HPLC fingerprint of corn silk wall-broken slices, construction method thereof, and application thereof

Abstract

The present invention discloses an HPLC fingerprint of corn silk broken wall slices and its construction method and application. The present invention uses methanol as an extraction solvent and ultrasonically extracts different batches of corn silk broken wall slices to prepare a test solution. Then, using ferulic acid as a reference substance and acetonitrile-0.3% phosphoric acid aqueous solution as a mobile phase system, the test solution is subjected to HPLC elution and spectrum construction to obtain an HPLC fingerprint of corn silk broken wall slices. Using the method of the present invention, 18 common fingerprint peaks can be determined, and at the same time, the separation between the peaks is good, the peak distribution is uniform, and the baseline is stable. The results of the methodological investigation show that the method of the present invention has good specificity and good repeatability, stability and durability. The method of the present invention can more objectively and comprehensively evaluate the overall quality of corn silk broken wall slices, laying a foundation for the quality control of corn silk broken wall slices.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis and detection, and in particular to an HPLC fingerprint of corn silk wall-broken decoction pieces, a construction method thereof, and applications thereof. Background Art

[0002] Corn silk (Stigma mydis) is the dried style and stigma of maize. It was first recorded in the Compendium of Materia Medica in my country. It has a sweet, light flavor and a neutral nature. It enters the bladder, liver, and gallbladder meridians, and has diuretic and anti-edema properties, clears the liver and gallbladder, and clears heat and reduces internal heat. Modern pharmacological research has shown that corn silk has multiple biological activities, including anti-tumor, antioxidant, hypoglycemic, and anti-inflammatory properties. However, research on its chemical composition is still incomplete, and its appearance and chemical composition vary considerably due to regional climates and corn varieties.

[0003] Corn silk broken wall slices are made from corn silk slices, which are processed into powder with D90 < 45μm (above 300 mesh) using traditional Chinese medicine wall-breaking technology. Water or ethanol of different concentrations is added to form a uniform dry granular slice with 30-100 mesh and all the ingredients of the original slice. Compared with traditional corn silk slices, corn silk broken wall slices have the advantages of uniform quality, high drug utilization rate, good stability and convenient and quick application. However, breaking the wall will bring about changes in the solubility of chemical components such as active ingredients or index ingredients, making the identification and quality inspection methods of traditional corn silk powder or corn silk formula granules not fully applicable to corn silk broken wall slices.

[0004] Therefore, in order to more comprehensively monitor the quality of corn silk broken wall slices, improve the quality evaluation system of corn silk broken wall slices, and establish a fingerprint of corn silk broken wall slices with good separation and comprehensive chromatographic peaks, it is of great significance. Summary of the Invention

[0005] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides an HPLC fingerprint of corn silk broken wall slices, a construction method thereof, and an application thereof.

[0006] The first object of the present invention is to provide a pretreatment method for detecting corn silk wall-broken pieces by high performance liquid chromatography.

[0007] The second object of the present invention is to provide a high performance liquid chromatography analysis method.

[0008] The third object of the present invention is to provide the application of any of the above-mentioned pretreatment methods and / or high performance liquid chromatography analysis methods in establishing the HPLC fingerprint of corn silk broken wall slices and / or quality control of corn silk broken wall slices.

[0009] The fourth object of the present invention is to provide a method for constructing an HPLC fingerprint of corn silk broken wall slices.

[0010] The fifth object of the present invention is to provide a quality detection method for corn silk broken wall slices.

[0011] Therefore, the present invention claims the following:

[0012] A pretreatment method for detecting corn silk wall-broken decoction pieces by high performance liquid chromatography comprises the following steps:

[0013] S1. Mix the sample to be tested with methanol and perform sonication, cool, add methanol to make up for the weight loss, separate the solid and liquid, and remove the methanol in the liquid;

[0014] S2. The product of step S1 was dissolved in water, extracted with ethyl acetate, and the ethyl acetate layer was removed to obtain a residue;

[0015] S3. Dissolve the residue in methanol, filter through a microporous filter membrane, and obtain the filtrate.

[0016] The corn silk broken wall slices are provided by Zhongshan Zhongzhi Chinese Medicine Pieces Co., Ltd., and the sample meets the quality standard requirements of the "Guangdong Province Chinese Medicine Broken Wall Pieces Quality Standard Research Specifications (Trial)".

[0017] Preferably, in step S1, the mass volume ratio of the sample to be tested to methanol is 1 g: (12-13) mL.

[0018] More preferably, in step S1, the mass volume ratio of the sample to be tested to methanol is 1 g:12.5 mL.

[0019] Preferably, in step S1, the ultrasonication time is 25 to 35 minutes.

[0020] More preferably, in step S1, the ultrasonication time is 30 minutes.

[0021] Preferably, the frequency of the ultrasound is 35-45 kHz, and the power is 490-630 W.

[0022] More preferably, the frequency of the ultrasound is 40 kHz and the power is 560 W.

[0023] Preferably, in step S2, the extraction is performed 2 to 3 times.

[0024] More preferably, in step S2, the extraction is performed twice.

[0025] Preferably, the mass volume ratio of the sample to be tested to water is 1 g: (7-8) mL.

[0026] More preferably, the mass volume ratio of the sample to be tested to water is 1 g:7.5 mL.

[0027] Preferably, in step S2, the volume ratio of water to ethyl acetate is (0.8-1.2) mL:1 mL.

[0028] More preferably, in step S2, the volume ratio of water to ethyl acetate is 1 mL:1 mL.

[0029] Preferably, in step S3, the pore size of the microporous filter membrane is 0.45 μm.

[0030] A high performance liquid chromatography analysis method comprises the following steps:

[0031] Reference solution: ferulic acid solution;

[0032] Test solution: Prepare using any of the above pretreatment methods;

[0033] Chromatographic conditions: Octadecylsilane bonded silica gel was used as the column filler, acetonitrile was used as mobile phase A, and 0.28-0.32% by volume phosphoric acid solution was used as mobile phase B. In the gradient elution procedure, the volume percentage of mobile phase A in the mobile phase system varied as follows:

[0034] From 0 to 8 min, mobile phase A was increased from 10% to 19%;

[0035] 8-13 min, mobile phase A 19%;

[0036] From 13 to 55 min, mobile phase A was increased from 19% to 45%;

[0037] From 55 to 65 min, mobile phase A was increased from 45% to 68%;

[0038] 65-80 min, mobile phase A is 68%;

[0039] From 80 to 85 min, mobile phase A was reduced from 68% to 10%;

[0040] The detection wavelength is 273 nm, and the theoretical plate number calculated based on ferulic acid should be no less than 10,000.

[0041] Preferably, the preparation method of the ferulic acid solution is: fully mixing ferulic acid and methanol, and the mass volume ratio of the ferulic acid to methanol is (245-255) μg:1 mL.

[0042] More preferably, the mass volume ratio of ferulic acid to methanol is 250 μg:1 mL.

[0043] Preferably, the injection volume of the reference solution and the test solution is 8 to 12 μL.

[0044] More preferably, the injection volume of the reference solution and the test solution is 10 μL.

[0045] Preferably, the chromatographic column is a SHISEIDO CAPCELL PAK MGⅢC18 chromatographic column with a specification of 5 μm and 250 mm×4.6 mm.

[0046] Preferably, the volume fraction of the phosphoric acid solution is 0.3%.

[0047] Preferably, the column temperature of the chromatographic column is 28-32°C.

[0048] More preferably, the column temperature of the chromatographic column is 30°C.

[0049] Preferably, the flow rate of the gradient elution is 0.8 to 1.2 mL / min.

[0050] More preferably, the flow rate of the gradient elution is 1 mL / min.

[0051] Application of any of the above-mentioned pretreatment methods and / or high performance liquid chromatography analysis methods in establishing the HPLC fingerprint of corn silk broken wall slices and / or quality control of corn silk broken wall slices.

[0052] A method for constructing an HPLC fingerprint of corn silk broken wall slices comprises using any of the above-mentioned high performance liquid chromatography analysis methods to detect different batches of corn silk broken wall slice samples, recording the chromatograms, importing the chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, screening and confirming common peaks, and obtaining the HPLC fingerprint of the corn silk broken wall slices.

[0053] The HPLC fingerprint of the corn silk broken wall slices contains 18 fingerprint peaks, with peak 5 as the S peak, and the specified values ​​of the relative retention times are:

[0054] Peak 1 is 0.441, Peak 2 is 0.624, Peak 3 is 0.689, Peak 4 is 0.956, Peak 5 is 1.000, Peak 6 is 1.129, Peak 7 is 1.216, Peak 8 is 1.451, Peak 9 is 1.519, Peak 10 is 1.672, Peak 11 is 2.549, Peak 12 is 2.642, Peak 13 is 2.918, Peak 14 is 3.445, Peak 15 is 4.125, Peak 16 is 4.161, Peak 17 is 4.188, and Peak 18 is 4.257.

[0055] The different batches are no less than 15 batches.

[0056] A quality testing method for corn silk broken wall slices comprises the following steps:

[0057] S1. Take the broken wall pieces of corn silk to be tested, and detect using any of the above-mentioned high performance liquid chromatography analysis methods, record the chromatogram, and obtain the HPLC spectrum of the broken wall pieces of corn silk to be tested;

[0058] S2. The HPLC fingerprint of the above-mentioned corn silk broken wall slices and the HPLC fingerprint of the corn silk broken wall slices sample to be tested are imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for comparison. The qualified ones are judged to be qualified if the following two conditions are met:

[0059] The HPLC spectrum of the corn silk broken wall decoction piece sample to be tested presents a fingerprint peak whose relative retention time is within ±10% of the specified value in the HPLC fingerprint spectrum of the corn silk broken wall decoction piece;

[0060] The similarity between the HPLC spectrum of the corn silk broken wall slice sample to be tested and the HPLC fingerprint spectrum of the corn silk broken wall slice sample is calculated, and the similarity between the HPLC spectrum of the corn silk broken wall slice sample to be tested and the HPLC fingerprint spectrum of the corn silk broken wall slice sample is not less than 0.85;

[0061] The HPLC fingerprint of the corn silk broken wall slices contains 18 fingerprint peaks, with peak 5 as the S peak, and the specified values ​​of the relative retention times are:

[0062] Peak 1 is 0.441, Peak 2 is 0.624, Peak 3 is 0.689, Peak 4 is 0.956, Peak 5 is 1.000, Peak 6 is 1.129, Peak 7 is 1.216, Peak 8 is 1.451, Peak 9 is 1.519, Peak 10 is 1.672, Peak 11 is 2.549, Peak 12 is 2.642, Peak 13 is 2.918, Peak 14 is 3.445, Peak 15 is 4.125, Peak 16 is 4.161, Peak 17 is 4.188, and Peak 18 is 4.257.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] The present invention discloses an HPLC fingerprint of corn silk cracked decoction pieces, a method for constructing the fingerprint, and applications thereof. Using the method of the present invention, 18 common fingerprint peaks can be identified, with good separation between the peaks, uniform peak distribution, and a stable baseline. Methodological investigation results show that the method of the present invention has good specificity, as well as good repeatability, stability, and durability. The method of the present invention can more objectively and comprehensively evaluate the overall quality of corn silk cracked decoction pieces, laying a foundation for quality control of corn silk cracked decoction pieces. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The effect of different extraction solvents on the chromatogram of the test sample.

[0066] Figure 2The effects of different extraction methods on the chromatograms of the test samples are shown in Figure 2.

[0067] Figure 3 The effect of different extraction material-liquid ratios on the chromatogram of the test sample.

[0068] Figure 4 The effect of different extraction times on the chromatogram of the test sample.

[0069] Figure 5 The effects of different extraction methods on the chromatograms of the test samples are shown in Figure 2.

[0070] Figure 6 The effect of different extraction times on the chromatogram of the test sample.

[0071] Figure 7 It is the isotropic absorption diagram of the test solution and the chromatogram at 273 nm.

[0072] Figure 8 The effect of different mobile phase systems on the chromatogram of the test sample.

[0073] Figure 9 The effect of different elution gradients on the chromatogram of the test sample.

[0074] Figure 10 The effect of different elution flow rates on the chromatogram of the test sample.

[0075] Figure 11 The effect of different column temperatures on the chromatogram of the test sample.

[0076] Figure 12 The effect of different injection volumes on the chromatogram of the test sample.

[0077] Figure 13 A new reference profile generated for the analytical profiles of 20 batches of corn silk cracked slices.

[0078] Figure 14 This is the HPLC reference fingerprint of corn silk broken wall slices.

[0079] Figure 15 This is the identification diagram of the reference peak (Peak 5) of the test sample.

[0080] Figure 16 For the exclusive results.

[0081] Figure 17 The instrument precision result.

[0082] Figure 18 For reproducible results.

[0083] Figure 19 The stability result.

[0084] Figure 20The durability results of different chromatographic columns are shown in Figure 2.

[0085] Figure 21 Durability results for different instruments.

[0086] Figure 22 The chromatogram result is that of comparative example 1.

[0087] Figure 23 The chromatogram result is the comparative example 2.

[0088] Figure 24 The chromatogram result is the comparative example 3. DETAILED DESCRIPTION

[0089] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0090] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0091] Reference substance information used in the examples of the present invention: Ferulic acid, batch number 110773-201915, content 99.4%, purchased from China Food and Drug Inspection Institutes.

[0092] Information on the test samples used in the embodiments of the present invention: The pilot samples of corn silk broken wall slices were provided by Zhongshan Zhongzhi Chinese Medicine Pieces Co., Ltd., and the samples met the quality standard requirements of the "Guangdong Province Traditional Chinese Medicine Broken Wall Slices Quality Standard Research Specification (Trial)", and the batch numbers were 2203010A, 2203020A, 2203030A, 2203040A, 2203050A, 20220401, 20220402, 20220403, 20220404, 20220406, 20220407, 20220408, 20220410, 20220412, 20220413, 20220414, 20220415, 20220416, 20220417, and 20220418, respectively.

[0093] Example 1 Effect of the test sample preparation method on the HPLC fingerprint of corn silk broken wall slices

[0094] 1. Extraction solvent

[0095] 1. Experimental Methods

[0096] Preparation of the test sample: Take 2 g of corn silk broken wall slices, accurately weigh 5 portions, place in a stoppered conical flask, accurately add 25 mL of methanol, 30% methanol aqueous solution (v / v), 50% methanol aqueous solution (v / v), 70% methanol aqueous solution (v / v) and ethanol respectively, weigh the weight, and extract by ultrasonication for 30 min (power 560 W, frequency 40 kHz). After taking out, let cool, make up the weight with extraction solvent, filter with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the product.

[0097] Chromatographic conditions: A SHISEIDO CAPCELL PAK MGⅢC18 column (5 μm, 250 mm×4.6 mm) was used, with acetonitrile as mobile phase A and 0.1% aqueous phosphoric acid (v / v) as mobile phase B; flow rate 1.0 mL / min; column temperature 30°C; detection wavelength 273 nm; sample injection volume 10 μL, gradient elution conditions: 0 min–10 min–20 min–55 min–60 min–85 min–90 min, with a variation of mobile phase A of 3%–5%–20%–50%–70%–95%–3%.

[0098] 2. Experimental Results

[0099] The results are as follows Figure 1 As shown in the figure, the number of peaks obtained by each extraction solvent is basically the same, and the types of component peaks obtained are consistent, but the response value of the main component peak obtained by methanol extraction is significantly higher than that of ethanol. There are relatively few interfering small peaks in the methanol-extracted sample, and the baseline is stable. Therefore, pure methanol is selected as the extraction solvent for corn wall-broken slices.

[0100] 2. Extraction Method

[0101] 1. Experimental Methods

[0102] Preparation of test sample: Take 2 g of corn silk broken wall slices, accurately weigh 2 portions, place in a stoppered conical flask, accurately add 25 mL of methanol, weigh the weight, heat reflux extraction for 30 min and ultrasonic treatment for 30 min (power 560 W, frequency 40 kHz), cool, weigh again, make up the loss with methanol, shake well, filter, and take the filtrate.

[0103] The chromatographic conditions were in accordance with those in “1. Extraction solvent”.

[0104] 2. Experimental Results

[0105] The results are as follows Figure 2 As shown in the results, the number of peaks in the chromatograms of the test samples obtained by the two extraction methods is basically the same, the types of main peaks are consistent, the chromatographic peak area in the chromatogram of the sample obtained by reflux extraction is slightly larger than that of the ultrasonic extraction sample, and there are no other significant differences. In view of the convenience of ultrasonic extraction operation, ultrasonic extraction was selected as the extraction method for corn silk broken wall slices.

[0106] 3. Extraction material-liquid ratio

[0107] 1. Experimental Methods

[0108] Preparation of the test sample: Take one portion of 1g (No. 1) and four portions of 2g (No. 2-5) of corn silk broken wall slices, accurately weigh them, place them in a stoppered conical flask, accurately add 10mL of methanol to No. 1 and No. 2, accurately add 15mL of methanol to No. 3 and No. 4, accurately add 25mL of methanol to No. 5 and weigh the weight, ultrasonically extract for 30min (power 560W, frequency 40KHz), take out and cool, make up the weight with methanol, filter No. 1-3 with a 0.45μm microporous membrane, and take the filtrate to obtain the test solution 1-3;

[0109] Filter No. 4 and No. 5, evaporate the filtrate to dryness, dissolve the residue in methanol to make the volume 2mL, filter with a 0.45μm microporous filter membrane, and take the filtrate to obtain the test solution 4~5.

[0110] The chromatographic conditions were in accordance with those in “1. Extraction solvent”.

[0111] 2. Experimental Results

[0112] The results are as follows Figure 3 As shown, the five components of the test solution were fully extracted, and the peak response values ​​of the main components were relatively uniform. Therefore, 2 g of sample was added with 25 mL of methanol for extraction for 30 min, and the filtrate was evaporated to dryness and the methanol residue was diluted to 2 mL as the extraction material-liquid ratio for the preparation of the test solution.

[0113] 4. Extraction time

[0114] 1. Experimental Methods

[0115] Preparation of test sample: Take 2 g of corn silk broken wall slices, accurately weigh 5 portions, place in stoppered conical flasks, accurately add 25 mL of methanol to each portion, weigh the weight, and ultrasonically extract for 30, 45, 60, 90, and 120 min (power 560 W, frequency 40 kHz), respectively. After taking out, cool, make up with methanol, filter, evaporate the filtrate to dryness, dissolve the residue with methanol to 2 mL, filter with a 0.45 μm microporous filter membrane, and take the filtrate.

[0116] The chromatographic conditions were in accordance with those in “1. Extraction solvent”.

[0117] 2. Experimental Results

[0118] The results are as follows Figure 4 As shown in the figure, with the extension of extraction time, the extraction efficiency is slightly different (the peak area of ​​the main component peak is slightly increased). Considering the time saving and extraction efficiency, 30 min was finally selected as the extraction time for the preparation of the test solution.

[0119] 5. Extraction Method

[0120] 1. Experimental Methods

[0121] Preparation of the test sample: Take 2 g of corn silk broken wall slices, accurately weigh 2 portions, place in stoppered conical flasks, accurately add 25 mL of methanol to each portion, weigh the weight, and ultrasonically extract for 30 min (power 560 W, frequency 40 kHz). After taking out, let it cool, make up the weight with methanol, filter, evaporate the filtrate No. 1 to dryness, dissolve the residue in methanol to 2 mL, filter with a 0.45 μm microporous membrane, and take the filtrate to obtain the product;

[0122] Filtrate No. 2 was evaporated to dryness, and the residue was dissolved in 15 mL of water, extracted twice with ethyl acetate, 15 mL each time, and the ethyl acetate layers were combined and evaporated to dryness. The residue was dissolved in methanol and the volume was adjusted to 2 mL. The residue was filtered through a 0.45 μm microporous filter membrane and the filtrate was taken to obtain the product.

[0123] The chromatographic conditions were in accordance with those in “1. Extraction solvent”.

[0124] 2. Experimental Results

[0125] The results are as follows Figure 5 As shown in the figure, the number of peaks obtained by each extraction solvent is not much different, and the types of component peaks obtained are basically the same, but the response value of the main component peak obtained by ethyl acetate extraction is significantly higher than that by methanol extraction, and there are relatively few interfering small peaks in the ethyl acetate extraction sample, and the baseline is stable. Therefore, methanol extraction followed by water dissolution and then ethyl acetate extraction are selected as the extraction method for corn silk wall-broken slices.

[0126] 6. Extraction times

[0127] 1. Experimental Methods

[0128] Preparation of the test sample: Take 2g of corn silk broken wall slices, accurately weigh three portions, place them in stoppered conical flasks, accurately add 25mL of methanol to each portion, weigh the weight, and ultrasonically extract for 30min (power 560W, frequency 40kHz). After removal, cool, add methanol to the weight, filter, and evaporate the filtrate to dryness. Dissolve the residue in 15mL of water, extract No. 1 once with ethyl acetate, No. 2 twice with ethyl acetate, and No. 3 three times with ethyl acetate, each time 15mL. Combine the ethyl acetate layers and evaporate to dryness. Dissolve the residue in methanol to a volume of 2mL, filter through a 0.45μm microporous membrane, and obtain the filtrate.

[0129] The chromatographic conditions were in accordance with those in “1. Extraction solvent”.

[0130] 2. Experimental Results

[0131] The results are as follows Figure 6As shown in the figure: with the increase of the number of extractions, the extraction efficiency is slightly different (the peak area of ​​the main component peak is slightly increased). Considering the saving of reagents and extraction efficiency, two extractions were finally selected as the number of extractions for the preparation of the test solution.

[0132] Example 2 A pre-treatment method for HPLC fingerprint of corn silk broken wall slices

[0133] Take 2 g of broken wall pieces of corn silk, accurately weigh them, place them in a stoppered conical flask, accurately add 25 mL of methanol, weigh them, sonicate for 30 minutes, cool to room temperature, make up the weight with methanol, shake well, filter, evaporate the filtrate to dryness, dissolve the residue with 15 mL of water, extract twice with ethyl acetate, each time with 15 mL, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with methanol to make up the volume to 2 mL, filter with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the product.

[0134] Example 3 Effect of Chromatographic Conditions on HPLC Fingerprints of Broken Wall Corn Silk Pieces

[0135] 1. Detection wavelength

[0136] 1. Experimental Methods

[0137] The corn silk broken wall slices test solution was prepared according to the pretreatment method of Example 2.

[0138] Scan the test solution at all wavelengths and select the optimal detection wavelength from its isolinear absorption plot. The criteria for evaluation are: high analytical signal response, high peak information content, uniform peak distribution and stable baseline, and good peak separation at this wavelength.

[0139] 2. Experimental Results

[0140] The results are as follows Figure 7 As shown in the figure, the sample peak at 273 nm has more complete information and a larger response value, so 273 nm is selected as the detection wavelength.

[0141] 2. Mobile Phase System

[0142] 1. Experimental Methods

[0143] The corn silk broken wall slices test solution was prepared according to the pretreatment method of Example 2.

[0144] The chromatographic conditions were in accordance with "I. Extraction Solvent" in Example 1, except that the mobile phase systems were set as follows: acetonitrile-0.1% acetic acid aqueous solution (v / v) (S1); acetonitrile-0.1% formic acid aqueous solution (v / v) (S2); acetonitrile-0.3% phosphoric acid aqueous solution (v / v) (S3); methanol-0.3% phosphoric acid aqueous solution (v / v) (S4); methanol-water (S5); and acetonitrile-water (S6). The selection criteria were a large number of peaks in the sample spectrum, uniform peak distribution, good peak shape and main component peak separation, and a stable baseline.

[0145] 2. Experimental Results

[0146] The results are as follows Figure 8 As shown, the mobile phase acetonitrile-0.3% phosphoric acid aqueous solution (v / v) obtained more peak information of the test sample, a relatively stable baseline, and good separation and peak shape. Therefore, the mobile phase system was finally determined to be acetonitrile-0.3% phosphoric acid aqueous solution (v / v).

[0147] 3. Elution Gradient

[0148] 1. Experimental Methods

[0149] The corn silk broken wall slices test solution was prepared according to the pretreatment method of Example 2.

[0150] Chromatographic conditions: A SHISEIDO CAPCELL PAK MGⅢ C18 column (5 μm, 250 mm × 4.6 mm) was used; the flow rate was 1.0 mL / min; the column temperature was 25°C; the detection wavelength was 273 nm; the sample injection volume was 10 μL; acetonitrile was used as mobile phase A, and 0.3% aqueous phosphoric acid was used as mobile phase B. Elution was performed according to the elution gradient shown in Table 1. Sample selection criteria were a stable baseline, a large number of peaks, a uniform peak distribution, and high resolution of the main component peaks.

[0151] Table 1 Mobile phase elution gradient

[0152]

[0153] 2. Experimental Results

[0154] The results are as follows Figure 9 As shown, the number of peaks in each gradient is basically the same, but the baseline of gradient 12 is stable, the peak distribution is uniform, and the main component peak separation effect is good, so gradient 12 is selected as the elution gradient.

[0155] 4. Mobile phase elution flow rate

[0156] 1. Experimental Methods

[0157] The corn silk broken wall slices test solution was prepared according to the pretreatment method of Example 2.

[0158] Chromatographic conditions: Use a SHISEIDO CAPCELL PAK MGⅢC18 column (5μm, 250mm×4.6mm); column temperature 30℃; detection wavelength 273nm; sample injection volume 10μL; acetonitrile as mobile phase A, 0.3% phosphoric acid aqueous solution (v / v) as mobile phase B, elution with gradient 12 in "III. Elution gradient", and set flow rates to 0.8, 0.9, 1.0, 1.1, and 1.2mL / min, respectively.

[0159] 2. Experimental Results

[0160] The results are as follows Figure 10 As shown in the figure, the effect of flow rate on elution is not significant. When the flow rate is 1.0 mL / min, the resolution, symmetry, and peak shape are good, and the elution time is moderate. Therefore, 1.0 mL / min is selected as the elution flow rate.

[0161] 5. Column temperature

[0162] 1. Experimental Methods

[0163] The corn silk broken wall slices test solution was prepared according to the pretreatment method of Example 2.

[0164] Chromatographic conditions: Use a SHISEIDO CAPCELL PAK MGⅢC18 column (5μm, 250mm×4.6mm); flow rate 1.0mL / min; detection wavelength 273nm; sample injection volume 10μL; acetonitrile as mobile phase A, 0.3% phosphoric acid aqueous solution (v / v) as mobile phase B, elution with gradient 12 in "III. Elution gradient", and set the column temperature to 20, 25, 30, 35 and 40℃, respectively.

[0165] 2. Experimental Results

[0166] The results are as follows Figure 11 As shown in the figure, the separation and peak shape of the chromatographic peaks at 30℃ are good and the elution time is moderate, so the column temperature of 30℃ is used as the elution column temperature.

[0167] 6. Injection Volume

[0168] 1. Experimental Methods

[0169] The corn silk broken wall slices test solution was prepared according to the pretreatment method of Example 2.

[0170] Chromatographic conditions: A SHISEIDO CAPCELL PAK MGⅢC18 column (5 μm, 250 mm × 4.6 mm) was used; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 273 nm; acetonitrile was used as mobile phase A, 0.3% aqueous phosphoric acid (v / v) was used as mobile phase B, and the gradient 12 in “III. Elution Gradient” was used for elution. The injection volumes were set to 5, 10, 15, 20, and 25 μL, respectively.

[0171] 2. Experimental Results

[0172] The results are as follows Figure 12 As shown in the figure, the separation and peak shape of the chromatographic peaks are good and the elution time is moderate under the injection volume of 10 μL, so the injection volume of 10 μL is used as the elution injection volume.

[0173] Example 4: Method for constructing an HPLC fingerprint of corn silk broken wall slices

[0174] 1. Preparation of Reference Solution

[0175] Take an appropriate amount of ferulic acid reference substance, accurately weigh it, and add methanol to make a reference substance solution containing 250.0 μg of ferulic acid per 1 mL.

[0176] 2. Preparation of test solution

[0177] Take 2 g of broken wall slices of corn silk from 20 batches respectively, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of methanol, weigh, ultrasonicate for 30 min, cool to room temperature, make up with methanol, shake well, filter, evaporate the filtrate to dryness, dissolve the residue with 15 mL of water, extract twice with ethyl acetate, 15 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with methanol to 2 mL, filter with a 0.45 μm microporous filter membrane, and take the filtrate to obtain.

[0178] 3. Chromatographic conditions and system suitability

[0179] Octadecylsilane bonded silica gel was used as the packing material (5 μm, 250 mm × 4.6 mm); acetonitrile was used as the mobile phase A, and 0.3% aqueous phosphoric acid (v / v) was used as the mobile phase B. Gradient elution was performed according to the conditions in Table 2. The detection wavelength was 273 nm; the column temperature was 30°C; and the flow rate was 1.0 mL / min. The number of theoretical plates calculated based on ferulic acid should be no less than 10,000.

[0180] Table 2 Mobile phase elution gradient

[0181] Time (min) Mobile phase A (%) Mobile phase B (%) 0～8 10→19 90→81 8～13 19→19 81→81 13～55 19→45 81→55 55～65 45→68 55→32 65～80 68→68 32→32 80～85 68→10 32→90

[0182] 4. Assay

[0183] Accurately pipette 10 μL of each reference solution and test solution into a liquid chromatograph, measure, and record the chromatogram. Use the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" developed by the Chinese Pharmacopoeia Committee to generate a reference chromatogram (average method). Screen and identify common peaks, and generate an HPLC fingerprint for corn silk wall-broken slices using software.

[0184] 5. Chromatographic peak identification and reference peak selection

[0185] A total of 18 chromatographic peaks were identified as the common fingerprint peaks of corn silk broken wall slices ( Figure 13 and Figure 14 ), the separation effect of peak 5 was good, and the peak position and peak height were in the middle, so peak 5 was determined to be the reference peak (S peak). The retention time and DAD spectrum of the ferulic acid reference peak corresponded one to one with peak 5 in the test sample and reference spectrum respectively ( Figure 15 ), so peak 5 was determined to be ferulic acid.

[0186] Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software developed by the Chinese Pharmacopoeia Commission, similarity analysis was performed between the chromatograms of 20 sample batches and the generated reference chromatograms (selecting a time window of 0.1) and the relative retention times of shared peaks were calculated. The results show that the similarity between the chromatograms of all 20 sample batches and the reference chromatograms was greater than 0.887. Considering that the raw medicinal materials for broken-wall slices are subject to certain inter-batch variability due to climate factors such as temperature and rainfall during the cultivation process in ecological planting bases, a limit of 0.85 was proposed, indicating that "according to the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine, the similarity between the fingerprints of the test sample and the reference fingerprints calculated must not be less than 0.85."

[0187] Table 3 Similarity of fingerprints of 20 batches of corn silk broken wall slices

[0188]

[0189]

[0190] Table 4 Relative retention time of common peaks in fingerprint of corn silk broken wall slices

[0191]

[0192] Taking peak 5 as the reference peak (S peak), the relative retention times of characteristic peaks 1 to 18 are within ±10% of the specified values; the specified values ​​are 0.441 for peak 1, 0.624 for peak 2, 0.689 for peak 3, 0.956 for peak 4, 1.000 for peak 5, 1.129 for peak 6, 1.216 for peak 7, 1.451 for peak 8, 1.519 for peak 9, 1.672 for peak 10, 2.549 for peak 11, 2.642 for peak 12, 2.918 for peak 13, 3.445 for peak 14, 4.125 for peak 15, 4.161 for peak 16, 4.188 for peak 17, and 4.257 for peak 18.

[0193] Example 5 Methodological Investigation

[0194] 1. Specificity Investigation

[0195] 1. Experimental Methods

[0196] Take corn silk broken wall slices with batch number 2203010A, prepare test solution and blank test solution according to the method of Example 4, inject the sample, analyze according to the chromatographic conditions of Example 4, and record the chromatogram.

[0197] 2. Experimental Results

[0198] The results are as follows Figure 16 As shown in the figure, the dissolving solvent and elution solvent used to prepare the test sample had basically no absorption under the chromatographic conditions and had no interference with the chromatographic analysis of the test sample, indicating that the established method had good specificity.

[0199] 2. Instrument Precision Inspection

[0200] 1. Experimental Methods

[0201] Corn silk broken wall slices with batch number 2203010A were prepared into a test solution according to the method of Example 4. The sample was injected six times continuously and analyzed according to the chromatographic conditions of Example 4. The chromatograms were recorded. The data of the six obtained chromatograms were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) of the Chinese Pharmacopoeia Committee.

[0202] 2. Experimental Results

[0203] The results are shown in Tables 5 to 7 and Figure 17 As shown in the figure, the similarity of the full spectrum of the six spectra all reached 1.000, the RSD values ​​of the relative retention times of the 18 main component peaks were all less than 0.133%, and the RSD values ​​of the relative peak areas were all less than 1.812%. The inspection results show that the instrument precision meets the requirements.

[0204] Table 5 Similarity of spectra of instrument precision investigation

[0205] Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 Precision 6 Precision 1 1.000 1.000 1.000 1.000 1.000 1.000 Precision 2 1.000 1.000 1.000 1.000 1.000 1.000 Precision 3 1.000 1.000 1.000 1.000 1.000 1.000 Precision 4 1.000 1.000 1.000 1.000 1.000 1.000 Precision 5 1.000 1.000 1.000 1.000 1.000 1.000 Precision 6 1.000 1.000 1.000 1.000 1.000 1.000

[0206] Table 6 Relative retention time of the spectrum of instrument precision investigation

[0207]

[0208]

[0209] Table 7 Relative peak area of ​​instrument precision investigation spectrum

[0210]

[0211] 3. Repeatability Investigation

[0212] 1. Experimental Methods

[0213] Six portions of corn silk broken wall slices with batch number 2203010A were prepared into test solutions according to the method of Example 4, analyzed according to the chromatographic conditions of Example 4, and the chromatograms were recorded. The six obtained chromatograms were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) of the Chinese Pharmacopoeia Committee.

[0214] 2. Experimental Results

[0215] The results are shown in Tables 8 to 10 and Figure 18 As shown in the figure, the similarity of the six spectra reached 0.999, the RSD values ​​of the relative retention times of the 18 principal component peaks were all less than 0.327%, and the RSD values ​​of the relative peak areas were all less than 2.866%. The results of the investigation showed that the repeatability of the method met the requirements.

[0216] Table 8 Similarity of repeatability test spectra

[0217] Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 Repeatability 1 1.000 1.000 0.999 0.999 0.999 1.000 Repeatability 2 1.000 1.000 0.999 0.999 0.999 1.000 Repeatability 3 0.999 0.999 1.000 1.000 1.000 1.000 Repeatability 4 0.999 0.999 1.000 1.000 1.000 0.999 Repeatability 5 0.999 0.999 1.000 1.000 1.000 0.999 Repeatability 6 1.000 1.000 1.000 0.999 0.999 1.000

[0218] Table 9 Relative retention time of repeatability test spectra

[0219]

[0220] Table 10 Relative peak areas of repeatability test spectra

[0221]

[0222] 4. Stability Investigation

[0223] 1. Experimental Methods

[0224] Corn silk broken wall slices with batch number 2203010A were prepared into test solutions according to the method of Example 4. Samples were injected at 0, 2, 4, 8, 12, and 24 hours after preparation, and analyzed according to the chromatographic conditions of Example 4, and chromatograms were recorded. The six obtained chromatograms were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) of the Chinese Pharmacopoeia Commission.

[0225] 2. Experimental Results

[0226] The results are shown in Tables 11 to 13 and Figure 19 As shown, the full spectrum similarity of the six spectra was above 0.999, the RSD values ​​of the relative retention times of the 18 principal component peaks were all less than 0.180%, and the RSD values ​​of the relative peak areas were all less than 1.887%. The results of the investigation showed that the sample was stable within 24 hours and met the methodological requirements.

[0227] Table 11 Similarity of stability test spectra

[0228] 0h 2h 4h 8h 12h 24h 0h 1.000 1.000 1.000 1.000 1.000 0.999 2h 1.000 1.000 1.000 1.000 1.000 1.000 4h 1.000 1.000 1.000 1.000 1.000 1.000 8h 1.000 1.000 1.000 1.000 1.000 1.000 12h 1.000 1.000 1.000 1.000 1.000 1.000 24h 0.999 1.000 1.000 1.000 1.000 1.000

[0229] Table 12 Relative retention time of stability test spectrum

[0230]

[0231] Table 13 Relative peak areas of stability test spectra

[0232]

[0233]

[0234] 5. Durability

[0235] 1. Different chromatographic columns

[0236] (1) Experimental methods

[0237] The corn silk broken wall pieces of batch number 2203010A were prepared into test solution according to the method of Example 4, respectively using Kromasil 100-5-C18 (5 μm, 250 mm × 4.6 mm), YMC Triart C18 (5 μm, 250 mm × 4.6 mm), Four different chromatographic columns, a T3 C18 (5 μm, 250 mm × 4.6 mm) and a Shiseido CAPCell PAK C18 MGIII (5 μm, 250 mm × 4.6 mm), were used for analysis according to the chromatographic conditions described in Example 4, and chromatograms were recorded. Data analysis of the four obtained chromatograms was performed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) developed by the Chinese Pharmacopoeia Commission.

[0238] (2) Experimental results

[0239] The results are shown in Table 14 and Figure 20 As shown: the similarity of the four spectra is above 0.991, indicating that this method is basically applicable to different chromatographic columns. However, the results of the investigation suggest that different chromatographic columns can be used for analysis, although the overall chromatographic Figure 1 However, the RSD values ​​of the relative retention times of the 18 main component peaks vary greatly, so there are still certain differences in the retention time, peak area and separation of the main chromatographic peaks. It is recommended to use the same brand and model of chromatographic columns when analyzing samples by fingerprint analysis to reduce the error in sample quality evaluation caused by chromatographic column differences.

[0240] Table 14 Similarity of spectra observed on different chromatographic columns

[0241]

[0242] 2. Different instruments

[0243] (1) Experimental methods

[0244] Corn silk cracked decoction pieces with batch number 2203010A were prepared into a test solution according to the method of Example 4. The solution was analyzed on a Waters 2695 / 2998, an Agilent 1260 II, and a Thermo Fisher Scientific U3000 liquid chromatograph using a Shiseido CAPCell PAK C18 MG III (250 mm × 4.6 mm, 5 μm) column according to the chromatographic conditions of Example 4, and the chromatograms were recorded. The three obtained chromatograms were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) of the Chinese Pharmacopoeia Commission.

[0245] (2) Experimental results

[0246] The results are shown in Table 15 and Figure 21 As shown: the similarity of the three spectra is 0.998, which meets the methodological requirements. However, it can be seen from the spectra that different instruments analyze the Figure 1 However, there are still some differences in the separation of the main chromatographic peaks. It is recommended to use the same liquid chromatograph when analyzing samples by fingerprint analysis to reduce the error in sample quality evaluation caused by instrument differences.

[0247] Table 15 Similarity of spectra observed by different instruments

[0248] Spectrum Agilent 1260Ⅱ Thermo U3000 Waters2695 / 2998 Agilent 1260Ⅱ 1.000 0.999 0.998 Thermo U3000 0.999 1.000 0.999 Waters2695 / 2998 0.998 0.999 1.000

[0249] Comparative Example 1

[0250] 1. Experimental Methods

[0251] Preparation of the test solution: Take corn silk broken wall slices and prepare the test solution according to the test solution preparation method in "HPLC fingerprint of corn silk" by Xie Caixia et al. (published on May 5, 2015). The specific experimental steps are as follows:

[0252] Accurately weigh 6.0 g of corn silk broken wall slices, add 180 mL of 60% ethanol aqueous solution (v / v), reflux at 80°C for 2 h, filter, take the supernatant and evaporate to dryness in a water bath, dissolve it in methanol and make up to volume in a 10 mL volumetric flask, ultrasonicate for 30 min, and filter through a 0.45 μm microporous filter membrane.

[0253] The chromatographic conditions were the same as in Example 4.

[0254] 2. Experimental Results

[0255] The results are as follows Figure 22 As shown, the interference peaks in the early stage of the chromatogram obtained by the chromatographic conditions of the present invention are smaller than those in the comparative file 1, the baseline is relatively flat, and the peak shape is better.

[0256] Comparative Example 2

[0257] 1. Experimental Methods

[0258] The corn silk broken wall slices test solution was prepared according to the method of Example 4.

[0259] Chromatographic conditions: Elution was performed according to the chromatographic conditions in "HPLC Fingerprint of Corn Silk" by Xie Caixia et al. (published on May 5, 2015). The specific experimental steps are as follows:

[0260] An Agilent ZORBAX SB-C column (4.6 mm × 250 mm, 5 μm) was used with a detection wavelength of 273 nm, a column temperature of 30°C, an injection volume of 10 μL, a flow rate of 0.8 mL / min, methanol as mobile phase A, and 0.2% aqueous phosphoric acid (v / v) as mobile phase B. The gradient elution conditions were: 0-45 min, 10%-40% A, 45-85 min, 40%-60% A, and 85-100 min, 60%-70% A.

[0261] 2. Experimental Results

[0262] The results are as follows Figure 23 As shown, there is no interference from other peaks in the early stage, the baseline is relatively flat, and the peak shape is symmetrical. However, the separation of some peaks in the second half of the chromatogram is not ideal, and the detection and analysis takes a long time.

[0263] Comparative Example 3

[0264] 1. Experimental Methods

[0265] Corn silk broken wall slices were taken and the test sample preparation method and chromatographic conditions in "HPLC fingerprint of corn silk" by Xie Caixia et al. (published on May 5, 2015) were used for the experiment. The specific experimental steps are as follows:

[0266] Accurately weigh 6.0 g of corn silk broken wall slices, add 180 mL of 60% ethanol aqueous solution (v / v), reflux at 80°C for 2 h, filter, take the supernatant and evaporate to dryness in a water bath, dissolve it in methanol and make up to volume in a 10 mL volumetric flask, ultrasonicate for 30 min, and filter through a 0.45 μm microporous filter membrane.

[0267] An Agilent ZORBAX SB-C column (4.6 mm × 250 mm, 5 μm) was used with a detection wavelength of 273 nm, a column temperature of 30°C, an injection volume of 10 μL, a flow rate of 0.8 mL / min, methanol as mobile phase A, and 0.2% aqueous phosphoric acid (v / v) as mobile phase B. The gradient elution conditions were: 0-45 min, 10%-40% A, 45-85 min, 40%-60% A, and 85-100 min, 60%-70% A.

[0268] 2. Experimental Results

[0269] The results are as follows Figure 24 As shown, this method has low chromatographic peak response, large early solvent peaks and many impurity peaks, few common peaks, long detection time, and poor display effect of the entire spectrum.

[0270] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of corn silk broken wall slices, characterized in that: The construction method comprises the following steps: Preparation of test solution: S1. After mixing the sample with methanol, ultrasonicate it, cool it, use methanol to make up for the weight loss, separate the solid and liquid, and remove the methanol in the liquid; S2. The product of step S1 was dissolved in water, extracted with ethyl acetate, and the ethyl acetate layer was removed to obtain a residue; S3. Dissolve the residue with methanol, filter through a microporous membrane, and take the filtrate to obtain the test solution; Reference solution: ferulic acid solution; Chromatographic conditions: Octadecylsilane bonded silica gel was used as the column filler, acetonitrile was used as mobile phase A, and 0.28-0.32% phosphoric acid solution was used as mobile phase B. In the gradient elution procedure, the volume percentage of mobile phase A in the mobile phase system varied as follows: From 0 to 8 min, mobile phase A was increased from 10% to 19%; 8-13 min, mobile phase A 19%; From 13 to 55 min, mobile phase A increased from 19% to 45%; From 55 to 65 min, mobile phase A increased from 45% to 68%; 65-80 min, mobile phase A 68%; From 80 to 85 min, mobile phase A decreased from 68% to 10%; The detection wavelength is 273 nm, and the theoretical plate number calculated based on ferulic acid should be no less than 10,000; The above-mentioned construction method was used to detect different batches of corn silk broken wall slices, record the chromatograms, import the chromatograms into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, screen and confirm the common peaks, and obtain the HPLC fingerprint of corn silk broken wall slices.

2. The construction method according to claim 1, wherein In step S1, the mass volume ratio of the sample to be tested to methanol is 1 g: (12-13) mL.

3. The construction method according to claim 1, wherein In step S1, the ultrasonication time is 25 to 35 minutes.

4. The construction method according to claim 1, characterized in that In step S2, the extraction is performed 2 to 3 times.

5. The construction method according to claim 1, characterized in that The column temperature of the chromatographic column is 28-32°C.

6. The construction method according to claim 1, wherein The flow rate of the gradient elution is 0.8-1.2 mL / min.

7. Application of the construction method according to any one of claims 1 to 6 in establishing HPLC fingerprints of corn silk broken wall slices and / or quality control of corn silk broken wall slices.

8. A quality detection method for corn silk broken wall slices, characterized in that: The following steps are involved: S1. Take the tested corn silk broken wall slices sample, detect using the construction method according to any one of claims 1 to 6, record the chromatogram, and obtain the HPLC spectrum of the tested corn silk broken wall slices sample; S2. The HPLC fingerprint of the crushed corn silk slices according to claim 1 and the HPLC fingerprint of the crushed corn silk slices to be tested are introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system for comparison. The qualified ones are judged to be qualified if they meet the following two conditions: The HPLC spectrum of the corn silk broken wall slice sample to be tested presents a fingerprint peak whose relative retention time is within ±10% of the specified value in the HPLC fingerprint spectrum of the corn silk broken wall slice; The similarity between the HPLC spectrum of the corn silk broken wall slice sample to be tested and the HPLC fingerprint spectrum of the corn silk broken wall slice sample is calculated, and the similarity between the HPLC spectrum of the corn silk broken wall slice sample to be tested and the HPLC fingerprint spectrum of the corn silk broken wall slice sample is not less than 0.85; The HPLC fingerprint of the corn silk broken wall slices contains 18 fingerprint peaks, with peak 5 as the S peak, and the specified values ​​of the relative retention times are: Peak 1 is 0.441, Peak 2 is 0.624, Peak 3 is 0.689, Peak 4 is 0.956, Peak 5 is 1.000, Peak 6 is 1.129, Peak 7 is 1.216, Peak 8 is 1.451, Peak 9 is 1.519, Peak 10 is 1.672, Peak 11 is 2.549, Peak 12 is 2.642, Peak 13 is 2.918, Peak 14 is 3.445, Peak 15 is 4.125, Peak 16 is 4.161, Peak 17 is 4.188, and Peak 18 is 4.257.

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