Construction method of high performance liquid chromatography characteristic atlas of bidens pilosa and application thereof

Abstract

The application provides a construction method of bidens pilosa high-performance liquid characteristic atlas and application thereof, and comprises the following steps: mixing bidens pilosa samples and methanol, and performing ultrasonic treatment to obtain a test sample solution; the test sample solution is determined by using a high-performance liquid chromatography method to obtain the bidens pilosa high-performance liquid characteristic atlas; and the chromatography conditions comprise: a C18 column, a mobile phase A of acetonitrile, a mobile phase B of 0.1% phosphoric acid solution, and gradient elution. The method firstly identifies a chromatographic peak 4-coumaric acid, and provides a more scientific identification basis for identifying bidens pilosa medicinal materials, decoction pieces, standard decoction and formula granules. The method has good stability, good repeatability and high accuracy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical detection, and particularly relates to a construction method of a high-performance liquid chromatography (HPLC) characteristic spectrum of Bidens pilosa and application thereof. BACKGROUND

[0002] Bidens pilosa is an annual herbaceous plant of the family Asteraceae, also known as Bidens pilosa, sticky grass, and three-leaf Bidens pilosa. It is a commonly used medicine in China and has the effects of clearing heat and resolving toxins and promoting blood circulation to remove blood stasis. It is often used for upper respiratory tract infection, sore throat, malaria, acute jaundice hepatitis, and the like. Bidens pilosa contains complex and diverse components, and the main active component is flavonoids.

[0003] In order to ensure the uniformity and stability of the quality of Bidens pilosa medicinal materials, decoction pieces, standard decoction and formula granules, it is urgently needed to establish a new characteristic spectrum construction method to control the quality thereof. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a construction method of a high-performance liquid chromatography (HPLC) characteristic spectrum of Bidens pilosa and application thereof. The method identifies chromatographic peak 4-p-coumaric acid for the first time, and provides a more scientific identification basis for identifying Bidens pilosa medicinal materials, decoction pieces, standard decoction and formula granules. The method has good stability, good repeatability and high accuracy.

[0005] The present application provides a construction method of a high-performance liquid chromatography (HPLC) characteristic spectrum of Bidens pilosa, comprising the following steps:

[0006] Mixing Bidens pilosa samples and methanol, and ultrasonic treatment to obtain a test solution;

[0007] Determining the test solution by high-performance liquid chromatography (HPLC) to obtain a high-performance liquid chromatography (HPLC) characteristic spectrum of Bidens pilosa;

[0008] The chromatographic conditions include a C18 column, a mobile phase A of acetonitrile, a mobile phase B of 0.1% phosphoric acid solution, and gradient elution.

[0009] In the present application, the 70% methanol and the 0.1% phosphoric acid solution are all volume fractions; the 0.1% phosphoric acid solution is a 0.1% aqueous phosphoric acid solution.

[0010] In the present application, the Bidens pilosa samples include Bidens pilosa medicinal materials, decoction pieces, standard decoction and formula granules. If the sample is Bidens pilosa medicinal materials or decoction pieces, the preparation of the test solution further comprises: boiling Bidens pilosa medicinal materials or decoction pieces in water, filtering to obtain residues. The mass-to-volume ratio of Bidens pilosa medicinal materials or decoction pieces to water is 1g:(45-55)mL. The boiling time is 25-35 min. In a specific embodiment, the mass-to-volume ratio of Bidens pilosa medicinal materials or decoction pieces to water is 1g:50mL, and the boiling time is 30 min. Mixing the residues and methanol, and ultrasonic treatment to obtain a test solution.

[0011] The methanol used in the present application is 70% methanol; the volume ratio of the Bidens pilosa sample and the 70% methanol used in the present application is 1g:(24-26)mL; the power of the ultrasonic treatment is 580-620W, the frequency of the ultrasonic treatment is 35-45kHz, and the time of the ultrasonic treatment is 20-30min. In specific embodiments, the volume ratio of the Bidens pilosa sample and the 70% methanol is 1g:25mL, the power of the ultrasonic treatment is 600W, the frequency of the ultrasonic treatment is 40kHz, and the time of the ultrasonic treatment is 20min.

[0012] After obtaining the test sample solution, the present application determines the Bidens pilosa high-performance liquid chromatography characteristic spectrum by using high-performance liquid chromatography to determine the test sample solution.

[0013] The gradient elution used in the present application includes:

[0014] 0-5min, A phase: 10%, B phase: 90%;

[0015] 5-15min, A phase: 10-16%, B phase: 90-84%;

[0016] 15-20min, A phase: 16%, B phase: 84%;

[0017] 20-60min, A phase: 16-28%, B phase: 84-72%.

[0018] In the present application, the flow rate of the mobile phase is 0.8-1.2mL / min, the column temperature is 25-35℃, and the wavelength is 320nm; the theoretical plate number is not less than 5000 calculated according to the 4-coumaric acid peak. The injection amount of the test sample solution is 9-11μL; the injection amount of the reference solution is 9-11μL. In specific embodiments, the flow rate of the mobile phase is 1.0mL / min, and the column temperature is 30℃. The injection amount of the test sample solution is 10μL, and the injection amount of the reference solution is 10μL. The experimental results show that: at 30℃, the chromatogram peak shape is relatively symmetrical and the retention time is relatively appropriate; at a flow rate of 1.0ml / min, the chromatogram peak shape is relatively symmetrical and the retention time is appropriate; at a detection wavelength of 320nm, the chromatogram peak information is relatively large, and the baseline is relatively flat.

[0019] The present application also includes preparing a reference solution:

[0020] After boiling the Bidens pilosa control medicinal material, filtering, evaporating to dryness, mixing the residue with methanol, and ultrasonic treatment, a control medicinal material reference solution is obtained;

[0021] The 4-coumaric acid control and methanol are mixed to obtain a 10μg / mL control reference solution.

[0022] The quality of the control medicinal material of bidens pilosa in the application and the volume ratio of water are 1g:(45-55)mL. The water boiling time is 25-35min. After filtration, the filtrate is evaporated to dryness; the residue is mixed with methanol and ultrasonically treated; the power of ultrasonic treatment is 580-620W, the frequency of ultrasonic treatment is 35-45kHz, and the time of ultrasonic treatment is 20-30min. The application is cooled after ultrasonic treatment, shaken uniformly, filtered, and the filtrate is taken as the reference solution of the control medicinal material.

[0023] The application further mixes 4-coumaric acid control and methanol to obtain a 10ug / mL reference solution of the control. In the application, 70% methanol is used in the reference solution of the control.

[0024] The chromatographic column in the application is ZORBAX SB C18 4.6*250mm, 5um.

[0025] The application precisely sucks the reference solution and the test solution into a liquid chromatograph for determination, and the high-performance liquid chromatography characteristic spectrum of bidens pilosa is obtained. The application adopts the Chinese Pharmacopoeia 2020 edition general 0512 for determination.

[0026] The application adopts the traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of bidens pilosa medicinal materials and decoction pieces, and obtains the HPLC standard characteristic spectrum of bidens pilosa medicinal materials and decoction pieces composed of 6 characteristic peaks, wherein the peak corresponding to the 4-coumaric acid reference is the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak should be within ±10% of the specified value; the specified value is: 0.36 (peak 1), 0.56 (peak 2), 0.60 (peak 3), 0.64 (peak 4), 1.77 (peak 6).

[0027] The application adopts the traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of bidens pilosa standard decoction, and obtains the HPLC standard characteristic spectrum of bidens pilosa standard decoction composed of 6 characteristic peaks, wherein the peak corresponding to the 4-coumaric acid reference is the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak should be within ±10% of the specified value; the specified value is: 0.34 (peak 1), 0.56 (peak 2), 0.61 (peak 3), 0.64 (peak 4), 1.86 (peak 6).

[0028] The application adopts traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of bidens pilosa formula granules, and obtains the HPLC standard characteristic spectrum of the bidens pilosa formula granules composed of six characteristic peaks, wherein the peak corresponding to the 4-coumaric acid reference is an S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak should be within ±10% of the specified value; the specified value is: 0.36 (peak 1), 0.57 (peak 2), 0.60 (peak 3), 0.64 (peak 4), 1.78 (peak 6).

[0029] The application also provides a method for identifying bidens pilosa medicinal materials, decoction pieces, standard decoctions and formula granules thereof, which is detected by the method in the above technical scheme, and the detection result is analyzed.

[0030] The application provides a method for constructing a bidens pilosa high-performance liquid chromatography characteristic spectrum, which comprises the following steps: mixing a bidens pilosa sample and methanol, and performing ultrasonic treatment to obtain a test sample solution; determining the test sample solution by using high-performance liquid chromatography to obtain a bidens pilosa high-performance liquid chromatography characteristic spectrum; and the chromatographic conditions comprise: a C18 column, a mobile phase A of acetonitrile, a mobile phase B of 0.1% phosphoric acid solution, and gradient elution. The method identifies the chromatographic peak 4-coumaric acid for the first time, and provides a more scientific identification basis for identifying bidens pilosa medicinal materials, decoction pieces, standard decoctions and formula granules. The method has good stability, good repeatability and high accuracy. The experimental results show that: in the stability investigation, the RSD of the retention time of the characteristic peak is 0.07%-0.15%; in the repeatability investigation, the relative retention time RSD of the six samples is 0.06%-0.07%; and in the precision experiment, the relative retention time RSD of each characteristic peak is 0.05%-0.14%. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a chromatogram of bidens pilosa formula granules at different wavelengths;

[0032] Figure 2 It is a chromatogram of column temperature investigation;

[0033] Figure 3 It is a flow rate investigation;

[0034] Figure 4 It is an extraction solvent investigation;

[0035] Figure 5 It is an extraction flow rate investigation;

[0036] Figure 6 It is an extraction time investigation;

[0037] Figure 7 It is an investigation of the amount of solvent added;

[0038] Figure 8 It is a chromatographic peak identification;

[0039] Figure 9 is the 4-coumaric acid control spectrum;

[0040] Figure 10 is the 4-coumaric acid spectrum in the test solution;

[0041] Figure 11 is the different instrument investigation;

[0042] Figure 12 is the column durability investigation;

[0043] Figure 13 is the 3 batches of bidens pilosa formula granules characteristic spectrum;

[0044] Figure 14 is the control characteristic spectrum of bidens pilosa formula granules;

[0045] Figure 15 is the 16 batches of bidens pilosa medicinal materials characteristic spectrum;

[0046] Figure 16 is the 16 batches of bidens pilosa decoction pieces characteristic spectrum;

[0047] Figure 17 is the control characteristic spectrum of bidens pilosa decoction pieces;

[0048] Figure 18 is the control characteristic spectrum of bidens pilosa medicinal materials;

[0049] Figure 19 is the bidens pilosa standard decoction characteristic superimposed spectrum;

[0050] Figure 20 is the control characteristic spectrum of bidens pilosa standard decoction. DETAILED DESCRIPTION

[0051] In order to further illustrate the present application, the construction method of bidens pilosa high performance liquid characteristic spectrum and its application provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the present application.

[0052] Example 1 Construction of bidens pilosa formula granules HPLC characteristic spectrum:

[0053] 1.1 Experimental instruments, materials, reagents and reagents

[0054] High performance liquid chromatograph: instrument-1 (Agilent 1260), instrument-2 (Shimadzu LC-20AD), instrument-3 (water se2695);

[0055] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0056] Ultrapure water machine: Cell type 1810A (Shanghai Molier Scientific Instrument Co., Ltd.);

[0057] Ultrasonic cleaner: KQ5200DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0058] Chromatographic column: Column-1 was ZORBAX SB C18 4.6x250mm, 5μm, Column-2 was Kromasil 100-5-C18 4.6x250mm, 5μm.

[0059] Phosphoric acid, acetonitrile were chromatographically pure, water was ultrapure water, and the rest of the reagents were analytically pure.

[0060] 4-coumaric acid (China Food and Drug Inspection Research Institute, batch number: 112037-202102, content was 99.7%);

[0061] Bidens pilosa control medicinal materials (Chengdu Desit Biotechnology Co., Ltd., batch number: DSTYG003801);

[0062] Bidens pilosa medicinal materials and decoction pieces batch number: YC-1~16, YP-1~16;

[0063] Bidens pilosa standard decoction batch number: BT-1~16;

[0064] Bidens pilosa formula granules batch number: KL-1, KL-2, KL-3;

[0065] 1.2 Chromatographic conditions

[0066] Chromatographic conditions and system suitability test

[0067] Octadecylsilane-bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, which was gradient eluted according to the provisions in Table 1 below; the flow rate was 1.0 ml per minute; the column temperature was 30°C; and the detection wavelength was 320 nm. The theoretical plate number should not be less than 5000 calculated according to the 4-coumaric acid peak.

[0068] Table 1

[0069]

[0070] Preparation of Reference Solution: Take 1 g of Bidens pilosa L. reference medicinal material and place it in a conical flask. Add 50 ml of water and boil for 30 minutes. Filter and evaporate the filtrate to dryness. Add 25 ml of 70% methanol to the residue and sonicate (power 600 W, frequency 40 kHz) for 20 minutes. Let cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take an appropriate amount of 4-coumaric acid reference substance, accurately weigh it, and add 70% methanol to make a solution containing 10 μg per 1 ml. This is the reference substance solution.

[0071] Preparation of test solution: Take an appropriate amount of the product, take 1g, place it in a conical flask, add 25ml of 70% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate.

[0072] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0073] 1.3 Wavelength Selection

[0074] Based on the experimental conditions proposed above, the test solution was scanned in the full band using a diode array detector, and the chromatograms of the test solution at wavelengths of 260nm, 280nm, 300nm, 320nm, and 340nm were extracted respectively. Figure 1 ; Figure 1 The results show that when the detection wavelength is 320 nm, the chromatographic peak has a larger amount of information and the baseline is relatively flat, so the detection wavelength is determined to be 320 nm.

[0075] 1.4 Column temperature investigation

[0076] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 2 and Table 2; The results show that at 30℃, the chromatogram peak shape is more symmetrical and the retention time is more appropriate, so the column temperature was selected to be 30℃.

[0077] Table 2 Column temperature investigation-relative retention time ratio

[0078]

[0079] 1.5 Flow rate investigation

[0080] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min and 1.2ml / min were investigated. Figure 3 and Table 3:

[0081] Table 3 Flow rate investigation-relative retention time ratio

[0082]

[0083] The results show that when the flow rate is 1.0 ml / min, the chromatogram peak shape is relatively symmetrical and the retention time is appropriate, so the flow rate is selected as 1.0 ml / min.

[0084] In summary, the characteristic chromatogram of the Bidens pilosa formula granules is determined by the following conditions: octadecylsilane-bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.1% phosphoric acid solution as the mobile phase B, gradient elution according to the above Table 1; flow rate of 1.0 ml / min; column temperature of 30°C; detection wavelength of 320 nm. The theoretical plate number should not be less than 5000 calculated by the 4-coumaric acid peak.

[0085] 1.6 Preparation of test sample

[0086] 1.6.1 Investigation of extraction solvent

[0087] Take an appropriate amount of the product (batch number: KL-1), grind it finely, take 1 g, and put it in a conical flask. Add water, 30% methanol, 70% methanol, methanol, and 30% ethanol, respectively, and ultrasonically treat for 20 minutes. Take it out, cool it down, shake it up, filter it, and take the filtrate. See Figure 4 The results show that when the extraction solvent is 70% methanol, the baseline is stable and the chromatographic peak information is relatively rich. Therefore, 70% methanol is tentatively selected as the extraction solvent.

[0088] 1.6.2 Investigation of extraction method

[0089] Take an appropriate amount of the product (batch number: KL-1), grind it finely, take 1 g, and put it in a conical flask. Add 70% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, respectively. Shake it up, filter it, and take the filtrate. See Figure 5 The results show that the effects of the two extraction methods are similar, the peak shape of each characteristic peak is good, and the separation degree is moderate. Therefore, ultrasonic extraction is selected.

[0090] 1.6.3 Investigation of extraction time

[0091] Take an appropriate amount of the product (batch number: KL-1), grind it finely, take 1 g, and put it in a conical flask. Add 70% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, 40 minutes, and 60 minutes, respectively. Shake it up, filter it, and take the filtrate. See Figure 6 The results show that the extraction time has little effect on the dissolution of the effective ingredients, so the extraction time is determined as 20 minutes.

[0092] 1.6.4 Investigation of solvent addition amount

[0093] Take the product (batch number: KL-1) appropriate amount, grind, take 1g, placed in a conical flask, add 70% methanol 25ml, 50ml, 100ml, ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes, shake, filter, take the filtrate, that is. See Figure 7 .

[0094] The results show that the solvent added amount is 25ml, the size of each chromatographic peak is moderate, and the solvent added amount is selected as 25ml.

[0095] In summary, the preparation method of the test solution of the Bidens pilosa formula granules characteristic map is determined as follows: take the product appropriate amount, grind, take 1g, placed in a conical flask, add 70% methanol 25ml, ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes, take out, cool, shake, filter, take the filtrate, that is.

[0096] 1.7 Methodology

[0097] 1.7.1 Chromatographic peak identification

[0098] Preparation of test solution: take the product appropriate amount, grind, take 1g, placed in a conical flask, add 70% methanol 25ml, ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes, take out, cool, shake, filter, take the filtrate, as test solution.

[0099] Preparation of reference solution: take 1g of Bidens pilosa reference material, add water 50ml, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 70% methanol 25ml, ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes, cool, shake, filter, take the filtrate, as reference solution of control drug. Take 4-coumaric acid control appropriate amount, add methanol to prepare a solution containing 4-coumaric acid 10μg per 1ml, as reference solution of control.

[0100] Preparation of negative control solution: according to the above experimental conditions, prepare the negative control solution of Bidens pilosa formula granules.

[0101] The characteristic map peaks of Bidens pilosa formula granules were located. See Figures 8-10 . The results show that peak 5 is 4-coumaric acid. In the following methodology study, 6 characteristic peaks in the sample were investigated.

[0102] 1.7.2 Precision test

[0103] Take the test solution of the product (batch number: KL-1) 1 part. According to the proposed experimental method, 6 times of continuous sampling, 10μl each time, calculate the retention time and peak area of each characteristic peak. See Table 4:

[0104] Table 4 Precision investigation-retention time

[0105]

[0106] The results show that the relative retention time RSD of the characteristic peaks of the samples is 0.05% to 0.14%, and the instrument precision is good.

[0107] 1.7.3 Reproducibility Investigation

[0108] Six portions of the test solution of the product (batch number: KL-1) were prepared and determined according to the proposed experimental method. See Table 5.

[0109] Table 5 Reproducibility Investigation - Relative Retention Time

[0110]

[0111]

[0112] The results show that the relative retention time RSD of the six samples is 0.06% to 0.07%, indicating that the method has good reproducibility.

[0113] 1.7.4 Intermediate Precision Investigation

[0114] 1.7.4.1 Investigation of Different Instruments

[0115] Based on the above proposed experimental conditions, two portions of the product (batch number: KL-1) were prepared into test solutions and determined on instrument-1 (Agilent 1260), instrument-2 (Shimadzu LC-20AD), and instrument-3 (Waters e2695), respectively. See Table 6: Figure 11 and Table 6:

[0116] Table 6 Instrument Durability Investigation - Relative Retention Time

[0117]

[0118] The results show that when the test solution is detected by the above three instruments, the RSD of the relative retention time of each characteristic peak is 1.84% to 4.05%, but the separation effect of the chromatographic peaks by instrument 3 is poorer.

[0119] 1.7.4.2 Investigation of Different Personnel and Time

[0120] Based on the above proposed experimental conditions, two portions of the product (batch number: KL-1) were prepared into test solutions by different personnel (A, B) at different times (T1, T2) and determined. See Table 7:

[0121] Table 7 Personnel and Time Investigation - Relative Retention Time

[0122]

[0123]

[0124] The results show that the RSD of the relative retention time of each characteristic peak is 0.06% to 0.16% when the same sample is determined by different persons at different times, and the method is stable.

[0125] 1.7.5 Durability of the chromatographic column

[0126] Based on the above experimental conditions, the different brands of chromatographic column-1 (ZORBAX SB C18 4.6x250mm) and chromatographic column-2 (Kromasil 100-5-C18 4.6x250mm, 5μm) were used for the investigation, respectively. See Table 7 and Table 8: Figure 12

[0127] Table 8 Durability of the chromatographic column- relative retention time

[0128]

[0129] The results show that the RSD of the relative retention time of each characteristic peak is 0.08% to 3.35% when the test sample is determined by the above two brands of chromatographic column, indicating that the chromatographic column is durable.

[0130] 1.7.6 Stability investigation

[0131] Based on the above experimental conditions, the same test sample solution was taken and determined at 0h, 4h, 8h, 12h, 16h and 24h, respectively. See Table 9:

[0132] Table 9 Stability investigation- retention time

[0133]

[0134] The results show that the RSD of the retention time of the characteristic peak is 0.07% to 0.15%, and the sample solution is stable within 24h.

[0135] 1.8 Determination of the characteristic peak and establishment of the control chromatogram

[0136] Verification results of 3 batches of Bidens pilosa formula granules

[0137] The characteristic chromatogram analysis of 3 batches of samples was carried out by using this method, and the relative retention time was calculated. See Table 7 and Table 8: Figure 13

[0138] Table 10 Relative retention time of Bidens pilosa formula granules

[0139] ​​

[0140] According to the principle of relatively stable retention time and each batch sample can be detected and the peak is relatively high, 6 peaks with better durability are selected as characteristic peaks.

[0141] The relative retention time of each characteristic peak is stable, and within the range of average value ± 10%, so the relative retention time of each peak is temporarily set as ± 10%.

[0142] Finally, the test sample chromatogram should present 6 characteristic peaks, which should correspond to the retention time of the 6 characteristic peaks in the reference chromatogram of the control medicinal material, and peak 5 should correspond to the retention time of the 4-coumaric acid reference peak. The peak corresponding to the 4-coumaric acid reference peak is the S peak. The relative retention time of peaks 1, 2, 3, 4, 6 and S peak should be within ± 10% of the specified value, and the specified value is: 0.36 (peak 1), 0.57 (peak 2), 0.60 (peak 3), 0.64 (peak 4), 1.78 (peak 6).

[0143] The similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (2012 version) was used to synthesize 3 batches of bidens pilosa formula granules, and the control chromatogram of bidens pilosa formula granule characteristic chromatogram was established. See Figure 14 Among them, peak 5 (S) is 4-coumaric acid.

[0144] Example 2

[0145] Reference to the HPLC characteristic chromatogram method of bidens pilosa formula granules, the method of establishing the characteristic chromatogram of bidens pilosa medicinal material and decoction piece is as follows:

[0146] The chromatographic conditions and system suitability test were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.1% phosphoric acid solution as the mobile phase B, gradient elution according to the above table 1; flow rate 1.0 ml per minute; column temperature 30℃; detection wavelength 320 nm. The theoretical plate number calculated by 4-coumaric acid peak should not be less than 5000.

[0147] The reference solution was prepared as follows: 1 g of bidens pilosa control medicinal material was placed in a conical flask, 50 ml of water was added, and decoction was carried out for 30 minutes. The filtrate was evaporated to dryness, the residue was added with 25 ml of 70% methanol, ultrasonic treatment (power 600 W, frequency 40 kHz) was carried out for 20 minutes, and the filtrate was filtered. The filtrate was taken as the control medicinal material reference solution. The appropriate amount of 4-coumaric acid reference substance was accurately weighed and dissolved in 70% methanol to prepare a solution containing 10 μg per 1 ml as the reference substance reference solution.

[0148] The test solution was prepared by taking about 1.0 g of the powder (passed through a No. 3 sieve) into a conical flask, adding 50 ml of water, decocting for 30 minutes, cooling, filtering, evaporating the filtrate to dryness, adding 25 ml of 70% methanol to the residue, tightly capping, and ultrasonically treating (power 600 W, frequency 40 kHz) for 20 minutes. After cooling, the mixture was shaken well and filtered.

[0149] The determination method was as follows: 10 μl of the reference solution and the test solution, respectively, were injected into the liquid chromatograph, and determination was performed.

[0150] 2.1 Verification results of 16 batches of bidens pilosa medicinal materials and 16 batches of decoction pieces

[0151] The characteristic chromatograms of 16 batches of samples were determined by using the proposed method, and the relative retention times were calculated. See Figures 15-16 (Figures represent batch numbers S1→S16: YP-1-16) and Tables 11-12:

[0152] Table 11 Relative retention times of 16 batches of bidens pilosa medicinal materials

[0153]

[0154] Table 12 Relative retention times of 16 batches of bidens pilosa decoction pieces

[0155]

[0156]

[0157] The test chromatogram should exhibit 6 characteristic peaks, which should correspond to the 6 characteristic peaks in the reference chromatogram of the control medicinal material, and peak 5 should correspond to the retention time of the 4-coumaric acid reference. The peak corresponding to the 4-coumaric acid reference is the S peak. The relative retention times of peaks 1, 2, 3, 4, 6 and the S peak should be within ±10% of the specified values, which are: 0.36 (peak 1), 0.56 (peak 2), 0.60 (peak 3), 0.64 (peak 4), and 1.77 (peak 6).

[0158] The 16 batches of bidens pilosa medicinal materials and 16 batches of bidens pilosa decoction pieces were synthesized by using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) to establish the control chromatogram of the characteristic chromatogram of bidens pilosa medicinal materials and decoction pieces. See Figures 17-18 , wherein peak 5 (S): 4-coumaric acid.

[0159] Example 3 Establishment of the characteristic chromatogram method of bidens pilosa standard decoction

[0160] The method for establishing the characteristic chromatogram of bidens pilosa standard decoction was established by referring to the HPLC characteristic chromatogram method of bidens pilosa formula granules, and the details were as follows:

[0161] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the packing material; acetonitrile as the mobile phase A, 0.1% phosphoric acid solution as the mobile phase B, gradient elution according to the provisions in Table 1 above; flow rate 1.0 ml per minute; column temperature 30 °C; detection wavelength 320 nm. Theoretical plate number should not be less than 5000 calculated according to the peak of 4-coumaric acid.

[0162] Preparation of reference solution: 1 g of the control medicinal material of Bidens pilosa was placed in a conical flask, 50 ml of water was added, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 25 ml of 70% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 20 minutes, allowed to cool, shaken well, filtered, and the filtrate was taken as the reference solution of the control medicinal material. An appropriate amount of the 4-coumaric acid reference substance was precisely weighed, dissolved in 70% methanol to prepare a solution containing 10 μg per 1 ml, and the solution was taken as the reference solution of the reference substance.

[0163] Preparation of test solution: an appropriate amount of the test sample was precisely weighed, about 1 g, placed in a conical flask, 25 ml of 70% methanol was precisely added, tightly capped, weighed, ultrasonically treated (power 600 W, frequency 40 kHz) for 20 minutes, allowed to cool, weighed again, 70% methanol was added to make up the weight loss, shaken well, filtered, and the filtrate was obtained.

[0164] Determination method: 10 μl of each of the reference solution and the test solution was precisely taken and injected into the liquid chromatograph for determination.

[0165] 3.1 Validation results of 16 batches of Bidens pilosa standard decoction

[0166] Using the above method, the characteristic chromatograms of 16 batches of samples were analyzed, and the relative retention time and relative peak area ratio were calculated. See Figure 19 (from bottom to top, batch numbers are BT-1~16) and Table 13:

[0167] Table 13 Relative retention time of 16 batches of Bidens pilosa standard decoction

[0168]

[0169] In the test sample chromatogram, 6 characteristic peaks should be present, and peak 6 should correspond to the retention time of the 4-coumaric acid peak in the reference solution. The peak corresponding to the 4-coumaric acid reference substance peak is the S peak. The relative retention time of each characteristic peak to the S peak was calculated, and the relative retention time should be within ±10% of the specified value, 0.34 (peak 1), 0.56 (peak 2), 0.61 (peak 3), 0.64 (peak 4), 1.86 (peak 6).

[0170] The 16 batches of standard decoction of Bidens pilosa were synthesized by using Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and the control chromatogram of the characteristic chromatogram of the standard decoction of Bidens pilosa was established. Figure 20 Peak 5 (S): 4-coumaric acid.

[0171] From the above examples, the application provides a construction method of high performance liquid chromatography characteristic chromatogram of Bidens pilosa, which comprises the following steps: mixing the sample of Bidens pilosa and methanol, and ultrasonic treatment to obtain a test solution; determining the test solution by using high performance liquid chromatography to obtain the high performance liquid chromatography characteristic chromatogram of Bidens pilosa; and the chromatographic conditions comprise: C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution. The method identifies 4-coumaric acid for the first time, and provides a more scientific identification basis for identifying the medicinal materials, decoction pieces, standard decoction and formula granules of Bidens pilosa. The method has good stability, good repeatability and high accuracy. The experimental results show that: in the stability investigation, the RSD of the retention time of the characteristic peak is 0.07%-0.15%; in the repeatability investigation, the relative retention time RSD of the 6 samples is 0.06%-0.07%; and in the precision experiment, the relative retention time RSD of each characteristic peak is 0.05%-0.14%.

[0172] The above only describes the preferred embodiments of the application, and it should be noted that, for those skilled in the art, without departing from the principles of the application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. A method for constructing a high-performance liquid chromatography characteristic spectrum of Bidens pilosa L., comprising the following steps: The Bidens pilosa L. sample and methanol were mixed and ultrasonically treated to obtain a test solution; The test solution is measured by high performance liquid chromatography to obtain a high performance liquid chromatography characteristic spectrum of Bidens pilosa L. Chromatographic conditions included: C18 column, mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid solution, gradient elution, wavelength 320 nm; The gradient elution comprises: 0-5 min, phase A: 10%, phase B: 90%; 5-15 min, phase A: 10-16%, phase B: 90-84%; 15-20 min, phase A: 16%, phase B: 84%; 20~60min, phase A: 16~28%, phase B: 84~72%.

2. The construction method according to claim 1, characterized in that Also includes the preparation of reference solutions: Boil the Bidens pilosa L. control medicinal material in water, filter it, evaporate it to dryness, mix the residue with methanol, and perform ultrasonic treatment to obtain a control medicinal material reference solution; Mix the 4-coumaric acid reference substance with methanol to obtain a 10 μg / mL reference substance solution.

3. The construction method according to claim 1, characterized in that The methanol is 70% methanol; The volume ratio of the Bidens pilosa sample to 70% methanol is 1 g: (24-26) mL; The flow rate of the mobile phase was 0.8–1.2 mL / min, and the column temperature was 25–35 °C; The number of theoretical plates calculated based on the 4-coumaric acid peak is not less than 5000.

4. The construction method according to claim 1, characterized in that The power of the ultrasonic treatment is 580-620 W, the frequency of the ultrasonic treatment is 35-45 kHz, and the time of the ultrasonic treatment is 20-30 min.

5. The construction method according to claim 1, characterized in that The similarity of Bidens pilosa medicinal materials and decoction pieces was evaluated using a Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of Bidens pilosa medicinal materials and decoction pieces consisting of 6 characteristic peaks was obtained, wherein the peak corresponding to the 4-coumaric acid reference substance was the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: 0.36 (peak 1), 0.56 (peak 2), 0.60 (peak 3), 0.64 (peak 4), 1.77 (peak 6).

6. The construction method according to claim 1, characterized in that The similarity of the standard decoction of Bidens pilosa was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the standard decoction of Bidens pilosa was obtained, consisting of 6 characteristic peaks, among which the peak corresponding to the 4-coumaric acid reference was the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: 0.34 (peak 1), 0.56 (peak 2), 0.61 (peak 3), 0.64 (peak 4), and 1.86 (peak 6).

7. The construction method according to claim 1, characterized in that The similarity of Bidens pilosa granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of Bidens pilosa granules consisting of 6 characteristic peaks was obtained, wherein the peak corresponding to the 4-coumaric acid reference substance was the S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: 0.36 (peak 1), 0.57 (peak 2), 0.60 (peak 3), 0.64 (peak 4), 1.78 (peak 6).

8. The construction method according to claim 2, characterized in that: The injection volume of the test solution is 9~11μL; The injection volume of the reference solution was 9-11 μL.

9. A method for identifying Bidens pilosa medicinal materials, decoction pieces, standard decoctions and formula granules thereof, characterized in that: The method according to any one of claims 1 to 8 is used for detection, and the detection results are analyzed.

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