Construction method for and use of rhizoma pinellinae praeparata decoction piece, standard decoction and formula granule characteristic spectra

Abstract

A construction method for and a use of rhizoma pinellinae praeparata decoction piece, standard decoction and formula granule characteristic spectra. The construction method comprises the following steps: dissolving a test sample by using a solvent, and extracting to obtain a solution to tested; and measuring said solution by using high-performance liquid chromatography (HPLC) to obtain rhizoma pinellinae praeparata decoction piece, standard decoction and formula granule characteristic spectra, wherein chromatographic conditions of the HPLC are as follows: the chromatographic column is a C18 column; and gradient elution is performed by using acetonitrile as mobile phase A and a 0.1% phosphoric acid aqueous solution as mobile phase B. HPLC is used, gradient elution is performed by using acetonitrile and a 0.1% phosphoric acid aqueous solution as mobile phases, and 4-hydroxybenzoic acid is used as a reference object, so that rhizoma pinellinae praeparata decoction piece, standard decoction and formula granule HPLC characteristic spectra are established; the method has good repeatability and precision, is stable and reliable, and can control the quality of rhizoma pinellinae praeparata decoction pieces, a standard decoction and formula granules.

Description

A method for constructing characteristic maps of Pinellia ternata slices, standard decoctions, and formula granules and its application

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on August 4, 2023, with application number 202310982776.7 and invention name “A method for constructing a characteristic map of Pinellia ternata slices, standard decoctions, and formula granules and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of detection technology, and in particular relates to a method for constructing characteristic maps of Peking Pinellia ternata slices, standard decoctions, and formula granules, and applications thereof. Background Art

[0003] Pinellia ternata, a traditional Chinese medicine, is derived from the dried tubers of the plant Pinellia ternata, a member of the Araceae family. Its pungent and warm properties are known to dry dampness, resolve phlegm, and dissipate lumps and masses. Pinellia ternata is mildly toxic, and its processed forms are often used clinically. Commonly used ingredients include Qing Banxia, ​​Jiang Banxia, ​​French Banxia, ​​and Jing Banxia.

[0004] The 2010 edition of the "Chinese Pharmacopoeia" includes Qing Banxia, ​​Jiang Banxia, ​​and Fa Banxia. Except for Qing Banxia, ​​the quality standards of Jiang Banxia and Fa Banxia only involve the inspection of properties, microscopic structure, moisture content, and total ash content. The "Chinese Pharmacopoeia" does not stipulate the processing methods and quality standards of Beijing Banxia, ​​and cannot effectively control the quality of Beijing Banxia.

[0005] Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for constructing characteristic maps of Beijing Pinellia slices, standard decoctions, and formula granules and its application, which provides more scientific and technological means for the quality evaluation of Beijing Pinellia slices, standard decoctions and their formula granules.

[0007] The present invention provides a method for constructing characteristic maps of Pinellia tuber slices, standard decoctions, and formula granules, comprising the following steps:

[0008] Dissolve the test sample in a solvent and extract it to obtain a test solution;

[0009] The test solution is measured by high performance liquid chromatography to obtain characteristic spectra of the Peking Pinellia ternata slices, standard decoction, and formula granules;

[0010] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0011] The present invention dissolves the sample solution with a solvent and extracts it to obtain a test solution. The sample is a Beijing Pinellia ternate slice, a standard decoction, and a formula granule. Beijing Pinellia ternate slice is a processed product of the dried tuber of the Araceae plant Pinellia ternate (Thunb.) Breit. It has the effects of drying dampness and resolving phlegm, and relieving adverse reactions and vomiting. The Beijing Pinellia ternate standard decoction is a freeze-dried powder made from the Beijing Pinellia ternate slice according to a fixed preparation process; the Beijing Pinellia ternate formula granule is a formula granule made from the Beijing Pinellia ternate slice according to the main quality indicators of the standard decoction.

[0012] In the present invention, 0.1% phosphoric acid aqueous solution and 30% methanol are both volume fractions.

[0013] In the present invention, the extraction method is ultrasonic extraction; the power of the ultrasonic extraction is 580-620W, the frequency of the ultrasonic extraction is 35-45kHz; the time of the ultrasonic extraction is 25-35min; in a specific embodiment, the power of the ultrasonic extraction is 600W, the frequency of the ultrasonic extraction is 40kHz; the time of the ultrasonic extraction is 30min.

[0014] When the test sample is the decoction piece of Pinellia ternata, the specific process of dissolving the test sample is as follows:

[0015] Boil the Pinellia ternata slices with water, cool, filter, evaporate the filtrate to dryness, add 30% methanol, ultrasonicate, shake well, and filter to obtain the filtrate, which is the test solution. The mass ratio of the Pinellia ternata slices to the volume of water is (1.8-2.2) g:100 mL, and boil for 28-32 minutes. In a specific embodiment, the mass ratio of the Pinellia ternata slices to the volume of water is 2 g:100 mL, and boil for 30 minutes.

[0016] When the test sample is the standard decoction of Pinellia ternata or the formulated granules of Pinellia ternata, the specific process of dissolution of the test sample is as follows:

[0017] Dissolve the sample in 30% methanol, ultrasonicate, shake well, and filter to obtain the filtrate, which is the test solution. The ratio of the mass of the sample (g) to the volume of 30% methanol (mL) is 1:25.

[0018] In the present invention, the extraction solvent is 30% methanol. The chromatographic peaks obtained by using 30% methanol as the extraction solvent have large information content and good separation.

[0019] The present invention uses high performance liquid chromatography to measure the test solution to obtain characteristic spectra of Beijing Pinellia ternata slices, standard decoction, and formula granules. The chromatographic conditions of the high performance liquid chromatography are: a chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is a 0.1% phosphoric acid aqueous solution, and gradient elution is performed.

[0020] In the present invention, the gradient elution is specifically:

[0021] 0-5min, phase A: 0-2%, phase B: 100-98%;

[0022] 5-8 min, phase A: 2%, phase B: 98%;

[0023] 8-15 min, phase A: 2-6%, phase B: 98-94%;

[0024] 15-32 min, phase A: 6-14%, phase B: 94-86%;

[0025] 32~42min, phase A: 14~25%, phase B: 86~75%;

[0026] 42-60min, phase A: 25-50%, phase B: 75-50%.

[0027] 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. The present invention found that when the detection wavelength is 270 nm, the chromatogram peak information is larger and the chromatogram baseline is more stable, so the detection wavelength is 270 nm; the injection volume is 3 μL;

[0028] The present invention adopts The chromatogram of the 2.1 × 150 mm, 1.6 μm column was well separated, and the column temperature was 30 °C;

[0029] The theoretical plate number calculated based on the 4-hydroxybenzoic acid peak should be no less than 5000.

[0030] The present invention also includes preparing a reference solution:

[0031] Dissolve 4-hydroxybenzoic acid and glycyrrhizic acid reference substances in methanol to obtain reference substance solutions;

[0032] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the HPLC characteristic spectra of the characteristic spectra of the Pinellia ternata slices, standard decoction, and formula granules are qualitatively analyzed based on the chromatogram of the reference.

[0033] The invention adopts a Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of HPLC characteristic spectra of Pinellia ternata slices, and obtains an HPLC standard characteristic spectra of Pinellia ternata slices consisting of 13 characteristic peaks, wherein the peak corresponding to a 4-hydroxybenzoic acid reference substance is an S peak; in the standard characteristic spectra, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of a specified value; the specified value is: 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), and 2.92 (peak 13).

[0034] The present invention uses a Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of an HPLC characteristic spectrum of a standard decoction of Pinellia ternata, thereby obtaining an HPLC standard characteristic spectrum of the standard decoction of Pinellia ternata consisting of 13 characteristic peaks, wherein the peak corresponding to a 4-hydroxybenzoic acid reference substance is an S peak. In the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of a specified value; the specified value is: 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), and 2.94 (peak 13).

[0035] The invention adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of HPLC characteristic spectra of Beijing Banxia formula granules, and obtains an HPLC standard characteristic spectra of Beijing Banxia formula granules consisting of 13 characteristic peaks, wherein the peak corresponding to a 4-hydroxybenzoic acid reference substance is an S peak; in the standard characteristic spectra, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of a specified value; the specified value is: 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), and 2.93 (peak 13).

[0036] Quality judgment standard: Take samples of Beijing Pinellia slices, standard decoctions, and formula granules, and operate according to the same method as above to obtain the characteristic spectra of Beijing Pinellia slices, standard decoctions, and formula granules. Use the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) of the National Pharmacopoeia Committee to analyze the standard characteristic spectra of Beijing Pinellia slices, standard decoctions, and formula granules and the sample characteristic spectra, and the similarity is greater than 0.90.

[0037] The method provided by the present invention can effectively monitor the quality of different batches of Beijing Pinellia ternata slices, standard decoctions, and formula granules to ensure stable quality. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensive monitoring of product quality.

[0038] The characteristic spectra of Pinellia ternata slices, standard decoction and formula granules established by the present invention use rosmarinic acid as a reference, focus on the order of each characteristic peak and the correlation with medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the products, and the method is scientific and reliable.

[0039] The present invention provides a method for identifying Peking Pinellia ternata slices, standard decoctions and formula granules, which is characterized by adopting the method described in the above technical solution to perform detection and analyze the detection results.

[0040] The present invention adopts the method to identify 4-hydroxybenzoic acid, and can control the quality of Peking Pinellia ternata slices, standard decoctions and formula granules.

[0041] The present invention provides a method for constructing a characteristic spectrum of Pinellia ternata slices, standard decoctions, and formula granules, comprising the following steps: dissolving a test sample in a solvent and extracting the sample to obtain a test solution; determining the test solution by high-performance liquid chromatography to obtain a characteristic spectrum of the Pinellia ternata slices, standard decoctions, and formula granules; the HPLC conditions are as follows: a C18 column; mobile phase A is acetonitrile; mobile phase B is a 0.1% phosphoric acid aqueous solution; and gradient elution is performed. The present invention uses high-performance liquid chromatography, selects acetonitrile and 0.1% phosphoric acid aqueous solution as the mobile phase for gradient elution, and uses 4-hydroxybenzoic acid as a reference substance to establish an HPLC characteristic spectrum of the Pinellia ternata slices, standard decoctions, and formula granules. The method has good repeatability and precision, is stable, and is reliable, and can be used to control the quality of the Pinellia ternata slices, standard decoctions, and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the extraction methods of Pinellia ternata slices;

[0043] Figure 2 is an investigation of solvents for extracting Pinellia ternata slices;

[0044] Figure 3 shows the extraction time of Pinellia ternata slices;

[0045] Figure 4 shows the investigation of solvent addition amount for Pinellia ternata slices;

[0046] Figure 5 shows the chromatographic peak identification of Beijing Pinellia ternata slices;

[0047] Figure 6 shows the inspection of different instruments for Beijing Pinellia ternata slices;

[0048] Figure 7 shows the investigation of different chromatographic columns for Beijing Pinellia ternata slices;

[0049] Figure 8 is a characteristic spectrum of Beijing Pinellia ternata slices;

[0050] Figure 9 is a comparative characteristic spectrum of Beijing Pinellia ternata slices;

[0051] Figure 10 shows the investigation of the extraction methods of the standard decoction of Pinellia ternata;

[0052] Figure 11 shows the investigation of solvents for extracting the standard decoction of Pinellia ternata;

[0053] Figure 12 shows the extraction time of the standard decoction of Pinellia ternata;

[0054] Figure 13 shows the investigation of solvent addition amount in the standard decoction of Pinellia ternata;

[0055] Figure 14 shows the chromatographic peak identification of the standard decoction of Pinellia ternata;

[0056] Figure 15 shows the inspection of different instruments for the standard decoction of Pinellia ternata;

[0057] Figure 16 shows the investigation of different chromatographic columns for the standard decoction of Pinellia ternata;

[0058] Figure 17 is a characteristic spectrum of the standard decoction of Pinellia ternata;

[0059] Figure 18 is a comparative characteristic spectrum of the standard decoction of Pinellia ternata;

[0060] Figure 19 shows the mobile phase selection for Jingbanxia granules;

[0061] Figure 20 is a chromatogram of different wavelengths of Jingbanxia formula granules;

[0062] Figure 21 shows the column temperature of Jingbanxia formula granules;

[0063] Figure 22 shows the flow rate of the Jing Ban Xia formula granules;

[0064] Figure 23 is an investigation of the delayed effect of Jingbanxia formula granules;

[0065] Figure 24 shows the investigation of the extraction method of Pinellia ternata formula granules;

[0066] Figure 25 is an investigation of the extraction solvents of Jingbanxia formula granules;

[0067] Figure 26 shows the investigation of the extraction time of Jingbanxia formula granules;

[0068] Figure 27 shows the investigation of the amount of solvent added to the Jing Ban Xia formula granules;

[0069] Figure 28 shows the chromatographic peak identification of Jingbanxia formula granules;

[0070] Figure 29 shows the inspection of different instruments for Jingbanxia formula granules;

[0071] Figure 30 shows the investigation of different chromatographic columns for Jingbanxia formula granules;

[0072] Figure 31 is a characteristic spectrum of Jingbanxia formula granules;

[0073] Figure 32 is a comparative characteristic spectrum of Beijing Banxia formula granules. DETAILED DESCRIPTION

[0074] In order to further illustrate the present invention, the construction method of the characteristic map of the Pinellia ternata slices, standard decoction, and formula granules provided by the present invention and its application are described in detail below in combination with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0075] Example 1 Construction of UPLC Characteristic Spectrum of Pinellia Rhizoma Decoction Pieces

[0076] 1) Instruments, materials, reagents and test drugs

[0077] Ultra-high performance liquid chromatograph: Agilent 1290;

[0078] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0079] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0080] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0081] Chromatographic column: 2.1×150mm,1.6μm;

[0082] Methanol and acetonitrile were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade;

[0083] Acetonitrile and phosphoric acid were of chromatographic grade; water was ultrapure water; and other reagents were of analytical grade.

[0084] Parahydroxybenzoic acid reference substance (Shanghai Shidande Standard Technology Service Co., Ltd., batch number: 2169, content is 99.9%)

[0085] Glycyrrhizic acid reference substance (China Food and Drug Inspection Institute, batch number: 111709-201702, content calculated as 99.9%)

[0086] Pinellia ternata reference medicinal material (National Institute of Food and Drug Control, batch number: 121272-201806)

[0087] Peking Pinellia ternata slices (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).

[0088] 2) Characteristic spectrum determination method

[0089] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 1 below; flow rate, 0.3 ml / min; column temperature, 30°C; detection wavelength, 270 nm. The number of theoretical plates, calculated based on the 4-hydroxybenzoic acid peak, should be no less than 5000.

[0090] Table 1 Gradient elution program

[0091] Preparation of reference solution Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, weigh accurately, and add methanol to make solutions containing 20 μg per 1 ml, which are used as reference substance solutions.

[0092] 3) Chromatographic conditions and system suitability test

[0093] Based on the manifestation of quantity-mass transfer, the characteristic chromatographic conditions of Pinellia ternata slices are consistent with the system adaptability and the characteristic chromatographic methods of Pinellia ternata standard decoction and Pinellia ternata formula granules.

[0094] The chromatographic conditions and system suitability testing for the characteristic spectrum of Pinellia ternata slices were as follows: octadecylsilane bonded silica gel as the filler (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 ml / min; column temperature, 30°C; detection wavelength, 270 nm. The number of theoretical plates, calculated based on the 4-hydroxybenzoic acid peak, should be no less than 5000.

[0095] Preparation of test solution: Take about 2 g of the product powder (passed through No. 3 sieve), place it in a conical flask, add 50 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the product.

[0096] Determination method: Accurately aspirate 3μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0097] 4) Preparation of test solution

[0098] 4.1 Investigation of extraction methods

[0099] Comparing water decoction followed by ultrasonic extraction, reflux extraction, and ultrasonic extraction, the water decoction followed by ultrasonic extraction showed larger peak information and a greater number of peaks. Therefore, the water decoction followed by ultrasonic extraction was ultimately selected for the extraction method (see Figure 1).

[0100] 4.2 Extraction solvent investigation:

[0101] 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 produced a large amount of chromatographic peak information and good resolution. 30% methanol was easier to operate, so 30% methanol was selected as the extraction solvent (see Figure 2).

[0102] 4.3 Extraction time investigation

[0103] Comparing ultrasonic extraction times of 15, 30, and 60 minutes, the sample was completely extracted by 30 minutes. Therefore, the extraction time was determined to be 30 minutes. (See Figure 3.)

[0104] 4.4 Investigation of extraction solvent amount

[0105] Comparison of ultrasonic extraction solvent additions of 25ml, 50ml, and 100ml revealed that the chromatographic peak size was more appropriate when the extraction solvent volume was 25ml, so the extraction solvent volume was determined to be 25ml (see Figure 4).

[0106] 4.5 Finalize the test sample preparation method

[0107] Take about 2 g of the powder (passed through a No. 4 sieve) of this product, 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 filtrate to obtain the product.

[0108] 5) Methodological investigation

[0109] 5.1 Chromatographic peak identification

[0110] Preparation of test sample solution: Finalize the test sample preparation method according to 4.5 and prepare the test sample solution of Pinellia ternata slices.

[0111] Preparation of reference medicinal material reference solution: Take about 2 g of Pinellia ternata reference 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 filtrate.

[0112] Preparation of reference solution: Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, add methanol to make a solution containing 20 μg per 1 ml, which will serve as reference solution.

[0113] Preparation of Negative Control Solution: A negative control solution lacking Pinellia ternata slices was prepared according to the experimental conditions outlined above. Characteristic peaks of Pinellia ternata slices were located. (See Figure 5.) The results indicate that Peak 3 is 4-hydroxybenzoic acid and Peak 13 is glycyrrhizic acid. The peaks of the Pinellia ternata control and Pinellia ternata slices differ significantly and are therefore not included in the characteristic method. In the following methodological investigation, 13 characteristic peaks in the sample were examined.

[0114] 5.2 Precision test

[0115] Take the test solution of Pinellia ternata (Batch No.: YP-06) and inject 1 μl of the sample 6 times according to the proposed experimental method. Calculate the retention time of each characteristic peak. See Table 2:

[0116] Table 2 Precision investigation-retention time

[0117] The results showed that the RSD of each characteristic peak retention time was 0.03% to 0.21%, indicating that the instrument had good precision.

[0118] 5.3 Repeatability Study

[0119] Six portions of Pinellia ternata slices (batch number: YP-06) were accurately weighed and prepared and assayed according to the proposed experimental method. See Table 3.

[0120] Table 3 Repeatability study - relative retention time ratio

[0121] The results showed that the RSD of the relative retention time of each characteristic peak was 0.00%-0.48%, indicating that the method had good repeatability.

[0122] 5.4 Intermediate precision study

[0123] 5.4.1 Different people, different times

[0124] Based on the experimental conditions proposed above, different personnel and different time periods were employed to prepare the test solutions for the assays. See Table 4.

[0125] Table 4 Intermediate precision-relative retention time ratio

[0126] The results showed that the method had good precision when the same sample was measured by different personnel at different times.

[0127] 5.4.2 Different instruments

[0128] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. See Figure 6 and Table 5.

[0129] Table 5 Column durability investigation - relative retention time

[0130] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.62% and 6.16% when the three instruments were used for detection, and the durability of the different instruments was good.

[0131] 5.5 Column durability inspection

[0132] Based on the experimental conditions proposed above, the chromatographic column 1 ( 2.1×150mm,1.6μm); chromatographic column 2 (Phenomenex Omega PS C18 100A 2.1×150mm,1.6μm); Column 3 (ACQUITY BEH Shield RP18 (2.1×150mm, 1.7μm) was used for the study. See Figure 7 and Table 6.

[0133] Table 6 Column durability investigation - relative retention time

[0134] As shown in Figure 7, The chromatogram of 2.1×150mm, 1.6μm (column 1) is better, so it is recommended to use the column 2.1×150mm,1.6μm.

[0135] 5.6 Stability Investigation

[0136] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 7h, 13h, 20h, and 25h. See Table 7.

[0137] Table 7 Stability Study-Retention Time

[0138] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.03% and 0.20%, and the sample solution was relatively stable within 24 hours.

[0139] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above 13 characteristic peaks were included in the subsequent investigation.

[0140] 6) Verification of the characteristic spectrum of Pinellia ternata slices

[0141] The Beijing Pinellia slices were tested and the relative retention time was calculated. The results are shown in Figure 8 (the batch numbers from bottom to top 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) and Table 8.

[0142] Table 8 Relative retention time of characteristic spectra of Pinellia ternata slices

[0143] Based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights, 13 peaks with good reproducibility were selected as characteristic peaks. The results showed that the relative peak area RSDs of the characteristic peaks of Pinellia ternata slices varied significantly, making it impossible to specify relative peak areas. Therefore, these peaks were not included in the main text of the quality standard. The final regulations stipulated that the test sample chromatogram should exhibit 13 characteristic peaks, of which Peak 3 should correspond to the retention time of the reference peak of the reference substance. The peak corresponding to the 4-hydroxybenzoic acid reference peak is designated as the S peak. The relative retention time of each characteristic peak relative to the S peak should be calculated, and the 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), and 2.92 (peak 13).

[0144] Fifteen batches of Pinellia ternata slices were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and a comparative characteristic spectrum of the Pinellia ternata slices was established, as shown in Figure 9. Peak 3 (S): 4-hydroxybenzoic acid; Peak 13: glycyrrhizic acid; Chromatographic column: 2.1×150mm,1.6μm.

[0145] In summary, the characteristic spectrum method of decoction pieces is:

[0146] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 1 above; flow rate, 0.2 ml / min; column temperature, 30°C; detection wavelength, 270 nm. The number of theoretical plates, calculated based on the 4-hydroxybenzoic acid peak, should be no less than 5000.

[0147] Preparation of reference solution Take an appropriate amount of 4-hydroxybenzoic acid reference substance, weigh accurately, and add methanol to make a solution containing 20 μg per 1 ml, which is used as the reference solution.

[0148] Preparation of test solution: Take about 2 g of the product powder (passed through No. 4 sieve), place it in a 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 filtrate.

[0149] Determination method: Accurately aspirate 3μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0150] The test sample chromatogram should show 13 characteristic peaks, of which Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the 4-hydroxybenzoic acid reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The 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), and 2.92 (peak 13).

[0151] Example 2 UPLC Characteristic Spectrum Method for Standardized Decoction of Pinellia Rhizoma

[0152] 1. Test materials

[0153] Jingbanxia standard decoction (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);

[0154] 2. Chromatographic conditions

[0155] Use octadecylsilane bonded silica gel as the packing (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1 above; flow rate, 0.2 ml / min; column temperature, 30°C; detection wavelength, 270 nm. The number of theoretical plates, calculated based on the 4-hydroxybenzoic acid peak, should be no less than 5000.

[0156] 3. Preparation of Reference Solution

[0157] Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, weigh accurately, and add methanol to make solutions containing 20 μg per 1 ml, which serve as reference substance solutions.

[0158] 4. Preparation of Test Solution

[0159] Take about 1.0 g of this product, place it in a stoppered conical flask, add 50 ml of 30% methanol, stopper it tightly, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0160] 5. Assay

[0161] Accurately pipette 3 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0162] 6. Chromatographic conditions and system suitability test

[0163] The material composition of the standard decoction and the finished granules is consistent, and the characteristic chromatographic conditions are consistent with the system adaptability and the characteristic chromatographic method of the granules.

[0164] 7. Preparation of test solution

[0165] 7.1 Investigation of extraction methods

[0166] Approximately 1.0 g of this product (Batch No. BT-06) was placed in a stoppered conical flask. 50 ml of 30% methanol was added, respectively. The flask was sealed tightly. Refluxing and ultrasonic extraction (power 600 W, frequency 40 kHz) were tested for 30 minutes. The sample was cooled, shaken, filtered, and the filtrate was collected. (See Figure 10.) The results show that there is little difference in the extraction efficiency between reflux and ultrasonic extraction. Ultrasonic extraction is faster and simpler, so ultrasonic extraction was determined to be the extraction method for the sample.

[0167] 7.2 Extraction solvent investigation

[0168] Approximately 1.0 g of this product (Batch No. BT-06) was placed in a stoppered conical flask. Water, 30% methanol, 50% methanol, 80% methanol, and 50 ml of methanol were added, respectively. The flask was sealed and ultrasonicated (power 600 W, frequency 40 kHz) for 30 minutes. The flask was allowed to cool, shaken, filtered, and the filtrate was collected. See Figure 11. The results show that the chromatographic peaks obtained using water, 30%, and 50% methanol as the extraction solvent are large in information content and have good resolution. Using 30% methanol as the extraction solvent is easier to operate, so 30% methanol is tentatively selected as the extraction solvent.

[0169] 7.3 Extraction time investigation

[0170] Approximately 1.0 g of this product (Batch No. BT-06) was placed in a stoppered conical flask. 50 ml of 30% methanol was added, the flask was sealed, and ultrasonic treatment (power 600 W, frequency 40 kHz) was performed. The sample was extracted for 15 minutes, 30 minutes, and 60 minutes, respectively. The sample was allowed to cool, shaken, filtered, and the filtrate was collected. As shown in Figure 12, the results show that the sample was completely extracted at 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0171] 7.4 Investigation of solvent addition amount

[0172] Approximately 1.0 g of this product (Batch No. BT-06) was placed in a stoppered conical flask. 25 ml, 50 ml, and 100 ml of 30% methanol were added, respectively. The flask was sealed and ultrasonicated (power 600 W, frequency 40 kHz) for 30 minutes. The flask was cooled, shaken, filtered, and the filtrate was obtained. (See Figure 13.) The results show that when 25 ml of extraction solvent was added, the peak shape and resolution of the individual chromatographic peaks were better, and the peak sizes were appropriate, so 25 ml of solvent was selected.

[0173] In summary, the preparation method of the test solution of the characteristic spectrum of the standard decoction of Pinellia ternata is determined as follows: take about 1.0 g of the product, place it in a stoppered conical flask, add 25 ml of 30% methanol, seal it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, shake it evenly, filter it, and take the filtrate to obtain it.

[0174] 8. Methodological Investigation

[0175] 8.1 Chromatographic Peak Identification

[0176] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Beijing Pinellia standard decoction.

[0177] Preparation of reference solution: Take about 2 g of Pinellia ternata reference medicinal material powder, 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 filtrate.

[0178] Preparation of reference solution: Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, add methanol to make a solution containing 20 μg per 1 ml, which will serve as reference solution.

[0179] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the standard decoction lacking Pinellia ternata.

[0180] Peaks in the chromatogram of the standard decoction of Pinellia ternata were located. See Figure 14. The results indicate that peak 3 is 4-hydroxybenzoic acid and peak 13 is glycyrrhizic acid. The peaks in the Pinellia ternata control and the standard decoction differ significantly and are therefore not included in the characteristic method. In the following methodological investigation, 13 characteristic peaks in the sample were examined.

[0181] 8.2 Precision test

[0182] Take the test solution of Beijing Pinellia ternata standard decoction (Batch No.: BT-06) and inject 3 μl of the sample six times according to the proposed experimental method. Calculate the retention time and peak area of ​​each characteristic peak. See Table 9.

[0183] Table 9 Precision Investigation-Retention Time

[0184] The results showed that the RSD range of the relative retention time of each characteristic peak of the sample was 0.01% to 0.04%, and the instrument had good precision.

[0185] 8.3 Repeatability Study

[0186] Six portions of the standard decoction of Pinellia ternata (Batch No. JBX-BT-230206) were accurately weighed and prepared and assayed according to the proposed experimental method (see Table 10).

[0187] Table 10 Repeatability study - relative retention time

[0188] The results showed that the RSD of the relative retention time of the six samples ranged from 0.00% to 0.32%, indicating that the method had good repeatability.

[0189] 8.4 Intermediate Precision Examination

[0190] 8.4.1 Inspection of different instruments

[0191] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. See Figure 15 and Table 11:

[0192] Table 11 Different instruments investigated - relative retention time

[0193] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.50% and 6.56% when the three instruments were used for detection, and the durability of the different instruments was good.

[0194] 8.4.2 Inspection by different personnel and time

[0195] Based on the experimental conditions proposed above, two aliquots of the standard decoction of Pinellia ternata (Batch No. BT-06) were accurately weighed by different individuals (A and B) at different times (T1 and T2) to prepare test samples for determination (see Table 12).

[0196] Table 12 Personnel and time inspection-relative retention time

[0197] The results showed that when different people measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.62%, indicating that the method was stable.

[0198] 8.5.1 Column Durability Assessment

[0199] Based on the experimental conditions proposed above, chromatographic columns 1, 2, and 3 were investigated respectively (see Figure 16 and Table 13).

[0200] Table 13 Column durability investigation - relative retention time

[0201] As shown in Figure 16, The chromatogram of 2.1×150mm, 1.6μm (column 1) is better, so it is recommended to use the column 2.1×150mm,1.6μm.

[0202] 8.5.2 Stability Study

[0203] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 7h, 13h, 20h, and 25h. See Table 14.

[0204] Table 14 Stability Study-Retention Time

[0205] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.06% and 0.31%, and the sample solution was relatively stable within 25 hours.

[0206] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above 13 characteristic peaks were included in the subsequent investigation.

[0207] 8.9 Determination of characteristic peaks and establishment of reference maps

[0208] 8.9.1 Verification Results of 15 Batches of Standardized Decoction of Pinellia ternata

[0209] Using this method, characteristic chromatograms were analyzed for 15 batches of samples, and relative retention times and peak areas were calculated. See Figure 17 (Batch numbers from bottom to top: 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) and Table 15.

[0210] Table 15 Relative retention time of characteristic spectra of standard decoction of Pinellia ternata

[0211] Based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights, 13 peaks with good reproducibility were selected as characteristic peaks. The results showed that the relative peak area RSDs of the characteristic peaks of Pinellia ternata slices varied significantly, making it impossible to specify relative peak areas. Therefore, these peaks were not included in the main text of the quality standard. The final regulations stipulate that the test sample chromatogram should show 13 characteristic peaks, of which Peak 3 should correspond to the retention time of the reference peak of the reference substance. The peak corresponding to the 4-hydroxybenzoic acid reference peak is designated as the S peak. The relative retention time of each characteristic peak relative to the S peak should be calculated, and the relative retention time should be within ±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), and 2.94 (peak 13).

[0212] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) was used to synthesize 15 batches of Pinellia ternata slices, and a reference characteristic spectrum of the Pinellia ternata slices was established. See Figure 18, Peak 3 (S): 4-hydroxybenzoic acid; Peak 13: glycyrrhizic acid; Chromatographic column: 2.1×150mm,1.6μm.

[0213] In summary, the characteristic spectrum method of standard decoction is:

[0214] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 1 above; flow rate, 0.2 ml / min; column temperature, 30°C; detection wavelength, 270 nm. The number of theoretical plates, calculated based on the 4-hydroxybenzoic acid peak, should be no less than 5000.

[0215] Preparation of reference solution Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, weigh accurately, and add methanol to make solutions containing 20 μg per 1 ml, which are used as reference substance solutions.

[0216] Preparation of test solution: Take about 1.0 g of the product, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper it tightly, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0217] Determination method: Accurately aspirate 3μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0218] The test sample chromatogram should show 13 characteristic peaks, of which Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the 4-hydroxybenzoic acid reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±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), and 2.94 (peak 13).

[0219] Example 3 Method for Constructing UPLC Characteristic Spectrum of Pinellia Rhizoma Formula Granules

[0220] 1. Test materials

[0221] Jingbanxia formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: KL-01, KL-001, KL-002, KL-003).

[0222] 2. Chromatographic conditions

[0223] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 1 above; detection wavelength, 270 nm; flow rate, 0.2 ml / min; column temperature, 30°C. The number of theoretical plates, calculated based on the 4-hydroxybenzoic acid peak, should be no less than 5000.

[0224] 3. Preparation of Reference Solution

[0225] Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, weigh accurately, and add methanol to make solutions containing 20 μg per 1 ml, which serve as reference substance solutions.

[0226] 7.2.9.4 Preparation of test solution

[0227] Take an appropriate amount of this product, grind it into powder, take about 1.0g, place it in a stoppered conical flask, add 50ml of 30% methanol, stopper it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0228] 7.2.9.5 Determination method

[0229] Accurately pipette 3 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0230] 7.2.9.6 Chromatographic conditions and system suitability test

[0231] 7.2.9.6.1 Mobile phase selection

[0232] Based on the experimental conditions proposed above, the separation effects of four different mobile phases were investigated, namely: methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% acetic acid, and acetonitrile-water, as shown in Figure 19. The results showed that under the conditions of acetonitrile-0.1% phosphoric acid solution gradient elution, the chromatographic peak baseline was relatively stable, and the theoretical plate number, separation degree, and symmetry were all better. Therefore, acetonitrile-0.1% phosphoric acid solution gradient elution was used as the mobile phase for the characteristic spectrum determination method of the formulated particles.

[0233] 7.2.9.6.2 Wavelength selection

[0234] Based on the experimental conditions proposed above, the test solution was scanned across the entire wavelength range using a diode array detector (DAD). Chromatograms of the test solution were extracted at wavelengths of 210 nm, 230 nm, 250 nm, 270 nm, 290 nm, 310 nm, and 330 nm. As shown in Figure 20, the results show that the chromatographic peak information content is greater at a detection wavelength of 270 nm, and the chromatogram baseline is more stable. Therefore, the detection wavelength was determined to be 270 nm.

[0235] 7.2.9.6.3 Column temperature inspection

[0236] Based on the experimental conditions proposed above, column temperatures of 25°C, 30°C, and 35°C were tested. As shown in Figure 21, the results show that at a column temperature of 30°C, the chromatogram peaks are more symmetrical and the resolution is better. Therefore, the column temperature was determined to be 30°C.

[0237] 7.2.9.6.4 Flow rate investigation

[0238] Based on the experimental conditions proposed above, flow rates of 0.15 ml / min, 0.2 ml / min, and 0.25 ml / min were tested. As shown in Figure 22, the results show that at a flow rate of 0.2 ml / min, the chromatogram has a better peak shape and a stable baseline. Therefore, the flow rate was determined to be 0.2 ml / min.

[0239] 7.2.9.6.5 Delay Investigation

[0240] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Figure 23. The results show that the sample has almost no chromatographic peak after 60 minutes, so the sample detection time is set to 60 minutes.

[0241] In summary, the chromatographic conditions and system suitability tests for the characteristic spectrum of the formulated particles were determined as follows: octadecylsilane bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 1 above; detection wavelength 270 nm; flow rate 0.2 ml / min; column temperature 30°C. The number of theoretical plates calculated based on the p-hydroxybenzoic acid peak should be no less than 5000.

[0242] 7.2.9.7 Preparation of test solution

[0243] 7.2.9.7.1 Examination of extraction methods

[0244] Approximately 1.0 g of this product (Batch No. KL-01) was placed in a stoppered conical flask. 50 ml of 30% methanol was added, respectively. The flask was sealed tightly. The sample was extracted using either reflux or ultrasound (power 600 W, frequency 40 kHz). The extraction time was 30 minutes. The sample was cooled, shaken, filtered, and the filtrate was collected. (See Figure 24.) The results show that there is little difference in the extraction efficiency between reflux and ultrasound. The ultrasound method is faster and simpler, so ultrasound extraction was determined to be the extraction method for the sample.

[0245] 7.2.9.7.2 Examination of extraction solvent

[0246] Approximately 1.0 g of this product (batch number: KL-01) was placed in a stoppered conical flask. Water, 30% methanol, 50% methanol, 80% methanol, and 50 ml of methanol were added, respectively. The flask was sealed tightly and ultrasonicated (power 600 W, frequency 40 kHz) for 30 minutes. The flask was cooled, shaken, filtered, and the filtrate was collected to obtain the product. See Figure 25.

[0247] The results showed that the chromatographic peaks obtained using water, 30% and 50% methanol as extraction solvents had large information content and good separation; when 30% methanol was used as the extraction solvent, the operation was easier, so the extraction solvent was temporarily set as 30% methanol.

[0248] 7.2.9.7.3 Extraction time investigation

[0249] Approximately 1.0 g of this product (Batch No. KL-01) was placed in a stoppered conical flask. 50 ml of 30% methanol was added, the flask was sealed, and ultrasonic treatment (power 600 W, frequency 40 kHz) was performed. The extraction time for the sample was 15 min, 30 min, and 60 min, respectively. The sample was cooled, shaken, filtered, and the filtrate was collected. See Figure 26.

[0250] The results showed that the sample was completely extracted when the extraction time was 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0251] 7.2.9.7.4 Investigation of solvent addition amount

[0252] Approximately 1.0 g of this product (Batch No. KL-01) was placed in a stoppered conical flask. 25 ml, 50 ml, and 100 ml of 30% methanol were added, respectively. The flask was sealed and ultrasonicated (power 600 W, frequency 40 kHz) for 30 minutes. The flask was cooled, shaken, filtered, and the filtrate was obtained. (See Figure 27.) The results show that when 25 ml of extraction solvent was added, the peak shape and resolution of the individual chromatographic peaks were good, and the peak sizes were appropriate, so 25 mL of solvent was selected.

[0253] In summary, the preparation method of the test solution of the characteristic spectrum of the formula particles is determined as follows: take an appropriate amount of the product, grind it into powder, take about 1.0 g, put it in a stoppered conical flask, add 25 ml of 30% methanol, stopper it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, shake it evenly, filter it, and take the filtrate to obtain it.

[0254] 7.2.9.8 Methodological Review

[0255] 7.2.9.8.1 Chromatographic peak identification

[0256] Preparation of test solution: Prepare the test solution of formulated particles according to the experimental conditions proposed above.

[0257] Preparation of reference solution: Take about 2g of the powder of this product, 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, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate.

[0258] Preparation of reference solution: Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, add methanol to make a solution containing 20 μg per 1 ml, which will serve as reference solution.

[0259] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Qujing Banxia formula granules.

[0260] Locate the characteristic spectrum peaks of the formulated particles. See Figure 28;

[0261] The results showed that peak 3 was 4-hydroxybenzoic acid and peak 13 was glycyrrhizic acid. The characteristic peaks of the Pinellia ternata control and the granules were significantly different and were not included in the characteristic method. In the following methodological investigation, 13 characteristic peaks in the samples were examined.

[0262] 7.2.9.8.2 Precision test

[0263] Take the test solution of the formulated granules (batch number: KL-01) and inject 3 μl of the sample 6 times continuously according to the proposed experimental method. Calculate the retention time of each characteristic peak. See Table 16:

[0264] Table 16 Precision Investigation-Retention Time

[0265] The results showed that the RSD range of the relative retention time of each characteristic peak of the sample was 0.01% to 0.07%, and the instrument had good precision.

[0266] 7.2.9.8.3 Repeatability test

[0267] Accurately weigh 6 portions of the formulated granules (Batch No. KL-01) and prepare and measure them according to the proposed experimental method. See Table 17.

[0268] Table 17 Repeatability Study-Relative Retention Time

[0269] The results showed that the RSD of the relative retention time of the six samples ranged from 0.00% to 0.44%, indicating that the method had good repeatability.

[0270] 7.2.9.8.4 Intermediate precision assessment

[0271] 7.2.9.8.4.1 Inspection of different instruments

[0272] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. See Figure 29 and Table 18.

[0273] Table 18 Column durability investigation - relative retention time

[0274] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.00% and 6.46% when detected by the three instruments mentioned above, indicating that the durability of the different instruments was good.

[0275] 7.2.9.8.4.2 Inspection by different personnel and time

[0276] Based on the experimental conditions proposed above, two aliquots of the formulated granules (Batch No.: KL-01) were accurately weighed by different individuals (A and B) at different times (T1 and T2) to prepare test samples for measurement. See Table 19.

[0277] Table 19 Personnel and time inspection - relative retention time

[0278] The results showed that when different people measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.60%, indicating that the method was stable.

[0279] 7.2.9.8.4.3 Chromatographic column durability assessment

[0280] Based on the experimental conditions proposed above, chromatographic columns 1, 2, and 3 were investigated respectively. See Figure 30 and Table 20.

[0281] Table 20 Column durability investigation - relative retention time

[0282] As shown in Figure 30, The chromatogram of 2.1×150mm, 1.6μm (column 1) is better, so it is recommended to use the column 2.1×150mm,1.6μm.

[0283] 7.2.8.8.5 Stability assessment

[0284] Based on the experimental conditions proposed above, the same test solution was measured at 0h, 3h, 7h, 13h, 20h, and 25h. See Table 21.

[0285] Table 21 Stability Study-Retention Time

[0286] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.02% and 0.27%, and the sample solution was relatively stable within 25 hours.

[0287] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above 13 characteristic peaks were included in the subsequent investigation.

[0288] 7.2.9.9 Determination of characteristic peaks and establishment of reference maps

[0289] 7.2.9.9.1 Verification results of three batches of formulated granules

[0290] Using this method, characteristic spectrum analysis was performed on three batches of samples and relative retention times were calculated. See Figure 31 and Table 22:

[0291] Table 22 Relative retention time of formula particles

[0292] Based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights, 13 peaks with good reproducibility were selected as characteristic peaks. The results showed that the relative peak areas of the characteristic peaks in the Jing Ban Xia formula granules varied significantly, making it impossible to specify relative peak areas. Therefore, these peaks were not included in the main text of the quality standard. The final regulations stipulate that the test sample chromatogram should show 13 characteristic peaks, of which Peak 3 should correspond to the retention time of the reference peak of the reference substance. The peak corresponding to the 4-hydroxybenzoic acid reference peak is designated as the S peak. The relative retention time of each characteristic peak relative to the S peak should be calculated, and the 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), 1.51 (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), and 2.93 (peak 13).

[0293] Three batches of formula granules were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), and a reference characteristic spectrum of the formula granules was established. See Figure 32; Peak 3 (S): 4-hydroxybenzoic acid, Peak 13: glycyrrhizic acid; Chromatographic column: 2.1×150mm,1.6μm.

[0294] In summary, the characteristic spectrum method of the formulated particles is:

[0295] Chromatographic conditions and system suitability tests were performed using octadecylsilane bonded silica gel as the filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1 above; flow rate: 0.2 ml / min; column temperature: 30°C;

[0296] Preparation of reference solution Take appropriate amount of 4-hydroxybenzoic acid and glycyrrhizic acid reference substances, weigh accurately, and add methanol to make solutions containing 20 μg per 1 ml, which are used as reference substance solutions.

[0297] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0 g, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper it tightly, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0298] Determination method: Accurately aspirate 3μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0299] The test sample chromatogram should show 13 characteristic peaks, of which Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the 4-hydroxybenzoic acid reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The 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), 1.51 (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), and 2.93 (peak 13).

[0300] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing characteristic maps of Pinellia ternata slices, standard decoctions, and formula granules, comprising the following steps: Dissolve the test sample, extract it, and obtain the test solution; The test solution is measured by high performance liquid chromatography to obtain characteristic spectra of Beijing Banxia decoction pieces, standard decoction, and formula granules; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

2. The construction method according to claim 1, characterized in that: Also includes the preparation of reference solutions: Dissolve 4-hydroxybenzoic acid and glycyrrhizic acid reference substances in methanol to obtain reference substance solutions; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the HPLC characteristic spectrum of the characteristic spectrum of the Pinellia ternata slices, standard decoction, and formula granules are qualitatively identified according to the chromatogram of the reference.

3. The construction method according to claim 2, characterized in that: The concentration of the reference solution is 20 μg / mL.

4. The construction method according to claim 1, characterized in that: The gradient elution is specifically: 0-5min, phase A: 0-2%, phase B: 100-98%; 5-8min, phase A: 2%, phase B: 98%; 8-15min, phase A: 2-6%, phase B: 98-94%; 15-32 min, phase A: 6-14%, phase B: 94-86%; 32~42min, phase A: 14~25%, phase B: 86~75%; 42~60min, phase A: 25~50%, phase B: 75~50%.

5. The construction method according to claim 1, characterized in that: The extraction solvent is 30% methanol; the extraction method is ultrasonic extraction; The power of the ultrasonic extraction is 580-620W, the frequency of the ultrasonic extraction is 35-45kHz, and the time of the ultrasonic extraction is 25-35min; The ratio of the mass g of the test sample to the volume mL of the extracted solvent is (1~2):25mL.

6. The construction method according to claim 1, characterized in that: 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 theoretical plate number calculated based on the 4-hydroxybenzoic acid peak should not be less than 5000.

7. The construction method according to claim 1, characterized in that: The similarity of HPLC characteristic spectrum of Pinellia ternata slices was evaluated by adopting the similarity evaluation system of Chinese medicine chromatographic fingerprint spectrum, and the HPLC standard characteristic spectrum of Pinellia ternata slices consisting of 13 characteristic peaks was obtained, wherein the peak corresponding to the 4-hydroxybenzoic acid reference substance was the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of the specified value; the specified value was: 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).

8. The construction method according to claim 1, characterized in that: The similarity of the HPLC characteristic spectrum of the standard decoction of Pinellia ternata was evaluated by using a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, and a standard characteristic spectrum of the HPLC standard decoction of Pinellia ternata consisting of 13 characteristic peaks was obtained, wherein the peak corresponding to the 4-hydroxybenzoic acid reference substance was the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of the specified value; the specified value was: 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), and 2.94 (peak 13).

9. The construction method according to claim 1, characterized in that: The similarity of the HPLC characteristic spectrum of the Banxia formula granules is evaluated by using a similarity evaluation system for Chinese medicine chromatographic fingerprints, and a HPLC standard characteristic spectrum of the Banxia formula granules consisting of 13 characteristic peaks is obtained, wherein the peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: 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), and 2.93 (peak 13).

10. A method for identifying Peking Pinellia slices, standard decoctions, and formula granules, characterized in that: The method according to any one of claims 1 to 9 is used for detection, and the detection results are analyzed.

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