CN117233271A - Separation method of chemical components in compound preparation of Wen Dan decoction or construction method of fingerprint - Google Patents

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and particularly provides a separation method of chemical components in a compound preparation of a Wen Dan decoction or a construction method of a fingerprint, which comprises the following steps of (1) preparing a sample solution of the compound preparation of the Wen Dan decoction; (2) The method is characterized in that the sample solution of the compound preparation of the warm bile soup is detected by adopting a high performance liquid chromatography, octadecylsilane chemically bonded silica gel is used as a filler, acetonitrile-formic acid-containing aqueous solution is used as a mobile phase for gradient elution, and an elution program is obtained through repeated experiments.

Description

Separation method of chemical components in compound preparation of Wen Dan decoction or construction method of fingerprint

Technical Field

The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and particularly relates to a separation method of chemical components in a compound preparation of a Wen Dan decoction or a construction method of a fingerprint.

Background

The decoction for warming gallbladder is carried in Tang Dynasty (preparation of first-aid Qianjin Fang) (Tangsun Si far away), and is described in the original text: for severe illness with dysphoria and insomnia, it is advisable to take the decoction for warming gallbladder because of its cold. The prescription consists of two pinellia ternate, bamboo shavings, two immature bitter orange, three orange peels, four ginger and one liquorice; has the effects of regulating qi-flowing for eliminating phlegm, regulating stomach and promoting bile flow, and is mainly used for treating symptoms of gallbladder stagnation and phlegm disturbance, such as timidity, easy convulsion, dizziness and palpitation, vexation and insomnia, night dreaminess; or vomiting and singultus, dizziness and epilepsy. A white and greasy coating and a wiry and slippery pulse. As a classical phlegm-eliminating prescription, the traditional phlegm-eliminating prescription clinically surrounds the pathogenesis of the 'gall depression and phlegm disturbance', and is commonly used for treating senile diseases such as insomnia, coronary heart disease, hypertension, dizziness, depression and the like. In the recipe, pinellia tuber, rhizoma Pinelliae is pungent and warm in nature, and is a monarch drug for eliminating dampness and resolving phlegm, harmonizing stomach and relieving vomiting; the ministerial drugs are bamboo shavings, which are sweet and slightly cold, and are used for clearing heat, resolving phlegm, removing vexation and preventing vomiting; pinellia tuber and bamboo Ru Xiangwu, are prepared for warming and cooling, resolving phlegm and harmonizing stomach, and relieving vomiting and restlessness; dried orange peel is bitter and warm, regulates qi and moves stagnancy, and eliminates dampness and phlegm; bitter orange is bitter and slightly cold, and has the effects of reducing qi, removing stagnation, resolving phlegm and relieving distension; pericarpium Citri Reticulatae and fructus Aurantii Immaturus are combined together to warm and cool, and regulate qi and resolve phlegm; poria, spleen invigorating and dampness eliminating, du Shengtan; decocting rhizoma Zingiberis recens to harmonize spleen and stomach, and rhizoma Zingiberis recens and rhizoma Pinelliae Preparata have toxicity; using licorice as a guide, and harmonizing various medicines; has the effects of regulating qi, resolving phlegm, harmonizing stomach and promoting bile flow.

The decoction for warming gall is composed of 6 traditional Chinese medicines including pinellia tuber, bamboo shavings, immature bitter orange, dried orange peel, ginger and liquorice, and each medicine contains key components capable of effectively playing the efficacy of the decoction for warming gall, so that the control of each component in the prescription can more comprehensively evaluate the overall quality of the prescription. The standard sample loses the morphological characteristics of the original decoction pieces, and the quality of the standard sample is difficult to reflect by the qualitative identification and quantitative analysis of the pure index components.

Therefore, research on a new method for effectively, accurately and comprehensively detecting the quality of the compound preparation of the hot bile soup becomes a problem to be solved urgently.

Disclosure of Invention

Therefore, the invention aims to provide a separation method of chemical components in a compound preparation of the Wen Dan decoction or a construction method of a fingerprint, the method establishes the fingerprint of the variety according to the characteristics of the compound preparation of the Wen Dan decoction, realizes effective separation of various characteristic peaks, improves the number of the characteristic peaks, improves the separation effect, and can effectively, accurately and comprehensively detect the compound preparation of the Wen Dan decoction.

In particular, the invention discloses a separation method of chemical components in a compound preparation of a Wen Dan decoction or a construction method of a fingerprint, which comprises the following steps,

(1) Preparing a sample solution;

(2) Detecting the sample solution by adopting a high performance liquid chromatography method, and performing gradient elution by taking octadecylsilane chemically bonded silica as a filler and acetonitrile-formic acid-containing water solution as a mobile phase; the gradient elution procedure included: 0 to 25min to 40min to 50min to 75min to 84min to 90min to 105min to 110min, and the volume percentage of acetonitrile in the mobile phase is as follows: 0.5% → 13% → 15% → 17% → 20% → 33% → 40% → 48% → 90%.

The construction method according to any one of the present invention, wherein the step (1) comprises: weighing compound preparation of WENCHEN decoction, diluting with solvent, separating solid from liquid, and collecting the liquid to obtain sample solution.

The construction method according to any one of the present invention, wherein the step (1) further satisfies any one or more of the following a-C:

A. the volume ratio of the compound preparation of the warm biliary soup to the solvent is 3:5-20;

B. the solid-liquid separation is selected from centrifugation or filtration;

C. the solvent is selected from at least one of methanol, water and ethanol; preferably methanol.

According to the construction method of any one of the invention, the compound preparation of the warm biliary soup is a compound preparation prepared by taking pinellia ternate, bamboo shavings, immature bitter orange, dried orange peel (orange peel), ginger and liquorice as raw material medicines according to the conventional technical means. The compound preparation of the warm biliary soup comprises the following raw materials in parts by weight: 23-30 parts of pinellia ternate, 23-30 parts of bamboo shavings, 23-30 parts of immature bitter orange, 35-45 parts of orange peel, 50-60 parts of ginger and 10-15 parts of liquorice. The fingerprint can be constructed for all the compound preparations by adopting the method of the invention.

The compound preparation of the warm gall bladder is preferably solid preparation, semisolid preparation and liquid preparation of the warm gall bladder, and more preferably at least one of warm gall bladder decoction, warm gall bladder granule, warm gall Shang Jiaonang and warm gall tablet. The compound preparation of the wendan decoction can be prepared according to the conventional technical means, such as but not limited to the method specified in Chinese pharmacopoeia.

Preferably, the compound preparation of the warm biliary soup is a water decoction obtained by decocting 6 traditional Chinese medicines of pinellia tuber, bamboo shavings, immature bitter orange, dried orange peel, ginger and liquorice.

The construction method according to any one of the present invention, wherein the step (1) comprises: and (3) putting 3ml of the compound preparation of the warm gall soup into a 5ml-20ml measuring flask, diluting to a scale, sealing, shaking uniformly, and filtering to obtain the compound preparation.

According to the construction method of any one of the present invention, the step (2) further satisfies any one or more of the following 1) to 3):

1) The gradient elution procedure further comprises: 110 min-115 min-120 min-130 min, and the volume percentage of acetonitrile in the mobile phase is as follows: 90% → 90% → 0.5% → 0.5%;

2) The volume percentage of formic acid in the formic acid-containing aqueous solution is 0.05-0.15%;

3) The chromatographic conditions of high performance liquid chromatography also include: the detection wavelength is 235-240nm, preferably 237nm; the flow rate is 0.9-1.1ml/min, preferably 0.9-1.0ml/min; the column temperature is 25-33℃and preferably 30 ℃.

In some preferred embodiments, the construction method further comprises a step of preparing a reference solution by using at least one of glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol and hesperetin, and a step of detecting the reference solution by high performance liquid chromatography according to any one of the construction methods to obtain a reference fingerprint.

Any one or more of glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol and hesperetin can be used for preparing a reference substance solution, and when two or more reference substances are used, each reference substance can be mixed for preparing a mixed solution, or the reference substance solution can be prepared independently.

The construction method according to any one of the present invention, the preparation method of the reference solution comprises the steps of: adding solvent into the reference substances of glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol and hesperetin to prepare a reference substance solution containing 1-2500 mug of each reference substance per 1ml; preferably, the solvent is selected from methanol or aqueous methanol; the volume fraction of methanol in the aqueous methanol solution is not less than 30%.

Each 1ml of the reference solution contains 15.71-209.46 mug of glycyrrhizin, 32.02-640.37 mug of rutin, 90.79-2269.71 mug of naringin, 32.38-647.66 mug of hesperidin, 30-1761.12 mug of neohesperidin, 17.85-357.09 mug of glycyrrhizic acid, 5.45-54.48 mug of nobiletin, 4.15-49.81 mug of 6-gingerol and 4.24-42.43 mug of hesperetin.

The reference substance solutions can be prepared independently or mixed to prepare a mixed reference substance solution.

In some preferred embodiments, the method further comprises constructing a reference fingerprint of the compound preparation of the warm bile soup, introducing the fingerprint obtained by detecting samples of the compound preparation of the warm bile soup into a computing software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to form a common pattern diagram, and generating the reference fingerprint of the compound preparation of the warm bile soup. In some preferred embodiments, the step of marking the common characteristic peak is further included after the comparison fingerprint of the compound preparation of the Wenzhen decoction is generated by using the traditional Chinese medicine chromatographic fingerprint similarity evaluation software.

At least 10 batches of the compound preparation of the warm bile soup are adopted to obtain a comparison spectrogram, for example, 10 batches, 15 batches, 20 batches and 30 batches of the compound preparation of the warm bile soup are adopted.

In some preferred embodiments, the fingerprint of the compound preparation of the warm biliary soup has 30 common peaks, peak 14 is glycyrrhizin, peak 18 is naringin, peak 19 is naringin, peak 20 is hesperidin, peak 21 is neohesperidin, peak 26 is glycyrrhizic acid, peak 27 is nobiletin, peak 28 is 6-gingerol, and peak 29 is hesperetin.

In some preferred embodiments, the fingerprint of the compound preparation of the warm biliary soup has 30 common characteristic peaks, the neohesperidin chromatographic peak is taken as a reference peak and is marked as a peak S, and the relative retention time of each characteristic peak and the peak S is within a range of +/-10% of a specified value; the prescribed values of peak 1 to peak 30 are in this order: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, 0.396, 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, 1.494.

The invention also provides the application of the construction method of the finger print of the Wendan Shang Fufang preparation in quality detection of the Wendan soup compound preparation product.

The invention also provides a quality detection method of the compound preparation of the temperature-measuring bile soup, which comprises the step of constructing the fingerprint of the compound preparation of the temperature-measuring bile Shang Fufang preparation according to the construction method of the fingerprint of the temperature-measuring bile soup.

Specifically, the method comprises the step of comparing the fingerprint spectrum of the compound preparation product of the bile soup to be measured with the fingerprint spectrum of the compound preparation of the bile soup to be measured; the fingerprint spectrum of the compound preparation product of the warm biliary soup to be detected is obtained by using the compound preparation product of the warm biliary soup to be detected according to any one of the construction methods of the invention, and the reference fingerprint spectrum of the compound preparation of the warm biliary soup is the reference fingerprint spectrum of the compound preparation of the warm biliary soup.

If the similarity between the fingerprint of the compound preparation of the bile soup to be measured and the comparison fingerprint of the compound preparation of the bile soup is not lower than 0.85-0.95 (for example, 0.90), the quality is qualified; if the value is less than 0.85-0.95 (e.g. 0.90), the test result is not qualified; specifically, the similarity is obtained through traditional Chinese medicine chromatographic fingerprint similarity evaluation software.

In the present invention, 0.1% or 0.5% formic acid refers to an aqueous solution containing 0.1% or 0.5% formic acid, respectively, by volume.

The technical scheme of the invention has the following advantages:

1. the separation method of chemical components or the construction method of the fingerprint in the compound preparation of the warm bile soup uses octadecylsilane chemically bonded silica as a filler, uses acetonitrile-formic acid-containing aqueous solution as a mobile phase for gradient elution, and obtains an elution program through repeated experiments, under the elution condition of the invention, the number of common peaks (up to 30) is obviously increased, the separation degree of the common characteristic peaks is obviously improved, and the common peaks have good peak shapes and no interference, so that the quality detection of the compound preparation of the warm bile soup can be more comprehensively, clearly and effectively carried out.

2. The separation method of chemical components in the compound preparation of the Wen Dan decoction or the construction method of the fingerprint spectrum, which are disclosed by the invention, prepares the Wen Dan decoction by collecting decoction pieces of different producing areas, and the Wen Dan decoction established according to pharmacopoeia is representative and reference. The fingerprint method established on the basis of decoction of the temperature liner can characterize the information of the medicines of pinellia ternate, caulis bambusae in taeniam, immature bitter orange, dried orange peel, ginger and liquorice, and is suitable for evaluating other compound preparations of the decoction of the temperature liner in the market.

3. The separation method of chemical components in the compound preparation of the wintergreen decoction or the construction method of the fingerprint can realize the positioning of 1-9 common peaks in the fingerprint of the compound preparation of the wintergreen decoction by adopting at least one of glycyrrhizin, rutin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol and hesperetin to prepare the reference substance solution and constructing the fingerprint of the reference substance, so that the detected chromatograph is more accurate, stable and reliable, and the lateral description can realize the effective separation of 9 effective components in the compound preparation of the wintergreen decoction, namely glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol and hesperetin by adopting the chromatographic conditions of the invention, and the constructed fingerprint has high reliability.

4. According to the quality detection method of the compound preparation of the warm bile soup, the fingerprint of the compound preparation product of the warm bile Shang Fufang preparation is constructed according to the construction method of the fingerprint of the compound preparation of the warm bile soup, and the quality detection is carried out on the characteristic active ingredients of the compound preparation of the warm bile soup, so that the quality detection of the compound preparation of the warm bile soup is more comprehensive, and the effectiveness and the controllability of the quality of the compound preparation of the warm bile soup are ensured.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a fingerprint of sample 15 of Wen Dan Shang Jizhun in example 1;

FIG. 2 is a control fingerprint of a sample of example 1, a thermal liner Shang Jizhun;

FIG. 3 is a graph comparing gradient 1-gradient 2 under the optimized term for chromatographic conditions in example 2;

FIG. 4 is an HPLC plot of various flow rates in example 2;

FIG. 5 is an HPLC chart of example 2 for various column temperatures;

FIG. 6 is an HPLC plot of the different mobile phases of example 2;

FIG. 7 is an HPLC chart obtained in an examination experiment of the test sample solution preparation method in example 3;

FIG. 8 is a 3D plot of the sample solution of example 4;

FIG. 9 is a contour diagram of the sample solution in example 4;

FIG. 10 is a graph showing the comparison of the fingerprint spectrum of the Wen Dan decoction in example 5 and the decoction of each component decoction piece;

FIG. 11 is a fingerprint of a sample of example 5, a thermal liner Shang Jizhun;

FIG. 12 is a graph showing the comparison of the sample solution with each control in example 5;

FIG. 13 is a graph showing the results of the system applicability test in example 6;

FIG. 14 is a sample specific HPLC profile of example 6 middle temperature liner Shang Jizhun; sequentially preparing a blank solvent, a reference substance solution, a dried orange peel negative, a bran-fried immature bitter orange negative, a dried orange peel-bran-fried immature bitter orange negative and a sample solution of a reference sample of the warm biliary soup from top to bottom;

FIG. 15 is a graph showing the results of the stability test of the control solution in example 6;

FIG. 16 is a graph showing the results of the test sample solution stability test in example 6;

FIG. 17 is a graph showing the results of the repeatability test of example 6;

FIG. 18 is a graph showing the results of various numbered columns of example 6; 1 is WelchXtimate-C18 (4.6X250 mm,5 μm, ZD-LC-073); 2 is WelchXtime-C18 (4.6X250 mm,5 μm, ZD-LC-062); 3 is WelchXtimate-C18 (4.6X250 mm,5 μm, ZD-LC-085);

FIG. 19 is a graph showing the results of various flow rate tests in example 6; 1 is the flow rate of 1.0ml/min;2 is the flow rate of 0.9ml/min;3 is the flow rate of 1.0ml/min;

FIG. 20 is a graph showing the results of the acid tests of example 6 at various concentrations; 1 is acetonitrile-0.1% formic acid; 2 is acetonitrile-0.05% formic acid; 3 is acetonitrile-0.15% formic acid.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge. The following examples, examples and comparative examples were all prepared according to the chinese pharmacopoeia process to obtain the decoction of the Wen dan, specifically: 27.6g of purified pinellia tuber, 27.6g of bamboo shavings, 27.6g of bran-fried immature bitter orange, 41.4g of dried orange peel and 55.2g of ginger are respectively taken, 13.8g of fried licorice root is crushed into 1-5 mm coarse particles, the coarse particles are put into a 3L marmite, 1600ml of water is added, the mixture is boiled with strong fire, the mixture is boiled with slow fire until the volume reaches 400ml, and the liquid medicine is sieved by a 200-mesh sieve, so that a decoction is obtained, and is taken as a reference sample of the gallbladder warming soup.

Example 1 method for constructing fingerprint

The embodiment provides a separation method of chemical components in a compound preparation of a Wen Dan decoction or a construction method of a fingerprint, which comprises the following steps:

(1) Preparation of test solution: taking 3ml of decoction of the gallbladder, placing into a 10ml measuring flask, adding methanol to dilute to scale, sealing, shaking, and filtering.

(2) Preparation of reference solution: taking proper amount of neohesperidin reference substance, precisely weighing, and adding methanol to obtain solution containing 30 μg per 1 ml.

(3) High performance liquid chromatography detection

Detecting the sample solution and the reference solution by high performance liquid chromatography under the following chromatographic conditions: octadecylsilane chemically bonded silica is used as filler (Welch Xtime-C18; column length 25cm, inner diameter 4.6mm, particle size 5.0 μm); acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B, eluting according to the gradient in the following table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 237nm. The sample was introduced in an amount of 10. Mu.l.

TABLE 1 gradient of mobile phase elution

The common mode of the fingerprint spectrum of the Wen Dan decoction is established according to the method.

Respectively taking 15 batches of warm gall Shang Yangpin (warm gall decoction prepared by the method) to obtain the medicinal preparation fingerprint of the warm gall decoction according to the method, as shown in figure 1. Introducing the fingerprint detection AIA data of 15 batches of the chemical components of the Wen Dan decoction into a computing software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, wherein parameters are set as follows: shearing the data for 0-3min to obtain a solvent peak, wherein the S1 is used as a reference map, the generating method is a median, and the time window is 0.1; and (3) carrying out multipoint correction, namely carrying out peak-to-peak matching with the peak area accounting for more than 0% of the total peak area, calibrating 30 peaks sharing the peak, forming a sharing mode diagram, and establishing a control map. As shown in fig. 2. And selecting chromatographic peaks with good stability and proper response values in 15 batches of sample fingerprints as common peaks, and calibrating 30 common peaks in total. Peak 21, the chromatographic peak of neohesperidin, is suitable for absorption and stable in response, and baseline separation is achieved, so that the chromatographic peak of neohesperidin is selected as a reference peak and marked as peak S.

As can be seen from fig. 1 and fig. 2, the control fingerprint shows 30 common characteristic peaks, the neohesperidin chromatographic peak is taken as a reference peak and is marked as a peak S, and the relative retention time of each characteristic peak and the peak S is as follows: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, 0.396, 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, 1.494.

The fingerprint of the 15 batches of medicine preparation of the Wendan decoction takes a neohesperidin chromatographic peak as a reference peak, is marked as a peak S, and the relative retention time of each characteristic peak and the peak S is within the range of +/-10% of a specified value; the prescribed values of peak 1 to peak 30 are in this order: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, 0.396, 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, 1.494.

The chromatographic peaks with the same retention time as the chromatographic peaks of the reference object are shown in the fingerprint of the test sample, the chromatographic spectrum of the test sample is basically consistent with the reference fingerprint, 30 corresponding common peaks exist, the similarity is calculated according to a similarity evaluation system of the traditional Chinese medicine chromatographic fingerprint by the common peaks, the result is shown in Table 2, the similarity between the fingerprint of the test sample and the reference fingerprint is higher than 0.900, and the difference between the fingerprints of the reference samples in each batch is smaller according to the similarity evaluation.

TABLE 2 fingerprint similarity results of pharmaceutical preparations of the WenDan decoction of 15 batches

By the construction method in the embodiment, the fingerprint with good separation degree of each characteristic peak can be effectively obtained, S-peak neohesperidin is selected as an internal reference peak in the fingerprint, and the relative retention time of the common characteristic peaks No. 1-30 of the medicinal preparation of the Wendan decoction can be determined. Therefore, the medicine preparation of the temperature-gall-bladder soup can be comprehensively and rapidly detected, the comprehensive quality detection and the overall quality control of the medicine preparation of the temperature-gall-bladder soup are facilitated, and the use safety and the stability uniformity of the medicine are improved.

EXAMPLE 2 investigation of chromatographic conditions

1. Preparation of test solution: precisely sucking 3ml of the decoction, placing in a 10ml measuring flask, diluting with methanol to scale, sealing, shaking, and filtering.

2. Optimization of chromatographic conditions

The test solutions prepared in item 1 of this example were each subjected to HPLC detection as follows.

Method 1: octadecylsilane chemically bonded silica is used as filler (Welch Xtime-C18; column length 25cm, inner diameter 4.6mm, particle size 5.0 μm); acetonitrile is taken as a mobile phase A,0.1 percent formic acid is taken as a mobile phase B, and elution is carried out according to the gradient in the table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 237nm.

TABLE 3 gradient elution program table (gradient 1)

Method 2: octadecylsilane chemically bonded silica is used as filler (Welch Xtime-C18; column length 25cm, inner diameter 4.6mm, particle size 5.0 μm); acetonitrile is taken as a mobile phase A,0.1 percent formic acid is taken as a mobile phase B, and elution is carried out according to the gradient in the table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 237nm.

TABLE 4 gradient elution program table (gradient 2)

Method 3: octadecylsilane chemically bonded silica is used as filler (Welch Xtime-C18; column length 25cm, inner diameter 4.6mm, particle size 5.0 μm); acetonitrile is taken as a mobile phase A,0.1 percent formic acid is taken as a mobile phase B, and elution is carried out according to the gradient in the table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 237nm.

TABLE 5 gradient elution program table (gradient 3)

As shown in FIG. 3, the chromatogram of the sample in method 3 has higher separation degree of each chromatographic peak and better peak shape than other chromatograms, so that the method 3 is used as the optimal method for measuring the fingerprint of the reference sample.

3. Investigation of flow Rate

The sample solution prepared in item 1 of this example was tested by HPLC. Octadecylsilane chemically bonded silica was used as a filler, acetonitrile as mobile phase A,0.1% phosphoric acid solution as mobile phase B, and elution was carried out in accordance with the gradient shown in Table 1 at flow rates of 0.9ml, 1.0ml and 1.1ml per minute, respectively, at column temperatures of 30℃and detection wavelength of 237nm.

As shown in fig. 4. The flow rate is 1.1ml/min through comparison, the peak separation degree marked in the graph is reduced, the flow rate is not applicable, and 9.0-1.0ml/min is applicable, wherein 1.0ml/min is the optimal flow rate for detecting chemical components of the temperature liner soup.

4. Investigation of column temperature

The sample solution prepared in item 1 of this example was tested by HPLC. Octadecylsilane chemically bonded silica was used as a filler, acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, elution was carried out in accordance with the gradient shown in Table 1 at a flow rate of 1.0ml per minute, column temperatures of 25℃and 30℃and 33℃respectively, and detection wavelength of 237nm.

As shown in fig. 5. The comparison shows that the peak separation degree is reduced when the column temperature is 35 ℃, which indicates that the column temperature is not applicable and is applicable at 25-30 ℃, wherein 30 ℃ is the optimal temperature for detecting the chemical components of the temperature liner soup.

5. Investigation of mobile phase System

The sample solution prepared in item 1 of this example was tested by HPLC. Octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, water, 0.1% phosphoric acid solution, 0.1% formic acid solution and 0.1% acetic acid solution are respectively used as a mobile phase B, elution is carried out according to the gradient shown in the table 1, the flow rate is 1.0ml per minute, the column temperature is 30 ℃, and the detection wavelength is 237nm.

As shown in FIG. 6, when acetonitrile-0.1% formic acid solution and acetonitrile-0.1% phosphoric acid solution are used as mobile phases, the peak information amount is larger, the separation degree of chromatographic peaks is better, but part of chromatographic peaks marked in an acetonitrile-0.1% phosphoric acid solution system are dense, the separation degree is low, the acetonitrile-0.1% formic acid solution is easy to be influenced by the fluctuation of a column Wen Liusu and is unstable, so that the acetonitrile-0.1% formic acid solution is used as the optimal mobile phase for detecting chemical components of the wintergreen soup.

Example 3 investigation of sample solution preparation methods

Sample solution preparation: accurately sucking 3ml decoction of WENCHEN decoction, placing into a 10ml measuring flask, respectively adding methanol, 50% methanol, 70% methanol, ethanol, 50% ethanol, and 70% ethanol, diluting to scale, sealing, shaking, and filtering. And precisely sucking 10 μl of the filtrate, and injecting into a liquid chromatograph for measurement. The high performance liquid chromatography of example 1 was used for detection.

TABLE 6 test sample preparation investigation determination results

Note that: RT is retention time, RRT is relative retention time, A is peak area, RPA is relative peak area, and the same applies below.

The data are shown in Table 6 and the comparative graph is shown in FIG. 7. By comparing the fingerprints of different solvents, the effective separation of 30 peaks can be realized under different solvents, wherein the total parameters such as the information quantity of the methanol fingerprint peaks, the peak response value, the peak separation degree and the like are better than those of extraction of 50% methanol, 70% methanol, ethanol, 50% ethanol and 70% ethanol, and the methanol is used as a temporary extraction solvent for subsequent research by comprehensively considering.

Example 4 selection of detection wavelength

3ml of decoction of the warm gall soup is precisely sucked, the decoction is placed in a measuring flask with 10ml, methanol is respectively added to dilute to a scale, the mixture is sealed, shaking is carried out, filtering is carried out, a test sample solution is prepared, a diode array detector is adopted, the test sample solution is scanned at the wavelength of 190-400 nm, analysis is carried out at the wavelength band, as shown in fig. 8 and 9, each chromatographic peak response value at the wavelength of 190-240 nm is higher, the information quantity is large, but 203-230 nm is near the cut-off wavelength, more impurity peaks cannot guarantee simultaneous detection of more specific peaks of medicines, so 237nm is selected as the detection wavelength in consideration of the information quantity of the peak and the attribution of the specific chromatographic peaks of medicines.

Example 5 selection of Peak assignment and reference

(1) The medicinal flavor peak is attributed to

The single decoction piece decoction is prepared according to the following method. Decoction of rhizoma Pinelliae decoction pieces: 27.6g of purified pinellia tuber is taken and crushed into coarse particles with the size of 1-5 mm, the coarse particles are put into a 3L marmite, 1600ml of water is added, the mixture is boiled with strong fire, the mixture is decocted with slow fire until the volume of the mixture is 400ml, and the decoction is filtered through a 200-mesh sieve, so as to obtain the decoction for standby.

Decoction of caulis Bambusae in Taenia decoction pieces: 27.6g of bamboo shavings are taken, crushed into coarse particles with the size of 1-5 mm, put into a 3L marmite, added with 1600ml of water, boiled with strong fire, decocted with slow fire to 400ml, and the decoction is filtered through a 200-mesh sieve to obtain the decoction for standby.

Immature bitter orange decoction piece decoction liquid: 27.6g of bran-fried immature bitter orange is taken and crushed into coarse particles with the size of 1-5 mm, the coarse particles are put into a 3L marmite, 1600ml of water is added, the mixture is boiled with strong fire, the mixture is decocted with slow fire until the volume of the mixture is 400ml, and the decoction is filtered through a 200-mesh sieve, so that the decoction is obtained for standby.

Dried orange peel decoction piece decoction liquid: taking 41.4g of dried orange peel, crushing into coarse particles with the size of 1-5 mm, placing into a 3L marmite, adding 1600ml of water, boiling with strong fire, decocting with slow fire to 400ml, and sieving the liquid medicine with a 200-mesh sieve to obtain a decoction for later use.

Ginger decoction piece decoction liquid: taking 55.2g of ginger, crushing into coarse particles with the size of 1-5 mm, placing into a 3L marmite, adding 1600ml of water, boiling with strong fire, decocting with slow fire to 400ml, and sieving the liquid medicine with a 200-mesh sieve to obtain a decoction for later use.

Decoction of licorice decoction pieces: 13.8g of liquorice is taken and crushed into coarse particles with the size of 1-5 mm, the coarse particles are put into a 3L marmite, 1600ml of water is added, the mixture is boiled with strong fire, the mixture is decocted with slow fire until the volume of 400ml, and the decoction is filtered through a 200-mesh sieve, thus obtaining the decoction for standby.

The test solutions were prepared and tested according to the method of example 1, respectively, from the individual decoction pieces.

Comparing the fingerprint of the decoction with the fingerprint of each decoction piece, see figure 10.

Experimental results show that chromatographic peaks 1, 2, 3, 4, 6, 7, 8, 10, 12, 17, 18, 19, 20, 21, 23, 24, 27, 29 and 30 are assigned to bran-fried immature bitter orange, chromatographic peaks 2, 5, 6, 9, 8, 10, 18, 20, 23, 27 and 29 are assigned to dried orange peel, chromatographic peaks 12 and 13 are assigned to bamboo shavings, chromatographic peaks 28 and 30 are assigned to ginger, and chromatographic peaks 14, 15, 16, 20, 23, 24, 25 and 26 are assigned to fried licorice.

(2) Characteristic peak assignment

Taking proper amount of neohesperidin reference substance, precisely weighing, adding methanol to obtain solution containing 30 μg reference substance per 1 ml. Weighing appropriate amount of glycyrrhizin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg reference substance per 1 ml. And (3) taking a proper amount of naringin reference substance, precisely weighing, and adding methanol to prepare a solution containing 80 mug reference substance per 1 ml. Taking appropriate amount of naringin reference substance, precisely weighing, and adding methanol to obtain solution containing 500 μg reference substance per 1 ml. Taking appropriate amount of hesperidin reference substance, precisely weighing, and adding methanol to obtain solution containing 150 μg reference substance per 1 ml. Weighing appropriate amount of glycyrrhizic acid reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg reference substance per 1 ml. Taking a proper amount of nobiletin reference substance, precisely weighing, adding methanol to prepare a solution containing 10 mug reference substance per 1 ml. Taking a proper amount of 6-gingerol reference substance, precisely weighing, adding methanol to prepare a solution containing 10 mug reference substance per 1 ml. Taking appropriate amount of hesperetin reference substance, precisely weighing, and adding methanol to obtain solution containing 10 μg reference substance per 1 ml.

Detecting the reference substance solution and blank solvent (methanol) according to the chromatographic conditions of the embodiment 1, and comparing the fingerprint of the test sample of the Wen Dan decoction with the chromatogram of the reference substance solution. The results are shown in FIG. 12, wherein the No. 14 chromatographic peak is glycyrrhizin, the No. 18 chromatographic peak is naringin, the No. 19 chromatographic peak is naringin, the No. 20 chromatographic peak is hesperidin, the No. 21 chromatographic peak is neohesperidin, the No. 26 chromatographic peak is glycyrrhizic acid, the No. 27 chromatographic peak is nobiletin, the No. 28 chromatographic peak is 6-gingerol, and the No. 29 chromatographic peak is hesperetin.

(3) Selection of reference

Of the known chromatographic peaks, chromatographic peak 21, i.e., neohesperidin chromatographic peak, was suitable for absorption, stable in response, reached baseline separation, and retention time was at the intermediate position as seen in fig. 11, so that neohesperidin chromatographic peak was selected as reference peak, labeled peak S.

Example 6 methodological verification

(1) System applicability

The sample solution prepared in example 1 was sampled 6 times continuously in the same manner as in example 1. The results show that the continuous sample injection is carried out for 6 times, the retention time RSD of chromatographic peaks is less than or equal to 2.0 percent, the peak area, the theoretical plate number and the symmetry factor are moderate, the similarity is more than or equal to 0.900, and the system applicability is good, as shown in tables 7-12 and figure 13.

TABLE 7 System suitability test results-retention time

Table 8 results of System suitability test-Peak area

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Table 9 results of System suitability test-theoretical plate number

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Table 10 results of System suitability test-symmetry factor

Table 11 results of System suitability test-degree of separation

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Table 12 results of the System suitability test-similarity

(2) Specialization of

To examine whether the blank solvent (70% methanol solution), purified pinellia tuber negative, dried orange peel negative, bran-fried immature bitter orange negative, bamboo shavings negative, ginger negative, fried licorice negative, dried orange peel-bran-fried immature bitter orange double negative have interference on the fingerprint of the reference sample of the warm biliary soup. The preparation method comprises the following steps.

Negative decoction of purified pinellia ternate: 27.6g of caulis bambusae in taeniam, 27.6g of immature bitter orange fried with bran, 41.4g of dried orange peel, 55.2g of ginger and 13.8g of fried licorice are taken, crushed into coarse particles with the size of 1-5 mm, placed in a 3L marmite, 1600ml of water is added, the mixture is boiled with strong fire, the mixture is decocted with slow fire until the volume reaches 400ml, and the decoction is sieved by a 200-mesh sieve, so that the decoction is obtained.

Pericarpium Citri Reticulatae negative decoction: 27.6g of purified pinellia tuber, 27.6g of bamboo shavings, 27.6g of bran-fried immature bitter orange, 55.2g of ginger and 13.8g of fried licorice are taken, crushed into coarse particles with the size of 1-5 mm, placed in a 3L marmite, 1600ml of water is added, the mixture is boiled with strong fire, the mixture is decocted with slow fire until the volume reaches 400ml, and the decoction is sieved by a 200-mesh sieve, thus obtaining the decoction.

Bran-fried immature bitter orange negative decoction: 27.6g of purified pinellia tuber, 27.6g of bamboo shavings, 41.4g of dried orange peel, 55.2g of ginger and 13.8g of fried licorice are taken, crushed into coarse particles with the size of 1-5 mm, put into a 3L marmite, added with 1600ml of water, boiled with strong fire, decocted with slow fire to 400ml, and the decoction is sieved by a 200-mesh sieve, thus obtaining the decoction.

Bamboo shavings negative decoction: 27.6g of purified pinellia tuber, 27.6g of bran-fried immature bitter orange, 41.4g of dried orange peel, 55.2g of ginger and 13.8g of fried licorice root are taken, crushed into coarse particles with the size of 1-5 mm, put into a 3L marmite, added with 1600ml of water, boiled with strong fire, decocted with slow fire to 400ml, and the liquid medicine is sieved by a 200-mesh sieve, thus obtaining the decoction.

Ginger negative decoction: 27.6g of purified pinellia tuber, 27.6g of bamboo shavings, 27.6g of bran-fried immature bitter orange, 41.4g of dried orange peel and 13.8g of fried licorice root are taken, crushed into coarse particles with the size of 1-5 mm, placed in a 3L marmite, 1600ml of water is added, the mixture is boiled with strong fire, the mixture is decocted with slow fire until the volume reaches 400ml, and the decoction is sieved by a 200-mesh sieve, thus obtaining the decoction.

Parching Glycyrrhrizae radix to obtain a negative decoction: 27.6g of purified pinellia tuber, 27.6g of bamboo shavings, 27.6g of bran-fried immature bitter orange, 41.4g of dried orange peel and 55.2g of ginger are taken, crushed into coarse particles with the size of 1-5 mm, put into a 3L marmite, added with 1600ml of water, boiled with strong fire, decocted with slow fire to 400ml, and the liquid medicine is sieved by a 200-mesh sieve, thus obtaining the decoction.

Pericarpium Citri Reticulatae-fructus Aurantii Immaturus double-negative decoction: 27.6g of purified pinellia tuber, 27.6g of bamboo shavings, 55.2g of ginger and 13.8g of fried licorice root are taken, crushed into 1-5 mm coarse particles, put into a 3L marmite, added with 1600ml of water, boiled with strong fire, decocted with slow fire to 400ml, and the liquid medicine is sieved by a 200-mesh sieve, thus obtaining the decoction.

The 70% methanol solution, the sample solution prepared in example 1, the purified pinellia tuber negative solution, the dried orange peel negative solution, the bran-fried immature bitter orange negative solution, the bamboo shavings negative solution, the ginger negative solution, the fried licorice negative solution and the dried orange peel-bran-fried immature bitter orange double negative solution are respectively precisely sucked, and the results are shown in fig. 14 according to the high performance liquid chromatography in example 1, so that the blank solvent has no interference at the common peak position. Wherein the purified pinellia tuber negative solution, the dried orange peel negative solution, the bran-fried immature bitter orange negative solution, the bamboo shavings negative solution, the ginger negative solution, the fried licorice negative solution and the dried orange peel-bran-fried immature bitter orange double negative solution are prepared by adopting the same method as the preparation method of the sample solution in the embodiment 1 respectively.

(3) Stability of

3ml of a reference sample of the warm biliary soup is precisely sucked, the neohesperidin reference substance solution prepared in the example 1 is taken according to the preparation of the sample solution in the example 1, the sample injection measurement is carried out at 0, 6, 12, 24, 42, 60 and 72 hours according to the high performance liquid chromatography in the example 1 respectively, the S peak is taken as a reference, the relative retention time of the rest peaks is calculated, and the specific measurement results are shown in tables 13-15 and figures 15-16. The result shows that the retention time of the reference solution is basically consistent with the retention time of the chromatographic peak area within 72 hours (RSD is less than or equal to 2.0%), the retention time of each peak of the test solution is basically consistent with the retention time of each peak of the test solution (RSD is less than or equal to 2.0%), the similarity is more than or equal to 0.900, and the stability of the reference solution and the test solution within 72 hours is good.

TABLE 13 control solution stability test results-relative retention time

TABLE 14 test results of stability of test solutions-relative retention time

TABLE 15 stability test results-similarity

(4) Repeatability of

3ml of a reference sample of the warm biliary soup was precisely sucked, 6 samples were prepared and operated according to the preparation and measurement methods of the sample solution in example 1, the relative retention time of the remaining peaks was calculated with reference to the S peak, and specific measurement results are shown in tables 16 to 17 and fig. 17. The result shows that the relative retention time is basically consistent, the RSD is less than or equal to 2.0 percent, the similarity is more than or equal to 0.900 percent, and the method has good repeatability.

TABLE 16 repeatability test results-relative retention time

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TABLE 17 repeatability test results-similarity

(5) Intermediate precision

Different measurement times, different brands of high performance liquid chromatographs and different experimenters (A, B) were selected to accurately suck 3ml of reference sample of the warm biliary soup, and the operations were performed according to the preparation and measurement of the sample solution in example 1. And the relative retention time of the rest peaks was calculated with reference to the S peak, and the specific measurement results are shown in tables 18 to 19. The result shows that the relative retention time is basically consistent, the RSD is less than or equal to 2.0 percent, the similarity is more than or equal to 0.900 percent, and the method has good intermediate precision.

Table 18 results of intermediate precision test-relative retention time

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Table 19 intermediate precision test results-similarity

(6) Durability of

(6) Stability of

(1) Different chromatographic columns

The same sample solution prepared in the method of example 1 is taken, the influences of chromatographic columns with different numbers on the fingerprint of a reference sample of the Wen-Dan decoction are compared, and the rest of the chromatographic solutions are detected according to the chromatographic condition in the item (3) of example 1. The results are shown in the following table and FIG. 18, and the relative retention time is basically consistent, RSD is less than or equal to 3.0%, and the similarity is more than or equal to 0.900, which indicates that the method has good durability for different chromatographic columns.

TABLE 20 results of different numbered chromatography columns-relative retention time

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TABLE 21 results of different numbered chromatography columns-similarity

(2) Different flow rates

The same sample solution prepared in example 1 was taken, and the effect of different flow rates on the durability of the fingerprint of the reference sample of the Wen-Dan decoction was compared, and the other chromatographic conditions were as in example 1 item (3). The results are shown in the following table and FIG. 19, and the relative retention times are substantially consistent, RSD is less than or equal to 5.0%, and similarity is greater than or equal to 0.900, indicating that the method has good durability for different flow rates.

TABLE 22 different flow Rate test results-relative retention time

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TABLE 23 results of different flow Rate tests-similarity

(3) Different acid concentrations

The same sample solution prepared in example 1 was taken, and the effect of different flow rates on the durability of the fingerprint of the reference sample of the Wen-Dan decoction was compared, and the other chromatographic conditions were as in example 1 item (3). The results are shown in the following table and figure 20, and the main common chromatographic peaks in the fingerprint under different acid concentration conditions are sharp and symmetrical, the separation degree is good, the relative retention time is basically consistent, the RSD is less than or equal to 3.0%, the similarity is more than or equal to 0.900, and the method is good in durability on different acid concentrations.

TABLE 24 investigation of different acid concentrations results-relative retention time

TABLE 25 results of different acid concentration tests-similarity

The fingerprint construction method has the advantages of good system applicability, stability, reliability, good durability, high specificity, high instrument precision and good repeatability, makes up the defects of the existing quality control method of the Wen Dan decoction, and has guiding significance for quality detection and evaluation of the variety.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. A method for separating chemical components in a compound preparation of a Wen Dan decoction or a method for constructing a fingerprint is characterized by comprising the following steps,

(1) Preparing a sample solution;

(2) Detecting the sample solution by adopting a high performance liquid chromatography method, and performing gradient elution by taking octadecylsilane chemically bonded silica as a filler and acetonitrile-formic acid-containing water solution as a mobile phase; the gradient elution procedure included: 0 to 25min to 40min to 50min to 75min to 84min to 90min to 105min to 110min, and the volume percentage of acetonitrile in the mobile phase is as follows:

0.5％→13％→15％→17％→20％→33％→40％→48％→90％。

2. the method for separating chemical components from a compound preparation of a decoction for warming gallbladder or the method for constructing a fingerprint according to claim 1, wherein the step (1) comprises: weighing compound preparation of WENCHEN decoction, diluting with solvent, separating solid from liquid, and collecting the liquid to obtain sample solution.

3. The method for separating chemical components or the method for constructing fingerprint in the compound preparation of the Wen Dan Tang according to claim 2, wherein the step (1) further satisfies any one or more of the following A-C:

A. the volume ratio of the pre-dilution to the post-dilution is 3:5-20;

B. the solid-liquid separation is selected from centrifugation or filtration;

C. the solvent is selected from at least one of methanol, water and ethanol; preferably methanol.

4. The method for separating chemical components or the method for constructing a fingerprint in a compound preparation of a Wen Dan Tang according to any one of claims 1-3, wherein the step (2) further satisfies any one or more of the following 1) -3):

1) The gradient elution procedure further comprises: 110 min-115 min-120 min-130 min, and the volume percentage of acetonitrile in the mobile phase is as follows: 90% → 90% → 0.5% → 0.5%;

2) The volume percentage of formic acid in the formic acid-containing aqueous solution is 0.05-0.15%;

3) The chromatographic conditions of high performance liquid chromatography also include: the detection wavelength is 235-240nm, preferably 237nm; the flow rate is 0.9-1.1ml/min, preferably 0.9-1.0ml/min; the column temperature is 25-33℃and preferably 30 ℃.

5. The method for separating chemical components or the method for constructing a fingerprint in a compound preparation of a Wen Dan decoction according to any one of claims 1-4, wherein the method for constructing a Wen Dan decoction further comprises the step of preparing a reference substance solution by using at least one of glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol and hesperetin, and the step of detecting the reference substance solution by high performance liquid chromatography in the method for constructing a Wen Dan decoction according to any one of claims 1-4 to obtain the fingerprint of the reference substance.

6. The method for separating chemical components or constructing fingerprint patterns in a compound preparation of a Wen Dan Tang according to claim 5, wherein the method for preparing the reference solution comprises the following steps: adding solvent into the reference substances of glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol and hesperetin to prepare a reference substance solution containing 1-2500 mug of each reference substance per 1ml; preferably, the solvent is selected from methanol or aqueous methanol; the volume fraction of methanol in the aqueous methanol solution is not less than 30%.

7. The method for separating chemical components or the method for constructing a fingerprint of a compound preparation of a warm bile soup according to any one of claims 1 to 6, wherein the fingerprint of the compound preparation of a warm bile soup has 30 common peaks, peak 14 is glycyrrhizin, peak 18 is rutin naringin, peak 19 is naringin, peak 20 is hesperidin, peak 21 is neohesperidin, peak 26 is glycyrrhizic acid, peak 27 is nobiletin, peak 28 is 6-gingerol, and peak 29 is hesperetin.

8. The method for separating chemical components or the method for constructing a fingerprint of a compound preparation of a warm bile soup according to any one of claims 1 to 7, wherein the fingerprint of the compound preparation of a warm bile soup has 30 common characteristic peaks, a neohesperidin chromatographic peak is taken as a reference peak and is marked as a peak S, and the relative retention time of each characteristic peak and the peak S is within a range of +/-10% of a specified value; the prescribed values of peak 1 to peak 30 are in this order: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, 0.396, 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, 1.494.

9. Use of the separation method of chemical components in the compound preparation of the Wen Dan decoction or the construction method of fingerprint spectrum in quality detection of the compound preparation of the Wen Dan decoction according to any one of claims 1-8.

10. A quality detection method of a compound preparation of a temperature-measuring bile soup, which is characterized by comprising a separation method of chemical components in the compound preparation of the temperature-measuring bile soup or a construction method of a fingerprint to be measured according to any one of claims 1-8.