Detection method for specific chromatogram of ginger and licorice root decoction

Abstract

The invention belongs to the field of traditional Chinese medicine detection, and particularly relates to a method for detecting a specific chromatogram of ginger and liquorice decoction. The detection method specifically comprises the following steps: a, preparing a reference substance solution; b, preparing a test solution; and c, respectively sucking the reference solution and the test solution, and injecting into a liquid chromatograph for gradient elution. According to the method, the specific sample solvent is matched with the chromatographic conditions, so that the chromatographic peak separation degree in the characteristic chromatogram is high, the number of calibrated common peaks is large, the characteristic property is strong, the chemical quality general picture of the classic famous prescription ginger and liquorice decoction can be accurately, comprehensively and stably reflected, and the quality control of the traditional preparation and the modern preparation of the ginger and liquorice decoction is realized; the popularization and application values are realized.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection, specifically relating to a method for detecting the characteristic chromatogram of ginger and licorice decoction. Background Technology

[0002] Ginger and Licorice Decoction originates from Sun Simiao's "Essential Prescriptions Worth a Thousand Gold Pieces" from the Tang Dynasty. The original text states that it "treats pulmonary atrophy, cough with incessant drooling, dry throat, and thirst." In modern times, it is used to treat pulmonary fibrosis or atelectasis caused by the later stages of debilitating diseases such as lung cancer, emphysema, and bronchitis.

[0003] The key technical challenge in the research and development of modern formulations of Shengjiang Gancao Tang (Ginger and Licorice Decoction) lies in the equivalence evaluation system for ancient and modern formulations. Because the quality of the final product is affected by multiple stages in the production process of modern formulations based on classic formulas—including raw materials, processing of raw materials, extraction techniques, and formulation processes—qualitative identification of simple indicator components is insufficient to reflect their quality. To ensure the safety, efficacy, stability, and controllability of classic formulas and their formulations, a more stable and comprehensive chemical spectrum reflecting their composition is needed as an effective means of controlling and evaluating ancient and modern formulations of classic formulas. However, currently, there is no method that can simultaneously conduct quality control evaluation of traditional Shengjiang Gancao Tang formulations and achieve comprehensive quality control of modern formulations of Shengjiang Gancao Tang. Summary of the Invention

[0004] To address the above problems, this invention provides a method for detecting the characteristic chromatogram of ginger and licorice decoction, which employs HPLC detection and specifically includes the following steps: a. Preparation of reference solution: Dissolve the reference standard in methanol to prepare the reference solution; b. Preparation of the test solution: Take the sample to be tested, extract with methanol solution, filter, and take the filtrate to obtain the test solution; c. Inject the reference solution and the test solution separately into the liquid chromatograph. Chromatographic conditions: Column: octadecylsilane-bonded silica gel; Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; Flow rate: 0.9 mL / min; Column temperature: 25℃; Gradient elution program as follows: The reference standard includes glycyrrhizin; The sample to be tested was a preparation of ginger and licorice decoction; The ginger and licorice decoction is composed of ginger, licorice, ginseng and jujube in a mass ratio of 65~70:50~58:40~45:30~38.

[0005] Furthermore, the reference standards also include isoglycyrrhizin, glycyrrhizin, apiosyl isoglycyrrhizin, glycyrrhizic acid monoammonium salt and / or 6-gingerol.

[0006] Furthermore, the preparations of the ginger and licorice decoction include ginger and licorice decoction decoction, ginger and licorice decoction granules, ginger and licorice decoction powder, ginger and licorice decoction pills, ginger and licorice decoction tablets, and ginger and licorice decoction material reference. Furthermore, the ginger and licorice decoction is composed of ginger, licorice, ginseng, and jujube in a mass ratio of 69:55.2:41.4:36.

[0007] Further, the concentration of the reference solution in step a) is 100-150 μg of reference standard per 1 ml.

[0008] Further, in step b), the mass-to-volume ratio of the sample to be tested to the methanol solution is 0.1~1g:10mL; the extraction is ultrasonic extraction with a power of 600w, a frequency of 40kHz, and a time of 30min.

[0009] Furthermore, the concentration of the methanol solution is 70%.

[0010] Further, in step c), the chromatographic column is a ZORBAX SB-Aq 4.6×150mm 3.5μm; the wavelength of the chromatographic conditions is 230nm, the injection volume is 5μl, and the theoretical plate number calculated based on glycyrrhizin should not be less than 5000.

[0011] Furthermore, the characteristic spectrum of the ginger and licorice decoction should exhibit 12 characteristic peaks. The peak corresponding to the glycyrrhizin reference is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: peak 1: 0.346, peak 2: 0.571, peak 3: 0.781, peak 4: 0.979, peak 6: 1.118, peak 7: 1.158, peak 8: 1.187, peak 9: 1.312, peak 10: 1.455, peak 11: 1.574, peak 12: 1.617. Peak 7 is apigenin isoglycyrrhizin, peak 8 isoglycyrrhizin, peak 9 is glycyrrhizin, peak 11 is glycyrrhizic acid monoammonium salt, and peak 12 is 6-gingerol.

[0012] The detection method of the characteristic chromatogram of ginger and licorice decoction of the present invention, through the combination of specific sample solvents and chromatographic conditions, can achieve high resolution of chromatographic peaks in the characteristic chromatogram, identify a large number of common peaks, and have strong characteristicity. It can accurately, comprehensively and stably reflect the chemical quality overview of the classic formula ginger and licorice decoction, realize the quality control of traditional and modern preparations of ginger and licorice decoction, and has the value of promotion and application.

[0013] The ginger and licorice decoction described in this invention is a traditional Chinese medicine formula, which comes from Sun Simiao's "Essential Prescriptions Worth a Thousand Gold Pieces" in the Tang Dynasty. Its formula consists of five liang of ginger, three liang of ginseng, four liang of licorice, and fifteen jujubes. The above four ingredients are boiled in seven liters of water until three liters remain, and then divided into three warm doses.

[0014] The ginger and licorice decoction material standard used in this invention is based on the preparation method of ginger and licorice decoction recorded in the ancient medical book "Qianjin Yaofang" (Essential Prescriptions Worth a Thousand Gold Pieces). Except for the molding process, the other preparation methods are basically consistent with those recorded in "Qianjin Yaofang". The specific preparation method is as follows: Weigh the raw materials according to the formula of ginger and licorice decoction; place the herbs and water in a clay pot, soak for 30 minutes, boil over high heat until the liquid boils, then simmer over low heat until 600 ml remains, filter while hot, remove the residue, extract the liquid, freeze dry, and pulverize to obtain the physical dry extract powder corresponding to the material basis of ginger and licorice decoction.

[0015] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0016] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0017] Figure 1 Mobile phase selection diagram; Figure 2 Gradient selection graph; Figure 3 Chromatogram of the material reference sample for ginger and licorice decoction; Figure 4 Investigation of different column temperatures on the material reference sample of ginger and licorice decoction; Figure 5 Flow velocity study; Figure 6 Injection volume investigation; Figure 7 Delayedness investigation of the reference sample of ginger and licorice decoction; Figure 8 Extraction solvent investigation; Figure 9 Extraction time consideration; Figure 10 Investigation of solvent addition amount; Figure 11 Comparison of characteristic spectra of negative reference samples of ginger and licorice decoction lacking single herbs; Figure 12 Chromatographic peak identification; Figure 13 Characteristic spectrum verification diagrams of six batches of ginger and licorice decoction benchmark samples; Figure 14Characteristic spectrum of the reference sample of ginger and licorice decoction (peak 5 (S): glycyrrhizin; peak 7: apigenin isoglycyrrhizin; peak 8: isoglycyrrhizin; peak 9: glycyrrhizin; peak 11: glycyrrhizic acid monoammonium salt peak; peak 12: 6-gingerol) Detailed Implementation Example 1: Characteristic spectrum detection of ginger and licorice decoction 1) Preparation of the reference solution: Accurately weigh the reference standards of glycyrrhizin, isoglycyrrhizin, glycyrrhizin, apigenin isoglycyrrhizin, glycyrrhizic acid monoammonium salt, and 6-gingerol, and prepare a mixed solution containing 100-150 μg per 1 mL with methanol as the reference solution. 2) Preparation of the test solution: Take 0.2g of ginger and licorice decoction, place it in a stoppered conical flask, add 10ml of 70% methanol, seal tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the decoction. 3) Determination of characteristic spectra Accurately inject 5 μl of the reference solution and the test solution into the liquid chromatograph to obtain chromatograms; The chromatographic conditions are as follows: Chromatographic column: ZORBAX SB-Aq (4.6×150mm, 3.5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; column temperature: 25℃; flow rate: 0.9ml / min; detection wavelength: 230nm; theoretical plate number calculated based on glycyrrhizin peak should not be less than 5000.

[0018] 4) Analyze the characteristic map The characteristic spectrum of ginger and licorice decoction shows 12 characteristic peaks: peak 5 is glycyrrhizin, peak 7 is apigenin isoglycyrrhizin, peak 8 isoglycyrrhizin, peak 9 is glycyrrhizin, peak 11 is glycyrrhizic acid monoammonium salt, and peak 12 is 6-gingerol. The peak corresponding to the glycyrrhizin reference is the S peak. The relative retention times of the remaining characteristic peaks and the S peak are calculated. The relative retention times should be within ±10% of the specified values, which are: peak 1: 0.346, peak 2: 0.571, peak 3: 0.781, peak 4: 0.979, peak 6: 1.118, and peak 10: 1.455.

[0019] Example 2: Characteristic spectrum detection of ginger and licorice decoction 1) Preparation of the reference solution: Accurately weigh glycyrrhizin reference standard, add methanol to prepare a solution containing 100-150 μg per 1 mL, and use it as the reference solution; 2) Preparation of the test solution: Take 0.2g of ginger and licorice decoction, place it in a stoppered conical flask, add 10ml of 70% methanol, seal tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the decoction. 3) Determination of characteristic spectra Accurately inject 5 μl of the reference solution and the test solution into the liquid chromatograph to obtain chromatograms; The chromatographic conditions are as follows: Chromatographic column: ZORBAX SB-Aq (4.6×150mm, 3.5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; column temperature: 25℃; flow rate: 0.9ml / min; detection wavelength: 230nm; theoretical plate number calculated based on glycyrrhizin peak should not be less than 5000.

[0020] 4) Analyze the characteristic map The characteristic spectrum of ginger and licorice decoction shows 12 characteristic peaks. The peak corresponding to the glycyrrhizin reference is the S peak. The relative retention times of the remaining characteristic peaks and the S peak are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: peak 1: 0.346, peak 2: 0.571, peak 3: 0.781, peak 4: 0.979, peak 6: 1.118, peak 7: 1.158, peak 8: 1.187, peak 9: 1.312, peak 10: 1.455, peak 11: 1.574, peak 12: 1.617.

[0021] The following experimental examples illustrate the beneficial effects of the present invention.

[0022] Experimental Example 1: Characteristic Spectrum Study of Ginger and Licorice Decoction I. Experimental Instruments and Materials High-performance liquid chromatograph (HPLC): Agilent 1260 HPLC system; Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instruments Ltd.); Ultrapure water system: Cellular 1810A (Shanghai Moller Scientific Instruments Co., Ltd.); Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.); Chromatographic column: ZORBAX SB-Aq4.6×150mm 3.5-Micron; Acetonitrile and phosphoric acid were chromatographic grade, water was ultrapure water, and all other reagents were analytical grade. Glycyrrhizin reference standard (China National Institutes for Food and Drug Control, batch number: 111610-201106, content calculated as 93.7%); isoglycyrrhizin reference standard (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq20022803, content calculated as 100%); glycyrrhizin reference standard (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 070002-202108, content calculated as 99.96%); apigenin isoglycyrrhizin reference standard (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq20100907, content calculated as 100%); 6-gingerol reference standard (Chengdu Desite Biotechnology Co., Ltd., batch number: 111833-201806, content calculated as 99.9%); glycyrrhizic acid monoammonium salt reference standard (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDG000802, content calculated as 98.95%).

[0023] II. Preparation of the prescription for Ginger and Licorice Decoction and its material reference samples 1) Processing: Remove impurities from ginger, wash it, and cut it into thick slices before use; remove impurities from licorice, wash it, soak it thoroughly, cut it into thick slices, and dry it at a low temperature of 60℃; soak ginseng thoroughly, cut it into thin slices, and dry it at a low temperature of 60℃; remove impurities from jujubes, wash them, sun-dry them, and remove the pits before use.

[0024] 2) Weighing: Weigh 69.0g of ginger, 55.2g of licorice, 41.4g of ginseng, and 36.0g of jujube according to the prescription ratio, and add 1400ml of water.

[0025] 3) Decoction: Place the herbs and water in a clay pot, soak for 30 minutes, bring to a boil over high heat, then reduce to a simmer until the liquid is reduced to 600ml. Filter while hot, remove the dregs, and extract the liquid.

[0026] 4) Drying: Prepare the ginger and licorice decoction material standard according to the freeze-drying parameters below. The freeze-drying program of the vacuum freeze dryer is as follows: ① Pre-freezing: Temperature: -50℃, holding temperature: 5h; Pressure: atmospheric pressure; ② Sublimation drying: Temperature: -40℃, holding temperature: 2h; Pressure: 0.0pa; Temperature: -30℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: -20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: -10℃, temperature maintenance: 3h; Pressure: 0.0pa; Temperature: 0℃, temperature maintenance: 3h; Pressure: 0.0pa; ③ Drying: Temperature: 10℃, holding temperature: 3h; Pressure: 0.0pa; Temperature: 20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 30℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 40℃, temperature maintenance: 2h; pressure: 0.0pa.

[0027] The following reference samples of ginger and licorice decoction were prepared using the above method: SJGCT-01, SJGCT-02, SJGCT-03, SJGCT-04, SJGCT-05, and SJGCT-6.

[0028] III. Fingerprint Spectrum Exploration 1. Investigation of the mobile phase The raw ginger and licorice decoction reference sample (SJGCT-01) was extracted with 70% methanol by ultrasonication. The filtrate was collected, and the chromatogram was determined as follows: Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 3.5 μm); the mobile phases were acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% formic acid, acetonitrile-0.1% glacial acetic acid, and methanol-0.1% phosphoric acid, with a flow rate of 1.0 mL / min; the column temperature was 25 °C; the detection wavelength was 230 nm; and the gradient elution program was as follows: Preparation of the test solution: Take 0.2g of the ginger and licorice decoction material standard sample, place it in a stoppered conical flask, add 10ml of 70% methanol, stopper tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0029] Assay: Accurately pipette 5 μl of the test solution and inject it into the liquid chromatograph for determination. The results are shown in the figure. Figure 1 .from Figure 1It is evident that, compared to acetonitrile-0.1% phosphoric acid, the resolution of some chromatographic peak pairs was insufficient (Rs < 1.0) during gradient elution with acetonitrile-0.1% formic acid, acetonitrile-0.1% glacial acetic acid, and methanol-0.1% phosphoric acid as mobile phases over 60 min. This resulted in uncertainty in the integration and identification of some peaks, affecting the identification of common characteristic peaks. The mobile phase of acetonitrile-0.1% phosphoric acid showed better peak separation and a more stable baseline; therefore, acetonitrile-0.1% phosphoric acid was selected as the mobile phase for subsequent chromatographic condition investigation.

[0030] 2. Investigation of mobile phase gradient Based on the determined mobile phase, adjust the gradient elution program in the chromatographic conditions, such as gradient 1 to 4.

[0031] Gradient 1 Gradient 2 Gradient 3 Gradient 4 (feature) Chromatograms under different gradient elution programs are shown below. Figure 2 .from Figure 2 It is evident that gradient elution with gradient 1 results in an unstable baseline, exhibiting a step-like change; gradient elution with gradients 2-3 leads to insufficient resolution (Rs < 1.0) for many peak pairs, resulting in uncertainty in the integration and identification of some peaks, and even after 60 minutes of elution, not all peaks can be eluted. Gradient 4 (characteristic) produces chromatograms with good peak shapes and separation; therefore, gradient 4 was chosen for further investigation.

[0032] 3. Determining the detection wavelength point Based on the determined mobile phase and elution program, the test solution was scanned across the entire wavelength range using a diode array detector, and chromatograms of the test solution were extracted at wavelengths of 210 nm, 220 nm, 230 nm, 260 nm, and 300 nm. See [link to chromatogram]. Figure 3 .from Figure 3It is evident that some substances in the test solution exhibit weak UV absorption in the 260–300 nm wavelength range. This results in some components failing to form significant characteristic peaks in the chromatogram, leading to a relatively small number of detected peaks. In the 210–220 nm wavelength range, other peaks similar to peak 10 in the chromatogram exhibit strong UV absorption. This strong absorption significantly reduces the resolution between peak 10 and other peaks, thus affecting the accuracy of peak identification. A better peak shape is observed at a detection wavelength of 230 nm; therefore, the detection wavelength was determined to be 230 nm.

[0033] 4. Column temperature investigation Based on the determined mobile phase, elution program, and wavelength, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃. Figure 4 As shown.

[0034] The column temperature investigation results showed that when the column temperature was 30℃ and 35℃, the resolution of many chromatographic peak pairs was insufficient (Rs<1.0), which led to uncertainty in the integration and identification of some peaks, affecting the identification of common characteristic peaks. When the column temperature was 25℃, the chromatographic peaks in the chromatogram were symmetrical and the resolution was better. Therefore, 25℃ was finally determined as the column temperature for the characteristic chromatographic method of ginger and licorice decoction.

[0035] 5. Flow velocity assessment Based on the established mobile phase, elution program, wavelength, and column temperature, the effects were investigated at flow rates of 0.9 mL / min, 1 mL / min, and 1.1 mL / min. Figure 5 As shown.

[0036] The results showed that the chromatogram peak shape was good and the resolution was moderate at a flow rate of 0.9 ml / min. Therefore, the flow rate was determined to be 0.9 ml / min.

[0037] 6. Sample volume analysis Based on the established mobile phase, elution program, wavelength, column temperature, and flow rate, experiments were conducted with injection volumes of 5 μl, 10 μl, and 15 μl. Figure 6 As shown.

[0038] The results showed that the injection volume of 5–15 μl had no significant effect on the separation of the target chromatographic peaks, but the peak shape and resolution were better when the injection volume was 5 μl. Therefore, the injection volume was determined to be 5 μl.

[0039] 7. Delayed testing Based on the established chromatographic conditions, the chromatogram acquisition time was extended to 60 minutes. For example... Figure 7 As shown: the sample showed almost no chromatographic peaks after 60 minutes, so the sample detection time was set at 60 minutes.

[0040] 8. Investigation of extraction solvent Take 0.2g of the ginger and licorice decoction reference sample (SJGCT-01), place it in a stoppered conical flask, add 10ml each of water, 30% methanol, and 70% methanol, seal tightly, sonicate (600W, 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate. Analyze according to the determined chromatographic conditions. The results are as follows: Figure 8 As shown.

[0041] The results showed that 70% methanol was used as the extraction solvent, resulting in a large amount of chromatographic peak information. Therefore, 70% methanol was determined to be the extraction solvent for the test sample.

[0042] 9. Examination of extraction time Take 0.2g of the ginger and licorice decoction reference sample (SJGCT-01), place it in a stoppered conical flask, add 10ml of 70% methanol, seal tightly, and sonicate (power 600W, frequency 40kHz). The extraction time of the sample was investigated at 20 minutes, 30 minutes, and 40 minutes. After cooling, shake well, filter, and collect the filtrate. Detect the chromatographic solution according to the determined chromatographic conditions. The results are as follows: Figure 9 As shown.

[0043] The results showed that extraction was complete at an extraction time of 30 minutes. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0044] 10. Investigation of Solvent Addition Amount Take 0.2g of the ginger and licorice decoction reference sample (SJGCT-01), place it in a stoppered conical flask, add 10ml, 20ml, and 25ml of 70% methanol respectively, stopper tightly, weigh, sonicate (600W power, 40kHz frequency) for 30 minutes, shake well, filter, take the filtrate, and detect according to the determined chromatographic conditions. The results are shown in the figure. Figure 10 .

[0045] The results showed that the peak areas of the characteristic chromatograms were moderate when the solvent volume was 10 ml. Therefore, the solvent volume for the test sample was determined to be 10 ml.

[0046] 11. Determine the method for preparing the test sample. Take 0.2g of the raw ginger and licorice decoction material reference sample, accurately weigh it, place it in a stoppered conical flask, add 10mL of 70% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0047] 12. Methodological Examination 12.1 Chromatographic Peak Identification Preparation of test solution: Prepare the test solution of ginger and licorice decoction material reference sample according to the proposed test solution preparation method.

[0048] Preparation of reference solution: Accurately weigh the reference standards of glycyrrhizin, isoglycyrrhizin, glycyrrhizin, apigenin isoglycyrrhizin, glycyrrhizic acid monoammonium salt, and 6-gingerol, and add methanol to prepare a solution containing 100-150 μg per mL.

[0049] Preparation of negative control solution: Prepare negative ginger and licorice root standard sample lacking single herb according to the same preparation method as the ginger and licorice root standard sample, and then prepare the corresponding test solution according to the proposed test sample preparation method; A comparison of the characteristic peaks in the chromatograms of the ginger and licorice decoction reference sample and the characteristic peaks in the negative chromatogram of the ginger and licorice decoction reference sample lacking a single herb revealed that the characteristic peaks mainly originated from licorice, with a few originating from ginger. For example... Figure 11 As shown.

[0050] The characteristic peaks of the spectral spectrum of the ginger and licorice decoction reference sample were located. For example... Figure 12 As shown: Peak 5 is glycyrrhizin, Peak 7 is apigenin isoglycyrrhizin, Peak 8 isoglycyrrhizin, Peak 9 is glycyrrhizin, Peak 11 is glycyrrhizic acid monoammonium salt, and Peak 12 is 6-gingerol.

[0051] 12.2 Precision Test Take the test solution of the ginger and licorice decoction material reference sample (batch number: SJGCT-01), and inject it 6 times consecutively under the proposed chromatographic conditions, 5 μl each time. Calculate the retention time and peak area of ​​each characteristic peak. As shown in Tables 1 and 2.

[0052] Table 1 Precision test - retention time Table 2 Precision Study - Peak Area The results show that the retention time RSD of each characteristic peak is 0.01%-0.04%, and the peak area RSD is 0.15%-4.54%. The method has good precision.

[0053] 12.3 Repeatability Test Six portions of the ginger and licorice decoction material reference sample (batch number: SJGCT-01) were accurately weighed and prepared and measured according to the proposed experimental method. See Tables 3 and 4 for details.

[0054] Table 3 Repeatability Tests - Relative Retention Time Table 4 Repeatability Tests - Relative Peak Area The results show that the relative retention time RSD of each characteristic peak is 0.03%-0.17%, and the relative peak area RSD is 0.35%-3.49%. The method has good repeatability.

[0055] 12.4 Intermediate Precision Examination Based on the experimental conditions outlined above, two samples of the ginger and licorice decoction reference material (batch number: SJGCT-01) were precisely weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples for determination. The results are shown in Tables 5 and 6.

[0056] Table 5. Personnel and Time Assessment - Relative Retention Time Table 6. Personnel and Time Survey - Relative Peak Area Ratio The results show that the method has good stability when different people measure the same sample at different times.

[0057] 12.5 Stability Test Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 9h, 13h, and 24h, respectively. See Tables 7 and 8 for details.

[0058] Table 7 Stability Study - Retention Time Table 8 Stability Study - Peak Area The results showed that the corresponding characteristic peak retention time RSD was 0.05%–0.06%, the peak area RSD was 0.46%–5.6%, and the sample solution was stable within 24 hours.

[0059] The methodological results show that the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. Therefore, the 12 characteristic peaks from each test will be included in subsequent validation.

[0060] 12.6 Validation of Material Reference Samples from Different Batches of Ginger and Licorice Decoction The proposed method was used to determine the characteristic spectra of six batches of ginger and licorice decoction reference samples, and the relative retention time and relative peak area were calculated. Figure 13 As shown in Tables 9 and 10.

[0061] Table 9. Relative retention times of six batches of ginger and licorice decoction reference samples Table 10. Relative peak areas of six batches of ginger and licorice decoction reference samples Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak area, the 12 characteristic peaks showed good repeatability. When peak 5 was considered the S peak, the relative retention time RSD of the characteristic peaks in all six batches of ginger and licorice decoction reference samples was less than 1%. The results indicate that the 12 characteristic peaks are common characteristic peaks of ginger and licorice decoction.

[0062] 12.7 Determination of the specified limits for the relative retention times of each characteristic peak in the characteristic spectrum Table 11 summarizes the methodological investigation items and validation results: The 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System was used to synthesize the characteristic chromatograms of six batches of Shengjiang Gancao Tang (Ginger and Licorice Decoction) reference samples, establishing a comparative chromatogram of the Shengjiang Gancao Tang reference samples. For example... Figure 14 As shown.

[0063] Table 1. Summary of RSD% of Methodological Results – Relative Retention Time From Table 11 and Figure 14 The results show that the characteristic spectrum of ginger and licorice decoction presents 12 characteristic peaks, among which the peak corresponding to the glycyrrhizin reference is the S peak. The relative retention times of the remaining characteristic peaks are within the range of ±10% of the specified values, which are 0.346 (peak 1), 0.571 (peak 2), 0.781 (peak 3), 0.979 (peak 4), 1.118 (peak 6), 1.158 (peak 7), 1.187 (peak 8), 1.312 (peak 9), 1.455 (peak 10), 1.574 (peak 11), and 1.617 (peak 12).

[0064] In summary, this invention, through the combination of specific sample solvents and chromatographic conditions, enables high peak resolution, a large number of identified common peaks, and strong characteristic in the characteristic chromatogram. It can accurately, comprehensively, and stably reflect the chemical quality profile of the classic formula, Shengjiang Gancao Tang, and achieve quality control of both traditional and modern preparations of Shengjiang Gancao Tang, thus possessing value for widespread application.

Claims

1. A method for detecting the characteristic chromatogram of ginger and licorice decoction, characterized in that: It uses HPLC detection, and specifically includes the following steps: a. Preparation of reference solution: Dissolve the reference standard in methanol to prepare the reference solution; b. Preparation of the test solution: Take the sample to be tested, extract with methanol solution, filter, and take the filtrate to obtain the test solution; c. Inject the reference solution and the test solution separately into the liquid chromatograph. Chromatographic conditions: Column: octadecylsilane-bonded silica gel; Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; Flow rate: 0.9 mL / min; Column temperature: 25℃; Gradient elution program as follows: The reference standard includes glycyrrhizin; The sample to be tested was a preparation of ginger and licorice decoction; The ginger and licorice decoction is composed of ginger, licorice, ginseng and jujube in a mass ratio of 65~70:50~58:40~45:30~38.

2. The detection method according to claim 1, characterized in that: The reference standards also include isoglycyrrhizin, glycyrrhizin, apiosyl isoglycyrrhizin, glycyrrhizic acid monoammonium salt and / or 6-gingerol.

3. The detection method according to claim 1, characterized in that: The preparations of the ginger and licorice decoction include ginger and licorice decoction decoction, ginger and licorice decoction granules, ginger and licorice decoction powder, ginger and licorice decoction pills, ginger and licorice decoction tablets, and ginger and licorice decoction material reference.

4. The detection method according to claim 1 or 3, characterized in that: The ginger and licorice decoction is composed of ginger, licorice, ginseng and jujube in a mass ratio of 69:55.2:41.4:

36.

5. The detection method according to claim 1, characterized in that: The concentration of the reference solution in step a) is 100-150 μg of reference standard per 1 ml.

6. The detection method according to claim 1, characterized in that: In step b), the mass-to-volume ratio of the sample to the methanol solution is 0.1~1g:10mL; the extraction is ultrasonic extraction with a power of 600w, a frequency of 40kHz, and a time of 30min.

7. The detection method according to claim 1 or 6, characterized in that: The concentration of the methanol solution is 70%.

8. The detection method according to claim 1, characterized in that: Step c) The chromatographic column is a ZORBAX SB-Aq 4.6×150mm 3.5μm; the wavelength of the chromatographic conditions is 230nm, the injection volume is 5μl, and the theoretical plate number calculated based on glycyrrhizin should not be less than 5000.

9. The detection method according to claim 1, characterized in that: The characteristic chromatogram of the ginger and licorice decoction should show 12 characteristic peaks. The peak corresponding to the glycyrrhizin reference is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: peak 1: 0.346, peak 2: 0.571, peak 3: 0.781, peak 4: 0.979, peak 6: 1.118, peak 7: 1.158, peak 8: 1.187, peak 9: 1.312, peak 10: 1.455, peak 11: 1.574, peak 12: 1.

617.

10. The detection method according to claim 9, characterized in that: Peak 7 is apigenin isoglycyrrhizin, peak 8 isoglycyrrhizin, peak 9 is glycyrrhizin, peak 11 is glycyrrhizic acid monoammonium salt, and peak 12 is 6-gingerol.

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