Detection method of Chinese angelica potion composition

Abstract

The invention relates to the technical field of quality detection of traditional Chinese medicines, and particularly discloses a detection method of an angelica sinensis potion composition, which comprises the following steps: (1) preparing a test solution; (2) preparing a mixed reference substance solution and / or a paeoniflorin reference substance solution; (3) determining the test solution and the paeoniflorin reference substance solution and / or the mixed reference substance solution to obtain the content of paeoniflorin; (4) taking paeoniflorin as an internal standard substance, and calculating the contents of caffeic acid, ferulic acid, liquiritin, calycosin-7-glucoside, cimicifugin, senkyunolide I, rosmarinic acid, 5-O-methylvisammioside and glycyrrhizic acid by using an internal standard method; a chromatographic column of the liquid chromatograph takes octadecylsilane chemically bonded silica as a stationary phase, methanol as a mobile phase A and a phosphoric acid aqueous solution with the concentration of 0.06-0.2 vol% as a mobile phase B for gradient elution. The detection method is high in practicability, easy to operate, rapid and accurate in testing and low in cost.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine quality detection, and in particular to a detection method for an Angelica sinensis decoction composition. Background Art

[0002] Danggui Yinzi comes from Yan's Jisheng Prescriptions written by Yan Yonghe in the Song Dynasty. It is mainly used to treat "stagnation of heart blood, internal wind-heat syndrome, skin sores, swelling or itching, pus infiltration, or urticaria; or skin itching, especially at night, pale red tongue, thin fur, and fine pulse." It has the effects of nourishing blood, activating blood circulation, dispelling wind and relieving itching. The whole recipe is composed of 11 medicinal ingredients, including angelica, white peony root, chuanxiong, rehmannia root, stir-fried tribulus, saposhnikovia root, schizonepeta spicate, polygonum multiflorum, astragalus root, stir-fried licorice, and ginger. In the formula, angelica enters the liver and spleen meridians, replenishes blood and activates blood circulation; white peony enters the liver and spleen blood division, softens the liver, nourishes blood and restrains yin; Chuanxiong promotes qi and activates blood circulation, dispels wind, and replenishes blood without stagnating blood; Shengdi nourishes yin and nourishes blood; Polygonum multiflorum nourishes blood and dispels wind; Schizonepeta is pungent and fragrant to dispel wind, bitter and warm to clear blood, and Saposhnikovia divaricata is used to disperse wind inside and outside; Tribulus terrestris promotes blood circulation, dispels wind and relieves itching; Astragalus and licorice strengthen the spleen and replenish qi. The whole formula replenishes blood, activates blood circulation, and strengthens the spleen and replenishes qi to produce blood, replenishes the middle and promotes circulation; dispels wind and relieves itching, and strengthens the spleen and strengthens the defense to consolidate the exterior, replenishes the middle and relieves itching.

[0003] Modern clinical studies have shown that Danggui Yinzi is mainly used to treat chronic urticaria of blood deficiency, wind dryness and qi and blood deficiency type, and has a definite clinical efficacy. Studies have shown that Danggui Yinzi may treat chronic spontaneous urticaria by increasing the level of cell autophagy, inhibiting delayed allergic reactions, regulating immune function, and inhibiting allergic inflammatory reactions. However, the ingredients of Chinese herbal compound are diverse, and the mechanism of action involves multiple targets and multiple links. The pharmacological ingredients and mechanism of action of Danggui Yinzi are not yet fully understood. Studies have predicted that the more important active ingredients of Danggui Yinzi are: resveratrol, rhubarb, luteolin, 7-O-methylisoeugenol, astragalol, glycyrrhizin, naringenin, donkey herbaceous phenol, 7-methoxy-2-methyl isoflavone, meditartin, licorice chalcone A, wogonin, β-sitosterol, formononetin, quercetin, isorhamnetin and kaempferol through screening of the drug-active ingredient-target-disease network. Most of the above active ingredients are flavonoids, so it is speculated that flavonoids are the main active ingredients of Danggui Yinzi that play the role of nourishing blood, activating blood circulation, dispelling wind and relieving itching. The research on Danggui Yinzi mainly focuses on the summary of clinical application of traditional Chinese medicine, clinical trials and experimental research, and no quantitative research has been conducted on its main active ingredients. 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 composite material, which has multiple components to be detected, simple detection procedures and low detection costs.

[0005] In order to solve the above technical problems, the present invention provides a method for detecting an angelica decoction 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 licorice glycoside reference substance, the calycosin glucoside reference substance, the cimicifuga glycoside reference substance, the ligusticum chuanxiong lactone I reference substance, the rosmarinic acid reference substance, the 5-O-methylvisaminol glucoside reference substance and the glycyrrhizic acid reference substance with the second methanol aqueous solution to obtain a mixed reference substance solution; and / or dissolving or extracting the paeoniflorin reference substance with the 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 glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside and glycyrrhizic acid in the Danggui Yinzi composition were calculated by the internal standard method;

[0010] The chromatographic column of the liquid chromatograph uses 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] 0-5min, 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 is 4%, mobile phase B is 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, 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 subjected to ultrasonic treatment or heating reflux treatment 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 to this. Preferably it is 0.2-0.8g:15-50mL. It should be noted that the above ratio can be a ratio that is enlarged or reduced in proportion. 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 to this.

[0030] Exemplarily, the extraction time is 12 min, 27 min, 34 min, 38 min, 53 min or 75 min, but not limited thereto, preferably 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] Wherein, 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, cimicifuga glycoside reference substance, ligusticum chuanxiong 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 invention, in the mixed reference solution, the concentrations of the caffeic acid reference, paeoniflorin reference, ferulic acid reference, liquiritin reference, calycosin glucoside reference, cimicifuga glycoside reference, ligusticol I reference, rosmarinic acid reference, 5-O-methylvisaminol glycoside reference and glycyrrhizic acid reference 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, 180.6816 μg / mL, respectively.

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

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

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

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

[0040] Preferably, in some embodiments of the present invention, 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 invention, the flow rate of the liquid chromatograph is 0.18-0.22 mL / min, exemplified by 0.19 mL / min, 0.2 mL / min or 0.21 mL / min, but not limited thereto.

[0043] The detection wavelength of the liquid chromatograph is 220-300 nm, exemplarily 225 nm, 240 nm, 255 nm, 270 nm or 285 nm, but 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 to 35 min, and 254 nm for 35 to 60 min.

[0050] Among them, in some embodiments of the present invention, in step (4), paeoniflorin is first used as an internal standard, the relative correction factors of 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 general rule 0512 high performance liquid chromatography of Part IV of the Chinese Pharmacopoeia (2020 edition), but is not limited thereto.

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

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

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

[0054] (4.3) The contents of caffeic acid, ferulic acid, liquiritigenin, calycosin glucoside, cimicifuga glycoside, ligusticol I, rosmarinic acid, 5-O-methylvisaminol glucoside 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 one-measurement multiple-evaluation method (i.e., QAMS method) using paeoniflorin as an internal standard, and obtains the contents of 10 components in the Danggui Yinzi composition, including caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticol I, rosmarinic acid, 5-O-methylvisaminol glycoside, and glycyrrhizic acid, through one measurement, with strong practicality, simple operation, rapid and accurate testing, and cost saving. The method of the present invention is simple and easy to operate, and the measurement result is accurate, the method has good repeatability, good stability, and good durability, and provides a basis for establishing the quality evaluation of the Danggui Yinzi composition. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0058] Figure 2 This is the chromatogram obtained by the gradient elution investigation and determination 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 is a chromatogram obtained by measuring with different 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 It is a chromatogram of a blank solvent, a mixed reference substance, and Dangguiyinzi granules in the specificity investigation in Example 2 of the present invention;

[0065] Fig. 9 It 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] Fig.10 It 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] Fig.11 It is a chromatogram of the mixed reference substance, Dangguiyinzi granules, and the negative sample lacking white peony root in the specificity investigation in Example 2 of the present invention;

[0068] Fig.12 It is a chromatogram of the mixed reference substance, Dangguiyinzi granules, and the negative sample lacking saposhnikovia in the specificity investigation in Example 2 of the present invention;

[0069] Fig.13 It is a chromatogram of the mixed reference substance, Dangguiyinzi granules, and the negative sample lacking astragalus in the specificity investigation in Example 2 of the present invention;

[0070] Fig.14 It is a chromatogram of the mixed reference substance, Dangguiyinzi granules, and the negative sample lacking licorice in the specificity investigation in Example 2 of the present invention;

[0071] In the figure, peak 1 is caffeic acid, peak 2 is paeoniflorin, peak 3 is ferulic acid, peak 4 is liquiritigenin, peak 5 is calycosin glucoside, peak 6 is cimicifuga glycoside, peak 7 is ligusticum chuanxiong lactone I, peak 8 is rosmarinic acid, peak 9 is 5-O-methylvisaminol glycoside, and peak 10 is glycyrrhizic acid. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

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

[0074] 1. Instrument

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

[0076] 2. Reagents

[0077] Methanol was of chromatographic grade (Merck, Germany), phosphoric acid was of chromatographic grade (Tianjin Komiou Chemical Reagent Co., Ltd.), water was ultrapure water, and other reagents were of analytical grade.

[0078] 3. Drug testing

[0079] Ten batches of Dangguiyinzi granules were prepared in the laboratory.

[0080] Reference substances: Caffeic acid (China Food and Drug Inspection Institute, batch number 110885-201703, purity 99.7%), paeoniflorin (China Food and Drug Inspection Institute, batch number 110736-202044, purity 97.4%), ferulic acid (China Food and Drug Inspection Institute, batch number 110773-201915, purity 99.4%), licorice glycoside (China Food and Drug Inspection Institute, batch number 111610-202209, purity 95.2%), calycosin isoflavone glucoside (China Food and Drug Inspection Institute, batch number 111920-201907, purity 96.8%), Cimicifuga heracleifolia Glucoside (China Food and Drug Inspection Institute, batch number 111522-202214, purity 95.7%), ligusticum chuanxiong lactone I (China Food and Drug Inspection Institute, batch number 112071-202101, purity 99.2%), rosmarinic acid (China Food and Drug Inspection Institute, batch number 111871-202007, purity 98.1%), 5-O-methylvisaminol glycoside (China Food and Drug Inspection Institute, batch number 111523-201811, purity 97.4%), glycyrrhizic acid (China Food and Drug Inspection Institute, batch number 110731-202122, purity 94.4%).

[0081] Example 1 Determination of relative correction factor

[0082] 1. Preparation of chromatographic conditions, reference solution, and test solution

[0083] 1.1 Chromatographic conditions

[0084] 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 provisions in Table 1; detection wavelength: 230 nm (0-35 min, caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifuga glycoside, ligusticolide I), 254 nm (35-60 min, rosmarinic acid, 5-O-methylvisaminol glycoside, glycyrrhizic acid); column temperature: 30°C; flow rate: 0.2 mL / min; injection volume: 1 μL.

[0085] Table 1 Mobile phase gradient elution conditions

[0086]

[0087] 1.2 Preparation of mixed reference solution

[0088] Take appropriate amount of caffeic acid reference substance, paeoniflorin reference substance, ferulic acid reference substance, liquiritin reference substance, calycosin glucoside reference substance, cimicifuga glycoside reference substance, ligusticum chuanxiong lactone I reference substance, rosmarinic acid reference substance, 5-O-methylvisaminol 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, including angelica, white peony root, chuanxiong, rehmannia root, stir-fried tribulus, saposhnikovia root, schizonepeta spicate, polygonum multiflorum, astragalus root, stir-fried licorice root, and ginger. In the formula, angelica root nourishes blood, invigorates blood circulation, and regulates the body's defense system, and is the main herb. White peony root nourishes blood, astringes yin, and relieves pain; raw rehmannia root clears heat, nourishes yin, and produces body fluid, and is a key herb for cooling blood and nourishing blood; chuanxiong promotes qi and activates blood circulation, and is a herb for qi in the blood; astragalus root is good at invigorating qi, detoxifying, astringing sores, and promoting muscle growth, and is a holy herb for sores. The combination of the two can not only help nourish yin and blood, but also prevent greasy products from being digested. White peony root, raw rehmannia root, chuanxiong, and astragalus root are all ministers, helping the main herb to work together to invigorate qi, invigorate blood circulation, and nourish blood and moisten dryness. Saposhnikovia and Schizonepeta dispel pathogenic factors and relieve the symptoms of the disease, eliminate sores and clear rashes, and when combined with Astragalus, they can consolidate the surface of the body without retaining pathogenic factors, and dispel wind without hurting the body. Polygonum multiflorum replenishes essence and blood, nourishes yin and body fluid, and can relieve the poison of skin sores, rashes, scabies, and ringworms. Tribulus terrestris dispels wind, promotes qi, and relieves itching. These four herbs are adjuvants, dispel wind and relieve itching. Licorice is the adjuvant that harmonizes the other herbs. The whole formula is well matched, nourishes blood and yin without leaving stasis, dispels wind and dispels pathogenic factors without hurting the body, takes into account both the symptoms and the root causes, and uses both attack and supplementation to achieve the effects of nourishing blood, activating blood circulation, and dispelling wind and relieving itching. The main components of Angelica sinensis are ferulic acid, ligusticol I, etc.; the main component of white peony root is paeoniflorin; the main components of Ligusticum chuanxiong are ferulic acid, ligusticol I, caffeic acid, etc.; the main components of Saposhnikovia divaricata are cimicifuga glycoside, 5-O-methylvisaminol glycoside, etc.; the effective components of Schizonepeta spicate are caffeic acid, rosmarinic acid, etc.; the effective component of Astragalus membranaceus is calycosin glucoside; the main components of fried liquorice are liquiritirubin and glycyrrhizic acid. Therefore, caffeic acid, paeoniflorin, ferulic acid, liquiritirubin, calycosin glucoside, cimicifuga glycoside, ligusticol 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 the item "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 the item "1.1 Chromatographic conditions". The peak area of ​​each component was recorded. Caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticum chuanxiong lactone I, rosmarinic acid, 5-O-methylvisaminol glycoside, and glycyrrhizic acid were used as internal standards to calculate the relative correction factor f of 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 RSD of the relative correction factor of each component to be measured is less than 3%, and the result is more accurate, so 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 considerations

[0118] 4.1 Linear relationship investigation

[0119] Accurately pipette the mixed reference solution prepared under "1.2 Preparation of mixed reference solution", add 80% methanol aqueous solution to prepare mixed reference solutions of series concentrations, inject and measure according to the chromatographic conditions under "1.1 Chromatographic conditions", and record the chromatogram. Linear regression is performed with 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, which shows that the linear relationship of each component is good 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 under "1.2 Preparation of mixed reference solution", and inject and measure according to the chromatographic conditions under "1.1 Chromatographic conditions", inject 6 injections continuously, and record the peak area. The results are shown in Table 13. The RSDs of the peak areas of caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside and glycyrrhizic acid were all less than 3%, indicating that the instrument had good precision.

[0125] Table 13 Precision investigation 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 correction factors of the other 9 components were investigated on two HPLC systems, Waters H-class and Thermo Vanquish, and three chromatographic columns, CORTECS UPLC T3 (150mm×2.1mm, 1.6μm), ACQUITY UPLC HSST3 (150mm×2.1mm, 1.8μm), and ACQUITY UPLC BEH C18 (150mm×2.1mm, 1.7μm). The results are shown in Table 14. The RSD of each component was less than 3%, indicating that the replacement of instruments and chromatographic columns had no significant effect on the relative correction 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 chromatographic 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 RSD of each component was less than 3%, indicating that the fluctuation of 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 RSD of each component was 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] Using Waters H-class high performance liquid chromatograph and CORTECS UPLC T3 chromatographic column, 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 were investigated. The results are shown in Table 17. The RSD of each component was 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 components to be measured

[0146] In the application of QAMS, the commonly used chromatographic peak positioning methods include relative retention value method, retention time difference method, time correction method, reference extract method, etc. In this experiment, relative retention time was used to locate the chromatographic peak of the component to be tested. Peony root glycoside was used as the internal standard to investigate the relative retention time of the other 8 components under two high-performance liquid chromatography systems, Waters H-class and Thermo Vanquish, and three chromatographic columns, CORTECS UPLC T3 (150mm×2.1mm, 1.6μm), ACQUITY UPLC HSS T3 (150mm×2.1mm, 1.8μm), and ACQUITY UPLC BEH C18 (150mm×2.1mm, 1.7μm). The results are shown in Table 18. The RSD of each component using the relative retention value method was less than 3%, indicating that the relative retention value method is feasible for locating the components to be tested.

[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 provisions in Table 1; Detection wavelength: 230 nm (0-35 min, caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifuga glycoside, ligusticolide I), 254 nm (35-60 min, rosmarinic acid, 5-O-methylvisaminol glycoside, 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, weigh it accurately, 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, weigh it accurately, add 20mL of 80% methanol aqueous solution by volume, weigh it, ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol aqueous solution by volume, shake it well, filter it, and take the filtrate to obtain the test solution.

[0157] 2. Determination method

[0158] Pipette 1 μL of the test solution and the paeoniflorin reference solution, inject them into the high performance liquid chromatograph, 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-0.05 vol% phosphoric acid, methanol-0.05 vol% phosphoric acid, acetonitrile-0.1 vol% phosphoric acid, methanol-0.1 vol% phosphoric acid, acetonitrile-0.2 vol% phosphoric acid, methanol-0.2 vol% 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 peak shape of the chromatographic peak 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. 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 can be selected as mobile phase B as the best.

[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 From the results, it can be seen 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 are composed of 11 traditional Chinese medicinal materials, including Angelica sinensis, white peony root, Chuanxiong, Rehmannia glutinosa, stir-fried Tribulus terrestris, Saposhnikovia divaricata, Schizonepeta tenuifolia, Polygonum multiflorum, Astragalus membranaceus, stir-fried licorice, and ginger. The composition is complex, and the maximum absorption wavelength of each component is different. By consulting the literature of Chinese angelica, white peony root, Chuanxiong, and Rehmannia root, it is found that both Chinese angelica and Chuanxiong contain ferulic acid, with a maximum absorption wavelength of about 310nm; both Chinese angelica and Chuanxiong contain ligusticolide I, with a maximum absorption wavelength of about 280nm; the maximum absorption wavelength of paeoniflorin in white peony root is about 230nm; the maximum absorption wavelength of calycosin isoflavone glucoside in Astragalus is 260nm; the maximum absorption wavelength of caffeic acid in Chuanxiong and Nepeta spicate is about 320nm; the maximum absorption wavelength of cimicifugin glycoside and 5-O-methylvisaminol glycoside in Saposhnikovia root is about 250nm; the maximum absorption wavelength of rosmarinic acid in Nepeta spicate is about 300nm; the maximum absorption wavelength of liquiritin and glycyrrhizic acid in fried licorice is about 250nm. The maximum absorption wavelengths of each component are different, so the detection wavelength can be initially considered to be 220-300nm. Furthermore, considering the stability of the overall baseline of the comprehensive spectrum, it was decided to use the method of switching wavelengths. The elution time was 0-35 min, the wavelength was set to 230 nm, and caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, and ligusticum chuanxiong lactone I were detected; the elution time was 35-60 min, the wavelength was set to 254 nm, and rosmarinic acid, 5-O-methylvisamidol glycoside, and glycyrrhizic acid were detected. At the corresponding wavelengths, the baseline was stable and the separation effect of each component was good.

[0172] 3.4 Other chromatographic conditions

[0173] 3.4.1 Column temperature

[0174] Different column temperatures were investigated: column temperatures of 28°C, 30°C, 32°C, and 35°C were used respectively. Accurately pipette the test solution prepared under "1.3 Preparation of test solution" and inject the sample according to the chromatographic conditions under "1.1 Chromatographic conditions" to record the chromatogram.

[0175] Test results such as Figure 3 As shown in the figure, it can be seen that each chromatographic peak is well separated and has a good response. Therefore, 28-35°C can be used as the column temperature.

[0176] 3.4.2 Injection volume

[0177] Different injection volumes were investigated: 0.5μL, 1.0μL, 1.5μL and 2.0μL were used respectively. Accurately pipette the test solution prepared under "1.3 Preparation of test solution" and inject according to the chromatographic conditions under "1.1 Chromatographic conditions" to record the chromatogram.

[0178] Test results such as Figure 4As shown in the figure, it can be seen that each chromatographic peak is well separated and has a good response. Therefore, 0.5 to 2.0 μL can be used as the injection volume.

[0179] 3.4.3 Flow rate

[0180] Different flow rates were investigated: 0.18 mL / min, 0.2 mL / min and 0.22 mL / min were used respectively. Accurately pipette the test solution prepared under "1.3 Preparation of test solution" and inject the sample according to the chromatographic conditions under "1.1 Chromatographic conditions" to record the chromatogram.

[0181] Test results such as Figure 5 to Figure 7 As shown in the figure, it can be seen that each chromatographic peak is well separated and has a good response. Therefore, the flow rate can be 0.18-0.22 mL / min. In addition, the experiment found that when the flow rate reached 0.25 mL / min, the separation was poor and the chromatographic column was prone to explosion.

[0182] 4 Methodological considerations

[0183] 4.1 Specificity Investigation

[0184] Take negative samples lacking Angelica and Chuanxiong, negative samples lacking Angelica, negative samples lacking Angelica and Schizonepeta spicate, negative samples lacking White Peony, negative samples lacking Saposhnikovia, negative samples lacking Astragalus and negative samples lacking Licorice, and prepare negative sample solutions lacking Angelica and Chuanxiong, negative sample solutions lacking Angelica, negative sample solutions lacking Angelica and Schizonepeta spicate, negative sample solutions lacking White Peony, negative sample solutions lacking Saposhnikovia, negative sample solutions lacking Astragalus and negative sample solutions lacking Licorice respectively according to the test sample solution preparation method of Section 1.3 in Example 1.

[0185] Accurately pipette 1 μL each of the mixed reference solution, the test solution, the negative sample solution lacking Angelica sinensis and Chuanxiong, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Angelica sinensis and Nepeta tenuifolia, the negative sample solution lacking White Peony Root, the negative sample solution lacking Saposhnikovia divaricata, the negative sample solution lacking Astragalus membranaceus and the negative sample solution lacking Licorice, and inject into the liquid chromatograph. Sample injection and determination are carried out according to the chromatographic conditions under "1.1 Chromatographic Conditions" in Example 1. The results are shown in the table below. Figure 8 to Figure 14 The results showed that the chromatographic peaks of each component had good separation, the theoretical plate number was not less than 5000, the retention time was consistent with the chromatographic peak of the reference substance, and it was not detected in the corresponding negative samples, indicating that the method had good specificity.

[0186] 4.2 Repeatability study

[0187] Take an appropriate amount of the same batch of Danggui Yinzi granules, prepare 6 test solutions in parallel according to the method under "1.3 Preparation of test solution", and inject and measure according to the chromatographic conditions under "1.1 Chromatographic conditions". The results are shown in Table 22. The RSDs of the calculated contents of caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside and glycyrrhizic acid were all less than 3%, indicating that the method had good repeatability.

[0188] Table 22 Repeatability test results (%)

[0189]

[0190] 4.3 Stability investigation

[0191] The test solution under "1.3 Preparation of test solution" was accurately aspirated, and the sample was injected and measured according to the chromatographic conditions under "1.1 Chromatographic conditions" at 0h, 2h, 4h, 8h, 12h and 24h after preparation, respectively. The results are shown in Table 23. The RSDs of the peak areas of caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside and glycyrrhizic acid were all less than 3%, indicating that the sample was stable within 24 hours.

[0192] Table 23 Stability test results

[0193]

[0194] 4.4 Sample recovery test

[0195] Take 9 portions of Danggui Yinzi granules with known content from the same batch, each portion is about 0.2g, accurately weighed, placed in a stoppered conical bottle, divided into 3 groups, accurately add appropriate amount of mixed reference solution according to high, medium and low concentrations, prepare the test solution according to the method under "1.3 Preparation of test solution", inject and determine according to the chromatographic conditions under "1.1 Chromatographic conditions", calculate the recovery rate, and the results are shown in Table 24. The calculated sample recovery rates of caffeic acid, paeoniflorin, ferulic acid, liquiritin, calycosin isoflavone glucoside, cimicifuga glycoside, ligusticum chuanxiong lactone I, rosmarinic acid, 5-O-methylvisaminol glycoside and glycyrrhizic acid are within 85% to 105%, all of which meet the recovery rate limits specified in the pharmacopoeia, and the RSDs are all less than 3%, indicating that the method has good accuracy.

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

[0197]

[0198]

[0199]

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

[0201] Take an appropriate amount of 10 batches of Danggui Yinzi Granules samples, prepare the test solution according to the method under "1.3 Preparation of test solution", and inject and determine the content of each component according to the chromatographic conditions under "1.1 Chromatographic conditions". The external standard method and the one-measurement-multiple-evaluation method were used to determine the content of each component, respectively. The two groups of test results were analyzed by Pearson correlation coefficient (r) using SPSS20.0 software. 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, liquiritigenin, calycosin glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glycoside 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 of which were less than 3%, indicating that there was no significant difference in the calculation results of the two methods.

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

[0203]

[0204]

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

[0206]

[0207] In summary, the present invention uses the one-test-multiple-evaluation method to detect 10 batches of Danggui Yinzi granules. The method is simple, accurate, and easy to operate, so it can be used for content determination when the 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 a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the 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; (2) dissolving or extracting the caffeic acid reference substance, the paeoniflorin reference substance, the ferulic acid reference substance, the licorice glycoside reference substance, the calycosin glucoside reference substance, the cimicifuga glycoside reference substance, the ligusticum chuanxiong lactone I reference substance, the rosmarinic acid reference substance, the 5-O-methylvisaminol glucoside reference substance and the glycyrrhizic acid reference substance with the second methanol aqueous solution to obtain a mixed reference substance solution; and / or dissolving or extracting the paeoniflorin reference substance with the second methanol aqueous solution to obtain a paeoniflorin reference substance solution; (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; (4) Using paeoniflorin as the internal standard, the contents of caffeic acid, ferulic acid, liquiritin, calycosin glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside and glycyrrhizic acid in the Danggui Yinzi composition were calculated by the internal standard method; The chromatographic column of the liquid chromatograph uses 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: 0-5min, 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 is 4%, mobile phase B is 96%; The concentration of the first methanol aqueous solution is 50 to 90 vol%; The concentration of the second methanol aqueous solution is 50-90 vol%.

2. The method for detecting the Dangguiyinzi composition according to claim 1, characterized in that: 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 Danggui Yinzi composition according to claim 1, characterized in that: 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; and / or In step (1), the ratio of the Angelica 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, characterized in that: In the mixed reference solution, the concentrations of the caffeic acid reference, the paeoniflorin reference, the ferulic acid reference, the liquiritin reference, the calycosin glucoside reference, the cimicifuga glycoside reference, the ligusticum chuanxiong lactone I reference, the rosmarinic acid reference, the 5-O-methylvisaminol glycoside reference and the glycyrrhizic acid reference 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 to 120 μg / mL.

5. The method for detecting the Dangguiyinzi composition according to claim 1, characterized in that: The column length of the chromatographic column is 100-200 mm, the column diameter is 1-3 mm, and the particle size of the stationary phase is 1.6-1.8 μm; 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, characterized in that: 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; The column temperature of the chromatographic column is 30°C.

7. The method for detecting the Dangguiyinzi composition according to claim 1, characterized in that: The flow rate of the liquid chromatograph is 0.18-0.22 mL / min, and the detection wavelength is 220-300 nm; The injection volume of the test solution is 0.5 to 2.0 μL, the injection volume of the paeoniflorin reference solution is 0.5 to 2.0 μL; or the injection volume of the mixed reference solution is 0.5 to 2.0 μL.

8. The method for detecting the Dangguiyinzi composition according to claim 1 or 7, characterized in that: The flow rate of the liquid chromatograph is 0.2 mL / min; 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.

9. The method for detecting the Danggui Yinzi composition according to claim 1 or 7, characterized in that: The detection wavelength of the detector of the liquid chromatograph is: 230 nm for 0-35 min and 254 nm for 35-60 min.

10. The method for detecting the Dangguiyinzi composition according to claim 1, characterized in that: Step (4) comprises: (4.1) diluting the mixed reference substance solution in a gradient manner to obtain a plurality of mixed reference substance solutions to be tested with a series of concentrations; (4.2) using a liquid chromatograph to measure a plurality of the mixed reference solutions to be tested, and calculating relative correction factors of caffeic acid, ferulic acid, liquiritigenin, calycosin glucoside, cimicifuga glycoside, ligusticolide I, rosmarinic acid, 5-O-methylvisaminol glucoside and glycyrrhizic acid; (4.3) The contents of caffeic acid, ferulic acid, liquiritigenin, calycosin glucoside, cimicifuga glycoside, ligusticol I, rosmarinic acid, 5-O-methylvisaminol glucoside and glycyrrhizic acid in the Danggui Yinzi composition were calculated according to the relative correction factors.

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