Determination method and application of a reference sample of daotan decoction and fingerprint spectrum of multiple components

Abstract

The application belongs to the technical field of traditional Chinese medicine preparation, quality control and detection of components, and discloses a determination method and application of a fingerprint spectrum and multi-component content of a Dautan decoction reference sample. The determination method has good precision, repeatability and stability by optimizing the extraction solvent, extraction mode and chromatography mobile phase of the Dautan decoction reference sample. The obtained fingerprint spectrum has a clean background, good separation effect of common peaks, and the percentage of the area of common characteristic peaks in the total chromatographic peak area is greater than or equal to 85%. Moreover, the content of synephrine, hesperidin, neohesperidin and naringin can be determined. The determination method can effectively evaluate the quality of the Dautan decoction and make up for the blank of the prior art.

Description

Technical Field

[0001] This invention belongs to the technical field of quality control and detection of traditional Chinese medicine preparation and its components, and relates to a fingerprint spectrum of a reference sample of Daotan Decoction and a method and application for determining the content of multiple components; specifically, it relates to a method for constructing a fingerprint spectrum of Daotan Decoction, and a method for determining the content of synephrine, hesperidin, naringin and neohesperidin in Daotan Decoction. Background Technology

[0002] The reference sample of Daotan Decoction contains a variety of effective chemical components. For example, flavonoids such as hesperidin contained in tangerine peel and immature bitter orange can inhibit mucus secretion, promote airway ciliary transport and serous secretion, and exert expectorant effects through multiple mechanisms. Synephrine has significant physiological activities such as promoting lipid metabolism, raising blood pressure and strengthening the heart, constricting bronchi, and antidepressant effects. The gingerol fraction of ginger is the site of its lung-warming efficacy. Its mechanism of action involves reducing the expression of inflammatory factors in serum, reducing the overexpression of matrix metalloproteinases in lung tissue, and preventing oxidative stress in the trachea, thereby improving the pathological morphology of lung tissue and trachea.

[0003] There are currently no research reports on the quality testing of Daotan Decoction. Summary of the Invention

[0004] The primary objective of this invention is to provide a fingerprint spectrum of phlegm-expelling decoction and a method for determining the content of multiple components.

[0005] Another object of the present invention is to provide an application of the above-described measurement method.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for determining the fingerprint spectrum and multi-component content of a phlegm-expelling decoction reference sample, comprising the following steps:

[0008] (1) Construction of fingerprint spectrum:

[0009] A. Preparation of test solution: Take the phlegm-expelling decoction reference sample, add solvent, extract, make up to volume, filter, take the filtrate to obtain the test solution;

[0010] B. Preparation of reference solutions: Take 6-gingerol, naringin, hesperidin, naringin, tangeretin, nobiletin, 3,5,6,7,8,3'4'-heptamethoxyflavone, and neohesperidin standards, dissolve them in solvents and make up to volume to obtain reference solutions.

[0011] C. Chromatographic conditions: The chromatographic column was a C18 column; the mobile phase consisted of acetonitrile in phase A and 0.05–0.1% v / v phosphoric acid aqueous solution in phase B; the elution gradient was: 0–25 min, 20% A; 25–40 min, 20% → 50% A; 40–50 min, 50% → 85% A; the detection wavelength was 280 nm, the flow rate was 0.8–1.2 mL / min, the column temperature was 25–35 °C, and the injection volume was 5–20 μL.

[0012] D. Establishment of fingerprint chromatogram: Using the chromatographic conditions of step C, inject the test solution from step A and the reference solution from step B into the high performance liquid chromatograph; locate and assign the chromatographic peaks in the fingerprint chromatogram of the test sample according to the chromatogram of the reference sample to obtain the fingerprint chromatogram of the phlegm-relieving decoction reference sample.

[0013] (2) Determination of the content of hesperidin, naringin, and neohesperidin:

[0014] E. Chromatographic conditions: The chromatographic column is a C18 column; the mobile phase consists of acetonitrile in phase A and 0.05–0.1% v / v phosphoric acid aqueous solution in phase B; the elution gradient is 0–25 min, 20% A; the detection wavelength is 280 nm, the flow rate is 0.8–1.2 mL / min, the column temperature is 25–35 °C, and the injection volume is 5–20 μL.

[0015] F. Preparation of reference standard curves: Using the chromatographic conditions in step E, a series of reference standard solutions of hesperidin, naringin, and neohesperidin were prepared by high performance liquid chromatography to obtain the standard curves of naringin, hesperidin, and neohesperidin.

[0016] G. Determination of the contents of hesperidin, naringin and neohesperidin: The test solution prepared in step A was tested by high performance liquid chromatography under the chromatographic conditions of step E. The contents of hesperidin, naringin and neohesperidin in the reference sample of Daotan Decoction were obtained according to the reference standard curve.

[0017] (3) Determination of synephrine content:

[0018] H. Preparation of the test solution: Take the phlegm-expelling decoction reference sample, extract with water, dilute to volume, and filter; take the filtrate for column chromatography, elute with water, collect the eluent and dilute to volume with water to obtain the test solution;

[0019] I. Preparation of reference solution: Dissolve synephrine in water to obtain synephrine solutions of a series of concentrations;

[0020] J. Chromatographic conditions: The chromatographic column was a C18 column; the mobile phase was a mixture of methanol and potassium dihydrogen phosphate buffer at a volume ratio of 50:50, with the potassium dihydrogen phosphate buffer consisting of 0.6 g / L potassium dihydrogen phosphate, 1.0 g / L sodium dodecyl sulfate, and 1 mL / L glacial acetic acid; the detection wavelength was 275 nm; the flow rate was 0.8–1.2 mL / min; the column temperature was 25–35 °C; and the injection volume was 5–20 μL.

[0021] K. Determination of synephrine content: The series of synephrine solutions prepared in step I were detected by high performance liquid chromatography under the chromatographic conditions of step J to obtain the synephrine standard curve; the test solution prepared in step H was detected by high performance liquid chromatography under the chromatographic conditions of step J, and the synephrine content in the phlegm-relieving decoction reference sample was obtained according to the synephrine standard curve.

[0022] The reference sample of Daotan Decoction mentioned in step (1) is a sample obtained by extracting and processing the Daotan Decoction Chinese medicine composition.

[0023] The aforementioned expectorant decoction composition includes Pinellia ternata, Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum, Poria cocos, and ginger slices; preferably, it is prepared by mixing Pinellia ternata, Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum, Poria cocos, and ginger slices in a mass ratio of 6.19:1.55:1.55:1.55:1.55:10.

[0024] The preferred extraction method is decoction.

[0025] The preferred steps for decocting the ingredients are as follows: Mix Pinellia ternata, Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum, Poria cocos, ginger slices, and water, bring to a boil over high heat, and then simmer over low heat.

[0026] The preferred form of the prepared Pinellia ternata is prepared Pinellia ternata granules.

[0027] The preferred form of the *Citrus aurantium* is granules.

[0028] The orange-red color is preferably orange-red granules.

[0029] The prepared Arisaema is preferably prepared Arisaema granules.

[0030] The preferred form of the red poria is red poria granules.

[0031] The amount of water used is preferably 10 to 30 times the mass of the Chinese medicinal materials.

[0032] The preferred simmering time is 50-60 minutes.

[0033] The process described involves concentration followed by freeze drying.

[0034] The freeze-drying conditions are a temperature of -70 to -80°C and a pressure of 0 to 25 Pa; preferably, a temperature of -70 to -80°C and a pressure of 0 to 20 Pa.

[0035] The preferred freeze-drying time is: freezing time 8-18 hours and drying time 48-54 hours.

[0036] The sample was a lyophilized powder.

[0037] The solvent used in step (1)A is preferably a 30-70% methanol solution or a 30-70% ethanol solution; more preferably a 50% methanol solution or a 50% ethanol solution.

[0038] The amount of solvent used in step (1)A is preferably calculated as 0.005 to 0.01 g of the expectorant decoction reference sample / mL of solvent; more preferably, it is calculated as 0.008 g of the expectorant decoction reference sample / mL of solvent.

[0039] The extraction methods described in step (1)A include reflux extraction and ultrasonic extraction; ultrasonic extraction is preferred.

[0040] The preferred conditions for ultrasonic extraction are: power 200-400W, frequency 30-50kHz, and extraction time 20-40min; more preferably, power 300W, frequency 40kHz, and extraction time 30min.

[0041] The solvent described in step (1)B is the same as the solvent described in step (1)A.

[0042] The chromatographic column used in step (1) C is preferably COSMOSIL 5C18-MS-Ⅱ.

[0043] The preferred chromatographic conditions described in step (1) C are: detection wavelength 280 nm, flow rate 0.9–1.1 mL / min, column temperature 28–32 °C, and injection volume 10–15 μL; more preferably, the detection wavelength is 280 nm, flow rate is 1.0 mL / min, column temperature is 30 °C, and injection volume is 10 μL.

[0044] In step (1) D, peak 2 is naringin, peak 3 is naringin, peak 4 is hesperidin, peak 5 is neohesperidin, peak 10 is norihesperidin, peak 11 is 6-gingerol, peak 12 is 3,5,6,7,8,3',4'-heptamethoxyflavone, and peak 13 is citrusin.

[0045] In the fingerprint spectrum, peak 5 is used as the reference peak, and the relative retention times are as follows: peak 1 0.43, peak 2 0.61, peak 3 0.73, peak 4 0.82, peak 5 1.00, peak 6 1.71, peak 7 1.85, peak 8 1.90, peak 9 1.93, peak 10 2.13, peak 11 2.15, peak 12 2.18, peak 13 2.22.

[0046] The preferred chromatographic conditions described in step (2) E are: detection wavelength 280 nm, flow rate 0.9-1.1 mL / min, column temperature 28-32 °C, and injection volume 10-15 μL; more preferably, the preferred chromatographic conditions are: detection wavelength 280 nm, flow rate 1.0 mL / min, column temperature 30 °C, and injection volume 10 μL.

[0047] The standard curve for naringin mentioned in step (2)F is y = 154.25x + 0.2194; coefficient of determination: R 2 =1.

[0048] The standard curve for hesperidin mentioned in step (2)F is y = 155.88x + 0.0935; coefficient of determination: R 2 =1.

[0049] The standard curve for the neohesperidin described in step (2)F is y = 164.07x - 0.1125; coefficient of determination: R 2 =1.

[0050] In step (3)H:

[0051] The water is preferably purified water or ultrapure water.

[0052] The filtration process involves using a 0.22μm filter membrane.

[0053] The column chromatography described is polyamide column chromatography.

[0054] The polyamide has a mesh size of 60-90.

[0055] The chromatographic conditions described in step (3)J are: detection wavelength 275 nm, flow rate 0.9–1.1 mL / min, column temperature 28–32 °C, and injection volume 10–15 μL; more preferably, the detection wavelength is 275 nm, the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL.

[0056] The standard curve of Sinfeldt mentioned in step (3) K is y = 77.644x + 0.0745.

[0057] A fingerprint spectrum of a phlegm-expelling decoction reference sample was obtained using the above-mentioned determination method.

[0058] The fingerprint spectrum of the above-mentioned Daotan Decoction and the determination method of the content of multiple components, or the application of the above-mentioned fingerprint spectrum in the quality control of Daotan Decoction.

[0059] The present invention has the following advantages and effects compared with the prior art.

[0060] (1) The fingerprint spectrum obtained by the method of constructing the fingerprint spectrum of the reference sample of Daotan Decoction provided by the present invention has a clean background, good separation effect of common peaks, and the percentage of common characteristic peak area to total chromatographic peak area is ≥85%; and has good precision, repeatability and stability, providing a new method for the quality control of Daotan Decoction.

[0061] (2) This invention establishes a method for determining the content of the main active ingredients of the herbs Citrus aurantium and Citrus reticulata in the formula, namely synephrine, hesperidin, neohesperidin and naringin, which can effectively evaluate the quality of Daotan Decoction and fill the gap in the prior art. Attached Figure Description

[0062] Figure 1 This is a chromatogram of the extract obtained by extracting the lyophilized standard sample powder with anhydrous methanol.

[0063] Figure 2 This is a chromatogram of the extract obtained by extracting the lyophilized standard sample powder with 50% methanol.

[0064] Figure 3 This is a chromatogram of the extract obtained by extracting the lyophilized standard sample powder with anhydrous ethanol.

[0065] Figure 4 This is a chromatogram of the extract obtained by extracting the lyophilized standard sample powder with 50% ethanol.

[0066] Figure 5 This is a chromatogram of the extract obtained by extracting the lyophilized powder of the reference sample from pure water.

[0067] Figure 6 These are chromatograms of extracts obtained from freeze-dried reference sample powders using different extraction methods; the blue curve represents the extract obtained from ultrasonic extraction, and the black curve represents the extract obtained from reflux extraction.

[0068] Figure 7 The chromatograms are of the extracts obtained from the freeze-dried powder of the reference sample at the same extraction time; the red curve represents the extract obtained after 15 min of extraction, the blue curve represents the extract obtained after 30 min of extraction, and the black curve represents the extract obtained after 60 min of extraction.

[0069] Figure 8 These are chromatograms obtained using phosphoric acid solutions of different concentrations; the blue curve represents the 0.05% phosphoric acid solution, and the black curve represents the 0.1% phosphoric acid solution.

[0070] Figure 9 This is a specific chromatogram of Daotan Decoction; the dark red curve represents the blank solution, and the black curve represents the test solution.

[0071] Figure 10 This is a peak identification spectrum diagram.

[0072] Figure 11 This is a peak attribution chromatogram; among them, curve S1 is the test sample solution, curve S2 is the negative solution lacking ginger, curve S3 is the negative solution lacking processed arisaema, curve S4 is the negative solution lacking both processed arisaema and ginger, curve S5 is the negative solution lacking immature bitter orange, curve S6 is the negative solution lacking tangerine peel, curve S7 is the double negative solution lacking both tangerine peel and immature bitter orange, curve S8 is the negative solution lacking processed pinellia, and curve S9 is the negative solution lacking red poria.

[0073] Figure 12 This is a common pattern diagram of the reference samples for Daotan Decoction.

[0074] Figure 13 This is the fingerprint spectrum of the reference sample for Daotan Decoction; among them, peak 2 is naringin, peak 3 is naringin, peak 4 is hesperidin, peak 5 is neohesperidin, peak 10 is norihesperidin, peak 11 is 6-gingerol, peak 12 is 3,5,6,7,8,3',4'-heptamethoxyflavone, and peak 13 is hesperidin.

[0075] Figure 14 This is a specificity test diagram; among them, 1-naringin, 2-hesperidin, 3-neohesperidin; S1 is the reference sample of Daotan Decoction, S2 is the blank solvent, S3 is the negative solution lacking Citrus aurantium, S4 is the negative solution lacking Citrus reticulata, and S5 is the double negative solution lacking both Citrus aurantium and Citrus reticulata.

[0076] Figure 15 This is a chromatogram of the purity of naringin peak; the upper part is the test sample, and the lower part is the reference sample.

[0077] Figure 16 This is a spectrum of hesperidin peak purity; the top is the test sample, and the bottom is the reference sample.

[0078] Figure 17 This is a chromatogram of the purity of neohesperidin peak; the top is the test sample, and the bottom is the reference sample.

[0079] Figure 18 This is the standard curve of naringin.

[0080] Figure 19 This is the standard curve of hesperidin.

[0081] Figure 20 This is the standard curve of neohesperidin.

[0082] Figure 21This is a specific chromatogram of synephrine content; where S1 is blank solvent, S2 is synephrine reference solution, S3 is phlegm-releasing decoction reference sample solution, S4 is negative sample solution lacking immature bitter orange, S5 is negative sample solution lacking tangerine peel, and S6 is double negative sample solution lacking both tangerine peel and immature bitter orange.

[0083] Figure 22 This is a spectrum of the purity of synephrine peaks; the top is the test sample, and the bottom is the reference sample.

[0084] Figure 23 This is the Sievelin standard curve. Detailed Implementation

[0085] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0086] Example 1

[0087] 1. Instruments and Materials

[0088] 1.1 The instruments are shown in Table 1:

[0089] Table 1 Instruments

[0090]

[0091] 1.2 The materials are shown in Table 2:

[0092] Table 2 Materials

[0093]

[0094] 2. Preparation of lyophilized reference sample powder

[0095] Prescription: 6.19g of Pinellia ternata, 1.55g each of Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum, and Poria cocos slices, and 10g of fresh ginger slices.

[0096] Pre-processing: Mix and crush Pinellia ternata, Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum and Poria cocos, and pass all of them through a 4-mesh sieve. No more than 20% of the particles should pass through a No. 1 sieve.

[0097] Extraction: Put the coarse pieces of the above-mentioned medicinal slices and ginger slices into a decoction pot, add 600mL of water, decoct on an electric ceramic stove (2200W) for 3-10 minutes until boiling, then simmer on an electric ceramic stove (400W) for 50-60 minutes. Filter while hot (using a 200-mesh filter membrane) to obtain 285-315mL of decoction.

[0098] Concentration: Five portions of the decoction were prepared and combined. They were then concentrated under reduced pressure at 70℃ and -0.08 to -0.1 MPa until the ratio of feed amount to concentrate amount was 1g:2 to 3g, thus obtaining the concentrate.

[0099] Freeze-drying: The concentrated solution obtained above was freeze-dried at -70 to -80°C and 20 Pa to obtain the freeze-dried powder of the reference sample. The freezing time was 8 to 18 hours, and the drying time was 48 to 54 hours.

[0100] 3. Fingerprint pattern

[0101] 3.1 Investigation of Extraction Solvents

[0102] 3.1.1 Take 0.4 g of the lyophilized powder of the reference sample, accurately weigh it, and put it into a stoppered conical flask. Accurately add different extraction solvents (anhydrous methanol, 50% v / v methanol, anhydrous ethanol, 50% v / v ethanol, and 50 mL of water), weigh them, sonicate (power 300W, frequency 40kHz) for 30 min, cool, add the corresponding extraction solvent to make up the weight, filter (using a 0.22μm filter membrane), and take the filtrate to obtain the test solution.

[0103] 3.1.2 Chromatographic conditions

[0104] Instrument: Thermo Ultimate 3000;

[0105] Column: COSMOSIL 5C18-MS-Ⅱ (4.6×250mm, 5μm);

[0106] Mobile phase: Phase A is acetonitrile, and Phase B is a 0.05% v / v aqueous solution of phosphoric acid.

[0107] The elution gradient is shown in Table 3:

[0108] Table 3 Elution gradient

[0109]

[0110] Detection wavelength: 280 nm; flow rate: 1.0 mL / min; column temperature: 30 ℃; injection volume: 10 μL.

[0111] 3.1.3 Results

[0112] The results are as follows Figures 1-5 The results showed that, compared with anhydrous methanol, anhydrous ethanol, and pure water, the extraction effect of the reference sample extracted with 50% methanol and 50% ethanol was better, with more peaks than water and higher peak areas for each characteristic peak than with methanol and anhydrous ethanol. There was no significant difference between the two extraction solvents, 50% methanol and 50% ethanol, and 50% methanol was ultimately selected as the extraction solvent.

[0113] 3.2 Examination of Extraction Methods

[0114] Accurately weigh 0.4 g of the lyophilized reference sample powder into a stoppered conical flask, add 50 mL of 50% methanol, weigh again, and then sonicate (300 W, 40 kHz) and reflux for 30 min respectively. After cooling, add 50% methanol to make up the weight, filter, and collect the filtrate. Inject and detect according to the chromatographic conditions in “3.1.2”. The results are as follows. Figure 6 As shown.

[0115] The results showed that the chromatographic peaks of the reference samples prepared by different extraction methods were well separated, the baselines were stable, and there were no significant differences. Ultrasonic extraction was ultimately selected as the extraction method.

[0116] 3.3 Examination of extraction time

[0117] Accurately weigh 0.4 g of the lyophilized reference sample powder into a stoppered conical flask, add 50 mL of 50% methanol, weigh again, and sonicate (300 W, 40 kHz) for 15, 30, and 60 min respectively. After cooling, add 50% methanol to make up the weight, filter, and collect the filtrate. Inject and detect according to "3.1.2 Chromatographic Conditions". The results are as follows: Figure 7 As shown.

[0118] The results showed that the chromatographic peaks of the reference samples prepared at different extraction times were well separated, the baselines were stable, and there were no significant differences, indicating that the samples were completely extracted within 15 to 60 minutes. The final extraction time was selected as 30 minutes.

[0119] 3.4 Investigation of different phosphoric acid concentrations under chromatographic conditions

[0120] The reference sample lyophilized powder was used to prepare the test solution according to the preferred sample preparation method described above. Different concentrations (0.05%, 0.1%, v / v) of phosphoric acid solution were used for injection and detection according to the chromatographic conditions in "3.1.2". The results are as follows: Figure 8 As shown.

[0121] The results showed that the chromatographic peaks of the reference samples with different concentrations of phosphoric acid solution had good separation, stable baselines, and no significant differences. Finally, the concentration of phosphoric acid solution was selected as 0.05%.

[0122] 3.5 Method Validation

[0123] Preparation of reference solution: Take an appropriate amount of neohesperidin reference standard, accurately weigh it, and add 50% methanol to prepare a reference solution containing 0.2 mg of neohesperidin per 1 mL.

[0124] Test solution: Accurately weigh 0.4 g of the lyophilized reference sample powder into a stoppered conical flask, accurately add 50 mL of 50% methanol solution, weigh, sonicate (300 W power, 40 kHz frequency) for 30 min, cool, add 50% methanol solution to make up the weight, filter, and take the filtrate to obtain the test solution.

[0125] 3.6 Methodological Examination

[0126] 3.6.1 Specificity Test

[0127] Prepare the test solution and blank solvent according to section "3.5", and inject the sample under the chromatographic conditions described in section "3.1.2". Figure 9 The results showed that the blank solvent did not interfere with the characteristic peaks, and the method used to determine the characteristic peaks in this product is specific.

[0128] 3.7 Peak Identification and Peak Attribution

[0129] 3.7.1 Peak Identification

[0130] Take the test sample solution and the reference solutions of 6-gingerol, naringin, hesperidin, naringenin, tangeretin, 3,5,6,7,8,3'4'-heptamethoxyflavone, and neohesperidin, and inject them according to the chromatographic conditions in section "3.1.2". Identify each characteristic peak. The results are as follows: Figure 10 As shown.

[0131] 3.7.2 Peak Attribution

[0132] Take the test sample solution (Daotan Decoction) and the negative solution lacking Citrus reticulata peel (i.e., the solution lacking Citrus reticulata peel in "2. Preparation of Standard Sample Lyophilized Powder" and prepared according to the test sample solution, the same below), the negative solution lacking Citrus aurantium, the double negative solution lacking Citrus reticulata peel and Citrus aurantium, the negative solution lacking Pinellia ternata, the negative solution lacking processed Arisaema heterophyllum, the negative solution lacking Poria cocos, the negative solution lacking fresh ginger, and the double negative solution lacking processed Arisaema heterophyllum and fresh ginger. Inject the samples according to the chromatographic conditions in "3.1.2", and assign each characteristic peak to the medicinal flavor. The results are shown in the figure. Figure 11 As shown in Table 4.

[0133] Table 4. Summary Table of Peak Identification and Peak Attribution

[0134] The results showed that, based on the comparison of individual herbal determinations, peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 13 were identified as chromatographic peaks of Citrus aurantium; peak 11 was identified as chromatographic peak of Zingiber officinale; and peaks 4 and 12 were identified as orange-red peaks. The relative retention times of each chromatographic peak showed little difference.

[0135] 3.8 Instrument precision test

[0136] Take the same test solution and inject it according to the chromatographic conditions in section “3.1.2”. Inject it 6 times consecutively and calculate the relative retention time and RSD of each characteristic peak. The results are shown in Table 5 below.

[0137] Table 5 Relative retention times of characteristic peaks in precision tests

[0138] The results show that the relative retention time (RSD) of each characteristic peak is less than 0.5%, indicating that the instrument has good precision.

[0139] 3.9 Repeatability Test Take approximately 0.4 g of the powder (6 portions in total), accurately weigh them, prepare the test solution in parallel according to the procedure in section “3.5”, inject the sample according to the chromatographic conditions in section “3.1.2”, and use peak 5 (neohesperidin) as the reference peak to calculate the relative retention time and RSD of each characteristic peak. The results are shown in Table 6 below.

[0140] Table 6 Relative retention times of characteristic peaks in repeatability tests

[0141] The results show that the relative retention time (RSD) of each characteristic peak is less than 1.0%, indicating that the method has good repeatability.

[0142] 3.10 Stability Test Take the same test solution and place it at room temperature for 0, 5, 10, 20, 30 and 50 h respectively. Then inject it according to the chromatographic conditions in section “3.1.2”. Take peak 5 (neohesperidin) as the reference peak and calculate the relative retention time and RSD of each characteristic peak. The results are shown in Table 7 below.

[0143] Table 7 Relative retention times of characteristic peaks in stability tests

[0144] The results showed that the relative retention time (RSD) of each characteristic peak was less than 1.5%, indicating that the test solution was stable within 50 hours.

[0145] 3.11 Establishment and Analysis of Fingerprint Patterns

[0146] Accurately weigh approximately 0.4g (6 portions) of this product powder and prepare the test solution in parallel according to section "3.5". Inject the sample under the chromatographic conditions described in section "3.1.2", record the chromatogram, and import the obtained data into the "Similarity Evaluation System for Fingerprints of Traditional Chinese Medicine" (2012 edition). Using the chromatogram of the Daotan Decoction reference sample (S1) as the reference chromatogram, perform multi-point calibration for matching. Automatically match the chromatographic peaks of other Daotan Decoction reference samples (S2-S5) with the reference chromatogram to generate a control fingerprint chromatogram, and perform similarity analysis. The results are shown in [Figure 1]. Figure 12 and Figure 13 .

[0147] The results showed that the similarity of the lyophilized powders of the six batches of Daotan Decoction reference samples was ≥0.999, indicating good similarity and stable internal quality among the samples. Thirteen common peaks were identified based on the fingerprint chromatograms. After comparison with reference standards, eight chromatographic peaks were identified: naringin (peak 2), naringin (peak 3), hesperidin (peak 4), neohesperidin (peak 5), norihesperidin (peak 10), 6-gingerol (peak 11), 3,5,6,7,8,3',4'-heptamethoxyflavone (peak 12), and hesperidin (peak 13).

[0148] The relative retention times were determined by comparing the retention times of each common peak with those of the reference peak. Thirteen common peaks were identified, with peak 5 (neohesperidin) as the reference peak. The relative retention times of each common peak were as follows: peak 1 (0.43), peak 2 (0.61), peak 3 (0.73), peak 4 (0.82), peak 5 (1.00), peak 6 (1.71), peak 7 (1.85), peak 8 (1.90), peak 9 (1.93), peak 10 (2.13), peak 11 (2.15), peak 12 (2.18), and peak 13 (2.22).

[0149] 4. Content of naringin, hesperidin, and neohesperidin

[0150] 4.1 Solution Preparation

[0151] Test solution: Accurately weigh 0.4 g of the lyophilized reference sample powder into a stoppered conical flask, accurately add 50 mL of 50% methanol solution, weigh, sonicate (300 W power, 40 kHz frequency) for 30 min, cool, add 50% methanol solution to make up the weight, filter, and take the filtrate to obtain the test solution.

[0152] Preparation of hesperidin reference solution: Take an appropriate amount of hesperidin, accurately weigh it, and add 50% methanol to prepare a reference solution containing 0.1 mg of hesperidin per 1 mL.

[0153] Preparation of mixed reference solution of naringin and neohesperidin: Take appropriate amounts of naringin and neohesperidin, accurately weigh them, and add 50% methanol to prepare a mixed reference solution containing 0.2 mg of naringin and 0.2 mg of neohesperidin per 1 mL.

[0154] 4.2 Chromatographic conditions

[0155] Instrument: Thermo Ultimate 3000;

[0156] Column: COSMOSIL 5C18-MS-Ⅱ (4.6×250mm, 5μm);

[0157] Mobile phase: Phase A is acetonitrile, and Phase B is a 0.05% v / v aqueous solution of phosphoric acid.

[0158] The elution gradient is shown in Table 8 below (the first 25 minutes of the elution method for the characteristic chromatogram can be used as the elution gradient for the content determination).

[0159] Table 8 Elution Gradient

[0160]

[0161] Detection wavelength: 280 nm; Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 10 μL.

[0162] 4.3 Methodology Investigation

[0163] 4.3.1 Specificity Investigation

[0164] Prepare the test solution, negative sample solution without Citri Reticulatae Exocarpium, negative sample solution without Aurantii Fructus Immaturus, double-negative sample solution without both Citri Reticulatae Exocarpium and Aurantii Fructus Immaturus, and blank solvent for injection according to item "3.5".

[0165] The results showed that there were no chromatographic peaks in the chromatogram of the negative sample without Aurantii Fructus Immaturus at the retention times corresponding to naringin and neohesperidin; there were chromatographic peaks in the chromatogram of the negative sample without Citri Reticulatae Exocarpium at the retention times corresponding to hesperidin, naringin and neohesperidin; there were no chromatographic peaks in the chromatogram of the double-negative sample without both Citri Reticulatae Exocarpium and Aurantii Fructus Immaturus at the retention times corresponding to hesperidin, naringin and neohesperidin; therefore, it was indicated that naringin and neohesperidin were derived from Aurantii Fructus Immaturus, hesperidin was present in both the two medicinal flavors of Citri Reticulatae Exocarpium and Aurantii Fructus Immaturus, and the negative sample of the phlegm-resolving decoction without Citri Reticulatae Exocarpium and Aurantii Fructus Immaturus and the blank solvent had no interference on the determination of hesperidin, naringin and neohesperidin. The method for determining the contents of hesperidin, naringin and neohesperidin in this product had specificity, as Figure 14 shown.

[0166] 4.3.2 Peak Purity Inspection

[0167] Take the above test solution, inject it into the liquid chromatograph, and perform full wavelength detection with a DAD detector under the above chromatographic conditions to calculate the peak purity. The results showed that no impurity peaks were detected in the chromatograms of hesperidin, naringin and neohesperidin in the sample; it was indicated that under these chromatographic conditions, the chromatographic peak purities of naringin, hesperidin and neohesperidin met the requirements, as shown Figures 15-17 .

[0168] Result analysis: No impurity peaks were detected in the chromatograms of naringin, hesperidin and neohesperidin in the sample; it was indicated that under these chromatographic conditions, the chromatographic peak purities of naringin, hesperidin and neohesperidin met the requirements.

[0169] 4.3.3 Linear Relationship Investigation

[0170] Accurately weigh 20.64 mg of naringin reference standard and add 50% methanol to prepare a naringin reference standard solution with a concentration of approximately 2.0 mg / mL. Accurately measure 1 mL of the above reference standard solution and place it into volumetric flasks of 5 mL, 10 mL, 20 mL, 50 mL, and 100 mL, respectively. Add 50% methanol to the mark and shake well. A total of reference standard solutions with concentrations of approximately 1.983504, 0.3967008, 0.1983504, 0.0991752, 0.03967008, and 0.01983504 mg / mL were obtained.

[0171] Accurately weigh 20.33 mg of neohesperidin reference standard and add 50% methanol to prepare a neohesperidin reference standard solution with a concentration of approximately 2.0 mg / mL. Accurately measure 1 mL of the above reference standard solution and place it into volumetric flasks of 5 mL, 10 mL, 20 mL, 50 mL, and 100 mL, respectively. Dilute to the mark with methanol and shake well. A total of reference standard solutions with concentrations of approximately 2.024868, 0.4049736, 0.2024868, 0.1012434, 0.04049736, and 0.02024868 mg / mL were obtained.

[0172] Accurately weigh 10.64 mg of hesperidin reference standard and add 50% methanol to prepare a hesperidin reference standard solution with a concentration of approximately 1.0 mg / mL. Accurately measure 1 mL of the above reference standard solution and place it into volumetric flasks of 5 mL, 10 mL, 20 mL, 50 mL, and 100 mL, respectively. Dilute to the mark with methanol and mix well. A total of reference standard solutions with concentrations of approximately 1.034208, 0.2068416, 0.1034208, 0.0517104, 0.02068416, and 0.01034208 mg / mL were obtained.

[0173] Accurately pipette 5 μL of each of the above reference solutions and inject it into the liquid chromatograph. Determine the concentration by liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2025 Edition). Plot a standard curve with the concentration of the reference standard (mg / mL) on the x-axis and the peak area on the y-axis. Calculate the regression equation, correlation coefficient, and linear range. See [link to relevant documentation]. Figures 18-20 .

[0174] Figure 18 The regression equation for naringin is: y = 154.25x + 0.2194; the coefficient of determination is R0. 2 =1. The results showed that the linearity of naringin concentration was good in the range of 0.01983504–1.983504 mg / mL.

[0175] Figure 19 In the study, the regression equation for hesperidin was: y = 155.88x + 0.0935; the coefficient of determination was R0. 2=1. The results showed that the linearity of hesperidin concentration was good in the range of 0.01034208–1.034208 mg / mL.

[0176] Figure 20 The regression equation for neohesperidin is: y = 164.07x - 0.1125; the coefficient of determination is R0. 2 =1. The results showed that the neohesperidin concentration exhibited good linearity in the range of 0.02024868–2.024868 mg / mL.

[0177] 4.3.4 Precision Examination

[0178] Take the same test solution and inject it according to the chromatographic conditions in section "3.1.2". Inject it 6 times consecutively. The results show that the peak area RSD of naringin, hesperidin and neohesperidin is less than 2.0%, indicating that the instrument has good precision.

[0179] 4.3.5 Repeatability Test

[0180] Take approximately 0.4 g of the powder (6 portions in total), accurately weigh them, prepare the test solution in parallel according to section “4.1”, and inject the sample according to the chromatographic conditions in section “3.1.2”. The results show that the RSD of the contents of naringin, hesperidin and neohesperidin is less than 2.0%, indicating that the reproducibility of this method is good.

[0181] 4.3.6 Stability Test

[0182] The same test solution was placed at room temperature for 0, 5, 10, 20, 30 and 50 hours and then injected according to the chromatographic conditions in section “3.1.2”. The results showed that the peak areas RSD of naringin, hesperidin and neohesperidin were less than 2.0%, and the test solution had good stability within 50 hours.

[0183] 4.3.7 Accuracy Test

[0184] Repeatability testing revealed that the non-dried sample of Daotan Decoction contained 23.98 mg / g of naringin, 6.20 mg / g of hesperidin, and 36.67 mg / g of neohesperidin.

[0185] 4.3.7.1 Naringin

[0186] Accurately weigh 25.62 mg of naringin reference standard (batch number: 110722-202417, purity: 96.1%) and place it in a 50 mL volumetric flask. Dissolve and dilute to the mark with 50% v / v methanol solution to obtain naringin reference standard solution (0.4924164 mg / mL, calculated as pure product). Accurately transfer 5, 10, and 15 mL of this reference standard solution to 100 mL volumetric flasks and dilute to the mark with 50% v / v methanol solution to obtain naringin reference standard solution (0.02462082 mg / mL), naringin reference standard solution (0.04924164 mg / mL), and naringin reference standard solution (0.07386246 mg / mL), respectively.

[0187] Test solution: Accurately weigh 0.2 g of the lyophilized powder of the expectorant decoction reference sample into a stoppered conical flask. Accurately add 50 mL of the above reference solution, weigh, sonicate (300 W power, 40 kHz frequency) for 30 min, cool, add 50% methanol solution to make up the weight, filter, and collect the filtrate. Results: The RSD% of the 9 samples with 3 concentrations of naringin recovered was 1.80%, less than 2.0%, indicating good method accuracy. The average recovery rate was 98.70%, and the true recovery rate (95%) confidence interval was 97.60%–99.80%. The proposed recovery rate range was 95%–102%. Therefore, the confidence interval matched the acceptable standard, indicating that the method has good accuracy for naringin.

[0188] 4.3.7.2 Neohesperidin

[0189] Accurately weigh 35.52 mg of neohesperidin (batch number: 111857-202305, purity: 99.6%) and place it in a 50 mL volumetric flask. Dissolve it in 50% methanol and dilute to the mark to obtain the neohesperidin reference solution (0.7075584 mg / mL, calculated as pure product). Accurately transfer 5, 10, and 15 mL of this reference solution to 100 mL volumetric flasks and dilute to the mark with 50% v / v methanol to obtain the neohesperidin reference solutions (0.03537792 mg / mL, 0.07075584 mg / mL, and 0.10613376 mg / mL).

[0190] Test solution: Accurately weigh 0.2 g of the lyophilized powder of the expectorant decoction reference sample into a stoppered conical flask. Accurately add 50 mL of the above reference solution, weigh, sonicate (300 W, 40 kHz) for 30 min, cool, add 50% methanol solution to make up the weight, filter, and collect the filtrate. Results: The RSD% of the 9 samples with 3 concentrations of neohesperidin was 1.95%, less than 2.0%, indicating good method precision. The average recovery rate was 98.53%, and the confidence interval for the true 95% recovery was 97.34%–99.71%. The proposed recovery range was 95%–102%, therefore the confidence interval matched the acceptable standard, indicating that the method has good accuracy for neohesperidin.

[0191] 4.3.7.3 Hesperidin

[0192] Accurately weigh 13.32 mg of hesperidin (batch number: 110721-202220, purity: 97.2%) and place it in a 50 mL volumetric flask. Dissolve it in 50% methanol and dilute to the mark to obtain the hesperidin reference solution (0.2589408 mg / mL, calculated as pure product). Accurately transfer 5, 10, and 15 mL of this reference solution to 100 mL volumetric flasks, respectively, and dilute to the mark with 50% v / v methanol to obtain the hesperidin reference solutions (0.01294704 mg / mL, 0.02589408 mg / mL, and 0.03884112 mg / mL).

[0193] Test solution: Accurately weigh 0.2 g of the lyophilized powder of the expectorant decoction reference sample into a stoppered conical flask. Accurately add 50 mL of the above reference solution, weigh, sonicate (300 W, 40 kHz) for 30 min, cool, add 50% methanol solution to make up the weight, filter, and collect the filtrate. Results: The RSD% of the 9 samples with 3 concentrations of hesperidin recovered was 1.48%, less than 2.0%, indicating good method accuracy. The average recovery rate was 99.94%, and the true recovery rate (95%) confidence interval was 99.05%–100.83%. The proposed recovery rate range was 92%–105%. Therefore, the confidence interval matched the acceptable standard, indicating that the method has good accuracy for hesperidin.

[0194] 5. Synephrine content

[0195] 5.1 Solution Preparation

[0196] Test solution: Accurately weigh 1.0 g of the lyophilized standard sample powder into a stoppered conical flask, accurately add 50 mL of purified water, weigh, sonicate (300 W power, 40 kHz frequency) for 30 min, cool, weigh again, replenish the lost weight with purified water, shake well, filter, accurately measure 10 mL of the filtrate, pass it through a polyamide column (60-90 mesh, 2.5 g, 1.5 cm inner diameter, dry packing), elute with 25 mL of purified water, collect the eluent, transfer it to a 25 mL volumetric flask, add water to the mark, shake well, and the solution is ready.

[0197] Preparation of synephrine reference solution: Take an appropriate amount of synephrine, accurately weigh it, and add purified water to prepare a reference solution containing 30 µg per 1 mL.

[0198] 5.2 Chromatographic conditions

[0199] Instrument: Thermo Ultimate 3000;

[0200] Column: Thermo AQUASIL C18, 5μm 4.6×250mm;

[0201] Mobile phase: Methanol (phase A) and potassium dihydrogen phosphate buffer (phase B) were mixed at a volume ratio of 50:50 and eluted isocratically. The composition of the potassium dihydrogen phosphate buffer was as follows: 0.6 g of potassium dihydrogen phosphate, 1.0 g of sodium dodecyl sulfonate, and 1 mL of glacial acetic acid were dissolved in purified water and diluted to 1000 mL.

[0202] Detection wavelength: 275nm;

[0203] Flow rate: 1.0 mL / min;

[0204] Column temperature: 30℃;

[0205] Injection volume: 10 μL.

[0206] 5.3 Methodological Examination

[0207] 5.3.1 Specificity Examination

[0208] The test solution, negative sample solution lacking tangerine peel, negative sample solution lacking immature bitter orange, double negative sample solution lacking both tangerine peel and immature bitter orange, and blank solvent were prepared according to section "5.1". The results showed that the negative samples lacking tangerine peel and immature bitter orange did not have a peak at the retention time corresponding to synephrine, while the negative samples lacking both tangerine peel and immature bitter orange did have a peak at the retention time corresponding to synephrine. This proves that synephrine is a common component of both tangerine peel and immature bitter orange, indicating that the negative samples lacking tangerine peel and immature bitter orange, as well as the blank solvent, do not interfere with the determination of synephrine in the Daotan Decoction. Therefore, this method is specific for determining the content of synephrine in this product. (See section "5.1") Figure 21 .

[0209] 5.3.2 Peak Purity Examination

[0210] The above-mentioned test solution was injected into the liquid chromatograph, and the chromatographic conditions described above were used for full-wavelength detection with a DAD detector. Peak purity was calculated. The results showed that no impurity peaks were detected in the synephrine peak of the sample chromatogram; this indicates that the purity of the synephrine peak meets the requirements under these chromatographic conditions, as shown below. Figure 22 .

[0211] Results analysis: No impurity peaks were detected in the synephrine peak of the sample chromatogram; this indicates that the purity of the synephrine peak met the requirements under these chromatographic conditions.

[0212] 5.3.3 Examination of Linear Relationships

[0213] Accurately weigh 31.28 mg of synephrine reference standard (batch number: 110727-202511, purity: 99.8%), place it in a 50 mL volumetric flask, dissolve and dilute to the mark with purified water, and shake well to obtain a reference standard solution stock solution with a concentration of 0.6243488 mg / mL (calculated as pure product). Accurately transfer 1 mL to 5 mL, 10 mL, 20 mL, 25 mL, and 50 mL volumetric flasks respectively, and dilute to the mark with purified water. Prepare synephrine reference standard solutions with concentrations of 0.12486976, 0.06243488, 0.03121744, 0.024973952, and 0.012486976 mg / mL. Accurately pipette 10 μL of each of the above reference solutions and inject it into the liquid chromatograph. Determine the concentration using liquid chromatography (Chinese Pharmacopoeia 2025 Edition, Part IV, General Chapter 0512). Plot a standard curve with concentration (mg / mL) on the x-axis and peak area on the y-axis. See below. Figure 23 .

[0214] Figure 23 In the regression equation, Sinfferlin's equation is y = 77.644x + 0.0745, and the coefficient of determination is R0. 2 =1. The results showed that synephrine exhibited good linearity in the concentration range of 0.6243488 mg / mL to 0.012486976 mg / mL.

[0215] 5.3.4 Precision Test

[0216] The same test solution was injected under the chromatographic conditions in section "5.2" for six consecutive injections. The results showed that the RSD of the synephrine peak area was less than 3.0%, indicating that the instrument precision was good.

[0217] 5.3.5 Repeatability Test

[0218] Take approximately 0.6–1.6 g of the powder (6 portions in total), accurately weigh them, prepare the test solution in parallel according to section “5.1”, and inject the sample according to the chromatographic conditions in section “5.2”. The result shows that the RSD of the synephrine content is less than 2.0%, indicating that the repeatability of this method is good.

[0219] 5.3.6 Stability Test

[0220] The same test solution was placed at room temperature for 0, 12, 15, 24, 39, 57 and 72 hours and then injected according to the chromatographic conditions in section “5.2”. The results showed that the RSD of the synephrine peak area was less than 2.0%, and the test solution had good stability within 72 hours.

[0221] 5.3.7 Accuracy Test

[0222] Repeatability testing showed that the synephrine content in the non-dried sample of the Daotan Decoction reference sample (lyophilized powder) was 1.83 mg / g.

[0223] Using the spiking recovery method, accurately weigh 18.65 mg of synephrine reference standard (batch number: 110727-202110, purity: 99.8%) and place it in a 50 mL volumetric flask. Dilute to the mark with purified water and shake well to obtain a reference solution with a concentration of 0.372254 mg / mL. Accurately transfer 5, 10, and 15 mL of this reference solution to 200 mL volumetric flasks, respectively, and dilute to the mark with purified water to obtain synephrine reference solution (0.00930635 mg / mL), synephrine reference solution (0.0186127 mg / mL), and synephrine solution (0.02791905 mg / mL).

[0224] Test solution: Accurately weigh 0.5 g of the lyophilized powder of the phlegm-expelling decoction reference sample into a stoppered conical flask, accurately add 50 mL of the above reference solution, weigh, sonicate (300 W power, 40 kHz frequency) for 30 min, cool, weigh again, replenish the lost weight with purified water, shake well, filter, accurately measure 10-15 mL of the filtrate, pass it through a polyamide column (60-90 mesh, 2.5 g, 1.5 cm inner diameter, dry packing), elute with 25 mL of purified water, collect the eluent, transfer it to a 25 mL volumetric flask, add purified water to the mark, shake well, and the test solution is ready. The results showed that the RSD% of the nine synephrine assays with three concentrations was 2.82%, less than 3.0%, indicating good method precision. The average recovery rate was 95.44%, and the 95% confidence interval for the true recovery rate was 93.78%–97.08%, while the proposed recovery rate range was 92%–105%. Therefore, the confidence interval matched the acceptable standard, indicating that the method has good accuracy for synephrine assays.

[0225] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fingerprint spectrum and a method for determining the content of multiple components in a phlegm-expelling decoction reference sample, characterized in that... The steps include the following: (1) Construction of fingerprint spectrum: A. Preparation of test solution: Take the phlegm-expelling decoction reference sample, add solvent, extract, make up to volume, filter, take the filtrate to obtain the test solution; B. Preparation of reference solutions: Take 6-gingerol, naringin, hesperidin, naringin, tangeretin, nobiletin, 3,5,6,7,8,3'4'-heptamethoxyflavone, and neohesperidin standards, dissolve them in solvents and make up to volume to obtain reference solutions. C. Chromatographic conditions: The chromatographic column was a C18 column; the mobile phase consisted of acetonitrile in phase A and 0.05–0.1% v / v phosphoric acid aqueous solution in phase B; the elution gradient was: 0–25 min, 20% A; 25–40 min, 20% → 50% A; 40–50 min, 50% → 85% A; the detection wavelength was 280 nm, the flow rate was 0.8–1.2 mL / min, the column temperature was 25–35 °C, and the injection volume was 5–20 μL. D. Establishment of fingerprint chromatogram: Using the chromatographic conditions of step C, inject the test solution from step A and the reference solution from step B into the high performance liquid chromatograph; locate and assign the chromatographic peaks in the fingerprint chromatogram of the test sample according to the chromatogram of the reference sample to obtain the fingerprint chromatogram of the phlegm-relieving decoction reference sample. (2) Determination of the contents of hesperidin, naringin, and neohesperidin: E. Chromatographic conditions: The chromatographic column is a C18 column; the mobile phase consists of acetonitrile in phase A and 0.05–0.1% v / v phosphoric acid aqueous solution in phase B; the elution gradient is 0–25 min, 20% A; the detection wavelength is 280 nm, the flow rate is 0.8–1.2 mL / min, the column temperature is 25–35 °C, and the injection volume is 5–20 μL. F. Preparation of reference standard curves: Using the chromatographic conditions in step E, a series of reference standard solutions of hesperidin, naringin, and neohesperidin were prepared by high performance liquid chromatography to obtain the standard curves of naringin, hesperidin, and neohesperidin. G. Determination of the contents of hesperidin, naringin and neohesperidin: The test solution prepared in step A was tested by high performance liquid chromatography under the chromatographic conditions of step E. The contents of hesperidin, naringin and neohesperidin in the reference sample of Daotan Decoction were obtained according to the reference standard curve. (3) Determination of synephrine content: H. Preparation of the test solution: Take the phlegm-expelling decoction reference sample, extract with water, dilute to volume, and filter; take the filtrate for column chromatography, elute with water, collect the eluent and dilute to volume with water to obtain the test solution; I. Preparation of reference solution: Dissolve synephrine in water to obtain synephrine solutions of a series of concentrations; J. Chromatographic conditions: The chromatographic column was a C18 column; the mobile phase was a mixture of methanol and potassium dihydrogen phosphate buffer at a volume ratio of 50:50, with the potassium dihydrogen phosphate buffer consisting of 0.6 g / L potassium dihydrogen phosphate, 1.0 g / L sodium dodecyl sulfate, and 1 mL / L glacial acetic acid; the detection wavelength was 275 nm; the flow rate was 0.8–1.2 mL / min; the column temperature was 25–35 °C; and the injection volume was 5–20 μL. K. Determination of synephrine content: The series of synephrine solutions prepared in step I were detected by high performance liquid chromatography under the chromatographic conditions of step J to obtain the synephrine standard curve; the test solution prepared in step H was detected by high performance liquid chromatography under the chromatographic conditions of step J, and the synephrine content in the phlegm-relieving decoction reference sample was obtained according to the synephrine standard curve. The reference sample for Daotan Decoction mentioned in step (1) is a sample obtained by extracting and processing the Daotan Decoction herbal composition; The solvent mentioned in step (1)A is a 30-70% methanol solution or a 30-70% ethanol solution; The column chromatography described in step (3)H is polyamide column chromatography.

2. The method for determining the fingerprint spectrum and multi-component content of the phlegm-expelling decoction reference sample according to claim 1, characterized in that: The aforementioned expectorant decoction composition includes Pinellia ternata, Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum, Poria cocos, and ginger slices; The extraction method described is water decoction; The process described involves concentration followed by freeze drying.

3. The method for determining the fingerprint spectrum and multi-component content of the phlegm-expelling decoction reference sample according to claim 2, characterized in that: The aforementioned expectorant decoction composition is prepared by combining Pinellia ternata, Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum, Poria cocos, and ginger slices in a mass ratio of 6.19:1.55:1.55:1.55:1.55:

10. The steps for decocting are as follows: Mix Pinellia ternata, Citrus aurantium, Citrus reticulata peel, processed Arisaema heterophyllum, Poria cocos, ginger slices and water, bring to a boil over high heat and then simmer over low heat. The freeze-drying conditions are a temperature of -70 to -80°C and a pressure of 0 to 20 Pa.

4. The method for determining the fingerprint spectrum and multi-component content of the phlegm-expelling decoction reference sample according to claim 1, characterized in that: The amount of solvent used in step (1) A is calculated based on 0.005 to 0.01 g of the expectorant decoction reference sample / mL of solvent; The extraction method described in step (1)A is reflux extraction or ultrasonic extraction; The solvent described in step (1)B is the same as the solvent described in step (1)A; The chromatographic conditions described in step (1) C are: detection wavelength 280 nm, flow rate 0.9-1.1 mL / min, column temperature 28-32 °C, and injection volume 10-15 μL.

5. The method for determining the fingerprint spectrum and multi-component content of the phlegm-expelling decoction reference sample according to claim 1, characterized in that: In step (1) D, peak 2 is naringin, peak 3 is naringin, peak 4 is hesperidin, peak 5 is neohesperidin, peak 10 is norihesperidin, peak 11 is 6-gingerol, peak 12 is 3,5,6,7,8,3',4'-heptamethoxyflavone, and peak 13 is tangeretin. In the fingerprint spectrum, peak 5 is used as the reference peak, and the relative retention times are as follows: peak 1 0.43, peak 2 0.61, peak 3 0.73, peak 4 0.82, peak 5 1.00, peak 6 1.71, peak 7 1.85, peak 8 1.90, peak 9 1.93, peak 10 2.13, peak 11 2.15, peak 12 2.18, peak 13 2.

22.

6. The method for determining the fingerprint spectrum and multi-component content of the phlegm-expelling decoction reference sample according to claim 1, characterized in that: The chromatographic conditions described in step (2)E are: detection wavelength 280 nm, flow rate 0.9–1.1 mL / min, column temperature 28–32 °C, and injection volume 10–15 μL; The standard curve for naringin mentioned in step (2)F is y = 154.25x + 0.2194; coefficient of determination: R 2 =1; The standard curve for hesperidin mentioned in step (2)F is y = 155.88x + 0.0935; coefficient of determination: R 2 =1; The standard curve for the new hesperidin mentioned in step (2)F is y = 164.07x - 0.1125; coefficient of determination: R 2 =1.

7. The method for determining the fingerprint spectrum and multi-component content of the phlegm-expelling decoction reference sample according to claim 1, characterized in that: In step (3)H: The water mentioned is purified water or ultrapure water; The filtration process involves using a 0.22μm filter membrane.

8. The method for determining the fingerprint spectrum and multi-component content of the phlegm-expelling decoction reference sample according to claim 1, characterized in that: The chromatographic conditions described in step (3)J are: detection wavelength 275 nm, flow rate 0.9–1.1 mL / min, column temperature 28–32 °C, and injection volume 10–15 μL; The standard curve of Sinfeldt mentioned in step (3) K is y = 77.644x + 0.0745.

9. The application of the fingerprint spectrum and multi-component content determination method of Daotan Decoction according to any one of claims 1 to 8 in the quality control of Daotan Decoction.

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