A quality control method for Homalomena occulta samples

Abstract

The present invention discloses a quality control method for Homalomena occulta samples, which comprises the following steps: respectively sucking a reference medicinal material reference solution, a reference substance reference solution of reference substance and a test solution for ultra-high performance liquid chromatography analysis, constructing a characteristic chromatogram of the Homalomena occulta samples to be tested, wherein the characteristic chromatogram includes characteristic peak 2 with the same retention time as the reference peak of uridine reference substance and characteristic peak 3 with the same retention time as the reference peak of osbeckianic acid reference substance; carrying out qualitative analysis on the Homalomena occulta samples to be tested through the obtained characteristic chromatogram; and / or taking osbeckianic acid as an index for content determination to carry out quantitative analysis on the Homalomena occulta samples to be tested. The content index established by the present invention has stronger specificity, has a high content in the Homalomena occulta samples to be tested, and can accurately reflect the quality characteristics of the Homalomena occulta samples; the established methods for content determination and characteristic chromatogram can simultaneously distinguish true and false Homalomena occulta, and the results are relatively objective and accurate.

Description

Technical Field

[0001] The present invention relates to a method for controlling the quality of drugs, and particularly to a method for controlling the quality of Homalomena occulta samples. Background Art

[0002] Homalomena occulta is the dried rhizome of the plant Homalomena occulta (Lout.) Schott of the Araceae family. Its main chemical components include volatile oils, sesquiterpenoids, alkaloids, fatty acids, sugars, etc.

[0003] Traditional Chinese medicine formula granules are made from single-flavor traditional Chinese medicine decoction pieces through water extraction, concentration, drying, and granulation. Compared with traditional decoction pieces, they have the characteristics of being convenient to carry and easy to use. Homalomena occulta formula granules are made from Homalomena occulta decoction pieces through modern processes of extraction, concentration, drying, and granulation, retaining the clinical effects of Homalomena occulta while taking into account the portability of use.

[0004] The existing quality control methods for Homalomena occulta mainly focus on the quality control of the medicinal materials, and mostly focus on the study of its volatile oil components. For example, She Jinming et al. used the heuristic evolving latent projections (HELP) method combined with capillary gas chromatography-mass spectrometry (GC-MS) technology to analyze the volatile oil components in Homalomena occulta, and a total of 116 chromatographic peaks were resolved, and 94 components were identified. The main chemical component is linalool (She Jinming, Liu Bing, Wang Xianqing, etc. Analysis of volatile oil components in Homalomena occulta by HELP and GC-MS methods [J]. Journal of Chinese Medicinal Materials, 2010, 33(09): 1421-1424). Xie Lisha et al. established a gas chromatography method for determining the content of linalool in Homalomena occulta medicinal materials from 6 production areas in Guangxi (Xie Lisha, Meng Tianxiu, Ouyang Wei, etc. Determination of linalool content in Homalomena occulta produced in Guangxi by GC method [J]. Chinese Pharmacist, 2012, 15(05): 607-608). However, during the preparation of Homalomena occulta formula granules, the Homalomena occulta medicinal materials need to be decocted and extracted with water, resulting in a large loss of volatile oil components. Therefore, volatile oils cannot be used for the quality control of Homalomena occulta formula granules.

[0005] Currently, only the quality standards of Homalomena occulta formula granules issued by the drug regulatory bureaus of some provinces and municipalities can be referred to. As shown in the quality standards of Homalomena occulta formula granules issued by provinces such as Jiangxi, Gansu, and Hunan, standards such as thin-layer identification, characteristic fingerprints, and content determination of Homalomena occulta have been established, providing a basis for the quality control of Homalomena occulta formula granules. However, the characteristic peaks in these standards are few, and the identification of characteristic peaks is not comprehensive enough; the content indicators lack specificity; they cannot accurately and effectively reflect the quality level of Homalomena occulta. Summary of the Invention

[0006] Objective of the Invention: The objective of the present invention is to provide a quality control method for Homalomena occulta samples, so as to solve the problems existing in the existing quality control methods, such as lack of specificity, fewer characteristic peaks and incomplete identification.

[0007] Technical Solution: A quality control method for Homalomena occulta samples according to the present invention comprises the following steps:

[0008] (1) Prepare a test solution from the Homalomena occulta sample to be tested; prepare a reference solution of the reference crude drug from the Homalomena occulta reference crude drug; respectively prepare reference solutions of reference substances, and the reference solutions of reference substances include a uridine solution and an aurantiamide acetate solution;

[0009] (2) Respectively aspirate the reference solution of the reference crude drug, the reference solutions of reference substances and the test solution for ultra-high performance liquid chromatography analysis, and construct a characteristic chromatogram of the Homalomena occulta sample to be tested. The characteristic chromatogram includes characteristic peak 2 with the same retention time as the uridine reference substance peak and characteristic peak 3 with the same retention time as the aurantiamide acetate reference substance peak;

[0010] (3) Through the characteristic chromatogram obtained in step (2), conduct qualitative analysis of the Homalomena occulta sample to be tested; and / or take aurantiamide acetate as the content determination index to conduct quantitative analysis of the Homalomena occulta sample to be tested.

[0011] Preferably, in step (1), the reference solutions of reference substances further include a 5-hydroxymethylfurfural solution, a protocatechuic acid solution and a p-hydroxybenzoic acid solution; in step (2), the characteristic chromatogram further includes characteristic peak 4 with the same retention time as the 5-hydroxymethylfurfural reference substance peak, characteristic peak 7 with the same retention time as the protocatechuic acid reference substance peak and characteristic peak 8 with the same retention time as the p-hydroxybenzoic acid reference substance peak.

[0012] Preferably, the concentration of the uridine solution is 5 - 15 μg / ml; the concentration of the aurantiamide acetate solution is 5 - 15 μg / ml; the concentration of the 5-hydroxymethylfurfural solution is 5 - 15 μg / ml; the concentration of the protocatechuic acid solution is 5 - 15 μg / ml; the concentration of the p-hydroxybenzoic acid solution is 5 - 15 μg / ml.

[0013] Preferably, the characteristic chromatogram further includes characteristic peak 1, characteristic peak 5, characteristic peak 6 and characteristic peak 9. The relative retention time of characteristic peak 1 with respect to the aurantiamide acetate reference substance peak is 0.76 ± 0.076; the relative retention time of characteristic peak 5 with respect to the aurantiamide acetate reference substance peak is 2.34 ± 0.234; the relative retention time of characteristic peak 6 with respect to the aurantiamide acetate reference substance peak is 2.54 ± 0.254; the relative retention time of characteristic peak 9 with respect to the p-hydroxybenzoic acid reference substance peak is 1.08 ± 0.108.

[0014] The characteristic chromatogram in the present invention stipulates 9 characteristic peaks, which is more than the 5 in the publicly announced standard of Gaultheria yunnanensis formula granules; 5 characteristic peaks are identified, namely characteristic peak 2, characteristic peak 3, characteristic peak 4, characteristic peak 7 and characteristic peak 8, exceeding 50% of the total number of characteristic peaks. At the same time, using the control crude drug as a reference substance can better reflect the consistency between the formula granules and the crude drug. The chemical components presented in the characteristic chromatogram include nucleosides, phenolic acids and other components. Compared with the volatile oil components studied in the prior art, they have less loss after decoction and better water solubility, which is more conducive to the quality control of the formula granules prepared by water extraction, concentration, drying and granulation.

[0015] Preferably, in step (1), for every 0.2 - 1 g of the Gaultheria yunnanensis sample to be tested, 15 - 25 ml of the extraction agent is used, and extraction is carried out for 15 - 60 min to prepare the test solution; for every 1 - 3 g of the control crude drug of Gaultheria yunnanensis, 15 - 25 ml of the extraction agent is used, and extraction is carried out for 15 - 60 min to prepare the reference solution of the control crude drug. The extraction agent is methanol or a methanol aqueous solution with a volume concentration of 10 - 70%.

[0016] In some embodiments, 0.2 g - 1.0 g of Gaultheria yunnanensis formula granules are taken, ground fine, added with methanol or 10% - 70% methanol for extraction, and the subsequent filtrate is taken to obtain the test solution.

[0017] In some embodiments, 1 g - 3 g of the control crude drug of Gaultheria yunnanensis are taken, added with 10% - 50% methanol for extraction, and the subsequent filtrate is taken as the reference solution of the control crude drug.

[0018] Preferably, in step (3), the qualitative analysis method for the Gaultheria yunnanensis sample to be tested is: distinguish Gaultheria yunnanensis from its counterfeits according to the relative peak area of characteristic peak 2 relative to characteristic peak 3 and the content of osbeckianic acid. It is also possible to distinguish Gaultheria yunnanensis from its counterfeits by the presence or absence of characteristic peak 4 and / or characteristic peak 5 and the content limit of osbeckianic acid. The content determination index is osbeckianic acid, which has good water solubility and high peak response in the chromatogram. Compared with sucrose in other publicly announced standards, it has higher specificity and can more effectively control the quality standard of Gaultheria yunnanensis.

[0019] Preferably, the Gaultheria yunnanensis sample to be tested includes Gaultheria yunnanensis crude drug, Gaultheria yunnanensis decoction pieces, Gaultheria yunnanensis standard decoction, Gaultheria yunnanensis preparation intermediate or Gaultheria yunnanensis preparation finished product.

[0020] Preferably, in step (2), the conditions for the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is packed with octadecylsilyl silica gel; methanol is used as mobile phase A, and a 0.05%-0.2% phosphoric acid solution is used as mobile phase B, and gradient elution is adopted: 0-10 min, 0% A, 10-22 min, 0→8% A, 22-32 min, 8% A, 32-33 min, 8→0% A, 33-38 min, 0% A; the detection wavelength is 220-280 nm; the flow rate is 0.28-0.32 ml / min; the column temperature is 18°C-22°C; an ultraviolet detector is used.

[0021] Preferably, before preparing the test solution of the Rhizoma Homalomenae to be tested, qualitative analysis of the Rhizoma Homalomenae to be tested is carried out by thin-layer chromatography. Using the reference medicinal material of Rhizoma Homalomenae as a reference, it is judged whether the spots of the Rhizoma Homalomenae to be tested on the thin-layer plate correspond one by one to the spots of the reference medicinal material of Rhizoma Homalomenae. If so, steps (1)-(3) are carried out. If not, the Rhizoma Homalomenae to be tested is an unqualified sample

[0022] In some embodiments, the specific steps of the thin-layer chromatography method include taking 0.2 g to 1.0 g of the Rhizoma Homalomenae formula granules, extracting with an organic solvent, and taking the subsequent filtrate to obtain the test solution; taking 1 g to 3 g of the reference medicinal material of Rhizoma Homalomenae, extracting with water, filtering, evaporating the filtrate to dryness, and dissolving the residue with an organic solvent to obtain the reference solution of the reference medicinal material of Rhizoma Homalomenae. The developing agent includes chloroform and methanol. Preferably, the thin-layer plate is silica gel G, the developing agent is chloroform-methanol (10:1), and the color-developing agent is a 10% sulfuric acid ethanol solution.

[0023] The thin-layer identification method in the present invention uses the reference medicinal material as a reference. For the water-soluble components in the Rhizoma Homalomenae, through the optimization of the developing agent, more chromatographic spots are presented, and the optimized developing agent chloroform-methanol (10:1) reflects more comprehensive quality information, which conforms to the characteristics of the overall quality control of traditional Chinese medicine.

[0024] Thin-layer identification is convenient to operate, has simple equipment, and low cost. Different polar components can be analyzed by optimizing the developing agent. The characteristic spectrum method relies on liquid phase equipment, has less human interference, and higher detection sensitivity. It is mainly suitable for analyzing medium-polarity and low-polarity components, and the two complement each other.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. The established content index of the present invention is more specific, with a high content in the samples to be tested of *Homalomena occulta*, and can accurately reflect the quality characteristics of *Homalomena occulta* samples; the established content determination and characteristic chromatogram methods can distinguish the genuine and fake *Homalomena occulta* samples at the same time, and the results are objective and accurate. This method is simple to operate, has good reproducibility and stability after methodological verification, reliable recovery rate, low detection cost and high detection efficiency. 2. The characteristic chromatogram method established by the method of the present invention presents rich chromatographic peak information and a large number of characteristic peaks. The characteristic chromatogram contains 9 characteristic peaks, and more than half of the characteristic peaks are identified, which can more comprehensively control the quality of *Homalomena occulta* formula granules. 3. The present invention conducts quality control from multiple angles including thin-layer identification, characteristic chromatogram, content determination, and identification of genuine and fake products, taking into account both integrity and effectiveness. Description of the Drawings

[0026] Figure 1 It is the characteristic chromatogram of *Homalomena occulta* formula granules;

[0027] Figure 2 It is the characteristic chromatogram of *Homalomena occulta* intermediate;

[0028] Figure 3 It is the characteristic chromatogram of *Homalomena occulta* standard decoction;

[0029] Figure 4 It is the characteristic chromatogram of *Homalomena occulta* cut crude drug;

[0030] Figure 5 It is the characteristic chromatogram of *Homalomena occulta* medicinal material;

[0031] Figure 6 It is the chromatograms of reference substance, reference medicinal material and *Homalomena occulta* formula granules;

[0032] Figure 7 Full wavelength scanning chart of aurantiamide acetate;

[0033] Figure 8 UPLC chromatograms of *Homalomena occulta* formula granules at different detection wavelengths;

[0034] Figure 9 UPLC charts for investigating different extraction solvents;

[0035] Figure 10 UPLC charts for investigating different extraction methods;

[0036] Figure 11 UPLC charts for investigating different extraction times;

[0037] Figure 12 UPLC charts for investigating different extraction volumes;

[0038] Figure 13 Linear relationship chart of aurantiamide acetate reference substance;

[0039] Figure 14 Chromatographic column investigation of the characteristic fingerprint of Gaultheria yunnanensis formula granules;

[0040] Figure 15 Column temperature investigation of the characteristic fingerprint of Gaultheria yunnanensis formula granules;

[0041] Figure 16 Flow rate investigation of the characteristic fingerprint of Gaultheria yunnanensis formula granules;

[0042] Figure 17 Comparison of the control characteristic fingerprints between the standard decoctions of Gaultheria yunnanensis and its counterfeit (Alocasia macrorrhiza);

[0043] Figure 18 TLC chromatogram of Gaultheria yunnanensis formula granules (developer 1);

[0044] Figure 19 TLC chromatogram of Gaultheria yunnanensis formula granules (developer 2);

[0045] Figure 20 TLC chromatograms of Gaultheria yunnanensis formula granules with different sample application amounts;

[0046] Figure 21 TLC chromatogram of the specificity test of Gaultheria yunnanensis formula granules;

[0047] Figure 22 Thin layer identification chromatograms of samples of Gaultheria yunnanensis formula granules from different batches;

[0048] Figure 23 Results of the specificity test of Gaultheria yunnanensis formula granules using five reference substances;

[0049] Figure 24 Results of the integrity test of Gaultheria yunnanensis formula granules;

[0050] Figure 25 Results of the specificity test of Gaultheria yunnanensis formula granules using osbeckianic acid as the reference substance. Detailed implementation manners

[0051] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0052] Example 1: A quality control method for a Gaultheria yunnanensis formula sample, comprising the following steps:

[0053] Take 3 batches of Gaultheria yunnanensis formula granules, 10 batches of Gaultheria yunnanensis standard decoctions, and 2 batches of standard decoctions of counterfeit Gaultheria yunnanensis (Alocasia macrorrhiza) as Gaultheria yunnanensis samples, and conduct quality control determinations respectively according to the following steps:

[0054] 1. Thin layer identification:

[0055] For the 3 batches of Rhizoma Homalomenae formula granules, perform thin-layer identification first and then determine whether to enter Step 2 based on the results; for the 10 batches of Rhizoma Homalomenae standard decoctions and the 2 batches of Rhizoma Homalomenae counterfeit (Alocasia macrorrhiza) standard decoctions, do not perform thin-layer identification and directly enter Step 2 for characteristic fingerprint, content determination, and authenticity identification. Qualitative analysis of the test samples is carried out by thin-layer chromatography, using the reference crude drug as a reference to determine whether the spots of the test samples on the thin-layer plate correspond one by one to those of the reference crude drug; if so, perform characteristic fingerprint and content determination, otherwise the test sample is an unqualified sample.

[0056] Process of thin-layer identification for 3 batches of Rhizoma Homalomenae formula granules: Take 0.5 g of Rhizoma Homalomenae granules, grind them finely, add 20 ml of methanol, ultrasonically treat for 30 minutes, let it cool, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Additionally, take 2 g of the reference crude drug of Rhizoma Homalomenae, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of methanol, and prepare the reference crude drug solution in the same way. Pipette 5 μl of each of the above two solutions and spot them on the same silica gel G thin-layer plate. Use chloroform - methanol (10:1) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible.

[0057] The results are shown in Figure 22 , where 1 - 3: Rhizoma Homalomenae formula granules; S: reference crude drug of Rhizoma Homalomenae. It can be seen that the chromatograms of Rhizoma Homalomenae formula granules and the reference crude drug chromatogram show spots of the same color at the corresponding positions. All 3 batches of Rhizoma Homalomenae formula granules enter Step 2 for further determination.

[0058] 2. Characteristic fingerprint, content determination, and authenticity identification:

[0059] (1) Select Rhizoma Homalomenae samples and prepare the test solutions; use Rhizoma Homalomenae as the reference crude drug to prepare the reference solution of the reference crude drug; use uridine, osbeckianic acid, 5 - hydroxymethylfurfural, protocatechuic acid, and p - hydroxybenzoic acid as the reference substances for the reference standards, and prepare the reference solutions of the reference standards respectively;

[0060] Preparation of the reference solution: Take 2 g of the reference crude drug of Rhizoma Homalomenae, place it in a stoppered conical flask, add 25 ml of 10% methanol, stopper tightly, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate as the reference solution of the reference crude drug.

[0061] Preparation of the reference solution of the reference standards: Take appropriate amounts of uridine reference standard, osbeckianic acid reference standard, 5 - hydroxymethylfurfural reference standard, protocatechuic acid reference standard, and p - hydroxybenzoic acid reference standard, accurately weigh them, dissolve them in 10% methanol to make a mixed solution containing 10 μg of each per 1 ml as the reference solution of the reference standards.

[0062] Preparation of the test solution of Homalomena occulta formula granules: Take an appropriate amount of Homalomena occulta formula granules, grind them finely, take about 0.5 g, weigh accurately, place in a stoppered conical flask, accurately add 20 ml of 10% methanol, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, and take the subsequent filtrate, which is the required solution.

[0063] Preparation of the test solution of the standard decoction of Homalomena occulta and its counterfeit (Alocasia macrorrhiza): Take about 0.3 g of the standard decoction powder, weigh accurately, place in a stoppered conical flask, accurately add 15 ml of 10% methanol, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 10% methanol, shake well, and take the subsequent filtrate, which is the required solution.

[0064] (2) Perform ultra-high performance liquid chromatography analysis on the test solution and the reference solution prepared in step (1) respectively to obtain the corresponding ultra-high performance liquid chromatography diagrams; accurately pipette 2 μl each of the reference solution and the test solution, inject into the liquid chromatograph, and determine, which is the required result.

[0065] Among them, the chromatographic condition parameters are as follows:

[0066] Using octadecylsilane chemically bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); using methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; the flow rate is 0.3 ml per minute; the column temperature is 20 °C; the detection wavelength is 259 nm.

[0067]

[0068] Using the above ultra-high performance liquid chromatography method, the characteristic chromatogram of Homalomena occulta formula granules constructed with Homalomena occulta formula granules as the sample is as Figure 1 shown; the characteristic chromatogram of Homalomena occulta intermediate constructed with Homalomena occulta intermediate as the sample is as Figure 2 shown; the characteristic chromatogram of Homalomena occulta standard decoction constructed with Homalomena occulta standard decoction as the sample is as Figure 3 shown; the characteristic chromatogram of Homalomena occulta cut pieces constructed with Homalomena occulta cut pieces as the sample is as Figure 4 shown; the characteristic chromatogram of Homalomena occulta medicinal materials constructed with Homalomena occulta medicinal materials as the sample is as Figure 5 shown. In Figures 1 - 5 , peak 2: uridine; peak 3 (S1): osbeckianic acid; peak 4: 5-hydroxymethylfurfural; peak 7: protocatechuic acid; peak 8 (S2): p-hydroxybenzoic acid.

[0069] As Figure 6As shown, the characteristic chromatogram of Gaultheria yunnanensis formula granules shows 9 characteristic peaks in the chromatogram of the test solution, corresponding to the retention times of 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 2, peak 3, peak 4, peak 7, and peak 8 should respectively be consistent with the retention times of the reference peaks of uridine reference substance, osbeckianic acid reference substance, 5-hydroxymethylfurfural reference substance, protocatechuic acid reference substance, and p-hydroxybenzoic acid reference substance; the peak corresponding to the osbeckianic acid reference substance is peak S1, and calculate the relative retention times of peak 1, peak 5, and peak 6 with respect to peak S1; the peak corresponding to the p-hydroxybenzoic acid reference substance is peak S2, and calculate the relative retention time of peak 9 with respect to peak S2. The relative retention times should be within ±10% of the specified values, and the specified values are: 0.76 (peak 1), 2.34 (peak 5), 2.54 (peak 6), 1.08 (peak 9).

[0070] The determination results of the test solution are shown in Tables 1 to 4.

[0071] Table 1 Determination Results of Gaultheria yunnanensis Formula Granules Samples (Relative Retention Time)

[0072]

[0073]

[0074] Table 2 Determination Results of Gaultheria yunnanensis Standard Decoction Samples (Relative Retention Time)

[0075]

[0076] Table 3 Determination Results of Gaultheria yunnanensis Slices Samples (Relative Retention Time)

[0077]

[0078] Table 4 Determination Results of Gaultheria yunnanensis Medicinal Material Samples (Relative Retention Time)

[0079]

[0080]

[0081] (3) For the ultra-high performance liquid chromatogram obtained in step (2), using osbeckianic acid as the content determination index, perform quantitative analysis of the samples. Determine the content of osbeckianic acid in the samples, and the experimental results are shown in Tables 5 to 8.

[0082] Table 5 Determination of the Content of Osbeckianic Acid in Gaultheria yunnanensis Formula Granules

[0083]

[0084] Table 6 Determination of the Content of Osbeckianic Acid in Gaultheria yunnanensis and Alocasia macrorrhiza Standard Decoctions

[0085]

[0086] Determination of the Content of Osbeckianic Acid in the Rhizome of Homalomena occulta (Lour.) Schott Slices

[0087]

[0088]

[0089] Determination of the Content of Osbeckianic Acid in the Medicinal Materials of Homalomena occulta (Lour.) Schott and Alocasia macrorrhiza (L.) Schott

[0090]

[0091] From the above test results, from the qualitative analysis, the characteristic chromatograms of 3 batches of Homalomena occulta (Lour.) Schott formula granules, 10 batches of Homalomena occulta (Lour.) Schott standard decoctions, slices and medicinal materials meet the requirements of the characteristic chromatogram in step (2). From the quantitative analysis, the content of osbeckianic acid in 3 batches of Homalomena occulta (Lour.) Schott formula granules, 10 batches of Homalomena occulta (Lour.) Schott standard decoctions, slices and medicinal materials is stable. The content of osbeckianic acid in Alocasia macrorrhiza (L.) Schott is lower than the lower limit of the content of Homalomena occulta (Lour.) Schott, which can effectively distinguish the two, ensuring the effectiveness and safety of Homalomena occulta (Lour.) Schott formula granules and standard decoctions.

[0092] Example 2: Establishment of the Characteristic Chromatogram Method for Homalomena occulta (Lour.) Schott Formula Granules and Its Methodology Research

[0093] This example is used to confirm the optimal scheme for the establishment of the characteristic chromatogram in the quality control method of Homalomena occulta (Lour.) Schott samples, specifically including the comparison of chromatographic conditions, wavelength selection, and the preparation scheme of the test solution, and selecting the optimal chromatographic conditions and test solution preparation scheme; and investigating the specificity, integrity, precision, intermediate precision, stability, repeatability, and durability of the optimal scheme respectively.

[0094] 1 Instruments and Reagents

[0095] Agilent Tcchnologies 1290Inftnity ultra-high performance liquid chromatograph; Thermo Vanquish ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); thermostatic water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan Keyuan Instrument Factory); pure water system (Millipore Corporation); AS165W centrifuge (As One (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher Scientific); methanol (chromatographic grade, Thermo Fisher Scientific); phosphoric acid (chromatographic grade, aladdin); water is ultrapure water; other reagents are all analytical grade.

[0096] Uridine reference substance, 5-hydroxymethylfurfural reference substance, protocatechuic acid reference substance, and p-hydroxybenzoic acid reference substance were all purchased from the National Institutes for Food and Drug Control, with batch numbers 110887-202104, 111626-201912, 110809-201906, and 101149-202204 respectively, and purities of 99.6%, 99.2%, 95.5%, and 100% respectively. The osbeckianic acid reference substance was purchased from Chengdu Pusi Biotechnology Co., Ltd., with batch number PS013665 and purity of 98%. The reference crude drug of Homalomena occulta was purchased from the National Institutes for Food and Drug Control, with batch number 121571-201202. The formula granules of Homalomena occulta were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0097] Determination of detection wavelength

[0098] Take an appropriate amount of this product, grind it finely, about 0.5 g, weigh it accurately, place it in a stoppered conical flask, accurately add 20 ml of 10% methanol, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 10% methanol, shake well, filter, take the subsequent filtrate as the test solution, record the chromatograms at different wavelengths, as Figure 8 shown. The results showed that the chromatographic information detected for the formula granules of Homalomena occulta at 259 nm was relatively rich and the baseline was stable. Finally, 259 nm was selected as the detection wavelength.

[0099] 3 Determination of chromatographic conditions

[0100] Using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); using methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; the flow rate is 0.3 ml per minute; the column temperature is 20 °C; the detection wavelength is 259 nm.

[0101]

[0102] 4 Preparation of test solution

[0103] 4.1 Investigation of different extraction solvents

[0104] Take an appropriate amount of this product, grind it into powder, take about 0.5g, and place it in five parallel portions, accurately weigh it, put it in a stoppered conical bottle, accurately add 20ml of 10% methanol, 30% methanol, 50% methanol, 70% methanol and methanol, seal it, weigh it, and perform ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes respectively, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate. Accurately take 2μl of each test solution, inject it into the liquid chromatograph, measure it according to the above chromatographic conditions, and calculate the ratio of the characteristic peak area / sample weight. The results are shown in Table 9. Figure 9 .

[0105] Table 9 Comparison of extraction efficiency of different extraction solvents (peak area / sample weight)

[0106]

[0107]

[0108] The results showed that the extraction efficiency was higher when 10% methanol was used as the extraction solvent, so 10% methanol was selected as the extraction solvent.

[0109] 4.2 Investigation of different extraction methods

[0110] Take an appropriate amount of this product, grind it into powder, take about 0.5g, make three parallel parts, weigh accurately, put it in a stoppered conical bottle, accurately add 20ml of 10% methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz), shake it to extract, heat it under reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate. Accurately take 2μl of each test solution, inject it into the liquid chromatograph, measure it according to the above chromatographic conditions, calculate the characteristic peak area / sample weight, the results are shown in Table 10. Figure 10 .

[0111] Table 10 Comparison of extraction efficiency of different extraction methods (peak area / sample weight)

[0112]

[0113] The results showed that the number of chromatographic peaks obtained by the three treatment methods was consistent, and the extraction efficiency of the chromatographic peaks was similar. For the sake of ease of operation, ultrasonic treatment was selected as the extraction method.

[0114] 4.3 Investigation of different extraction times

[0115] Take an appropriate amount of this product, grind it finely, take about 0.5 g, divide it into four parallel portions, weigh accurately, place it in a stoppered conical flask, accurately add 20 ml of 10% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively, let it cool, weigh it again, make up the lost weight with 10% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution. Accurately pipette 2 μl of each test solution respectively, inject it into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the peak area of the characteristic peak / sample weight, and the results are shown in Table 11. Figure 11 。

[0116] Table 11 Comparison of extraction efficiency at different extraction times (peak area / sample weight)

[0117]

[0118] The results show that the number of chromatographic peaks obtained at each extraction time is the same. When ultrasonically treated for 15 minutes, the extraction is basically sufficient. To ensure complete extraction, the extraction time is selected as 30 minutes.

[0119] 4.4 Investigation of different extraction volumes

[0120] Take an appropriate amount of this product, grind it finely, take about 0.5 g, divide it into three parallel portions, weigh accurately, place it in a stoppered conical flask, accurately add 15 ml, 20 ml, and 25 ml of 10% methanol respectively, stopper it tightly, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 10% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution. Accurately pipette 2 μl of each test solution respectively, inject it into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the peak area of the characteristic peak / sample weight * extraction volume / 20, and the results are shown in Table 12. Figure 12 。

[0121] Table 12 Comparison of extraction efficiency at different extraction volumes (peak area / sample weight * extraction volume / 20)

[0122]

[0123] The results show that the number of chromatographic peaks obtained with the three extraction solvent volumes is the same, and there is no obvious difference in extraction efficiency. To ensure sufficient extraction and save solvents, the extraction volume is determined to be 20 ml.

[0124] 4.5 Determination of the preparation method of the test solution

[0125] According to the above research results, the preparation method of the test solution for the characteristic chromatogram of Homalomena occulta formula granules is determined as follows:

[0126] Take an appropriate amount of Homalomena occulta formula granules, grind them finely, take about 0.5 g, weigh accurately, place it in a stoppered conical flask, accurately add 20 ml of 10% methanol, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0127] 5 Methodology research of characteristic fingerprint

[0128] 5.1 Specificity investigation

[0129] The excipients added in Homalomena occulta formula granules are maltodextrin, silicon dioxide and magnesium stearate. In this experiment, the influence of the negative sample without Homalomena occulta on Homalomena occulta formula granules was investigated. Take the negative sample without Homalomena occulta and prepare the negative sample solution according to the preparation method of the test sample. Inject 2 μl according to the above chromatographic conditions, and the results are shown in Figure 23 . The results show that the solvent and excipients have no interference on the determination of the characteristic fingerprint of Homalomena occulta formula granules. It has specificity to determine Homalomena occulta formula granules by this method.

[0130] 5.2 Integrity investigation

[0131] Under the same chromatographic conditions, maintain the elution gradient when the methanol ratio is the highest, and double the elution time. The results are shown in Figure 24 . The results show that there are no obvious chromatographic peaks flowing out after the original gradient elution, indicating that this chromatographic condition basically meets the principle of the largest amount of information.

[0132] 5.3 Precision investigation

[0133] Take samples of the same batch number, prepare the test solution according to the preparation method of the test solution, inject continuously 6 times, 2 μl each time, record the retention time of its characteristic peaks, and calculate the relative retention time according to the requirements of the main text. The results are shown in Table 13.

[0134] Table 13 Results of precision experiment (relative retention time)

[0135]

[0136]

[0137] The results show that the RSD of the relative retention time of each characteristic peak is less than 1%, and the precision is good.

[0138] 5.4 Intermediate precision investigation

[0139] Take samples of the same batch number, and two experimenters A and B respectively prepare 3 portions according to the preparation method of the test solution, inject 2 μl at different times and on different instruments respectively, record the retention time of its characteristic peaks, and calculate the relative retention time according to the requirements of the main text. The results are shown in Table 14.

[0140] Table 14 Intermediate Precision Investigation (Relative Retention Time)

[0141]

[0142] The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, indicating good intermediate precision.

[0143] 5.5 Stability Investigation

[0144] Take samples of the same batch number, prepare test solution according to the preparation method of the test solution, inject 2 μl respectively at 0, 2, 4, 8, 12, 18, and 24 h, record the retention time of its characteristic peaks, calculate the relative retention time according to the requirements of the main text, and the results are shown in Table 15.

[0145] Table 15 Stability Investigation (Relative Retention Time)

[0146]

[0147]

[0148] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, indicating good stability of the test solution within 24 hours.

[0149] 5.6 Repeatability Investigation

[0150] Take samples of the same batch number, prepare 6 parallel groups, prepare test solution according to the preparation method of the test solution, inject 2 μl respectively, record the retention time of its characteristic peaks, calculate the relative retention time according to the requirements of the main text, and the results are shown in Table 16.

[0151] Table 16 Repeatability Test (Relative Retention Time)

[0152]

[0153] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, indicating good repeatability of the method.

[0154] 5.7 Robustness Investigation

[0155] (1) Chromatographic Column Investigation

[0156] Take samples of the same batch number and prepare the test solution according to the preparation method of the test solution in the main text. Use three different chromatographic columns, namely ACQUITY CORTECS T3 (Waters, 2.1 mm × 150 mm, 1.6 μm); Syncronis C18 (Thermo, 2.1 mm × 100 mm, 1.7 μm); Hypersil GLOD aQ (Thermo, 2.1 mm × 150 mm, 1.9 μm), to investigate their separation effects. The results are shown in Figure 14 , Table 17.

[0157] Table 17 Chromatographic Column Investigation (Relative Retention Time)

[0158]

[0159] The results show that better separation of the Homalomena occulta samples can be obtained on the ACQUITY CORTECS T3 (Waters, 2.1 mm × 150 mm, 1.6 μm) chromatographic column. The resolution of peak 7 is poor on the Hypersil GLOD aQ (Thermo, 2.1 mm × 150 mm, 1.9 μm) chromatographic column, and there is peak wrapping between peak 8 and peak 9 on the Syncronis C18 (Thermo, 2.1 mm × 100 mm, 1.7 μm) chromatographic column. The ACQUITY CORTECS T3 (Waters, 2.1 mm × 150 mm, 1.6 μm) chromatographic column was used in subsequent studies.

[0160] (2) Column Temperature Investigation

[0161] Take samples of the same batch number and prepare the test solution according to the preparation method of the test solution in the main text. Using the ACQUITY CORTECS T3 (Waters, 2.1 mm × 150 mm, 1.6 μm) as the chromatographic column, the separation effects of the samples were investigated at column temperatures of 18 °C, 20 °C, and 22 °C respectively. The results are shown in Figure 15 , Table 18.

[0162] Table 18 Column Temperature Investigation (Relative Retention Time)

[0163]

[0164] The results show that when the column temperature is between 18 °C and 22 °C, the relative retention times of each characteristic peak are within the specified range, and small column temperature variations can meet the requirements of system durability. A column temperature of 20 °C was used in subsequent studies.

[0165] (3) Flow Rate Investigation

[0166] Take samples of the same batch number and prepare the test solution according to the preparation method of the test solution in the main text. Using ACQUITY UPLC CORTECS T3 (Waters, 2.1 mm × 150 mm, 1.6 μm) as the chromatographic column, the separation effects of the samples were investigated at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min, respectively. The results are shown in Figure 16 , Table 19.

[0167] Table 19 Investigation of Flow Rate (Relative Retention Time)

[0168]

[0169] The results show that within the range of 0.28 ml / min to 0.32 ml / min for the flow rate, small flow rate variations can meet the system suitability requirements, and a flow rate of 0.30 ml / min was adopted in the subsequent research.

[0170] Example 3: Establishment of the Content Determination Method for Homalomena occulta Formula Granules and Its Methodology Research

[0171] 1 Instruments and Reagents

[0172] Same as Example 2. The Homalomena occulta formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0173] 2 Source of Reference Substances and Purity Inspection

[0174] Auriculoside (batch number: PS013665), purchased from Chengdu Pusi Biotechnology Co., Ltd., for content determination, with a content of 98% and no need for treatment before use.

[0175] 3 Determination of Detection Wavelength

[0176] Collect the spectrum of auriculoside at wavelengths from 190 to 400 nm, as shown in Figure 7 . The results show that auriculoside has a strong absorption around 256 nm. Combining with the chromatographic conditions of the characteristic fingerprint, 259 nm was finally selected as the detection wavelength.

[0177] 4 Determination of Chromatographic Conditions

[0178] Referring to the determination method of the Homalomena occulta characteristic fingerprint, a content determination method for auriculoside in Homalomena occulta formula granules was established and detected using the chromatographic conditions determined in Example 2.

[0179] 5 Preparation of Test Solution

[0180] 5.1 Investigation of Different Extraction Solvents

[0181] Take an appropriate amount of Gaultheria yunnanensis formula granules, grind them finely, take about 0.5 g, in parallel for 5 groups, 2 portions in each group, weigh accurately, place in a stoppered conical flask, accurately add 20 ml of 10% methanol, 30% methanol, 50% methanol, 70% methanol and methanol respectively, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, take the continuous filtrate, and you will get it. Accurately pipette 2 μl of each test solution respectively, inject into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the content of osbeckianic acid, and the results are shown in Figure 9 and Table 20.

[0182] Table 20 Comparison of Different Extraction Solvents

[0183]

[0184]

[0185] Conclusion: When 10% methanol, 30% methanol, 50% methanol, 70% methanol and methanol are used as extraction solvents respectively, the content of osbeckianic acid extracted with 10% methanol is relatively high and the symmetry factor is better. Therefore, 10% methanol is selected as the extraction solvent.

[0186] 5.2 Investigation of Different Extraction Methods

[0187] Take an appropriate amount of Gaultheria yunnanensis formula granules, grind them finely, take about 0.5 g, in parallel for 3 groups, 2 portions in each group, weigh accurately, place in a stoppered conical flask, accurately add 20 ml of 10% methanol, stopper tightly, weigh, perform ultrasonic treatment (power 250 W, frequency 40 kHz), heating under reflux and shaking extraction for 30 minutes respectively, let it cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, take the continuous filtrate, and you will get it. Accurately pipette 2 μl of each test solution respectively, inject into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the content of osbeckianic acid, and the results are shown in Figure 10 and Table 21.

[0188] Table 21 Comparison of Different Extraction Methods

[0189]

[0190] Conclusion: It can be seen from the results that the contents of osbeckianic acid extracted by the three extraction methods are not very different. Considering the simplicity of operation, ultrasonic treatment is selected as the extraction method.

[0191] 5.3 Investigation of Different Extraction Times

[0192] Take an appropriate amount of Gaultheria yunnanensis formula granules, grind them finely, take about 0.5 g, in parallel for 4 groups, 2 portions in each group, weigh accurately, place in a stoppered conical flask, accurately add 20 ml of 10% methanol, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, let it cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, take the subsequent filtrate, and you will get it. Accurately pipette 2 μl of each test solution respectively, inject into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the content of osbeckianic acid, and the results are shown in Figure 11 , Table 22.

[0193] Table 22 Comparison of Different Extraction Times

[0194]

[0195] Conclusion: As can be seen from the results, the contents of osbeckianic acid measured at different extraction times are close. To ensure complete extraction, the extraction time is determined to be 30 minutes.

[0196] 5.4 Investigation of Different Extraction Volumes

[0197] Take an appropriate amount of Gaultheria yunnanensis formula granules, grind them finely, take about 0.5 g, in parallel for 3 groups, 2 portions in each group, weigh accurately, place in a stoppered conical flask, accurately add 15 ml, 20 ml and 25 ml of 10% methanol respectively, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, take the subsequent filtrate, and you will get it. Accurately pipette 2 μl of each test solution respectively, inject into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the content of osbeckianic acid, and the results are shown in Figure 12 , Table 23.

[0198] Table 23 Comparison of Different Solvent Volumes for Extraction

[0199]

[0200]

[0201] As can be seen from the results: The extraction efficiencies of different extraction volumes are comparable. Considering comprehensively from the perspectives of sufficient extraction and solvent saving, the extraction volume is determined to be 15 ml.

[0202] 5.5 Determination of the Preparation Method of the Test Solution

[0203] According to the above research results, the final preparation method of the test solution for the content determination of Gaultheria yunnanensis formula granules is as follows:

[0204] Take an appropriate amount of Gaultheria yunnanensis formula granules, grind them finely, take about 0.5 g, weigh accurately, place it in a stoppered conical flask, accurately add 20 ml of 10% methanol, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0205] 6 Methodology verification

[0206] 6.1 Linearity

[0207] Precisely pipette 0.1 μl, 0.2 μl, 0.5 μl, 1.0 μl, 1.5 μl, 2.0 μl, 3.0 μl of the osbeckianic acid reference substance solution (concentration 9.51 μg / ml) into the liquid chromatograph, determine according to the above chromatographic conditions, with the peak area integral value as the ordinate and the injection volume (μg) of each component as the abscissa, plot the standard curve, and obtain the regression equation of osbeckianic acid. The results are shown in Figure 13 , Table 24 and Table 25.

[0208] Table 24 Regression equation

[0209]

[0210] Table 25 Relationship between injection volume of osbeckianic acid reference substance and peak area

[0211]

[0212] The results show that: within the range of 0.0009506 - 0.0285180 μg of the injection volume of osbeckianic acid, there is a good linear relationship between the injection volume and the peak area value.

[0213] 6.2 Precision test

[0214] 6.2.1 Instrument precision test

[0215] Take Gaultheria yunnanensis formula granules, prepare the sample test solution according to the above preparation method of the test solution, precisely pipette 2 μl, inject it into the liquid chromatograph, determine according to the above chromatographic conditions, inject continuously for 6 times, record the measured value of the osbeckianic acid peak area, calculate its RSD, and the results are shown in Table 26. The results show that: the instrument precision test is good.

[0216] Table 26 Instrument precision test

[0217]

[0218] 6.2.2 Repeatability test

[0219] Take an appropriate amount of Gaultheria yunnanensis formula granules, grind them finely, take about 0.5 g, weigh accurately, in parallel for 6 portions, and prepare sample test solutions respectively according to the above-mentioned preparation method of test solutions. Inject 2 μl respectively, determine the peak area value of osbeckianic acid, calculate its content and RSD, and the results are shown in Table 27. The results show that the RSD of the osbeckianic acid content is 0.29%, and the repeatability test is good.

[0220] Table 27 Results of repeatability test of Gaultheria yunnanensis formula granules

[0221]

[0222] 6.2.3 Intermediate precision test

[0223] Take Gaultheria yunnanensis formula granules, and prepare 3 portions of test solutions respectively by two experimenters A and B according to the above-mentioned preparation method of test solutions. Inject 2 μl respectively at different times and on different instruments, determine the peak area value of osbeckianic acid, calculate its content and RSD, and the results are shown in Table 28. The results show that the RSD of the osbeckianic acid content is 0.88%, and the intermediate precision test is good.

[0224] Table 28 Intermediate precision test

[0225]

[0226]

[0227] 6.3 Accuracy test

[0228] Take 0.25 g of the sample with known content (osbeckianic acid content: 0.15 mg / g), weigh accurately for 9 portions, add 5 ml, 10 ml, and 15 ml of osbeckianic acid (4.06 μg / ml) respectively to three portions each, prepare spiked recovery test solutions according to the above-mentioned preparation method of test solutions, according to the above chromatographic conditions, inject 2 μl respectively, and calculate the recovery rate and RSD according to the following formula, and the results are shown in Table 29. The results show that the recovery rate of osbeckianic acid is between 92.33% and 94.63%, and the accuracy test is good.

[0229] Table 29 Accuracy test

[0230]

[0231] 6.4 Specificity test

[0232] The excipients added in Gaultheria yunnanensis formula granules are maltodextrin, silicon dioxide and magnesium stearate. This experiment examines the influence of the negative sample without Gaultheria yunnanensis on the content determination of Gaultheria yunnanensis formula granules. Take the negative sample without Gaultheria yunnanensis and prepare the negative sample solution according to the preparation method of test samples.

[0233] Take the Qiannianjian formula granules test solution, negative control solution and Jinjinxiang acid reference solution and inject them into the liquid chromatograph. Figure 25 The results showed that there was no chromatographic peak at the retention time corresponding to the reference substance in the negative chromatogram, indicating that the excipients and solvents had no interference with the determination of cinnamaldehyde. This method was specific for determining the content of cinnamaldehyde in Qiannianjian formula granules.

[0234] 6.5 Integrity test

[0235] Under the same chromatographic conditions, keep the elution gradient when the methanol ratio is the highest, double the elution time, and record the chromatogram. Figure 24 The results showed that doubling the analysis time did not interfere with the determination of cinnamonic acid.

[0236] 6.6 Durability test

[0237] 6.6.1 Stability test

[0238] Take Qiannianjian formula granules and prepare the test solution according to the above test solution preparation method. Inject 2 μl at 0, 2, 4, 8, 10, 12, 18, and 24 hours respectively, measure the peak area value, and calculate its RSD. The results are shown in Table 30.

[0239] Table 30 Stability test results

[0240]

[0241] The results showed that the sample solution had good stability within 24 hours.

[0242] 6.6.2 Investigation of different flow rates

[0243] Take Qiannianjian formula granules and prepare the test solution according to the above test solution preparation method. The content of cinnamaldehyde at flow rates of 0.28ml / min, 0.30ml / min and 0.32ml / min is investigated. The results are shown in Figure 16 , Table 31.

[0244] Table 31 Investigation of different flow rates

[0245]

[0246] The results show that when the flow rate is in the range of 0.28ml / min to 0.32ml / min, small changes in the flow rate have no significant effect on the content determination of the sample, and the durability is good.

[0247] 6.6.3 Investigation of different column temperatures

[0248] Take the Rhizoma Homalomenae formula granule, prepare the test solution according to the above-mentioned preparation method of the test solution, and examine the content of osbeckianic acid at three temperatures of 18 °C, 20 °C, and 22 °C. The results are shown in Figure 15 and Table 32.

[0249] Table 32 Examination at different column temperatures

[0250]

[0251] The results show that: within the range of 18 °C to 22 °C of the column temperature, small changes in the column temperature have no significant impact on the determination of the content of the sample, and the durability is good.

[0252] 6.6.4 Chromatographic column examination

[0253] Take the Rhizoma Homalomenae formula granule, prepare the test solution according to the above-mentioned preparation method of the test solution, and examine the content of osbeckianic acid under 3 different chromatographic column conditions of ACQUITY UPLC CORTECS T3 (Waters, 2.1 mm × 150 mm, 1.6 μm); Syncronis C18 (Thermo, 2.1 mm × 100 mm, 1.7 μm); Hypersil GLOD aQ (Thermo, 2.1 mm × 150 mm, 1.9 μm). The results are shown in Figure 14 and Table 33.

[0254] Table 33 Examination of different chromatographic columns

[0255]

[0256] The results show that: for the three different types of chromatographic columns, the resolution of the target component is good, the content of osbeckianic acid is similar, and the durability is good.

[0257] Example 4: Identification study on the genuineness and fakeness of Rhizoma Homalomenae

[0258] 1 Instruments and reagents

[0259] Same as Example 2. The standard decoction of Rhizoma Homalomenae and the standard decoction of the fake Rhizoma Homalomenae (Alocasia macrorrhiza) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0260] 2 Determination of chromatographic conditions

[0261] Detection was carried out using the chromatographic conditions determined in Example 2. 3 Preparation of the test solution

[0262] Take about 0.3 g of the powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 15 ml of 10% methanol, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 10% methanol, shake well, and take the subsequent filtrate to obtain the solution.

[0263] 4 Sample Determination and Data Analysis

[0264] Take 10 batches of the standard decoction of *Homalomena occulta* and 2 batches of the standard decoction of the fake *Homalomena occulta* ( *Alocasia macrorrhiza*) respectively. Prepare the sample test solution according to the above-mentioned preparation method of the test solution. Precisely pipette 2 μl and inject it into the liquid chromatograph. According to the above-mentioned chromatographic conditions, determine the characteristic fingerprints of the standard decoctions of *Homalomena occulta* and *Alocasia macrorrhiza* respectively. Use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" recommended by the Pharmacopoeia Commission of the People's Republic of China to generate the reference characteristic fingerprint, and then import the characteristic fingerprints of *Homalomena occulta* and its fake into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" for comparative analysis.

[0265] The results are as Figure 17 shown. There are significant differences in the number of common peaks between *Homalomena occulta* and its fake (*Alocasia macrorrhiza*). The chromatogram of the standard decoction of *Homalomena occulta* shows 9 common characteristic peaks, while the chromatogram of the standard decoction of *Alocasia macrorrhiza* only shows 3 common characteristic peaks (Peak 1 - Peak 3) in *Homalomena occulta*. Peak 4 and Peak 5 do not exist in *Alocasia macrorrhiza*, and Peak 6 - Peak 9 only exist in some batches of the fake, and they are not common peaks; while Peak A and Peak B are the newly added common characteristic peaks of *Alocasia macrorrhiza*. Peak A only exists in some batches of *Homalomena occulta*, and Peak B does not exist in *Homalomena occulta*; in addition, the content of osbeckianic acid in Peak 3 of the standard decoction of *Homalomena occulta* is significantly higher than that of its fake *Alocasia macrorrhiza*; therefore, *Homalomena occulta* and its fake can be effectively distinguished by the presence or absence of Peak 4 and Peak 5 and the limit of the content index of osbeckianic acid.

[0266] Further analyze and compare the differences between the genuine and fake *Homalomena occulta* through the relative peak area and the content of osbeckianic acid. Taking Peak 3 as the reference peak S1, compare the relative peak area differences of Peak 1, Peak 2, Peak A, Peak B, Peak 6 and Peak 7 in the standard decoctions of *Homalomena occulta* and *Alocasia macrorrhiza*; taking Peak 8 as the reference peak S2, compare the relative peak area differences of Peak 9 in the standard decoctions of *Homalomena occulta* and *Alocasia macrorrhiza*. The results are as follows:[[]]

[0267] Table 34 Relative Peak Area Range of the Standard Decoction Samples of *Homalomena occulta* and *Alocasia macrorrhiza*

[0268]

[0269] Table 35 Content Range of Osbeckianic Acid in the Standard Decoction Samples of *Homalomena occulta* and *Alocasia macrorrhiza*

[0270]

[0271] Table 36 Relative Peak Area of the Standard Decoction Samples of *Homalomena occulta* and *Alocasia macrorrhiza*

[0272]

[0273]

[0274] The results showed that there were significant differences in the relative peak area of peak 2 to peak 3 and the content of osajinic acid in the standard decoctions of Homalomena occulta and Alocasia macrorrhiza, which could effectively distinguish the two.

[0275] Example 5: Establishment of TLC identification method for Homalomena occulta formula granules and its methodology research

[0276] 1 Test drugs

[0277] The control medicinal material of Homalomena occulta (batch number: 121571 - 201202) was purchased from the National Institutes for Food and Drug Control. The Homalomena occulta formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0278] 2 Preparation of solutions

[0279] 2.1 Preparation of test solution

[0280] Method 1: Take 0.5 g of Homalomena occulta formula granules, grind them finely, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0281] Method 2: Take 0.5 g of Homalomena occulta formula granules, grind them finely, add 20 ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0282] Method 3: Take 0.5 g of Homalomena occulta formula granules, grind them finely, add 20 ml of water to dissolve, extract twice with ethyl acetate by shaking, 20 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0283] 2.2 Preparation of control medicinal material solution

[0284] Method 1: Take 2 g of the control medicinal material of Homalomena occulta, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0285] Method 2: Take 2 g of the control medicinal material of Homalomena occulta, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0286] 2.3 Preparation of negative control solution

[0287] Take 0.5 g of the negative sample without Homalomena occulta, and prepare the negative control solution according to "Method 1 in 3.1".

[0288] 3 Determination of TLC identification method

[0289] 3.1 Investigation on Preparation Methods of Different Developing Solvents, Test Solution and Control Medicinal Material Solution

[0290] Take the test solutions prepared by the above three methods, and take the control medicinal material solutions prepared by the above two methods. Spot them on a silica gel G thin-layer plate respectively. Use cyclohexane-ethyl acetate (8:2) as the first developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are shown in Figure 18 , where 1-2: Homalomena occulta formula granules (Method 1); 3-4: Homalomena occulta formula granules (Method 2); 5-6: Homalomena occulta formula granules (Method 3); S1: Homalomena occulta control medicinal material (Method 1); S2: Homalomena occulta control medicinal material (Method 2).

[0291] Take the above three test solutions and two control medicinal material solutions and spot them on a silica gel G thin-layer plate respectively. Use chloroform-methanol (10:1) as the second developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are shown in Figure 19 , where 1-2: Homalomena occulta formula granules (Method 1); 3-4: Homalomena occulta formula granules (Method 2); 5-6: Homalomena occulta formula granules (Method 3); S1: Homalomena occulta control medicinal material (Method 1); S2: Homalomena occulta control medicinal material (Method 2).

[0292] As can be seen from the figure, since the formula granules are prepared by water extraction process, the first developing solvent is mainly for low-polarity components. Therefore, no spots corresponding to the control medicinal material could be detected in the above three test samples; spots corresponding to the control medicinal material could be detected in all the test samples with the second developing solvent, and the spots were clear and the resolution was good. Therefore, the second developing solvent was selected as the developing solvent for the thin-layer identification method of Homalomena occulta formula granules.

[0293] For the optimized thin-layer chromatogram of the second developing solvent, the chromatograms of Method 1 and Method 3 had little difference, the spots were clear and the resolution was good, while the spot information of Method 2 was incomplete. Compared with Method 3, Method 1 was more convenient to operate. Therefore, Method 1 was selected as the sample preparation method for the thin-layer identification of Homalomena occulta formula granules. There were differences between the two preparation methods of the control medicinal material solution. The control medicinal material after boiling in water corresponded better to the granules' spots. Therefore, Method 2 of the control medicinal material was selected as the preparation method of the control medicinal material for the thin-layer identification of Homalomena occulta formula granules.

[0294] 3.2 Investigation on Different Sample Sizes

[0295] Take the Homalomena occulta formula granule and the Homalomena occulta control crude drug, and prepare the test solution and the control crude drug solution respectively according to the determined preparation method. According to the above-determined thin-layer chromatography conditions, spot different amounts of samples on the same silica gel G thin-layer plate respectively, use chloroform-methanol (10:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are shown in Figure 20 , where 1: 2 μl of the test solution; 2: 5 μl of the test solution; 3: 8 μl of the test solution; 4: 10 μl of the test solution; S1: 2 μl of the control crude drug; S2: 5 μl of the control crude drug; S3: 8 μl of the control crude drug; S4: 10 μl of the control crude drug. It can be seen that when the spotting amounts of the test solution and the control crude drug solution are 5 μl, the chromatograms of the Homalomena occulta formula granule and the control crude drug have clear spots at the corresponding positions, with good resolution and no other interference. Therefore, the spotting amounts of the test solution and the control crude drug solution are selected as 5 μl.

[0296] 4 Specificity experiment of the sample

[0297] Take the test solution of the Homalomena occulta formula granule, the control crude drug solution of Homalomena occulta and the negative control solution, according to the above thin-layer chromatography conditions, spot on the same silica gel G thin-layer plate respectively, use chloroform-methanol (10:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are shown in Figure 21 , where 1-3: Homalomena occulta formula granule; 4: negative control; S: Homalomena occulta control crude drug. It can be seen that the Homalomena occulta formula granule chromatogram and the control crude drug chromatogram show the same color spots at the corresponding positions, while the negative control has no interference. It shows that the specificity of this thin-layer method is good.

Claims

1. A quality control method for Qiannianjian samples, characterized in that: The following steps are involved: (1) Prepare the test sample solution with the Qiannianjian test sample; prepare the control medicinal material reference solution with the Qiannianjian control medicinal material; prepare the control material reference solution separately, the control material reference solution includes uridine solution and cinnamonic acid solution; use 15-25 ml of extractant for every 0.2-1 g of Qiannianjian test sample, extract for 15-60 min to prepare the test solution; use 15-25 ml of extractant for every 1-3 g of Qiannianjian control medicinal material, extract for 15-60 min to prepare the control medicinal material reference solution; the extractant is a methanol aqueous solution with a volume concentration of 10-30%; (2) The reference medicinal material reference solution, the reference substance reference solution and the test sample solution were respectively taken for ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of the Qiannianjian sample to be tested, wherein the characteristic spectrum includes a characteristic peak 2 having a retention time consistent with the uridine reference substance peak and a characteristic peak 3 having a retention time consistent with the cypermethrin reference substance peak; the conditions of the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is ACQUITY CORTECS T3, Waters, 2.1 mm×150mm, 1.6μm chromatographic column; methanol as mobile phase A, 0.05%-0.2% phosphoric acid solution as mobile phase B, gradient elution: 0-10min, 0%A, 10-22min, 0→8%A, 22-32min, 8%A, 32-33min, 8→0%A, 33-38min, 0%A; detection wavelength 220-280nm; flow rate 0.28-0.32ml / min; column temperature 18℃-22℃; UV detector; (3) Performing a qualitative analysis of the Qiannianjian sample using the characteristic spectrum obtained in step (2); and performing a quantitative analysis of the Qiannianjian sample using cinnamonic acid as a content determination indicator; The Qiannianjian samples to be tested include Qiannianjian medicinal materials, Qiannianjian decoction pieces, Qiannianjian standard decoctions and Qiannianjian formula granules.

2. The quality control method of Qiannianjian samples according to claim 1, characterized in that: In step (1), the reference substance solution also includes a 5-hydroxymethylfurfural solution, a protocatechuic acid solution and a p-hydroxybenzoic acid solution; in step (2), the characteristic spectrum also includes a characteristic peak 4 having a retention time consistent with the 5-hydroxymethylfurfural reference substance peak, a characteristic peak 7 having a retention time consistent with the protocatechuic acid reference substance peak and a characteristic peak 8 having a retention time consistent with the p-hydroxybenzoic acid reference substance peak.

3. The quality control method of Qiannianjian samples according to claim 2, characterized in that: The concentration of the uridine solution is 5-15 μg / ml; the concentration of the cinnamyl acid solution is 5-15 μg / ml; the concentration of the 5-hydroxymethylfurfural solution is 5-15 μg / ml; the concentration of the protocatechuic acid solution is 5-15 μg / ml; and the concentration of the p-hydroxybenzoic acid solution is 5-15 μg / ml.

4. The quality control method of Qiannianjian samples according to claim 1, characterized in that: In step (3), the qualitative analysis method of the Qiannianjian sample to be tested is to distinguish Qiannianjian from Alocasia odora based on the relative peak area of ​​characteristic peak 2 to characteristic peak 3 and the content of cinnamomum camphora.

5. The quality control method of Qiannianjian samples according to claim 1, characterized in that: The Qiannianjian test sample is qualitatively analyzed by thin layer chromatography before preparing the test solution, and the Qiannianjian control medicinal material is used as a reference to determine whether the spots of the Qiannianjian test sample and the Qiannianjian control medicinal material on the thin layer plate correspond one to one. If so, steps (1)-(3) are performed; if not, the Qiannianjian test sample is an unqualified sample.

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