A liquid chromatography detection method for cross-seating traditional Chinese medicine fingerprint spectrum

Abstract

The application discloses a liquid chromatography detection method for Hengjing Chinese medicinal material fingerprint, which comprises the following steps: crushing Hengjing to obtain Hengjing powder, adding the Hengjing powder into a solvent, then shaking for 30 min, ultrasonicating for 30 min, filtering to obtain a filtrate, then performing liquid chromatography detection on the filtrate, wherein the liquid chromatography detection is performed under the following conditions: using octadecylsilane bonded silica gel as a filler, using a solution prepared by mixing acetonitrile, methanol and an ammonium acetate solution as a mobile phase, a detection wavelength of 254 nm, a flow rate of 1 mL / min, and a column temperature of 25-30 DEG C; collecting more than 10 kinds of Hengjing Chinese medicinal materials, performing detection according to the above steps, comparing the obtained detection results, selecting common characteristic peaks, and obtaining Hengjing Chinese medicinal material fingerprint. The application can identify the authenticity and quality of Hengjing Chinese medicinal material simply and accurately, improve the quality control of Hengjing Chinese medicinal material, and ensure the safety and stability of Hengjing medicinal material.

Description

TECHNICAL FIELD

[0001] The present application belongs to the detection technical field of Hengjingxi Chinese herbal medicine, and particularly relates to a liquid chromatography detection method for a Hengjingxi Chinese herbal medicine fingerprint. BACKGROUND

[0002] Hengjingxi is also called as Bianziwang, Chubangwang, Boheyhongchuo, and Dujiaofeng, is a plant of Clidemia genus in Clidemiaceae, and belongs to shrubs to small trees with a height of 1-5 meters. The young branches are tetrahedral with narrow wings. The leaves are opposite, thin leathery, and long oval or lanceolate. The flowers are bisexual, white with a little red. The drupes are ovate long oval with a short tip at the top. The flowering period is in summer, and the fruiting period is in autumn. Hengjingxi grows in mountainous sparse or dense forests, and is mainly distributed in Guangdong Province and Guangxi Zhuang Autonomous Region. Hengjingxi has the functions of expelling wind and dampness, strengthening bones and muscles, promoting blood circulation and relieving pain, and can be used for the treatment of rheumatic arthralgia, lumbago and leg pain, contusion and injury, jaundice hepatitis, irregular menstruation, and dysmenorrhea. With the development of traditional Chinese medicine industry, the quality requirements for Hengjingxi medicinal materials are obviously improved, and it is necessary to develop a quality control method for Hengjingxi medicinal materials, so that the authenticity and quality of Hengjingxi traditional Chinese medicine materials can be simply and accurately detected, and the quality and stability of traditional Chinese medicine can be controlled.

[0003] Fingerprint is a method of using modern analysis technology to characterize the chemical components in traditional Chinese medicine and its preparations, which can reflect the overall chemical characteristics of traditional Chinese medicine, rather than only a single component. Different traditional Chinese medicines have different fingerprints, which can be used to distinguish different medicinal materials and control the quality of medicinal materials. However, there are few reports on the fingerprint of Hengjingxi, and it is still necessary to establish a Hengjingxi fingerprint with certain stability and repeatability to assist in the quality control of Hengjingxi medicinal materials. SUMMARY

[0004] In view of the above problems, the present application discloses a liquid chromatography detection method for a Hengjingxi Chinese herbal medicine fingerprint, which can detect and analyze the fingerprint of Hengjingxi Chinese herbal medicine, simply and accurately identify the authenticity and quality of Hengjingxi Chinese herbal medicine, improve the quality control of Hengjingxi Chinese herbal medicine, and ensure the safety and stability of Hengjingxi medicinal materials.

[0005] The present application is implemented by using the following technical scheme:

[0006] A liquid chromatography detection method for a Hengjingxi Chinese herbal medicine fingerprint, comprising the following steps:

[0007] (1) Hengjingxi is crushed to obtain Hengjingxi powder, the Hengjingxi powder is added to a solvent, then shaken for 30 min, ultrasonically treated for 30 min, and filtered to obtain a filtrate; the solvent is any one of methanol and petroleum ether;

[0008] (2) taking the filtrate obtained in step (1) to perform liquid chromatography detection, wherein the liquid chromatography detection is performed under the following conditions: octadecylsilane-bonded silica gel (C18) is used as the filler, a solution prepared by mixing acetonitrile, methanol and an ammonium acetate solution is used as the mobile phase, the detection wavelength is 254 nm, the flow rate is 1 mL / min, and the column temperature is 25-30°C;

[0009] (3) collecting more than 10 kinds of Hengjingxi Chinese medicinal materials, and performing detection according to the steps described in steps (1)-(2), comparing the obtained detection results, and selecting common characteristic peaks to obtain the Hengjingxi Chinese medicinal material fingerprint, wherein the Hengjingxi Chinese medicinal material fingerprint comprises peaks 1-4, and the relative retention times of the peaks 1-4 are 8.093-8.49 min, 11.549-12.246 min, 12.268-12.962 min and 14.413-15.338 min, respectively.

[0010] Further, in step (1), the Hengjingxi is crushed to pass through a 80-100 mesh sieve to obtain Hengjingxi powder.

[0011] Further, in step (1), the mass and volume ratio of the Hengjingxi powder to the solvent is 1 g:5 mL, and the solvent is methanol.

[0012] Further, in step (1), the Hengjingxi powder is added to the solvent, and then shaken at a speed of 2000-3000 r / min for 30 min, followed by ultrasonic treatment for 30 min, and the shaking speed during the ultrasonic treatment is 2000-3000 r / min.

[0013] Further, in step (2), the mass concentration of the ammonium acetate solution is 0.5%, and the volume ratio of acetonitrile, methanol and ammonium acetate solution is 50:18:32.

[0014] The Hengjingxi Chinese medicinal material fingerprint can be used for identification of Hengjingxi Chinese medicinal materials, specifically, the to-be-identified medicinal materials are detected according to the steps (1)-(2), and then the obtained liquid chromatography results are compared with the Hengjingxi Chinese medicinal material fingerprint, if the peaks 1-4 are present, it can be preliminarily determined that the to-be-tested medicinal materials are Hengjingxi.

[0015] The technical solution has the following beneficial effects compared with the prior art:

[0016] 1. This invention employs liquid chromatography to detect and construct a fingerprint spectrum of *Hymenoplastyum* medicinal materials. Then, the corresponding liquid chromatogram of the purchased *Hymenoplastyum* medicinal materials is detected according to the detection method described in this invention. By comparing this chromatogram with the constructed *Hymenoplastyum* fingerprint spectrum, the authenticity and quality of the *Hymenoplastyum* medicinal materials can be accurately identified. This effectively screens out counterfeit or substandard medicinal materials, thereby improving the quality control of *Hymenoplastyum* medicinal materials and ensuring their safety and stability.

[0017] 2. The method of this invention is simple, with good accuracy, repeatability and durability. Moreover, the peaks in the obtained chromatograms are attractive and the resolution is appropriate, which is conducive to comparative analysis and improves analytical accuracy. At the same time, the analysis time required for a single sample is short when using this invention, which helps to improve the efficiency of analytical work. In addition, it is easy to operate and reduces the workload of the testing and analysis personnel. Attached Figure Description

[0018] Figure 1 This is the liquid chromatogram of sample No. 1 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0019] Figure 2 This is the liquid chromatogram of sample No. 2 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0020] Figure 3 This is the liquid chromatogram of sample No. 3 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0021] Figure 4 This is the liquid chromatogram of sample No. 4 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0022] Figure 5 This is the liquid chromatogram of sample No. 5 of the *Hypericum perforatum* medicinal material detected in Example 1.

[0023] Figure 6 This is the liquid chromatogram of sample No. 6 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0024] Figure 7 This is the liquid chromatogram of sample No. 7 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0025] Figure 8 This is the liquid chromatogram of sample No. 8 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0026] Figure 9 This is the liquid chromatogram of sample No. 9 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0027] Figure 10 This is the liquid chromatogram of sample No. 10 of the *Hymenoplastyum* medicinal material detected in Example 1.

[0028] Figure 11is the liquid chromatogram of the No. 11 transverse seat medicine sample detected in Example 1.

[0029] Figure 12 is the liquid chromatogram obtained when methanol solvent is used for detection in Experimental Example 1.

[0030] Figure 13 is the liquid chromatogram obtained when petroleum ether solvent is used for detection in Experimental Example 1.

[0031] Figure 14 is the liquid chromatogram obtained when the proportion of the mobile phase is 50:18:32 for detection in Experimental Example 2.

[0032] Figure 15 is the liquid chromatogram obtained when the proportion of the mobile phase is 67:12:21 for detection in Experimental Example 2.

[0033] Figure 16 is the liquid chromatogram obtained when the proportion of the mobile phase is 30:25:45 for detection in Experimental Example 2.

[0034] Figure 17 is the liquid chromatogram obtained when C18 chromatographic column is used for detection in Experimental Example 3.

[0035] Figure 18 is the liquid chromatogram obtained when C8 chromatographic column is used for detection in Experimental Example 3.

[0036] Figure 19 is the liquid chromatogram obtained when the chromatographic column produced by Shiseido Company is used for detection in Experimental Example 4.

[0037] Figure 20 is the liquid chromatogram obtained when the chromatographic column produced by Nap Company is used for detection in Experimental Example 4.

[0038] Figure 21 is the liquid chromatogram obtained when the chromatographic column produced by Shimadzu Company is used for detection in Experimental Example 4.

[0039] Figure 22 is the liquid chromatogram obtained when the chromatographic column produced by Filomena Company is used for detection in Experimental Example 4.

[0040] Figure 23 is the liquid chromatogram obtained when 25℃ column temperature is used for detection in Experimental Example 5.

[0041] Figure 24 is the liquid chromatogram obtained when 30℃ column temperature is used for detection in Experimental Example 5.

[0042] Figure 25 is the liquid chromatogram obtained when 35℃ column temperature is used for detection in Experimental Example 5.

[0043] Figure 26is the liquid chromatogram obtained when the column temperature is detected at 40℃ in Experimental Example 5.

[0044] Figure 27 is the liquid chromatogram obtained when the blank control is detected in Experimental Example 6.

[0045] Figure 28 is the liquid chromatogram obtained when parallel sample 1 is detected in Experimental Example 6.

[0046] Figure 29 is the liquid chromatogram obtained when parallel sample 2 is detected in Experimental Example 6.

[0047] Figure 30 is the liquid chromatogram obtained when parallel sample 3 is detected in Experimental Example 6.

[0048] Figure 31 is the liquid chromatogram obtained when parallel sample 4 is detected in Experimental Example 6.

[0049] Figure 32 is the liquid chromatogram obtained when parallel sample 5 is detected in Experimental Example 6.

[0050] Figure 33 is the liquid chromatogram obtained when parallel sample 6 is detected in Experimental Example 6.

[0051] Figure 34 is the liquid chromatogram obtained when parallel sample 7 is detected in Experimental Example 6.

[0052] Figure 35 is the liquid chromatogram obtained when the tester 1 is detected in Experimental Example 7.

[0053] Figure 36 is the liquid chromatogram obtained when the tester 2 is detected in Experimental Example 7. DETAILED DESCRIPTION

[0054] The present application is further illustrated by the following examples, but not as a limitation to the present application. The specific experimental conditions and methods not specified in the following examples, the technical means used are generally well known to those skilled in the art of conventional means.

[0055] Example 1: A liquid chromatographic detection method for the fingerprint of Hengjingxi traditional Chinese medicinal materials, comprising the following steps:

[0056] (1) Hengjingxi is crushed through 100 mesh sieve to obtain Hengjingxi powder, the Hengjingxi powder is added into solvent, then it is shaken at a speed of 3000 r / min for 30 min, then it is ultrasonically treated for 30 min, and the shaking speed during ultrasonic treatment is 2000 r / min, then it is filtered to obtain filtrate; the solvent is methanol; the mass of Hengjingxi powder and the volume of solvent are 1 g:5 mL;

[0057] (2) Take the filtrate obtained in step (1) and perform liquid chromatography detection. The liquid chromatography detection conditions are as follows: use octadecylsilane bonded silica gel as the packing material, use a solution prepared by mixing acetonitrile, methanol and ammonium acetate solution as the mobile phase, the detection wavelength is 254 nm, the flow rate is 1 mL / min, and the column temperature is 25 °C; the mass concentration of the ammonium acetate solution is 0.5%, and the volume ratio of the acetonitrile, methanol and ammonium acetate solution is 50:18:32.

[0058] (3) Collect 11 kinds of Chinese medicinal materials for the horizontal meridian (see Table 1) and test them according to the steps (1) to (2). The test results are shown in Table 2 and Appendix. Figures 1-11 The fingerprint spectrum of the Chinese medicinal materials of Hengjingxi was obtained by comparing the common characteristic peaks. The fingerprint spectrum of the Chinese medicinal materials of Hengjingxi includes peaks 1 to 4, and the relative retention times of peaks 1 to 4 are 8.093 to 8.49 min, 11.549 to 12.246 min, 12.268 to 12.962 min, and 14.413 to 15.338 min, respectively.

[0059] The application of the fingerprint spectrum of the Chinese medicinal material of the horizontal meridian in this embodiment is used for the identification of the Chinese medicinal material of the horizontal meridian. Specifically, the medicinal material to be identified is tested according to the method in steps (1) to (2), and then the obtained liquid chromatography results are compared with the fingerprint spectrum of the Chinese medicinal material of the horizontal meridian. If the 1 to 4 peaks are present, the medicinal material to be tested can be preliminarily determined to be the Chinese medicinal material of the horizontal meridian.

[0060] Table 1. Collection of Chinese medicinal materials from different transverse meridians

[0061]

[0062] Table 2. Retention time results (min) of Chinese medicinal materials with different transverse meridians.

[0063]

[0064] Experimental Example 1: The same transverse meridian herb sample was tested according to steps (1) and (2) in Example 1, wherein the mobile phase was a solution prepared by mixing acetonitrile, methanol, and ammonium acetate solution, with a volume ratio of acetonitrile, methanol, and ammonium acetate solution of 67:12:21. Methanol and petroleum ether were used as solvents, respectively. The test results are shown in Table 3 and... Figures 12-13 .

[0065] Table 3. Retention time results (min) using different solvents.

[0066]

[0067] According to Table 3 and Figures 12-13From the results in Table 4 and Table 5, it can be seen that methanol and petroleum ether can be used as the solvent in the method of the present application, and the obtained chromatogram can clearly obtain the characteristic peaks corresponding to peaks 1-4.

[0068] Experimental Example 2: The same horizontal cross seat medicine sample was detected according to the method described in steps (1) and (2) in Example 1, wherein different mobile phases were used for detection, i.e. the volume ratio of acetonitrile, methanol and ammonium acetate solution in the mobile phase was 50:18:32, 67:12:21, 30:25:45, respectively, and the detection results are shown in Table 4 and Table 5. Figures 14-16 .

[0069] Table 4 Retention time results (min) detected by different mobile phases

[0070]

[0071] According to the results in Table 4 and Table 5, it can be seen that the mobile phase ratio (50:18:32) used in the present application is relatively reasonable, the peak time is appropriate, the peak shape is good, and the separation degree is appropriate. Figures 14-16

[0072] Experimental Example 3: The same horizontal cross seat medicine sample was detected according to the method described in steps (1) and (2) in Example 1, wherein different chromatographic columns (C18 and C8) were used for detection, and the detection results are shown in Table 5 and Table 6. Figures 17-18 .

[0073] Table 5 Retention time results (min) detected by different chromatographic columns

[0074]

[0075] According to the results in Table 5 and Table 6, it can be seen that the C18 chromatographic column selected in the present application has good separation degree and has good detection effect. Figures 17-18

[0076] Experimental Example 4: The same horizontal cross seat medicine sample was detected according to the method described in steps (1) and (2) in Example 1, wherein C18 chromatographic columns produced by different manufacturers were used for detection, and the detection results are shown in Table 6 and Table 7. Figures 19-22 .

[0077] Table 6 Retention time results (min) detected by C18 chromatographic columns produced by different manufacturers

[0078]

[0079] According to the results in Table 6 and Table 7, it can be seen that the C18 column produced by Nupu Company is selected, the peak time is appropriate, the amount of solvent used is appropriate, the peak shape is good, and the separation degree is good. Figures 19-22

[0080] ​​​Experimental Example 5: The same horizontal and vertical seat medicinal material sample was detected according to the method described in the step (1) and step (2) in the embodiment 1, wherein different column temperatures were used, i.e. the column temperatures were 25℃, 30℃, 35℃ and 40℃ respectively, and the detection results were shown in Table 7 and Table 8. Figures 23-26 .

[0081] Table 7: Retention time results (min) detected by using different column temperatures

[0082]

[0083] According to the results in Table 7 and Table 8, it can be seen that only when the detection is carried out at the column temperature of 25-30℃ as described in the embodiment, good peak effect can be obtained, and the temperature is too high to affect the peak effect. Figures 23-26

[0084] Experimental Example 6: The horizontal and vertical seat medicinal material sample was detected according to the method described in the step (1) and step (2) in the embodiment 1, and was evenly divided into 7 samples for parallel test, and the test solution without the horizontal and vertical seat medicinal material sample was used as a blank control for detection, and the detection results were shown in Table 8 and Table 9. Figures 27-34 .

[0085] Table 8: Retention time results (min) detected by parallel test

[0086]

[0087] According to the results in Table 8 and Table 9, it can be seen that the method has good specificity, and the method is stable and has good repeatability. Figures 27-34

[0088] Experimental Example 7: Two experimental personnel were arranged to use two instruments of the same type to detect the same horizontal and vertical seat medicinal material sample according to the method described in the step (1) and step (2) in the embodiment 1, and the detection results were shown in Table 9 and Table 10. Figures 35-36 .

[0089] Table 9: Retention time results (min) detected by different experimental personnel

[0090]

[0091] According to the results in Table 9 and Table 10, it can be seen that the results of the detection by different experimental personnel according to the method of the embodiment have good repeatability and good accuracy. Figures 35-36

[0092] ​​​Furthermore, it should be understood that although the specification is described in terms of embodiments, not every embodiment includes every feature described. The specification can include implicit combinations of explicitly mentioned features and / or explicit combinations of implicitely mentioned features. Each embodiment depends on the explicit combinations of features and / or the implicit combinations of features made specifically within that embodiment, and each such embodiment can be combined with every other such embodiment to create further embodiments.

Claims

1. A liquid chromatography method for detecting the fingerprint spectrum of traditional Chinese medicinal materials, characterized in that: Includes the following steps: (1) Take the transverse warp mat and crush it to obtain transverse warp mat powder. Add the transverse warp mat powder to the solvent, shake it for 30 minutes, sonicate it for 30 minutes, and then filter it to obtain the filtrate. The solvent is either methanol or petroleum ether. (2) Take the filtrate obtained in step (1) for liquid chromatography detection. The liquid chromatography detection conditions are: using octadecylsilane bonded silica gel as the packing material, using a solution prepared by mixing acetonitrile, methanol and ammonium acetate solution as the mobile phase, the detection wavelength is 254nm, the flow rate is 1mL / min, and the column temperature is 25~30℃. The mass concentration of the ammonium acetate solution is 0.5%, and the volume ratio of the acetonitrile, methanol, and ammonium acetate solution is 50:18:

32. (3) Collect more than 10 kinds of Chinese medicinal materials from the Hengjingxi area and test them according to the steps in (1) to (2). Compare the test results and select the common characteristic peaks to obtain the fingerprint spectrum of the Chinese medicinal materials from the Hengjingxi area. The fingerprint spectrum of the Chinese medicinal materials from the Hengjingxi area includes peaks 1 to 4. The relative retention times of peaks 1 to 4 are 8.093 to 8.49 min, 11.549 to 12.246 min, 12.268 to 12.962 min, and 14.413 to 15.338 min, respectively.

2. The liquid chromatography detection method for fingerprint spectroscopy of traditional Chinese medicine materials according to claim 1, characterized in that: In step (1), the transverse warp mat is crushed and passed through an 80-100 mesh sieve to obtain transverse warp mat powder.

3. The liquid chromatography detection method for fingerprint spectroscopy of traditional Chinese medicine materials according to claim 1, characterized in that: In step (1), the mass ratio of the transverse warp powder to the volume ratio of the solvent is 1g:5mL, and the solvent is methanol.

4. The liquid chromatography detection method for the fingerprint spectrum of *Hypericum perforatum* medicinal materials according to claim 1, characterized in that: In step (1), the transverse warp powder is added to the solvent and then shaken for 30 minutes at a speed of 2000-3000 r / min, and then sonicated for 30 minutes, with the shaking speed during the sonication process being 2000-3000 r / min.

5. The application of the liquid chromatography detection method for the fingerprint spectrum of *Hypericum perforatum* medicinal materials as described in any one of claims 1 to 4, characterized in that: The fingerprint spectrum of the Chinese medicinal material of the Horizontal Meridian is used for the identification of the Chinese medicinal material of the Horizontal Meridian. Specifically, the medicinal material to be identified is tested according to the method in steps (1) to (2). Then, the obtained liquid chromatography results are compared with the fingerprint spectrum of the Chinese medicinal material of the Horizontal Meridian. If the 1 to 4 peaks are present, the medicinal material to be tested can be preliminarily determined to be the Horizontal Meridian.

Images