Detection methods of white earthworm products

Abstract

The present invention relates to the field of detection technology and discloses a method for detecting white earth ling products, comprising: subjecting a sample to be tested to high-performance liquid chromatography (HPLC) to obtain a fingerprint of the sample; comparing the fingerprint of the sample to be tested with a pre-established standard reference fingerprint of a white earth ling product; and performing gradient elution using a mobile phase comprising methanol and a phosphoric acid solution in the HPLC detection. The present invention utilizes suitable HPLC detection conditions to establish a fingerprint of white earth ling containing eight characteristic peaks, facilitating identification of white earth ling; and confirming that there are a large number of characteristic peaks of active ingredients, thereby providing more accurate quality detection results for white earth ling.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to a detection method for a white earthworm product. Background Art

[0002] Bai Tuling is a modern variety. It is sweet, light and flat in nature. It has the effects of removing dampness, detoxifying and unblocking joints. It is often used clinically to treat limb cramps and muscle and bone pain caused by damp-heat stranguria, leucorrhea, carbuncle and mercury poisoning.

[0003] Currently, the most commercially available white earthworm is Heterosmilax yunnanensis Gagnep., which has a significant cultivation base in Guizhou, Yunnan, and Sichuan. Due to the diverse varieties and origins of white earthworm products, product quality control is difficult. To effectively characterize the overall quality of white earthworms, it is necessary to establish a standard reference fingerprint for quality identification.

[0004] Patent document CN102608236A provides a method for detecting the medicinal material Smilax china. Using syringyl glucoside as a reference substance and acetonitrile-phosphoric acid as a mobile phase, liquid chromatography analysis is performed to construct a fingerprint of Smilax china. However, this method has poor separation efficiency and reproducibility for Smilax china, and is insufficient to fully and accurately reflect the quality characteristics of Smilax china. Summary of the Invention

[0005] To achieve this object, the present invention provides a method for detecting white earthworm products with better separation effect and repeatability, so as to achieve comprehensive and accurate identification of their quality.

[0006] The detection method of the white earthworm product of the present invention comprises the following steps:

[0007] The sample to be tested is subjected to high performance liquid chromatography (HPLC) to obtain a fingerprint of the sample to be tested;

[0008] The fingerprint of the sample to be tested is compared with the fingerprint of the standard control of the white earthworm product established in advance;

[0009] In the high performance liquid chromatography detection, the mobile phase includes methanol and phosphoric acid solution, and gradient elution is performed.

[0010] Preferably, the concentration of the phosphoric acid solution is 0.05-0.15%.

[0011] Preferably, the concentration of the phosphoric acid solution is 0.1%.

[0012] Preferably, the gradient elution is performed as follows:

[0013] 0-12min, the volume percentage of the mobile phase B is 100%;

[0014] 12-18 min, the volume percentage of the mobile phase B decreased from 100% to 96%;

[0015] 18-24min, the volume percentage of the mobile phase B decreased from 96% to 90%;

[0016] 24-36min, the volume percentage of the mobile phase B decreased from 90% to 87%;

[0017] 36-55min, the volume percentage of the mobile phase B is 87%.

[0018] Preferably, the wavelength of the HPLC detection is 220-360 nm.

[0019] Preferably, the wavelength of HPLC detection is 254 nm.

[0020] Preferably, the flow rate of the high performance liquid chromatography detection is 0.8-1.2 ml / min.

[0021] Preferably, the flow rate of HPLC detection is 1.0 ml / min.

[0022] Preferably, the chromatographic column for high performance liquid chromatography detection uses octadecylsilane bonded silica gel as a filler.

[0023] Preferably, the chromatographic column has a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 3.5 μm.

[0024] Preferably, the column temperature of the chromatographic column is 20-35°C.

[0025] Preferably, the column temperature of the chromatographic column is 30°C.

[0026] Preferably, the injection volume for HPLC detection is 10 μl; and the detection time is 55 min.

[0027] The present invention detects the sample to be tested according to the above-mentioned high performance liquid chromatography detection conditions to obtain a fingerprint of the sample to be tested. Preferably, the sample to be tested is dissolved in a methanol aqueous solution, ultrasonically extracted, filtered, and the filtrate is taken and subjected to high performance liquid chromatography detection.

[0028] Preferably, the power of ultrasonic extraction is 600W and the frequency is 40kHz.

[0029] Preferably, the concentration of the methanol aqueous solution is 30%-80%, preferably 30%.

[0030] Preferably, the amount of methanol aqueous solution added is 25-100 ml, preferably 25 ml.

[0031] Preferably, the ultrasonic extraction time is 30-90 min, preferably 30 min.

[0032] The present invention pre-establishes a standard control fingerprint of a white earthworm product, comprising the following steps:

[0033] Dissolve multiple white earthworm products separately to obtain multiple test solution;

[0034] The reference substance and each test solution were subjected to high performance liquid chromatography to obtain fingerprints of the reference substance and multiple white earthworm products;

[0035] According to the fingerprints of the reference object and multiple white earthworm products, the fingerprints of the multiple white earthworm products are synthesized to obtain a standard control fingerprint of the white earthworm product.

[0036] Specifically, the method for preparing the test solution of the Bai Tu Ling product is the same as that for the test sample, which will not be repeated here. According to the conditions of the above-mentioned high-performance liquid chromatography detection, the reference material and each test solution are subjected to high-performance liquid chromatography detection. Based on the fingerprints of the reference material and multiple Bai Tu Ling products, the common characteristic peaks in the fingerprints are calibrated. The fingerprints of multiple Bai Tu Ling products are synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) to establish a standard control fingerprint of the Bai Tu Ling product.

[0037] In some embodiments provided by the present invention, the standard reference fingerprint of the white earthworm product contains 8 characteristic peaks, wherein peak 2 is protocatechuic acid, peak 4 is 4-hydroxybenzoic acid, peak 7 is vanillic acid, and peak 8 is syringic acid glucoside.

[0038] Preferably, the Bai Tuling products include Bai Tuling formula granules, Bai Tuling standard decoction, Bai Tuling medicinal materials or Bai Tuling decoction pieces.

[0039] The present invention establishes different standard control fingerprints for each type of Bai Tuling products, specifically including: the standard control fingerprint of Bai Tuling formula granules, the standard control fingerprint of Bai Tuling standard decoction, the standard control fingerprint of Bai Tuling medicinal materials and the standard control fingerprint of Bai Tuling decoction pieces. The establishment methods are similar and will not be repeated in the present invention.

[0040] Preferably, the reference substances are protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside.

[0041] In some embodiments provided herein, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside are added to methanol to obtain a mixed solution containing 30 μg of each of protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside per 1 ml, and high performance liquid chromatography detection is performed.

[0042] It can be seen from the above technical solutions that the present invention has the following advantages:

[0043] The present invention adopts appropriate high-performance liquid chromatography detection conditions to establish a standard control fingerprint of a white earth ling product containing 8 characteristic peaks, which is convenient for identifying the white earth ling product; and can confirm that there are more characteristic peaks of effective ingredients, so that the quality detection result of the white earth ling product is more accurate; methanol-phosphoric acid solution is selected as the mobile phase to ensure that the separation degree of the white earth ling product is appropriate, and a chromatogram with higher peaks and better separation effect can be obtained.

[0044] In addition, the detection method of the Bai Tuling products of the present invention has good repeatability and is applicable to different batches of Bai Tuling medicinal materials, Bai Tuling decoction pieces, Bai Tuling standard decoctions and Bai Tuling formula granules. It has a wide range of applications and can provide a comprehensive and accurate detection basis for the quality identification of Bai Tuling products. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0046] Figure 1 This is the 3D chromatogram of Bai Tuling formula granules at different detection wavelengths;

[0047] Figure 2 This is the chromatogram of Bai Tuling formula granules at different detection wavelengths;

[0048] Figure 3 This is the chromatogram of Bai Tuling formula granules at different column temperatures;

[0049] Figure 4 This is the chromatogram of Bai Tuling formula granules at different flow rates;

[0050] Figure 5 The chromatograms of Bai Tuling formula granules at different measurement times;

[0051] Figure 6 This is the chromatogram of Bai Tuling formula granules extracted by different methods;

[0052] Figure 7 This is the chromatogram of Bai Tuling formula granules extracted with different solvents;

[0053] Figure 8 This is the chromatogram of Bai Tuling formula granules at different extraction times;

[0054] Figure 9 This is the chromatogram of Bai Tuling formula granules when different amounts of solvent were added;

[0055] Figure 10 The chromatograms of Bai Tuling formula granules, Bai Tuling reference medicinal materials and reference substances are shown;

[0056] Figure 11A This is the spectrum of syringyl glucoside; Figure 11B is the spectrum of 4-hydroxybenzoic acid; Figure 11C is the spectrum of protocatechuic acid; Figure 11D is the spectrum of vanillic acid; Figure 11E This is the spectrum of methyl azoxymethanol primrose glycoside;

[0057] Figure 12 This is the chromatogram of Bai Tuling formula granules measured by different instruments;

[0058] Figure 13 The fingerprints of three batches of Bai Tuling granules;

[0059] Figure 14 It is the comparison map of the fingerprint of Bai Tuling formula granules;

[0060] Figure 15 This is the 3D chromatogram of Bai Tuling standard decoction at different detection wavelengths;

[0061] Figure 16 The chromatograms of Bai Tuling standard decoction at different detection wavelengths;

[0062] Figure 17 This is the chromatogram of Bai Tuling standard decoction at different column temperatures;

[0063] Figure 18 This is the chromatogram of Bai Tuling standard decoction at different flow rates;

[0064] Figure 19 The chromatograms of Bai Tuling standard decoction at different measurement times;

[0065] Figure 20 This is the chromatogram of Bai Tuling standard decoction extracted in different ways;

[0066] Figure 21 This is the chromatogram of Bai Tuling standard decoction with different extraction solvents;

[0067] Figure 22 This is the chromatogram of the white turmeric standard decoction at different extraction times;

[0068] Figure 23 This is the chromatogram of the extraction solvent of Bai Tuling standard decoction with different addition amounts;

[0069] Figure 24 The chromatograms of Bai Tuling standard decoction, Bai Tuling reference medicinal material and reference substance are shown;

[0070] Figure 25 The chromatograms of Bai Tuling standard decoction measured by different instruments;

[0071] Figure 26 The fingerprints of 21 batches of Bai Tuling standard decoctions;

[0072] Figure 27 It is the comparison map of the fingerprint of the standard decoction of Bai Tuling;

[0073] Figure 28 This is the 3D chromatogram of Bai Tuling medicinal material at different detection wavelengths;

[0074] Figure 29 This is the chromatogram of Bai Tuling medicinal material at different detection wavelengths;

[0075] Figure 30 The chromatograms of Bai Tuling standard decoction at different measurement times;

[0076] Figure 31 This is the chromatogram of Bai Tuling medicinal material when it is extracted in different ways;

[0077] Figure 32 The chromatograms of Bai Tuling medicinal material, Bai Tuling reference medicinal material and reference substance;

[0078] Figure 33 This is the chromatogram of Bai Tuling medicinal material measured by different instruments;

[0079] Figure 34 The fingerprints of 21 batches of Bai Tuling medicinal materials;

[0080] Figure 35 The fingerprints of 21 batches of Bai Tuling decoction pieces;

[0081] Figure 36 It is the comparison map of Bai Tuling medicinal material fingerprint map;

[0082] Figure 37 It is the comparison map of the fingerprint of Bai Tuling decoction pieces;

[0083] Figure 38 The fingerprints of Bai Tuling formula granules, Bai Tuling standard decoction, Bai Tuling medicinal materials and Bai Tuling decoction pieces are compared;

[0084] Figure 39 This is a comparison chart of the elution effects of methanol and acetonitrile as mobile phase A;

[0085] Figure 40 This is the elution effect diagram of methanol as mobile phase A;

[0086] Figure 41 This is the elution effect diagram when acetonitrile is used as mobile phase A. DETAILED DESCRIPTION

[0087] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0088] Example 1

[0089] In this example, a standard reference fingerprint of Bai Tu Ling formula granules was established and tested. A chromatographic column was filled with octadecylsilane bonded silica gel, with methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, using gradient elution as specified in the table below. The flow rate was 1.0 ml per minute, the column temperature was 30°C, and the detection wavelength was 254 nm. The number of theoretical plates, calculated based on the syringyl glucoside peak, must not be less than 5000.

[0090]

[0091] Preparation of the reference solution: Take 1g of the Bai Tu Ling reference medicinal material, add 50ml of water, boil for 30 minutes, cool, filter, and evaporate the filtrate to dryness. Add 25ml of 30% methanol to the residue and sonicate (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and use the filtrate as the reference medicinal material solution. Separately, take an appropriate amount of the syringyl glucoside reference substance, accurately weigh it, and add methanol to a solution containing 30μg per 1ml. This will serve as the reference substance solution.

[0092] Preparation method of the test solution: Take an appropriate amount of Bai Tuling formula granules, grind them into powder, take about 0.2g, accurately weigh, place in a stoppered conical flask, accurately add 25ml of 30% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0093] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0094] The test solution chromatogram should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.15 (peak 1), 0.42 (peak 2), 0.48 (peak 3), 0.63 (peak 4), 0.76 (peak 5), 0.87 (peak 6), and 0.96 (peak 7).

[0095] The method of this embodiment is screened, and the specific process is as follows:

[0096] 1. Instruments and test drugs

[0097] Electronic balances: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.); Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.); Ultrasonic cleaner: KQ5200DB (600 W, 40 kHz; Kunshan Ultrasonic Instrument Co., Ltd.); Methanol (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.); Laboratory-made ultrapure water was used for the mobile phase; all other waters were laboratory-made pure water; Protocatechuic acid (China Food and Drug Inspection Institute, batch number: 110809-201906, content calculated as 97.7%). Methyl azoxymethanol primulosyl glycoside (OST, batch number: U40-1309310-02, content is 98%); 4-hydroxybenzoic acid (China Food and Drug Inspection Institute, batch number: 101149-202204, content is 100%); vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, content is 99.8%); syringic acid glucoside (OST, batch number: U40-7008658-02, content is 99.5%); white turmeric reference medicinal material (China Food and Drug Inspection Institute, batch number: 121537-201502); white turmeric formula granules (Sichuan New Green Pharmaceutical).

[0098] 2. Chromatographic conditions and system suitability test

[0099] (1) Detection wavelength investigation

[0100] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 220 nm, 240 nm, 254 nm, 280 nm, 300 nm, 320 nm, 340 nm, and 360 nm were extracted respectively ( Figure 1-2 ).

[0101] The results showed that when the detection wavelength was 254 nm, the chromatographic peak information was richer, the number of chromatographic peaks was greater, and the chromatogram baseline was more stable, so the detection wavelength was determined to be 254 nm.

[0102] (2) Column temperature investigation

[0103] Under the experimental conditions proposed above, the column temperatures were investigated at 20℃, 25℃, 30℃ and 35℃ respectively. The results are shown in Figure 3 .

[0104] The results showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was good. Therefore, the detection column temperature of the fingerprint of Bai Tuling formula granules was determined to be 30℃.

[0105] (3) Flow rate investigation

[0106] Under the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min and 1.2ml / min were investigated respectively. The results are shown in Figure 4 .

[0107] The results showed that when the flow rate was 1.0 ml / min, the chromatogram peak shape was better and the separation was moderate, so the flow rate was determined to be 1.0 ml / min.

[0108] (4) Delay test

[0109] Under the experimental conditions proposed above, the chromatogram acquisition time was extended to 120 minutes, and the results were as follows: Figure 5 shown.

[0110] The results showed that the sample had basically no chromatographic peak after 55 minutes, so the measurement time was determined to be 55 minutes.

[0111] (5) Determination of chromatographic conditions and system suitability test

[0112] The chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was methanol, and the mobile phase B was 0.1% phosphoric acid solution, with gradient elution as specified in the table below. The column temperature was 30°C; the flow rate was 1.0 ml / min; the fingerprint detection wavelength was 254 nm; the injection volume was 10 μl, and the measurement time was 55 minutes. The number of theoretical plates, calculated based on the syringoside peak, must not be less than 5000.

[0113] 3. Investigation of the preparation method of test solution

[0114] (1) Investigation of extraction methods

[0115] Accurately weigh 0.2 g of Bai Tu Ling formula granules, place in a stoppered conical flask, add 10 ml of 80% methanol, reflux and ultrasonically treat (power 600 W, frequency 40 kHz) for 60 minutes, cool, shake well, filter, and take the filtrate to obtain; sample injection and determination according to the above chromatographic conditions, the results are shown in Figure 6 .

[0116] The results showed that the effects of ultrasonic extraction and reflux extraction were the same for the test sample solution. Since ultrasonic extraction was simpler to operate, ultrasonic extraction was determined to be the extraction method for the test sample.

[0117] (2) Investigation of extraction solvent

[0118] Accurately weigh 0.2 g of Bai Tu Ling formula granules, add 25 ml each of 30% methanol, 50% methanol, 80% methanol, methanol, 50% ethanol, 80% ethanol, ethanol, and water, respectively, and ultrasonically treat (power 600 W, frequency 40 kHz) for 60 minutes, let cool, shake well, filter, and take the filtrate to obtain; sample injection and determination according to the above chromatographic conditions, the results are shown in FIG. Figure 7 .

[0119] The results showed that when the extraction solvent was 30% methanol, the obtained chromatogram peak shape was better and the chromatographic peak information was larger, so the extraction solvent for the test sample was determined to be 30% methanol.

[0120] (3) Extraction time investigation

[0121] Accurately weigh 0.2 g of Bai Tuling Bai Tuling granules, place them in a stoppered conical flask, add 25 ml of 30% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, 60 minutes, and 90 minutes, respectively. Let cool, shake well, filter, and take the filtrate to obtain the product; the sample was injected and determined according to the above chromatographic conditions. The experimental results are shown in the table. Figure 8 .

[0122] The results showed that the effects were consistent when the extraction time was 30 minutes, 60 minutes, and 90 minutes, so the extraction time of the test sample was selected as 30 minutes.

[0123] (4) Investigation of the amount of extraction solvent added

[0124] Accurately weigh 0.2 g of Bai Tuling formula granules and place them in a stoppered conical flask. Add 25 ml, 50 ml, and 100 ml of 30% methanol, respectively. Ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain the product. Sample injection and determination are carried out according to the above chromatographic conditions. The experimental results are shown in FIG. Figure 9 .

[0125] The results showed that when the amount of extraction solvent added was 25 ml, the size of each chromatographic peak was appropriate, so the amount of extraction solvent added was selected to be 25 ml.

[0126] (5) Determination of the preparation method of the test solution

[0127] Take an appropriate amount of this product, grind it into powder, take about 0.2g, weigh it accurately, put it into a stoppered conical flask, accurately add 25ml of 30% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0128] 4. Methodological Investigation

[0129] 4.1 Chromatographic peak identification

[0130] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Bai Tuling formula granules.

[0131] Preparation of reference solution: Take 1 g of Bai Tuling reference medicinal material, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper it tightly, and sonicate (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate as the reference medicinal material solution;

[0132] Take appropriate amounts of protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside reference substances, accurately weigh them, and add methanol to prepare a mixed solution containing 30 μg each of protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside per 1 ml, which is used as the reference substance solution.

[0133] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Bai Tuling formula granules.

[0134] 10 μl of the test solution and reference solution were respectively injected into the high performance liquid chromatograph for sample determination; the chromatograms of Bai Tuling formula granules, Bai Tuling reference medicinal materials and reference substances were obtained, and the characteristic peaks of the fingerprint of Bai Tuling formula granules were located. The results are shown in the figure. Figure 10-1 1.

[0135] The nine common peaks that were consistent with the retention time of the Bai Tuling reference medicinal material were selected as the characteristic peaks of the fingerprint of the Bai Tuling formula granules; the characteristic peaks of the reference substance were compared with the characteristic peaks of the Bai Tuling formula granules, and according to the spectrum of the reference substance ( Figure 11A -E) for identification; the results showed that characteristic peaks with consistent retention times with methyl azoxymethanol primulosidoside, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, and syringyl glucoside could be found in the fingerprint of Bai Tuling formula granules, and peak 2 was determined to be methyl azoxymethanol primulosidoside; peak 3 was determined to be protocatechuic acid; peak 5 was determined to be 4-hydroxybenzoic acid; peak 8 was determined to be vanillic acid; and peak 9 (S) was determined to be syringyl glucoside.

[0136] In order to comprehensively and accurately establish the standard fingerprint of Bai Tuling formula granules, the 9 characteristic peaks identified in the fingerprint were investigated.

[0137] 4.2 Precision experiment

[0138] Take the Bai Tuling formula granules, grind them into powder, take 0.2g, and inject 6 times continuously according to the test solution preparation method and the proposed chromatographic conditions and detection method, each time 10μl; calculate the retention time and peak area of ​​each characteristic peak, the results are shown in Table 1 and Table 2.

[0139] Table 1 Precision investigation - characteristic peak retention time

[0140]

[0141] Table 2 Precision investigation - characteristic peak area

[0142]

[0143] The results showed that the RSD of the retention time of each characteristic peak in the precision investigation was between 0.06% and 0.34%, and the RSD of the peak area of ​​each characteristic peak was between 0.91% and 18.51%, indicating good precision.

[0144] 4.3 Repeatability study

[0145] Grind the Bai Tuling formula granules into powder, accurately weigh 6 portions, 0.2 g each, and prepare and measure them according to the proposed experimental method. Calculate the relative retention time and relative peak area of ​​each characteristic peak of the 6 samples. The results are shown in Tables 3 and 4.

[0146] Table 3 Repeatability study - relative retention time of characteristic peaks

[0147]

[0148] Table 4 Repeatability study - relative peak area of ​​characteristic peaks

[0149]

[0150] The results showed that the RSD of the characteristic peaks relative to the retention time was between 0.07% and 0.36%, and the RSD of the characteristic peaks relative to the peak area was between 5.90% and 27.46%, indicating good repeatability.

[0151] 4.4 Inspection by different personnel and time

[0152] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed 2 portions of Bai Tuling formula granules at different times (T1, T2), each 0.2 g, to prepare the test solution, conduct the measurement, and calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 5 and 6.

[0153] Table 5 Relative retention time of characteristic peaks under investigation by different personnel and time

[0154]

[0155] Table 6 Relative peak areas of characteristic peaks for different personnel and time periods

[0156]

[0157] The results showed that under different sample preparation personnel and different sample preparation time conditions, the RSDs of the relative retention times of the characteristic peaks were between 0.16% and 0.84%; the RSDs of the relative peak areas of the characteristic peaks were between 0.16% and 0.84%.

[0158] 4.5 Investigation of different instruments

[0159] Grind the Bai Tu Ling formula granules, accurately weigh 0.2 g, prepare the test solution, and measure it on three different types of high performance liquid chromatographs; calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Table 7, Table 8 and Figure 12 .

[0160] Table 7 Instrument investigation - relative retention time of characteristic peaks

[0161]

[0162] Table 8 Instrument Investigation - Relative Peak Area of ​​Characteristic Peaks

[0163]

[0164] The results showed that when detected by three different instruments, the RSDs of the relative retention times of the characteristic peaks were between 0.25% and 1.57%, and the RSDs of the relative peak areas of the characteristic peaks were between 3.92% and 30.48%, indicating that the different instruments had good durability.

[0165] 4.6 Stability investigation

[0166] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h respectively; the retention time and peak area of ​​each characteristic peak were calculated. The results are shown in Tables 9 and 10.

[0167] Table 9 24-hour stability study - characteristic peak retention time

[0168]

[0169] Table 10 24-hour stability study - characteristic peak area

[0170]

[0171]

[0172] The results showed that the RSDs of the retention times of the characteristic peaks were between 0.42% and 0.70%, and the RSDs of the peak areas of the characteristic peaks were between 1.35% and 24.11%. The test solution was stable within 24 hours.

[0173] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above nine characteristic peaks were included in the subsequent investigations.

[0174] 4.7 Investigation Results

[0175] The results of multiple surveys are summarized in Tables 11 and 12.

[0176] Table 11 Summary of RSD% results for each item of the methodology - retention time / relative retention time

[0177]

[0178] Table 12 Summary of RSD% of each method result - Peak area / Relative peak area

[0179]

[0180] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method is good. Since Peak 2 showed a split peak during the investigation, indicating that Peak 2 was contained, Peak 2 was deleted, leaving Peak 1, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, and Peak 9, a total of 8 characteristic peaks, as the characteristic peaks of the fingerprint of Bai Tuling Formula Granules; Peak 2 is protocatechuic acid; Peak 4 is 4-hydroxybenzoic acid; Peak 7 is vanillic acid; and Peak 8 (S) is syringoyl glucoside.

[0181] 4.8 Determination of characteristic peaks and establishment of standard reference patterns

[0182] (1) Verification results of three batches of Bai Tuling formula granules

[0183] The proposed method was used to determine the fingerprint of three batches of Bai Tu Ling formula granules; the relative retention time and relative peak area of ​​each characteristic peak of the three batches of Bai Tu Ling formula granules were calculated. The results are shown in Table 13, Table 14 and Figure 13 .

[0184] Table 13 Relative retention time of three batches of Bai Tuling formula granules

[0185]

[0186]

[0187] Table 14 Relative peak areas of three batches of Bai Tuling granules

[0188]

[0189] Based on the principles of stable relative retention time, detectable characteristic peaks in all batches of samples, and relatively high peak values ​​of characteristic peaks, a total of 8 peaks with good durability, namely Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, and Peak 8, were selected as characteristic peaks; based on the results of the methodological investigation and the verification results of 3 batches of Bai Tuling formula granules, the theoretical plate number calculated based on the syringic acid glucoside peak was tentatively determined to be no less than 5000.

[0190] (2) Establishment of relative retention time limit values

[0191] The summary of the methodology inspection items and verification results are shown in Tables 15-16:

[0192] Table 15 Summary of RSD% results for each item of the methodology - relative retention time / retention time

[0193]

[0194] Table 16 Summary of RSD% of each item results of the method - Peak area / Relative peak area

[0195]

[0196] The relative retention time of each characteristic peak is stable, and the relative retention time values ​​are all within the range of ±10% of the average value, so the specified value range of the relative retention time of each peak is temporarily set at ±10%; the relative peak areas of each batch vary too much to be specified, so the relative peak areas are not included in the main text.

[0197] Final regulations: The fingerprint of the Bai Tu Ling formula granules in this example should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside peak is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are as follows: Peak 1 is 0.15, Peak 2 is 0.42, Peak 3 is 0.48, Peak 4 is 0.63, Peak 5 is 0.76, Peak 6 is 0.87, and Peak 7 is 0.96.

[0198] The fingerprints of three batches of Bai Tuling formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) and a standard reference fingerprint of Bai Tuling formula granules was established. The results are shown in Figure 14 .

[0199] Conclusion: The fingerprint detection method of Bai Tuling formula granules in this example can accurately analyze and detect Bai Tuling formula granules.

[0200] 5. Determination of the detection method for the fingerprint of Bai Tuling formula granules

[0201] (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; methanol was used as the mobile phase A; 0.1% phosphoric acid solution was used as the mobile phase B; gradient elution was performed according to the requirements in the table below; the flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelength was 254 nm. The number of theoretical plates calculated based on the syringoside peak was not less than 5000.

[0202]

[0203] (2) Preparation of reference solution: Take 1 g of the reference medicinal material Bai Tu Ling, add 50 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Separately, take an appropriate amount of the reference substance syringyl glucoside, accurately weigh it, and add methanol to make a solution containing 30 μg per 1 ml, which is used as the reference solution.

[0204] (3) Preparation of test solution: Take an appropriate amount of Bai Tuling granules, grind them into powder, take about 0.2 g, accurately weigh, place in a stoppered conical flask, accurately add 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate.

[0205] (4) Detection method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure.

[0206] The test sample chromatogram should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside reference peak is the S peak. Calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.15 (peak 1), 0.42 (peak 2), 0.48 (peak 3), 0.63 (peak 4), 0.76 (peak 5), 0.87 (peak 6), and 0.96 (peak 7).

[0207] Example 2

[0208] The method for detecting the fingerprint of the Bai Tu Ling formula granules in Example 1 is also applicable to the Bai Tu Ling standard decoction. The details are as follows:

[0209] The chromatographic column was filled with octadecylsilane bonded silica gel, with methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was performed as specified in the table below. The flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelength was 254 nm. The number of theoretical plates, calculated based on the syringoside peak, must be no less than 5000.

[0210]

[0211]

[0212] Preparation of the reference solution: Take 1g of the Bai Tu Ling reference medicinal material, add 50ml of water, boil for 30 minutes, cool, filter, and evaporate the filtrate to dryness. Add 25ml of 30% methanol to the residue and sonicate (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and use the filtrate as the reference medicinal material solution. Separately, take an appropriate amount of the syringyl glucoside reference substance, accurately weigh it, and add methanol to a solution containing 30μg per 1ml. This will serve as the reference substance solution.

[0213] Preparation method of the test solution: Take about 0.2g of freeze-dried powder of Bai Tuling standard decoction, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 30% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0214] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0215] The test solution chromatogram should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.15 (peak 1), 0.42 (peak 2), 0.48 (peak 3), 0.63 (peak 4), 0.77 (peak 5), 0.88 (peak 6), and 0.95 (peak 7).

[0216] The method of this embodiment is screened, and the specific process is as follows:

[0217] 1. Instruments and test drugs

[0218] Electronic balances: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.); Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.); Ultrasonic cleaner: KQ5200DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0219] Methanol (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), and water used in the mobile phase were laboratory-made ultrapure water, and the rest were laboratory-made pure water. Protocatechuic acid (China Food and Drug Inspection Institute, batch number: 110809-201906, content is 97.7%); methyl azoxymethanol primulosidoside (OST, batch number: U40-1309310-02, content is 98%); 4-hydroxybenzoic acid (China Food and Drug Inspection Institute, batch number: 101149-202204, content is 100%); vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, content is 99.8%); syringyl glucoside (OST, batch number: U40-7008658-02, content is 99.5%); white turmeric reference medicinal material (China Food and Drug Inspection Institute, batch number: 121537-201502);

[0220] The freeze-dried powder of the standard decoction of Bai Tuling used in this embodiment was prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd. (Batch No.: BTLBT01, BTLBT02, BTLBT03, BTLBT04, BTLBT05, BTLBT06, BTLBT07, BTLBT08, BTLBT09, BTLBT10, BTLBT11, BTLBT12, BTLBT13, BTLBT14, BTLBT15, BTLBT16, BTLBT17, BTLBT18, BTLBT19, BTLBT20, BTLBT21).

[0221] 2. Chromatographic conditions and system suitability test

[0222] (1) Detection wavelength investigation

[0223] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning on the test solution of Bai Tuling standard decoction, and the chromatograms of the test solution at wavelengths of 220nm, 240nm, 254nm, 280nm, 300nm, 320nm, 340nm, and 360nm were extracted respectively. The results are shown in Figure 15-16 .

[0224] The results showed that when the detection wavelength was 254 nm, the chromatographic peak information was richer, the number of chromatographic peaks was greater, and the chromatogram baseline was more stable, so the detection wavelength was determined to be 254 nm.

[0225] (2) Column temperature investigation

[0226] Based on the experimental conditions proposed above, the column temperatures of 20℃, 25℃, 30℃ and 35℃ were investigated respectively. Figure 17 .

[0227] The results of column temperature investigation showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was better, so 30℃ was finally determined as the column temperature for the fingerprint detection method of Bai Tuling standard decoction.

[0228] (3) Flow rate investigation

[0229] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 18 .

[0230] The results showed that when the flow rate was 1.0 ml / min, the chromatogram peak shape was better and the separation was moderate. Therefore, the flow rate was determined to be 1.0 ml / min.

[0231] (4) Delay investigation

[0232] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 120 minutes. Figure 19 shown.

[0233] The results showed that the chromatogram peaks were completely collected at 55 minutes, so the chromatogram acquisition time was determined to be 55 minutes.

[0234] (5) Determination of chromatographic conditions and system suitability test

[0235] The chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was methanol, and the mobile phase B was 0.1% phosphoric acid solution, with gradient elution as specified in the table below. The column temperature was 30°C; the flow rate was 1.0 ml / min; the fingerprint detection wavelength was 254 nm; the injection volume was 10 μl, and the measurement time was 55 minutes. The number of theoretical plates, calculated based on the syringoside peak, must not be less than 5000.

[0236]

[0237] 3. Investigation of the preparation method of test solution

[0238] (1) Investigation of extraction methods

[0239] Take about 0.2g of freeze-dried powder of Bai Tuling standard decoction, accurately weigh it, place it in a stoppered conical flask, add 10ml of 30% methanol, reflux and ultrasonicate (power 600W, frequency 40kHz) for 60 minutes respectively, let it cool, shake it well, filter it, and take the filtrate. Figure 20 .

[0240] The results showed that ultrasonic extraction and reflux extraction of the sample solution of Bai Tuling standard decoction had the same effect. Because ultrasonic extraction is more convenient, ultrasonic extraction was determined to be the extraction method for the sample.

[0241] (2) Investigation of extraction solvent

[0242] Take about 0.2g of freeze-dried powder of Bai Tuling standard decoction, add 10ml each of 30% methanol, 50% methanol, 80% methanol, 80% ethanol, 50% ethanol, methanol, ethanol, and water, respectively, and ultrasonically treat (power 600W, frequency 40kHz) for 60 minutes, let cool, shake well, filter, and take the filtrate. Figure 21 .

[0243] The results showed that when the extraction solvent was 30% methanol, the obtained chromatogram peak shape was better and the chromatographic peak information was larger, so the extraction solvent for the test sample was determined to be 30% methanol.

[0244] (3) Extraction time investigation

[0245] Take about 0.2g of freeze-dried powder of Bai Tuling standard decoction, accurately weigh it, place it in a stoppered conical flask, add 10ml of 30% methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, 60 minutes, and 90 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate. Figure 22 .

[0246] The results showed that the extraction time of 30 minutes, 60 minutes and 90 minutes for the test sample of Bai Tuling standard decoction was the same, so the extraction time of 30 minutes was selected as the test sample.

[0247] (4) Investigation of the amount of extraction solvent added

[0248] Take about 0.2g of freeze-dried powder of Bai Tuling standard decoction, accurately weigh it, place it in a stoppered conical flask, accurately add 30% methanol 5ml, 10ml, 15ml, and 25ml respectively, stopper it tightly, ultrasonicate it (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 23 .

[0249] The results showed that when the extraction solvent was 25 ml, the size of each chromatographic peak was appropriate, so the solvent volume was selected as 25 ml.

[0250] (5) Determination of the preparation method of the test solution

[0251] Take about 0.2g of freeze-dried powder of Bai Tuling standard decoction, place it in a stoppered conical flask, accurately add 25ml of 30% methanol, seal it tightly, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0252] 4. Methodological Investigation

[0253] 4.1 Chromatographic peak identification

[0254] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Bai Tuling standard decoction.

[0255] Preparation of reference solution: Take 1 g of Bai Tuling reference medicinal material, add 50 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake, filter, and take the filtrate as the reference medicinal material solution;

[0256] Take appropriate amounts of protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside reference substances, accurately weigh them, and add methanol to prepare a mixed solution containing 30 μg each of protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside per 1 ml, which is used as the reference substance solution.

[0257] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Bai Tuling standard decoction.

[0258] 10 μl of the test solution and reference solution were respectively injected into the high performance liquid chromatograph for sample determination; the chromatograms of the Bai Tuling standard decoction, Bai Tuling reference medicinal material and reference substance were obtained, and the characteristic peaks of the fingerprint of the Bai Tuling standard decoction were located. The results are shown in the figure. Figure 24 .

[0259] The nine common peaks with the retention time of the reference medicinal material of Bai Tuling were selected as the characteristic peaks of the fingerprint of the Bai Tuling standard decoction; the characteristic peaks of the reference substance were compared with the characteristic peaks of the Bai Tuling standard decoction, and the fingerprint of the reference substance was obtained according to the spectrum of the reference substance ( Figure 11A -E) for identification; the results showed that characteristic peaks with consistent retention times with those of methylazoxymethanol primulosidoside, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, and syringyl glucoside could be found in the fingerprint of Bai Tuling standard decoction, and peak 2 was determined to be methylazoxymethanol primulosidoside; peak 3 was determined to be protocatechuic acid; peak 5 was determined to be 4-hydroxybenzoic acid; peak 8 was determined to be vanillic acid; and peak 9(S) was determined to be syringyl glucoside.

[0260] In order to establish the fingerprint of Bai Tuling standard decoction comprehensively and accurately, the nine characteristic peaks identified in the fingerprint were investigated.

[0261] 4.2 Precision experiment

[0262] The test solution was prepared by taking the freeze-dried powder of Bai Tuling standard decoction. According to the proposed experimental method, the sample was injected continuously 6 times, each time 10 μl, and the retention time and peak area of ​​each characteristic peak were calculated. The results are shown in Tables 17-18.

[0263] Table 17 Precision investigation - characteristic peak retention time

[0264]

[0265]

[0266] Table 18 Precision investigation - characteristic peak area

[0267]

[0268] The results showed that the RSD values ​​of the retention time and peak area of ​​each characteristic peak met the requirements and the instrument had good precision.

[0269] 4.3 Repeatability study

[0270] Accurately weigh 6 portions of freeze-dried powder of Bai Tuling standard decoction, prepare and measure according to the proposed experimental method, calculate the relative retention time and relative peak area of ​​the characteristic peaks, and the results are shown in Tables 19-20.

[0271] Table 19 Repeatability study - relative retention time of characteristic peaks

[0272]

[0273] Table 20 Repeatability study - relative peak area of ​​characteristic peaks

[0274]

[0275] The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak met the requirements, indicating that the method had good repeatability.

[0276] 4.4 Investigation with different instruments

[0277] Based on the experimental conditions proposed above, two portions of freeze-dried powder of Bai Tuling standard decoction were accurately weighed to prepare test solutions. The solutions were measured on three different types of high performance liquid chromatographs, and the relative retention times and peak areas of the characteristic peaks were calculated. The results are shown in Table 1. Figure 25 , Table 21-22.

[0278] Table 21 Instrument investigation - relative retention time of characteristic peaks

[0279]

[0280] Table 22 Instrument Investigation—Relative Peak Area of ​​Characteristic Peaks

[0281]

[0282] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.25% and 1.58% when detected by the three instruments mentioned above, indicating that the different instruments had good durability.

[0283] 4.5 Inspection by different personnel and time

[0284] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed 2 portions of 0.2 g each of freeze-dried powder of Bai Tuling standard decoction at different times (T1, T2) to prepare test solutions, conduct measurements, and calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 23-24.

[0285] Table 23 Different personnel and time investigation - relative retention time of characteristic peaks

[0286]

[0287] Table 24 Different personnel and time investigation - relative peak area of ​​characteristic peaks

[0288]

[0289]

[0290] The results showed that under different sample preparation personnel and different sample preparation time conditions, the RSDs of the relative retention times of the characteristic peaks were between 0.24% and 1.07%, and the RSDs of the relative peak areas of the characteristic peaks were between 6.86% and 24.66%.

[0291] 4.6 Stability investigation

[0292] Based on the experimental conditions proposed above, a sample solution was prepared and measured at 0h, 2h, 4h, 8h, 16h, and 24h respectively; the retention time and peak area of ​​each characteristic peak were calculated. The results are shown in Tables 25-26.

[0293] Table 25 24-hour stability study - characteristic peak retention time

[0294]

[0295] Table 26 24-hour stability study - characteristic peak area

[0296]

[0297] The results showed that the RSD of the characteristic peak retention time was 0.27%-1.06%, the RSD of the peak area was 0.98%-13.38%, and the sample solution was relatively stable within 24 hours.

[0298] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above nine characteristic peaks were included in the subsequent investigations.

[0299] 4.7 Investigation Results

[0300] The results of multiple surveys are summarized in Tables 27-28.

[0301] Table 27 Summary of RSD% results for each item of the methodology - retention time / relative retention time

[0302]

[0303] Table 28 Summary of RSD% of each item results of the method - Peak area / Relative peak area

[0304]

[0305] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method is good. Since Peak 2 showed a split peak during the investigation, indicating that Peak 2 was a peak envelope, Peak 2 was deleted, leaving Peak 1, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, and Peak 9, a total of 8 characteristic peaks, as the characteristic peaks of the fingerprint of the Bai Tu Ling standard decoction; Peak 2 is protocatechuic acid; Peak 4 is 4-hydroxybenzoic acid; Peak 7 is vanillic acid; Peak 8 (S) is syringoside.

[0306] 4.8 Determination of characteristic peaks and establishment of reference fingerprints

[0307] (1) Verification results of 21 batches of Bai Tuling standard decoction

[0308] The proposed method was used to determine the fingerprints of 21 batches of Bai Tu Ling standard decoctions; the relative retention time and relative peak area of ​​each characteristic peak of the 21 batches of Bai Tu Ling standard decoctions were calculated. The results are shown in Tables 29, 30 and Figure 26 .

[0309] Table 29 Verification results of 21 batches of Bai Tuling standard decoction

[0310]

[0311]

[0312] Table 30 Relative peak areas of 21 batches of Bai Tuling standard decoction

[0313]

[0314]

[0315] Based on the principles of stable relative retention time, detectable characteristic peaks in all batches of samples, and relatively high peak values ​​of characteristic peaks, a total of 8 peaks with good durability were selected as characteristic peaks; based on the results of methodological investigation and verification results of 21 batches of standard soups, the theoretical plate number calculated based on the syringic acid glucoside peak was tentatively set to no less than 5000.

[0316] (2) Establishment of relative retention time limit values

[0317] The summary of the methodology inspection items and verification results are shown in Tables 31-32:

[0318] Table 31 Summary of RSD% results for each item of the methodology - relative retention time

[0319]

[0320] Table 32 Summary of RSD% results for each item of the methodology - relative peak area

[0321]

[0322] The relative retention time of each characteristic peak is stable, and the relative retention time values ​​are all within the range of ±10% of the average value, so the specified value range of the relative retention time of each peak is temporarily set at ±10%; the relative peak areas of each batch vary too much to be specified, so the relative peak areas are not included in the main text.

[0323] Final regulations: The fingerprint of the test solution of the freeze-dried powder of the standard decoction of Bai Tuling in this example should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside peak is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are as follows: Peak 1 is 0.15, Peak 2 is 0.42, Peak 3 is 0.48, Peak 4 is 0.63, Peak 5 is 0.77, Peak 6 is 0.88, and Peak 7 is 0.95.

[0324] The fingerprints of 21 batches of Bai Tuling standard decoctions were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison of the fingerprints of Bai Tuling standard decoctions was established. The results are shown in Figure 27 .

[0325] Conclusion: The fingerprint detection method of Bai Tuling standard decoction in this example can accurately analyze and detect Bai Tuling standard decoction.

[0326] 5. Determination of the detection method of the fingerprint of Bai Tuling standard decoction

[0327] (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; methanol was used as the mobile phase A; 0.1% phosphoric acid solution was used as the mobile phase B; gradient elution was performed according to the requirements in the table below; the flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelength was 254 nm. The number of theoretical plates calculated based on the syringoside peak was not less than 5000.

[0328]

[0329] (2) Preparation of reference solution: Take 1 g of the reference medicinal material Bai Tu Ling, add 50 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Separately, take an appropriate amount of the reference substance syringyl glucoside, accurately weigh it, and add methanol to make a solution containing 30 μg per 1 ml, which is used as the reference solution.

[0330] (3) Preparation of test solution: Take an appropriate amount of standard decoction of Bai Tuling, grind it into powder, take about 0.2 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate.

[0331] (4) Detection method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure.

[0332] The test solution chromatogram should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.15 (peak 1), 0.42 (peak 2), 0.48 (peak 3), 0.63 (peak 4), 0.77 (peak 5), 0.88 (peak 6), and 0.95 (peak 7).

[0333] Example 3

[0334] The method for detecting the fingerprint of Bai Tu Ling formula granules in Example 1 is also applicable to Bai Tu Ling medicinal materials and decoction pieces. The details are as follows:

[0335] The chromatographic column was filled with octadecylsilane bonded silica gel, with methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was performed as specified in the table below. The flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelength was 254 nm. The number of theoretical plates, calculated based on the syringoside peak, must be no less than 5000.

[0336]

[0337] Preparation of the reference solution: Take 1 g of the Bai Tu Ling reference medicinal material, add 50 mL of water, boil for 30 minutes, cool, filter, and evaporate the filtrate to dryness. Add 25 mL of 30% methanol to the residue and sonicate (power 600 W, frequency 40 kHz) for 30 minutes. Cool, shake well, filter, and use the filtrate as the reference medicinal material solution. Separately, accurately weigh an appropriate amount of the syringyl glucoside reference substance and add methanol to a solution containing 30 μg per mL. This will serve as the reference substance solution.

[0338] Preparation method of test solution: take the powder of Bai Tuling medicinal material or decoction piece, pass it through No. 3 sieve, accurately weigh 1g, add 50ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25ml of 30% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate.

[0339] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0340] The test solution chromatogram should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.15 (peak 1), 0.42 (peak 2), 0.48 (peak 3), 0.63 (peak 4), 0.76 (peak 5), 0.87 (peak 6), and 0.95 (peak 7).

[0341] The method of this embodiment is screened, and the specific process is as follows:

[0342] 1. Instruments and test drugs

[0343] Electronic balances: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.); Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.); Ultrasonic cleaner: KQ5200DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0344] Methanol (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), and water used in the mobile phase were laboratory-made ultrapure water, and the rest were laboratory-made pure water. Protocatechuic acid (China Food and Drug Inspection Institute, batch number: 110809-201906, content is 97.7%); methyl azoxymethanol primulosidoside (OST, batch number: U40-1309310-02, content is 98%); 4-hydroxybenzoic acid (China Food and Drug Inspection Institute, batch number: 101149-202204, content is 100%); vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, content is 99.8%); syringyl glucoside (OST, batch number: U40-7008658-02, content is 99.5%); white turmeric reference medicinal material (China Food and Drug Inspection Institute, batch number: 121537-201502);

[0345] The Bai Tuling medicinal material used in this embodiment was prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd. (Batch Nos.: BTL01, BTL02, BTL03, BTL04, BTL05, BTL06, BTL07, BTL08, BTL09, BTL10, BTL11, BTL12, BTL13, BTL14, BTL15, BTL16, BTL17, BTL18, BTL19, BTL20, and BTL21);

[0346] The Bai Tuling decoction pieces used in this embodiment were prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd. (Batch Nos.: BTL001, BTL002, BTL003, BTL004, BTL005, BTL006, BTL007, BTL008, BTL009, BTL010, BTL011, BTL012, BTL013, BTL014, BTL015, BTL016, BTL017, BTL018, BTL019, BTL020, and BTL021);

[0347] 2. Chromatographic conditions and system suitability test

[0348] (1) Detection wavelength investigation

[0349] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 220nm, 240nm, 254nm, 280nm, 300nm, 320nm, 340nm, and 360nm were extracted respectively. Figures 28-29 .

[0350] The results showed that when the detection wavelength was 254 nm, the chromatographic peak information was richer, the number of chromatographic peaks was greater, and the chromatogram baseline was more stable, so the detection wavelength was determined to be 254 nm.

[0351] (2) Column temperature and flow rate investigation

[0352] According to the results of the column temperature and flow rate investigation of Bai Tuling formula granules, the column temperature for the determination of Bai Tuling medicinal material fingerprint was proposed to be 30℃ and the flow rate was 1.0ml / min for subsequent investigation.

[0353] (3) Delay investigation

[0354] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 120 minutes. The results are as follows: Figure 30 shown.

[0355] The results showed that the sample had basically no chromatographic peak after 55 minutes, so the sample detection time was set at 55 minutes.

[0356] (4) Determination of chromatographic conditions and system suitability test

[0357] The chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was methanol, and the mobile phase B was 0.1% phosphoric acid solution, with gradient elution as specified in the table below. The column temperature was 30°C; the flow rate was 1.0 ml / min; the fingerprint detection wavelength was 254 nm; the injection volume was 10 μl, and the measurement time was 55 minutes. The number of theoretical plates, calculated based on the syringoside peak, must not be less than 5000.

[0358]

[0359] 3. Investigation of the preparation method of test solution

[0360] According to the results of the preparation investigation of the test sample solution of Bai Tuling standard decoction and formula granules, the extraction solvent and extraction time of Bai Tuling medicinal material were designed to be consistent with those of Bai Tuling standard decoction and formula granules, and the extraction method was investigated based on this condition.

[0361] (1) Investigation of extraction methods

[0362] Extraction method 1 (boiling): Take 1.0 g of the medicinal material of White Tuling (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of water, boil it in water for 30 minutes, cool it, filter it, evaporate the filtrate to dryness, dissolve it in 25 ml of 30% methanol, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, filter it, and take the filtrate as the test solution.

[0363] Extraction method 2 (ultrasound): Take 1.0g of Bai Tuling medicinal material (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 30% methanol, stopper it tightly, weigh it, ultrasonicate it for 30 minutes, let it cool, weigh it again, make up the lost weight with 30% methanol, shake it well, filter it, and take the filtrate; the results are shown in the figure. Figure 31 .

[0364] The results showed that when the Bai Tuling medicinal material was boiled and extracted by ultrasonic, the boiling method made the peak shapes of the characteristic peaks better separated, so the extraction method of the test sample solution was determined to be boiling extraction.

[0365] (2) Determination of the preparation method of the test solution

[0366] Take about 1g of Bai Tuling medicinal material powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, and treat it with 25ml of 30% methanol ultrasonically (power 600W, frequency 40kHz) for 30 minutes, filter, and take the filtrate to obtain.

[0367] 4. Methodological Investigation

[0368] 4.1 Chromatographic peak identification

[0369] Preparation of reference solution: Take 1 g of the control medicinal material of Bai Tuling, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol to the residue, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material solution;

[0370] Take appropriate amounts of protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside reference substances, accurately weigh them, and add methanol to prepare a mixed solution containing 30 μg each of protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside per 1 ml, which is used as the reference substance solution.

[0371] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Bai Tuling medicinal material.

[0372] Preparation of test solution: Take about 1 g of Bai Tuling medicinal material powder (passed through No. 3 sieve) and prepare the test solution of Bai Tuling medicinal material in the same way as the preparation method of the control medicinal material solution.

[0373] 10 μl of the test solution and reference solution were respectively injected into the high performance liquid chromatograph for sample determination; the chromatograms of Bai Tuling medicinal material, Bai Tuling reference medicinal material and reference substance were obtained, and the characteristic peaks of the Bai Tuling medicinal material fingerprint were located. The results are shown in FIG. Figure 32 .

[0374] The nine common peaks with the retention time of the reference medicinal material of Bai Tuling were selected as the characteristic peaks of the fingerprint of Bai Tuling medicinal material; the characteristic peaks of the reference substance were compared with the characteristic peaks of Bai Tuling medicinal material, and the peaks of the fingerprint of the reference substance were compared with the characteristic peaks of the Bai Tuling medicinal material. Figure 11A -E) was identified; the results showed that characteristic peaks with consistent retention times with methylazoxymethanol primulosidoside, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, and syringyl glucoside could be found in the fingerprint of Bai Tuling medicinal material, and peak 2 was determined to be methylazoxymethanol primulosidoside; peak 3 was determined to be protocatechuic acid; peak 5 was determined to be 4-hydroxybenzoic acid; peak 8 was determined to be vanillic acid; and peak 9 (S) was determined to be syringyl glucoside.

[0375] In order to establish the standard fingerprint of Bai Tuling medicinal material comprehensively and accurately, the 9 characteristic peaks identified in the fingerprint were investigated.

[0376] 4.2 Precision investigation

[0377] Take the Bai Tuling medicinal material test solution and inject it continuously six times according to the proposed experimental method, with 10 μl each time. Calculate the retention time and peak area of ​​each characteristic peak. See Tables 33 and 34.

[0378] Table 33 Precision investigation - characteristic peak retention time

[0379]

[0380] Table 34 Precision investigation - characteristic peak area

[0381]

[0382] The results showed that the RSD of the retention time of each characteristic peak of the sample was 0.24% to 0.63%, and the instrument had good precision.

[0383] 4.3 Repeatability study

[0384] Take 6 portions of the test solution of Bai Tuling medicinal material, prepare and measure it according to the proposed experimental method, and calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 35 and 36.

[0385] Table 35 Repeatability study - relative retention time of characteristic peaks

[0386]

[0387]

[0388] Table 36 Repeatability study - relative peak area of ​​characteristic peaks

[0389]

[0390] The results showed that the RSD of the relative retention time of the six samples was 0.11% to 0.31%, indicating that the method had good repeatability.

[0391] 4.4 Investigation with different instruments

[0392] Based on the experimental conditions proposed above, 3 portions of Bai Tuling medicinal material powder were accurately weighed to prepare test solutions. The solutions were measured on three different types of high performance liquid chromatographs, and the relative retention times and peak areas of the characteristic peaks were calculated. The results are shown in Table 1. Figure 33 , Tables 37-38.

[0393] Table 37 Instrument Investigation—Relative Retention Time of Characteristic Peaks

[0394]

[0395] Table 38 Instrument Investigation—Relative Peak Area of ​​Characteristic Peaks

[0396]

[0397] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.35% and 1.10% when detected by the three instruments mentioned above, indicating that the different instruments had good durability.

[0398] 4.5 Inspection by different personnel and time

[0399] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed two portions of Bai Tuling medicinal material powder at different times (T1, T2) to prepare test solutions, conduct measurements, and calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 39-40.

[0400] Table 39 Different personnel and time investigation - relative retention time of characteristic peaks

[0401]

[0402] Table 40 Different personnel and time investigation - relative peak area of ​​characteristic peaks

[0403]

[0404] The results showed that when different people measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks were 0.28% to 1.75%, indicating that the method was stable.

[0405] 4.6 Stability investigation

[0406] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h respectively; the retention time and peak area of ​​each characteristic peak were calculated. The results are shown in Tables 41-42.

[0407] Table 41 24-hour stability study - characteristic peak retention time

[0408]

[0409]

[0410] Table 42 24-hour stability study - characteristic peak area

[0411]

[0412] The results showed that the RSDs of the characteristic peak retention times were between 0.37% and 1.11%, and the sample solutions were stable within 24 hours.

[0413] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above nine characteristic peaks were included in the subsequent investigations.

[0414] 4.7 Investigation Results

[0415] The results of multiple surveys are summarized in Tables 43-44.

[0416] Table 43 Summary of RSD% results for each item of the methodology - retention time / relative retention time

[0417]

[0418] Table 44 Summary of RSD% of each item results of the method - Peak area / Relative peak area

[0419]

[0420] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. Since Peak 2 showed a split peak during the investigation, indicating that Peak 2 was a peak envelope, Peak 2 was deleted, leaving Peak 1, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, and Peak 9, a total of 8 characteristic peaks, as characteristic peaks of the Bai Tu Ling medicinal material fingerprint for subsequent investigations; Peak 2 is protocatechuic acid; Peak 4 is 4-hydroxybenzoic acid; Peak 7 is vanillic acid; and Peak 8 (S) is syringoyl glucoside.

[0421] 4.8 Determination of characteristic peaks and establishment of reference fingerprints

[0422] (1) Verification results of 21 batches of Bai Tuling medicinal materials

[0423] The proposed method was used to determine the fingerprints of 21 batches of Bai Tuling medicinal materials, and the obtained fingerprints were tested; the relative retention time and relative peak area of ​​each characteristic peak of the 21 batches of Bai Tuling medicinal materials were calculated. The results are shown in Tables 45-46 and Figure 34 .

[0424] Table 45 Relative retention time of 21 batches of Bai Tuling medicinal materials

[0425]

[0426] Table 46 Relative peak areas of 21 batches of Bai Tuling medicinal materials

[0427]

[0428]

[0429] Based on the principles of stable relative retention time, detectable characteristic peaks in all batches of samples, and relatively high peak values ​​of characteristic peaks, a total of 8 peaks with good durability, namely Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, and Peak 8, were selected as characteristic peaks; based on the results of methodological investigation and verification results of 21 batches of medicinal materials, the theoretical plate number calculated based on the syringic acid glucoside peak was tentatively determined to be no less than 5000.

[0430] (2) Verification results of 21 batches of Bai Tuling decoction pieces

[0431] The proposed method was used to determine the fingerprints of 21 batches of Bai Tuling decoction pieces, and the obtained fingerprints were tested; the relative retention time and relative peak area of ​​each characteristic peak of the 21 batches of Bai Tuling decoction pieces were calculated. The results are shown in Tables 47-48 and Figure 35 .

[0432] Table 47 Relative retention time of 21 batches of Bai Tuling decoction pieces

[0433]

[0434]

[0435] Table 48 Relative peak areas of 21 batches of Bai Tuling decoction pieces

[0436]

[0437]

[0438] Based on the principles of stable relative retention time, detectable characteristic peaks in each batch of samples and relatively high peak values ​​of characteristic peaks, a total of 8 peaks with good durability, namely Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7 and Peak 8, were selected as characteristic peaks; according to the results of methodological investigation and verification results of 21 batches of medicinal pieces, the theoretical plate number calculated based on the syringic acid glucoside peak was tentatively determined to be no less than 5000.

[0439] Final Regulation: The chromatograms of the test solutions of the Bai Tu Ling medicinal material and Bai Tu Ling decoction pieces in this example should each exhibit eight characteristic peaks, with retention times corresponding to those of the eight characteristic peaks in the chromatogram of the control medicinal material reference. The peak corresponding to the syringyl glucoside peak is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are as follows: Peak 1 is 0.15, Peak 2 is 0.42, Peak 3 is 0.48, Peak 4 is 0.63, Peak 5 is 0.76, Peak 6 is 0.87, and Peak 7 is 0.95.

[0440] The fingerprints of 21 batches of Bai Tuling medicinal materials were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison map of Bai Tuling medicinal material fingerprints was established. The results are shown in Figure 36 .

[0441] The fingerprints of 21 batches of Bai Tuling decoction pieces were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison of the fingerprints of Bai Tuling decoction pieces was established. Figure 37 .

[0442] Conclusion: The fingerprint detection method of Bai Tuling medicinal material in this embodiment can accurately analyze and detect Bai Tuling medicinal material; the fingerprint detection method of Bai Tuling decoction pieces in this embodiment can accurately analyze and detect Bai Tuling decoction pieces.

[0443] 5. Determination of the detection method for the fingerprint of Bai Tuling medicinal materials and decoction pieces

[0444] (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; methanol was used as the mobile phase A; 0.1% phosphoric acid solution was used as the mobile phase B; gradient elution was performed according to the requirements in the table below; the flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelength was 254 nm. The number of theoretical plates calculated based on the syringoside peak was not less than 5000.

[0445]

[0446] (2) Preparation of reference solution: Take 1 g of the reference medicinal material Bai Tu Ling, add 50 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Separately, take an appropriate amount of the reference substance syringyl glucoside, accurately weigh it, and add methanol to make a solution containing 30 μg per 1 ml, which is used as the reference solution.

[0447] (3) Preparation of test solution: Take 1 g of the powder of the medicinal material or slices of Bai Tuling (passed through a No. 3 sieve) and prepare the test solution in the same way as the control medicinal material solution.

[0448] (4) Detection method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure.

[0449] The test solution chromatogram should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the syringyl glucoside reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.15 (peak 1), 0.42 (peak 2), 0.48 (peak 3), 0.63 (peak 4), 0.76 (peak 5), 0.87 (peak 6), and 0.95 (peak 7).

[0450] See Figure 38 , which is a comparison of the fingerprints of Bai Tuling formula granules, Bai Tuling standard decoction, Bai Tuling medicinal materials, and Bai Tuling decoction pieces. As can be seen from the figure, Bai Tuling formula granules, Bai Tuling standard decoction, Bai Tuling medicinal materials, and Bai Tuling decoction pieces exhibit eight common characteristic peaks, of which Peak 2 is the characteristic peak of protocatechuic acid, Peak 4 is the characteristic peak of 4-hydroxybenzoic acid, Peak 7 is the characteristic peak of vanillic acid, and Peak 8 is the characteristic peak of syringyl glucoside. Therefore, Tuling formula granules are correlated with Bai Tuling standard decoction, Bai Tuling medicinal materials, and Bai Tuling decoction pieces.

[0451] Comparative Example 1

[0452] (1) Comparative selection of different mobile phases A

[0453] Use octadecylsilane bonded silica gel as the filler; replace methanol with acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B. Perform gradient elution according to the table below. The flow rate is 1.0 ml / min, the column temperature is 30°C, and the detection wavelength is 254 nm. The number of theoretical plates calculated based on the syringoside peak must be no less than 5000.

[0454]

[0455] Based on the experimental conditions proposed above, the separation effects of two different mobile phases were investigated: methanol-0.1% phosphoric acid and acetonitrile-0.1% phosphoric acid. Figures 39-41 .

[0456] As can be seen from the figure, methanol is more effective than acetonitrile in elution, with better separation, smaller front solvent peaks, more distinct overall peak heights, and richer chromatographic peak information. Therefore, methanol was selected as the organic mobile phase.

[0457] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0458] Note that the above embodiments are merely a few examples of implementations of the present invention and the underlying technical principles, and are not intended to limit the present invention. A person skilled in the art, based on the principles of the present invention, can make various obvious changes, readjustments, and substitutions to the technical solutions described in the above embodiments without departing from the scope of the present invention. Therefore, this specification should not be construed as limiting the present invention.

Claims

1. A method for detecting white earthworm products, characterized in that: include: The sample to be tested is dissolved in methanol-water solution, subjected to ultrasonic extraction, filtered, and the filtrate is taken for high performance liquid chromatography detection to obtain the fingerprint of the sample to be tested; The fingerprint of the sample to be tested is compared with the fingerprint of the standard control of the white earthworm product established in advance; In the high performance liquid chromatography detection, the mobile phase includes: mobile phase A methanol and mobile phase B phosphoric acid solution, and gradient elution is performed; The gradient elution was performed as follows: 0-12 min, the volume percentage of the mobile phase B is 100%; 12-18 min, the volume percentage of the mobile phase B decreased from 100% to 96%; 18-24 min, the volume percentage of the mobile phase B decreased from 96% to 90%; 24-36 min, the volume percentage of the mobile phase B decreased from 90% to 87%; 36-55 min, the volume percentage of the mobile phase B is 87%; The method of pre-establishing a standard control fingerprint of the white earthworm product comprises the following steps: Dissolve multiple white earthworm products separately to obtain multiple test solution; The reference substance and each test solution were subjected to high performance liquid chromatography to obtain fingerprints of the reference substance and multiple white earthworm products; According to the fingerprints of the reference material and multiple white earthworm products, the standard control fingerprint of the white earthworm products is synthesized; The reference substances are protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, syringyl glucoside, and methyl azoxymethanol primulosidoside; The wavelength of the high performance liquid chromatography detection is 254 nm.

2. The method for detecting white earthworm products according to claim 1, wherein The concentration of the phosphoric acid solution is 0.05-0.15%.

3. The method for detecting white earthworm products according to claim 1, wherein The flow rate of the high performance liquid chromatography detection is 0.8-1.2 ml / min.

4. The method for detecting white earthworm products according to claim 1, wherein The chromatographic column for high performance liquid chromatography detection uses octadecylsilane bonded silica gel as a filler.

5. The method for detecting the white earthworm product according to claim 4, wherein: The column temperature of the chromatographic column is 20-35°C.

6. The method for detecting white earthworm products according to claim 1, wherein: The Bai Tuling products include Bai Tuling formula granules, Bai Tuling standard decoction, Bai Tuling medicinal materials or Bai Tuling decoction pieces.

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