Method for detecting characteristic chromatogram of compound gardenia root granules, standard characteristic chromatogram of compound gardenia root granules and application of standard characteristic chromatogram

Abstract

The invention relates to a method for detecting a characteristic spectrum of compound gardenia root granules and application of a standard characteristic spectrum of the compound gardenia root granules. According to the method for detecting the characteristic spectrum of the compound gardenia root particles, an ultra-high performance liquid chromatograph equipped with a diode array detector (DAD) is used for detection, and a chromatogram obtained under selected chromatographic conditions is sensitive in characteristic peak response, large in response value, good in separation degree, high in sensitivity, stable in base line and low in noise. In addition, the blank solvent does not interfere with the test of the test solution, and the specificity is good. The precision, the stability and the reproducibility of the method are good. The method for establishing the standard characteristic chromatogram of the compound gardenia root granules and the standard characteristic chromatogram of the compound gardenia root granules are obtained by adopting the method for detecting the characteristic chromatogram of the compound gardenia root granules, and can be used for controlling the quality of the compound gardenia root granules.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine analysis, and relates to a method for detecting a characteristic spectrum of compound gardenia root particles, a method for establishing a standard characteristic spectrum comprising the method, and a standard characteristic spectrum of compound gardenia root particles. Background Art

[0002] Compound Gardenia Root Granules are mainly composed of 15 Chinese herbs, including Gardenia Root, Artemisia Capillaris, Tortoise Shell, Turtle Shell, Astragalus, Citron, Salvia Miltiorrhiza, Curcuma Radix, Bupleurum, Panax Notoginseng, Corydalis, Cortex Moutan, Patrinia Saposhnikoviae, Radix Pseudostellariae, and Rubia Cordata. Astragalus, Tortoise Shell, and Turtle Shell are supplemented with Curcuma Radix, Bupleurum, Salvia Miltiorrhiza, and Cortex Moutan to replenish the internal organs and blood circulation, replenish the yin fluid, and promote blood circulation, soften and disperse the nodules; Rubia Cordata cools the blood, promotes blood circulation and removes blood stasis, and can clear the blood stasis and prevent blood from moving; Gardenia Root and Artemisia Capillaris are supplemented with the toxins of the Qi and the evil of the blood, and they work together to strengthen the body, eliminate evil, and remove blood stasis. It is used for chronic hepatitis, liver fibrosis, and early liver cirrhosis, which are caused by Qi deficiency and blood stasis, liver depression and Qi stagnation, and damp-heat syndrome.

[0003] Compound Gardenia Root Granules is a preparation independently developed by Xiamen Hospital of Traditional Chinese Medicine. The original quality standard used thin layer chromatography to identify chlorogenic acid, ginsenoside Rg1 and notoginsenoside R1. In recent years, as the in-hospital preparations have been transformed towards Class 1 new Chinese medicines, the Drug Evaluation Center has put forward higher requirements for product quality. In this case, it is particularly important to adopt effective and more demanding quality control methods.

[0004] The characteristic spectrum of traditional Chinese medicine refers to the chromatogram or spectrum that can characterize the characteristics of Chinese medicinal materials or Chinese patent medicines after processing through certain analytical methods. The characteristic methodology breaks through the traditional linear thinking and focuses on the characteristic analysis of macroscopic regularities. Compared with the current traditional quality control methods of traditional Chinese medicine, the characteristic spectrum method emphasizes the comprehensive chromatographic characteristics represented by the relatively stable ratio and peak sequence of multiple components. Therefore, the characteristic spectrum method can simultaneously display the "similarity" and "difference" between different samples. As a research method, the characteristic spectrum of traditional Chinese medicine has the characteristics of fully reflecting the integrity and fuzziness of traditional Chinese medicine.

[0005] The main chemical components of the main drug Gardenia Root of Compound Gardenia Root Granules are sterols, organic acids, volatile oils, sugars and other components; the main chemical components of the auxiliary drug Artemisia Capillaris are coumarins, flavonoids, organic acids, volatile oils and other components; Tortoise shell and turtle shell are both animal drugs with complex components, mainly containing animal gelatin, fat, calcium, phosphorus and the like; the main chemical components of Astragalus are flavonoids, polysaccharides, saponins, amino acids, trace elements and other components; Salvia miltiorrhiza mainly contains diterpenes, triterpenes, lactones, nitrogen-containing compounds, phenolic acids and other components; the main components of Curcuma include sesquiterpenes, monoterpenes, diterpenes, curcumin, alkaloids and other components; the main chemical components of Bupleurum are Bupleurosaponins, Bupleurum polysaccharides, volatile oils, flavonoids and other components; the main chemical components of Panax notoginseng are triterpenoid saponins, flavonoids, amino acids, polysaccharides, volatile oils and other components. It can be seen that the medicinal taste of Compound Gardenia Root Granules is complex and contains many chemical components, and it is very difficult to study its standard characteristic spectrum. Therefore, there is currently no method for detecting the characteristic spectrum of Compound Gardenia Root Granules, establishing a method for the standard characteristic spectrum of Compound Gardenia Root Granules, and using it for quality control. Summary of the invention

[0006] In order to more comprehensively and effectively control the quality of the compound gardenia root granules, the inventors carefully studied and adopted the UPLC (ultra-high performance liquid chromatography)-DAD method and studied the extraction solvent, extraction time, chromatographic conditions, chromatographic column, detection wavelength and other conditions, and obtained a method for detecting the characteristic spectrum of the compound gardenia root granules, and thus obtained a method for establishing a standard characteristic spectrum of the compound gardenia root granules and a standard characteristic spectrum of the compound gardenia root granules, which can be used for quality control of the compound gardenia root granules, thereby completing the present invention.

[0007] In one aspect, the present invention provides a method for detecting the characteristic spectrum of compound gardenia root particles, wherein the method is detected by an ultra-high performance liquid chromatograph equipped with a diode array detector (DAD), and the chromatographic conditions are as follows:

[0008] Chromatographic column: Agilent ZORBAX SB Aq RRHD (1.8 μm, 150 × 2.1 mm);

[0009] Mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B);

[0010] Gradient elution: 0-13min, 4%-6% A; 13-21min, 6%-10%A; 21-27min, 10%-15%A; 27-42min, 15%-23%A; 42-50min, 23%-28 %A; 50-55min, 28%-31%A; 55-65min, 31%-39%A; 65-66min, 39%-95%A; 66-71min, 95%A;

[0011] Flow rate: 0.2ml / min;

[0012] Column temperature: 30°C;

[0013] Injection volume: 1-2 μl, preferably 2 μl;

[0014] Detection wavelength: 230nm.

[0015] In the present invention, the percentage in gradient elution refers to volume percentage, for example, 21% means 21% by volume.

[0016] In the method for detecting the characteristic spectrum of compound gardenia root particles, the compound gardenia root particle test solution used for detection can be prepared as follows: add the compound gardenia root particle fine powder and the ethanol-water mixed solution into a stoppered container, weigh and then perform ultrasonic treatment, cool and then weigh again, make up the lost weight with the same solvent, and obtain the test solution after filtering.

[0017] Another aspect of the present invention provides a method for establishing a standard characteristic spectrum of compound gardenia root granules, the method comprising:

[0018] (1) Preparation of test solution:

[0019] Add the compound gardenia root granule powder and ethanol-water mixed solution into a stoppered container, weigh them, perform ultrasonic treatment, cool them, weigh them again, make up the lost weight with the same solvent, and filter them to obtain the test solution;

[0020] (2) Preparation of reference solution:

[0021] Take gallic acid, deacetylcaryophyllene methyl ester, gardenia glycoside B, neochlorogenic acid, caffeic acid, cryptochlorogenic acid, chlorogenic acid, 1.3-dicaffeoylquinic acid, 3.4-dicaffeoylquinic acid, 3.5-dicaffeoylquinic acid, 4.5-dicaffeoylquinic acid, and salvianolic acid B, add methanol to prepare a mixed reference solution;

[0022] (3) UPLC detection

[0023] The test solution and the reference solution of (1) and (2) are detected by the method for detecting the characteristic spectrum of the compound gardenia root granules according to the present invention to obtain the characteristic chromatogram of the test sample and the characteristic chromatogram of the reference;

[0024] (4) Generate standard feature maps

[0025] A standard characteristic spectrum was generated based on the characteristic chromatogram of the test sample, and the chromatographic peaks of the main components in the compound gardenia root granules with good separation were selected as characteristic peaks;

[0026] (5) Identification and attribution of characteristic peaks

[0027] The common peaks in the characteristic spectrum of compound gardenia root granules were assigned and identified.

[0028] In the above method, there is no particular limitation on the method for generating a standard characteristic spectrum based on the characteristic chromatogram of the test sample, and a conventional method in the art can be used. For example, the measured chromatogram of the compound gardenia root granules test sample can be introduced into the "Chinese medicine chromatographic fingerprint similarity evaluation system" (2012.130723 version) promulgated by the State Pharmacopoeia Committee, one of the chromatograms of the test sample can be set as a reference spectrum, the standard spectrum generation method can be, for example, the median method, and a multi-point correction method can be used to establish a standard characteristic spectrum of the compound gardenia root granules.

[0029] In the above method, the identification and attribution of characteristic peaks are completed by comparing the chromatograms of single medicinal materials and reference solution.

[0030] In the method for establishing the standard characteristic spectrum of compound gardenia root granules according to the present invention, there is no particular restriction on the number of test samples used to establish the standard characteristic spectrum, and those skilled in the art can easily select a suitable number of test samples. Generally speaking, the number of test samples can be more than 10, or more than 15, such as 20, and the upper limit is, for example, less than 30, less than 20, etc.

[0031] In the preparation process of the compound gardenia root granule test solution, there is no particular restriction on the particle size of the compound gardenia root granule fine powder, but it is preferably a particle size that can at least pass through the No. 3 sieve specified in the Chinese Pharmacopoeia, for example, the compound gardenia root granule fine powder can pass through the No. 3 sieve or the No. 4 sieve. The volume ratio of the two components in the ethanol-water mixed solution can be ethanol: water = 1: 1, that is, 50% ethanol. The solid-liquid ratio of the compound gardenia root granule fine powder to the ethanol-water mixed solution is 1: 50 (g / ml) in terms of weight / volume.

[0032] In one embodiment, the test solution is prepared as follows: take about 0.5 g of the compound gardenia root granule powder passed through a No. 3 sieve, accurately weigh it, place it in a stoppered conical bottle, accurately add 25 ml of 50% ethanol, seal it, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake it well, and filter it.

[0033] In the preparation process of the above-mentioned reference solution, there is no particular restriction on the concentration of the reference solution, but it is preferred that the content ratio of each component therein is substantially equivalent to that of the test solution. In one embodiment, the reference solution is prepared as follows: take gallic acid, deacetylcarbamate methyl ester, gardenia glycoside B, neochlorogenic acid, caffeic acid, cryptochlorogenic acid, chlorogenic acid, 1.3-dicaffeoylquinic acid, 3.4-dicaffeoylquinic acid, 3.5-dicaffeoylquinic acid, 4.5-dicaffeoylquinic acid, and salvianolic acid B in appropriate amounts, accurately weigh, and add methanol to make 23.11 μg of gallic acid, 62.60 μg of deacetylcarbamate methyl ester, and gardenia glycoside B per 1 ml. A mixed solution of 107.72 μg, 100.24 μg of neochlorogenic acid, 80.15 μg of caffeic acid, 99.88 μg of cryptochlorogenic acid, 100.20 μg of chlorogenic acid, 1.3-dicaffeoylquinic acid 100.17 μg, 98.10 μg of 3.4-dicaffeoylquinic acid, 101.22 μg of 3.5-dicaffeoylquinic acid, 103.25 μg of 4.5-dicaffeoylquinic acid, and 215.22 μg of salvianolic acid B.

[0034] Another aspect of the present invention provides a standard characteristic spectrum of compound gardenia root granules, which includes 12 characteristic peaks, wherein, with peak 7 as a reference, the relative retention time of each peak is as follows:

[0035] Table 2 Relative retention time of standard characteristic spectrum of compound gardenia root granules

[0036]

[0037] The RSD value of each characteristic relative retention time is no greater than 1%, preferably no greater than 0.5%.

[0038] After comparison, the above-mentioned peak No. 1 is gallic acid, peak No. 2 is deacetylcaryophylloside methyl ester, peak No. 3 is jasminoidin B, peak No. 4 is neochlorogenic acid, peak No. 5 is caffeic acid, peak No. 6 is cryptochlorogenic acid, peak No. 7 is chlorogenic acid, peak No. 8 is 1.3-dicaffeoylquinic acid, peak No. 9 is 3.4-dicaffeoylquinic acid, peak No. 10 is 3.5-dicaffeoylquinic acid, peak No. 11 is 4.5-dicaffeoylquinic acid, and peak No. 12 is salvianolic acid B; among them, peaks 2 and 3 are derived from Gardenia jasminoides root, peak No. 5 is derived from Artemisia capillaris, and peak No. 12 is derived from Salvia miltiorrhiza.

[0039] In an embodiment, the above-mentioned standard characteristic spectrum of compound gardenia root granules is obtained by the method for establishing a standard characteristic spectrum of compound gardenia root granules according to the present invention.

[0040] In an embodiment, the standard characteristic spectrum of the compound gardenia root granules according to the present invention is generally as follows Fig.14 shown.

[0041] In another aspect, the present invention provides a method for detecting the quality of a compound gardenia root granule product, comprising:

[0042] (1) using the method for detecting the characteristic spectrum of the compound gardenia root granules according to the present invention to obtain the characteristic spectrum of the compound gardenia root granules product;

[0043] (2) The characteristic spectrum of the obtained compound gardenia root granules product is compared with the standard characteristic spectrum. If the relative retention time of the characteristic peaks of the product is within ±10% of the relative retention time of each peak of the standard characteristic spectrum, it means that the compound gardenia root granules product is of qualified quality.

[0044] In the implementation mode, the characteristic spectrum of the obtained compound gardenia root granule product only needs to be compared with the relative retention time of the standard characteristic spectrum. The characteristic spectrum of the compound gardenia root granule product and the standard characteristic spectrum of the compound gardenia root granule can also be further introduced into the "Chinese medicine chromatographic fingerprint similarity evaluation system" promulgated by the State Pharmacopoeia Committee for comparison, and the similarity between the sample spectrum and the standard spectrum is calculated to further reflect the quality of the product.

[0045] As confirmed by the experimental results, according to the method for detecting the characteristic spectrum of the compound gardenia root particles of the present invention, the obtained chromatogram has a large number of peaks, high sensitivity, stable baseline, low noise, sharp response, large response value, and good separation. In addition, the blank solvent does not interfere with the test of the test solution, and the specificity is good. The precision, stability and reproducibility of the method are good. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0047] Figure 1 It is a liquid chromatogram showing the test solution obtained by extracting the compound gardenia root granule powder with different extraction solvents, wherein 1, 50% ethanol; 2, 70% methanol; 3, ethanol; 4, 50% methanol; 5, methanol; 6, 70% ethanol;

[0048] Figure 2 It is a liquid chromatogram showing the test solution obtained by extracting compound gardenia root granule powder by different extraction methods, including 1. ultrasound; 2. reflux.

[0049] Figure 3 It is a liquid chromatogram showing the test solution obtained by extracting the compound gardenia root granule powder using different extraction times, including 1, 60min, 2, 40min, and 3, 30min.

[0050] Figure 4It is the liquid chromatogram of the same test solution measured at different detection wavelengths, including 1, 300nm, 2, 320nm, 3, 280nm, and 4, 230nm.

[0051] Figure 5 It is a liquid chromatogram showing the measurement of the same test solution using different mobile phases, wherein 1, 0.05% phosphoric acid aqueous solution-acetonitrile; 2, 0.1% phosphoric acid aqueous solution-acetonitrile;

[0052] Figure 6 It is the liquid chromatogram of the test solution obtained from the compound gardenia root granule powder with different sampling amounts, including 1, 2.0g; 2, 0.5g.

[0053] Figure 7 It is a liquid chromatogram showing the measurement of the same test solution using different chromatographic columns, including: 1. Waters UPLC CORTECS C18 (1.6μm, 150×2.1mm); 2. Agilent ZORBAX SB-C18 RRHD (1.8μm, 150×2.1mm); 3. Agilent ZORBAX SB Aq RRHD (1.8μm, 150×2.1mm).

[0054] Figure 8 It is a liquid chromatogram showing the determination of the same test solution using different gradient elution systems.

[0055] Fig. 9 It is a graph showing the results of a specificity test, wherein A is a blank solvent, B is a mixed control, and C is a sample.

[0056] Fig.10 is a graph showing the results of a precision test.

[0057] Fig.11 is a graph showing the results of the stability test.

[0058] Fig.12 is a graph showing the results of a reproducibility test.

[0059] Fig.13 This is the UPLC characteristic spectrum of batch 12 of Compound Gardenia Root Granules.

[0060] Fig.14 This is the UPLC standard characteristic spectrum of Compound Gardenia Root Granules.

[0061] Fig.15 This is the UPLC chromatogram of the compound gardenia root granules mixed reference substance, where A is the compound gardenia root granules sample and B is the mixed reference substance.

[0062] Fig.16This is an attribution and identification diagram for the common peaks in the characteristic spectrum of compound gardenia root granules. DETAILED DESCRIPTION

[0063] 1. Instruments and test drugs

[0064] XS105 electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), BL-1000T electronic balance (Xiamen Bailunsi Electronic Technology Co., Ltd.), HHS-11-4 digital display constant temperature water bath (Xiamen Nabai Technology Co., Ltd.), KQ-600DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), Agilent 1290infinity II ultra high performance liquid chromatograph (Agilent Technologies (China) Co., Ltd.).

[0065] Gallic acid (batch number 110831-201906, China Food and Drug Inspection Institute), deacetyl oxalic acid methyl ester (batch number 202303, China Food and Drug Inspection Institute), chlorogenic acid (batch number 110753-201817, China Food and Drug Inspection Institute), caffeic acid (batch number 110885-200102, China Food and Drug Inspection Institute), 1.3-dicaffeoylquinic acid (batch number 111717-201402, China Food and Drug Inspection Institute), 3.5-dicaffeoylquinic acid (batch number 111782-202208, China Food and Drug Inspection Institute), Food and Drug Inspection Institute), 4.5-dicaffeoylquinic acid (batch number 111894-202205, China Food and Drug Inspection Institute), salvianolic acid B (batch number 111562-202318, China Food and Drug Inspection Institute); Gardenia glycoside B (batch number 23101, Zhuhai Anzhe Biotechnology Co., Ltd.); neochlorogenic acid (batch number 18031001, Chengdu Mansite Biotechnology Co., Ltd.); cryptochlorogenic acid (batch number 21061801, Chengdu Gelip Biotechnology Co., Ltd.); 3.4-dicaffeoylquinic acid (batch number 0518-RE-0009, CATO).

[0066] 2. Liquid phase conditions

[0067] Chromatographic column: Agilent ZORBAX SB Aq RRHD (1.8μm, 150×2.1mm); mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution: 0-13min, 4%-6% A; 13-21min, 6%-10% A; 21-27min, 10%-15% A; 27-42min, 15%-23% A; 42-50min, 23%-28% A; 50-55min, 28%-31% A; 55-65min, 31%-39% A; 65-66min, 39%-95% A; 66-71min, 95% A; flow rate: 0.20ml / min; column temperature: 30℃; injection volume: 2μl; detection wavelength: 230nm.

[0068] In the following experiments, except for the selected conditions and the conditions specifically listed, other liquid phase conditions were the same as those described above.

[0069] 3. Solution preparation

[0070] Preparation of test solution:

[0071] About 0.5 g of compound gardenia root granules powder was accurately weighed and placed in a stoppered conical flask. 25 ml of 50% ethanol was accurately added, the flask was sealed, and the weight was determined. Ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. The flask was cooled and weighed again. The lost weight was supplemented with 50% ethanol. The filtrate was shaken and filtered, and the filtrate was taken for sampling and analysis.

[0072] Preparation of reference solution:

[0073] Take appropriate amounts of gallic acid, deacetylated oxalic acid methyl ester, gardenia glycoside B, neochlorogenic acid, caffeic acid, cryptochlorogenic acid, chlorogenic acid, 1.3-dicaffeoylquinic acid, 3.4-dicaffeoylquinic acid, 3.5-dicaffeoylquinic acid, 4.5-dicaffeoylquinic acid, and salvianolic acid B, weigh them accurately, and add methanol to make a solution containing 23.11 μg of gallic acid, 62.60 μg of deacetylated oxalic acid methyl ester, and gardenia glycoside B per 1 ml. A mixed solution of 107.72 μg, 100.24 μg of neochlorogenic acid, 80.15 μg of caffeic acid, 99.88 μg of cryptochlorogenic acid, 100.20 μg of chlorogenic acid, 1.3-dicaffeoylquinic acid 100.17 μg, 98.10 μg of 3.4-dicaffeoylquinic acid, 101.22 μg of 3.5-dicaffeoylquinic acid, 103.25 μg of 4.5-dicaffeoylquinic acid, and 215.22 μg of salvianolic acid B.

[0074] In the following experiments, except for the selected conditions and the conditions specifically listed, other solution preparation conditions are the same as here.

[0075] 4. Investigation of extraction solvent

[0076] Preparation of test solution:

[0077] Take 2.0 g of compound gardenia root granule powder (batch number 230315, passed through No. 3 sieve), accurately weigh, a total of 6 portions, put into a stoppered conical flask, accurately add 25 ml of 6 different solvents including methanol, 70% methanol, ethanol, 70% ethanol, 50% methanol, and 50% ethanol, weigh the weight, ultrasonically treat for 40 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and obtain the test solution.

[0078] Accurately pipette 2 μl of each of the above test solutions and inject them into liquid chromatography for determination. Specific liquid phase conditions are as follows: chromatographic column: Agilent ZORBAX SB Aq RRHD (1.8 μm, 150×2.1 mm); mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), gradient elution: 0-5 min, 7%-10% A, 5-10 min, 10%-15% A, 10-20 min, 15%-17% A, 20-30 min, 17%-21% A, 30-35 min, 21%-95% A, 35-45 min, 95% A; flow rate: 0.20 ml / min; column temperature: 30°C; injection volume: 2 μl; detection wavelength: 280 nm.

[0079] Results Figure 1 The results showed that when 70% methanol, 70% ethanol, 50% methanol, and 50% ethanol solutions were used as extraction solvents, the number of peaks of the compound gardenia root granules was large and the peak area was large. The total peak area of ​​the chromatographic peaks with better separation was used as a reference. The results are shown in Table 3. The total peak area of ​​50% ethanol was large, so 50% ethanol was selected as the extraction solvent.

[0080] Table 3 Extraction solvent investigation results

[0081] Extraction solvent Sampling volume (g) Total peak area (muA) 70% ethanol 2.0020 3457.745 50% ethanol 2.0014 3544.602 70% methanol 2.0013 3378.335 50% methanol 2.0015 3399.392

[0082] 5. Investigation of extraction methods

[0083] Preparation of test solution:

[0084] Take 2.0 g of compound gardenia root granule powder (batch number 230315, passed through No. 3 sieve), accurately weigh, and place 2 portions in a stoppered conical flask. Accurately add 25 ml of 50% ethanol to each portion and weigh the weight. Set one portion to ultrasonically treat at 25% power for 40 minutes, and reflux the other portion for 40 minutes. Cool and weigh again. Make up the lost weight with the corresponding solvent, shake well, filter, and obtain the test solution.

[0085] Accurately pipette 2 μl of each of the above test solutions and inject them into liquid chromatography for determination. Specific liquid phase conditions are as follows: chromatographic column: Agilent ZORBAX SB Aq RRHD (1.8 μm, 150×2.1 mm); mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), gradient elution: 0-5 min, 7%-10% A, 5-10 min, 10%-15% A, 10-20 min, 15%-17% A, 20-30 min, 17%-21% A, 30-35 min, 21%-95% A, 35-45 min, 95% A; flow rate: 0.20 ml / min; column temperature: 30°C; injection volume: 2 μl; detection wavelength: 280 nm.

[0086] Results Figure 2 The results showed that there was no significant difference in the number of peaks of the compound gardenia root granules when ultrasound and reflux were used as extraction methods. The total peak area of ​​the chromatographic peaks with better separation was used as a reference. The results are shown in Table 4. The total peak area of ​​ultrasound as the extraction method was larger, so ultrasound was selected as the best extraction method.

[0087] Table 4 Results of investigation on extraction methods

[0088] Extraction method Sampling volume (g) Total peak area (muA) Ultrasound 2.0014 3690.570 Reflow 2.0012 3459.727

[0089] 6. Extraction time investigation

[0090] Preparation of test solution:

[0091] Take 2.0 g of compound gardenia root granule powder (batch number 230315, passed through No. 3 sieve), accurately weigh, and place in 3 portions, put in stoppered conical flasks, accurately add 25 ml of 50% ethanol to each portion, weigh the weight, ultrasonically treat for 30 minutes, 40 minutes, and 60 minutes respectively, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and obtain the test solution.

[0092] Accurately pipette 2 μl of each of the above test solutions and inject them into liquid chromatography for determination. Specific liquid phase conditions are as follows: chromatographic column: Agilent ZORBAX SB Aq RRHD (1.8 μm, 150×2.1 mm); mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), gradient elution: 0-5 min, 7%-10% A, 5-10 min, 10%-15% A, 10-20 min, 15%-17% A, 20-30 min, 17%-21% A, 30-35 min, 21%-95% A, 35-45 min, 95% A; flow rate: 0.20 ml / min; column temperature: 30°C; injection volume: 2 μl; detection wavelength: 280 nm.

[0093] Results Figure 3Taking the total peak area of ​​the chromatographic peaks with better separation as a reference, the results are shown in Table 5. Different extraction times have little effect on the number and peak area of ​​chromatographic peaks, so 30 min is preferred as the extraction time.

[0094] Table 5 Extraction time investigation results

[0095] Extraction method Sampling volume (g) Total peak area (muA) 30 minutes 2.0019 3535.859 40 minutes 2.0014 3562.123 60 minutes 2.0006 3582.844

[0096] 7. Investigation of different detection wavelengths

[0097] Preparation of test solution:

[0098] Take 2.0 g of compound gardenia root granule powder (batch number 230315, passed through No. 3 sieve), accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 50% ethanol, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and get the test solution.

[0099] Accurately pipette 2 μl of the above test solution and inject it into the liquid chromatograph for determination. The specific liquid phase conditions are as follows: chromatographic column: Agilent ZORBAX SB Aq RRHD (1.8 μm, 150×2.1 mm); mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), gradient elution: 0-5 min, 5%-6% A, 5-10min, 6%-10%A, 10-13min, 10%A, 13-20min, 10%-12%A, 20-30min, 12%-20%A, 30-40min, 20%-22%A, 40-50min, 22%-35%A, 50-55min, 35%-95%A, 55-60min, 95%A; flow rate: 0.20ml / min; column temperature: 30℃; injection volume: 2μl; the detection wavelength adopts the full wavelength scanning mode, and 320nm, 300nm, 280nm, and 230nm are set as the observation wavelengths.

[0100] Results Figure 4 The chromatographic information at 230nm wavelength is rich, and the absorption of each peak of Compound Gardenia Root Granules is good, so 230nm is selected as the optimal detection wavelength.

[0101] 8. Different mobile phases

[0102] The preparation of the test solution is the same as in step 7 above.

[0103] Accurately pipette 2 μl of the above test solution, respectively, use 0.05% phosphoric acid aqueous solution-acetonitrile and 0.1% phosphoric acid aqueous solution-acetonitrile as the mobile phase system, and use the following gradients. In addition, the above test solution is measured under the same chromatographic conditions. The results are as follows: Figure 5Specific liquid phase conditions: chromatographic column: Agilent ZORBAX SB-Aq RRHD (1.8μm, 150×2.1mm); flow rate: 0.20ml / min; column temperature: 30℃; injection volume: 2μl; detection wavelength: 230nm.

[0104] Figure 5 The mobile phase system is: 1. 0.05% phosphoric acid aqueous solution-acetonitrile; 2. 0.1% phosphoric acid aqueous solution-acetonitrile;

[0105] The gradients are:

[0106] Mobile phase system 1: 0-5min, 5%-10% acetonitrile; 5-20min, 10%-18% acetonitrile; 20-30min, 18%-20% acetonitrile; 30-40min, 20%-30% acetonitrile; 40-45min, 30%-40% acetonitrile; 45-55min, 40%-95% acetonitrile; 55-60min, 95% acetonitrile.

[0107] Mobile phase system 2: 0-5min, 5%-6% acetonitrile; 5-10min, 6%-10% acetonitrile; 10-12min, 10% acetonitrile; 12-19min, 10%-13% acetonitrile; 19-30min, 13%-15% acetonitrile; 30-40min, 15%-20% acetonitrile; 40-50min, 20%-25% acetonitrile; 50-55min, 25%-30% acetonitrile; 55-65min, 30% acetonitrile; 65-70min, 30%-95% acetonitrile; 70-75min, 95% acetonitrile.

[0108] from Figure 5 It can be seen that when the mobile phase system is 0.1% phosphoric acid aqueous solution-acetonitrile, the peak shapes of each chromatographic peak are better, so 0.1% phosphoric acid aqueous solution-acetonitrile is selected as the final mobile phase system.

[0109] 9. Comparison of different sampling volumes

[0110] Preparation of test solution:

[0111] Take 2.0g and 0.5g of compound gardenia root granule powder (batch number 230315, passed through No. 3 sieve) respectively, accurately weigh, put into stoppered conical flasks, accurately add 25ml of 50% ethanol respectively, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter to obtain the test solution.

[0112] Accurately pipette 2 μl of the above test solution and inject it into the liquid chromatograph. The specific liquid phase conditions are as follows: chromatographic column: Agilent ZORBAX SB-Aq RRHD (1.8 μm, 150×2.1 mm); mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B); flow rate: 0.20 ml / min; column temperature: 30°C; injection volume: 2 μl; detection wavelength: 230 nm; and the following gradient injection was used for determination.

[0113] The gradients are:

[0114] Sampling amount 2.0g: 0-5min, 5%-6%A; 5-10min, 6%-12%A; 10-20min, 12%-20%A; 20-25min, 20%A; 25-35min ,20%-25%A; 35-45min, 25%-35%A; 45-50min, 35%-40%A; 50-55min, 40%-95%A; 55-60min, 95%A.

[0115] Sampling amount: 0.5g: 0-10min, 4%-5%A; 10-15min, 5%-10%A; 15-25min, 10%-12%A; 25-30min, 12%-15%A; 30-40min, 15%-20%A; 40-50min, 20%-25%A; 50-60min, 25%-30%A; 60-70min, 30%-35%A.

[0116] from Figure 6 It can be seen that when the sampling volume is 0.5g, the chromatogram baseline of the compound gardenia root granules is relatively stable. Reducing the concentration of the test sample can effectively improve the separation of individual chromatographic peaks, so the sampling volume of the compound gardenia root granules is adjusted to 0.5g.

[0117] 10. Different chromatographic columns

[0118] Preparation of test solution:

[0119] Take 0.5 g of compound gardenia root granule powder (batch number 230315, passed through No. 3 sieve), accurately weigh, place in a stoppered conical flask, accurately add 25 ml of 50% ethanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and obtain the test solution.

[0120] Accurately pipette 2μl of the above test solution and use chromatographic column 1: Waters UPLC CORTECS C18 (1.6μm, 150×2.1mm); chromatographic column 2: Agilent ZORBAX SB-C18 RRHD (1.8μm, 150×2.1mm); chromatographic column 3: Agilent ZORBAX SB Aq RRHD (1.8μm, 150×2.1mm). In addition to using the following gradient, the above test solution was measured under the same chromatographic conditions. The specific chromatographic conditions are: mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B); flow rate: 0.20ml / min; column temperature: 30℃; injection volume: 2μl; detection wavelength: 230nm;

[0121] The gradients are:

[0122] Column 1-2: 0-6min, 4%-6%A; 6-18min, 6%-12%A; 18-32min, 12%-20%A; 32-40min, 20%A; 40-45min ,20%-25%A; 45-55min, 25%-30%A; 55-60min, 30%-31%A; 60-61min, 31%-95%A; 61-66min, 95%A.

[0123] Column 3: 0-6min, 4%-6%A; 6-18min, 6%-12%A; 18-32min, 12%-20%A; 32-42min, 20%-25% A; 42-50min, 25%-30%A; 50-55min, 30%-31%A; 55-56min, 31%-95%A; 56-61min, 95%A.

[0124] from Figure 7 It can be seen that when using chromatographic column 3, the separation of the front and back peaks of the chromatogram of the compound gardenia root granules is good, so Agilent ZORBAX SB Aq RRHD (1.8μm, 150×2.1mm) was selected as the final chromatographic column.

[0125] 11. Gradient system adjustment

[0126] Prepare the test solution according to the test solution preparation method in 10 above.

[0127] In addition to using the following gradient systems, the above test solution was measured by liquid chromatography according to the liquid phase conditions in 9 above:

[0128] Gradient system 1: 0-12min, 4%-5%A; 12-15min, 5%-10%A; 15-25min, 10%-12%A; 25-29min, 12%-15%A; 29-3 9min, 15%-21%A; 39-45min, 21%A; 45-55min, 21%-25%A; 55-65min, 25%-30%A; 65-70min, 35%A.

[0129] Gradient system 2: 0-5min, 4%-6% A; 5-10min, 6%-10% A; 10-12min, 10%-11% A; 12-19min, 11%-13% A; 19-30min, 13%-15% A; 30-40min, 15%-20% A; 40-50min, 20%-25% A; 50-55min, 25%-30% A; 55-75min, 30%.

[0130] Gradient system 3: 0-6min, 4%-6%A; 6-18min, 6%-12%A; 18-32min, 12%-20%A; 32-40min, 20%A; 40-45min, 20%-25%A; 45-55min, 25%-30%A; 55-60min, 30%-31%A; 60-61min, 31%-95%A; 61-66min, 95%A.

[0131] Gradient system 4: 0-5min, 4%-6%A; 5-14min, 6%-9%A; 14-23min, 9%-13%A; 23-42min, 13%-23%A; 42-50 min, 23%-28%A; 50-55min, 28%-31%A; 55-60min, 31%A; 60-61min, 31%-95%A; 61-66min, 95%.

[0132] Gradient system 5: 0-13min, 4%-6% A; 13-21min, 6%-10%A; 21-27min, 10%-15%A; 27-42min, 15%-23%A; 42-50min, 23%-28 %A; 50-55min, 28%-31%A; 55-65min, 31%-39%A; 65-66min, 39%-95%A; 66-71min, 95%A;

[0133] Results Figure 8 The results show that the best gradient system is gradient system 5.

[0134] 12. Methodological investigation

[0135] Prepare the test solution according to the test solution preparation method in 10 above.

[0136] Liquid phase conditions:

[0137] Chromatographic column: Agilent ZORBAX SB Aq RRHD (1.8μm, 150×2.1mm); mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution: 0-13min, 4%-6% A; 13-21min, 6%-10% A; 21-27min, 10%-15% A; 27-42min, 15%-23% A; 42-50min, 23%-28% A; 50-55min, 28%-31% A; 55-65min, 31%-39% A; 65-66min, 39%-95% A; 66-71min, 95% A; flow rate: 0.2ml / min; column temperature: 30℃; injection volume: 2μl; detection wavelength: 230nm.

[0138] 12.1 Specificity Experiment

[0139] Take blank solvent (i.e. acetonitrile-0.1% phosphoric acid aqueous solution) and compound gardenia root granule test solution respectively, inject them according to the above liquid phase conditions, and record the chromatogram. Fig. 9 .

[0140] The results showed that the blank solvent had no interference with the test solution and had good specificity.

[0141] 12.2 Precision test

[0142] Take the same sample solution of Compound Gardenia Root Granules, inject it 6 times continuously according to the above liquid phase conditions, record the chromatogram, and the results are shown in Fig.10 (Among them, precision experiments 1-6 are Fig.10 With peak 7 as reference, the relative retention times and relative peak areas of the 12 characteristic peaks were calculated. The results are shown in Tables 6 and 7.

[0143] Table 6 Precision test results (relative peak area)

[0144]

[0145]

[0146] Table 7 Precision test results (relative retention time)

[0147]

[0148] It can be seen from Tables 6 and 7 that the RSD of the relative retention time of the 12 main chromatographic peaks is between 0.0144-0.7302%, and the RSD of the relative peak area is between 0.5062-4.8227%, and the RSD is less than 5%. The above experimental results show that the precision of the instrument is good.

[0149] 12.3 Stability test

[0150] Take the same sample solution of Compound Gardenia Root Granules and inject it at 0, 2, 4, 8, 12, and 24 hours according to the above chromatographic conditions. Record the chromatograms. The results are shown in Fig.11 (Among them, the experiments of 0, 2, 4, 8, 12, and 24 h were Fig.11 With peak 7 as reference, the relative retention times and relative peak areas of the 12 characteristic peaks were calculated. The results are shown in Tables 8 and 9.

[0151] Table 8 Stability test results (relative retention time)

[0152]

[0153]

[0154] Table 9 Stability test results (relative peak area)

[0155]

[0156] As can be seen from Tables 8 and 9, the RSD of the relative retention time of the 12 main chromatographic peaks is between 0.0449-0.6202%, and the RSD of the relative peak area is between 0.5469-4.5059%, and the RSD is less than 5%, indicating that the test sample is basically stable within 24 hours.

[0157] 12.4 Repeatability test

[0158] Take 6 portions of the same batch of compound gardenia root granules powder, accurately weigh them, prepare 6 portions of test solution in parallel according to the test solution preparation method, inject and analyze them according to the above chromatographic conditions, record the chromatograms, and the results are shown in Fig.16 (Among them, reproducibility experiments 1-6 are Fig.16 With peak 13 as reference, the relative retention times and relative peak areas of 15 main chromatographic peaks were calculated. The results are shown in Tables 10 and 11.

[0159] Table 10 Repeatability test results (relative retention time)

[0160]

[0161]

[0162] Table 11 Repeatability test results (relative peak area)

[0163]

[0164] It can be seen from Tables 10 and 11 that the RSD of the relative retention time of the 12 main characteristic peaks is between 0.0825-0.3210%, and the RSD of the relative peak area is between 0.0997-4.1984%, and the RSD is less than 5.0%. The above experimental results show that the repeatability of the experiment is good.

[0165] 13. Establishment of feature map

[0166] 13.1 Characteristic Peak Calibration

[0167] For 12 batches (batch numbers: 20231031-1, 20230808-1, 20230802-1, 20230613-2, 20230615-2, 20230525-1, 20231102--1, 20231027-1, 20240501-B, 20240502-B, 20240503-B, 20230807-1) of Compound Gardenia Granules, the test solutions were prepared according to the test solution preparation method in 10 above.

[0168] The chromatographic conditions described in 12 were used for determination. The chromatograms of the 12 batches of compound Zhizihen granules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.130723 version) promulgated by the State Pharmacopoeia Committee, and the chromatogram of the batch number 20231031-1 was set as the reference spectrum. The standard spectrum generation method was the median method. At the same time, the multi-point correction method was used to establish the standard characteristic spectrum of the compound Zhizihen granules. According to the characteristic spectrum analysis of the compound Zhizihen granules, the chromatographic peaks of the main components in the compound Zhizihen granules with good separation were selected as characteristic peaks, and 12 common characteristic peaks were determined. The results are shown in Fig.13 and 14 .

[0169] 13.2 Reference peak selection: Peak 7 (chlorogenic acid) in the chromatogram has good separation, appropriate and stable response value, and easy access to reference substances, so this peak is selected as the reference peak (S).

[0170] Taking peak 7 as a reference, the relative retention time and relative peak area of ​​the characteristic peaks in the chromatograms of 12 batches of compound gardenia root granules were calculated. The results are shown in Tables 12 and 13.

[0171] Table 12 Relative peak area analysis of characteristic peaks in UPLC characteristic spectra of 12 batches of compound Gardenia Root Granules

[0172]

[0173] Table 12 (continued)

[0174]

[0175] Table 13 Relative retention time analysis of characteristic peaks in UPLC characteristic spectra of 12 batches of compound Gardenia root granules

[0176]

[0177] Table 13 (continued)

[0178]

[0179] The results showed that the RSD values ​​of the relative retention time of each common peak were all less than 0.32%, indicating that the retention time of each common peak was relatively uniform and stable. The relative peak area RSD% value ranged from 8.63% to 29.46%, with a large span, indicating that there were certain differences in the chemical component content of different batches of compound gardenia root granules.

[0180] 13.3 Similarity Evaluation

[0181] The chromatograms of the above 12 batches of compound Gardenia Root Granules test samples were imported into the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” (2012.130723 version) issued by the Chinese Pharmacopoeia Committee to generate standard characteristic spectra, and the similarity between each test sample spectrum and the standard spectrum was calculated. The results are shown in Table 14.

[0182] Table 14 Similarity evaluation results

[0183]

[0184]

[0185] Table 14 (continued)

[0186]

[0187] The similarities between the test sample spectra and the standard spectra were 1.000, 1.000, 0.996, 0.995, 0.998, 0.997, 0.999, 0.996, 0.998, 0.988, 0.997 and 0.999, respectively, all greater than 0.9, indicating that the quality of these 12 batches of compound gardenia root granules was relatively stable and uniform.

[0188] 14. Identification and attribution of characteristic peaks

[0189] By comparing the single herbal medicine and the reference substance, the characteristic peaks in the characteristic spectrum of the compound gardenia root granules were assigned and identified. Fig.15 and 16, where A is the sample picture of compound gardenia root granules, and B is the mixed reference picture.

[0190] By comparison, it was found that peak 1 was gallic acid, peak 2 was deacetylcaryophylloside methyl ester, peak 3 was jasminoidin B, peak 4 was neochlorogenic acid, peak 5 was caffeic acid, peak 6 was cryptochlorogenic acid, peak 7 was chlorogenic acid, peak 8 was 1.3-dicaffeoylquinic acid, peak 9 was 3.4-dicaffeoylquinic acid, peak 10 was 3.5-dicaffeoylquinic acid, peak 11 was 4.5-dicaffeoylquinic acid, and peak 12 was salvianolic acid B; and peaks 2 and 3 were derived from Gardenia jasminoides root, peak 5 was derived from Artemisia capillaris, and peak 12 was derived from Salvia miltiorrhiza.

[0191] The characteristic spectrum of traditional Chinese medicine is a multi-index quality control model that can more comprehensively reflect the chemical composition information in compound preparations, providing new ideas and methods for the quality evaluation of traditional Chinese medicines, and has become a widely accepted quality evaluation model for traditional Chinese medicines at home and abroad. The UPLC characteristic spectrum method of compound gardenia root granules established by the present invention is simple, stable and reliable. A total of 12 characteristic peaks are calibrated, and the main components are attributed to medicinal materials. The similarity of 12 batches of compound gardenia root granules is evaluated, and the correlation coefficients of each are between 0.988-1.000, indicating that the chemical composition of compound gardenia root granules between different batches is consistent, and the preparation process is stable and controllable.

[0192] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for detecting the characteristic spectrum of compound gardenia root particles, the method using an ultra-high performance liquid chromatograph equipped with a diode array detector for detection, the chromatographic conditions are as follows: Chromatographic column: Agilent ZORBAX SB-Aq RRHD, 1.8 μm, 150 × 2.1 mm; Mobile phase: acetonitrile A-0.1% phosphoric acid aqueous solution B; Gradient elution: 0-13min, 4%-6%A; 13-21min, 6%-10%A; 21-27min, 10%-15%A; 27-42min, 15%-23%A; 42-50m in, 23%-28%A; 50-55min, 28%-31%A; 55-65min, 31%-39%A; 65-66min, 39%-95%A; 66-71min, 95%A; Flow rate: 0.20ml / min; Column temperature: 30°C; Injection volume: 1-2 μl; Detection wavelength: 230nm.

2. The method according to claim 1, characterized in that: In the chromatographic conditions, the injection volume was 2 μl.

3. The method according to claim 1, characterized in that The compound gardenia root granule test solution used for testing is prepared as follows: add the compound gardenia root granule fine powder and ethanol-water mixed solution into a stoppered container, weigh and then perform ultrasonic treatment, cool and then weigh again, make up the lost weight with the same solvent, and obtain the test solution after filtering.

4. A method for establishing a standard characteristic spectrum of compound Gardenia Root Granules, the method comprising: (1) Preparation of test solution: Add the compound gardenia root granule powder and ethanol-water mixed solution into a stoppered container, weigh them, perform ultrasonic treatment, cool them, weigh them again, make up the lost weight with the same solvent, and filter them to obtain the test solution; (2) Preparation of reference solution: Take gallic acid, deacetylcaryophyllene methyl ester, gardenia glycoside B, neochlorogenic acid, caffeic acid, cryptochlorogenic acid, chlorogenic acid, 1.3-dicaffeoylquinic acid, 3.4-dicaffeoylquinic acid, 3.5-dicaffeoylquinic acid, 4.5-dicaffeoylquinic acid, and salvianolic acid B, add methanol to prepare a mixed reference solution; (3) UPLC detection The test solution and the reference solution of (1) and (2) are detected by the method of claim 1 to obtain a characteristic chromatogram of the test sample and a characteristic chromatogram of the reference solution; (4) Generate standard feature maps A standard characteristic spectrum was generated based on the characteristic chromatogram of the test sample, and the chromatographic peaks of the main components in the compound gardenia root granules with high peaks and good separation were selected as characteristic peaks; (5) Identification and attribution of characteristic peaks The characteristic peaks in the characteristic spectrum of compound gardenia root granules were attributed and identified.

5. The method according to claim 4, characterized in that The particle size of the compound gardenia root granular fine powder is at least the particle size that can pass through the No. 3 sieve specified in the Chinese Pharmacopoeia; and / or the ethanol-water mixed solution is 50% ethanol; and / or the solid-to-liquid ratio of the compound gardenia root granular fine powder to 50% ethanol, in terms of weight / volume, is 1:50 g / ml.

6. The method according to claim 4, characterized in that The test solution was prepared as follows: 0.5 g of the compound gardenia root granule powder passed through a No. 3 sieve was accurately weighed, placed in a stoppered conical flask, 25 ml of 50% ethanol was accurately added, the flask was sealed, and the weight was determined. Ultrasonic treatment was performed for 30 minutes, the flask was cooled, and the weight was determined again. The lost weight was supplemented with 50% ethanol, and the flask was shaken and filtered.

7. The method according to claim 4, characterized in that The reference solution was prepared as follows: gallic acid, deacetylated methyl oxalate, gardenia glycoside B, neochlorogenic acid, caffeic acid, cryptochlorogenic acid, chlorogenic acid, 1.3-dicaffeoylquinic acid, 3.4-dicaffeoylquinic acid, 3.5-dicaffeoylquinic acid, 4.5-dicaffeoylquinic acid, and salvianolic acid B were accurately weighed, and methanol was added to prepare a solution containing 23.11 μg of gallic acid and 62.60 μg of deacetylated methyl oxalate per ml. g, a mixed solution of jasminoidin B 107.72 μg, neochlorogenic acid 100.24 μg, caffeic acid 80.15 μg, cryptochlorogenic acid 99.88 μg, chlorogenic acid 100.20 μg, 1.3-dicaffeoylquinic acid 100.17 μg, 3.4-dicaffeoylquinic acid 98.10 μg, 3.5-dicaffeoylquinic acid 101.22 μg, 4.5-dicaffeoylquinic acid 103.25 μg, and salvianolic acid B 215.22 μg.

8. A standard characteristic spectrum of compound gardenia root granules, characterized in that: It includes 12 characteristic peaks, among which peak 7 is taken as the reference peak. The relative retention time of each peak is as follows: Peak 1, relative retention time 0.21; Peak 2, relative retention time 0.37; Peak 3, relative retention time 0.43; Peak 4, relative retention time 0.50; Peak 5, relative retention time 0.92; Peak 6, relative retention time 0.96; Peak 7, relative retention time 1.00; Peak 8, relative retention time 1.37; Peak 9, relative retention time 1.71; Peak 10, relative retention time 1.77; Peak 11, relative retention time 1.85; Peak 12, relative retention time 2.05; Among them, the RSD value of the relative retention time of each characteristic peak is no more than 1%.

9. The use of the standard characteristic spectrum of the compound gardenia root granules according to claim 8, characterized in that: Used to test the product quality of compound gardenia root granules.

10. A method for detecting the quality of compound gardenia root granules, comprising the following steps: (1) using the method according to any one of claims 1 to 7 to obtain a characteristic spectrum of the compound gardenia root granule product; (2) The characteristic spectrum of the obtained compound gardenia root granules product is compared with the standard characteristic spectrum. If the relative retention time of the characteristic peaks of the product is within ±10% of the relative retention time of each peak of the standard characteristic spectrum, it means that the compound gardenia root granules product is of qualified quality.

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