Detection method for angelica drink composition

Abstract

The present application relates to the technical field of traditional Chinese medicine quality detection, and specifically discloses a detection method for Angelica drink composition, which comprises the following steps: (1) preparing a test sample solution; (2) preparing a mixed reference sample solution and / or a paeonol reference sample solution; (3) determining the test sample solution and the paeonol reference sample solution and / or the mixed reference sample solution to obtain the content of paeonol; (4) taking paeonol as an internal standard substance, and calculating the contents of caffeic acid, ferulic acid, glycyrrhizin, calycosin-7-glucoside, epimedoside, senkyunol I, rosmarinic acid, 5-O-methylvisamminol glycoside and glycyrrhizic acid by using an internal standard method; the chromatographic column of a liquid chromatograph is made of octadecylsilane bonded silica gel as a stationary phase, methanol as a mobile phase A, and a 0.06-0.2 vol% phosphoric acid aqueous solution as a mobile phase B for gradient elution. The detection method is practical, simple to operate, fast in testing, accurate and low in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection of traditional Chinese medicines, and in particular to a detection method for a Danggui Yinzi composition. Background Art

[0002] Danggui Yinzi, originating from Yan's Jisheng Prescriptions, a book written by Yan Yonghe during the Song Dynasty, is primarily used to treat "cardiac blood stagnation and internal wind-heat syndrome, with symptoms including skin sores, swelling, itching, pus-filled rashes, or itching worse at night, accompanied by a pale red tongue with a thin coating and a thready, stringy pulse." It nourishes and invigorates blood circulation, dispels wind, and relieves itching. The recipe consists of 11 herbs: angelica, white peony root, Chuanxiong root, Rehmannia root, stir-fried Tribulus terrestris, Saposhnikovia root, Schizonepeta spicata, Polygonum multiflorum, Astragalus root, stir-fried licorice root, and ginger. In this formula, Angelica sinensis enters the liver and spleen meridians, replenishing blood and promoting blood circulation; White Peony Root enters the liver and spleen blood divisions, softening the liver, nourishing blood, and consolidating yin; Chuanxiong promotes qi and blood circulation, dispelling wind, replenishing blood without stagnating it; Raw Rehmannia nourishes yin and nourishes blood; Polygonum multiflorum nourishes blood and dispels wind; Schizonepeta tenuifolia is pungent and fragrant, dispersing wind, while its bitter and warm properties clear blood, and Saposhnikovia divaricata dispels internal and external wind; Tribulus terrestris promotes blood circulation, dispels wind, and relieves itching; Astragalus and Licorice root strengthen the spleen and replenish qi. The entire formula replenishes blood and promotes blood circulation, while also strengthening the spleen and replenishing qi to generate blood, replenishing the middle and promoting circulation; dispelling wind and relieving itching, while also strengthening the spleen and strengthening the defensive system to consolidate the exterior, replenishing the middle and dispersing itching.

[0003] Modern clinical research indicates that Danggui Yinzi is primarily used to treat chronic urticaria caused by blood deficiency, wind-dryness, and qi and blood deficiency, with proven clinical efficacy. Other studies suggest that Danggui Yinzi may treat chronic spontaneous urticaria by enhancing autophagy, inhibiting delayed hypersensitivity reactions, regulating immune function, and suppressing allergic inflammatory responses. However, due to the diverse composition of traditional Chinese medicine compounds and their mechanisms of action involving multiple targets and pathways, the active ingredients and mechanisms of action of Danggui Yinzi remain incompletely understood. A study, through screening of the drug-active ingredient-target-disease network, predicted the following as the most important active ingredients of Danggui Yinzi: resveratrol, emodin, luteolin, 7-O-methylisoeugenol, astragalol, glycyrrhizin, naringenin, scutellarin, 7-methoxy-2-methylisoflavone, meditartin, licoricechalcone A, wogonin, β-sitosterol, formononetin, quercetin, isorhamnetin, and kaempferol. Most of these active ingredients are flavonoids, suggesting that flavonoids are the primary active ingredients in Danggui Yinzi, which promotes blood-nourishing, blood-activating, wind-dispelling, and itching-relieving effects. Research on Danggui Yinzi has primarily focused on summarizing its clinical applications in Traditional Chinese Medicine, conducting clinical trials, and conducting experimental studies, but quantitative studies of its primary active ingredients have yet to be conducted. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting a Danggui Yinzi composition, which can detect multiple components, has a simple determination process and low detection cost.

[0005] In order to solve the above technical problems, the present invention provides a method for detecting an angelica saccharin composition, which comprises:

[0006] (1) extracting the Danggui Yinzi composition with a first methanol aqueous solution to obtain a test solution;

[0007] (2) dissolving or extracting the caffeic acid reference substance, the paeoniflorin reference substance, the ferulic acid reference substance, the liquiritin reference substance, the calycosin isoflavone glucoside reference substance, the cimicifuga glycoside reference substance, the ligusticum lactone I reference substance, the rosmarinic acid reference substance, the 5-O-methylvisamidoside reference substance, and the glycyrrhizic acid reference substance with a second methanol aqueous solution to obtain a mixed reference substance solution; and / or dissolving or extracting the paeoniflorin reference substance with a second methanol aqueous solution to obtain a paeoniflorin reference substance solution;

[0008] (3) injecting the test solution and the paeoniflorin reference solution and / or the mixed reference solution into a liquid chromatograph to determine the content of paeoniflorin in the Danggui Yinzi composition;

[0009] (4) Using paeoniflorin as the internal standard, the contents of caffeic acid, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5-O-methylvisamidoside and glycyrrhizic acid in the Dangguiyinzi composition were calculated by the internal standard method;

[0010] The chromatographic column of the liquid chromatograph is composed of octadecylsilane bonded silica gel as the stationary phase, methanol as the mobile phase A, and a 0.06-0.2 vol% phosphoric acid aqueous solution as the mobile phase B for gradient elution. The gradient elution curve is:

[0011] 0-5 min, mobile phase A from 4% to 21%, mobile phase B from 96% to 79%;

[0012] 5-25 min, mobile phase A from 21% to 32%, mobile phase B from 79% to 68%;

[0013] 25-33 min, mobile phase A from 32% to 34%, mobile phase B from 68% to 66%;

[0014] 33-37 min, mobile phase A from 34% to 40%, mobile phase B from 66% to 60%;

[0015] 37-40 min, mobile phase A from 40% to 42%, mobile phase B from 60% to 58%;

[0016] 40-42 min, mobile phase A from 42% to 60%, mobile phase B from 58% to 40%;

[0017] 42-55 min, mobile phase A from 60% to 95%, mobile phase B from 40% to 5%;

[0018] 55-56 min, mobile phase A from 95% to 4%, mobile phase B from 5% to 96%;

[0019] 56-60 min, mobile phase A: 4%, mobile phase B: 96%;

[0020] The concentration of the first methanol aqueous solution is 50-90 vol%, illustratively, 55 vol%, 65 vol%, 75 vol%, 85 vol% or 88 vol%, but not limited thereto, and preferably 60-90 vol%.

[0021] The concentration of the second methanol aqueous solution is 50-90 vol%, illustratively, 55 vol%, 65 vol%, 75 vol%, 85 vol% or 88 vol%, but not limited thereto, preferably 60-90 vol%.

[0022] For example, the concentration of the phosphoric acid aqueous solution is 0.08 vol%, 0.10 vol%, 0.12 vol%, 0.14 vol%, 0.16 vol% or 0.18 vol%, but is not limited thereto, preferably 0.1 to 0.2 vol%, and more preferably 0.1 to 0.15 vol%.

[0023] Preferably, in some embodiments of the present invention, the mobile phase B is a 0.1 vol% phosphoric acid aqueous solution.

[0024] Preferably, in some embodiments of the present invention, the concentration of the first methanol aqueous solution is 80 vol%.

[0025] Preferably, in some embodiments of the present invention, the concentration of the second methanol aqueous solution is 80 vol%.

[0026] Specifically, in some embodiments of the present invention,

[0027] Specifically, in some embodiments of the present invention, in step (1), the Danggui Yinzi composition is mixed with the first methanol aqueous solution, and ultrasonically treated or heated under reflux for 10 to 80 minutes to obtain a test solution.

[0028] The dosage form of the Danggui Yinzi composition can be decoction, powder, pill, tablet, granule, etc., but is not limited thereto.

[0029] Among them, the ratio of the angelica drink composition to the first methanol aqueous solution is 0.2-1g:15-60mL. Exemplary are 0.23g:19mL, 0.35g:23mL, 0.41g:37mL, 0.67g:40mL or 0.85g:57mL, but not limited thereto. Preferably, it is 0.2-0.8g:15-50mL. It should be noted that the above ratios can be proportionally enlarged or reduced, for example, the ratio of the angelica drink composition to the first methanol aqueous solution is 0.23kg:19L or 0.23mg:19μL, but not limited thereto.

[0030] Exemplarily, the extraction time is 12 min, 27 min, 34 min, 38 min, 53 min or 75 min, but is not limited thereto, and preferably is 10 min to 70 min.

[0031] Specifically, when ultrasonic treatment is used for extraction, the ultrasonic power is 200-800W and the ultrasonic frequency is 20-60kHz.

[0032] Preferably, in some embodiments of the present invention, in step (1), an ultrasonic extraction process is used for extraction, with an ultrasonic power of 200 to 500 W, an ultrasonic frequency of 20 to 50 kHz, and an ultrasonic time of 10 to 60 min.

[0033] Preferably, in some embodiments of the present invention, in step (1), the ratio of the Danggui Yinzi composition to the first methanol aqueous solution is 0.2-0.6 g: 15-25 mL.

[0034] In some embodiments of the present invention, in the mixed reference solution, the concentrations of the caffeic acid reference substance, paeoniflorin reference substance, ferulic acid reference substance, liquiritin reference substance, calycosin glucoside reference substance, cimicifugo glycoside reference substance, ligusticum lactone I reference substance, rosmarinic acid reference substance, 5-O-methylvisaminol glycoside reference substance and glycyrrhizic acid reference substance are 210-220 μg / mL, 185-195 μg / mL, 205-215 μg / mL, 140-150 μg / mL, 140-150 μg / mL, 145-155 μg / mL, 215-225 μg / mL, 85-95 μg / mL, 160-170 μg / mL and 175-185 μg / mL, respectively.

[0035] Preferably, in some embodiments of the present application, the concentrations of the caffeic acid reference substance, paeonol reference substance, ferulic acid reference substance, glycyrrhizin reference substance, calycosin-7-glucoside reference substance, prim-O-glucosylcimifugin reference substance, senkyunol I reference substance, rosmarinic acid reference substance, 5-O-methylvisamminol reference substance and glycyrrhizic acid reference substance in the mixed reference substance solution are 212.4607 μg / mL, 189.8326 μg / mL, 209.0382 μg / mL, 146.8270 μg / mL, 144.6192 μg / mL, 150.3447 μg / mL, 218.9344 μg / mL, 90.4482 μg / mL, 166.4566 μg / mL and 180.6816 μg / mL, respectively.

[0036] Specifically, in some embodiments of the present application, the concentration of the paeonol reference substance in the paeonol reference substance solution is 80-120 μg / mL.

[0037] Preferably, in some embodiments of the present application, the concentration of the paeonol reference substance in the paeonol reference substance solution is 100 μg / mL.

[0038] In some embodiments of the present application, the column length of the chromatographic column is 100-200 mm, for example, 100 mm, 150 mm or 200 mm, but is not limited thereto. The column diameter of the chromatographic column is 1-3 mm, for example, 1.0 mm, 2.1 mm or 3.0 mm, but is not limited thereto. The particle size of the stationary phase is 1.6-1.8 μm, for example, 1.6 μm, 1.7 μm or 1.8 μm, but is not limited thereto.

[0039] The column temperature of the chromatographic column is 28-35°C, for example, 28°C, 30°C, 32°C or 34°C, but is not limited thereto.

[0040] Preferably, in some embodiments of the present application, the column length of the chromatographic column is 150 mm, the column diameter is 2.1 mm, and the particle size of the stationary phase is 1.6 μm.

[0041] The column temperature of the chromatographic column is 30°C.

[0042] In some embodiments of the present application, the flow rate of the liquid chromatograph is 0.18-0.22 mL / min, for example, 0.19 mL / min, 0.2 mL / min or 0.21 mL / min, but is not limited thereto.

[0043] The detection wavelength of the liquid chromatograph is 220-300 nm, for example, 225 nm, 240 nm, 255 nm, 270 nm or 285 nm, but is not limited thereto.

[0044] The injection volume of the test solution is 0.5 to 2.0 μL, exemplified by 0.5 μL, 1.0 μL, 1.5 μL or 2.0 μL, but not limited thereto.

[0045] The injection volume of the mixed reference solution is 0.5 μL to 2.0 μL, exemplified by 0.5 μL, 1.0 μL, 1.5 μL or 2.0 μL, but not limited thereto.

[0046] The injection volume of the paeoniflorin reference solution is 0.5 μL to 2.0 μL, exemplified by 0.5 μL, 1.0 μL, 1.5 μL or 2.0 μL, but not limited thereto.

[0047] Preferably, in some embodiments of the present invention, the flow rate of the liquid chromatograph is 0.2 mL / min;

[0048] The injection volume of the test solution is 1.0 μL, the injection volume of the paeoniflorin reference solution is 1.0 μL; or the injection volume of the mixed reference solution is 1.0 μL.

[0049] Preferably, in some embodiments of the present invention, the detection wavelength of the detector of the liquid chromatograph is: 230 nm for 0-35 min, and 254 nm for 35-60 min.

[0050] Among them, among some embodiments of the present invention, in step (4), paeoniflorin is first used as an internal standard, the relative correction factors of the other standards are calculated, and then the content of each component is calculated. The method for establishing the relative correction factor can refer to the "Chinese Pharmacopoeia" (2020 edition) Part IV General Chapter 0512 High Performance Liquid Chromatography, but is not limited thereto.

[0051] Preferably, in some embodiments of the present invention, step (4) comprises:

[0052] (4.1) diluting the mixed reference solution in a gradient manner to obtain a plurality of mixed reference solutions to be tested at a series of concentrations;

[0053] (4.2) using a liquid chromatograph to measure a plurality of the mixed reference solutions, and calculating relative correction factors for caffeic acid, ferulic acid, liquiritin, calycosin glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside, and glycyrrhizic acid;

[0054] (4.3) The contents of caffeic acid, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticum lactone I, rosmarinic acid, 5-O-methylvisamidoside and glycyrrhizic acid in the Danggui Yinzi composition were calculated according to the relative correction factors.

[0055] The implementation of the present invention has the following beneficial effects:

[0056] The present invention adopts a single-measurement, multiple-evaluation method (i.e., QAMS method) using paeoniflorin as an internal standard. Through a single measurement, the contents of ten components in the Danggui Yinzi composition, including caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5-O-methylvisamidoside, and glycyrrhizic acid, are determined. The method is highly practical, simple to operate, and provides rapid, accurate, and cost-effective testing. The method is simple and easy to operate, and the measurement results are accurate. The method has good reproducibility, stability, and durability, providing a basis for establishing a quality evaluation method for Danggui Yinzi compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a chromatogram obtained by measuring different mobile phases in Example 2 of the present invention;

[0058] Figure 2 This is a chromatogram obtained by gradient elution investigation and measurement in Example 2 of the present invention;

[0059] Figure 3 This is a chromatogram obtained by measuring at different column temperatures in Example 2 of the present invention;

[0060] Figure 4 This is a chromatogram obtained by measuring different sample injection volumes in Example 2 of the present invention;

[0061] Figure 5 This is the chromatogram obtained when the flow rate is 0.18 mL / min in Example 2 of the present invention;

[0062] Figure 6 This is the chromatogram obtained when the flow rate is 0.20 mL / min in Example 2 of the present invention;

[0063] Figure 7 This is the chromatogram obtained when the flow rate is 0.22 mL / min in Example 2 of the present invention;

[0064] Figure 8 This is a chromatogram of the blank solvent, mixed reference substance, and Dangguiyinzi granules in the specificity investigation in Example 2 of the present invention;

[0065] Figure 9 This is a chromatogram of the mixed reference substance, Danggui Yinzi granules, Danggui deficiency, and Chuanxiong negative samples in the specificity investigation in Example 2 of the present invention;

[0066] Figure 10 This is a chromatogram of the mixed reference substance, Dangguiyinzi granules, Chuanxiong-deficient, and Schizonepeta spicata negative samples in the specificity investigation in Example 2 of the present invention;

[0067] Figure 11 is the chromatogram of the mixed reference substance, Angelica drink son granules, and the sample without white peony root in the specificity investigation in embodiment 2 of the present application;

[0068] Figure 12 is the chromatogram of the mixed reference substance, Angelica drink son granules, and the sample without feng wind in the specificity investigation in embodiment 2 of the present application;

[0069] Figure 13 is the chromatogram of the mixed reference substance, Angelica drink son granules, and the sample without Huangqi in the specificity investigation in embodiment 2 of the present application;

[0070] Figure 14 is the chromatogram of the mixed reference substance, Angelica drink son granules, and the sample without liquorice in the specificity investigation in embodiment 2 of the present application;

[0071] In the figure, peak 1 is caffeic acid, peak 2 is paeoniflorin, peak 3 is ferulic acid, peak 4 is glycyrrhizin, peak 5 is calycosin-7-glucoside, peak 6 is prim-O-glucosylcimifugin, peak 7 is senkyunolide I, peak 8 is rosmarinic acid, peak 9 is 5-O-methylvisamminol, and peak 10 is glycyrrhizic acid. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0073] The instruments and medicines used in the embodiments of the present application are as follows:

[0074] 1. Instruments

[0075] Waters H-class high performance liquid chromatograph (Waters Corporation, USA); Thermo Vanquish high performance liquid chromatograph (Thermo Fisher Corporation, USA); ME204E one-thousandth balance, XP26 one-hundred-thousandth balance (METTLER TDLEDO Corporation, Switzerland); KQ-500DE digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q Direct ultrapure water system (Merck Corporation, Germany).

[0076] 2. Reagents

[0077] Methanol is chromatographically pure (Merck Corporation, Germany), phosphoric acid is chromatographically pure (Tianjin Kemio Chemical Reagent Co., Ltd.), water is ultrapure water, and other reagents are analytically pure.

[0078] 3. Test reagents

[0079] Ten batches of Angelica drink son granules were prepared in the laboratory.

[0080] Reference substances: caffeic acid (China National Institute for Food and Drug Control, batch number 110885-201703, purity 99.7%), paeoniflorin (China National Institute for Food and Drug Control, batch number 110736-202044, purity 97.4%), ferulic acid (China National Institute for Food and Drug Control, batch number 110773-201915, purity 99.4%), glycyrrhizin (China National Institute for Food and Drug Control, batch number 111610-202209, purity 95.2%), calycosin-7-glucoside (China National Institute for Food and Drug Control, batch number 111920-201907, purity 96.8%), epimediumin (China National Institute for Food and Drug Control, batch number 111522-202214, purity 95.7%), senkyunolide I (China National Institute for Food and Drug Control, batch number 112071-202101, purity 99.2%), rosmarinic acid (China National Institute for Food and Drug Control, batch number 111871-202007, purity 98.1%), 5-O-methylvisamminol (China National Institute for Food and Drug Control, batch number 111523-201811, purity 97.4%), glycyrrhizic acid (China National Institute for Food and Drug Control, batch number 110731-202122, purity 94.4%).

[0081] Determination of relative correction factors of Example 1

[0082] 1. Chromatographic conditions, preparation of reference substance solution, preparation of test sample solution

[0083] 1.1 Chromatographic conditions

[0084] Chromatographic column: CORTECS UPLC T3 (150 mm x 2.1 mm, 1.6 μm); mobile phase: methanol as mobile phase A, 0.1 vol% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the provisions in Table 1; detection wavelength: 230 nm (0-35 min, caffeic acid, paeoniflorin, ferulic acid, glycyrrhizin, calycosin-7-glucoside, epimediumin, senkyunolide I), 254 nm (35-60 min, rosmarinic acid, 5-O-methylvisamminol, glycyrrhizic acid); column temperature: 30 °C; flow rate: 0.2 mL / min; injection volume: 1 μL.

[0085] Table 1 Gradient elution conditions of mobile phase

[0086]

[0087] 1.2 Preparation of mixed reference substance solution

[0088] Take appropriate amounts of caffeic acid reference substance, paeoniflorin reference substance, ferulic acid reference substance, liquiritin reference substance, calycosin isoflavone glucoside reference substance, cimicifuga glycoside reference substance, ligusticum chuanxiong lactone I reference substance, rosmarinic acid reference substance, 5-O-methylvisamin glycoside reference substance and glycyrrhizic acid reference substance, accurately weigh them, and add 80 vol% methanol aqueous solution to prepare mixed reference substance solutions with concentrations of 212.4607 μg / mL, 189.8326 μg / mL, 209.0382 μg / mL, 146.8270 μg / mL, 144.6192 μg / mL, 150.3447 μg / mL, 218.9344 μg / mL, 90.4482 μg / mL, 166.4566 μg / mL and 180.6816 μg / mL, respectively.

[0089] 2. Determination method

[0090] Pipette 1 μL of the mixed reference solution, inject it into the high performance liquid chromatograph, measure it, and calculate the relative correction factor of each component according to the measurement results.

[0091] 3 Determination of correction factors

[0092] 3.1 Selection of components to be tested

[0093] Danggui Yinzi Granules are composed of 11 herbs: angelica, white peony root, Chuanxiong, Rehmannia root, stir-fried Tribulus terrestris, siler, schizonepeta spicate, Polygonum multiflorum, astragalus root, stir-fried licorice root, and ginger. Angelica is the main herb in this formula, tonifying blood and promoting blood circulation, regulating the vital organs and defense system. White peony root nourishes blood, astringes yin, and relieves pain; raw rehmannia root clears heat, nourishes yin, and promotes fluid production, making it a key herb for cooling and nourishing the blood. Chuanxiong promotes qi and blood circulation, acting as a qi-boosting herb. Astragalus root is excellent at tonifying qi, expelling toxins, healing sores, and promoting tissue regeneration, making it a panacea for sore treatment. The combination of these two herbs not only enhances the nourishing effects of yin and blood, but also prevents the indigestion of greasy ingredients. White peony root, raw rehmannia root, Chuanxiong, and astragalus root serve as auxiliary herbs, complementing the main herb in tonifying qi, promoting blood circulation, nourishing blood, and moistening dryness. Saposhnikovia divaricata and Schizonepeta tenuifolia dispel pathogenic factors and resolve exterior symptoms, eliminating sores and rashes. Combined with Astragalus membranaceus, they strengthen the exterior, preventing pathogenic factors from lingering, and dispelling wind without harming the body. Polygonum multiflorum replenishes essence and blood, nourishes yin and fluid, and can resolve the toxins of skin sores, rashes, scabies, and ringworm. Tribulus terrestris dispels wind, promotes qi circulation, and relieves itching. These four herbs serve as adjuvants, dispelling wind and relieving itching. Licorice root harmonizes the other herbs. The formula is well-combined, nourishing blood and yin without retaining stasis, dispelling wind and pathogenic factors without harming the body. It addresses both the symptoms and the root cause, combining attack and supplementation to achieve the combined effects of nourishing blood, activating blood circulation, and dispelling wind and relieving itching. The main components of Angelica sinensis are ferulic acid, ligusticolide I, etc.; the main component of white peony root is paeoniflorin; the main components of Chuanxiong are ferulic acid, ligusticolide I, caffeic acid, etc.; the main components of Saposhnikovia divaricata are cimicifuga glycoside, 5-O-methylvisaminol glycoside, etc.; the active components of Schizonepeta tenuifolia are caffeic acid, rosmarinic acid, etc.; the active component of Astragalus membranaceus is calycosin glucoside; the main components of stir-fried liquorice are liquiritin and glycyrrhizic acid. Therefore, caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glycoside, and glycyrrhizic acid were selected as quality evaluation indicators.

[0094] 3.2 Selection of internal standard components

[0095] Under the chromatographic conditions specified in the present invention, 0.2 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 10.0 mL of the mixed reference solution prepared under "1.2 Preparation of Mixed Reference Solution" were accurately drawn and placed in 10 mL volumetric flasks, respectively. 80% methanol was added to the scale and the solution was shaken to prepare mixed reference solutions of different concentrations to be tested. The samples were injected and measured according to the chromatographic conditions under "1.1 Chromatographic Conditions". The peak area of ​​each component was recorded. Caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glycoside, and glycyrrhizic acid were used as internal standards to calculate the relative correction factor f for each component to be tested. s / k , f s / k =f s / f k =(A s ×C k ) / (A k ×C s ), where A s is the peak area of ​​the internal standard, C s is the mass concentration of the internal standard, A k is the peak area of ​​the component to be measured, C k is the mass concentration of the component to be measured, and the results are shown in Tables 2 to 11. The results show that when paeoniflorin is used as the internal standard, the RSDs of the relative correction factors of each component to be measured are all less than 3%, and the results are relatively accurate. Therefore, paeoniflorin is used as the internal standard.

[0096] Table 2 Relative correction factors of each component 1 (n=6)

[0097]

[0098] Table 3 Relative correction factors 2 for each component (n=6)

[0099]

[0100] Table 4 Relative correction factors of each component 3 (n=6)

[0101]

[0102] Table 5 Relative correction factors of each component 4 (n=6)

[0103]

[0104] Table 6 Relative correction factors of each component 5 (n=6)

[0105]

[0106]

[0107] Table 7 Relative correction factors of each component 6 (n=6)

[0108]

[0109] Table 8 Relative correction factors of each component 7 (n=6)

[0110]

[0111] Table 9 Relative correction factors of each component 8 (n=6)

[0112]

[0113] Table 10 Relative correction factors of each component 9 (n=6)

[0114]

[0115] Table 11 Relative correction factors of each component 10 (n=6)

[0116]

[0117] 4 Methodological Investigation

[0118] 4.1 Linear Relationship Investigation

[0119] Accurately pipette the mixed reference solution prepared under "1.2 Preparation of Mixed Reference Solution" and add 80% methanol in water to prepare mixed reference solutions of varying concentrations. Inject and measure according to the chromatographic conditions under "1.1 Chromatographic Conditions," and record the chromatograms. Linear regression was performed using the mass concentration of the mixed reference solution as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 12, demonstrating good linearity for each component within its respective concentration range.

[0120] Table 12 Linear relationship investigation results (n=6)

[0121]

[0122]

[0123] 4.2 Precision investigation

[0124] Accurately pipette an appropriate amount of the mixed reference solution described in "1.2 Preparation of Mixed Reference Solution" and inject the sample according to the chromatographic conditions described in "1.1 Chromatographic Conditions." Make six consecutive injections and record the peak areas. The results are shown in Table 13. The calculated RSDs for the peak areas of caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5-O-methylvisamidoside, and glycyrrhizic acid were all less than 3%, indicating good instrument precision.

[0125] Table 13 Precision inspection results

[0126]

[0127] 4.3 System durability investigation

[0128] 4.3.1 Investigation of different instruments and chromatographic columns

[0129] Using paeoniflorin as the internal standard, the relative calibration factors of the other nine components were investigated on two HPLC systems, Waters H-class and Thermo Vanquish, and three chromatographic columns, CORTECS UPLC T3 (150 mm × 2.1 mm, 1.6 μm), ACQUITY UPLC HSST3 (150 mm × 2.1 mm, 1.8 μm), and ACQUITY UPLC BEH C18 (150 mm × 2.1 mm, 1.7 μm). The results are shown in Table 14. The RSDs of each component were less than 3%, indicating that the replacement of instruments and chromatographic columns had no significant effect on the relative calibration factors of each component.

[0130] Table 14 Effects of different instruments and chromatographic columns on relative correction factors

[0131]

[0132] 4.3.2 Investigation of different column temperatures

[0133] A Waters H-class high performance liquid chromatograph and a CORTECS UPLC T3 column were used to investigate the effects of column temperatures of 28°C, 30°C, 32°C, and 35°C on the relative correction factors of each component. The results are shown in Table 15. The RSDs of each component were all less than 3%, indicating that fluctuations in column temperature had no significant effect on the relative correction factors of each component.

[0134] Table 15 Effect of different column temperatures on relative correction factors

[0135]

[0136] 4.3.3 Investigation of different flow rates

[0137] A Waters H-class high performance liquid chromatograph and a CORTECS UPLC T3 column were used at a flow rate of 0.18 mL min. -1 、0.20mL·min -1 、0.22mL·min -1 The results of the effects on the relative correction factors of each component are shown in Table 16. The RSDs of each component were all less than 3%, indicating that different flow rates had no significant effect on the relative correction factors of each component.

[0138] Table 16 Effect of different flow rates on relative correction factors

[0139]

[0140]

[0141] 4.3.4 Investigation of different injection volumes

[0142] A Waters H-class high performance liquid chromatograph and a CORTECS UPLC T3 column were used to investigate the effects of injection volumes of 0.5 μL, 1.0 μL, 1.5 μL, and 2.0 μL on the relative correction factors of each component. The results are shown in Table 17. The RSDs of each component were all less than 3%, indicating that different injection volumes had no significant effect on the relative correction factors of each component.

[0143] Table 17 Effect of different injection volumes on relative correction factors

[0144]

[0145] 5. Positioning of chromatographic peaks of the components to be measured

[0146] In QAMS applications, commonly used chromatographic peak location methods include relative retention value, retention time difference, time correction, and reference extract methods. In this experiment, relative retention time was used to locate the chromatographic peaks of the target components. Using paeoniflorin as the internal standard, the relative retention times of eight other components were investigated on two HPLC systems: a Waters H-class and a Thermo Vanquish, and three chromatographic columns: a CORTECS UPLC T3 (150 mm × 2.1 mm, 1.6 μm), an ACQUITY UPLC HSS T3 (150 mm × 2.1 mm, 1.8 μm), and an ACQUITY UPLC BEH C18 (150 mm × 2.1 mm, 1.7 μm). The results are shown in Table 18. The RSDs for each component using the relative retention method were all less than 3%, demonstrating the feasibility of using this method for locating target components.

[0147] Table 18 Relative retention time of each component

[0148]

[0149]

[0150] Example 2 Detection Method of Danggui Yinzi Composition

[0151] 1. Preparation of chromatographic conditions, reference solution, and test solution

[0152] 1.1 Chromatographic conditions

[0153] Chromatographic column: CORTECS UPLC T3 (150 mm × 2.1 mm, 1.6 μm); Mobile phase: Methanol as mobile phase A, 0.1 vol% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the specifications in Table 1; Detection wavelength: 230 nm (0-35 min, caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifugoside, and ligusticolide I), 254 nm (35-60 min, rosmarinic acid, 5-O-methylvisamidoside, and glycyrrhizic acid); Column temperature: 30°C; Flow rate: 0.2 mL / min; Injection volume: 1 μL. 1.2 Preparation of paeoniflorin reference solution

[0154] Take an appropriate amount of paeoniflorin, accurately weigh it, and add 80 vol% methanol aqueous solution to prepare a paeoniflorin reference solution with a concentration of 100 μg / mL.

[0155] 1.3 Preparation of test solution

[0156] Take an appropriate amount of Danggui Yinzi granules, grind them into powder, take about 0.4g, accurately weigh, accurately add 20mL of 80% methanol aqueous solution by volume, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 80% methanol aqueous solution by volume, shake well, filter, and take the filtrate to obtain the test solution.

[0157] 2. Determination method

[0158] Pipette 1 μL each of the test solution and the paeoniflorin reference solution, inject them into the high performance liquid chromatography instrument, and measure them. Calculate the relative correction factor and content of each component according to the measurement results.

[0159] 3. Determination of chromatographic conditions

[0160] 3.1 Selection of mobile phase

[0161] In this embodiment, acetonitrile-0.05vol% phosphoric acid, methanol-0.05vol% phosphoric acid, acetonitrile-0.1vol% phosphoric acid, methanol-0.1vol% phosphoric acid, acetonitrile-0.2vol% phosphoric acid, methanol-0.2vol% phosphoric acid and other mobile phases were selected for investigation. The chromatograms are shown in FIG. Figure 1 . As can be seen from the figure, when acetonitrile is used as mobile phase A, the chromatographic peak shape is narrow and the separation effect is poor, while when methanol is used as mobile phase A, the peak shape is relatively good and the separation effect is better, so methanol is selected as mobile phase A. As for the concentration of mobile phase B phosphoric acid aqueous solution, when 0.05vol% phosphoric acid is used, the chromatographic peaks are difficult to separate, while when 0.1vol% and 0.2vol% phosphoric acid are used, good separation can be achieved; therefore, 0.06vol% to 0.2vol% phosphoric acid is used as mobile phase B. In addition, it can be seen from the figure that when 0.1vol% phosphoric acid is used, the chromatographic peaks are well separated, there are few impurity peaks, and the peak shape is good. Therefore, 0.1vol% phosphoric acid is the best choice as mobile phase B.

[0162] 3.2 Gradient elution procedure investigation

[0163] Through comparative tests of various gradient elution conditions, the elution gradient was optimized and the final gradient elution program was determined. The gradient elution conditions are shown in Tables 19 to 21, and the chromatograms are shown in Figure 2 The results show that under the gradient elution condition 3 in Table 21, the baseline is stable and the separation effect of each component is good.

[0164] Table 19 Mobile phase gradient elution conditions 1

[0165]

[0166] Table 20 Mobile phase gradient elution conditions 2

[0167]

[0168] Table 21 Mobile phase gradient elution conditions 3

[0169]

[0170] 3.3 Selection of detection wavelength

[0171] Danggui Yinzi Granules is composed of 11 kinds of Chinese herbal medicines, including Angelica sinensis, Radix Paeoniae Alba, Chuanxiong Rhizome, Rehmannia glutinosa, Fried Tribulus terrestris, Saposhnikovia divaricata, Schizonepeta tenuifolia, Polygonum multiflorum, Astragalus membranaceus, Fried Glycyrrhiza uralensis, and Zingiber officinale. The components are complex, and the maximum absorption wavelengths of the components are different. According to the literature of Angelica sinensis, Radix Paeoniae Alba, Chuanxiong Rhizome, Rehmannia glutinosa, etc., Angelica sinensis and Chuanxiong Rhizome both contain ferulic acid, and the maximum absorption wavelength is about 310 nm; Angelica sinensis and Chuanxiong Rhizome both contain Ligustilide I, and the maximum absorption wavelength is about 280 nm; the maximum absorption wavelength of paeoniflorin in Radix Paeoniae Alba is about 230 nm; the maximum absorption wavelength of calycosin-7-glucoside in Astragalus membranaceus is about 260 nm; the maximum absorption wavelength of caffeic acid in Chuanxiong Rhizome and Schizonepeta tenuifolia is about 320 nm; the maximum absorption wavelength of magnolol and honokiol in Saposhnikovia divaricata is about 250 nm; the maximum absorption wavelength of rosmarinic acid in Schizonepeta tenuifolia is about 300 nm; the maximum absorption wavelength of glycyrrhizin and glycyrrhizic acid in Fried Glycyrrhiza uralensis is about 250 nm. The maximum absorption wavelengths of the components are different, so the detection wavelength can be initially considered to be 220-300 nm. Further, considering the smooth baseline of the overall chromatogram, it is decided to use the switching wavelength mode, with the elution time being 0-35 min, the wavelength being set at 230 nm, and the detection of caffeic acid, paeoniflorin, ferulic acid, glycyrrhizin, calycosin-7-glucoside, magnolol, and honokiol; the elution time being 35-60 min, the wavelength being set at 254 nm, and the detection of rosmarinic acid, 5-O-methylvisamminol, and glycyrrhizic acid. Under the corresponding wavelength, the baseline is smooth, and the separation effect of each component is good.

[0172] 3.4 Other chromatographic conditions

[0173] 3.4.1 Column temperature

[0174] Different column temperatures were investigated: the column temperatures were 28 ℃, 30 ℃, 32 ℃, and 35 ℃, respectively. The test solution prepared under “1.3 Preparation of test solution” was precisely taken, and the chromatographic conditions in “1.1 Chromatographic conditions” were used for sample injection and determination, and the chromatogram was recorded.

[0175] The test results are shown in Table 3.4.1-1. Figure 3 As can be seen from the figure, each chromatographic peak is well separated and has good response. Therefore, 28-35 ℃ can be used as the column temperature.

[0176] 3.4.2 Injection volume

[0177] Different injection volumes were investigated: the injection volumes were 0.5 μL, 1.0 μL, 1.5 μL, and 2.0 μL, respectively. The test solution prepared under “1.3 Preparation of test solution” was precisely taken, and the chromatographic conditions in “1.1 Chromatographic conditions” were used for sample injection and determination, and the chromatogram was recorded.

[0178] The test results are shown in Table 3.4.2-1. Figure 4As shown in the figure, each chromatographic peak is well separated, and the response is good. Therefore, 0.5-2.0 μL can be used as the injection amount.

[0179] 3.4.3 Flow rate

[0180] The flow rate was investigated: the flow rates were 0.18 mL / min, 0.2 mL / min and 0.22 mL / min, respectively. The test solution prepared under "1.3 Preparation of test solution" was precisely pipetted, and the chromatogram was recorded under the chromatographic conditions in "1.1 Chromatographic conditions".

[0181] The test results are shown in Figures 5 to 7 As shown in the figure, each chromatographic peak is well separated, and the response is good. Therefore, 0.18-0.22 mL / min can be used as the flow rate. In addition, it was found during the experiment that when the flow rate reached 0.25 mL / min, the separation degree was poor, and the chromatographic column was prone to burst.

[0182] 4 Methodology investigation

[0183] 4.1 Investigation of specificity

[0184] The negative samples of Angelica sinensis and Ligusticum chuanxiong, the negative sample of Angelica sinensis, the negative samples of Angelica sinensis and Schizonepeta tenuifolia, the negative sample of Radix Paeoniae Alba, the negative sample of Saposhnikovia divaricata, the negative sample of Astragalus membranaceus and the negative sample of Glycyrrhiza uralensis were prepared according to the preparation method of test solution in Example 1, Section 1.3, to prepare the negative sample solution of Angelica sinensis and Ligusticum chuanxiong, the negative sample solution of Angelica sinensis, the negative sample solution of Angelica sinensis and Schizonepeta tenuifolia, the negative sample solution of Radix Paeoniae Alba, the negative sample solution of Saposhnikovia divaricata, the negative sample solution of Astragalus membranaceus and the negative sample solution of Glycyrrhiza uralensis.

[0185] 1 μL of the mixed reference solution, the test solution, the negative sample solution of Angelica sinensis and Ligusticum chuanxiong, the negative sample solution of Angelica sinensis, the negative sample solution of Angelica sinensis and Schizonepeta tenuifolia, the negative sample solution of Radix Paeoniae Alba, the negative sample solution of Saposhnikovia divaricata, the negative sample solution of Astragalus membranaceus and the negative sample solution of Glycyrrhiza uralensis was precisely pipetted and injected into the liquid chromatograph, and the sample was determined by injection under the chromatographic conditions in "1.1 Chromatographic conditions" in Example 1. The results are shown in Figures 8 to 14 . The results show that each component chromatographic peak has good separation degree, the theoretical plate number is not less than 5000, the retention time is consistent with that of the reference substance, and no detection is made in the corresponding negative sample, indicating that the method has good specificity.

[0186] 4.2 Investigation of repeatability

[0187] Take the same batch of Dangui Yinzi granules, prepare 6 test solution according to the method of "1.3 Preparation of test solution", and determine by injecting sample under the chromatographic conditions of "1.1 Chromatographic conditions". The results are shown in Table 22. The RSD of the contents of caffeic acid, paeoniflorin, ferulic acid, glycyrrhizin, calycosin-7-glucoside, prim-O-glucosylcimifugin, senkyunolide I, rosmarinic acid, 5-O-methylvisamminol glucoside and glycyrrhizic acid are all less than 3%, indicating that the method has good repeatability.

[0188] Table 22 Repeatability test results (%)

[0189]

[0190] 4.3 Stability test

[0191] Precisely take the test solution under "1.3 Preparation of test solution", and determine by injecting sample under the chromatographic conditions of "1.1 Chromatographic conditions" at 0h, 2h, 4h, 8h, 12h and 24h after preparation. The results are shown in Table 23. The RSD of the peak areas of caffeic acid, paeoniflorin, ferulic acid, glycyrrhizin, calycosin-7-glucoside, prim-O-glucosylcimifugin, senkyunolide I, rosmarinic acid, 5-O-methylvisamminol glucoside and glycyrrhizic acid are all less than 3%, indicating that the sample is stable within 24 hours.

[0192] Table 23 Stability test results

[0193]

[0194] 4.4 Spiking recovery test

[0195] Take the same batch of Dangui Yinzi granules with known content, 9 portions of about 0.2g each, and accurately weigh, place in a conical flask with a stopper, and divide into 3 groups. Precisely add mixed control solution at high, medium and low concentrations respectively, prepare test solution according to the method of "1.3 Preparation of test solution", and determine by injecting sample under the chromatographic conditions of "1.1 Chromatographic conditions". Calculate the recovery rate, and the results are shown in Table 24. The spiking recovery rates of caffeic acid, paeoniflorin, ferulic acid, glycyrrhizin, calycosin-7-glucoside, prim-O-glucosylcimifugin, senkyunolide I, rosmarinic acid, 5-O-methylvisamminol glucoside and glycyrrhizic acid are all within 85%-105%, which meet the recovery limit specified in the Pharmacopoeia, and the RSD is less than 3%, indicating that the method has good accuracy.

[0196] Table 24 Spiking recovery test results (n=9)

[0197]

[0198]

[0199]

[0200] Comparison of the determination results between 5QAMS method and external standard method (ESM)

[0201] Take appropriate amounts of 10 batches of Danggui Yinzi granules samples, prepare test solutions according to the method under "1.3 Preparation of test solution", and inject and determine the contents of each component according to the chromatographic conditions under "1.1 Chromatographic conditions". The external standard method and the single measurement multiple evaluation method were used to determine the contents of each component, respectively. SPSS20.0 software was used to perform Pearson correlation coefficient (r) analysis on the two groups of test results. The results are shown in Tables 25 and 26. The correlation coefficient r between the two methods was 1.000, and the content results calculated by the two methods were basically consistent. The relative standard deviations of caffeic acid, ferulic acid, liquiritin, calycosin glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside, and glycyrrhizic acid were 0.00%-0.99%, 0.81%-2.99%, 0.57%-2.84%, 1.19%-2.91%, 0.06%-0.36%, 0.34%-0.80%, 0.54%-2.96%, 0.56%-2.99%, and 0.59%-1.98%, respectively, all less than 3%, indicating that there was no significant difference in the calculation results between the two methods.

[0202] Table 25 Comparison of the results of the external standard method and the one-measurement-multiple-evaluation method 1 (%, n=4)

[0203]

[0204]

[0205] Table 26 Comparison of the results of the external standard method and the one-measurement-multiple-evaluation method 2 (%, n=4)

[0206]

[0207] In summary, the present invention uses a one-test, multiple-evaluation method to detect 10 batches of Danggui Yinzi granules. This method is simple, accurate, and easy to operate, so it can be used for content determination when a reference substance is in short supply. At the same time, it can reduce solvent consumption and analysis time, and is more conducive to environmental protection.

[0208] The above is a preferred embodiment of the invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for detecting a Dangguiyinzi composition, characterized in that: include: (1) extracting the Danggui Yinzi composition with a first methanol aqueous solution to obtain a test solution; wherein the Danggui Yinzi composition comprises the following components: Danggui, White Peony Root, Chuanxiong, Rehmannia Glutinosa, stir-fried Tribulus Terrestris, Saposhnikovia Divaricata, Schizonepeta Tenuifolia, Polygonum Multiflorum, Astragalus, stir-fried Licorice Root, and ginger; (2) Caffeic acid reference substance, paeoniflorin reference substance, ferulic acid reference substance, liquiritin reference substance, calycosin isoflavone glucoside reference substance, cimicifuga glycoside reference substance, ligusticum lactone I reference substance, rosmarinic acid reference substance, 5- O -Methylvisaminol glycoside reference substance and glycyrrhizic acid reference substance are dissolved or extracted with a second methanol aqueous solution to obtain a mixed reference substance solution; and the paeoniflorin reference substance is dissolved or extracted with a second methanol aqueous solution to obtain a paeoniflorin reference substance solution; (3) Injecting the test solution, the paeoniflorin reference solution, and the mixed reference solution into a liquid chromatograph to determine the content of paeoniflorin in the Danggui Yinzi composition; (4) Using paeoniflorin as the internal standard, the internal standard method was used to calculate the content of caffeic acid, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5- O - Contents of methyl visamidoside and glycyrrhizic acid; The chromatographic column of the liquid chromatograph is CORTECS UPLC T3, which has a column length of 150 mm, a column diameter of 2.1 mm, and a particle size of the stationary phase of 1.6 μm. The liquid chromatograph uses methanol as mobile phase A and a 0.06-0.2 vol% phosphoric acid aqueous solution as mobile phase B for gradient elution. The gradient elution curve is: 0-5 min, mobile phase A from 4% to 21%, mobile phase B from 96% to 79%; 5-25 min, mobile phase A from 21% to 32%, mobile phase B from 79% to 68%; 25-33 min, mobile phase A from 32% to 34%, mobile phase B from 68% to 66%; 33-37 min, mobile phase A from 34% to 40%, mobile phase B from 66% to 60%; 37-40 min, mobile phase A from 40% to 42%, mobile phase B from 60% to 58%; 40-42 min, mobile phase A from 42% to 60%, mobile phase B from 58% to 40%; 42-55 min, mobile phase A from 60% to 95%, mobile phase B from 40% to 5%; 55-56 min, mobile phase A from 95% to 4%, mobile phase B from 5% to 96%; 56-60 min, mobile phase A: 4%, mobile phase B: 96%; The concentration of the first methanol aqueous solution is 50-90 vol%; The concentration of the second methanol aqueous solution is 50-90 vol%; The detection wavelengths of the detector of the liquid chromatograph are: 230 nm for 0-35 min and 254 nm for 35-60 min.

2. The method for detecting the Dangguiyinzi composition according to claim 1, wherein: The mobile phase B is a 0.1 vol% phosphoric acid aqueous solution; and / or The concentration of the first methanol aqueous solution is 80 vol%; and / or The concentration of the second methanol aqueous solution is 80 vol%.

3. The method for detecting the Dangguiyinzi composition according to claim 1, wherein: In step (1), ultrasonic extraction is performed with an ultrasonic power of 200-500 W, an ultrasonic frequency of 20-50 kHz, and an ultrasonic time of 10-60 min; and / or In step (1), the ratio of the Angelica sinensis decoction composition to the first methanol aqueous solution is 0.2-0.6 g: 15-25 mL.

4. The method for detecting the Dangguiyinzi composition according to claim 1, wherein: In the mixed reference solution, the caffeic acid reference substance, paeoniflorin reference substance, ferulic acid reference substance, liquiritin reference substance, calycosin isoflavone glucoside reference substance, cimicifugo glycoside reference substance, ligusticum lactone I reference substance, rosmarinic acid reference substance, 5- O The concentrations of the -methylvisaminol glucoside reference substance and the glycyrrhizic acid reference substance are: 210-220 μg / mL, 185-195 μg / mL, 205-215 μg / mL, 140-150 μg / mL, 140-150 μg / mL, 145-155 μg / mL, 215-225 μg / mL, 85-95 μg / mL, 160-170 μg / mL, and 175-185 μg / mL, respectively; and / or In the paeoniflorin reference substance solution, the concentration of the paeoniflorin reference substance is 80-120 μg / mL.

5. The method for detecting the Dangguiyinzi composition according to claim 1, wherein: The column temperature of the chromatographic column is 28-35°C.

6. The method for detecting the Dangguiyinzi composition according to claim 1 or 5, wherein: The column temperature of the chromatographic column is 30°C.

7. The method for detecting the Dangguiyinzi composition according to claim 1, wherein: The flow rate of the liquid chromatograph is 0.18~0.22mL / min The injection volume of the test solution is 0.5~2.0 μL, the injection volume of the paeoniflorin reference solution is 0.5~2.0 μL; the injection volume of the mixed reference solution is 0.5~2.0 μL.

8. The method for detecting the Dangguiyinzi composition according to claim 1 or 7, wherein: The flow rate of the liquid chromatograph was 0.2 mL / min; The injection volume of the test solution was 1.0 μL, the injection volume of the paeoniflorin reference solution was 1.0 μL; and the injection volume of the mixed reference solution was 1.0 μL.

9. The method for detecting the Dangguiyinzi composition according to claim 1, wherein: Step (4) includes: (4.1) diluting the mixed reference solution in a gradient manner to obtain a plurality of mixed reference solutions to be tested at a series of concentrations; (4.2) Using liquid chromatography, the plurality of mixed reference solutions to be tested were respectively measured, and the caffeic acid, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifugoside, ligusticolide I, rosmarinic acid, 5- O -Relative correction factors for methylvisamin and glycyrrhizic acid; (4.3) According to the relative correction factors, the caffeic acid, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifugoside, ligusticum lactone I, rosmarinic acid, 5- O -The content of methyl visamidoside and glycyrrhizic acid.

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