An analytical detection method for Lingguizhugan decoction

Abstract

The present invention provides an analytical detection method for Ling Gui Zhu Gan Decoction. The method uses ultra-high performance liquid chromatography (UPLC) for detection and can be used for fingerprint detection of 14 components in Ling Gui Zhu Gan Decoction, including apiosyl liquiritin, liquiritin, apiosyl isoliquiritin, isoliquiritin, glycyrrhizin, cinnamic acid, cinnamaldehyde, o-methoxycinnamic acid, glycyrrhizic acid, atractylodes lactone III, atractylodes lactone II, pachymosin B, policocoric acid C and dehydropachymosic acid. The method can also be used for content control of liquiritin, cinnamic acid, cinnamaldehyde and glycyrrhizic acid. The method has strong applicability and is of great significance for comprehensive and objective evaluation of Ling Gui Zhu Gan Decoction.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular, relates to an analysis and detection method for Ling Gui Zhu Gan Tang. Background Art

[0002] Ling Gui Zhu Gan Tang is a classic ancient prescription known for its efficacy in warming the yang, transforming dampness, strengthening the spleen, and eliminating dampness. Because traditional Chinese medicines (TCMs) are complex in composition, studying their fingerprints and content can comprehensively reveal the types and quantities of their chemical components, enabling a holistic description and evaluation of drug quality.

[0003] Patent CN201910542587.1 discloses a quality detection method for Ling Gui Zhu Gan Tang, including the preparation of Ling Gui Zhu Gan Tang test solution, the preparation of mixed reference solution, the establishment of linear regression equation and content determination. The content determination adopts HPLC method, chromatographic column: YMC-Pack ODS-A, specification 250mm×4.6mm, 5μm, flow rate 1mL / min, column temperature 30℃, detection wavelength: 237nm for liquiritin, ammonium glycyrrhizate and atractylodes lactone I, and 290nm for cinnamic acid; injection volume: 10μL; mobile phase A is acetonitrile, phase B is 0.1% volume concentration phosphoric acid aqueous solution, and gradient elution is used to accurately analyze the active ingredients liquiritin, cinnamic acid, ammonium glycyrrhizate and atractylodes lactone I in Ling Gui Zhu Gan Tang. The document "Quantitative Analysis of Eleven Bioactive Constituents of a Traditional Herbal Medicine, Yeonggyechulgam-tang using, Liquid Chromatographv-Electrosprav Ionization Tandem Mass Spectrometry" by Chang-Seob Seo et al., "Natural Product Sciences" discloses a method for quantitatively analyzing 11 active ingredients in the Chinese herbal medicine Ling Gui Shu Gan Tang using liquid chromatography tandem mass spectrometry: the chromatographic separation conditions are Acquity UPLC BEH A C column (2.1×100 mm, 1.7 μm, Waters, Milford, MA, USA) was used as the mobile phase, consisting of 0.1% formic acid in water and acetonitrile. The column temperature was 45°C, the flow rate was 0.3 mL / min, and the injection volume was 2.0 μL. Gradient elution was used to detect apigenin (1), liquiritin (2), liquiritigenin (3), coumarin (4), cinnamic acid (5), cinnamaldehyde (6), ammonium glycyrrhizate (7), atractylodes lactone III (8), atractylodes lactone II (9), atractylodes lactone I (10), and pachymic acid (11). This method does not disclose the detection wavelength, and the characteristic peaks of Poria cocos are poorly characterized. In addition, the detection and quantification limits for compounds 1-11 were 0.06-4.73 ng / mL and 0.17-14.20 ng / mL, respectively, making it unsuitable for the detection and separation of higher concentrations of substances. Moreover, the study found that the dissolution rate of the characteristic components of Poria in Ling Gui Zhu Gan Decoction was extremely low. In order to achieve the characteristic peaks of the four medicinal materials Poria, cinnamon twig, Atractylodes macrocephala and licorice in the sample fingerprint, it is necessary to study the analytical detection method.

[0004] In summary, there are few analytical detection methods for Ling Gui Zhu Gan Tang in the existing technology, and the detection substances are mainly concentrated in the detection of glycyrrhizin, cinnamic acid, ammonium glycyrrhizate and atractylodes lactone. There are few methods for the joint detection of the characteristic peaks of the four medicinal materials Poria cocos, cinnamon twig, Atractylodes macrocephala and licorice. Summary of the Invention

[0005] The present invention aims to provide an analytical detection method for Ling Gui Zhu Gan Decoction. The method adopts ultra-high performance liquid chromatography (UPLC) for detection and can be used for fingerprint detection of apiosyl glycyrrhizin, glycyrrhizin, apiosyl isoliquiritin, isoliquiritin, cinnamic acid, cinnamaldehyde, o-methoxycinnamic acid, glycyrrhizic acid, atractylodes lactone III, atractylodes lactone II, tuckahoe lactone B, dehydrotrimoxanic acid, poliporic acid C, dehydrotrimoxanic acid and dehydrotuckahoe acid in Ling Gui Zhu Gan Decoction. The method can also be used for content control of glycyrrhizin, cinnamic acid, cinnamaldehyde and glycyrrhizic acid. The method has strong applicability and is of great significance for comprehensive and objective evaluation of Ling Gui Zhu Gan Decoction.

[0006] In order to achieve the above object, the technical solution adopted in the present invention is:

[0007] A method for analyzing and detecting Linggui Zhugan Decoction is provided, employing ultra-high performance liquid chromatography for detection. The chromatographic conditions are as follows: using an ACQUITY UPLC BEH C18, CORTECS UPLC T3, or Shim-Pack GIST C18 column, acetonitrile as mobile phase B1, 0.05% to 0.15% aqueous phosphoric acid as mobile phase A1, a column temperature of 30° C., a flow rate of 0.2 ml per minute, and gradient elution according to the following procedure:

[0008] Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> 0 82 18 39 43 57 5 82 18 51 42 58 10 76 24 53 37.5 62.5 20 75 25 61 20 80 23 62 38 61.1 82 18 31 58 42 66 82 18 .

[0009] Preferably, the analytical detection method is used to detect one or more fingerprints of apiosyl liquiritin, liquiritin, apiosyl isoliquiritin, isoliquiritin, glycyrrhizin, cinnamic acid, cinnamaldehyde, o-methoxycinnamic acid, glycyrrhizic acid, atractylodes lactone III, atractylodes lactone II, poria acid B, poria acid C, and dehydropachymic acid in Lingguizhugan Decoction.

[0010] Preferably, the detection wavelength of the analytical detection method is 235 nm, 266 nm, 242 nm or 237 nm.

[0011] A method for analyzing and detecting Linggui Zhugan Decoction specifically comprises the following steps:

[0012] (1) Preparation of reference solution: Take an appropriate amount of reference substance, weigh accurately, and add methanol to make a mixed solution as the reference solution;

[0013] (2) Preparation of test solution: Place the test sample in a stoppered conical flask, add the extraction solvent, seal the flask, ultrasonicate for 30 minutes, cool, shake well, centrifuge for 5 minutes, take the supernatant and concentrate it in vacuo at 50°C to near dryness, dissolve it in the extraction solvent in batches, filter, and take the filtrate as the test solution;

[0014] (3) Chromatographic conditions: ACQUITY UPLC BEH C18, CORTECS UPLC T3, or Shim-PackGIST C18 columns were used, acetonitrile or methanol was used as mobile phase B1, 0.05%, 0.10%, or 0.15% aqueous phosphoric acid solution was used as mobile phase A1, column temperature was 25-40°C, flow rate was 0.1-0.3 ml / min, detection wavelength was 235 nm, 266 nm, 242 nm, or 237 nm, and gradient elution was performed according to the following procedure:

[0015] Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> 0 82 18 39 43 57 5 82 18 51 42 58 10 76 24 53 37.5 62.5 20 75 25 61 20 80 23 62 38 61.1 82 18 31 58 42 66 82 18 ;

[0016] (4) Accurately pipette 3 μl of the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatography instrument, and measure them.

[0017] Preferably, the extraction solvent is one of water, ethanol and methanol.

[0018] A method for analyzing and detecting Linggui Zhugan Decoction is provided, using ultra-high performance liquid chromatography. The chromatographic conditions are as follows: octadecylsilane bonded silica gel as a filler, ACQUITY UPLC BEH C18, 100 mm × 2.1 mm, 1.7 μm, acetonitrile as mobile phase B1, 0.1% phosphoric acid as mobile phase A1, column temperature 25-40° C., flow rate 0.1-0.3 ml / min, and gradient elution according to the following procedure:

[0019] Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> 0 82 18 5 82 18 10 76 24 20 75 25 23 62 38 31 58 42 31.1 82 18 35 82 18 .

[0020] Preferably, the analytical detection method is used for the quantitative detection of glycyrrhizin, cinnamic acid, cinnamaldehyde and glycyrrhizic acid in Lingguizhugan Decoction.

[0021] A method for analyzing and detecting Linggui Zhugan Decoction specifically comprises the following steps:

[0022] (1) Preparation of reference solution: Take appropriate amounts of glycyrrhizin reference substance, cinnamic acid reference substance, cinnamaldehyde reference substance, and ammonium glycyrrhizate reference substance, accurately weigh them, and add methanol to prepare a solution containing 40 μg of glycyrrhizin, 6 μg of cinnamic acid, 5 μg of cinnamaldehyde, and 200 μg of ammonium glycyrrhizate per 1 ml, which is used as the reference solution;

[0023] (2) Preparation of test solution: 0.1 g to 0.5 g of the test sample was placed in a stoppered conical flask, and 25% to 100% methanol was added as the extraction solvent at a liquid-to-solid ratio of 50 to 500 times. The flask was sealed, and ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 5 to 50 minutes. The flask was cooled, shaken, and centrifuged (3500 rpm) for 5 minutes. The supernatant was filtered, and the filtrate was used as the test solution.

[0024] (4) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the filler (ACQUITY UPLC BEH C18, 100 mm × 2.1 mm, 1.7 μm), acetonitrile was used as the mobile phase B1, 0.1% phosphoric acid was used as the mobile phase A1, the column temperature was 25-40°C, the flow rate was 0.1-0.3 ml / min, the detection wavelength was 266 nm, and the gradient elution was performed according to the following program:

[0025] Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> 0 82 18 5 82 18 10 76 24 20 75 25 23 62 38 31 58 42 31.1 82 18 35 82 18

[0026] (4) Determination method: Accurately pipette 2 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography instrument, and determine.

[0027] The beneficial effects achieved by the technical solution of the present invention are: (1) providing an analytical detection method for Ling Gui Shu Gan Decoction, which can not only be used for fingerprint spectrum detection of 14 components in Ling Gui Shu Gan Decoction, including apiosyl glycyrrhizin, glycyrrhizin, apiosyl isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, cinnamic acid, cinnamaldehyde, o-methoxycinnamic acid, glycyrrhizic acid, atractylodes lactone III, atractylodes lactone II, poria acid B, poria acid C, and dehydroporia acid, but can also be used for quantitative detection of glycyrrhizin, cinnamic acid, cinnamaldehyde and glycyrrhizic acid, and has strong practicality and is suitable for promotion and application; (2) selecting appropriate detection conditions according to the characteristics of the active ingredients contained in Ling Gui Shu Gan Decoction, especially verifying and analyzing the fingerprint spectrum detection of the characteristic components of Poria, providing a more comprehensive, objective and accurate evaluation method for the quality analysis of Ling Gui Shu Gan Decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the sample profile of Example 2 with acetonitrile-0.1% phosphoric acid as the mobile phase;

[0029] Figure 2 This is the sample spectrum of Example 2 with methanol-0.1% phosphoric acid as the mobile phase;

[0030] Figure 3 This is an overlay of the 266 nm chromatograms of Example 3 using 0.1% phosphoric acid and 0.6% formic acid as the mobile phase;

[0031] Figure 4 This is an overlay of the 235 nm chromatograms of Example 3 using 0.1% phosphoric acid and 0.6% formic acid as the mobile phase;

[0032] Figure 5 The 266 nm chromatograms under different pH conditions in Example 4 are overlaid;

[0033] Figure 6 The chromatograms of the test products of different chromatographic columns in Example 5;

[0034] Figure 7 Example 6 ACQUITY 266nm spectrum of the test sample under BEH C18 column;

[0035] Figure 8 Example 6 ACQUITY 235nm spectrum of the test sample under BEH C18 column;

[0036] Figure 9 This is the 266nm spectrum of the test sample under the Shim-Pack GIST C18 chromatographic column in Example 6;

[0037] Figure 10 This is the 235nm spectrum of the test sample under the Shim-Pack GIST C18 chromatographic column in Example 6;

[0038] Figure 11 This is a comparison of the extraction solvents of different test samples in Example 7;

[0039] Figure 12 This is the 242 nm spectrum of the unconcentrated reference sample of Example 8;

[0040] Figure 13 This is the 242nm spectrum of the concentrated benchmark sample of Example 8;

[0041] Figure 14 This is the 237nm spectrum of the concentrated benchmark sample of Example 8;

[0042] Figure 15 The superimposed spectra of the test samples at different column temperatures in Example 11;

[0043] Figure 16 This is the superimposed graph of the test sample at different flow rates in Example 12;

[0044] Figure 17 This is the superimposed spectrum of the test sample under different ratios of methanol extraction solvent in Example 13;

[0045] Figure 18 The superimposed graphs of the test samples at different extraction times in Example 14;

[0046] Figure 19 The superimposed graphs of the test samples under different sampling amounts in Example 15;

[0047] Figure 20 This is the superimposed spectrum of the test samples at different liquid-to-solid ratios in Example 16;

[0048] Figure 21 This is a linear standard curve diagram of glycyrrhizin, cinnamic acid, cinnamaldehyde and glycyrrhizic acid in Example 17. DETAILED DESCRIPTION

[0049] In order to better understand the technical solutions and advantages of the present invention, the present invention is further described below through specific implementation methods.

[0050] Example 1

[0051] Preparation of reference solution: Take appropriate amount of apiolactone, liquiritin, apiolactone isoliquiritin, isoliquiritin, glycyrrhizin, cinnamic acid, cinnamaldehyde, o-methoxycinnamic acid, glycyrrhizic acid, atractylodes lactone III, atractylodes lactone II, tuckahoe acid B, poliporic acid C, and dehydropachymic acid reference substances, accurately weigh them, and add methanol to make a mixed solution containing 50 μg of apiolactone, 500 μg of liquiritin, 50 μg of isoliquiritin, 50 μg of glycyrrhizin, 50 μg of cinnamic acid, 10 μg of cinnamaldehyde, 50 μg of o-methoxycinnamic acid, 500 μg of glycyrrhizic acid, 50 μg of atractylodes lactone III, 20 μg of atractylodes lactone II, 10 μg of tuckahoe acid B, 10 μg of poliporic acid C, and 10 μg of dehydropachymic acid per ml, as the reference substance solution.

[0052] Preparation of test solution: Take about 2.0 g of the test sample, place it in a stoppered conical flask, add 20 ml of extraction solvent methanol, stopper it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, shake it well, centrifuge it (3500 r / min) for 5 minutes, take the supernatant and concentrate it in vacuo at 50°C to near dryness, dissolve it in 4 ml of extraction solvent methanol in batches, filter it, and take the filtrate as the test solution.

[0053] Determination method: Accurately pipette 3 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography, and determine.

[0054] Chromatographic conditions: an ACQUITY UPLC BEH C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm was used. Acetonitrile was used as the mobile phase B1, and 0.1% phosphoric acid was used as the mobile phase A1. The column temperature was 30°C, and the flow rate was 0.2 ml / min. The elution program was as follows.

[0055] Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> 0 82 18 39 43 57 5 82 18 51 42 58 10 76 24 53 37.5 62.5 20 75 25 61 20 80 23 62 38 61.1 82 18 31 58 42 66 82 18

[0056] The fingerprint of the reference sample was detected using a PDA full-wavelength scan. Based on the full-wavelength scan results from 190nm to 400nm for 15 characteristic peaks, including apiosylliquiritin, liquiritin, apiosylisoliquiritin, isoliquiritin, cinnamic acid, cinnamaldehyde, o-methoxycinnamic acid, glycyrrhizic acid, glycyrrhizin, atractylodes lactone III, atractylodes lactone II, pachymic acid B, policolic acid C, and dehydropachymic acid, the wavelengths where each characteristic peak had absorption and relatively good resolution were selected. Ultimately, the wavelengths used for the fingerprint detection of the reference sample were determined to be 266nm and 235nm.

[0057] Table 1

[0058]

[0059]

[0060] Example 2

[0061] The detection method was the same as in Example 1, with acetonitrile and methanol as the mobile phases B1, and the detection wavelength was 237 nm. Figure 1 、 2 The results show that using acetonitrile as mobile phase B1 resulted in a relatively large number of peaks in the chromatogram, relatively good separation between apiosin and liquiritin, and a relatively good peak shape for glycyrrhizic acid. Using methanol as mobile phase B1 resulted in a relatively small number of peaks in the chromatogram, with the apiosin and liquiritin peaks overlapping and unable to be identified, and interference from other peaks surrounding the glycyrrhizic acid peak.

[0062] Example 3

[0063] The detection method was the same as in Example 1. The mobile phase A1 used 0.1% phosphoric acid and 0.6% formic acid, respectively. The detection wavelengths were 266 nm and 235 nm. The results were as follows: Figure 3 、 Figure 4 shown.

[0064] Example 4

[0065] The detection method is the same as in Example 1, with phosphoric acid aqueous solution as mobile phase A1, pH values ​​of 2.16, 2.14 and 2.02, i.e. 0.05%, 0.10% and 0.15% phosphoric acid solutions, and the detection wavelength is 266nm. The results are as follows Figure 5 The chromatographic results show that when the mobile phase has different pH values ​​(2.16, 2.14, and 2.02), the tailing factors and separation degrees of the components in the fingerprint of the reference sample are not significantly different.

[0066] Example 5

[0067] The detection method is the same as in Example 1, using ACQUITY BEH C18 and CORTECS UPLC T3 columns were used for detection, and the detection wavelength was 237 nm. The chromatograms of the test samples with different columns are as follows: Figure 6 As shown in the chromatogram, ACQUITY Compared with the CORTECS UPLC T3 column, the BEH C18 column has a larger number of total peaks in the chromatogram, and the separation of some chromatographic peaks is relatively good. In the selection of the chromatographic column for the fingerprint of the benchmark sample, the number of chromatographic peaks is an important basis, so the ACQUITY BEH C18 column is better.

[0068] Example 6

[0069] The detection method was the same as in Example 1, using ACQUITY BEH C18 and Shim-Pack GIST C18 columns, the results are shown in Table 2. Figure 7 、 8 , 9, and 10. The results show that the Shim-Pack GIST C18 column has good separation of most characteristic peaks, and the tailing factors of components with higher peak areas (apigenin, liquiritin) are relatively good. After comprehensive comparison, the Shim-Pack GIST C18 column is better.

[0070] Table 2

[0071]

[0072] Example 7

[0073] The detection method is the same as in Example 1, with methanol, ethanol and purified water as the extraction solvents for the sample solution, and the detection wavelength is 266nm. The results of the applicability parameters of the fingerprint spectrum system for the sample under different extraction solvents are shown in Table 3, and the comparison of the extraction solvents of the sample is shown in Table 3. Figure 11 The results showed that compared with methanol, the total number of chromatographic peaks in the chromatogram using ethanol as the extraction solvent was relatively small, and the peak areas were relatively small. Compared with methanol, the overall baseline of the chromatogram using water as the extraction solvent was less stable, and the peak areas of most characteristic peaks were relatively small. The total number of peaks in the chromatogram using methanol as the extraction solvent was greater, and the peak areas of each peak were relatively high.

[0074] Table 3

[0075]

[0076] Example 8

[0077] Through preliminary research, it was found that the dissolution rate of the characteristic components of Poria cocos in the benchmark sample was extremely low. Therefore, in order to achieve the characteristic peaks of the four medicinal materials Poria cocos, cinnamon twig, Atractylodes macrocephala and licorice in the fingerprint of the benchmark sample, it is necessary to investigate the sample concentration.

[0078] The preparation of the unconcentrated reference sample solution was the same as that of the test solution in Example 1, and the preparation of the reference solution was the same as that in Example 1.

[0079] Preparation of concentrated reference sample solution: Take about 5.0 g of the test sample, place it in a stoppered conical flask, add 20 ml of extraction solvent methanol, stopper it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, shake it well, centrifuge it (3500 r / min) for 5 minutes, take the supernatant and concentrate it in a vacuum at 50°C to near dryness, dissolve it in 10 ml of extraction solvent methanol in batches, filter it, and take the filtrate as the concentrated reference sample solution.

[0080] Chromatographic conditions: An ACQUITY UPLC BEH C18 column was used, with acetonitrile as mobile phase B1 and 0.1% phosphoric acid as mobile phase A1. The column temperature was 30°C, the flow rate was 0.2 ml / min, and the detection wavelengths were 242 nm and 237 nm. The elution program was as shown in the table. Accurately pipette 3 μl of each reference solution and test solution into the ultra-high performance liquid chromatograph for measurement to obtain the chromatogram.

[0081] Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> 0 82 18 39 43 57 5 82 18 51 42 58 10 76 24 53 37.5 62.5 20 75 25 61 20 80 23 62 38 61.1 82 18 31 58 42 66 82 18

[0082] The test results are as follows Figure 12 、 13 As shown in Figures 1 and 14, in the unconcentrated reference sample spectrum, except for the five characteristic peaks of Poria cocos, the characteristic peaks of other medicinal materials can all be found with corresponding peaks; when the concentrated reference sample solution was tested, there was no flat-top peak in the spectrum, and the five characteristic peaks of Poria cocos could be detected, but the peak areas were small (all below 100).

[0083] Example 9

[0084] The chromatographic conditions of Example 1 were used, with detection wavelengths of 235 nm and 266 nm, to conduct verification experiments on specificity, precision, repeatability, stability, and sample determination similarity.

[0085] Specificity: Solvent blank, reference solution and test solution were selected for specificity experiment. The results showed that the retention time of positive sample was consistent with that of reference solution, and there was no interference between reagent blank sample and negative sample near the retention time of target substance. The specificity met the method validation requirements.

[0086] Precision: Six consecutive injections of the same sample solution were performed, and the chromatograms were recorded. After full-spectrum peak matching, the similarity of the chromatographic peaks of the six sample solutions was above 0.99, indicating that the method has good precision and meets the methodological validation requirements for characteristic chromatograms.

[0087] Repeatability: The repeatability was determined based on the full-peak matching similarity of the repeatable sample chromatograms calculated by the Chinese medicine chromatographic fingerprint similarity evaluation system software. After full-spectrum peak matching, the chromatographic peak similarities of the six test sample solutions were all above 0.99, indicating that the method had good repeatability and met the methodological validation requirements of the characteristic spectrum.

[0088] Stability: Take the same sample solution and inject it at 0h, 3h, 6h, 9h, 15h, 21h, and 27h after preparation. Stability is determined based on the full peak matching similarity of the repetitive sample chromatograms calculated by the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software. The results show that after Mark peak matching, the chromatographic peak similarity of the sample solution is above 0.99 within 27h, indicating that the sample solution is stable within 27 hours.

[0089] Sample determination similarity: 10 batches of reference samples were prepared and tested. After Mark peak matching, the chromatographic peak similarities of the 10 batches of test solution were all above 0.99, and the fit was good.

[0090] Example 10

[0091] Preparation of reference solution: Take appropriate amount of glycyrrhizin reference substance, cinnamic acid reference substance, cinnamaldehyde reference substance, and ammonium glycyrrhizate reference substance, accurately weigh them, and add methanol to make a solution containing 40 μg of glycyrrhizin, 6 μg of cinnamic acid, 5 μg of cinnamaldehyde, and 200 μg of ammonium glycyrrhizate per 1 ml, which is used as the reference substance solution.

[0092] Preparation of test solution: Take about 0.2 g of the test sample, place it in a stoppered conical flask, add 20 ml of 75% methanol as the extraction solvent, stopper it tightly, and ultrasonically treat it (power 250 W, frequency 40 kHz) for 10 minutes. Let it cool, shake it well, and centrifuge it (3500 r / min) for 5 minutes. Filter the supernatant and take the filtrate as the test solution.

[0093] Determination method: Accurately aspirate 2 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography instrument, and determine.

[0094] Chromatographic conditions: octadecylsilane bonded silica gel as the packing agent (ACQUITY UPLC BEH C18, 100 mm × 2.1 mm, 1.7 μm), acetonitrile as the mobile phase B1, 0.1% phosphoric acid as the mobile phase A1, column temperature 30°C, flow rate 0.2 ml / min, and gradient elution according to the following program.

[0095] Time (min) <![CDATA[A1%]]> <![CDATA[B1%]]> 0 82 18 5 82 18 10 76 24 20 75 25 23 62 38 31 58 42 31.1 82 18 35 82 18

[0096] PDA full wavelength scanning was used to detect the content determination spectrum of the reference sample. According to the full wavelength scanning results of 190nm to 400nm for the four target peaks of glycyrrhizin, cinnamic acid, cinnamaldehyde, and glycyrrhizic acid, the results of the maximum absorption wavelength of the chromatographic peak are shown in Table 4. The response values ​​of each target peak at a wavelength of 266nm were good.

[0097] Table 4

[0098] name Maximum absorption wavelength Remark Peak 1 192nm, 216nm, 275nm, 311nm Liquiritin Peak 2 216nm, 277nm Cinnamic acid Peak 3 220nm, 291nm Cinnamaldehyde Peak 4 252nm, 320nm Glycyrrhizic acid

[0099] Example 11

[0100] The detection method was the same as in Example 10. The column temperatures were set at 25°C, 30°C, 35°C, and 40°C, respectively. The detection wavelength was 266 nm. The results of the system suitability parameters of the target peak at different column temperatures are shown in Table 5. The superimposed spectra at different column temperatures are shown in Table 5. Figure 15 Since the sample was obtained by vacuum drying, the chromatographic peak corresponding to cinnamaldehyde was not detected in the spectrum. The results showed that there was no significant difference in the separation degree and tailing factor among liquiritin, cinnamic acid and glycyrrhizic acid.

[0101] Table 5

[0102]

[0103] Example 12

[0104] The detection method is the same as that in Example 10, the flow rates are set to 0.1 ml / min, 0.2 ml / min, and 0.3 ml / min, respectively, and the detection wavelength is 266 nm. The results of the target peak system suitability parameters at different flow rates are shown in Table 6. Figure 16 The results showed that when the flow rate was 0.1 ml / min, the separation of liquiritin was the best, but the chromatographic baseline was relatively unstable; when the flow rate was 0.3 ml / min, the baseline was stable, but the chromatographic peaks of liquiritin and apigenin overlapped; when the flow rate was 0.2 ml / min, the separation of liquiritin met the quantitative requirements and the chromatographic baseline was stable.

[0105] Table 6

[0106]

[0107] Example 13

[0108] The detection method is the same as that in Example 10, with a detection wavelength of 266 nm. The preparation method of the test solution is as follows: take about 0.5 g of the reference sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of the extraction solvent, ultrasonically treat it (250 W, 40 kHz) for 30 minutes, let it cool, make up the weight, shake it well and filter it to obtain the solution. The extraction solvent is 25%, 50%, 75% and 100% methanol. The test sample content determination results under different proportions of methanol extraction solvent are shown in Table 7. Figure 17 Because different extraction solvents have different effects on different components, the four components were assigned an average weight (each accounting for 0.25) and their total scores were compared. The results showed that 75% methanol was the best.

[0109] Table 7

[0110]

[0111] Example 14

[0112] The detection method is the same as that in Example 10. The extraction times for the test solution preparation are 5, 10, 15, 20, 30, 40, and 50 minutes, respectively. The detection wavelength is 266 nm. The results of the test sample content determination at different extraction times are shown in Table 8. Figure 18 The results showed that when the extraction time was 5 minutes, the relative average deviation between parallel samples was greater than 3%, which may be due to the short ultrasonication time, which still caused some solids in the test sample to be not completely dispersed. When the extraction time was 5 to 50 minutes, the coefficient of variation of each quantitative component was less than 3%, indicating that under this liquid-to-solid ratio, the extraction time had little effect on the extraction amount of each quantitative component.

[0113] Table 8

[0114]

[0115] Example 15

[0116] The detection method is the same as that in Example 10, the sampling amounts are 0.1 g, 0.2 g and 0.5 g respectively, the detection wavelength is 266 nm, and the results of the test sample content determination under different sampling amounts are shown in Table 9, Figure 19 The results showed that the relative average deviation of the parallel samples was less than 3%, which was in compliance with the regulations; the coefficient of variation of the three different sampling amounts was calculated, and the results showed that the coefficient of variation of each quantitative component was less than 2%.

[0117] Table 9

[0118]

[0119] Example 16

[0120] The detection method is the same as that in Example 10, the liquid-to-solid ratio (times) is 50, 100, 250, 350 and 500 respectively, the detection wavelength is 266 nm, and the test results of the test sample content under different liquid-to-solid ratios are shown in Table 10. Figure 20 The data showed that the relative average deviation of parallel samples met the parallel sample requirements except for the 500-fold liquid-to-solid ratio, which was greater than 3%. Comparing the differences in the coefficient of variation of each component, the coefficient of variation of cinnamic acid, cinnamaldehyde, and glycyrrhizic acid was greater than 3%. Comparing the content values ​​of each component extracted with different liquid-to-solid ratios, it was found that the content of each component extracted with a 100-fold liquid-to-solid ratio was higher.

[0121] Table 10

[0122]

[0123] Example 17

[0124] The detection conditions were the same as those in Example 10, with a detection wavelength of 266 nm, and specificity, precision, repeatability, linearity, accuracy, and stability experiments were performed.

[0125] Specificity: Solvent blank, reference solution and test solution were selected for specificity experiment. The results showed that the retention time of positive sample was consistent with that of reference solution, and there was no interference between reagent blank sample and negative sample near the retention time of target substance. The specificity met the method validation requirements.

[0126] Precision: Six consecutive injections of the same sample solution were performed, and the chromatograms were recorded. The results showed that the RSD% values ​​for the peak areas of the quantitative components of glycyrrhizin, cinnamic acid, cinnamaldehyde, and glycyrrhizic acid were all less than 1%, indicating good instrument precision.

[0127] Repeatability: Six parallel sample solutions were prepared from the same batch of test sample. The results showed that the RSD% values ​​for the quantitative components of glycyrrhizin, cinnamic acid, cinnamaldehyde, and glycyrrhizic acid were all less than 2.0%, meeting the requirements of the pharmacopoeia. This indicates that the method has good repeatability and meets the methodological validation requirements for content determination.

[0128] Linearity: Weigh 20.52 mg of liquiritin, 6.004 mg of cinnamic acid, 204.2 mg of cinnamaldehyde, and 19.74 mg of ammonium glycyrrhizate, and dilute with methanol to a mixed control mother solution containing 82.8 μg of liquiritin, 12.008 μg of cinnamic acid, 20.42 μg of cinnamaldehyde, and 394.80 μg of ammonium glycyrrhizate per ml. Dilute and measure in sequence, record and integrate the chromatogram, calculate the peak area, and the linear standard curve is shown in the figure below. Figure 21 The concentration and peak area data of each quantitative component are shown in Table 11. The linear standard curve is plotted with concentration as the horizontal axis and peak area as the vertical axis. The results show that glycyrrhizin, cinnamic acid, cinnamaldehyde, and ammonium glycyrrhizate have a good linear relationship within the linear range, R 2 All are above 0.999.

[0129] Table 11

[0130]

[0131] Accuracy: Weigh 6 portions of the test sample with determined content, each portion is about 0.2 g, accurately weighed, and placed in a conical flask. Appropriate amounts of glycyrrhizin, cinnamic acid, cinnamaldehyde, and ammonium glycyrrhizate are added in parallel, and the chromatogram is recorded. The results show that the recovery rate of each quantitative component is in the range of 92% to 105%, and the RSD% value is less than 2.0%, indicating that this method has good accuracy and meets the methodological validation requirements for content determination.

[0132] Stability: Take the same sample solution and inject it at 0h, 1h, 3h, 6h, 9h, 12h, 18h, and 24h after preparation, and record the chromatogram. The results show that the peak area RSD% value of each quantitative component is less than 2.0%, indicating that the sample solution is basically stable within 24 hours.

[0133] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing and detecting Ling Gui Zhu Gan Tang, characterized in that: The specific steps include: (1) Preparation of reference solution: Take an appropriate amount of reference substance, weigh accurately, and add methanol to make a mixed solution as the reference solution; (2) Preparation of test solution: 5 g of the test sample was placed in a stoppered conical flask, 20 ml of methanol was added, the flask was sealed, and ultrasonic treatment was performed for 30 minutes. The flask was cooled, shaken, and centrifuged for 5 minutes. The supernatant was concentrated to near dryness in a vacuum at 50°C, and the supernatant was dissolved in 10 ml of methanol in portions. The filtrate was filtered and used as the test solution. (3) Chromatographic conditions: ACQUITY UPLC BEH C18 column, acetonitrile as mobile phase B1, 0.10% phosphoric acid aqueous solution as mobile phase A1, column temperature 30°C, flow rate 0.2 ml / min, detection wavelengths 242 nm and 237 nm, gradient elution according to the following procedure: ； (4) Accurately pipette 3 μl of the reference solution and the test solution, inject them into the ultra-high performance liquid chromatography instrument, and measure them to obtain the fingerprint of Ling Gui Zhu Gan Tang; Among them, in the fingerprint spectrum, the components represented by its characteristic peaks include apiosyl glycyrrhizin, glycyrrhizin, apiosyl isoliquiritin, isoliquiritin, glycyrrhizin, cinnamic acid, cinnamaldehyde, o-methoxycinnamic acid, glycyrrhizic acid, atractylodes lactone III, atractylodes lactone II, pachymosic acid B, policocormic acid C, and dehydropachymosic acid.

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