WO2019072247A1 - Ultra-high-performance liquid chromatography detection method for traditional chinese medicine composition - Google Patents

Abstract

An ultra-high-performance liquid chromatography detection method for a traditional Chinese medicine (TCM) composition. The detection method comprises the following steps: step A, preparation of a test sample solution: collecting the content of a TCM composition capsule and adding an organic solvent for extraction so as to obtain the test sample solution; step B, preparation of a control solution: collecting chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin and adding an organic solvent to dissolve so as to obtain the control solution; and step C, detection: using ultra-high-performance liquid chromatography with methanol (A) and water containing phosphate (B) as mobile phases for gradient elution. The ultra-high-performance liquid chromatography can quantitatively determine seven chemical components in a composition, such that the quality of the composition is evaluated by means of said fast multi-component chemical analysis, thereby achieving better characterization of quality differences between batches of TCM composition capsules.

Description

Ultra high performance liquid chromatography method for detecting traditional Chinese medicine composition Technical field

The invention belongs to the technical field of traditional Chinese medicine, and in particular relates to an ultra-high performance liquid chromatography detection method for a traditional Chinese medicine composition.

Background technique

The quality control method of traditional Chinese medicine compound preparation is mainly to qualitatively and quantitatively analyze the microscopic identification, examination and content determination of individual raw materials in the preparation. However, traditional Chinese medicine compound preparations often include multi-flavored medicines, and some preparations even include animal medicines or mineral medicines, and the chemical composition thereof is far more complicated than single-flavor medicine. In fact, more and more pharmaceutics researchers recognize that the use of microscopic identification, thin-layer chromatographic identification and quantitative analysis of a single indicator component is far from comprehensive and effective in reflecting the consistency and effectiveness of traditional Chinese medicine compound preparations. Since the first edition of the Chinese Pharmacopoeia in 2015, there has been a certain improvement in the quality control standards for traditional Chinese medicine compound preparations. For example, the quality control standard is unified for different quality control standards for the same preparation; thin layer identification is added for some traditional Chinese medicine preparations without thin layer identification (or thin layer identification is less, and thin layer identification is not for monarch) Indicators; determination of the content of multiple index components that are related to the efficacy of the original single indicator component; thin-layer scanning method for the determination of the content of the preparation has been reduced; enhanced gas chromatography for the application of volatiles The traditional Chinese medicine compound preparations of sexual chemical composition, etc.; the improvement of these quality control methods undoubtedly reflects the continuous improvement of the quality standards and quality control power of the traditional Chinese medicine compound preparations. However, the quality evaluation and control of traditional Chinese medicine compound preparations is still a bottleneck that plagues the development and internationalization of traditional Chinese medicine. It is extremely urgent to carry out research on new quality assessment methods and methods for Chinese medicine compound preparations.

The traditional Chinese medicine composition of the invention is gathered in the famous name of the three dynasties, from forsythia, honeysuckle, ramie, fried almond, gypsum, radix scutellaria, Mianma Guanzhong, Houttuynia, patchouli, rhubarb, Rhodiola, menthol, licorice The composition of 13 traditional Chinese medicines is complicated, and the preparation process includes water decoction, alcohol extract and volatile oil. In the quality standard contained in the 2015 edition of the Chinese Pharmacopoeia, only the determination of forsythin is required. As a large compound preparation of traditional Chinese medicine, this is obviously not enough. How to better characterize the difference in the quality of traditional Chinese medicine compositions between batches, and to evaluate the quality of traditional Chinese medicine compositions by multi-component chemical rapid analysis is an urgent problem to be solved.

Summary of the invention

The object of the present invention is to provide a method for detecting a traditional Chinese medicine composition for the problem of the existing Chinese medicine compound reagent in characterizing the quality evaluation of drugs between different batches, and quantitatively determining by ultra performance liquid chromatography (UPLC). The main representative chemical components in the preparations are evaluated by multi-component chemical rapid analysis to better characterize the differences in the quality of traditional Chinese medicine compositions between batches.

In order to achieve the above object, the present invention provides a method for detecting a traditional Chinese medicine composition, the method comprising the following steps:

Step a, preparing a test solution: taking a Chinese medicine composition, for example, a capsule content of a traditional Chinese medicine composition, and extracting by adding an organic solvent, thereby obtaining a test solution;

Step b, preparation of the reference solution: taking chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin, dissolved in an organic solvent, that is, a control Product solution

Step c, detection: using ultra performance liquid chromatography, using methanol (A)-phosphoric acid aqueous solution (B) as a mobile phase gradient elution;

The raw material composition of the traditional Chinese medicine composition is: 200-300 parts by weight of forsythia, 200-300 parts by weight of honeysuckle, 50-100 parts by weight of ramie, 50-100 parts by weight of fried almond, 200-300 parts by weight of gypsum, Banlangen 200 to 300 parts by weight, 200 to 300 parts by weight of Kumba, 200 to 300 parts by weight of Houttuynia, 50 to 100 parts by weight of patchouli, 20 to 70 parts by weight of rhubarb, 50 to 100 parts by weight of Rhodiola, 5 to 10 parts by weight of menthol and 50 to 100 parts by weight of licorice.

According to the detection method provided by the present invention, preferably, the raw material composition of the traditional Chinese medicine composition is: 255 parts by weight of forsythia, 255 parts by weight of honeysuckle, 85 parts by weight of ramie, 85 parts by weight of fried almond, 255 parts by weight of gypsum, 255 parts by weight of Radix Isatidis, 255 parts by weight of Phytosanitary, 255 parts by weight of Houttuynia cordata, 85 parts by weight of patchouli, 51 parts by weight of rhubarb, 85 parts by weight of Rhodiola, 7.5 parts by weight of menthol, and 85 parts by weight of licorice.

It will be understood by those skilled in the art that the raw materials of the traditional Chinese medicine composition can be made into different types of preparations. In the preparation of the preparation of the traditional Chinese medicine composition, different dosage forms may be selected for preparation, for example, but not limited to emulsifiable concentrate, wettable powder, suspending agent, granule, water-dispersible granule, microemulsion, water emulsion or micro Capsules, and so on. The choice of each dosage form will depend on the materials or application of the traditional Chinese medicine composition; the corresponding preparation procedures are well known to those skilled in the art.

According to the detection method provided by the present invention, preferably, the raw material of the traditional Chinese medicine composition is made into a traditional Chinese medicine composition for detection.

Preferably, the preparation method of the traditional Chinese medicine composition comprises the following steps:

(1) extracting volatile oil from patchouli and adding water to collect volatile oil; filtering the aqueous solution to obtain a filtrate;

(2) extracting forsythia, ramie, houttuynia, rhubarb with 70 vol% ethanol, filtering the extract, and recovering the ethanol to obtain an alcohol extract;

(3) Add honeysuckle, gypsum, Banlangen, Mianmaguanzhong, licorice, Rhodiola to boil, add frying almonds, and add the filtrate obtained by decoction to the filtrate (1). The patchouli oil and the filtered filtrate are concentrated to a relative concentration of 1.10 to 1.15, ethanol is added to make the alcohol content 70 vol%, refrigerated at 2-4 ° C for 20-24 hours, filtered, and the filtrate is recovered and the step is recovered. 2) The obtained alcohol extracts of four traditional Chinese medicines such as Forsythia are combined, concentrated to a relative density of 1.10 to 1.15, spray-dried; then mixed with starch to make granules, dried, sieved, and sieved to a fine powder;

(4) The menthol and the volatile oil of the patchouli obtained in the step (1) are dissolved in ethanol, sprayed into the fine powder obtained in the step (3), and mixed; and the particles obtained after the step (3) are mixed, preferably. Also includes the operation of sealing for 20-30min;

Further preferably, the method further comprises the steps of: filling the preparation obtained in the step (4) into a capsule to prepare a capsule of the traditional Chinese medicine composition.

The traditional Chinese medicine composition or the traditional Chinese medicine composition capsule of the present invention can also be prepared by specifically referring to the preparation method of the traditional Chinese medicine composition described in the patent document No. 03143211.5.

For example, in some embodiments, the preparation method of the traditional Chinese medicine composition capsule comprises the following steps: the above thirteen flavors, patchouli and water distilled extract volatile oil, collecting volatile oil, filtering the water extract, and standby; forsythia , ramie, houttuynia, rhubarb with 70% ethanol extraction twice, the first 2 hours, the second 1.5 hours, the extract was filtered, combined, recovered ethanol, spare; honeysuckle, gypsum, Banlangen, Mian Ma Guan Add sautéed licorice, Rhodiola and Rhodiola to boiling, add fried almonds, and cook twice, the first 1.5 hours, the second time for 1 hour, the decoction is filtered, the filtrate is combined, and the patch is added to the patchouli oil. The aqueous solution used is concentrated to a relative concentration of 1.10 to 1.15 (60 ° C), ethanol is added to make the alcohol content 70%, and refrigerated at 4 ° C for 24 hours, filtered, and the filtrate is recovered to recover ethanol, and the above-mentioned forsythia and other four flavors are reserved. The alcohol extracts are combined, concentrated to a relative density of 1.10~1.15 (60 ° C), spray dried, mixed with an appropriate amount of starch, granulated, dried, sieved, sieved out a suitable amount of fine powder, menthol, patchouli volatile oil Dissolve with the right amount of ethanol, spray Powder, mixing, and mixing the granules, closed for 30 minutes into the capsule to prepare 1000, that is, too.

According to the detection method provided by the present invention, preferably, in the detection process of step c, ultra-high performance liquid chromatography is used, and the methanol (A)-phosphoric acid aqueous solution (B) is used as a mobile phase gradient elution; respectively, the reference substance is aspirated. The solution and the test solution are injected into an ultra-high performance liquid chromatograph for determination of chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin The content.

According to the detection method provided by the present invention, preferably, the gradient elution procedure is:

Time/min

A/%

0.00	12 to 16
8.00	18~20
15.00	22~25
20.00	28~30
25.00	32~35
30.00	38～43
35.00	48～52
40.00	55~60

More preferably, the gradient elution procedure is:

Time/min	A/%
0.00	15
8.00	20
15.00	twenty four
20.00	30
25.00	34
30.00	40
35.00	50
40.00	60

Among them, "A" as mentioned above means methanol in the mobile phase. The percentage in the mobile phase of the present invention is a volume percentage, and the percentage of the aqueous phosphoric acid solution is a volume percentage.

Preferably, the aqueous phosphoric acid solution has a concentration of 0.1% to 1%; more preferably 0.1% to 0.5%; still more preferably 0.1%.

According to the detection method provided by the present invention, preferably, the organic solvent in the step a is selected from any one or more of methanol, ethanol, acetone, dichloromethane and chloroform, preferably a 60% methanol solution.

Preferably, the method of extracting in step a includes ultrasonic extraction or reflux extraction; more preferably, in the preparation of the test solution, the content of the capsule of the traditional Chinese medicine composition is mixed with the organic solvent of step a, Ultrasonic treatment or reflux extraction, followed by filtration, the resulting filtrate is the test solution.

Preferably, the concentration of the test solution in step a is 10-20 mg/mL, more preferably 14 mg/mL. The concentration of the test solution as used herein refers to the volume ratio of the contents of the capsule of the traditional Chinese medicine composition or the capsule of the traditional Chinese medicine composition to the organic solvent used.

According to the detection method provided by the present invention, preferably, the organic solvent in the step b is selected from any one or more of methanol, ethanol, acetone, dichloromethane and chloroform, more preferably methanol.

Preferably, the concentration of chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin in the reference solution of step b is 55-65 μg/ mL, 70-80 μg/mL, 65-72 μg/mL, 120-130 μg/mL, 60-70 μg/mL, 75-85 μg/mL, and 48-58 μg/mL. More preferably, the concentration of chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin in the reference solution is 60 μg/mL, 75 μg, respectively. /mL, 68 μg/mL, 125 μg/mL, 64 μg/mL, 80 μg/mL, 53 μg/mL. Here, the concentration of the reference solution refers to the volume ratio of the weight of chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin to the organic solvent used.

According to the detection method provided by the present invention, preferably, the detected detection wavelength is 200 to 250 nm, more preferably 210 nm; the injection amount is 1.0 to 2.0 μL; the flow rate is 0.10 to 0.50 mL/min; and the column temperature is 20 to 40 ° C.

In the present invention, the structural formula of the component to be tested is as shown in Table 1:

Table 1 Structure of the component to be tested

Compared with the prior art, the technical solution of the present invention has the beneficial effects that: (1) it is not enough to use only one component as a quality evaluation index in the traditional Chinese medicine composition; the present invention adopts ultra high performance liquid chromatography (UPLC). Simultaneous determination of 7 chemical components (chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin) in the preparation of traditional Chinese medicine composition, accuracy And high accuracy; the quality of the preparation is evaluated by multi-component chemical rapid analysis, which can better characterize the difference in the quality of the traditional Chinese medicine composition between batches; (2) the detection method of the present invention is increased relative to the method of the Pharmacopoeia The components of chlorogenic acid, isochlorogenic acid B and isochlorogenic acid C, which are strongly related to biological activity, have established a method for detecting seven components in traditional Chinese medicine compositions, which can more fully characterize the composition of traditional Chinese medicines. (3) The invention utilizes the strong separation ability of ultra-high performance liquid chromatography, and can better separate the target peak and the impurity peak in the complex Chinese medicine composition, and obtain better resolution.

DRAWINGS

1 is a UPLC chromatogram of the test solution of Example 1 according to chromatographic condition 1;

2 is a UPLC chromatogram of the test solution of Example 1 according to chromatographic condition 2;

3 is a UPLC chromatogram of the test solution of Example 1 according to chromatographic condition 3;

4 is a UPLC chromatogram of the test solution of Example 1 according to chromatographic condition 4;

Figure 5 is a UPLC chromatogram of the mixed reference solution of Example 1 according to chromatographic condition 4;

Figure 6 is a UPLC chromatogram of the test solution according to the chromatographic condition 5 in Example 3;

Figure 7 is a UPLC chromatogram of the mixed reference solution in Example 3 according to chromatographic condition 5;

Figure 8 is a UPLC chromatogram of the test solution according to chromatographic condition 6 in Example 4;

Figure 9 is a UPLC chromatogram of the mixed reference solution in Example 4 according to chromatographic condition 6;

Figure 10 is a UPLC chromatogram of the test solution according to the chromatographic condition 7 in Example 5;

Figure 11 is a UPLC chromatogram of the mixed reference solution in Example 5 according to chromatographic conditions 7.

In Figure 1-11, peak 1 is chlorogenic acid; peak 2 is caffeic acid; peak 3 is forsythiaside A; peak 4 is isochlorogenic acid B; peak 5 is rutin; The peak is isochlorogenic acid C; the peak No. 7 is forsythin.

Detailed ways

The present invention will be described in detail with reference to the preferred embodiments of the invention.

The invention provides a method for detecting a traditional Chinese medicine composition, the method comprising the following steps:

Step a, preparation of the test solution: taking the content of the traditional Chinese medicine composition, for example, the capsule of the traditional Chinese medicine composition, and extracting by adding an organic solvent, thereby obtaining a test solution;

Step b, preparation of the reference solution: taking chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin, dissolved in an organic solvent, that is, a control Product solution

Step c, detection: using ultra-high performance liquid chromatography, using methanol (A)-phosphoric acid aqueous solution (B) as a flow to the sample to be tested for gradient elution; separately taking the reference solution and the test solution, injecting super The detection is carried out in a high performance liquid chromatograph, and the components to be tested include chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin;

The raw material composition of the traditional Chinese medicine composition is: 200-300 parts by weight of forsythia, 200-300 parts by weight of honeysuckle, 50-100 parts by weight of ramie, 50-100 parts by weight of fried almond, 200-300 parts by weight of gypsum, Banlangen 200 to 300 parts by weight, 200 to 300 parts by weight of Kumba, 200 to 300 parts by weight of Houttuynia, 50 to 100 parts by weight of patchouli, 20 to 70 parts by weight of rhubarb, 50 to 100 parts by weight of Rhodiola, 5 to 10 parts by weight of menthol and 50 to 100 parts by weight of licorice.

In a preferred embodiment, the raw material composition of the traditional Chinese medicine composition is: 255 parts by weight of forsythia, 255 parts by weight of honeysuckle, 85 parts by weight of ramie, 85 parts by weight of fried almond, 255 parts by weight of gypsum, 255 parts by weight of radix isatimus, 255 parts by weight of Guanzhong, 255 parts by weight of Houttuynia cordata, 85 parts by weight of patchouli, 51 parts by weight of rhubarb, 85 parts by weight of Rhodiola, 7.5 parts by weight of menthol, and 85 parts by weight of licorice.

In some examples, the raw material of the traditional Chinese medicine composition is tested after being made into a traditional Chinese medicine composition. For the preparation method of the traditional Chinese medicine composition or the Chinese medicine composition capsule, the specific content described in the patent document No. 03143211.5 can be specifically referred to.

In some examples of the invention, the gradient elution procedure is:

Time/min	A/%
0.00	12 to 16
8.00	18~20
15.00	22~25
20.00	28~30
25.00	32~35
30.00	38～43
35.00	48～52
40.00	55~60

In some preferred embodiments, the gradient elution procedure is:

Time/min	A/%
0.00	14 to 16
8.00	18~20
15.00	23~25
20.00	28~30
25.00	34～35
30.00	40~43

35.00	50～52
40.00	55~60

In a preferred embodiment, the gradient elution procedure is:

Time/min	A/%
0.00	15
8.00	20
15.00	twenty four
20.00	30
25.00	34
30.00	40
35.00	50
40.00	60

The concentration of the aqueous phosphoric acid solution is 0.1% to 1%, for example, 0.2%, 0.4%, 0.6%, 0.8%, 0.9%; preferably 0.1% to 0.5%. In a preferred embodiment, the concentration of the aqueous phosphoric acid solution is 0.1%. In the present invention, "A" as seen above means methanol in the mobile phase, the percentage occurring in the mobile phase is a volume percentage, and the content percentage of "A" is 100 vol% based on the total volume of A + B. The percentage of the concentration in the aqueous phosphoric acid solution is a volume percentage. Further, the concentrations of the methanol solution and the ethanol solution appearing in the present invention are all volume concentrations.

In some examples of the invention, the organic solvent of step a is selected from any one or more of methanol, ethanol, acetone, dichloromethane, and chloroform, preferably 60 vol% aqueous methanol.

The method of extracting in step a includes ultrasonic extraction or reflux extraction. In some examples, the test solution is prepared by mixing the traditional Chinese medicine composition with the organic solvent of step a, by sonication and filtration, and the obtained filtrate is the test solution. The process of ultrasonic extraction is used, for example, taking the capsule content of the traditional Chinese medicine composition, grinding it into a fine powder by a mortar, taking about 0.35 g, accurately weighing, placing it in a 25 mL volumetric flask, precisely adding 60% methanol 20 mL, and sealing After weighing, weighed for 40 minutes (power 250W, frequency 40kHz); let cool, weighed the weight, make up the lost weight with 60% methanol, shake it, filter it with 0.22μm microporous membrane, take The filtrate is continuously supplied, that is, the test solution is obtained. The process of reflux extraction is adopted, for example, taking the capsule content of the traditional Chinese medicine composition, grinding it into a fine powder by a mortar, taking about 0.35 g, accurately weighing, placing it in a 25 mL volumetric flask, precisely adding 60% methanol 20 mL, and sealing After weighing, heat and reflux for 30 minutes; let cool, weigh the weight, make up the lost weight with 60% methanol, shake well, filter with 0.22μm microporous membrane, take the filtrate, then you can test Product solution.

In some preferred embodiments of the present invention, the concentration of the test solution in step a is 10-20 mg/mL, for example, 12 mg/mL, 13 mg/mL, 15 mg/mL, 17 mg/mL, 19 mg/mL, preferably. It is 14 mg/mL.

In some examples of the invention, the organic solvent of step b is selected from any one or more of methanol, ethanol, acetone, dichloromethane, and chloroform, preferably methanol.

In some preferred embodiments of the present invention, the reference solution in step b has a concentration of chlorogenic acid of 55-65 μg/mL, a concentration of caffeic acid of 70-80 μg/mL, and a concentration of rutin of 65-72 μg. /mL, the concentration of isochlorogenic acid B is 120-130 μg/mL, the concentration of isochlorogenic acid C is 60-70 μg/mL, the concentration of forsythiaside A is 75-85 μg/mL, and the concentration of forsythin It is 48 to 58 μg/mL. Preferably, the concentration of chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin in the reference solution is 60 μg/mL, 75 μg/ respectively. mL, 68 μg/mL, 125 μg/mL, 64 μg/mL, 80 μg/mL, and 53 μg/mL.

In some preferred embodiments of the present invention, the detection wavelength detected in the step c is 200 to 250 nm, for example, 220 nm, 230 nm, 240 nm, preferably 210 nm; the reference solution and the test solution as the detection target The injection amount is 1.0 to 2.0 μL, for example, 1.2 μL, 1.5 μL, and 1.8 μL; the flow rate is 0.10 to 0.50 mL/min, for example, 0.2 mL/min, 0.3 mL/min, 0.4 mL/min; and the column temperature is 20 ~40 ° C, for example, 30 ° C, 35 ° C.

Example 1

The composition of traditional Chinese medicine composition: Forsythia 255g, Honeysuckle 255g, Castor Yellow 85g, Fried Bitter Almond 85g, Gypsum 255g, Radix Isatidis 255g, Mianma Guanzhong 255g, Houttuynia 255g, Patchouli 85g, Rhubarb 51g, Red View 85g, 7.5g of menthol, 85g of licorice.

The raw material of the traditional Chinese medicine composition is made into a capsule of a traditional Chinese medicine composition for detection.

The preparation method of the traditional Chinese medicine composition capsule comprises the following steps:

(1) weighing the thirteen flavor Chinese herbal medicine raw materials of the above traditional Chinese medicine composition by weight;

(2) The volatile oil is extracted by adding 6 times of mass water to the extract of patchouli, and the oil extraction time is 4 hours, and the volatile oil is collected and used; after the water extract is filtered, the residue is discarded, and the filtrate is reserved;

(3) Forsythia, ramie, houttuynia, rhubarb were extracted twice with 8 times the mass of 70 vol% ethanol, the first extraction was 2 hours, the second extraction was 1.5 hours, and the extracts obtained after two extractions were filtered. The filtrates are combined, and the ethanol is recovered to obtain an alcohol extract for use;

(4) Add honeysuckle, gypsum, Banlangen, Mianma Guanzhong, licorice, Rhodiola and 9 times of quality water to boiling, add fried almonds, cook twice, first time for 1.5 hours, second After decoction for 1 hour, the decoction obtained by two decoctions was filtered, and the filtrate was combined. The filtrate obtained by adding the patch of the patchouli extract obtained in the step (2) was concentrated to a clear concentration of 1.10 (60 ° C), and ethanol was added. The alcohol content was 70 vol%, and the product was refrigerated at 4 ° C for 24 hours, filtered, and the ethanol was recovered to an alcohol-free taste; the obtained filtrate was further combined with the spare alcohol extract obtained in the step (3), and concentrated to a relative density of 1.15 (60 ° C). Clearing paste, spray drying; then mixing with appropriate amount of starch to make granules, drying, sieving, sieving out a suitable amount of fine powder;

(5) The menthol and the volatile oil of the patchouli obtained in the step (2) are dissolved with an appropriate amount of ethanol (to the extent that the volatile oil can be dissolved), sprayed into the fine powder obtained in the step (4), mixed, and sieved with the step (4). The granules were mixed, sealed for 30 minutes, and filled into capsules to prepare 1000 capsules of the traditional Chinese medicine composition.

<Detection method>

Step a, the preparation of the test solution is shown in the preparation of the liquid medicine of 2.2;

Step b, the preparation of the reference solution is shown in the liquid preparation of 2.2;

Step c, detection: using ultra-high performance liquid chromatography, using methanol (A)-phosphoric acid aqueous solution (B) as a mobile phase for gradient elution; taking the reference solution into an ultra-high performance liquid chromatograph for detection, drawing The standard curve of each reference solution is taken; the test solution is aspirated, injected into an ultra-high performance liquid chromatograph for detection, and the peak area of each component is taken into a standard curve to determine chlorogenic acid, caffeic acid, rutin, The content of isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin.

1 experimental material

1.1 Experimental equipment

Acquity Ultra Performance Liquid Chromatograph (Waters, USA);

One in ten thousand electronic analytical balances (METTLER TOLEDO Instruments Shanghai Co., Ltd.);

Ultrasound (Nanjing Xinchen Biotechnology Co., Ltd.);

Milli-Q Ultrapure Water Preparation System (Synergy, USA);

Micropipette (Eppendorf, Germany).

1.2 drugs and reagents

40 batches of traditional Chinese medicine composition capsules (No. S01-S40), provided by Shijiazhuang Yiling Pharmaceutical; its raw material formula and preparation method are as described above;

The contents of the capsule of the traditional Chinese medicine composition of No. S40 were placed under high temperature (60 ° C), high humidity (RH 95%), and light (4500 lx) conditions for 20 days to obtain samples A1, A2, and A3 (No. S41-S43);

Chlorogenic acid (batch No. 151217, 99.68% for content determination), caffeic acid (batch No. 151103 for 99.40% for content determination), isochlorogenic acid B (batch number 15060708, for 98.82% for content determination) ), isochlorogenic acid C (batch 151028, 98.70% for content determination), forsythin (batch 15102801, 98.54% for content determination), forsythiaside A (lot 16082602, for content determination) Used in 99.17%), rutin (lot number 151023, 98.94% for content determination), provided by Chengdu Pfeide Biotechnology Co., Ltd.;

Chromatographic grade methanol, acetonitrile, chromatographic grade phosphoric acid (85% w/w) were purchased from Thermo Company, USA.

2 experimental methods

2.1 chromatographic conditions

1 The column was a Waters Acquity HSS T3 column (2.1 × 100 mm, 1.7 μm), and the mobile phase was a mixture of methanol (A) and 0.1% (v/v) aqueous phosphoric acid (B). The gradient elution procedure was:

0.00–8.00 minutes: 5-10% A; 8.01–15.00 minutes: 10-20% A; 15.01–20.00 minutes: 20-25% A; 20.01–25.00 minutes: 25-28% A; 25.01–30.00 minutes: 28 ～30%A; 30.01–35.00 minutes: 30～45%A; 35.01–40.00 minutes: 45～50%A; detection wavelength 210nm, injection volume 3.0μL, flow rate 0.20mL/min, column temperature 30°C, column The balance time is 7 minutes.

2 The column was a Waters Acquity HSS T3 column (2.1×100 mm, 1.7 μm), and the mobile phase was a mixture of methanol (A) and 0.1% (v/v) aqueous phosphoric acid (B). The gradient elution procedure was:

0.00–8.00 minutes: 5-12% A; 8.01–15.00 minutes: 12-22% A; 15.01–20.00 minutes: 22-28% A; 20.01–25.00 minutes: 28-30% A; 25.01–30.00 minutes: 30 ～48%A; 30.01–35.00 minutes: 48～52%A; 35.01–40.00 minutes: 52～58%A; detection wavelength 210nm, injection volume 2.0μL, flow rate 0.20mL/min, column temperature 30°C, column The balance time is 7 minutes.

3 The column was a Waters Acquity HSS T3 column (2.1×100 mm, 1.7 μm), and the mobile phase was a mixture of methanol (A) and 0.1% (v/v) aqueous phosphoric acid (B). The gradient elution procedure was:

0.00–8.00 minutes: 10-20% A; 8.01–15.00 minutes: 20-23% A; 15.01–20.00 minutes: 23-30% A; 20.01–25.00 minutes: 30–33% A; 25.01–30.00 minutes: 33 ～39%A; 30.01–35.00 minutes: 39～48%A; 35.01–40.00 minutes: 48～60%A; detection wavelength 210nm, injection volume 2.0μL, flow rate 0.20mL/min, column temperature 30°C, column The balance time is 7 minutes.

4 The column was a Waters Acquity HSS T3 column (2.1 x 100 mm, 1.7 μm), and the mobile phase was a mixture of methanol (A) and 0.1% (v/v) aqueous phosphoric acid (B). The gradient elution procedure was:

0.00–8.00 minutes: 15-20% A; 8.01–15.00 minutes: 20-24% A; 15.01–20.00 minutes: 24-30% A; 20.01–25.00 minutes: 30-34% A; 25.01–30.00 minutes: 34 ～40%A; 30.01–35.00 minutes: 40～50%A; 35.01–40.00 minutes: 50～60%A; detection wavelength 210nm, injection volume 2.0μL, flow rate 0.20mL/min, column temperature 30°C, column The balance time is 7 minutes.

2.2 Preparation of liquid medicine

Preparation of reference solution: accurately weigh chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin as reference substance, accurately weigh, set In a 25 mL brown volumetric flask, add 6 μg of chlorogenic acid, 75 μg of caffeic acid, 68 μg of rutin, 125 μg of isochlorogenic acid B, 64 μg of isochlorogenic acid C, 80 μg of forsythiaside A and 1 mL of methanol. A solution of 53 μg of forsythin, that is, a reference solution.

Preparation of the test solution: Take the content of the capsule of the traditional Chinese medicine composition, research into a fine powder, about 0.35g, accurately weighed, placed in a 25mL volumetric flask, precision added 60vol% methanol 20mL, densely plug, weigh Determine the weight, sonicate for 40 minutes (power 250W, frequency 40kHz) for extraction, let cool, then weigh the weight, make up the lost weight with 60vol% methanol, shake well, filter with 0.22μm microporous membrane, take out the filtrate , that is, the test solution is available.

3 experimental results

3.1 chromatographic conditions and investigation results

Accurately draw 2.0 μL of the test solution (No. S40) obtained from 2.2, inject into an ultra-high performance liquid chromatograph, and observe the chromatographic conditions 1, 2, 3 and 4, and record the chromatogram, see Figure 1-4.

Accurately draw 2.0 μL of the control solution obtained from 2.2, inject into an ultra-high performance liquid chromatograph, and observe according to chromatographic conditions 4, and record the chromatogram, see Figure 5.

According to the chromatographic conditions 1, 2, and 3, the components to be tested cannot be completely separated. According to the chromatographic conditions 4, 7 of the tested components (chlorogenic acid, caffeic acid, rutin, and isochlorogenic) The resolution of the acid B, isochlorogenic acid C, forsythiaside A and forsythin, the number of theoretical plates and the tailing factor all meet the requirements for content determination.

3.2 Detection methods and test results

Accuracy investigation: The same reference mixture solution was used as a standard solution for measurement, and the sample was continuously injected for 6 times, and the chromatographic condition was measured according to the chromatographic condition 4, and the chromatographic peak area was recorded. The results showed that the RSD of the peak areas of seven tested components such as chlorogenic acid, caffeic acid, forsythiaside A, isochlorogenic acid B, rutin, isochlorogenic acid C and forsythin were less than 5.0%. It shows that the precision of the instrument is good.

Linear relationship investigation: Precision extraction of the above mixed reference solution 0.4μL, 0.6μL, 0.8μL, 1.0μL, 1.5μL, 2.0μL, 3.0μL into the ultra-high performance liquid chromatograph, according to chromatographic conditions 4, record the chromatographic peak area . The standard curve is drawn by taking the peak area as the ordinate (Y) and the injection amount (μg) as the abscissa (X). Results The correlation coefficient was greater than 0.999, indicating that chlorogenic acid was 0.024~0.1788μg, caffeic acid was 0.0302～0.2268μg, forsythiaside A was 0.0317～0.2376μg, isochlorogenic acid B was 0.0504～0.378μg, rutin. The linear relationship is in the range of 0.027 to 0.2028 g, isochlorogenic acid C at 0.0252 to 0.1896 μg, and forsythin in the range of 0.021 to 0.1596 μg.

Stability investigation: Take the same test solution (No. S40), inject them within 0h, 3h, 6h, 12h, 24h after preparation, and test according to chromatographic conditions 4, record the peak area of each chromatographic peak, calculate the control The RSD of the peak area of the seven tested components in the solution. The results showed that the RSD of the peak area of the four tested materials in 2.4h was 2.45%, 3.08%, 3.17%, 1.89%, 3.44%, 4.02% and 3.78%, respectively, indicating that the test solution was stable within 24 hours. Good sex.

Repeatability investigation: Take the same batch (numbered S40) of the content of the capsule of traditional Chinese medicine composition 0.35g, a total of 6 parts, accurately weighed, prepare the test solution according to 2.2 method, determine according to chromatographic condition 4 and calculate the content . Results The average content of 7 tested substances in 6 samples was 2.93 mg/g chlorogenic acid, 1.41 mg/g caffeic acid, 2.17 mg/g of forsythiaside A, and 6.66 mg of isochlorogenic acid. g, rutin 1.08 mg / g, isochlorogenic acid C 1.31 mg / g forsythin 2.37 mg / g; indicating good repeatability of the method.

Accuracy investigation: take the same batch of known Chinese medicine composition capsules (No. S40) content 0.16 ~ 0.18g, placed in a 25mL brown volumetric flask, respectively added to calculate the same amount of standard substance, The test solution was prepared according to the method of 2.2, and the detection was carried out according to the chromatographic condition 4, and the recovery rate was calculated. The results showed that except for the recovery rate of caffeic acid (90.79%) and rutin (88.91%), the recovery rate of the other five tested materials was better, indicating that the method was more accurate.

3.3 Determination of the content of 7 active components in the capsule of traditional Chinese medicine composition:

Take different batches of traditional Chinese medicine composition capsules, prepare the test solution according to 2.2 method, test according to chromatographic condition 4, and calculate the content. The results are shown in Table 2.

Table 2 Results of determination of the content of 7 active components in the capsule of traditional Chinese medicine composition

The Chinese Pharmacopoeia stipulates that the qualified standard for the determination of capsules of traditional Chinese medicine composition is: containing at least 0.17 mg/granule of forsythin, and not less than 0.49 mg/g by the amount of 0.35 g/granule. The results of the measurements in Table 2 indicate that 40 different batches of traditional Chinese medicine composition capsules (No. S01-S40) are all qualified; while the destructive samples A1, A2, A3 (No. S41-S43) have the content of forsythin. Qualified, but the variation of the content of each component is large, the variation coefficient of chlorogenic acid, caffeic acid, forsythiaside A, isochlorogenic acid B, rutin, isochlorogenic acid C, forsythin content is 26%, 16%, 30%, 27%, 35%, 36%, 28%. It can be seen that it is not enough to use only one component of forsythin as a quality evaluation index. By the detection method of the invention, the main representative chemical components in the preparation can be quantitatively determined, and the rapid analysis by multi-component chemistry is realized. To evaluate the quality of the formulation.

Example 2

Correlation analysis between quality chemical evaluation and biological evaluation of traditional Chinese medicine composition: The present invention utilizes PCR technology to measure IL-6 and COX-2 in mouse lung tissue, and finds that the traditional Chinese medicine composition capsule of the present invention can significantly reduce virality The expression of IL-6 and COX-2 in the lung tissue of pneumonia model mice was dose-dependent, and therefore, IL-6 and COX-2 were used as indicators for the biomass evaluation of the capsule quality of the traditional Chinese medicine composition of the present invention.

The content of seven chemical components (chlorogenic acid, rutin, forsythiaside A, forsythiaside, isochlorogenic acid C, isochlorogenic acid B, caffeic acid) in the traditional Chinese medicine composition is an independent variable. The potency is the dependent variable. For the 40 batches of the traditional Chinese medicine composition numbered S01-S40, the statistical analysis samples were taken to investigate the inhibition of COX-2 biopotency, inhibition of macrophage secretion of IL-6 biopotency and 7 chemical components. Correlation analysis of the results of the content determination was performed using multivariate statistical SPSS statistical software. The results are shown in Table 3.

Table 3 Correlation analysis between biological activity and chemical content

In Table 3, r represents correlation and p is a statistical indicator.

From the perspective of anti-inflammatory biopotency against COX-2 activity, the related components were chlorogenic acid, isochlorogenic acid B, isochlorogenic acid C and other acid components, and were positively correlated (p<0.05). It is suggested that the higher the content of these three components, the higher the level of inhibition of COX-2 enzyme activity. Therefore, in the case where the detection method of the present invention can realize the content and quality detection of the seven components, the detection of the index component which increases the correlation activity of the chemical composition of the capsule of the traditional Chinese medicine composition further improves the traditional Chinese medicine to a certain extent. The ability to assess the consistency of composition quality. From the anti-inflammatory biopotency of inhibiting the secretion of IL-6 by macrophages, the related component was forsythin, and it was positively correlated (p<0.05), suggesting that the higher the content of forsythin, the higher its The level of inhibition of IL-6 secretion by cells also tends to increase. It is suggested that forsythin is an effective component for exerting anti-inflammatory effects, and its content has a certain influence on the quality of traditional Chinese medicine compositions.

Compared with the method of "Pharmacopoeia", the detection method of the invention increases the component indexes of chlorogenic acid, isochlorogenic acid B and isochlorogenic acid C which are highly correlated with biological activity, and establishes 7 components in the traditional Chinese medicine composition. The detection method can more fully characterize the quality of the traditional Chinese medicine composition.

Example 3

The preparation method and detection method of the composition of the traditional Chinese medicine composition and the capsule of the traditional Chinese medicine composition are the same as those in the first embodiment; the detection is performed according to the chromatographic condition 5: the column is a Waters Acquity HSS T3 column (2.1×100 mm, 1.7 μm). The mobile phase is a mixture of methanol (A) and 0.1% (v/v) aqueous phosphoric acid (B). The gradient elution procedure is:

0.00–8.00 minutes: 12-20% A; 8.01–15.00 minutes: 20-22% A; 15.01–20.00 minutes: 22-28% A; 20.01–25.00 minutes: 28-35% A; 25.01–30.00 minutes: 35 ～43%A; 30.01–35.00 minutes: 43～50%A; 35.01–40.00 minutes: 50～60%A; detection wavelength 210nm, injection volume 2.0μL, flow rate 0.20mL/min, column temperature 30°C, column The balance time is 7 minutes.

Preparation of reference solution: accurately weigh chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin reference substance, accurately weighed, placed In a 25 mL brown volumetric flask, a solution containing 60 μg, 75 μg, 68 μg, 125 μg, 64 μg, 80 μg, and 53 μg per 1 mL was added to obtain a reference solution.

Preparation of the test solution: Take the content of the traditional Chinese medicine composition capsule (No. S40), research into a fine powder, about 0.35g, accurately weighed, placed in a 25mL volumetric flask, precisely added 60% methanol 20mL, Concealed, weighed, sonicated for 40 minutes (power 250W, frequency 40kHz), let cool, then weighed, with 60% methanol to make up the lost weight, shake, filtered through a 0.22μm microporous membrane, The filtrate is taken to obtain the test solution.

Determination: Precision extraction of the test solution (No. S40) and mixed control solution mixed solution of 2.0μL, injected into the ultra-high performance liquid chromatograph, record the chromatogram; the spectrum of the test solution and the reference solution are shown in Figure 6 and 7.

Example 4

The preparation method and detection method of the prescription of the traditional Chinese medicine composition and the capsule of the traditional Chinese medicine composition are the same as those in the first embodiment; the detection is performed according to the chromatographic condition 6: the column is a Waters Acquity HSS T3 column (2.1×100 mm, 1.7 μm). The mobile phase is a mixture of methanol (A) and 0.1% (v/v) aqueous phosphoric acid (B). The gradient elution procedure is:

0.00–8.00 minutes: 16-18% A; 8.01–15.00 minutes: 18 to 25% A; 15.01–20.00 minutes: 25 to 28% A; 20.01–25.00 minutes: 28 to 32% A; 25.01–30.00 minutes: 32 ～42%A; 30.01–35.00 minutes: 42～48%A; 35.01–40.00 minutes: 48～58%A; detection wavelength 210nm, injection volume 2.0μL, flow rate 0.20mL/min, column temperature 30°C, column The balance time is 7 minutes.

Preparation of reference solution: accurately weigh chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin reference substance, accurately weighed, placed In a 25 mL brown volumetric flask, a solution containing 60 μg, 75 μg, 68 μg, 125 μg, 64 μg, 80 μg, and 53 μg per 1 mL was added to obtain a reference solution.

Preparation of the test solution: Take the content of the capsule of the traditional Chinese medicine composition (No. S40), research into a fine powder, take about 0.35g, accurately weigh it, place it in a 25mL volumetric flask, and accurately add 60mL methanol to 20mL. Concealed, weighed, sonicated for 40 minutes (power 250W, frequency 40kHz), let cool, then weighed, with 60% methanol to make up the lost weight, shake, filtered through a 0.22μm microporous membrane, The filtrate is taken to obtain the test solution.

Determination: Precision extraction of the test solution (No. S40) and mixed control solution mixed solution of 2.0μL, injected into the ultra-high performance liquid chromatograph, record the chromatogram; the spectrum of the test solution and the reference solution are shown in Figure 8 and 9.

Example 5

The preparation method and the detection method of the prescription of the traditional Chinese medicine composition and the capsule of the traditional Chinese medicine composition are the same as those in the first embodiment; the difference is that the chromatography column is the waters Acquity HSS T3 column (2.1×100 mm, 1.7 μm). The mobile phase is a mixture of methanol (A) and 0.1% (v/v) aqueous phosphoric acid (B). The gradient elution procedure is:

0.00–8.00 minutes: 14-20% A; 8.01–15.00 minutes: 20-25% A; 15.01–20.00 minutes: 25-30% A; 20.01–25.00 minutes: 30-35% A; 25.01–30.00 minutes: 35 ～42%A; 30.01–35.00 minutes: 42～51%A; 35.01–40.00 minutes: 51～60%A; detection wavelength 210nm, injection volume 2.0μL, flow rate 0.20mL/min, column temperature 30°C, column The balance time is 7 minutes.

Preparation of reference solution: accurately weigh chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin reference substance, accurately weighed, placed In a 25 mL brown volumetric flask, a solution containing 60 μg, 75 μg, 68 μg, 125 μg, 64 μg, 80 μg, and 53 μg per 1 mL was added to obtain a reference solution.

Preparation of the test solution: Take the content of the capsule of the traditional Chinese medicine composition (No. S40), research into a fine powder, take about 0.35g, accurately weigh it, place it in a 25mL volumetric flask, and accurately add 60mL methanol to 20mL. Concealed, weighed, sonicated for 40 minutes (power 250W, frequency 40kHz), let cool, then weighed, with 60% methanol to make up the lost weight, shake, filtered through a 0.22μm microporous membrane, The filtrate is taken to obtain the test solution.

Determination: Precision extraction of the test solution (No. S40) and mixed control solution mixed solution of 2.0μL, injected into the ultra-high performance liquid chromatograph, record the chromatogram; the spectrum of the test solution and the reference solution are shown in Figure 10 and 11.

The results showed that the components to be tested could not be completely separated according to the chromatographic conditions 1, 2, and 3. In the case of examining the chromatographic conditions in Example 1, seven of the components to be tested (chlorogenic acid, caffeic acid) The separation degree, theoretical plate number and tailing factor of rutin, chlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin all meet the requirements for content determination, and the system has good applicability. Example 3-5 was examined according to the chromatographic conditions 5-7. The separation degree, the number of theoretical plates, and the tailing factor of the seven components in the test results all met the requirements for content determination.

The embodiments of the present invention have been described above, and the foregoing description is illustrative, not limiting, and not limited to the disclosed embodiments. Numerous modifications and changes will be apparent to those skilled in the art without departing from the scope of the invention.

Claims (13)

1. A method for detecting a traditional Chinese medicine composition, characterized in that the detection method comprises the following steps:

   Step a, preparing a test solution: taking a Chinese medicine composition, for example, a capsule content of a traditional Chinese medicine composition, and extracting by adding an organic solvent, thereby obtaining a test solution;

   Step b, preparation of the reference solution: taking chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin, dissolved in an organic solvent, that is, a control Product solution

   Step c, detection: using ultra performance liquid chromatography, using methanol (A)-phosphoric acid aqueous solution (B) as a mobile phase gradient elution;

   The raw material composition of the traditional Chinese medicine composition is: 200-300 parts by weight of forsythia, 200-300 parts by weight of honeysuckle, 50-100 parts by weight of ramie, 50-100 parts by weight of fried almond, 200-300 parts by weight of gypsum, Banlangen 200 to 300 parts by weight, 200 to 300 parts by weight of Kumba, 200 to 300 parts by weight of Houttuynia, 50 to 100 parts by weight of patchouli, 20 to 70 parts by weight of rhubarb, 50 to 100 parts by weight of Rhodiola, 5 to 10 parts by weight of menthol and 50 to 100 parts by weight of licorice.
2. The detecting method according to claim 1, wherein the raw material composition of the traditional Chinese medicine composition is: 255 parts by weight of forsythia, 255 parts by weight of honeysuckle, 85 parts by weight of ramie, 85 parts by weight of fried almond, gypsum 255. Parts by weight, 255 parts by weight of Radix, 255 parts by weight of hunters, 255 parts by weight of Houttuynia, 85 parts by weight of patchouli, 51 parts by weight of rhubarb, 85 parts by weight of Rhodiola, 7.5 parts by weight of menthol, licorice 85 Parts by weight.
3. The detecting method according to claim 1 or 2, wherein in the detecting process of the step c, ultra-high performance liquid chromatography is employed, and the methanol (A)-phosphoric acid aqueous solution (B) is used as a mobile phase gradient elution; The reference solution and the test solution are aspirated and injected into an ultra-high performance liquid chromatograph for determination of chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside A and forsythin content.
4. The detecting method according to any one of claims 1 to 3, wherein the gradient elution procedure is:

   Time/min A/% 0.00 12 to 16 8.00 18~20 15.00 22~25 20.00 28~30 25.00 32~35

   30.00 38～43 35.00 48～52 40.00 55~60 .
5. The detecting method according to claim 4, wherein the gradient elution program is:

   Time/min A/% 0.00 15 8.00 20 15.00 twenty four 20.00 30 25.00 34 30.00 40 35.00 50 40.00 60 .
6. The detecting method according to claim 4, wherein the concentration of the aqueous phosphoric acid solution is from 0.1% to 1%; preferably from 0.1% to 0.5%.
7. The detecting method according to claim 1 or 2, wherein the organic solvent in the step a is selected from any one or more of methanol, ethanol, acetone, dichloromethane and chloroform, preferably 60%. Methanol solution.
8. The detecting method according to claim 1 or 2 or 7, wherein the method of extracting in step a comprises ultrasonic extraction or reflux extraction; preferably, in the preparation of the test solution, the traditional Chinese medicine composition is The organic solvent in step a is mixed, extracted by sonication or reflux, and then filtered, and the obtained filtrate is the test solution.
9. The detection method according to claim 1 or 2 or 8, wherein the concentration of the test solution in step a is 10 to 20 mg/mL, more preferably 14 mg/mL.
10. The detecting method according to claim 1 or 2, wherein the organic solvent in the step b is selected from any one or more selected from the group consisting of methanol, ethanol, acetone, dichloromethane and chloroform, preferably methanol.
11. The detection method according to claim 1 or 2, wherein the reference solution is chlorogenic acid, caffeic acid, rutin, isochlorogenic acid B, isochlorogenic acid C, forsythiaside in the reference solution. The concentrations of A and forsythin are 55-65 μg/mL, 70-80 μg/mL, 65-72 μg/mL, 120-130 μg/mL, 60-70 μg/mL, 75-85 μg/mL, and 48-58 μg/ mL.
12. The detecting method according to any one of claims 1 to 11, wherein the detected detection wavelength is 200 to 250 nm, preferably 210 nm; the injection amount is 1.0 to 2.0 μL; and the flow rate is 0.10 to 0.50 mL. /min; The column temperature is 20 to 40 °C.
13. The detection method according to any one of claims 1 to 12, wherein the raw material of the traditional Chinese medicine composition is made into a traditional Chinese medicine composition for detection; preferably, the preparation method of the traditional Chinese medicine composition comprises the following steps :

    (1) extracting volatile oil from patchouli and adding water to collect volatile oil; filtering the aqueous solution to obtain a filtrate;

    (2) extracting forsythia, ramie, houttuynia, rhubarb with 70 vol% ethanol, filtering the extract, and recovering the ethanol to obtain an alcohol extract;

    (3) Add honeysuckle, gypsum, Banlangen, Mianmaguanzhong, licorice, Rhodiola to boil, add frying almonds, and add the filtrate obtained by decoction to the filtrate (1). The patchouli oil and the filtered filtrate are concentrated to a relative concentration of 1.10 to 1.15, ethanol is added to make the alcohol content 70 vol%, refrigerated at 2-4 ° C for 20-24 hours, filtered, and the filtrate is recovered and the step is recovered. 2) The obtained alcohol extracts are combined, concentrated to a relative density of 1.10 to 1.15, spray-dried; then mixed with starch to form granules, dried, sieved, and sieved to a fine powder;

    (4) The menthol and the volatile oil of the patchouli obtained in the step (1) are dissolved in ethanol, sprayed into the fine powder obtained in the step (3), and mixed; and the particles obtained after the step (3) are mixed, preferably. Also includes the operation of sealing for 20-30min;

    Further preferably, the method further comprises the steps of: filling the preparation obtained in the step (4) into a capsule to prepare a capsule of the traditional Chinese medicine composition.

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