurately weigh 0.15 g of the test sample, place it in the same Erlenmeyer flask, accurately add 50 ml of 70% methanol, sonicate for 20 minutes, remove, allow to room temperature, dilute to the mark with 70% methanol, and shake well. Perform three parallel treatments.

[0463] Accurately pipette 50%, 100%, and 150% spiked solutions, injecting two syringes into each solution into the liquid chromatograph for determination and calculation of the recovery rate (%). The results showed good recovery (90%–108%), and the experimental results are shown in Table 24.

[0464] Table 24 Results of the sample recovery experiment

[0465]

[0466] 4.5.7 Detection Limits Assessment

[0467] (1) Cirsium glycoside reference standard

[0468] Detection limit solution: Accurately pipette the reference solution and dilute it stepwise with 70% ethanol solution to achieve a signal-to-noise ratio (S / N) of piperazine of 2:1 or 3:1. Take a 5.2479 μg / ml piperazine reference solution, dilute it, and inject 10 μl into the liquid chromatograph for determination. When the S / N is approximately 3.3, the concentration of the diluted solution is 0.052479 μg / ml. Therefore, the minimum detectable limit of piperazine is approximately 0.052479 μg / ml.

[0469] (2) Xiangqinglan formula granules test sample

[0470] Detection limit solution: Accurately pipette the test solution and serially dilute with 70% methanol to achieve a signal-to-noise ratio (S / N) of piperazine of 2:1 or 3:1. Prepare the solution according to the test solution preparation method. Accurately pipette the test solution and serially dilute with 70% methanol. Inject 10 μl of the solution into the liquid chromatograph for determination. When the S / N is 2.8, the concentration of piperazine in the test solution is 0.1310 μg / mL. Therefore, the minimum detectable limit is 0.1310 μg / mL. Results are shown in Table 25. Figure 41 .

[0471] Table 25 Detection Limits

[0472]

[0473] 4.5.8 Limit of Quantitation Assessment

[0474] (1) Cirsium glycoside reference standard

[0475] Limit of Quantification Solution: Accurately pipette the reference solution and serially dilute it with 70% ethanol solution to achieve a signal-to-noise ratio of 10:1 for piperazine. Take a 20.9916 μg / ml piperazine reference solution, dilute it, and pipette 10 μl of the solution into the liquid chromatograph for determination via six consecutive injections. When the signal-to-noise ratio (S / N) is approximately 10.1, the diluted concentration is 0.209916 μg / ml. Therefore, the minimum quantitation concentration is 0.209916 μg / ml.

[0476] (2) Xiangqinglan formula granules test sample

[0477] Limit of Quantification (LOQ) Solution: Accurately pipette the test solution and serially dilute with 70% methanol to achieve a signal-to-noise ratio (S / N) of piperazine of 0.2619 μg / mL. Prepare the solution according to the test solution preparation method. Accurately pipette the test solution and serially dilute with 70% methanol. Inject 10 μL of the solution into the liquid chromatograph six times consecutively for determination. The results show that when the S / N is approximately 11.9, the concentration of piperazine in the test solution is 0.2619 μg / mL. Therefore, the minimum quantitation concentration is 0.2619 μg / mL. The results are shown in Table 26. Figure 42 .

[0478] Table 26 Limits of Quantification

[0479]

[0480] 4.5.9 Summary

[0481] In summary, the entire analytical method was tested for linearity, precision, repeatability, intermediate precision, stability, accuracy, limit of quantitation, and limit of detection. The results all met the requirements, indicating that the established method can be well used for the determination of citronellol content.

[0482] The proposed limit range for the content of granules in the 5-fragrant orchid formula.

[0483] 5.1 Content determination of three batches of pilot-scale samples

[0484] Following the proposed content analysis method, the content of citronellol in 6 batches of finished products was determined, and the results are shown in Table 27.

[0485] Table 27. Content of citronella in 6 batches of finished products

[0486]

[0487] The content of citronella glycoside in six batches of Xiangqinglan formula granules was determined, and the average content was 3.827 mg / g.

Claims

1. A method for quality testing of Xiangqinglan formula granules, characterized in that, It includes the following steps: separating the test solution 1 containing Xiangqinglan formula particles by gradient elution using high performance liquid chromatography; In the high-performance liquid chromatography, mobile phase A is methanol, and mobile phase B is an aqueous solution of phosphoric acid; the column temperature is 30-40℃.

2. The quality testing method for Xiangqinglan formula granules as described in claim 1, characterized in that, The quality testing method for the Xiangqinglan formula granules meets one or more of the following conditions: (1) The Xiangqinglan formula granules include citronellol and / or rosmarinic acid; (2) The preparation method of the test solution 1 containing Xiangqinglan formula particles includes the following steps: extracting and processing the mixture containing Xiangqinglan formula particles and solvent, filtering, and using the filtrate as test solution 1; (3) In the high performance liquid chromatography, reference solution 1 and reference medicinal material reference solution 1 are also prepared; (4) In the high performance liquid chromatography, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1%-0.5%, for example 0.2%, 0.3% or 0.4%; (5) In the high performance liquid chromatography, the column temperature is 32-38℃, for example 35℃; (6) In the high performance liquid chromatography, the flow rate of the mobile phase is 0.3-1.0 mL / min, preferably 0.5-0.8 mL / min, for example 0.6 mL / min; (7) In the high-performance liquid chromatography, the injection volume is 10-20 μL, for example 15 μL; and, (8) In the high performance liquid chromatography, the detection wavelength is 200-400nm, for example 328nm.

3. The quality testing method for Xiangqinglan formula granules as described in claim 1, characterized in that, In the high-performance liquid chromatography, gradient elution is as follows: 、 、 or, The percentages in the table represent the volume percentage of each component in the total volume of mobile phase A and mobile phase B, respectively.

4. The quality testing method for Xiangqinglan formula granules as described in claim 2, characterized in that, The quality testing method for the Xiangqinglan formula granules meets one or more of the following conditions: (1) In the preparation method of the test solution 1, the particle size of the Xiangqinglan formula particles is that they pass through a No. 6 sieve; (2) In the preparation method of the test solution 1, the solvent is methanol and / or an aqueous solution of ethanol; the volume percentage of ethanol in the aqueous solution of ethanol is preferably 40%-80%, for example 70%; (3) In the preparation method of the test solution 1, the mass-volume ratio of the Xiangqinglan formula granules and the solvent is 0.6g:(10-80)mL, preferably 0.6g:(20-70)mL, for example 0.6g:40mL or 0.6g:50mL; (4) In the preparation method of the test solution 1, the extraction treatment is ultrasonic treatment or reflux treatment; and, (5) In the preparation method of the test solution 1, the extraction time is 0.2-2h, preferably 0.5-1.5h, for example 1h.

5. The quality testing method for Xiangqinglan formula granules as described in claim 2, characterized in that, The quality testing method for the Xiangqinglan formula granules meets one or more of the following conditions: (1) The reference solution 1 comprises reference 1 and a solvent, wherein reference 1 is argiol and / or rosmarinic acid; the solvent is preferably an aqueous solution of methanol and / or ethanol; the volume percentage of ethanol in the aqueous solution is preferably 40%-80%, for example 70%; When reference standard 1 includes citronellol, the mass concentration of citronellol in the reference standard solution 1 is preferably 40-70 μg / mL, for example 60 μg / mL; When reference standard 1 includes rosmarinic acid, the mass concentration of rosmarinic acid in the reference standard solution 1 is preferably 100-130 μg / mL, for example 120 μg / mL; (2) The preparation method of the reference solution 1 of the reference medicinal material includes the following steps: extracting and processing the mixture including the reference medicinal material 1 and the solvent, filtering, and using the filtrate as the reference solution 1 of the reference medicinal material; In the reference solution 1, the solvent is preferably an aqueous solution of methanol and / or ethanol; the volume percentage of ethanol in the aqueous solution is preferably 40%-80%, for example, 70%. The preferred mass-to-volume ratio of the reference medicinal material 1 to the solvent is 0.5g:(10-30)mL, for example, 0.5g:20mL; The preferred extraction and processing method is ultrasonic treatment or reflux treatment; The extraction process is preferably performed for 0.5-1.5 hours, for example, 1 hour.

6. The method for quality testing of Xiangqinglan formula granules as described in claim 1, characterized in that, The fingerprint spectrum obtained after gradient elution separation of the test solution 1 of the Xiangqinglan formula granules by high performance liquid chromatography includes two characteristic peaks. Taking the citronellol peak as the reference peak, the average relative retention time of each characteristic peak is: Peak 4 is rosmarinic acid, with an average relative retention time of 0.558 and an RSD of 0.05%. Preferably, the fingerprint spectrum obtained after gradient elution separation of the test solution 1 of the Xiangqinglan formula granules by high performance liquid chromatography includes 9 characteristic peaks. Taking the citronellol peak as the reference peak, the average relative retention times of each characteristic peak are as follows: The mean relative retention time of peak 1 was 0.348, with an RSD of 0.02%. The mean relative retention time of peak 2 was 0.383, with an RSD of 0.03%. The mean relative retention time of peak 3 was 0.498, with an RSD of 0.03%. Peak 4 is rosmarinic acid, with an average relative retention time of 0.558 and an RSD of 0.05%. The mean relative retention time of peak 5 was 0.619, with an RSD of 0.04%. The mean relative retention time of peak 6 was 0.647, with an RSD of 0.04%. The mean relative retention time of peak 7 was 0.739, with an RSD of 0.04%. The mean relative retention time of peak 9 was 1.017, with an RSD of 0.02%.

7. The method for quality testing of Xiangqinglan formula granules as described in claim 1, characterized in that, The quality testing method for the Xiangqinglan formula granules satisfies steps (1) and / or (2) below: (1) Thin-layer chromatography was used to detect the test solution 2 containing the Xiangqinglan formula particles; (2) The content of piracetam in the test solution 3 of the Xiangqinglan formula granules was determined by high performance liquid chromatography.

8. The method for quality testing of Xiangqinglan formula granules as described in claim 7, characterized in that, The quality testing method for the Xiangqinglan formula granules meets one or more of the following conditions. (1) In step (1), the preparation method of the test solution 2 containing Xiangqinglan formula particles includes the following steps: ultrasonic treatment, filtration and drying of Xiangqinglan formula particle solution to obtain residue; the solution of the residue is the test solution 2; In step (1), the solvent 2-1 in the Xiangqinglan formula granule solution is preferably an aqueous solution of methanol and / or ethanol; the volume percentage of ethanol in the aqueous solution is preferably 40%-80%, for example 70%. In step (1), the preferred mass-to-volume ratio of the Xiangqinglan formula granules to solvent 2-1 in the Xiangqinglan formula granule solution is 5g:(40-60)mL, for example, 5g:50mL. In step (1), the ultrasonic treatment time is preferably 5-30 min, for example 10 min; In step (1), the solvent 2-2 in the residue solution is preferably the same type as the solvent 2-1; the volume ratio of solvent 2-1 to solvent 2-2 is preferably (4-6):1, for example 5:

1. (2) In step (1), the preparation of reference solution 2 and reference medicinal material reference solution 2 is also carried out; In step (1), the reference solution 2 preferably includes reference standard 2 and a solvent; the reference standard 2 is preferably citronellol; the solvent is preferably an aqueous solution of methanol and / or ethanol; the volume percentage of ethanol in the aqueous solution is preferably 40%-80%, for example 70%; In step (1), when the reference standard 2 includes citronellol, the mass concentration of citronellol in the reference standard solution 2 is preferably 0.1-0.3 mg / mL, for example 0.2 mg / mL; In step (1), the preparation method of the reference medicinal material reference solution 2 preferably includes the following steps: ultrasonic treatment, filtration, and drying of the solution containing reference medicinal material 2 and solvent 2-3 to obtain residue; the solution of the residue and solvent 2-4 is the reference medicinal material reference solution 2; In step (1), the solution containing reference medicinal material 2 and solvent 2-3 preferably contains methanol and / or an aqueous solution of ethanol; the volume percentage of ethanol in the aqueous solution is preferably 40%-80%, for example 70%. In step (1), the mass-to-volume ratio of the reference medicinal material 2 and solvent 2-3 in the solution is preferably 1g:(40-60)mL, for example 1g:50mL; In step (1), the ultrasonic treatment time is preferably 5-30 min, for example 10 min; In step (1), the type of solvent 2-4 in the solution of residue and solvent 2-4 is preferably the same as that of solvent 2-3; the volume ratio of solvent 2-3 to solvent 2-4 is preferably (4-6):1, for example 5:1; (3) In step (1), the thin-layer chromatography method includes the following steps: spotting the test solution 2, the reference medicinal material solution 2 and the reference solution 2 on the same silica gel thin-layer plate, developing, removing, drying and examining under ultraviolet light; In step (1), the spotting volume of the test solution 2, the reference medicinal material solution 2 and the reference solution 2 in the thin-layer chromatography is preferably 1-10 μL, more preferably 2-5 μL, for example 3 or 4 μL. In step (1), the wavelength of the test in the thin-layer chromatography is preferably at an ultraviolet wavelength, for example at 254 nm or 365 nm. In step (1), during the thin-layer chromatography, it is preferable to spray a 5% aluminum trichloride ethanol solution during inspection. In step (1), the silica gel thin-layer chromatogram is preferably of type H plate, GF254 or G plate, for example G plate; In step (1), the developing solvent used in the thin-layer chromatography is preferably an aqueous solution of ethyl acetate and formic acid, or an aqueous solution of ethyl acetate, formic acid and toluene. When the developing solvent used is an aqueous solution of ethyl acetate and formic acid, the volume ratio of ethyl acetate, formic acid and water is preferably (6-10):1:(5-7), for example 8:0.5:3; When the developing solvent used is an aqueous solution of ethyl acetate, toluene, and formic acid, the volume ratio of ethyl acetate, toluene, formic acid, and water is preferably (14-17):(0.5-2.5):1:(0.5-0.7), for example, 16:2:1:0.6 or 16:0.6:1:0.6; In step (1), the development temperature in the thin-layer chromatography is preferably 10-40℃, for example 25℃; In step (1), the developing humidity in the thin-layer chromatography is preferably 10%-90%, for example 40%.

9. The method for quality testing of Xiangqinglan formula granules as described in claim 7, characterized in that, The quality testing method for the Xiangqinglan formula granules meets one or more of the following conditions: (1) In step (2), the preparation method of the test solution 3 containing Xiangqinglan formula particles includes the following steps: extracting and processing the Xiangqinglan formula particle solution, filtering it, and using the filtrate as the test solution 3; In step (2), the solvent 3-1 in the Xiangqinglan formula granule solution is preferably methanol, an aqueous ethanol solution, or an aqueous methanol solution; in the aqueous ethanol solution, the volume percentage of ethanol is preferably 40%-80%, for example, 70%; in the aqueous methanol solution, the volume percentage of methanol is preferably 40%-80%, for example, 70%. In step (2), the mass-to-volume ratio of the fragrant orchid formula granules to solvent 3-1 is preferably 0.3g:(20-110)mL, more preferably 0.3g:(25-100)mL, for example 0.3g:50mL or 0.3g:80mL; In step (2), the extraction process is preferably performed by ultrasonic treatment or reflux treatment. In step (2), the extraction time is preferably 10-30 min, for example 20 min; (2) In step (2), the preparation of reference solution 3 and reference medicinal material reference solution 3 is also carried out; In step (2), the reference solution 3 preferably includes a general reference standard 3 and a solvent 3-2; the reference standard 3 is preferably piracetam; the solvent 3-2 is preferably methanol, an aqueous ethanol solution, or an aqueous methanol solution; the volume percentage of ethanol in the aqueous ethanol solution is preferably 40%-80%, for example, 70%; the volume percentage of methanol in the aqueous methanol solution is preferably 40%-80%, for example, 70%. When the reference standard 3 is citronellol, the mass concentration of citronellol in the reference standard solution 3 is preferably 20-30 μg / mL, for example 26 μg / mL; (3) In step (2), the mobile phase A in the high performance liquid chromatography is acetonitrile; (4) In step (2), the mobile phase B in the high performance liquid chromatography is an aqueous phosphoric acid solution; the volume percentage of phosphoric acid in the aqueous phosphoric acid solution is preferably 0.1%-0.5%, for example 0.2%, 0.3% or 0.4%; (5) In step (2), the volume ratio of mobile phase A to mobile phase B in the high performance liquid chromatography is (20-25):(75-80), for example 22:78, 23:77, 25:75 or 20:80; (6) In step (2), the detection wavelength in the high performance liquid chromatography is 200-400 nm, for example 333 nm; (7) In step (2), the column temperature of the chromatographic column in the high performance liquid chromatography is 25-40℃, for example 30℃; (8) In step (2), the flow rate of the mobile phase in the high-performance liquid chromatography is 0.5-2 mL / min, for example, 1.0 mL / min; and, (9) In step (2), the injection volume in the high performance liquid chromatography is 10-20 μL, for example 15 μL.

10. The application of a quality testing method for Xiangqinglan formulation granules as described in any one of claims 1-9 in evaluating and / or controlling their quality.

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