Method for constructing HPLC (High Performance Liquid Chromatography) characteristic chromatograms of nandina medicinal material decoction pieces, standard decoction and formula granules

Abstract

The invention provides a construction method of HPLC (high performance liquid chromatography) characteristic chromatograms of common nandina herb medicinal slices, standard decoction and formula granules, which comprises the following steps: A) dissolving and extracting raw materials of a test sample by adopting a solvent to obtain a solution to be detected; b) measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain HPLC characteristic chromatograms of the common nandina herb medicinal decoction pieces, the standard decoction and the formula granules; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; a mobile phase A is an acetonitrile solution, a mobile phase B is a 0.1% phosphoric acid aqueous solution, and gradient elution is carried out. According to the method, a high performance liquid chromatography method is adopted, an acetonitrile-0. 1% phosphoric acid solution is selected as a mobile phase for gradient elution, chlorogenic acid is taken as a reference substance, and the HPLC characteristic chromatogram method of the nandina medicinal material decoction pieces, standard decoction and formula granules is established. And more scientific technical means are provided for controlling the medicinal quality of common nandina herb decoction pieces, standard decoction and formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for constructing HPLC characteristic spectra of Radix Gastrodiae dasyphyllae medicinal material pieces, standard decoctions and formula granules. Background Art

[0002] Tianzhuzi is the dried mature fruit of Nandina domesties., a plant of the Berberidaceae family. When the fruit matures in autumn, the fruit branches are cut off, the fruit is picked, and dried in the sun. The chemical components mainly contain alkaloids, flavonoids, lignans, volatile oils, phenolic acids and other components. It has the effects of clearing away heat and dampness, promoting blood circulation and activating collaterals, relieving cough and relieving asthma. Its cough-relieving effect is particularly prominent. It is used to treat colds and fever, lung heat cough, damp-heat jaundice, acute gastroenteritis, urinary tract infection, trauma, cough, asthma and other diseases, but Tianzhuzi is often used in the form of preparations in clinical practice. No literature has studied the quality standards of Tianzhuzi formula granules.

[0003] In order to ensure the uniformity and stability of the quality of Gastrodia dahurica slices, standard decoctions, and formula granules, a new characteristic spectrum method is needed to control its quality. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic spectra of Gastrodia elata medicinal material pieces, standard decoctions, and formula granules. The method of the present invention is accurate and reliable.

[0005] The present invention provides a method for constructing HPLC characteristic spectra of Radix Gastrodiae decoction pieces, standard decoctions and formula granules, comprising:

[0006] A) Dissolve the test sample raw material with a solvent, extract, and obtain a test solution;

[0007] B) the test solution is measured by high performance liquid chromatography to obtain HPLC characteristic spectra of the Chinese baicalensis decoction pieces, standard decoction and formula granules;

[0008] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an acetonitrile solution, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the gradient elution is performed;

[0009] The gradient elution is specifically:

[0010] 0-8min, phase A: 6%-8%, phase B: 94-92%;

[0011] 8-22min, phase A: 8%-12%, phase B: 92%-88%;

[0012] 22-46 min, phase A: 12%-18%, phase B: 88%-82%;

[0013] 46-65min, phase A: 18%-36%, phase B: 82%-64%;

[0014] 65-70min, phase A: 36%-40%, phase B: 64%-60%.

[0015] The test sample raw material of the present invention is one or more of the following: Radix Gastrodiae, Radix Gastrodiae decoction pieces, Radix Gastrodiae standard decoction or Radix Gastrodiae formula granules.

[0016] Provided is a method for constructing HPLC characteristic spectra of Gastrodia elata medicinal material slices, standard decoctions and formula granules. Firstly, the test sample raw material is dissolved and extracted with a solvent to obtain a test solution.

[0017] When the raw materials are guava seeds and guava slices, step A) is to decoct the guava seeds and guava slices in water, filter, dissolve the residue in 50% methanol, ultrasonicate, cool, and filter to obtain a test solution; the ratio of the mass g of the test raw material, the volume mL of water, and the volume mL of 50% methanol is 0.5:25:50; and the decoction time is 30 min.

[0018] When the raw materials are guava seeds granules and guava seeds decoction, step A) is to ultrasonically treat the guava seeds granules and guava seeds decoction with 50% methanol, cool, and filter to obtain a test solution;

[0019] The power of the ultrasound is 600W, the frequency is 40kHz, and the ultrasound time is 30min.

[0020] The ratio of the mass g of the test sample raw material to the volume mL of the solvent is 0.1:50.

[0021] The above raw materials of the present invention can all be subjected to quality control and qualitative and quantitative detection by the method of the present invention.

[0022] The present invention also includes preparing chlorogenic acid, dissolving it in 50% methanol to obtain a reference solution;

[0023] The present invention also includes preparing a control medicinal material solution: the control medicinal material of dahurian baicalensis is decocted in water, filtered, the residue is dissolved in 50% methanol, ultrasonicated, cooled, filtered, and a test solution is obtained; the mass g of the test material, the volume mL of water, and the volume mL of 50% methanol are 1:50:10; the decocting time is 30 minutes; the ultrasonic power is 600W, the frequency is 40kHz; and the ultrasonic time is 30 minutes.

[0024] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the herbal pieces of Gastrodia elata, standard decoction and formula granules are qualitatively analyzed according to the chromatogram of the reference.

[0025] The concentration of the reference solution of the present invention is preferably as follows: chlorogenic acid is 50 μg / mL.

[0026] The test solution is measured by high performance liquid chromatography to obtain HPLC characteristic spectra of the herbal pieces of Gastrodia elata, standard decoction and formula granules.

[0027] The chromatographic conditions of the high performance liquid chromatography method are: the chromatographic column is a C18 column; the specification of the chromatographic column is 250×4.6mm 5μm; the column temperature is 5°C; under the above chromatographic column temperature, the chromatographic peaks of the present invention are symmetrical, the separation is good, and the peaks are more complete.

[0028] The chromatographic columns of the present invention include but are not limited to: ChromstarTM C18 250×4.6mm5μm; ZORBAX SB-Aq 250×4.6mm5μm; C18250×4.6mm5μm.

[0029] The mobile phase A of the invention is acetonitrile solution, the mobile phase B is 0.1% phosphoric acid aqueous solution, and the gradient elution is performed.

[0030] Specifically, the gradient elution is specifically:

[0031] 0-8min, phase A: 6%-8%, phase B: 94-92%;

[0032] 8-22min, phase A: 8%-12%, phase B: 92%-88%;

[0033] 22-46 min, phase A: 12%-18%, phase B: 88%-82%;

[0034] 46-65min, phase A: 18%-36%, phase B: 82%-64%;

[0035] 65-70min, phase A: 36%-40%, phase B: 64%-60%.

[0036] The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 5000.

[0037] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0038] The flow rate of the mobile phase of the present invention is 1.0 mL / min.

[0039] The present invention found that the chromatographic peaks were well separated and the separation degree was moderate under the above flow rate, which is the most preferred solution.

[0040] The injection volume was 10 μL.

[0041] The detection wavelength is 300 nm. The inventors found that at 300 nm, the chromatographic peak information volume is larger, the chromatogram baseline is more stable, and the peak area of ​​each peak is larger.

[0042] The present invention adopts a Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of HPLC characteristic spectra of Gastrodia elata medicinal material slices, standard decoctions, and formula granules, and obtains HPLC standard characteristic spectra of Gastrodia elata medicinal material slices, standard decoctions, and formula granules consisting of 10 characteristic peaks, wherein Peak 2: 5-hydroxymethylfurfural; Peak 3: neochlorogenic acid; Peak 4 (S): chlorogenic acid; Peak 5: cryptochlorogenic acid.

[0043] In the characteristic spectrum of the Tianzhuzi formula granules, cinnamic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.30 (peak 1), 0.39 (peak 2), 0.65 (peak 3), 1.12 (peak 5), 1.39 (peak 6), 1.67 (peak 7), 2.54 (peak 8), 2.60 (peak 9), and 2.70 (peak 10).

[0044] The present invention provides a method for identifying characteristic spectra of Radix Gastrodiae dasyphyllae medicinal material pieces, standard decoctions and formula granules, and adopts the above-mentioned method to perform detection and analyze the detection results.

[0045] The invention provides a method for constructing an HPLC characteristic spectrum of a Chinese baicalensis medicinal material slice, a standard decoction and a formula granule, comprising: A) dissolving a test sample raw material with a solvent, extracting, and obtaining a test solution; B) determining the test solution with a high performance liquid chromatography method, and obtaining the HPLC characteristic spectrum of the Chinese baicalensis medicinal material slice, the standard decoction and the formula granule; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; a mobile phase A is an acetonitrile solution, a mobile phase B is a 0.1% phosphoric acid aqueous solution, Gradient elution; the gradient elution is specifically: 0-8min, phase A: 6%-8%, phase B: 94-92%; 8-22min, phase A: 8%-12%, phase B: 92%-88%; 22-46min, phase A: 12%-18%, phase B: 88%-82%; 46-65min, phase A: 18%-36%, phase B: 82%-64%; 65-70min, phase A: 36%-40%, phase B: 64%-60%. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution, takes chlorogenic acid as a reference, establishes the HPLC characteristic spectrum method of Tianzhuzi medicinal material slices, standard decoctions, and formula granules, and provides more and more scientific technical means for controlling the medicinal quality of Tianzhuzi medicinal material slices, standard decoctions, and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1Chromatogram of Tianzhuzi granules;

[0047] Figure 2 Investigation of different column temperatures for Tianzhuzi formula granules;

[0048] Figure 3 Flow rate investigation;

[0049] Figure 4 Study on the delayed release of Tianzhuzi formula granules;

[0050] Figure 5 Investigation of extraction methods;

[0051] Figure 6 Investigation of extraction solvents;

[0052] Figure 7 Investigation of solvent addition amount;

[0053] Figure 8 Extraction time investigation;

[0054] Fig. 9 Chromatographic peak identification;

[0055] Fig.10 Different instruments;

[0056] Fig.11 Different chromatographic columns;

[0057] Fig.12 Comparative characteristic spectrum of Tianzhuzi formula granules;

[0058] Fig.13 Verification diagram of characteristic spectra of 3 batches of Tianzhuzi formula granules;

[0059] Fig.14 Comparative characteristic spectrum of Chinese baicalensis medicinal materials;

[0060] Fig.15 Characteristics of the medicinal material of Fructus Tiliae;

[0061] Fig.16 Comparative characteristic spectrum of Tianzhuzi decoction pieces;

[0062] Fig.17 Characteristic spectrum of Tianzhuzi decoction pieces;

[0063] Fig.18 Comparative characteristic spectrum of standard decoction of Fructus Gastrodiae;

[0064] Fig.19 Characteristic spectrum of standard decoction of Fructus Gastrodiae;

[0065] Fig. 20 It is the result diagram of comparative example 1;

[0066] Fig.21 It is the result diagram of comparative example 2;

[0067] Fig. 22 This is the result diagram of Comparative Example 3. DETAILED DESCRIPTION

[0068] The present invention provides a method for constructing HPLC characteristic spectra of Tianzhuzi medicinal material slices, standard decoctions, and formula granules. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0069] To further illustrate the present invention, a method for constructing an HPLC characteristic spectrum of a Fructus Gastrodiae decoction piece, a standard decoction, and a formula granule provided by the present invention is described in detail below in conjunction with examples.

[0070] Example 1

[0071] 1.1 Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 300 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 5000.

[0072]

[0073] Preparation of reference solution Take 1g of Fructus dasyphyllae, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 10ml of methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 50% methanol to make a solution containing 50μg per 1ml, as the reference solution of the reference substance.

[0074] Preparation of the test solution: Take 0.1 g of Tianzhuzi granules, grind them into powder, put them in a stoppered conical flask, add 50 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0075] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0076] 1.2 Experimental instruments and materials

[0077] High performance liquid chromatograph: Shimadzu LC-20AD high performance liquid chromatograph, Agilent 1260 high performance liquid chromatograph, Waterse2695 high performance liquid chromatograph;

[0078] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0079] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0080] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0081] Chromatographic columns: ChromstarTM C18 250×4.6mm5μm; ZORBAX SB-Aq 250×4.6mm5μm; C18250×4.6mm5μm;

[0082] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade.

[0083] Chlorogenic acid (China Food and Drug Inspection Institute, batch number: 110753-202119, purity: 96.3%);

[0084] Neochlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDX001504, purity: 98.80%),

[0085] Chlorogenic acid (Chengdu Desit Biotechnology Co., Ltd., batch number: DST220104-035, purity: 99.30%),

[0086] 5-Hydroxymethylfurfural reference substance (China Food and Drug Inspection Institute, batch number: 111626-202316, purity: 98.4%);

[0087] Fructus dianthus reference drug (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYT005901);

[0088] Tianzhuzi formula granules: TZZ-KL-01, TZZ-KL-02, TZZ-KL-03;

[0089] Tianzhuzi medicinal materials: TZZ-YC-01, TZZ-YC-02, TZZ-YC-03, TZZ-YC-04, TZZ-YC-05, TZZ-YC-06, TZZ-YC-07, TZZ-YC-08, TZZ-YC-09, TZZ-YC-10, TZ Z-YC-11, TZZ-YC-12, TZZ-YC-13, TZZ-YC-14, TZZ-YC-15, TZZ-YC-16, TZZ-YC-17, TZZ-YC-18, TZZ-YC-19, TZZ-YC-20, TZZ-YC-21;

[0090] Tianzhuzi decoction pieces: TZZ-YP-01, TZZ-YP-02, TZZ-YP-03, TZZ-YP-04, TZZ-YP-05, TZZ-YP-06, TZZ-YP-07, TZZ-YP-08, TZZ-YP-09, TZZ-YP-10, TZ Z-YP-11, TZZ-YP-12, TZZ-YP-13, TZZ-YP-14, TZZ-YP-15, TZZ-YP-16, TZZ-YP-17, TZZ-YP-18, TZZ-YP-19, TZZ-YP-20, TZZ-YP-21;

[0091] Tianzhuzi standard decoction: TZZ-BT-01, TZZ-BT-02, TZZ-BT-03, TZZ-BT-04, TZZ-BT-05, TZZ-BT-06, TZZ-BT-07, TZZ-BT-08, TZZ-BT-09, TZZ-BT-10, T ZZ-BT-11, TZZ-BT-12, TZZ-BT-13, TZZ-BT-14, TZZ-BT-15, TZZ-BT-16, TZZ-BT-17, TZZ-BT-18, TZZ-BT-19, TZZ-BT-20, TZZ-BT-21.

[0092] The medicinal materials come from Hubei, Anhui and Zhejiang; the decoction pieces, standard soups and granules are produced by Xinlv.

[0093] 1.2.1 Determination of detection wavelength point

[0094] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution at wavelengths of 220nm, 240nm, 260nm, 280nm, 300nm, 320nm, and 340nm were extracted respectively. Figure 1 . Figure 1 Chromatogram of Tianzhuzi granules.

[0095] The results showed that when the detection wavelength was 300 nm, the chromatographic peak had more information and the chromatogram baseline was more stable, so the detection wavelength was determined to be 300 nm.

[0096] 1.2.2 Column temperature investigation

[0097] Based on the experimental conditions proposed in 1.1 above, the column temperatures of 20℃, 25℃, and 30℃ were investigated. Figure 2 As shown in Table 1. Figure 2 Investigation on different column temperatures for formula granules of Gastrodia elata.

[0098] Table 1 Column temperature investigation - characteristic peak relative retention time ratio

[0099]

[0100] The results of column temperature investigation showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of Tianzhuzi formula granules.

[0101] 1.2.3 Flow rate investigation

[0102] Based on the experimental conditions proposed in 1.1 above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 3 As shown in Table 2. Figure 3 Flow rate inspection.

[0103] Table 2 Flow rate-relative retention time

[0104]

[0105] The results show that when the flow rate is 1.0 ml / min, the chromatogram peak shape is better and the separation is moderate. Therefore, the flow rate is determined to be 1.0 ml / min.

[0106] 1.2.4 Delay test

[0107] Based on the above proposed experimental conditions, the chromatogram acquisition time was extended to 70 minutes. Figure 4 shown. Figure 4 Study on the delayed release of formula granules of Gastrodia elata.

[0108] The results showed that the sample had almost no chromatographic peak after 70 minutes, so the sample detection time was set at 70 minutes.

[0109] 1.2.5 Investigation of extraction methods

[0110] Take 0.1g of the formula granules (batch number: TZZ-KL-01), grind them into powder, put them into a stoppered conical flask, and examine the extraction methods of the test sample when they are refluxed and ultrasonic respectively. The extraction time is 30 minutes, cool, shake well, filter, and take the filtrate to obtain, such as Figure 5 shown. Figure 5 Investigation of extraction methods.

[0111] The results showed that the ultrasonic extraction and reflux extraction of the test sample had the same effect. Because the ultrasonic extraction operation is simpler, the ultrasonic extraction method was determined to be the extraction method of the test sample.

[0112] 1.2.6 Investigation of extraction solvent

[0113] Take 0.1 g of the formula granules (batch number: TZZ-KL-01), grind them into powder, put them into a stoppered conical flask, add 25 ml of methanol, 70% methanol, 50% methanol, 30% methanol, ethanol, 50% ethanol, and water, respectively, and treat them with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain, such as Figure 6 shown. Figure 6 Investigation of extraction solvents.

[0114] The results showed that when the extraction solvent was 50% methanol, the peak shapes of the characteristic peaks were good and the separation was moderate. The extraction solvent was temporarily set to 50% methanol.

[0115] 1.2.7 Investigation of solvent addition amount

[0116] Take 0.1g of the formula granules (batch number: TZZ-KL-01), grind them into powder, put them into a stoppered conical flask, add 15ml, 25ml, 50ml, and 100ml of 50% methanol respectively, and treat them with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain, such as Figure 7 shown. Figure 7 The amount of solvent added was investigated; the results showed that when the amount of extraction solvent added was 50 ml, the peak shapes and separation of each chromatographic peak were better, so 50 ml of solvent was selected.

[0117] 1.2.8 Extraction time investigation

[0118] Take 0.1g of the formula granules (batch number: TZZ-KL-01), grind them into powder, put them into a stoppered conical flask, add 50ml of 50% methanol, and treat them with ultrasound (power 600W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively, shake well, filter, and take the filtrate to obtain the product. Figure 8 . Figure 8 Extraction time investigation.

[0119] The results showed that when the extraction time was 30 minutes, the chromatogram peak shape and separation were good, and the extraction was complete. Therefore, the extraction time was determined to be 30 minutes.

[0120] 1.2.9 Determine the preparation method of the test product

[0121] Take 0.1 g of the formula granules, grind them into powder, put them in a stoppered conical flask, add 50 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0122] 1.3 Methodological Review

[0123] 1.3.1 Chromatographic peak identification

[0124] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Gastrodia elata formula granules.

[0125] Preparation of reference medicinal material solution: Take 1g of Tianzhuzi reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate to dryness, add 10ml of methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, and 5-hydroxymethylfurfural reference substances, accurately weigh, and add methanol to make a solution containing 50μg per 1ml as the reference substance solution.

[0126] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the formula granules lacking Gastrodia elata.

[0127] The characteristic spectrum peaks of the Tianzhuzi formula granules were located. Fig. 9 shown. Fig. 9 Chromatographic peak identification. Fig. 9 It can be seen that peak 2 is 5-hydroxymethylfurfural, peak 3 is neochlorogenic acid, peak 4 is chlorogenic acid, and peak 5 is cryptochlorogenic acid.

[0128] 1.3.2 Precision test

[0129] Take the test solution of Tianzhuzi formula granules, inject 6 times continuously according to the proposed experimental method, 10 μL each time, and calculate the retention time and peak area of ​​each characteristic peak. See Table 3 and Table 4.

[0130] Table 3 Precision investigation-retention time

[0131]

[0132]

[0133] The results showed that the RSD of the retention time of each characteristic peak was 0.01%-0.04%, and the RSD of the peak area was 0.61%-1.94%. This method has good precision.

[0134] 1.3.3 Repeatability study

[0135] 6 portions of Tianzhuzi formula granules were accurately weighed and prepared and tested according to the proposed experimental method.

[0136] Table 4 Repeatability study - relative retention time

[0137]

[0138] The results showed that the relative retention time RSD of each characteristic peak was 0.01%-0.06%, and the relative peak area RSD was 0.51%-1.49%. This method has good repeatability.

[0139] 1.3.4 Intermediate precision study

[0140] 1.3.4.1 Investigation with different instruments

[0141] Based on the experimental conditions proposed above, three portions of the formula granules of Fructus Gastrodiae were accurately weighed to prepare the test solution, which was then measured on Agilent 1260, Shimadzu LC-20AD, and Waters e2695 high performance liquid chromatographs. Fig.10 , as shown in Table 5. Fig.10 Different instruments.

[0142] Table 5 Instrument durability investigation - relative retention time ratio

[0143]

[0144] The results showed that when the three instruments were used to detect the test products, the RSD range of the relative retention time of each characteristic peak was 0.56%-6.29%, and the durability of the instruments was good.

[0145] 1.3.4.2 Investigation by different personnel and time

[0146] Based on the experimental conditions proposed above, different personnel (A, B) weighed two portions of the formula granules of Gynura ovata at different times (T1, T2) to prepare the test samples for determination, as shown in Table 6.

[0147] Table 6 Personnel and time inspection-relative retention time

[0148]

[0149]

[0150] The results show that the method is stable when different people measure the same sample at different times.

[0151] 1.3.4.3 Durability test

[0152] 1.3.4.3.1 Column durability test

[0153] On the basis of the experimental conditions proposed above, the chromatographic columns were ChromstarTMC18 250×4.6mm5μm; ZORBAX SB-Aq 250×4.6mm5μm; C18 250×4.6mm5μm.

[0154] Conduct an investigation. Fig.11 , as shown in Table 7. Fig.11 Different chromatographic columns.

[0155] Table 7 Column durability investigation - relative retention time

[0156]

[0157] Depend on Fig.11 It can be seen that the chromatographic peak separation of each chromatographic column is good and the peaks are clear, and the chromatographic columns have good durability.

[0158] 1.3.4.3.2 Stability test

[0159] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 12h, and 24h, respectively. See Table 8.

[0160] Table 8 Stability Study-Retention Time

[0161]

[0162] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.02% and 0.07%, the RSD of the peak area was between 0.77% and 1.97%, and the sample solution was stable within 24 hours.

[0163] Example 2

[0164] 2.1 Characteristic spectrum of Tianzhuzi formula granules:

[0165] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 300 nm. The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 5000.

[0166]

[0167] Preparation of reference solution Take 1g of Fructus dasyphyllae, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 10ml of methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 50% methanol to make a solution containing 50μg per 1ml, as the reference solution of the reference substance.

[0168] Preparation of test solution: Take 0.1 g of the product, grind it into powder, put it in a stoppered conical flask, add 50 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0169] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0170] The characteristic spectrum of the test sample should show 10 characteristic peaks, and the retention time should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, among which Peak 4 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.30 (peak 1), 0.39 (peak 2), 0.65 (peak 3), 1.12 (peak 5), 1.39 (peak 6), 1.67 (peak 7), 2.54 (peak 8), 2.60 (peak 9), 2.70 (peak 10). Fig.12 Comparative characteristic spectrum of Tianzhuzi formula granules; Peak 2: 5-hydroxymethylfurfural; Peak 3: neochlorogenic acid; Peak 4 (S): chlorogenic acid; Peak 5: cryptochlorogenic acid.

[0171] 2.2 Verification results of three batches of Tianzhuzi formula granules

[0172] The proposed method was used to measure the characteristic spectra of three batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.13 , as shown in Table 9. Fig.13 Verification diagrams of characteristic spectra of three batches of Tianzhuzi formula granules (from bottom to top: TZZ-KL-01, TZZ-KL-02, TZZ-KL-03).

[0173] Table 9 Relative retention time of three batches of Tianzhuzi formula granules

[0174]

[0175]

[0176] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 10 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 4 was used as the S peak, the relative retention time RSD of the characteristic peaks of the three batches of Tianzhuzi formula granules was less than 1%.

[0177] Example 3

[0178] Establishment of relative retention time limit values

[0179] The methodology investigation items and verification results are summarized in Table 10:

[0180] Table 10 Summary of RSD% results of each item of methodology Standard - relative retention time

[0181]

[0182] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0183] Final regulations: The RSD of the relative retention time of each characteristic peak meets the requirements in all the above investigations, and the method is good. The characteristic spectrum of the test product should show 10 characteristic peaks, among which the peak corresponding to the chlorogenic acid reference is the S peak.

[0184] Example 4

[0185] 4.1 Characteristic spectrum of Chinese baicalensis

[0186] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 300 nm. The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 5000.

[0187]

[0188] Preparation of reference solution Take 1g of Fructus dasyphyllae, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 10ml of methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 50% methanol to make a solution containing 50μg per 1ml, as the reference solution of the reference substance.

[0189] Preparation of the test solution: Take 0.5 g of Gastrodia elata powder (passed through No. 2 sieve), add 25 ml of water, boil for 30 minutes, filter, evaporate to dryness, add 50 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0190] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0191] The characteristic spectrum of the test sample should show 10 characteristic peaks, and the retention time should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, among which Peak 4 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.30 (peak 1), 0.39 (peak 2), 0.65 (peak 3), 1.12 (peak 5), 1.39 (peak 6), 1.67 (peak 7), 2.54 (peak 8), 2.60 (peak 9), 2.70 (peak 10). Fig.14 Comparative characteristic spectrum of Fructus dasyphyllae; Peak 2: 5-hydroxymethylfurfural; Peak 3: neochlorogenic acid; Peak 4 (S): chlorogenic acid; Peak 5: cryptochlorogenic acid.

[0192] 4.2 Results of 21 batches of Radix Glehniae medicinal materials certification

[0193] The proposed method was used to measure the characteristic spectra of 21 batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.15 , as shown in Table 11. Fig.15 Characteristic atlas of medicinal material Ganoderma lucidum.

[0194] Table 11 Relative retention time of 21 batches of Fructus dianthus

[0195]

[0196]

[0197] Example 5

[0198] 5.1 Characteristic spectrum of Chinese baicalensis decoction pieces

[0199] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 300 nm. The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 5000.

[0200]

[0201] Preparation of reference solution Take 1g of Fructus dasyphyllae, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 10ml of methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 50% methanol to make a solution containing 50μg per 1ml, as the reference solution of the reference substance.

[0202] Preparation of the test solution: Take 0.5 g of Tianzhuzi decoction powder (passed through No. 2 sieve), add 25 ml of water, boil for 30 minutes, filter, evaporate to dryness, add 50 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0203] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0204] The characteristic spectrum of the test sample should show 10 characteristic peaks, and the retention time should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, among which Peak 4 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.30 (peak 1), 0.39 (peak 2), 0.65 (peak 3), 1.12 (peak 5), 1.39 (peak 6), 1.67 (peak 7), 2.54 (peak 8), 2.60 (peak 9), 2.70 (peak 10). Fig.16 Comparative characteristic spectrum of Tianzhuzi decoction pieces; Peak 2: 5-hydroxymethylfurfural; Peak 3: neochlorogenic acid; Peak 4 (S): chlorogenic acid; Peak 5: cryptochlorogenic acid.

[0205] 5.2 Results of the 21 batches of Radix Glehniae decoction pieces certification

[0206] The proposed method was used to measure the characteristic spectra of 21 batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.17 , as shown in Table 12. Fig.17 Characteristic spectrum of Gastrodia elata decoction pieces.

[0207] Table 12 Relative retention time of 21 batches of Tianzhuzi decoction pieces

[0208]

[0209]

[0210] Example 6

[0211] 6.1 Characteristic spectrum of standard decoction of Fructus dasyphyllae

[0212] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 300 nm. The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 5000.

[0213]

[0214] Preparation of reference solution Take 1g of Fructus dasyphyllae, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 10ml of methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 50% methanol to make a solution containing 50μg per 1ml, as the reference solution of the reference substance.

[0215] Preparation of test solution

[0216] Take an appropriate amount of standard decoction of Gastrodia elata, take about 0.1g, weigh accurately, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it tightly, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain it.

[0217] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0218] The characteristic spectrum of the test sample should show 10 characteristic peaks, and the retention time should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, among which Peak 4 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.30 (peak 1), 0.39 (peak 2), 0.65 (peak 3), 1.12 (peak 5), 1.39 (peak 6), 1.67 (peak 7), 2.54 (peak 8), 2.60 (peak 9), 2.70 (peak 10). Fig.18 Comparative characteristic spectrum of standard decoction of Fructus Gastrodiae; Peak 2: 5-hydroxymethylfurfural; Peak 3: neochlorogenic acid; Peak 4 (S): chlorogenic acid; Peak 5: cryptochlorogenic acid.

[0219] 6.2 Verification results of 21 batches of standard decoction of Fructus dasyphyllae

[0220] The proposed method was used to measure the characteristic spectra of 21 batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.19 , as shown in Table 13. Fig.19 Characteristic spectrum of standard decoction of Fructus Gastrodiae.

[0221] Table 13 Relative retention time of 21 batches of standard decoction of Fructus Gastrodiae

[0222]

[0223]

[0224] Comparative Example 1

[0225] The pretreatment means are the same as those in Example 1, except that the detection conditions are different: octadecylsilane bonded silica gel is used as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 1.0 ml per minute; the column temperature is 25° C.; and the detection wavelength is 254 nm.

[0226]

[0227] Results Fig. 20 , Fig. 20 This is the result diagram of Comparative Example 1. Fig. 20 It can be seen that the chromatographic peak separation is poor, the baseline is uneven, and the peaks are incomplete.

[0228] Comparative Example 2

[0229] The pretreatment method is the same as that in Example 1, except that the detection conditions are different: octadecylsilane bonded silica gel is used as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); acetonitrile is used as mobile phase A, 0.1% formic acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 1.0 ml per minute; the column temperature is 25° C.; the detection wavelength is 254 nm

[0230]

[0231] Results Fig.21 , Fig.21 This is the result diagram of Comparative Example 2. Fig.21 It can be seen that the chromatographic peak separation is poor, the baseline is uneven, and the characteristic spectrum graphics are poor.

[0232] Comparative Example 3

[0233] The pretreatment means are the same as those in Example 1, except that the detection conditions are different: octadecylsilane bonded silica gel is used as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); acetonitrile is used as mobile phase A, 0.1% formic acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 1.0 ml per minute; the column temperature is 25° C.; and the detection wavelength is 254 nm.

[0234]

[0235]

[0236] Results Fig. 22 , Fig. 22 This is the result diagram of Comparative Example 3. Fig. 22 It can be seen that the chromatographic peak has an uneven baseline, a wide peak shape, and a poor characteristic spectrum graph.

[0237] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic spectra of Radix Gastrodiae decoction pieces, standard decoctions, and formula granules, comprising: A) Dissolve the test sample raw material with a solvent, extract, and obtain a test solution; B) the test solution is measured by high performance liquid chromatography to obtain HPLC characteristic spectra of the Chinese baicalensis decoction pieces, standard decoction and formula granules; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an acetonitrile solution, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the gradient elution is performed; The gradient elution is specifically: 0-8min, phase A: 6%-8%, phase B: 94-92%; 8-22min, phase A: 8%-12%, phase B: 92%-88%; 22-46 min, phase A: 12%-18%, phase B: 88%-82%; 46-65min, phase A: 18%-36%, phase B: 82%-64%; 65-70min, phase A: 36%-40%, phase B: 64%-60%.

2. The method according to claim 1, characterized in that The invention also includes preparing a reference solution: taking chlorogenic acid and dissolving it in 50% methanol to obtain a reference solution; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the herbal pieces of Gastrodia elata, standard decoction and formula granules are qualitatively analyzed according to the chromatogram of the reference.

3. The method according to claim 2, characterized in that The concentration of the reference solution is specifically: 50 μg / mL of chlorogenic acid.

4. The method according to claim 1, characterized in that: The chromatographic column has a specification of 250×4.6 mm 5 μm; the column temperature is 30° C.; and the theoretical plate number calculated based on the chlorogenic acid peak should be no less than 5000.

5. The method according to claim 1, characterized in that The flow rate of the mobile phase was 1.0 mL / min; the injection volume was 10 μL; and the detection wavelength was 300 nm.

6. The method according to claim 1, characterized in that The similarity of HPLC characteristic spectra of Gastrodia elata slices, standard decoction and formula granules was evaluated by using the similarity evaluation system of Chinese medicine chromatographic fingerprints, and the HPLC standard characteristic spectra of Gastrodia elata slices, standard decoction and formula granules consisting of 10 characteristic peaks were obtained, including Peak 2: 5-hydroxymethylfurfural; Peak 3: neochlorogenic acid; Peak 4 (S): chlorogenic acid; Peak 5: cryptochlorogenic acid.

7. The method according to claim 6, characterized in that In the characteristic spectra of the Gastrodia elata medicinal material slices, standard decoction, and formula granules, chlorogenic acid is used as the reference peak S peak to calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.30 (peak 1), 0.39 (peak 2), 0.65 (peak 3), 1.12 (peak 5), 1.39 (peak 6), 1.67 (peak 7), 2.54 (peak 8), 2.60 (peak 9), and 2.70 (peak 10).

8. The method according to claim 4, characterized in that The test sample raw material in step A) is one or more of the following: guava seeds, guava slices, guava standard decoction or guava formula granules; When the raw materials are guava seeds and guava slices, step A) is to decoct the guava seeds and guava slices in water, filter, dissolve the residue in 50% methanol, perform ultrasound, cool, and filter to obtain a test solution; the ratio of the mass g of the test raw material, the volume mL of water, and the volume mL of 50% methanol is 0.5:25:50; and the decoction time is 30 min.

9. The method according to claim 1, characterized in that: When the raw materials are guava seeds granules and guava seeds decoction, step A) is to ultrasonically treat the guava seeds granules and guava seeds decoction with 50% methanol, cool, and filter to obtain a test solution; The ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 30min. The ratio of the mass g of the test sample raw material to the volume mL of the solvent is 0.1:

50.

10. A method for identifying characteristic spectra of Radix Gastrodiae Pseudostellariae decoction pieces, standard decoctions, and formula granules, characterized in that: The method according to any one of claims 1 to 9 is used for detection, and the detection results are analyzed.

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