A method for constructing a characteristic spectrum of a fried pinellia ternata and a method for determining the content of xanthine

Abstract

The application provides a construction method of bran-fried pinellia ternata tablet characteristic spectrum, comprising the following steps: A) mixing a test sample raw material and an ammonia solution, ultrasonic treatment, filtering to obtain a to-be-tested solution; B) using high performance liquid chromatography to determine the to-be-tested solution to obtain a bran-fried pinellia ternata tablet HPLC characteristic spectrum; the high performance liquid chromatography chromatographic conditions are as follows: a C 18 column is used as the chromatographic column; a mobile phase A is acetonitrile, a mobile phase B is water, and gradient elution is used. The construction method provided by the application has the characteristics of comprehensive detection and simple operation, can effectively identify bran-fried pinellia ternata tablet formula granules, and can ensure that the quality of the bran-fried pinellia ternata tablet formula granules is uniform and stable. The method is also suitable for pinellia ternata tablet medicinal materials, bran-fried pinellia ternata tablet decoction pieces and standard decoction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a construction method of a characteristic spectrum of bran-roasted pinellia tuber and a determination method of xanthine content. BACKGROUND

[0002] The bran-roasted pinellia tuber is one of common varieties of fermented processed products of traditional Chinese medicines, has the functions of relieving cough and reducing sputum, and has the effects of treating cough and sputum, nausea and vomiting, food accumulation and diarrhea. The bran-roasted pinellia tuber is prepared by using microorganisms (mainly molds, yeasts and bacteria) in the natural environment for mixed solid-state fermentation. The bran-roasted pinellia tuber has the defects of impure fermentation strains, unstable product quality and uncontrollability. At present, there is no research on the bran-roasted pinellia tuber, the bran-roasted pinellia tuber decoction pieces, the bran-roasted pinellia tuber standard decoction, the bran-roasted pinellia tuber granules, the content determination and the characteristic spectrum. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a construction method of a characteristic spectrum of bran-roasted pinellia tuber and a determination method of xanthine content. The construction method is used for quality control of the bran-roasted pinellia tuber, the bran-roasted pinellia tuber decoction pieces, the standard decoction and the granules, so as to ensure the uniformity and stability of the quality. The content determination method is accurate, has good separation degree and good stability.

[0004] The present application provides a construction method of a characteristic spectrum of bran-roasted pinellia tuber, which comprises the following steps:

[0005] A) mixing a test sample and an ammonia solution, ultrasonic treatment, filtration, to obtain a to-be-tested solution;

[0006] B) determining the to-be-tested solution by high performance liquid chromatography (HPLC) to obtain a bran-roasted pinellia tuber HPLC characteristic spectrum;

[0007] The HPLC chromatography condition is that a C18 column is used as the chromatography column, and acetonitrile is used as the mobile phase A and water is used as the mobile phase B for gradient elution.

[0008] The present application establishes an HPLC characteristic spectrum method for detection of the bran-roasted pinellia tuber, the bran-roasted pinellia tuber decoction pieces, the standard decoction, the granules and related preparations thereof. In the process of establishing the characteristic spectrum of the bran-roasted pinellia tuber, seven common characteristic peaks are confirmed, two components are identified, and the relative retention time and the relative peak area are researched, so as to ensure the stability of the chemical composition and the safety in use.

[0009] The present application mixes a test sample and an ammonia solution, ultrasonic treatment, filtration, to obtain a to-be-tested solution. The test sample is the bran-roasted pinellia tuber, the bran-roasted pinellia tuber decoction pieces, the bran-roasted pinellia tuber standard decoction and the bran-roasted pinellia tuber granules.

[0010] In the present application, the volume concentration of the ammonia solution is 0.030-0.035%; in a specific embodiment, the concentration of the ammonia solution is 0.032%. The mass of the test sample raw material and the volume of the ammonia solution are in a ratio of (1.8-2.3) g:25 mL; in a specific embodiment, the mass of the test sample raw material and the volume of the ammonia solution are in a ratio of 2 g:25 mL. The power of the ultrasonic treatment is 550-650 W, the frequency of the ultrasonic treatment is 35-45 kHz, and the time of the ultrasonic treatment is 25-35 min; in a specific embodiment, the power of the ultrasonic treatment is 600 W, the frequency of the ultrasonic treatment is 40 kHz, and the time of the ultrasonic treatment is 30 min.

[0011] In the present application, the 0.45-μm microporous filter head is used for filtering to obtain the test liquid.

[0012] In a specific embodiment, the preparation process of the test sample solution is as follows: an appropriate amount of the product is taken, about 2 g is accurately weighed and placed in a conical flask with a plug, 25 ml of 0.032% ammonia solution is accurately added, ultrasonic treatment (power 600 W, frequency 40 kHz) is performed for 30 minutes, it is cooled, the weight is re-weighed, the lost weight is made up with 0.032% ammonia solution, it is shaken uniformly, it is filtered, and the filtrate is taken, i.e. the test sample solution is obtained.

[0013] The present application also includes the preparation of a reference solution:

[0014] The pinellia tuber powder control medicinal material and 0.030-0.035% ammonia solution are mixed, ultrasonic treatment is performed, and filtration is performed to obtain a control medicinal material reference solution;

[0015] The xanthine and guanine control samples are respectively mixed with 0.030-0.035% ammonia solution to prepare 10 μg / mL of a control sample reference solution.

[0016] In a specific embodiment, the preparation of the reference solution is as follows:

[0017] The pinellia tuber powder control medicinal material and 0.032% ammonia solution are mixed, ultrasonic treatment is performed, and filtration is performed to obtain a control medicinal material reference solution;

[0018] The xanthine and guanine control samples are respectively mixed with 0.032% ammonia solution to prepare 10 μg / mL of a control sample reference solution.

[0019] In the present application, the test liquid is determined by high performance liquid chromatography to obtain a pinellia tuber powder HPLC characteristic spectrum; the high performance liquid chromatography chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is performed.

[0020] The same sample solution is continuously sampled for 6 times by the proposed method, 10 μL each time, and the retention time of each characteristic peak indicates that the instrument precision is good. The same sample is weighed for 6 times, prepared and determined according to the proposed experiment, which indicates that the method repeatability is good. The determination is carried out on different instrument models of high performance liquid chromatograph, and the results show that different instruments all meet the requirements. The determination is also carried out on different personnel and different time, and the results show that the method stability is good. The durability of the same specification and different models of C18 column is investigated, and the results show that the column durability is good. The same sample solution is determined at 0 h, 2 h, 4 h, 8 h, 12 h, 16 h and 24 h, which indicates that the sample solution is stable within 24 h.

[0021] In the present application, the gradient elution program is as follows:

[0022] 0-15 min, mobile phase A: 1-4%, mobile phase B: 99-96%;

[0023] 15-25 min, mobile phase A: 4-5%, mobile phase B: 96-95%;

[0024] 25-35 min, mobile phase A: 5-10%, mobile phase B: 95-90%;

[0025] 35-40 min, mobile phase A: 10%, mobile phase B: 90%.

[0026] In the present application, the specifications of the chromatographic column are as follows: column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm, and column temperature is 25-35 ℃; in specific embodiments, the chromatographic column models used are as follows: chromatographic column 1: Agilent 5TC-C18(2) 4.6*250 mm, 5 μm; chromatographic column 2: Kromasil 100-5-C18 4.6*250 mm, 5 μm; chromatographic column 3: Waters XBridge C18 4.6*250 mm, 5 μm.

[0027] The detection wavelength is 260-280 nm; the flow rate of the mobile phase is 0.8-1.2 mL / min; the theoretical plate number calculated according to the xanthine peak should not be less than 5000; and the injection amount is 8-12 μl.

[0028] The application adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of the fried pinellia tuber powder, and obtains a HPLC standard characteristic spectrum of the fried pinellia tuber powder composed of seven peaks, wherein peak 1 and peak 2 should correspond to the retention time of the corresponding control product reference peak respectively, the peak corresponding to the xanthine reference peak is an S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is: 1.10 (peak 3), 1.24 (peak 4), 1.41 (peak 5), 2.00 (peak 6), and 2.16 (peak 7).

[0029] The application provides a method for determining the content of xanthine in the fried pinellia tuber powder, comprising the following steps:

[0030] S1, mixing the fried pinellia tuber powder sample with an ammonia solution, ultrasonic treatment, and filtering to obtain a to-be-tested solution;

[0031] S2, mixing the xanthine control product and 0.030-0.035% ammonia solution to obtain a 10 μg / mL control product reference solution;

[0032] S3, under the same detection conditions, obtaining the chromatograms of the chemical components in the to-be-tested solution in step S1 and the control product reference solution in step S2 by using high performance liquid chromatography, and the specific chromatographic parameters are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is adopted;

[0033] S4, based on the detection conditions in step S3, the content of xanthine in the fried pinellia tuber powder is calculated by an external standard method according to the concentration of the control product reference, the peak area of the control product reference solution in the chromatogram, and the peak area of the component corresponding to the control product reference solution in the chromatogram of the fried pinellia tuber powder.

[0034] The determination method has the characteristics of comprehensive detection and simple operation, can effectively identify the fried pinellia tuber powder formula granules, pinellia tuber medicine, fried pinellia tuber powder decoction pieces, and fried pinellia tuber powder standard decoction, and can ensure the uniformity and stability of the quality of the fried pinellia tuber powder formula granules, pinellia tuber medicine, fried pinellia tuber powder decoction pieces, and fried pinellia tuber powder standard decoction.

[0035] In the application, the injection concentration of the xanthine solution is 1.09075-218.15 μg / ml, the regression equation of xanthine is Y=36.4178X+7.2240, R 2 =0.9999.

[0036] In the application, the detection conditions in step S3 are as follows: gradient elution, and the specific program is as follows:

[0037] 0~15min, mobile phase A: 1~4%, mobile phase B: 99~96%;

[0038] 15~25min, mobile phase A: 4~5%, mobile phase B: 96~95%;

[0039] 25~35min, mobile phase A: 5~10%, mobile phase B: 95~90%;

[0040] 35~40min, mobile phase A: 10%, mobile phase B: 90%.

[0041] The chromatographic column specifications are that the column length is 250mm, the inner diameter is 4.6mm, the particle size is 5um, the column temperature is 30 DEG C, the detection wavelength is 270nm, the flow rate of the mobile phase is 1.0mL / min; the theoretical plate number should be not less than 5000 calculated by the xanthine peak; the injection amount is 8~12ul.

[0042] The application provides a construction method of bran-fried pinellia tablet characteristic spectrum, comprising the following steps: A) mixing the test product raw material and ammonia solution, ultrasonic treatment, filtering to obtain a test solution; B) using high performance liquid chromatography to determine the test solution to obtain the bran-fried pinellia tablet HPLC characteristic spectrum; the high performance liquid chromatography chromatographic conditions are that the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is carried out. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Different instruments are used for bran-fried pinellia tablet formula granules;

[0044] Figure 2 Different chromatographic columns are used for bran-fried pinellia tablet formula granules;

[0045] Figure 3 The chromatographic peak of bran-fried pinellia tablet formula granules is identified;

[0046] Figure 4 The xanthine standard curve is shown in the figure;

[0047] Figure 5 The verification spectrum of 3 batches of bran-fried pinellia tablet formula granules is shown in the figure;

[0048] Figure 6 The verification spectrum of 3 batches of pinellia tablet medicinal materials is shown in the figure;

[0049] Figure 7 The verification spectrum of 3 batches of bran-fried pinellia tablet decoction pieces is shown in the figure;

[0050] Figure 8 Validation chromatogram of 3 batches of standard decoction of bran-fried pinellia preparation;

[0051] Figure 9 Control chromatogram of bran-fried pinellia preparation formula granules;

[0052] Figure 10 Control chromatogram of pinellia preparation medicinal materials;

[0053] Figure 11 Control chromatogram of bran-fried pinellia preparation slices;

[0054] Figure 12 Control chromatogram of standard decoction of bran-fried pinellia preparation. DETAILED DESCRIPTION

[0055] In order to further illustrate the present application, the construction method of a characteristic chromatogram of bran-fried pinellia preparation and the determination method of xanthine content provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the present application.

[0056] Example 1

[0057]

Characteristic chromatogram

[0058] 1. Experimental instruments and materials

[0059] High-performance liquid chromatograph: instrument 1, instrument 2, instrument 3, and instrument 4;

[0060] Electronic balance: ME204E / 02, MS205DU, and XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0061] Ultrapure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0062] Ultrasonic cleaner: KQ5200DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0063] Chromatographic column: chromatographic column 1, chromatographic column 2, and chromatographic column 3; the column temperature was 30℃, the detection wavelength was 270nm, and the flow rate of the mobile phase was 1.0mL / min.

[0064] Methanol and acetonitrile were chromatographically pure, water was ultrapure water, and the rest of the reagents were analytically pure.

[0065] Xanthine (China Food and Drug Inspection Research Institute, batch number: 140662-200802);

[0066] Guanine (China Food and Drug Inspection Research Institute, batch number: 140631-202008, content 98.9%);

[0067] Banxia Qu control drug (Chengdu Desit Biotechnology Co., Ltd., batch number: DSTYB010901);

[0068] Fuzhao Banxia Qu formula granules FCBXQ-01, FCBXQ-02, FCBXQ-03;

[0069] Banxia Qu medicinal materials: BXQ-YC-01, BXQ-YC-02, BXQ-YC-03;

[0070] Fuzhao Banxia Qu decoction pieces: BXQ-YP-01, BXQ-YP-02, BXQ-YP-03;

[0071] Fuzhao Banxia Qu standard decoction: BXQ-BT-01, BXQ-BT-02, BXQ-BT-03.

[0072] Chromatographic conditions and system suitability test With octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); with acetonitrile as mobile phase A, and water as mobile phase B, gradient elution according to the provisions in Table 1 below; the detection wavelength is 270 nm. The theoretical plate number should not be less than 5000 calculated by the xanthine peak.

[0073] Table 1

[0074]

[0075] Preparation of reference solution Take Banxia Qu control drug 2 g, add 0.032% ammonia solution 25 ml in a conical flask with a plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the reference solution of the control drug.

[0076] Take xanthine reference substance in an appropriate amount, accurately weigh, add 0.032% ammonia solution to prepare a solution containing 10 μg per 1 ml, and obtain it.

[0077] Take guanine reference substance in an appropriate amount, accurately weigh, add 0.032% ammonia solution to prepare a solution containing 10 μg per 1 ml, and obtain it as the reference solution of the reference substance.

[0078] Preparation of test solution Take the product (FCBXQ-01) in an appropriate amount, take about 2 g, accurately weigh, add 0.032% ammonia solution 25 ml in a conical flask with a plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 0.032% ammonia solution, shake well, filter, and take the filtrate, namely the test solution.

[0079] Determination method: accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, namely the test solution.

[0080]

Content Determination

[0081] Chromatographic conditions and system suitability test with octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as the mobile phase A, water as the mobile phase B, gradient elution according to the provisions in Table 1 above; detection wavelength 270 nm. The theoretical plate number should not be less than 5000 calculated by the xanthine peak.

[0082] Preparation of reference solution: Take xanthine reference substance, accurately weigh, add 0.032% ammonia solution to make a solution containing 10 μg per 1 ml, and obtain.

[0083] Preparation of test solution: Take the product (FCBXQ-01), accurately weigh about 2 g, accurately weigh, add 0.032% ammonia solution 25 ml in a conical flask with a plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 0.032% ammonia solution, shake well, filter, take the filtrate, and obtain.

[0084] Determination method: accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain.

[0085] 2. Characteristic chromatographic methodological verification

[0086] 2.1 Precision test

[0087] Take the test solution of the bran-fried pinellia tablet (batch number: FCBXQ-01), according to the proposed experimental method, continuously sample 6 times, 10 μL each time, calculate the retention time and peak area of each characteristic peak. As shown in Table 2.

[0088] Table 2 Precision test - retention time

[0089]

[0090]

[0091] The results show that the instrument has good precision.

[0092] 2.2 Reproducibility test

[0093] Accurately weigh 6 portions of the bran-fried pinellia tablet (batch number: FCBXQ-01), prepare and determine according to the proposed experimental method. As shown in Table 3.

[0094] Table 3 Reproducibility test - relative retention time ratio

[0095]

[0096] The results show that the method has good repeatability.

[0097] 2.3 Intermediate precision investigation

[0098] 2.3.1 Investigation of different instruments

[0099] On the basis of the above proposed test conditions, the test sample solution was prepared by precisely weighing the Fenqiaobanxiaqu formula granules (batch number: FCBXQ-01), and was determined on instrument 1, instrument 2, instrument 3, and instrument 4 high performance liquid chromatographs, respectively. As shown in Table 4. Figure 1

[0100] Table 4 Investigation of instrument durability- relative retention time ratio

[0101]

[0102] The results show that different instruments meet the requirements.

[0103] 2.3.2 Investigation of different personnel and time

[0104] On the basis of the above proposed test conditions, the test sample was prepared by precisely weighing the Fenqiaobanxiaqu formula granules (batch number: FCBXQ-01) by different personnel (A, B) at different times (T1, T2), and was determined. As shown in Table 5:

[0105] Table 5 Investigation of personnel and time- relative retention time ratio

[0106]

[0107] The results show that the determination of the same sample by different personnel at different times has good method stability.

[0108] 2.4 Durability investigation

[0109] 2.4.1 Investigation of column durability

[0110] On the basis of the above proposed experimental conditions, three columns were used for analysis investigation, including column 1: Agilent 5TC-C18 (2) 4.6*250mm, 5μm; column 2: Kromasil 100-5-C18 4.6*250mm, 5μm; and column 3: Waters XBridge C18 4.6*250mm, 5μm. As shown in Table 6. Figure 2

[0111] Table 6 Investigation of column durability- relative retention time ratio

[0112] ​​

[0113] The results show that the chromatographic column has good durability.

[0114] 2.4.2 Stability Investigation

[0115] Based on the above experimental conditions, the same test solution was taken and measured at 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h. The results are shown in Table 7.

[0116] Table 7 Stability Investigation - Retention Time

[0117]

[0118]

[0119] The results show that the sample solution is stable within 24 hours.

[0120] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 7 characteristic peaks are included in the subsequent investigation.

[0121] 2.5 Chromatographic Peak Identification

[0122] Preparation of Test Solution: According to the above experimental conditions, the standard decoction test solution of bran-fried pinellia tablet was prepared.

[0123] Preparation of Reference Solution: Take 2 g of pinellia tablet control drug, place it in a conical flask with a plug, add 25 ml of 0.032% ammonia solution, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the control drug reference solution.

[0124] Take an appropriate amount of xanthine and guanine reference substance, accurately weigh, and add 0.032% ammonia solution to prepare a solution containing 10 μg per 1 ml as the reference substance solution.

[0125] Preparation of Negative Control Solution: According to the above experimental conditions, the standard decoction negative control solution of bran-fried pinellia tablet was prepared.

[0126] The characteristic peaks of bran-fried pinellia tablet formula granules were located. See Figure 3 The results show that peak 1 is guanine and peak 2 is xanthine. In the following methodological investigation, 7 characteristic peaks in the sample were investigated.

[0127] 3. Content Determination Verification

[0128] 3.1 Precision Investigation

[0129] Take the reference solution and inject it continuously for 6 times, record the peak area of xanthine, and calculate the RSD value. The results are shown in Table 8.

[0130] Table 8 Precision test results

[0131]

[0132]

[0133] The results show that the sample injection precision of the method is good.

[0134] 3.2 Linear relationship

[0135] Take the appropriate amount of xanthine 4.363 mg, put it in a 20 ml volumetric flask, and prepare a reference solution containing ephedrine hydrochloride 218.15 μg / ml with methanol as the solvent, then dilute it to a concentration of 1.09075 μg / ml, 5.45375 μg / ml, 10.9075 μg / ml, 54.5375 μg / ml, 109.075 μg / ml, 218.15 μg / ml. Take 10 μl of the solution and the mother liquor respectively, get the peak area, take the sample size (X, μg) as the abscissa and the peak area (Y) as the ordinate to draw the response curve, the results are shown in Table 9, Figure 4 .

[0136] Table 9 Xanthine standard curve analysis results

[0137]

[0138] The results show that the xanthine concentration range is 1.09075-218.15 μg / ml, showing a good linear relationship.

[0139] 3.3 Reproducibility experiment

[0140] Take about 2 g of bran-fried pinellia tablet (batch number: FCBXQ-01), accurately weigh 6 portions, and prepare the test sample solution according to the determined method by the same operator, calculate the xanthine content of the 6 test samples, the results are shown in Table 10.

[0141] Table 10 Reproducibility experiment results

[0142]

[0143] The results show that the method has good reproducibility.

[0144] 3.4 Stability experiment

[0145] Take the same test sample solution (batch number: FCBXQ-01), and measure the xanthine chromatographic peak area at 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h, respectively, the results are shown in Table 11.

[0146] Table 11 Stability experiment results

[0147]

[0148]

[0149] The results show that the stability of the test solution is good within 24 hours.

[0150] 3.5 Intermediate precision

[0151] 3.5.1 Different personnel and time investigation

[0152] The same test sample (batch number: FCBXQ-01) was prepared by different personnel (A, B) at different times (I, II) to analyze the content of xanthine in the test sample, and the results are shown in Table 12.

[0153] Table 12 Intermediate precision experiment results

[0154]

[0155] The results show that the RSD value of the content of xanthine is 0.5%, and the intermediate precision of the method is good.

[0156] 3.5.2 Spiked recovery

[0157] About 1 g of test sample with known content (batch number: FCBXQ-01, xanthine content 0.128 mg / g) was precisely weighed and divided into 6 portions, and 25 ml of xanthine (purity: 100%) mixed control solution was precisely added, the test sample solution was prepared according to the proposed method and determined, and the recovery rate was calculated, and the results are shown in Table 13. The calculation formula is as follows:

[0158]

[0159] Table 13 Xanthine spiked recovery experiment results

[0160]

[0161]

[0162] The results show that the average recovery rate of xanthine is 97%. The accuracy of the method is good.

[0163] 3.5.3 Durability investigation

[0164] 3.5.3.1 Different instrument investigation

[0165] The test sample solution was prepared according to the determined method, 2 g of the product powder (batch number: FCBXQ-01) was precisely weighed and determined, and was investigated on instrument 1, instrument 2, instrument 3 and instrument 4 high performance liquid chromatograph respectively, and the content of xanthine was calculated. The results are shown in Table 14.

[0166] Table 14 Results of different instrument experiments

[0167]

[0168] The results show that the instrument is durable.

[0169] 3.5.3.2 Investigation of different chromatographic columns

[0170] Different brands of C18 were used: Column 1: Agilent 5TC-C18 (2) 4.6*250mm, 5μm; Column 2: Kromasil 100-5-C18 4.6*250mm, 5μm; Column 3: Waters XBridge C18 4.6*250mm, 5μm. The same test sample (batch number: FCBXQ-01) was detected by the three chromatographic columns, and the results are shown in Table 15.

[0171] Table 15 Results of investigation of column durability

[0172]

[0173]

[0174] The results show that the analysis chromatographic indicators of different chromatographic columns are good. The theoretical plate number should not be less than 5000 calculated by the xanthine peak.

[0175] Example 2 3 batches of bran-fried pinellia tablet granules verification results

[0176] The proposed method was used to determine the characteristic spectrum, content, relative retention time, and xanthine content of 3 batches of samples. The results are shown in Table 16 and Table 17. Figure 5

[0177] Table 16 Relative retention time of 3 batches of Rouqiaobaniaocuo Qu Formula Granules

[0178]

[0179] Table 17 Xanthine content of 3 batches of Rouqiaobaniaocuo Qu Formula Granules

[0180]

[0181] The results of each investigation item and verification of the methodology are shown in Table 18:

[0182] Table 18 Results of each item of the methodology RSD% summary standard-relative retention time

[0183]

[0184] ​From the above table, different instruments and different chromatographic columns have great influence on the relative retention time of each characteristic peak. In order to increase the reproducibility and applicability of the method, the relative retention time of each peak is temporarily set as 10%.

[0185] Example 3 3 batches of pinellia tablet medicinal material verification results

[0186] The characteristic spectrum and content of 3 batches of samples were determined by the proposed method, and the relative retention time and xanthine content were calculated. The results are shown in Table 19, Table 20. Figure 6

[0187] Table 19 Relative retention time of 3 batches of bran-roasted pinellia tablet

[0188]

[0189] Table 20 Xanthine content of 3 batches of pinellia tablet

[0190]

[0191] Example 4 3 batches of bran-fried pinellia tablet decoction pieces verification results

[0192] The characteristic spectrum and content of 3 batches of samples were determined by the proposed method, and the relative retention time and xanthine content were calculated. The results are shown in Table 21 and Table 22. Figure 7

[0193] Table 21 Relative retention time of 3 batches of bran-roasted pinellia tablet

[0194]

[0195] Table 22 Xanthine content of 3 batches of bran-roasted pinellia tablet

[0196]

[0197] Example 5 3 batches of bran-fried pinellia tablet standard decoction verification results

[0198] The characteristic spectrum and content of 3 batches of samples were determined by the proposed method, and the relative retention time and xanthine content were calculated. The results are shown in Table 23 and Table 24. Figure 8

[0199] Table 23 Relative retention time of 3 batches of bran-roasted pinellia tablet

[0200]

[0201] Table 24 Xanthine content of 3 batches of bran-roasted pinellia tablet

[0202]

[0203] ​​​

[0204] Example 6: Characteristic chromatogram of formula granules

[0205] Chromatographic conditions and system suitability test The chromatographic conditions and system suitability test were as follows: octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as the mobile phase A, water as the mobile phase B, gradient elution according to the provisions in Table 1 above; detection wavelength 270 nm. The theoretical plate number should not be less than 5000 calculated according to the xanthine peak.

[0206] Preparation of reference solution Take 2 g of pinellia ternate processed control medicinal material, add 25 ml of 0.032% ammonia water solution into a conical flask with a plug, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the control medicinal material reference solution. Take an appropriate amount of xanthine reference substance, accurately weigh, add 0.032% ammonia water solution to prepare a solution containing 10 μg per 1 ml, namely the reference substance reference solution. Further take an appropriate amount of guanine reference substance, accurately weigh, add 0.032% ammonia water solution to prepare a solution containing 10 μg per 1 ml, as the reference substance reference solution.

[0207] Preparation of test solution Take an appropriate amount of bran-fried pinellia ternate processed granules, take about 2 g, accurately weigh, add 25 ml of 0.032% ammonia water solution into a conical flask with a plug, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 0.032% ammonia water solution, shake well, filter, and take the filtrate, namely the test solution.

[0208] Determination method accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, namely the determination result.

[0209] Figure 9 The control characteristic chromatogram of bran-fried pinellia ternate processed granules; from Figure 9 It can be seen that 7 characteristic peaks should appear in the test chromatogram, and should correspond to the retention time of the 7 characteristic peaks in the control medicinal material reference chromatogram, wherein peak 1 and peak 2 should correspond to the retention time of the corresponding reference substance peaks. The peak corresponding to the xanthine reference peak is the S peak, and the relative retention time of the remaining characteristic peaks to the S peak should be within ±10% of the specified value, and the specified value is: 1.10 (peak 3), 1.24 (peak 4), 1.41 (peak 5), 2.00 (peak 6), 2.16 (peak 7).

[0210] Example 7: Characteristic chromatogram of medicinal materials

[0211] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filling agent (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as the mobile phase A, water as the mobile phase B, gradient elution according to the provisions in Table 1 above; detection wavelength 270 nm. Theoretical plate number should not be less than 5000 calculated according to the xanthine peak.

[0212] Preparation of reference solution: take 2 g of pinellia ternate medicinal material, add 25 ml of 0.032% ammonia water solution in a conical flask with a plug, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the subsequent filtrate as the reference solution of the control medicinal material. Take the xanthine reference substance, accurately weigh, add 0.032% ammonia water solution to prepare a solution containing 10 μg per 1 ml, and obtain it. Take the guanine reference substance, accurately weigh, add 0.032% ammonia water solution to prepare a solution containing 10 μg per 1 ml, and obtain it as the reference solution of the reference substance.

[0213] Preparation of test solution: take pinellia ternate medicinal material, accurately weigh about 2 g, add 25 ml of 0.032% ammonia water solution in a conical flask with a plug, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 0.032% ammonia water solution, shake well, filter, and take the subsequent filtrate, and obtain it.

[0214] Determination: accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain it.

[0215] Figure 10 The control characteristic chromatogram of pinellia ternate medicinal material; from Figure 10 It can be seen that 7 characteristic peaks should appear in the test chromatogram, and should correspond to the retention time of the 7 characteristic peaks in the reference chromatogram of the control medicinal material. The peaks corresponding to the xanthine reference peaks are S peaks, and the relative retention time of the remaining characteristic peaks to the S peaks should be within ±10% of the specified value, and the specified value is: 1.10 (peak 3), 1.24 (peak 4), 1.41 (peak 5), 2.00 (peak 6), 2.16 (peak 7).

[0216] Example 8: Characteristic chromatogram of decoction pieces

[0217] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filling agent (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as the mobile phase A, water as the mobile phase B, gradient elution according to the provisions in Table 1 above; detection wavelength 270 nm. Theoretical plate number should not be less than 5000 calculated according to the xanthine peak.

[0218] Reference solution preparation: Take 2 g of Pinelliae Praeparatae, put it in a conical flask with a plug, add 25 ml of 0.032% ammonia solution, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the reference solution of the control medicinal material. Take the xanthine reference substance, accurately weigh, add 0.032% ammonia solution to make a solution containing 10 μg per 1 ml, and obtain it. Take the guanine reference substance, accurately weigh, add 0.032% ammonia solution to make a solution containing 10 μg per 1 ml, and obtain it as the reference solution of the reference substance.

[0219] Test solution preparation: Take Pinelliae Praeparatae Decoction Pieces, accurately weigh about 2 g, put it in a conical flask with a plug, accurately add 25 ml of 0.032% ammonia solution, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 0.032% ammonia solution, shake well, filter, and take the filtrate.

[0220] Determination method: accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain it.

[0221] Figure 11 The control characteristic chromatogram of Pinelliae Praeparatae Decoction Pieces; from Figure 11 It can be seen that 7 characteristic peaks should appear in the test sample chromatogram, and should correspond to the retention time of the 7 characteristic peaks in the reference chromatogram of the control medicinal material, wherein peak 1 and peak 2 should correspond to the retention time of the corresponding reference substance peaks, respectively. The peak corresponding to the xanthine reference peak is the S peak, and the relative retention time of the other characteristic peaks to the S peak is calculated, which should be within ±10% of the specified value, and the specified value is: 1.10 (peak 3), 1.24 (peak 4), 1.41 (peak 5), 2.00 (peak 6), and 2.16 (peak 7).

[0222] Example 9 Characteristic chromatogram of standard decoction

[0223] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as the mobile phase A, water as the mobile phase B, gradient elution according to the provisions in Table 1 above; detection wavelength 270 nm. The theoretical plate number should not be less than 5000 according to the xanthine peak.

[0224] Reference solution preparation: 2g of pinellia ternate prepared with wheat bran was placed in a conical flask with a stopper, 25ml of 0.032% ammonia water was added, and ultrasonic treatment (power 600W, frequency 40kHz) was performed for 30 minutes, and then filtration was performed, and the filtrate was used as the reference solution of the control medicinal material.

[0225] Test sample solution preparation: an appropriate amount of standard decoction of pinellia ternate prepared with wheat bran was taken, about 2g was accurately weighed, placed in a conical flask with a stopper, 25ml of 0.032% ammonia water was accurately added, ultrasonic treatment (power 600W, frequency 40kHz) was performed for 30 minutes, and then the weight was re-weighed, the lost weight was made up with 0.032% ammonia water, and then shaking was performed, filtration was performed, and the filtrate was obtained.

[0226] Determination method: 10ul of the reference solution and the test sample solution were accurately taken and injected into a liquid chromatograph, and determination was performed, and then the characteristic chromatogram of pinellia ternate prepared with wheat bran was obtained.

[0227] Figure 12 The characteristic chromatogram of the standard decoction of pinellia ternate prepared with wheat bran is provided. Figure 12 It can be seen that 7 characteristic peaks should be present in the test sample chromatogram, and should correspond to the retention time of the 7 characteristic peaks in the reference chromatogram of the control medicinal material, wherein peak 1 and peak 2 should correspond to the retention time of the corresponding reference peaks, respectively. The peak corresponding to the xanthine reference peak is the S peak, and the relative retention time of the other characteristic peaks to the S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 1.10 (peak 3), 1.24 (peak 4), 1.41 (peak 5), 2.00 (peak 6), and 2.16 (peak 7).

[0228] From the above examples, it can be seen that the present application provides a construction method of a characteristic chromatogram of pinellia ternate prepared with wheat bran, which comprises the following steps: A) mixing a test sample raw material and an ammonia water solution, performing ultrasonic treatment, filtering, and obtaining a to-be-tested liquid; B) determining the to-be-tested liquid by using a high performance liquid chromatography method, and obtaining a HPLC characteristic chromatogram of pinellia ternate prepared with wheat bran; and the chromatography conditions of the high performance liquid chromatography method are that a C18 column is used as the chromatography column, acetonitrile is used as the mobile phase A, water is used as the mobile phase B, and gradient elution is performed. The construction method provided by the present application has the characteristics of comprehensive detection and simple operation, can effectively identify pinellia ternate prepared with wheat bran formula granules, and can ensure that the quality of the pinellia ternate prepared with wheat bran formula granules is uniform and stable. The method is also suitable for pinellia ternate medicinal materials, pinellia ternate prepared with wheat bran decoction pieces, standard decoctions, and formula granules.

[0229] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for constructing a characteristic map of stir-fried Pinellia ternata. Includes the following steps: A) Mix the test sample raw material and ammonia solution, sonicate, filter, and obtain the test solution; B) The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of stir-fried Pinellia ternata. The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is water, and gradient elution is used. The gradient elution procedure is as follows: 0~15min, mobile phase A: 1~4%, mobile phase B: 99~96%; 15-25 min, mobile phase A: 4-5%, mobile phase B: 96-95%; 25-35 min, mobile phase A: 5-10%, mobile phase B: 95-90%; 35-40 min, mobile phase A: 10%, mobile phase B: 90%.

2. The construction method according to claim 1, characterized in that, This also includes the preparation of the reference solution: Mix Pinellia ternata reference material with 0.030~0.035% ammonia solution, sonicate, filter, and obtain reference material solution; Xanthine and guanine reference standards were respectively mixed with 0.030-0.035% ammonia solution to prepare a 10 μg / mL reference solution.

3. The construction method according to claim 1, characterized in that, The volume concentration of the ammonia solution is 0.030~0.035%; The mass ratio of the test sample raw material to the volume ratio of the ammonia solution is (1.8~2.3) g: 25 mL; The ultrasonic treatment power is 550~650W, the ultrasonic treatment frequency is 35~45kHz, and the ultrasonic treatment time is 25~35min.

4. The construction method according to claim 1, characterized in that, The specifications of the chromatographic column are as follows: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm, and column temperature 25~35℃. Detection wavelength: 260~280nm; The flow rate of the mobile phase is 0.8~1.2 mL / min; The theoretical plate number, calculated based on the xanthine peak, should be no less than 5000.

5. The construction method according to claim 1, characterized in that, The similarity of stir-fried Pinellia ternata with wheat bran was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. A standard characteristic HPLC chromatogram of stir-fried Pinellia ternata with wheat bran, consisting of seven peaks, was obtained. Peaks 1 and 2 should correspond to the retention times of their respective reference peaks. The peak corresponding to the xanthine reference peak is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S were calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 1.10 (peak 3), 1.24 (peak 4), 1.41 (peak 5), 2.00 (peak 6), and 2.16 (peak 7).

6. A method for determining the xanthine content in stir-fried Pinellia ternata, comprising the following steps: S1. Mix the stir-fried Pinellia ternata sample with an ammonia solution, sonicate, filter, and obtain the test solution; S2. Mix xanthine reference standard with 0.030~0.035% ammonia solution to obtain a 10 μg / mL reference standard solution; S3. Using high performance liquid chromatography (HPLC), chromatograms of the chemical components in the test solution described in step S1 and the reference solution described in step S2 were obtained under the same detection conditions. The specific chromatographic parameters are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is water, and gradient elution is used. S4. Based on the concentration of the reference standard, the peak area of ​​the reference standard solution in the chromatogram, and the peak area of ​​the component in the stir-fried Pinellia ternata corresponding to the reference standard solution in the chromatogram, and based on the detection conditions in step S3, calculate the xanthine content in the stir-fried Pinellia ternata using the external standard method.

7. The determination method according to claim 6, characterized in that, The injection concentration of xanthine solution ranged from 1.09075 to 218.15 μg / ml. The regression equation for xanthine was Y = 36.4178 X + 7.2240, R0 2 =0.9999.

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