Method for establishing fingerprint of Danti granules

Abstract

The present invention proposes a method for establishing an HPLC-DAD-ELSD fingerprint of Danxi granules, which includes preparing a test sample, determining chromatographic conditions, and using HPLC-DAD-ELSD for measurement to obtain an HPLC-DAD-ELSD fingerprint of Danxi granules. The fingerprint has the characteristics of good separation and peak shape of common peaks, good repeatability, and strong operability, and can effectively control the quality of Danxi granules.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicine preparations, and in particular to a method for establishing a HPLC-DAD-ELSD fingerprint spectrum of Danxi granules. Background Art

[0002] The quality standards of Danxi Granules are included in the 2020 edition of the "Chinese Pharmacopoeia". Danxi Granules were originally named "Piantanling Granules" and are an exclusive product of Jiuzhitang Co., Ltd. The whole formula is composed of 12 medicinal materials: Salvia miltiorrhiza, Achyranthes bidentata, Gastrodia elata, Paeonia suffruticosa, Paeonia lactiflora, Chuanxiong, Rehmannia glutinosa, Epimedium, Viscum album, Gardenia jasminoides, Cassia seed, and Cannabis sativa seed. It has the effects of nourishing yin and calming the liver, extinguishing wind and unblocking the meridians, clearing away heat and relieving restlessness. The compound preparation of Danxi Granules is based on the holistic view and syndrome differentiation and treatment. Clinically, it has its unique prevention and treatment advantages for hemiplegia caused by Yin deficiency and wind movement type and Qi deficiency and blood stasis type of stroke. Clinical data show that it can relieve cerebral edema, promote the absorption of intracranial hematoma, anti-thrombosis, and improve blood rheology. It can significantly improve the patient's neurological function, promote the absorption of intracranial hematoma, and reduce the disability rate. In the prescription, Salvia miltiorrhiza and Achyranthes bidentata are the main herbs, which can invigorate blood circulation, remove blood stasis, relieve pain, nourish the liver and kidneys, and strengthen the tendons and bones; Red peony root, Rehmannia glutinosa, and Paeonia lactiflora are the assistant herbs, which can clear away heat and cool blood, nourish yin and promote the production of body fluid, and relieve pain; Epimedium, Viscum album, Gastrodia elata, Ligusticum chuanxiong, and Gardenia jasminoides are the adjuvant herbs, which can enhance the effects of the main herbs in nourishing the liver and kidneys, strengthening the tendons and bones, invigorating blood circulation, relieving pain, clearing away heat, and relieving restlessness. Cassia seed and Cannabis sativa seed in the prescription can clear the liver and improve eyesight, and moisten the intestines and promote bowel movements.

[0003] The quality standard for Danxi Granules establishes thin-layer chromatography identification of Danshen (Salvia miltiorrhiza), Achyranthes bidentata (Achyranthes bidentata), Epimedium brevicornum (Horny goat weed), Gardenia jasminoides (Gardenia jasminoides), and Cassia seed, as well as HPLC content determination of salvianolic acid B and gastrodin. While the TLC and content determination procedures are complex and pretreatment methods are cumbersome, they present numerous influencing factors. While there are also reports of simultaneous HPLC determination of eight components in Danxi Granules, this approach has limited comprehensive quality control and evaluation. Currently, there are no reports of studies on Danxi Granule fingerprints. This present invention establishes a Danxi Granule HPLC-DAD-ELSD fingerprint using HPLC, and analyzes it using a Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. This provides a valuable foundation for overall quality control of Danxi Granule compound preparations. Summary of the Invention

[0004] To address the above-mentioned technical problems, the present invention provides a method for establishing an HPLC-DAD-ELSD fingerprint of Danxi Granules, as well as a fingerprint obtained by this method. This fingerprint comprehensively reflects the overall chemical information of the Danxi Granules HPLC-DAD-ELSD fingerprint, and can be used to evaluate and control the quality of Danxi Granules, ensuring their stability, safety, and efficacy.

[0005] The present invention provides a method for establishing a HPLC-DAD-ELSD fingerprint of Danxi granules, comprising the following steps:

[0006] (1) Preparation of reference solution:

[0007] Accurately weigh appropriate amounts of gallic acid, geniposide, salvianolic acid B, icariin, paeonol, and tanshinone IIA reference substances into a brown volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and prepare reference solution 1; accurately weigh appropriate amounts of danshensu, paeoniflorin, ginsenoside Ro, and cassia bark reference substances into a brown volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and prepare reference solution 2;

[0008] (2) Preparation of test solution:

[0009] Take Danxi granules, grind them into powder, weigh it accurately, put it into a stoppered conical flask, add methanol, and extract it by ultrasonic for 40-50 minutes. Make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution;

[0010] (3) HPLC-DAD-ELSD tandem detection:

[0011] The reference solution and the test solution obtained in steps (1) and (2) were respectively drawn and injected into a high performance liquid chromatograph, and the DAD and ELSD dual channels were used for serial determination, and the chromatogram was recorded; wherein the chromatographic conditions were: C 18 Reversed-phase column, mobile phase A: acetonitrile, mobile phase B: 0.05-0.1% formic acid in water; flow rate: 0.8-1.0 mL / min, column temperature: 30-35°C, injection volume: 10 μl, analysis time: 135 min, DAD detection: 240-260 nm; ELSD conditions: drift tube temperature: 40-55°C, gas flow rate: 1.5 L / min, gain: 8-10; elution was performed using the following gradient elution method:

[0012]

[0013] (4) Establishment of HPLC-DAD-ELSD fingerprint

[0014] 10 μl of each of the reference solution and the test solution obtained in steps (1) and (2) were taken and injected into the high performance liquid chromatograph respectively. According to the high performance liquid chromatography method, DAD and ELSD dual-channel serial determination was performed and the chromatogram was recorded. The Chinese medicine chromatographic fingerprint similarity evaluation system was used for analysis to establish a dual-channel fingerprint.

[0015] In the method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules of the present invention, the method for preparing the reference solution in step (1) is preferably:

[0016] Accurately weigh appropriate amounts of gallic acid, geniposide, salvianolic acid B, icariin, paeonol, and tanshinone IIA reference substances, put them into a brown volumetric flask, and add methanol to prepare reference substance solutions 1 with concentrations of 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.05 mg / ml, 0.10 mg / ml, and 0.015 mg / ml, respectively; accurately weigh appropriate amounts of danshensu, paeoniflorin, ginsenoside Ro, and cassia bark reference substances, put them into a brown volumetric flask, and add methanol to prepare reference substance solutions 2 with concentrations of 0.10 mg / ml, 0.015 mg / ml, 0.015 mg / ml, and 0.015 mg / ml, respectively.

[0017] The preparation method of the test solution in step (2) is preferably:

[0018] Take Danxi granules of this product, grind them into powder, take about 2.0g of powder, weigh it accurately, put it into a stoppered conical flask, add 50ml of methanol, weigh it, and extract it ultrasonically (power 250W, frequency 40kHz) for 45 minutes, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0019] Step (3) HPLC-DAD-ELSD tandem detection is preferably:

[0020] The reference solution and the test solution obtained in steps (1) and (2) were respectively drawn and injected into a high performance liquid chromatograph, and the DAD and ELSD dual channels were used for serial determination, and the chromatogram was recorded; wherein, the chromatographic conditions were: CAPCELL PAK C 18 MGⅡ column (5 μm, 4.6 mm × 250 mm); mobile phase: acetonitrile (A)-0.05% formic acid aqueous solution (B); flow rate: 0.8 mL / min; column temperature: 35°C, injection volume: 10 μl, analysis time: 135 min, detection by DAD at 254 nm; ELSD conditions: drift tube temperature: 45°C, gas flow rate: 1.5 L / min, gain: 9; gradient elution:

[0021]

[0022] The establishment of step (4) HPLC-DAD-ELSD fingerprint is preferably:

[0023] 10 μl of each of the reference solution and the test solution obtained in steps (1) and (2) were taken and injected into the high performance liquid chromatograph respectively. According to the high performance liquid chromatography method, DAD and ELSD dual-channel serial determination was performed and the chromatogram was recorded. The Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 version) was used for analysis to establish a dual-channel fingerprint [(reference spectrum: S1, control spectrum generation method: average, time window width: 0.2 min), generate the control spectrum, and establish the fingerprint spectrum by multi-point correction method). The chromatogram fingerprint is shown in Figure 1-5 ]. The fingerprint showed a total of 17 common peaks, and the similarity results are shown in Table 2. A total of 10 peaks were marked under DAD (254 nm), peak 1 was gallic acid (retention time 8.672 min), peak 2 was geniposide (retention time 30.072 min), peak 3 was salvianolic acid B (retention time 61.214 min), peak 4 was icariin (retention time 71.975 min), peak 5 was paeonol (retention time 79.625 min), and peak 9 was tanshinone ⅡA (retention time 116.740 min). Seven peaks were marked under ELSD, peak 1 was tanshinone (retention time 13.261 min), peak 3 was paeoniflorin (retention time 36.343 min), peak 6 was ginsenoside Ro (retention time 81.188 min), and peak 7 was cassia bark (retention time 84.741 min).

[0024] Based on the previously reported "Simultaneous Determination of Eight Components in Danxi Granules by High-Performance Liquid Chromatography," a high-performance liquid chromatography (HPLC) method was established for the simultaneous determination of gastrodin, geniposide, salvianolic acid B, icariin, aurantin, tanshinone I, cryptotanshinone, and tanshinone IIA in Danxi Granules. Methods: The test samples were extracted with 70% methanol. An Agilent ZORBAXSB-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase consisted of acetonitrile-0.2% aqueous phosphoric acid in a gradient elution system at a flow rate of 1.0 mL / min. -1 Column temperature: 30℃, detection wavelengths: 220, 238, 280nm. The eight components determined by this method are Gastrodia elata, Gardenia jasminoides, Salvia miltiorrhiza, Epimedium, and Cassia seed.

[0025] The technical solution of the present invention establishes a fingerprint of Danxi Granules, marks 17 common peaks, and identifies 10 peaks of six medicinal materials (6 common peaks are identified under DAD - Peak 1: gallic acid in Paeonia suffruticosa and Paeonia lactiflora; Peak 2: geniposide in Gardenia jasminoides; Peak 3: salvianolic acid B in Paeonia suffruticosa; Peak 4: icariin in Epimedium; Peak 5: paeonol in Paeonia suffruticosa; Peak 9: tanshinone IIA in Salvia miltiorrhiza; 4 common peaks are identified under ELSD - Peak 1: tanshinone in Salvia miltiorrhiza; Peak 3: paeoniflorin in Paeonia lactiflora; Peak 6: ginsenoside Ro in Achyranthes bidentata; Peak 7: citronellol in Cassia seed), which more completely and comprehensively reflects the multiple components of Danxi Granules.

[0026] The beneficial effects of the present invention are:

[0027] (1) Traditional Chinese medicine fingerprint is currently one of the effective methods for comprehensively evaluating the consistency and stability of the quality of traditional Chinese medicine and its preparations. The multi-index fingerprint control mode of traditional Chinese medicine compound preparations has been gradually applied to its quality standards. The technical solution of this application is based on the theory of TCM compatibility of "monarch, minister, adjuvant and envoy", combined with the principle of confirming the components contained in the monarch medicine as the main component of traditional Chinese medicine quality markers, while taking into account the components contained in the minister, adjuvant and envoy medicines, and comprehensively evaluating and analyzing the 17 components in the compound as target components. Based on the analysis of 43 batches of samples, the technical solution explored multiple method conditions and established a multi-index component control mode of HPLC-DAD-ELSD fingerprint spectrum for Danxixi granules, providing data support for the material basis, batch difference research and overall quality control of Danxixi granules compound preparations.

[0028] The technical solution uses high-performance liquid chromatography and DAD and ELSD dual-channel detectors in series for simultaneous detection, which more comprehensively reflects the multi-component characteristics and batch differences of Danxi Granules, provides a basis for further raw material control and process impact research, improves the quality consistency and stability of Danxi Granules, and thus effectively ensures the quality and clinical efficacy of Danxi Granules.

[0029] (2) The fingerprint establishment method of the present invention is simple in pre-treatment of the sample, the characteristic components are retained intact, and the sample solution is stable;

[0030] (3) The HPLC method has high precision, good reproducibility, and certain specificity; the characteristic peaks in the obtained fingerprint spectrum are well separated;

[0031] (4) The fingerprint established by the present invention marks a total of 17 common peaks, has a large amount of information, and more completely retains the chemical components in the test solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: HPLC-DAD-ELSD chromatogram, wherein Figure A is the HPLC-DAD mixed reference substance (1 gallic acid, 2 gardenoside, 3 salvianolic acid B, 4 icariin, 5 paeonol, 9 tanshinone ⅡA), Figure B is the HPLC-ELSD mixed reference substance (11 danshensu, 13 paeoniflorin, 16 ginsenoside Ro, 17 aurantium dulcisin), Figure C is the HPLC-DAD sample chromatogram, and Figure D is the HPLC-ELSD sample chromatogram.

[0033] Figure 2 : HPLC-DAD fingerprints of 43 batches of Danxi granules.

[0034] Figure 3 : HPLC-ELSD fingerprints of 43 batches of Danxi granules.

[0035] Figure 4 : HPLC-DAD reference fingerprint of Danxi Granules, including 1 gallic acid, 2 gardeniaside, 3 salvianolic acid B, 4 icariin, 5 paeonol, and 9 tanshinone ⅡA.

[0036] Figure 5 : HPLC-ELSD control fingerprint of Danxi granules, including 1 danshensu, 3 paeoniflorin, 6 ginsenoside Ro, and 7 aurantin. DETAILED DESCRIPTION

[0037] Example 1

[0038] 1. Instruments and reagents

[0039] Instruments: Shimadzu LC-20AD high performance liquid chromatograph, SPD-M20A detector, ELSD-LT II evaporative light scattering detector.

[0040] Reference substances and reference medicinal materials: gallic acid (batch number: 110831-201906, 91.5%), geniposide (batch number: 110749-201718, 97.6%), salvianolic acid B (batch number: 110562-201917, 96.6%), icariin (batch number: 110737-202017, 98.1%), paeonol (batch number: 110708-201908, 99.8%), dan Shenone ⅡA (batch number: 110766-202022, 98.9%), paeoniflorin (batch number: 110736-201943, 95.1%), danshensu (batch number: 20101, 99.3%), ginsenoside Ro (batch number: 20051, 96.0%), aurantium cassiae (batch number: 20031, 98.0%), red peony root (batch number: 121093-201804), Gardenia jasminoides (120986- 201610), Danshen (120923-201816), Epimedium (121632-201502), Achyranthes bidentata (batch number: 121066-201809), Cassia seed (batch number: 121011-201807), Gastrodia elata (batch number: 120944-201611), Paeonia suffruticosa (batch number: 121490-201603), Chuanxiong (batch number: 120918-201813), Raw Rehmannia root (batch number: 121180-201506), mistletoe (batch number: 121075-201604), hemp seed (batch number: 12109790-202006), danshensu, ginsenoside Ro, and cassia bark reference substances were purchased from Zhuhai Anzhe Biotechnology Co., Ltd., and the remaining reference substances and reference medicinal materials were purchased from the China Food and Drug Inspection Institutes. The reagents methanol and formic acid were of analytical grade, as were water (ultrapure water) and acetonitrile (chromatographic grade).

[0041] Sample: Danxi Granules (Specification: 10g per bag) 43 batches of samples provided by Jiuzhitang Co., Ltd. Sample information is shown in Table 1.

[0042] Table 1 Sample information

[0043]

[0044]

[0045] 2 Methods and Results

[0046] 2.1 Solution Preparation

[0047] 2.1.1 Preparation of Reference Solution: Accurately weigh appropriate amounts of gallic acid, geniposide, salvianolic acid B, icariin, paeonol, and tanshinone IIA reference substances into a brown volumetric flask, and add methanol to prepare reference solution 1 with concentrations of 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.05 mg / ml, 0.10 mg / ml, and 0.015 mg / ml, respectively. Accurately weigh appropriate amounts of danshensu, paeoniflorin, ginsenoside Ro, and cassia bark reference substances into a brown volumetric flask, and add methanol to prepare reference solution 2 with concentrations of 0.10 mg / ml, 0.015 mg / ml, 0.015 mg / ml, and 0.015 mg / ml, respectively.

[0048] 2.1.2 Preparation of test solution: Take Danxi granules of this product, grind them into powder, take about 2.0g of powder, weigh it accurately, put it into a stoppered conical flask, add 50ml of methanol, weigh it, and extract it by ultrasonic (power 250W, frequency 40kHz) for 45 minutes, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0049] 2.1.3 Control medicinal material solutions are prepared according to the prescribed quantity and preparation process in accordance with the method under "2.1.2" to prepare control medicinal material solutions of Salvia miltiorrhiza, Achyranthes bidentata, Gastrodia elata, Paeonia suffruticosa, Paeonia lactiflora, Ligusticum chuanxiong, Rehmannia root, Epimedium, Morus alba, Gardenia jasminoides, Cassia seed, and Cannabis sativa seed.

[0050] 2.2 Chromatographic conditions CAPCELL PAKC 18 MGⅡ column (5μm, 4.6mm×250mm), mobile phase: acetonitrile (A)-0.05% formic acid aqueous solution (B), gradient elution, using the following gradient elution mode:

[0051]

[0052] Flow rate: 0.8 mL / min; column temperature 35°C, injection volume 10 μl, analysis time 135 min, DAD (254 nm) detection; ELSD conditions: drift tube temperature 45°C, gas flow 1.5 L / min, gain 9. Chromatograms and fingerprints are shown in Figure 1-3 .

[0053] 2.3 HPLC-DAD-ELSD fingerprint establishment and similarity evaluation

[0054] 2.3.1 Precision Test The same test solution (No. S1) was injected and measured six times continuously under the above chromatographic conditions. The RSDs of the retention time and relative peak area of each common peak measured under DAD (254 nm) and ELSD were all less than 2.0%, indicating good instrument precision.

[0055] 2.3.2 Stability Test The same test solution (No. S1) was taken and measured in sequence under the above chromatographic conditions at 0, 15, 30, 45, 60h, 75h, and 90h. The RSDs of the retention time and relative peak area of each common peak under DAD (254nm) and ELSD were all less than 2.0%, indicating that the test solution was stable within 90h.

[0056] 2.3.3 Repeatability test Six portions of Danxi granules (No. S1) from the same batch were taken and processed according to the method under "2.1.2". They were measured successively under the above-mentioned chromatographic conditions. The RSDs of the retention time and relative peak area of each common peak under DAD (254 nm) and ELSD were both less than 2.0%, indicating that the method had good repeatability.

[0057] 2.3.4 Fingerprint establishment and similarity analysis Take appropriate amount of 43 batches of Danxi granules samples, prepare the test solution according to the method under "2.1.2", inject and measure according to the law, and use the Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 version) for analysis, establish a dual-channel fingerprint (reference spectrum: S1, reference spectrum generation method: average, time window width: 0.2min), generate a reference spectrum, and establish a fingerprint by multi-point correction method) chromatographic fingerprint is shown in Figure 2. Figure 1-5 A total of 17 shared peaks were identified. The similarity results are shown in Table 2, and the shared peak area results are shown in Table 3. Peaks 1-10 are shared peaks identified by DAD (1: gallic acid, 2: geniposide, 3: salvianolic acid B, 4: icariin, 5: paeonol, 9: tanshinone IIA), and peaks 11-17 are shared peaks identified by ELSD (11: danshensu, 13: paeoniflorin, 16: ginsenoside Ro, 17: aurantin). The similarity values under DAD (254 nm) ranged from 0.906 to 0.998, and under ELSD, from 0.957 to 0.999. The similarity results are shown in Table 2. The overall similarity distribution was above 0.9.

[0058] Table 2 Similarity results

[0059]

[0060] Table 3 Total peak area results

[0061]

[0062]

Claims

1. A method for establishing an HPLC-DAD-ELSD fingerprint of Danxi granules, comprising the following steps: (1) Preparation of reference solution: Accurately weigh appropriate amounts of gallic acid, geniposide, salvianolic acid B, icariin, paeonol, and tanshinone IIA reference substances into a brown volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and prepare reference solution 1; accurately weigh appropriate amounts of danshensu, paeoniflorin, ginsenoside Ro, and cassia bark reference substances into a brown volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and prepare reference solution 2; (2) Preparation of test solution: Take Danxi granules, grind them into powder, weigh it accurately, put it into a stoppered conical flask, add methanol, and extract it by ultrasonic for 40-50 minutes. Make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution; (3) HPLC-DAD-ELSD tandem detection: The reference solution and the test solution obtained in steps (1) and (2) were respectively drawn and injected into a high performance liquid chromatograph, and the DAD and ELSD dual channels were used for serial determination to record the chromatogram; wherein, Chromatographic conditions: C 18 Reversed-phase column, mobile phase A: acetonitrile, mobile phase B: 0.05-0.1% formic acid in water; flow rate: 0.8-1.0 mL / min, column temperature: 30-35°C, injection volume: 10 μl, analysis time: 135 min, DAD detection: 240-260 nm; ELSD conditions: drift tube temperature: 40-55°C, gas flow rate: 1.5 L / min, gain: 8-10; elution was performed using the following gradient elution method: (4) Establishment of HPLC-DAD-ELSD fingerprint 10 μl of each of the reference solution and the test solution obtained in steps (1) and (2) were taken and injected into the high performance liquid chromatograph respectively. According to the high performance liquid chromatography method, DAD and ELSD dual-channel serial determination was performed and the chromatogram was recorded. The Chinese medicine chromatographic fingerprint similarity evaluation system was used for analysis to establish a dual-channel fingerprint.

2. The method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules according to claim 1, characterized in that: The preparation method of the reference solution in step (1) is as follows: Accurately weigh appropriate amounts of gallic acid, geniposide, salvianolic acid B, icariin, paeonol, and tanshinone IIA reference substances, put them into a brown volumetric flask, and add methanol to prepare reference substance solutions 1 with concentrations of 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.05 mg / ml, 0.10 mg / ml, and 0.015 mg / ml, respectively; accurately weigh appropriate amounts of danshensu, paeoniflorin, ginsenoside Ro, and cassia bark reference substances, put them into a brown volumetric flask, and add methanol to prepare reference substance solutions 2 with concentrations of 0.10 mg / ml, 0.015 mg / ml, 0.015 mg / ml, and 0.015 mg / ml, respectively.

3. The method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules according to claim 1, characterized in that: The preparation method of the test solution in step (2) is: Take Danxi granules, grind them into powder, take about 2.0g of powder, weigh it accurately, put it into a stoppered conical flask, add 50ml of methanol, weigh the weight, and extract it ultrasonically for 45 minutes at a power of 250W and a frequency of 40kHz. Make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

4. The method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules according to claim 1, characterized in that: The chromatographic column in step (3) is CAPCELL PAK C 18 MGⅡ chromatographic column, the column specifications are 250mm×4.6mm, 5μm, and the filler is octadecylsilane bonded silica gel.

5. The method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules according to claim 1, characterized in that: In step (3), the mobile phase B is 0.05% formic acid solution, the total flow rate is 0.8 ml / min, and the column temperature is 35°C.

6. The method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules according to claim 1, characterized in that: In the step (3), the detection wavelength under DAD is 254 nm, and evaporative light detection (ELSD) is connected in series, and the drift tube temperature is 45°C.

7. The method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules according to claim 1, characterized in that: The HPLC-DAD-ELSD fingerprint of the Danxi granules showed a total of 17 common peaks, 10 peaks were marked under DAD, peak 1 was gallic acid, peak 2 was gardenoside, peak 3 was salvianolic acid B, peak 4 was icariin, peak 5 was paeonol, and peak 9 was tanshinone ⅡA; 7 peaks were marked under ELSD, peak 1 was tanshinone, peak 3 was paeoniflorin, peak 6 was ginsenoside Ro, and peak 7 was cassia bark extract.

8. The method for establishing the HPLC-DAD-ELSD fingerprint of Danxi granules according to claim 1 comprises the following steps: (1) Preparation of reference solution: Accurately weigh appropriate amounts of gallic acid, geniposide, salvianolic acid B, icariin, paeonol, and tanshinone IIA reference substances into a brown volumetric flask, and add methanol to prepare reference substance solutions 1 with concentrations of 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.05 mg / ml, 0.10 mg / ml, and 0.015 mg / ml, respectively; accurately weigh appropriate amounts of danshensu, paeoniflorin, ginsenoside Ro, and cassia bark reference substances into a brown volumetric flask, and add methanol to prepare reference substance solutions 2 with concentrations of 0.10 mg / ml, 0.015 mg / ml, 0.015 mg / ml, and 0.015 mg / ml, respectively; (2) Preparation of test solution: Take Danxi granules, grind them into powder, take about 2.0g of powder, weigh accurately, place in a stoppered conical flask, add 50ml of methanol, weigh the weight, and extract by ultrasonication at a power of 250W and a frequency of 40kHz for 45 minutes. Make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution; (3) HPLC-DAD-ELSD tandem detection: The reference solution and the test solution obtained in steps (1) and (2) were respectively drawn and injected into a high performance liquid chromatograph, and the DAD and ELSD dual channels were used for serial determination to record the chromatogram; wherein, Chromatographic conditions: CAPCELLPAKC 18 MGⅡ column, 5 μm, 4.6 mm × 250 mm; mobile phase: acetonitrile A-0.05% formic acid aqueous solution B; flow rate: 0.8 mL / min; column temperature: 35°C, injection volume: 10 μl, analysis time: 135 min, detection by DAD at 254 nm; ELSD conditions: drift tube temperature: 45°C, gas flow rate: 1.5 L / min, gain: 9; gradient elution: (4) Establishment of HPLC-DAD-ELSD fingerprint 10 μl of each of the reference solution and the test solution obtained in steps (1) and (2) were taken and injected into the HPLC instrument respectively. The chromatograms were recorded according to the HPLC method using the dual channels of DAD and ELSD. The Chinese medicine chromatographic fingerprint similarity evaluation system was used for analysis, and a dual-channel fingerprint was established; the HPLC-DAD-ELSD fingerprint of the Danxi granules showed a total of 17 common peaks; a total of 10 peaks were marked under DAD, peak 1 was gallic acid, peak 2 was gardenoside, peak 3 was salvianolic acid B, peak 4 was icariin, peak 5 was paeonol, and peak 9 was tanshinone IIA; 7 peaks were marked under ELSD, peak 1 was danshensu, peak 3 was paeoniflorin, peak 6 was ginsenoside Ro, and peak 7 was cassia bark extract.

Images