Construction method and application of characteristic chromatograms of Pinelliae Rhizoma preparata, standard decoction and formula granules

Abstract

The present invention provides a method for constructing characteristic chromatograms of Pinelliae Rhizoma Praeparatum, standard decoction, and formula granules, and its application, including the following steps: dissolving the test sample with a solvent, extracting to obtain a test solution; measuring the test solution by high performance liquid chromatography to obtain the characteristic chromatograms of Pinelliae Rhizoma Praeparatum, standard decoction, and formula granules; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is performed. The present invention uses high performance liquid chromatography, selects acetonitrile - 0.1% phosphoric acid aqueous solution as the mobile phase for gradient elution, uses 4-hydroxybenzoic acid as the reference substance, and establishes the HPLC characteristic chromatograms of Pinelliae Rhizoma Praeparatum, standard decoction, and formula granules, with good repeatability and precision, stable and reliable method, and can control the quality of Pinelliae Rhizoma Praeparatum, standard decoction, and formula granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a method for constructing characteristic chromatograms of Pinelliae Rhizoma preparata, standard decoction, and formula granules, and its application. Background Art

[0002] The Chinese medicinal material Pinelliae Rhizoma is the dried tuber of the plant Pinellia ternata (Thunb.) Breit. of the Araceae family. It is pungent and warm in nature, and has the functions of drying dampness and resolving phlegm, and reducing masses and dissipating binds. Pinelliae Rhizoma is slightly toxic, and its processed products are mostly used clinically, commonly including Rhizoma Pinelliae Praeparata cum Alumine, Rhizoma Pinelliae Praeparata cum Zingibere, Rhizoma Pinelliae Praeparata, Pinelliae Rhizoma preparata, etc.

[0003] The 2010 edition of the Chinese Pharmacopoeia included Rhizoma Pinelliae Praeparata cum Alumine, Rhizoma Pinelliae Praeparata cum Zingibere, and Rhizoma Pinelliae Praeparata. Except for Rhizoma Pinelliae Praeparata cum Alumine, the quality standards of Rhizoma Pinelliae Praeparata cum Zingibere and Rhizoma Pinelliae Praeparata only involved the inspection of character, microscopy, moisture, and total ash. The processing method and quality standard of Pinelliae Rhizoma preparata are not specified in the Chinese Pharmacopoeia, and the quality of Pinelliae Rhizoma preparata cannot be effectively controlled. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing characteristic chromatograms of Pinelliae Rhizoma preparata, standard decoction, and formula granules, and its application. This method provides more scientific and technological means for the quality evaluation of Pinelliae Rhizoma preparata, standard decoction, and its formula granules.

[0005] The present invention provides a method for constructing characteristic chromatograms of Pinelliae Rhizoma preparata, standard decoction, and formula granules, comprising the following steps:

[0006] Dissolve and extract the test sample with a solvent to obtain a test solution;

[0007] Determine the test solution by high performance liquid chromatography to obtain the characteristic chromatograms of Pinelliae Rhizoma preparata, standard decoction, and formula granules;

[0008] The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid aqueous solution, with gradient elution.

[0009] In the present invention, the test sample solution is dissolved and extracted with a solvent to obtain a test solution. The test sample is Pinelliae Rhizoma preparata, standard decoction, and formula granules. Pinelliae Rhizoma preparata is the processed product of the dried tuber of the plant Pinellia ternate (Thunb.) Breit. of the Araceae family. It has the functions of drying dampness and resolving phlegm, and lowering adverse qi and stopping vomiting. The standard decoction of Pinelliae Rhizoma preparata is the freeze-dried powder prepared from Pinelliae Rhizoma preparata according to a fixed preparation process; the formula granules of Pinelliae Rhizoma preparata are the formula granules processed from Pinelliae Rhizoma preparata according to the main quality indicators of the standard decoction.

[0010] In the present invention, both 0.1% phosphoric acid aqueous solution and 30% methanol are volume fractions.

[0011] In the present invention, the extraction method is ultrasonic extraction; the power of the ultrasonic extraction is 580 - 620 W, the frequency of the ultrasonic extraction is 35 - 45 kHz; the time of the ultrasonic extraction is 25 - 35 min; in a specific embodiment, the power of the ultrasonic extraction is 600 W, the frequency of the ultrasonic extraction is 40 kHz; the time of the ultrasonic extraction is 30 min.

[0012] When the test sample is Pinelliae Rhizoma preparatum slices, the specific process of dissolving the test sample is as follows:

[0013] Boil Pinelliae Rhizoma preparatum slices in water, let it cool, filter, evaporate the filtrate to dryness, add 30% methanol, perform ultrasonic treatment, shake well, filter, and obtain the filtrate, which is the test solution to be measured. The mass - to - volume ratio of Pinelliae Rhizoma preparatum slices to water is (1.8 - 2.2) g:100 mL, and boil for 28 - 32 min; in a specific embodiment, the mass - to - volume ratio of Pinelliae Rhizoma preparatum slices to water is 2 g:100 mL, and boil for 30 min.

[0014] When the test sample is the standard decoction of Pinelliae Rhizoma preparatum or Pinelliae Rhizoma preparatum formula granules, the specific process of dissolving the test sample is as follows:

[0015] Dissolve the test sample with 30% methanol, perform ultrasonic treatment, shake well, filter, and obtain the filtrate, which is the test solution to be measured. The mass (g) of the test sample to the volume (mL) of 30% methanol ratio is 1:25.

[0016] In the present invention, the extraction solvent is 30% methanol; the chromatographic peaks obtained by using 30% methanol as the extraction solvent in the present invention have a large amount of information and good resolution.

[0017] In the present invention, the test solution is determined by high - performance liquid chromatography to obtain the characteristic chromatograms of Pinelliae Rhizoma preparatum slices, standard decoctions, and formula granules. The chromatographic conditions of the high - performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is performed.

[0018] In the present invention, the gradient elution is specifically as follows:

[0019] 0 - 5 min, mobile phase A: 0 - 2%, mobile phase B: 100 - 98%;

[0020] 5 - 8 min, mobile phase A: 2%, mobile phase B: 98%;

[0021] 8 - 15 min, mobile phase A: 2 - 6%, mobile phase B: 98 - 94%;

[0022] 15 - 32 min, mobile phase A: 6 - 14%, mobile phase B: 94 - 86%;

[0023] 32 - 42 min, mobile phase A: 14 - 25%, mobile phase B: 86 - 75%;

[0024] 42 - 60 min, Phase A: 25 - 50%, Phase B: 75 - 50%.

[0025] In the present invention, the flow rate of the mobile phase is 0.2 mL / min, the chromatogram peak shape is good, and the baseline is stable; it is found in the present invention that when the detection wavelength is 270 nm, the chromatographic peak information amount is larger and the chromatogram baseline is more stable, so the detection wavelength is 270 nm; the injection volume is 3 μL;

[0026] The present invention adopts The chromatogram of the T3 2.1×150 mm, 1.6 μm chromatographic column has good separation, and the column temperature is 30 °C;

[0027] The theoretical plate number calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000.

[0028] The present invention also includes preparing a reference solution:

[0029] Dissolve 4-hydroxybenzoic acid and glycyrrhizic acid reference substances in methanol respectively to obtain a reference substance reference solution;

[0030] Determine the reference solution by high performance liquid chromatography to obtain the chromatogram of the reference substance; and qualitatively analyze the components of the HPLC characteristic chromatograms of Pinelliae Rhizoma Praeparatum, standard decoction, and formula granules according to the chromatogram of the reference substance.

[0031] The present invention uses a similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarity of the HPLC characteristic chromatogram of Pinelliae Rhizoma Praeparatum, and obtains an HPLC standard characteristic chromatogram of Pinelliae Rhizoma Praeparatum composed of 13 characteristic peaks, where the peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the said standard characteristic chromatogram, calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.93 (peak 1), 0.96 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.50 (peak 6), 1.63 (peak 7), 1.68 (peak 8), 1.72 (peak 9), 2.43 (peak 10), 2.58 (peak 11), 2.83 (peak 12), 2.92 (peak 13).

[0032] The present invention uses the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint to evaluate the similarity of the HPLC characteristic fingerprint of the standard decoction of Pinelliae Rhizoma Praeparatum. An HPLC standard characteristic fingerprint composed of 13 characteristic peaks is obtained, and the peak corresponding to the reference substance of 4-hydroxybenzoic acid is the S peak. In the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.93 (peak 1), 0.97 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.51 (peak 6), 1.64 (peak 7), 1.69 (peak 8), 1.73 (peak 9), 2.45 (peak 10), 2.60 (peak 11), 2.85 (peak 12), 2.94 (peak 13).

[0033] The present invention uses the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint to evaluate the similarity of the HPLC characteristic fingerprint of the formulated granules of Pinelliae Rhizoma Praeparatum. An HPLC standard characteristic fingerprint composed of 13 characteristic peaks is obtained, and the peak corresponding to the reference substance of 4-hydroxybenzoic acid is the S peak. In the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.93 (peak 1), 0.96 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.51 (peak 6), 1.63 (peak 7), 1.69 (peak 8), 1.73 (peak 9), 2.44 (peak 10), 2.59 (peak 11), 2.84 (peak 12), 2.93 (peak 13).

[0034] Quality judgment criteria: Take the samples of the cut crude drugs, standard decoction, and formulated granules of Pinelliae Rhizoma Praeparatum, and operate according to the same method as above to obtain the characteristic fingerprints of the cut crude drugs, standard decoction, and formulated granules of Pinelliae Rhizoma Praeparatum. Use the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition) of the Pharmacopoeia Commission of the People's Republic of China to analyze the standard characteristic fingerprints and sample characteristic fingerprints of the cut crude drugs, standard decoction, and formulated granules of Pinelliae Rhizoma Praeparatum, and the similarity is greater than 0.90.

[0035] The method provided by the present invention can effectively monitor the quality of the cut crude drugs, standard decoction, and formulated granules of different batches of Pinelliae Rhizoma Praeparatum, making their quality stable. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensively monitoring the quality of products.

[0036] The characteristic fingerprints of the cut crude drugs, standard decoction, and formulated granules of Pinelliae Rhizoma Praeparatum established by the present invention use rosmarinic acid as the reference substance, pay attention to the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, and can comprehensively evaluate the overall quality characteristics of the products. The method is scientific and reliable.

[0037] The present invention provides a method for identifying the cut crude drugs, standard decoction, and formulated granules of Pinelliae Rhizoma Praeparatum, which is characterized in that the method described in the above technical solution is used for detection and the detection results are analyzed.

[0038] The present invention uses the above method to identify 4-hydroxybenzoic acid, and can control the quality of Pinelliae Rhizoma preparata slices, standard decoctions, and formula granules.

[0039] The present invention provides a method for constructing characteristic chromatograms of Pinelliae Rhizoma preparata slices, standard decoctions, and formula granules, comprising the following steps: dissolving and extracting a test sample with a solvent to obtain a test solution; measuring the test solution by high performance liquid chromatography to obtain characteristic chromatograms of Pinelliae Rhizoma preparata slices, standard decoctions, and formula granules; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid aqueous solution, with gradient elution. The present invention uses high performance liquid chromatography, selects acetonitrile and 0.1% phosphoric acid aqueous solution as mobile phases for gradient elution, and uses 4-hydroxybenzoic acid as a reference substance to establish HPLC characteristic chromatograms of Pinelliae Rhizoma preparata slices, standard decoctions, and formula granules, with good repeatability and precision, and the method is stable and reliable, and can control the quality of Pinelliae Rhizoma preparata slices, standard decoctions, and formula granules. Description of the Drawings

[0040] Figure 1 Investigation on the extraction method of Pinelliae Rhizoma preparata slices;

[0041] Figure 2 Investigation on the extraction solvent of Pinelliae Rhizoma preparata slices;

[0042] Figure 3 Investigation on the extraction time of Pinelliae Rhizoma preparata slices;

[0043] Figure 4 Investigation on the solvent addition amount of Pinelliae Rhizoma preparata slices;

[0044] Figure 5 Identification of chromatographic peaks of Pinelliae Rhizoma preparata slices;

[0045] Figure 6 Investigation on different instruments of Pinelliae Rhizoma preparata slices;

[0046] Figure 7 Investigation on different chromatographic columns of Pinelliae Rhizoma preparata slices;

[0047] Figure 8 Characteristic chromatogram of Pinelliae Rhizoma preparata slices;

[0048] Figure 9 Reference characteristic chromatogram of Pinelliae Rhizoma preparata slices;

[0049] Figure 10 Investigation on the extraction method of Pinelliae Rhizoma preparata standard decoction;

[0050] Figure 11 Investigation on the extraction solvent of Pinelliae Rhizoma preparata standard decoction;

[0051] Figure 12For the investigation of the extraction time of the standard decoction of Pinelliae Rhizoma Praeparatum cum Alumine;

[0052] Figure 13 For the investigation of the solvent addition amount of the standard decoction of Pinelliae Rhizoma Praeparatum cum Alumine;

[0053] Figure 14 For the identification of chromatographic peaks of the standard decoction of Pinelliae Rhizoma Praeparatum cum Alumine;

[0054] Figure 15 For the investigation of different instruments of the standard decoction of Pinelliae Rhizoma Praeparatum cum Alumine;

[0055] Figure 16 For the investigation of different chromatographic columns of the standard decoction of Pinelliae Rhizoma Praeparatum cum Alumine;

[0056] Figure 17 For the characteristic chromatogram of the standard decoction of Pinelliae Rhizoma Praeparatum cum Alumine;

[0057] Figure 18 For the control characteristic chromatogram of the standard decoction of Pinelliae Rhizoma Praeparatum cum Alumine;

[0058] Figure 19 For the selection of the mobile phase of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0059] Figure 20 For the chromatograms of different wavelengths of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0060] Figure 21 For the investigation of the column temperature of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0061] Figure 22 For the investigation of the flow rate of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0062] Figure 23 For the investigation of the delay of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0063] Figure 24 For the investigation of the extraction method of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0064] Figure 25 For the investigation of the extraction solvent of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0065] Figure 26 For the investigation of the extraction time of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0066] Figure 27 For the investigation of the solvent addition amount of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0067] Figure 28 For the identification of chromatographic peaks of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0068] Figure 29 For the investigation of different instruments of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0069] Figure 30 For the investigation of different chromatographic columns of the formula granules of Pinelliae Rhizoma Praeparatum cum Alumine;

[0070] Figure 31 It is the characteristic spectrum of Jingbanxia formula granules;

[0071] Figure 32 It is the reference characteristic spectrum of Jingbanxia formula granules. Specific implementation manners

[0072] To further illustrate the present invention, the following describes in detail a method for constructing characteristic chromatograms of Jingbanxia cut crude drugs, standard decoctions, and formula granules provided by the present invention and their applications in combination with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0073] Example 1 Construction of UPLC characteristic chromatogram of Jingbanxia cut crude drugs

[0074] 1) Instruments, materials, reagents, and test drugs

[0075] Ultra-high performance liquid chromatograph: Agilent 1290;

[0076] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler Toledo Instruments Co., Ltd.);

[0077] Ultra-pure water machine: Cell type 1810A (Shanghai Mole Scientific Instruments Co., Ltd.);

[0078] Ultrasonic cleaner: Model KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.);

[0079] Chromatographic column: T3 2.1×150mm, 1.6μm;

[0080] Methanol and acetonitrile are chromatographically pure, water is ultra-pure water, and the rest of the reagents are all analytically pure;

[0081] Acetonitrile and phosphoric acid are chromatographically pure; water is ultra-pure water, and other reagents are all analytically pure.

[0082] p-Hydroxybenzoic acid reference substance (Shanghai Standard Technology Service Co., Ltd., batch number: 2169, content calculated as 99.9%)

[0083] Glycyrrhizic acid reference substance (National Institutes for Food and Drug Control, batch number: 111709-201702, content calculated as 99.9%)

[0084] Pinellia ternata Thunb. reference crude drug (National Institutes for Food and Drug Control, batch number: 121272-201806)

[0085] Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. M. Feng Decoction Pieces (Batch Numbers: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15).

[0086] 2) Determination Method of Characteristic Chromatogram

[0087] Chromatographic Conditions and System Suitability Test Use octadecylsilane chemically bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 1 below; the flow rate is 0.3 ml per minute; the column temperature is 30 °C; the detection wavelength is 270 nm. The number of theoretical plates calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000.

[0088] Table 1 Gradient Elution Program

[0089]

[0090] Preparation of Reference Substance Solution Weigh an appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances accurately, and dissolve them separately in methanol to prepare solutions containing 20 μg per 1 ml as the reference substance solution for the reference substances.

[0091] 3) Chromatographic Conditions and System Suitability Test

[0092] Based on the manifestation of mass and mass transfer, the characteristic chromatographic conditions and system adaptability of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. M. Feng Decoction Pieces are consistent with the characteristic chromatogram methods of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. M. Feng Standard Decoction and Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. M. Feng Formula Granules.

[0093] The chromatographic conditions and system suitability test for the characteristic chromatogram of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. M. Feng Decoction Pieces are determined as follows: use octadecylsilane chemically bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 1 above; the flow rate is 0.3 ml per minute; the column temperature is 30 °C; the detection wavelength is 270 nm. The number of theoretical plates calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000.

[0094] Preparation of Test Solution Take about 2 g of the powder of this product (passed through No. 3 sieve), place it in a conical flask, add 50 ml of 30% methanol, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0095] Determination Method Accurately pipette 3 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and perform the determination to obtain the results.

[0096] 4) Investigation on the Preparation of Test Solution

[0097] 4.1 Investigation of extraction methods

[0098] By comparing ultrasonic extraction, reflux extraction and ultrasonic extraction after water decoction, it was found that the chromatographic peak information was larger and the number of peaks was more after ultrasonic extraction following water decoction. Finally, the method of ultrasonic extraction after water decoction was selected for extraction. See Figure 1 .

[0099] 4.2 Investigation of extraction solvents:

[0100] Methanol, water, 30% methanol, 50% methanol and 80% methanol were investigated as ultrasonic extraction solvents. The results showed that using water and 30% methanol as extraction solvents gave large chromatographic peak information and good resolution, and 30% methanol was easier to operate. Therefore, 30% methanol was selected as the extraction solvent. See Figure 2 .

[0101] 4.3 Investigation of extraction time

[0102] By comparing ultrasonic extraction for 15 min, 30 min and 60 min, it was found that the sample was completely extracted at 30 min. Therefore, the extraction time was determined to be 30 min. See Figure 3 .

[0103] 4.4 Investigation of extraction solvent volume

[0104] The amounts of ultrasonic extraction solvent added, 25 ml, 50 ml and 100 ml, were compared. When the extraction solvent volume was 25 ml, the chromatographic peak size was appropriate. Therefore, the extraction solvent volume was determined to be 25 ml. See Figure 4 .

[0105] 4.5 Final determination of the preparation method of test samples

[0106] Take about 2 g of the powder of this product (sieved through No. 4 sieve), place it in a stoppered conical flask, add 100 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the continuous filtrate to obtain the solution.

[0107] 5) Methodology investigation

[0108] 5.1 Chromatographic peak identification

[0109] Preparation of test solution: Prepare the test solution of Pinelliae Rhizoma Praeparatum according to the final determined preparation method of test samples in 4.5.

[0110] Preparation of reference solution of control medicinal material: Take about 2 g of Pinelliae Rhizoma control medicinal material, place it in a stoppered conical flask, add 100 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the continuous filtrate to obtain the solution.

[0111] Preparation of reference substance solution: Appropriate amounts of reference substances of 4-hydroxybenzoic acid and glycyrrhizic acid were separately dissolved in methanol to prepare solutions containing 20 μg per 1 ml as reference substance reference solutions.

[0112] Preparation of negative control solution: According to the above-mentioned experimental conditions, a negative control solution without Pinelliae Rhizoma Praeparatum was prepared. The characteristic chromatogram peaks of Pinelliae Rhizoma Praeparatum were located. See Figure 5 . The results showed that peak 3 was 4-hydroxybenzoic acid and peak 13 was glycyrrhizic acid. There were significant differences in the peaks between the reference medicinal material of Pinelliae Rhizoma and Pinelliae Rhizoma Praeparatum, which were not included in the characteristic method. In the following methodological investigations, 13 characteristic peaks in the samples were investigated.

[0113] 5.2 Precision test

[0114] Take the test solution of Pinelliae Rhizoma Praeparatum (batch number: YP-06), inject 1 μl each time continuously for 6 times according to the proposed experimental method, and calculate the retention time of each characteristic peak. See Table 2:

[0115] Table 2 Precision investigation - Retention time

[0116]

[0117] The results showed that the RSD of the retention time of each characteristic peak in terms of precision was 0.03% - 0.21%. The precision of this instrument was good.

[0118] 5.3 Repeatability investigation

[0119] Accurately weigh 6 portions of Pinelliae Rhizoma Praeparatum (batch number: YP-06), prepare and determine them according to the proposed experimental method. See Table 3.

[0120] Table 3 Repeatability investigation - Relative retention time ratio

[0121]

[0122]

[0123] The results showed that the RSD of the relative retention time of each characteristic peak in terms of repeatability was 0.00% - 0.48%. This method had good repeatability.

[0124] 5.4 Intermediate precision investigation

[0125] 5.4.1 Different personnel and different times

[0126] On the basis of the above-mentioned experimental conditions, weigh Pinelliae Rhizoma Praeparatum (batch number: YP-06), prepare test solutions by different personnel at different times, and conduct determinations. See Table 4.

[0127] Table 4 Intermediate precision - Relative retention time ratio

[0128]

[0129] The results show that when the same sample is measured by different personnel at different times, the method precision is good.

[0130] 5.4.2 Different Instruments

[0131] On the basis of the above-mentioned established experimental conditions, Instruments 1, 2, and 3 were investigated respectively. See Figure 6 , Table 5.

[0132] Table 5 Chromatographic Column Durability Investigation - Relative Retention Time

[0133]

[0134] The results show that when detected with the above three instruments, the RSD of the relative retention time of each characteristic peak is between 0.62% and 6.16%; the durability of different instruments is good.

[0135] 5.5 Chromatographic Column Durability Investigation

[0136] On the basis of the above-mentioned established experimental conditions, Chromatographic Column 1 ( T3 2.1×150mm, 1.6μm); Chromatographic Column 2 (Phenomenex Omega PS C18 100A

[0137] 2.1×150mm, 1.6μm); Chromatographic Column 3 (ACQUITY BEH Shield RP18 2.1×150mm, 1.7μm) were investigated respectively. See Figure 7 , Table 6.

[0138] Table 6 Chromatographic Column Durability Investigation - Relative Retention Time

[0139]

[0140]

[0141] From Figure 7 it can be seen that the chromatogram of T3 2.1×150mm, 1.6μm (Chromatographic Column 1) has better separation, so it is recommended to use Chromatographic Column T3 2.1×150mm, 1.6μm.

[0142] 5.6 Stability Investigation

[0143] On the basis of the above-mentioned experimental conditions, the same test solution was taken and determined at 0 h, 3 h, 7 h, 13 h, 20 h, and 25 h respectively. See Table 7.

[0144] Table 7 Stability study - Retention time

[0145]

[0146] The results showed that the RSD of the retention time of the corresponding characteristic peaks was between 0.03% and 0.20%, and the sample solution was relatively stable within 24 hours.

[0147] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in all the above investigations, and the method was good. The above 13 characteristic peaks were included in the subsequent investigations.

[0148] 6) Verification of the characteristic chromatogram of Pinelliae Rhizoma preparata

[0149] The Pinelliae Rhizoma preparata was inspected, and the relative retention time was calculated. The results are shown in Figure 8 (From bottom to top, the batch numbers are: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15), Table 8.

[0150] Table 8 Relative retention time of the characteristic chromatogram of Pinelliae Rhizoma preparata

[0151]

[0152]

[0153] According to the principle of stable relative retention time and that all batches of samples could be detected with relatively high peaks, a total of 13 peaks with good repeatability were selected as characteristic peaks. The results showed that the RSD of the relative peak area of the characteristic peaks of Pinelliae Rhizoma preparata was too large to stipulate the relative peak area, so it was not included in the main text of the quality standard. Finally, it was stipulated that: 13 characteristic peaks should be presented in the chromatogram of the test solution. Among them, peak 3 should correspond to the retention time of the reference substance peak of the reference substance, and the peak corresponding to the reference substance peak of 4-hydroxybenzoic acid reference substance was the S peak. The relative retention time of each characteristic peak and the S peak was calculated, and its relative retention time should be within the range of ±10% of the specified value. The specified values were: 0.93 (peak 1), 0.96 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.50 (peak 6), 1.63 (peak 7), 1.68 (peak 8), 1.72 (peak 9), 2.43 (peak 10), 2.58 (peak 11), 2.83 (peak 12), 2.92 (peak 13).

[0154] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), 15 batches of Pinelliae Rhizoma preparata slices were synthesized to establish a reference fingerprint for the characteristic fingerprint of Pinelliae Rhizoma preparata slices, as shown in Figure 9 , Peak 3 (S): 4-Hydroxybenzoic acid; Peak 13: Glycyrrhizic acid; Chromatographic column: T3 2.1×150mm, 1.6μm.

[0155] In summary, the method for the characteristic fingerprint of the slices is as follows:

[0156] Chromatographic conditions and system suitability test Using octadecylsilane chemically bonded silica gel as the filler (column length 100mm, inner diameter 2.1mm, particle size 1.6μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 1 above; the flow rate was 0.2ml per minute; the column temperature was 30°C; the detection wavelength was 270nm. The number of theoretical plates calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000.

[0157] Preparation of the reference substance solution Weigh an appropriate amount of 4-hydroxybenzoic acid reference substance accurately, dissolve it in methanol to make a solution containing 20μg per 1ml, and use it as the reference substance solution for the reference substance.

[0158] Preparation of the test solution Take about 2g of the powder of this product (sieved through No. 4 sieve), place it in a conical flask, add 100ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the subsequent filtrate, that is, obtain.

[0159] Determination method Accurately pipette 3μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and determine, that is, obtain.

[0160] In the chromatogram of the test solution, 13 characteristic peaks should be presented. Among them, Peak 3 should correspond to the retention time of the reference substance peak of the reference substance, and the peak corresponding to the 4-hydroxybenzoic acid reference substance peak of the reference substance is the S peak. Calculate the relative retention time of each characteristic peak to the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are 0.93 (Peak 1), 0.96 (Peak 2), 1.16 (Peak 4), 1.21 (Peak 5), 1.50 (Peak 6), 1.63 (Peak 7), 1.68 (Peak 8), 1.72 (Peak 9), 2.43 (Peak 10), 2.58 (Peak 11), 2.83 (Peak 12), 2.92 (Peak 13).

[0161] Example 2 UPLC characteristic fingerprint method for the standard decoction of Pinelliae Rhizoma preparata

[0162] 1. Test materials

[0163] Standard Decoction of Pinellia ternata (Thunb.) Breit. var. pekinensis (Baill.) Hsiao et Kuang (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15);

[0164] 2. Chromatographic conditions

[0165] Using octadecylsilane chemically bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 1 above; the flow rate was 0.2 ml per minute; the column temperature was 30 °C; the detection wavelength was 270 nm. The number of theoretical plates calculated with respect to the 4-hydroxybenzoic acid peak should be not less than 5000.

[0166] 3. Preparation of reference substance solution[[ID=IO]] M

[0167] Appropriately weigh an appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, and accurately weigh them. Respectively dissolve them in methanol to prepare solutions containing 20 μg per 1 ml as the reference substance reference solutions.

[0168] 4. Preparation of test solution

[0169] Take about 1.0 g of this product, place it in a stoppered conical flask, add 50 ml of 30% methanol, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate, that is obtained.

[0170] 5. Assay method

[0171] Precisely pipette 3 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and determine, then it is obtained.

[0172] 6. Chromatographic conditions and system suitability test [[ID=Z8]]

[0173] The material composition basis of the standard decoction and the finished product granules is consistent, and the characteristic chromatographic conditions, system suitability, and the characteristic chromatogram method of the granules are kept consistent.

[0174] 7. Investigation on the preparation of test solution

[0175] 7.1 Investigation on extraction method

[0176] Take about 1.0 g of this product (batch number: BT-06), place it in a stoppered conical flask, add 50 ml of 30% methanol respectively, tightly stopper, and investigate the extraction method of the test solution for reflux and ultrasonic (power 600 W, frequency 40 kHz) respectively. The extraction time is 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate, that is obtained. See Figure 10, The results showed that there was little difference in the extraction effects between refluxing and ultrasonic extraction of the test sample, and the ultrasonic method was rapid and simple. Therefore, the ultrasonic extraction method was determined as the extraction method for the test sample.

[0177] 7.2 Investigation of extraction solvents

[0178] Take about 1.0 g of this product (batch number: BT-06), place it in a stoppered conical flask, add water, 30% methanol, 50% methanol, 80% methanol, and 50 ml of methanol respectively, stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. See Figure 11 , The results showed that when water, 30%, and 50% methanol were used as extraction solvents, the chromatographic peak information was large and the resolution was good; when 30% methanol was used as the extraction solvent, the operation was easier. Therefore, the extraction solvent was tentatively set as 30% methanol.

[0179] 7.3 Investigation of extraction time

[0180] Take about 1.0 g of this product (batch number: BT-06), place it in a stoppered conical flask, add 50 ml of 30% methanol, stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz), and investigate the extraction time of the test sample at 15 min, 30 min, and 60 min respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. See Figure 12 , The results showed that when the extraction time was 30 min, the test sample had been completely extracted. Therefore, the extraction time was determined as 30 min.

[0181] 7.4 Investigation of solvent addition amount

[0182] Take about 1.0 g of this product (batch number: BT-06), place it in a stoppered conical flask, add 25 ml, 50 ml, and 100 ml of 30% methanol respectively for investigation, stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. See Figure 13 . The results showed that when the addition amount of the extraction solvent was 25 ml, the peak shapes and resolutions of each chromatographic peak were good, and the peak sizes were appropriate. Therefore, the solvent amount was selected as 25 ml.

[0183] In summary, the preparation method of the test sample solution for the characteristic chromatogram of the standard decoction of Pinellia ternata Thunb. var. typica Engl. was determined as: Take about 1.0 g of this product, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0184] 8. Methodological investigation

[0185] 8.1 Chromatographic peak identification

[0186] Preparation of test solution: Prepare the test solution of the standard decoction of Pinelliae Rhizoma according to the above-mentioned determined experimental conditions.

[0187] Preparation of reference solution: Weigh about 2 g of the powder of the reference medicinal material of Pinelliae Rhizoma, put it into a stoppered conical flask, add 100 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the subsequent filtrate to obtain the reference solution.

[0188] Preparation of control solution: Separately weigh appropriate amounts of the reference substances of 4-hydroxybenzoic acid and glycyrrhizic acid, and prepare solutions containing 20 μg per 1 ml with methanol respectively as the control reference solutions.

[0189] Preparation of negative control solution: Prepare the negative control solution without the standard decoction of Pinelliae Rhizoma according to the above-mentioned determined experimental conditions.

[0190] Locate the peaks in the chromatogram of the standard decoction of Pinelliae Rhizoma. See Figure 14 . The results showed that peak 3 was 4-hydroxybenzoic acid and peak 13 was glycyrrhizic acid. There were significant differences in the peaks between the reference medicinal material of Pinelliae Rhizoma and the standard decoction of Pinelliae Rhizoma, and they were not included in the characteristic method. In the following methodological investigations, 13 characteristic peaks in the samples were investigated.

[0191] 8.2 Precision test

[0192] Take the test solution of the standard decoction of Pinelliae Rhizoma (batch number: BT-06), inject 3 μl each time continuously for 6 times according to the determined experimental method, and calculate the retention time and peak area of each characteristic peak. See Table 9.

[0193] Table 9 Precision investigation - Retention time

[0194]

[0195] The results showed that the RSD range of the relative retention time of each characteristic peak in the sample was 0.01% - 0.04%, indicating good precision of the instrument.

[0196] 8.3 Repeatability investigation

[0197] Precisely weigh 6 portions of the standard decoction of Pinelliae Rhizoma (batch number: JBX-BT-230206), prepare and determine them according to the determined experimental method. See Table 10.

[0198] Table 10 Repeatability investigation - Relative retention time

[0199]

[0200] As shown in the results, the RSD range of the relative retention time of 6 samples was 0.00% - 0.32%, indicating good repeatability of this method.

[0201] 8.4 Intermediate precision investigation

[0202] 8.4.1 Investigation with different instruments

[0203] On the basis of the above - determined experimental conditions, instruments 1, 2, and 3 were investigated respectively. See Figure 15 , Table 11:

[0204] Table 11 Investigation with different instruments - relative retention time

[0205]

[0206] The results showed that when detected with the above three instruments, the RSD of the relative retention time of each characteristic peak was between 0.50% and 6.56%; the durability of different instruments was good.

[0207] 8.4.2 Investigation with different personnel and at different times

[0208] On the basis of the above - determined experimental conditions, two portions of the standard decoction of Pinelliae Rhizoma Praeparata (batch number: BT - 06) were accurately weighed by different personnel (A, B) at different times (T1, T2) respectively, and the test samples were prepared for determination. See Table 12.

[0209] Table 12 Investigation with personnel and at different times - relative retention time

[0210]

[0211] The results showed that when the same sample was determined by different personnel at different times, the RSD of the relative retention time of each characteristic peak was between 0.00% and 0.62%, and the method had good stability.

[0212] 8.5.1 Chromatographic column durability investigation

[0213] On the basis of the above - determined experimental conditions, chromatographic columns 1, 2, and 3 were investigated respectively. See Figure 16 , Table 13.

[0214] Table 13 Chromatographic column durability investigation - relative retention time

[0215]

[0216] It can be seen from Figure 16 that the chromatogram of T3 2.1×150mm, 1.6μm (chromatographic column 1) had better separation, so it was recommended to use chromatographic column T3 2.1×150mm, 1.6μm.

[0217] 8.5.2 Stability investigation

[0218] On the basis of the above-determined experimental conditions, the same test solution was taken and measured at 0 h, 3 h, 7 h, 13 h, 20 h, and 25 h respectively. See Table 14.

[0219] Table 14 Stability Study - Retention Time

[0220]

[0221] The results showed that the RSD of the retention time of the corresponding characteristic peaks was between 0.06% and 0.31%, and the sample solution was relatively stable within 25 hours.

[0222] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in all the above investigations, and the method was good. The above 13 characteristic peaks were included in the subsequent investigations.

[0223] 8.9 Determination of Characteristic Peaks and Establishment of Reference Chromatogram

[0224] 8.9.1 Verification Results of 15 Batches of Standard Decoctions of Pinelliae Rhizoma Praeparatum

[0225] Using this method, characteristic chromatograms of 15 batches of samples were analyzed, and the relative retention time and relative peak area were calculated. See Figure 17 (From bottom to top, the batch numbers are: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15), Table 15.

[0226] Table 15 Relative Retention Time of Characteristic Chromatogram of Standard Decoction of Pinelliae Rhizoma Praeparatum

[0227]

[0228] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 13 peaks with good repeatability were selected as characteristic peaks. The results showed that the RSD of the relative peak area of the characteristic peaks of Pinelliae Rhizoma Praeparatum slices was too different to specify the relative peak area, so it was not included in the main text of the quality standard. Finally, it was stipulated that: 13 characteristic peaks should be presented in the chromatogram of the test solution. Among them, peak 3 should correspond to the retention time of the reference substance peak of the reference substance, and the peak corresponding to the reference substance peak of 4-hydroxybenzoic acid reference substance was the S peak. Calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.93 (peak 1), 0.97 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.51 (peak 6), 1.64 (peak 7), 1.69 (peak 8), 1.73 (peak 9), 2.45 (peak 10), 2.60 (peak 11), 2.85 (peak 12), 2.94 (peak 13).

[0229] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), the fingerprints of 15 batches of Rhizoma Pinelliae Praeparatum were synthesized, and the reference fingerprint of the characteristic fingerprint of Rhizoma Pinelliae Praeparatum was established. See Figure 18 , Peak 3 (S): 4-Hydroxybenzoic acid; Peak 13: Glycyrrhizic acid; Chromatographic column: T3 2.1×150mm, 1.6μm.

[0230] In summary, the method for the characteristic fingerprint of the standard decoction is as follows:

[0231] Chromatographic conditions and system suitability test Using octadecylsilane chemically bonded silica gel as the filler (column length 100mm, inner diameter 2.1mm, particle size 1.6μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 1 above; the flow rate was 0.2ml per minute; the column temperature was 30°C; the detection wavelength was 270nm. The number of theoretical plates calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000.

[0232] Preparation of the reference substance solution Weigh an appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances accurately, and dissolve them separately in methanol to prepare solutions containing 20μg per 1ml as the reference substance solution for the reference substances.

[0233] Preparation of the test solution Take about 1.0g of this product, place it in a stoppered conical flask, add 25ml of 30% methanol, stopper it tightly, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate, that is, obtain the test solution.

[0234] Determination method Accurately pipette 3μl of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and determine, that is, obtain the result.

[0235] There should be 13 characteristic peaks in the chromatogram of the test solution. Among them, the retention time of Peak 3 should correspond to that of the reference substance peak of the reference substance. The peak corresponding to the 4-hydroxybenzoic acid reference substance of the reference substance is the S peak. Calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.93 (Peak 1), 0.97 (Peak 2), 1.16 (Peak 4), 1.21 (Peak 5), 1.51 (Peak 6), 1.64 (Peak 7), 1.69 (Peak 8), 1.73 (Peak 9), 2.45 (Peak 10), 2.60 (Peak 11), 2.85 (Peak 12), 2.94 (Peak 13).

[0236] Example 3 Method for constructing the UPLC characteristic fingerprint of Rhizoma Pinelliae Praeparatum formula granules

[0237] 1. Test materials

[0238] Jingbanxia Formula Granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: KL-01, KL-001, KL-002, KL-003).

[0239] 2. Chromatographic conditions

[0240] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, and perform gradient elution according to the regulations in Table 1 above; the detection wavelength is 270 nm. The flow rate is 0.2 ml / min; the column temperature is 30 °C. The number of theoretical plates calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000.

[0241] 3. Preparation of reference substance solution

[0242] Weigh appropriate amounts of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances accurately, and dissolve them separately in methanol to make solutions containing 20 μg per 1 ml as the reference substance solutions.

[0243] 7.2.9.4 Preparation of test solution

[0244] Take an appropriate amount of this product, grind it finely, take about 1.0 g, place it in a stoppered conical flask, add 50 ml of 30% methanol, tightly stopper it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0245] 7.2.9.5 Determination method

[0246] Precisely pipette 3 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and perform the determination to obtain the results.

[0247] 7.2.9.6 Chromatographic conditions and system suitability test

[0248] 7.2.9.6.1 Selection of mobile phase

[0249] On the basis of the above-determined experimental conditions, the separation effects of 4 different mobile phases were investigated, namely: methanol - 0.1% phosphoric acid, acetonitrile - 0.1% phosphoric acid, acetonitrile - 0.1% acetic acid, acetonitrile - water, as shown in Figure 19 , and the results showed that the baseline of the chromatographic peaks was relatively stable under the gradient elution conditions of acetonitrile - 0.1% phosphoric acid solution, and the number of theoretical plates, resolution, and symmetry were all better. Therefore, the gradient elution of acetonitrile - 0.1% phosphoric acid solution was used as the mobile phase for the determination method of the characteristic chromatogram of the formula granules.

[0250] 7.2.9.6.2 Wavelength selection

[0251] On the basis of the above - specified experimental conditions, the test solution was scanned over the full wavelength range using a diode array detector, and the chromatograms of the test solution at wavelengths of 210 nm, 230 nm, 250 nm, 270 nm, 290 nm, 310 nm, and 330 nm were extracted respectively. See Figure 20 , and the results showed that when the detection wavelength was 270 nm, the chromatographic peak had more information and the chromatogram baseline was more stable. Therefore, the detection wavelength was determined to be 270 nm.

[0252] 7.2.9.6.3 Column temperature investigation

[0253] On the basis of the above - specified experimental conditions, investigations were carried out at column temperatures of 25 °C, 30 °C, and 35 °C respectively. See Figure 21 , and the results showed that when the column temperature was 30 °C, the chromatogram peak shape was relatively symmetric and the resolution was better. Therefore, the column temperature was determined to be 30 °C.

[0254] 7.2.9.6.4 Flow rate investigation

[0255] On the basis of the above - specified experimental conditions, investigations were carried out at flow rates of 0.15 ml / min, 0.2 ml / min, and 0.25 ml / min respectively. See Figure 22 , and the results showed that when the flow rate was 0.2 ml / min, the chromatogram peak shape was good and the baseline was stable. Therefore, the flow rate was determined to be 0.2 ml / min.

[0256] 7.2.9.6.5 Delay investigation

[0257] On the basis of the above - specified experimental conditions, a delay test was carried out. The results are shown in Figure 23 , and the results showed that there were basically no chromatographic peaks after 60 min for the sample. Therefore, the sample detection time was set at 60 min.

[0258] To sum up, the chromatographic conditions and system suitability test for the characteristic chromatogram of the formula granules were determined as follows: Using octadecylsilane chemically bonded silica as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, and performing gradient elution according to the regulations in Table 1 above; the detection wavelength was 270 nm. The flow rate was 0.2 ml / min; the column temperature was 30 °C. The number of theoretical plates calculated based on the p - hydroxybenzoic acid peak should be not less than 5000.

[0259] 7.2.9.7 Preparation investigation of the test solution

[0260] 7.2.9.7.1 Extraction method investigation

[0261] Take about 1.0 g of this product (batch number: KL-01), place it in a stoppered conical flask, add 50 ml of 30% methanol respectively, stopper tightly, and investigate the extraction methods of the test samples by reflux and ultrasonic wave (power 600 W, frequency 40 kHz). The extraction time is 30 minutes. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample solution. See Figure 24 , and the results show that there is little difference in the extraction effects when refluxing and ultrasonic extracting the test samples, and the ultrasonic method is fast and simple. Therefore, the extraction method of the test sample is determined to be ultrasonic extraction.

[0262] 7.2.9.7.2 Investigation of extraction solvents

[0263] Take about 1.0 g of this product (batch number: KL-01), place it in a stoppered conical flask, add 50 ml of water, 30% methanol, 50% methanol, 80% methanol, and methanol respectively, stopper tightly, and perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample solution. See Figure 25 .

[0264] The results show that when water, 30%, and 50% methanol are used as extraction solvents, the chromatographic peak information is large and the resolution is good; when 30% methanol is used as the extraction solvent, the operation is easier. Therefore, the extraction solvent is tentatively set as 30% methanol.

[0265] 7.2.9.7.3 Investigation of extraction time Take about 1.0 g of this product (batch number: KL-01), place it in a stoppered conical flask, add 50 ml of 30% methanol, stopper tightly, and perform ultrasonic treatment (power 600 W, frequency 40 kHz). Investigate the extraction time of the test sample at 15 min, 30 min, and 60 min respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample solution. See Figure 26 .

[0266] The results show that when the extraction time is 30 min, the test sample has been completely extracted. Therefore, the extraction time is determined to be 30 min.

[0267] 7.2.9.7.4 Investigation of solvent addition amount

[0268] Take about 1.0 g of this product (batch number: KL-01), place it in a stoppered conical flask, and investigate by adding 25 ml, 50 ml, and 100 ml of 30% methanol respectively. Stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample solution. See Figure 27 , and the results show that when the addition amount of the extraction solvent is 25 ml, the peak shapes and resolutions of each chromatographic peak are good, and the peak sizes are appropriate. Therefore, the solvent amount is selected as 25 mL.

[0269] To sum up, the preparation method of the test solution for the characteristic chromatogram of formula granules was determined as follows: Take an appropriate amount of this product, grind it finely, take about 1.0 g, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper it tightly, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0270] 7.2.9.8 Methodological investigation

[0271] 7.2.9.8.1 Chromatographic peak identification

[0272] Preparation of the test solution: Prepare the test solution of formula granules according to the above-mentioned determined experimental conditions.

[0273] Preparation of the reference solution: Take about 2 g of the powder of this product, place it in a conical flask, add 100 ml of water, decoct for 30 minutes, let it cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0274] Preparation of the control solution: Separately take appropriate amounts of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, and prepare solutions containing 20 μg per 1 ml with methanol respectively as the control reference solution.

[0275] Preparation of the negative control solution: Prepare the negative control solution of formula granules without Pinellia ternata according to the above-mentioned determined experimental conditions.

[0276] Locate the peaks in the characteristic chromatogram of formula granules. See Figure 28 ;

[0277] The results showed that peak 3 was 4-hydroxybenzoic acid and peak 13 was glycyrrhizic acid. There were significant differences between the characteristic peaks of the Pinellia ternata control crude drug and the granules, so it was not included in the characteristic method. In the following methodological investigation, 13 characteristic peaks in the sample were investigated.

[0278] 7.2.9.8.2 Precision test

[0279] Take the test solution of formula granules (batch number: KL-01), inject 3 μl each time continuously for 6 times according to the determined experimental method, and calculate the retention time of each characteristic peak. See Table 16:

[0280] Table 16 Precision investigation - Retention time

[0281]

[0282]

[0283] The results showed that the relative standard deviation (RSD) range of the relative retention time of each characteristic peak in the sample was 0.01% - 0.07%, indicating good precision of the instrument.

[0284] 7.2.9.8.3 Repeatability Investigation

[0285] Accurately weigh 6 portions of the formulated granules (batch number: KL-01), and prepare and determine them according to the proposed experimental method. See Table 17.

[0286] Table 17 Repeatability Investigation - Relative Retention Time

[0287]

[0288] As shown in the results, the RSD range of the relative retention time of the 6 samples is 0.00% - 0.44%, indicating that the method has good repeatability.

[0289] 7.2.9.8.4 Intermediate Precision Investigation

[0290] 7.2.9.8.4.1 Investigation with Different Instruments

[0291] On the basis of the above proposed experimental conditions, investigate Instruments 1, 2, and 3 respectively. See Figure 29 , Table 18.

[0292] Table 18 Column Durability Investigation - Relative Retention Time

[0293]

[0294] The results show that when detected with the above three instruments, the RSD of the relative retention time of each characteristic peak is between 0.00% - 6.46%, indicating good durability of different instruments.

[0295] 7.2.9.8.4.2 Investigation with Different Personnel and at Different Times

[0296] On the basis of the above proposed experimental conditions, accurately weigh two portions of the formulated granules (batch number: KL-01) respectively by different personnel (A, B) at different times (T1, T2), prepare the test samples, and conduct the determination. See Table 19.

[0297] Table 19 Personnel and Time Investigation - Relative Retention Time

[0298]

[0299]

[0300] The results show that when the same sample is determined by different personnel at different times, the RSD of the relative retention time of each characteristic peak is 0.00% - 0.60%, indicating good stability of the method.

[0301] 7.2.9.8.4.3 Column Durability Investigation

[0302] On the basis of the above - determined experimental conditions, the chromatographic columns 1, 2, and 3 were investigated respectively. See Figure 30 , Table 20.

[0303] Table 20 Durability Investigation of Chromatographic Column - Relative Retention Time

[0304]

[0305] It can be seen from Figure 30 that the chromatogram of T3 2.1×150mm, 1.6μm (chromatographic column 1) has a better separation. Therefore, the chromatographic column T3 2.1×150mm, 1.6μm is recommended.

[0306] 7.2.8.8.5 Stability Investigation

[0307] On the basis of the above - determined experimental conditions, the same test solution was taken and measured at 0h, 3h, 7h, 13h, 20h, and 25h respectively. See Table 21.

[0308] Table 21 Stability Investigation - Retention Time

[0309]

[0310] The results show that the RSD of the retention time of the corresponding characteristic peaks is between 0.02% and 0.27%, and the sample solution is relatively stable within 25 hours.

[0311] To sum up, the RSD of the relative retention time of each characteristic peak meets the requirements in the above - mentioned investigations, and this method is good. The above - mentioned 13 characteristic peaks were included in the subsequent investigations.

[0312] 7.2.9.9 Determination of Characteristic Peaks and Establishment of Reference Chromatogram

[0313] 7.2.9.9.1 Verification Results of 3 Batches of Formula Granules

[0314] Using this method, characteristic fingerprint analysis was carried out on 3 batches of samples, and the relative retention time was calculated. See Figure 31 , Table 22:

[0315] Table 22 Relative Retention Time of Formula Granules

[0316]

[0317]

[0318] According to the principle of stable relative retention time, detectable in each batch of samples, and relatively high peaks, a total of 13 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative peak area RSD of the characteristic peaks of Jingbanxia formula granules was too different to stipulate the relative peak area, so it was not included in the main text of the quality standard. Finally, it was stipulated that 13 characteristic peaks should be presented in the chromatogram of the test sample. Among them, the retention time of peak 3 should correspond to that of the reference substance peak, and the peak corresponding to the 4-hydroxybenzoic acid reference substance peak was the S peak. Calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.93 (peak 1), 0.96 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.51 (peak 6), 1.63 (peak 7), 1.69 (peak 8), 1.73 (peak 9), 2.44 (peak 10), 2.59 (peak 11), 2.84 (peak 12), 2.93 (peak 13).

[0319] Using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition), the characteristic chromatograms of 3 batches of formula granules were synthesized to establish the reference characteristic chromatogram of the formula granule characteristic chromatogram. See Figure 32 ; Peak 3 (S): 4-hydroxybenzoic acid, Peak 13: glycyrrhizic acid; Chromatographic column: T3 2.1×150mm, 1.6μm.

[0320] In summary, the method for the characteristic chromatogram of formula granules is as follows:

[0321] Chromatographic conditions and system suitability test Using octadecylsilane chemically bonded silica gel as the filler (column length 100mm, inner diameter 2.1mm, particle size 1.6μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 1 above; the flow rate was 0.2ml per minute; the column temperature was 30°C;

[0322] Preparation of reference substance solution Weigh an appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances accurately, and dissolve them in methanol respectively to make solutions containing 20μg per 1ml as the reference substance reference solution.

[0323] Preparation of test sample solution Take an appropriate amount of this product, grind it finely, take about 1.0g, place it in a stoppered conical flask, add 25ml of 30% methanol, stopper it tightly, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0324] Determination method Accurately pipette 3μl of the reference substance solution and the test sample solution respectively, inject them into the liquid chromatograph for determination to obtain the results.

[0325] There should be 13 characteristic peaks in the chromatogram of the test sample. Among them, the retention time of peak 3 should correspond to that of the reference peak of the reference substance. The peak corresponding to the reference peak of the 4-hydroxybenzoic acid reference substance is the S peak. Calculate the relative retention times of each characteristic peak with respect to the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are: 0.93 (peak 1), 0.96 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.51 (peak 6), 1.63 (peak 7), 1.69 (peak 8), 1.73 (peak 9), 2.44 (peak 10), 2.59 (peak 11), 2.84 (peak 12), 2.93 (peak 13).

[0326] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing characteristic chromatograms of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. C. Hsia pieces, standard decoctions, and formula granules, comprising the following steps: Dissolve the test sample, extract it to obtain a test solution; the extraction solvent is 30% methanol; the extraction method is ultrasonic extraction; the power of the ultrasonic extraction is 580 - 620 W, the frequency of the ultrasonic extraction is 35 - 45 kHz; the time of the ultrasonic extraction is 25 - 35 min; the mass ratio of the test sample to the volume of the extraction solvent is (1 - 2) g : 25 mL; Determine the test solution by high performance liquid chromatography to obtain characteristic chromatograms of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. C. Hsia pieces, standard decoctions, and formula granules; The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid aqueous solution, with gradient elution; The specific gradient elution is as follows: 0 - 5 min, phase A: 0 - 2%, phase B: 100 - 98%; 5 - 8 min, phase A: 2%, phase B: 98%; 8 - 15 min, phase A: 2 - 6%, phase B: 98 - 94%; 15 - 32 min, phase A: 6 - 14%, phase B: 94 - 86%; 32 - 42 min, phase A: 14 - 25%, phase B: 86 - 75%; 42 - 60 min, phase A: 25 - 50%, phase B: 75 - 50%; It also includes preparing a reference substance solution: Dissolve 4-hydroxybenzoic acid and glycyrrhizic acid reference substances in methanol respectively to obtain a reference substance solution; the concentration of the reference substance solution is 20 μg / mL; Determine the reference substance solution by high performance liquid chromatography to obtain the chromatogram of the reference substance; and qualitatively analyze the components of the HPLC characteristic chromatograms of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. C. Hsia pieces, standard decoctions, and formula granules according to the chromatogram of the reference substance.

2. The construction method according to claim 1, wherein The flow rate of the mobile phase is 0.2 mL / min, the detection wavelength is 270 nm; the injection volume is 3 μL; the column temperature is 30 °C; the number of theoretical plates calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000.

3. The construction method according to claim 1, wherein Use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarity of the HPLC characteristic chromatogram of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. C. Hsia pieces, and obtain the HPLC standard characteristic chromatogram of Pinellia ternata Thunb. var. pinellioides (Pritz.) Hsiao et K. C. Hsia pieces composed of 13 characteristic peaks, where the peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the standard chromatogram, calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.93 (peak 1), 0.96 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.50 (peak 6), 1.63 (peak 7), 1.68 (peak 8), 1.72 (peak 9), 2.43 (peak 10), 2.58 (peak 11), 2.83 (peak 12), 2.92 (peak 13).

4. The construction method according to claim 1, characterized in that The similarity evaluation system for traditional Chinese medicine chromatographic fingerprint was used to evaluate the similarity of the HPLC characteristic fingerprint of the standard decoction of Pinelliae Rhizoma Praeparata cum Alumine. The HPLC standard characteristic fingerprint of the standard decoction of Pinelliae Rhizoma Praeparata cum Alumine consisting of 13 characteristic peaks was obtained, and the peak corresponding to the reference substance of 4-hydroxybenzoic acid was the S peak. In the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values. The specified values were: 0.93 (peak 1), 0.97 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.51 (peak 6), 1.64 (peak 7), 1.69 (peak 8), 1.73 (peak 9), 2.45 (peak 10), 2.60 (peak 11), 2.85 (peak 12), 2.94 (peak 13).

5. The construction method according to claim 1, characterized in that, The similarity evaluation system for traditional Chinese medicine chromatographic fingerprint was used to evaluate the similarity of the HPLC characteristic fingerprint of the formulated granules of Pinelliae Rhizoma Praeparata cum Alumine. The HPLC standard characteristic fingerprint of the formulated granules of Pinelliae Rhizoma Praeparata cum Alumine consisting of 13 characteristic peaks was obtained, and the peak corresponding to the reference substance of 4-hydroxybenzoic acid was the S peak. In the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values. The specified values were: 0.93 (peak 1), 0.96 (peak 2), 1.16 (peak 4), 1.21 (peak 5), 1.51 (peak 6), 1.63 (peak 7), 1.69 (peak 8), 1.73 (peak 9), 2.44 (peak 10), 2.59 (peak 11), 2.84 (peak 12), 2.93 (peak 13).

6. A method for identifying Pinelliae Rhizoma preparata, standard decoction, and formula granules of Pinelliae Rhizoma preparata from Jingzhou, characterized in that, The method according to any one of claims 1 to 5 was used for detection, and the detection results were analyzed.

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