Construction method for HPLC content determination of Cinochrysum wilsonii medicinal herb pieces, standard decoction and formula granules

Abstract

The present invention provides a method for constructing HPLC content determination of large lung tendon herb slices, standard decoctions, and formula granules, including: A) dissolving the test sample raw material in a solvent, extracting, and obtaining a test liquid; B) determining the test liquid by high performance liquid chromatography to obtain HPLC characteristic patterns of large lung tendon herb slices, standard decoctions, and formula granules; the HPLC chromatographic conditions are as follows: the chromatographic column is a C 18 column; the mobile phase A is a methanol solution, and the mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution. The present invention adopts high performance liquid chromatography, selects methanol 0.1% phosphoric acid solution as the mobile phase for gradient elution, and uses rosmarinic acid and 4 caffeoylquinic acid as reference substances to establish large lung tendon herb slices, standard decoctions, and formula granules HPLC content determination methods, which provide more and more scientific technical means for controlling the medicinal quality of large lung tendon herb slices, standard decoctions, and formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a construction method for HPLC content determination of dafeijincao medicinal material slices, standard decoctions and formula granules. Background Art

[0002] Big lung grass is the dried whole herb of the Umbelliferae plant Sanicμla lamelligera Hance., which has the effects of dispelling cold and relieving cough, promoting blood circulation and relieving dysmenorrhea.

[0003] Therefore, in order to ensure the uniformity and stability of the quality of Dafeijincao decoction pieces, standard decoctions, and formula granules, it is necessary to establish a detection method for Dafeijincao decoction pieces, standard decoctions, and formula granules, which is conducive to the overall evaluation of the scientificity and rationality of the process of Dafeijincao decoction pieces, standard decoctions, and formula granules, and can better control their internal quality as a whole to ensure clinical efficacy. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC content determination of Dafeijincao medicinal material slices, standard decoctions, and formula granules. The method of the present invention is accurate and reliable.

[0005] The present invention provides a method for constructing HPLC content determination of Dafeijincao medicinal material slices, standard decoctions, and formula granules, comprising:

[0006] A) Dissolve the test sample in a solvent and extract to obtain a test solution;

[0007] B) The test solution is measured by high performance liquid chromatography to obtain HPLC characteristic patterns of the Dafeijincao herbal medicine slices, standard decoction, and formula granules;

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

[0009] The test sample raw materials of the present invention are one or more of the following: dafeijin herbal medicine, dafeijin herbal decoction pieces, dafeijin herbal standard decoction or dafeijin herbal formula granules.

[0010] Provided is a method for constructing HPLC content determination of large lung tendon herb slices, standard decoctions, and formula granules. First, the test sample raw material is dissolved in a solvent and extracted to obtain a test solution.

[0011] Specifically, the test sample raw materials are dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the product.

[0012] The extraction of the present invention is heating reflux extraction or ultrasonic extraction; the time of the heating water flow extraction is preferably 15 to 45 minutes; more preferably 20 to 40 minutes; most preferably 30 minutes.

[0013] The ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 30min.

[0014] The ratio of the mass g of the test sample raw material to the volume mL of the solvent is 0.5:50.

[0015] The above raw materials of the present invention can all be subjected to quality control and qualitative and quantitative detection by the method of the present invention.

[0016] The present invention further comprises preparing a reference solution: taking rosmarinic acid and 4-caffeoylquinic acid respectively, dissolving them in 70% methanol to obtain a reference solution;

[0017] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the large lung tendon herb slices, standard decoction, and formula granules are qualitatively analyzed based on the chromatogram of the reference.

[0018] The concentrations of the reference solution of the present invention are preferably: 21 to 520 μg / mL for rosmarinic acid, and 18 to 455 μg / mL for 4-caffeoylquinic acid.

[0019] Specifically, in the Dafeijincao herbal slices, Dafeijincao standard decoction, and Dafeijincao formula granules, the rosmarinic acid concentration ranged from 20.78242 to 519.5605 μg / ml, with a linear relationship of y = 3,553.4968x - 6,136.5358, and R² = 0.9999; the 4-caffeoylquinic acid concentration ranged from 18.217184 to 455.4296 μg / ml, with a linear relationship of y = 3,648.4793x - 5,517.6844, and R² = 0.9999. The results showed that both rosmarinic acid and 4-caffeoylquinic acid showed good linear relationships.

[0020] The test solution is determined by high performance liquid chromatography to obtain HPLC characteristic spectra of the dafeijincao herbal medicine slices, standard decoction and formula granules.

[0021] The HPLC conditions are as follows: the chromatographic column is a C18 column; the chromatographic column is an Agilent 5TC-C 18 、Kromasil C 18 , Agilent ZORBAX Eclipse XDB C 18250×4.6mm 5μm; column temperature is 25-35°C; at the above chromatographic column temperature, the chromatographic peaks of the present invention are symmetrical, the separation is good, and the peaks are more complete.

[0022] The mobile phase A of the present invention is a methanol solution, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0023] Specifically, the gradient elution is as follows:

[0024] 0-5min, phase A: 15%-40%, phase B: 85-60%;

[0025] 5-35min, phase A: 40%-65%, phase B: 60%-35%.

[0026] The theoretical plate number calculated based on the genistein peak should be no less than 4000.

[0027] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0028] The flow rate of the mobile phase of the present invention is 0.8-1.2 mL / min, more preferably 1.0 mL / min.

[0029] The present invention found that the chromatographic peaks were well separated and the separation degree was moderate under the above flow rate, which is the most preferred solution.

[0030] The injection volume was 10 μL.

[0031] The detection wavelength is 330 nm. The inventors found that at 330 nm, the chromatographic peak information volume is larger, the chromatogram baseline is more stable, and the peak area of each peak is larger.

[0032] The present invention provides a method for identifying characteristic patterns of lung tendon herb slices, standard decoctions, and formula granules, and adopts the above-mentioned method to perform detection and analyze the detection results.

[0033] The present invention provides a method for constructing an HPLC content determination method for Dafeijincao medicinal material slices, standard decoctions, and formula granules, comprising: A) dissolving a test sample raw material with a solvent and extracting it to obtain a test solution; B) determining the test solution by high-performance liquid chromatography to obtain an HPLC characteristic spectrum of the Dafeijincao medicinal material slices, standard decoctions, and formula granules; the chromatographic conditions of the high-performance liquid chromatography method are: a chromatographic column is a C18 column; mobile phase A is a methanol solution, and mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution. The present invention adopts high-performance liquid chromatography, selects methanol-0.1% phosphoric acid solution as the mobile phase for gradient elution, and uses rosmarinic acid and 4-caffeoylquinic acid as reference substances to establish an HPLC content determination method for Dafeijincao medicinal material slices, standard decoctions, and formula granules, providing more and more scientific technical means for controlling the medicinal quality of Dafeijincao medicinal material slices, standard decoctions, and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The chromatograms of different flow rates of Example 1 are shown;

[0035] Figure 2 The chromatograms of different column temperatures of Example 1 are shown;

[0036] Figure 3 The chromatograms of different injection amounts of Example 3 are shown;

[0037] Figure 4 This is a graph showing the specific results of Example 3.

[0038] Figure 5 This is the rosmarinic acid standard curve diagram of Example 3;

[0039] Figure 6 This is the standard curve of 4-caffeoylquinic acid in Example 3.

[0040] Figure 7 This is the specific result of Example 8.

[0041] Figure 8 This is the standard curve of rosmarinic acid in Example 8;

[0042] Figure 9 This is the standard curve of 4-caffeoylquinic acid in Example 8;

[0043] Figure 10 This is the specific result diagram;

[0044] Figure 11 This is the standard curve diagram of rosmarinic acid in Example 12;

[0045] Figure 12 This is the standard curve of 4-caffeoylquinic acid in Example 12;

[0046] Figure 13 This is a chromatogram of the lung meridian grass used in Example 14 to identify the fake large lung tendon grass;

[0047] Figure 14 It is the chromatogram of Comparative Example 2;

[0048] Figure 15 It is the chromatogram of Comparative Example 3;

[0049] Figure 16 It is the chromatogram of Comparative Example 4. DETAILED DESCRIPTION

[0050] The present invention provides a method for constructing HPLC content determination of Radix Phellodendri decoction pieces, standard decoctions, and formula granules. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0051] To further illustrate the present invention, the following describes in detail a method for constructing HPLC content determination of Dafeijincao herbal medicine slices, standard decoctions, and formula granules provided by the present invention in combination with examples.

[0052] Experimental instruments and materials

[0053] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph, Shimadzu-20AD high performance liquid chromatograph;

[0054] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0055] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0056] Ultrasonic cleaner: KQ5200DB (600 W, 40 kHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0057] Columns: Agilent 5TC-C18(2), Kromasil C18, Agilent ZORBAX Eclipse XDB C 18 .

[0058] 2 Reagents and test drugs

[0059] Methanol (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), phosphoric acid (chromatographic grade, Tianjin Komiou Chemical Reagent Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), and the water used in the mobile phase were laboratory-made ultrapure water, and the rest were laboratory-made pure water.

[0060] Rosmarinic acid reference substance (China Food and Drug Control Institutes, batch number: 111871-201706);

[0061] 4-Caffeoylquinic acid reference substance (China Food and Drug Control Institutes, batch number: 110885-201703);

[0062] Large lung tendon herbal medicine: 010094-1708001, XLS202109284, XLS202109285, XLS202109286, XLS202109287, XLS202109288, XLS202109289, XLS202109290, XLS202109291, XLS202109292, XL S202109293, XLS202109294, XLS202109295, XLS202109296, XLS202109297, XLS2021 09298, XLS202109299, XLS202109300, XLS202109301, XLS202109302,

[0063] Pulmonaria officinalis slices: DFJC-210901, DFJC-210902, DFJC-210903, DFJC-210904, DFJC-210905, DFJC-210906, DFJC-210907, DFJC-210908, DFJC-210909, DFJC-210910, DFJC-210911, DFJC-210912, DFJC-210913, DFJC-210914, DFJC-210915, DFJC-210916, DFJC-210917, DFJC-210918, DFJC-210919, DFJC-210920, DFJC-210921.

[0064] Standard decoction of Dafeijincao: DFJC-BT-210901, DFJC-BT-210902, DFJC-BT-210903, DFJC-BT-210904, DFJC-BT-210905, DFJC-BT-210906, DFJC-BT-210907, DFJC-BT-210908, DFJC-BT-210909, DFJC-BT-210910, DFJ C-BT-210911, DFJC-BT-210912, DFJC-BT-210913, DFJC-BT-210914, DFJC-BT-210915, DFJC-BT-2 10916, DFJC-BT-210917, DFJC-BT-210918, DFJC-BT-210919, DFJC-BT-210920, DFJC-BT-210921.

[0065] Formula granules of large lung grass: 1902061, SY2112001, SY2112002, SY2112003.

[0066] Example 1 Investigation of chromatographic conditions

[0067] 1.1 Proposed chromatographic conditions

[0068] Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B. Gradient elution was performed according to the requirements in Table 1; the detection wavelength was 330 nm.

[0069] Table 1

[0070]

[0071] 3.2 Flow rate investigation

[0072] Under the above proposed chromatographic conditions, the separation effects of rosmarinic acid and 4-caffeoylquinic acid peaks were investigated at flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively. The results are shown in Table 1. Figure 1 . Figure 1 Chromatograms at different flow rates.

[0073] The results showed that retention time and other indicators were suitable at flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min. Therefore, flow rates of 0.8 mL / min to 1.2 mL / min were suitable. For the convenience of the experiment, the flow rate in the following experiments was set at 1.0 mL / min.

[0074] 3.3 Column temperature investigation

[0075] Under the above proposed chromatographic conditions, the analysis of the test solution at column temperatures of 25℃, 30℃, and 35℃ was investigated, with the separation effect of rosmarinic acid and 4-caffeoylquinic acid chromatographic peaks as evaluation indicators. The results are shown in Figure 2 . Figure 2 Chromatograms at different column temperatures;

[0076] The results showed that the retention time and other indicators were suitable when the column temperature was 25℃, 30℃, and 35℃. In other words, the method performed well when the column temperature was between 25℃ and 35℃. For the convenience of the experiment, the column temperature in the following experiments was set at 30℃.

[0077] 3.4 Sample injection volume investigation

[0078] Under the above chromatographic conditions, 5 μL, 10 μL, and 15 μL were injected respectively, and the separation effect of rosmarinic acid and 4-caffeoylquinic acid chromatographic peaks was used as the evaluation index. The results are shown in Figure 3 . Figure 3 Chromatograms of different injection volumes. The results show that the retention time and other indicators are relatively suitable when the injection volume is 5μL, 10μL, and 15μL. In other words, the method has good indicators when the injection volume is 5-15μL. For the convenience of the experiment, the injection volume is tentatively set to 10μL.

[0079] In summary, the method for determining the content of Radix Pseudostellariae was performed using octadecylsilane bonded silica gel as a filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the detection wavelength was 330 nm.

[0080] Example 2 Preparation of test solution

[0081] 2.1 Investigation of extraction solvent

[0082] Approximately 0.5 g of the medicinal material (batch number: 010094-1708001) was accurately weighed and placed in a stoppered conical flask. The extraction solvents for the test sample were methanol, 50% methanol, 70% methanol, ethanol, 30% ethanol, and 50 ml of water, respectively. The flask was sealed and weighed. Ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. The flask was allowed to cool and weighed again. The weight loss was made up with the extraction solvent, shaken well, filtered, and the filtrate was collected. 10 μl of each extraction solution was injected into a chromatograph and analyzed for rosmarinic acid content under different extraction methods. The results are shown in Table 2.

[0083] Table 2 Analysis results of different extraction solvents

[0084]

[0085] The results showed that 70% methanol had the highest extraction efficiency and was used as the extraction solvent.

[0086] 2.2 Investigation of extraction methods

[0087] Take about 0.5 g of the medicinal material (batch number: 010094-1708001), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 70% methanol, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, and you have it.

[0088] Example 3 Methodological Investigation

[0089] 3.1 Specificity Experiment

[0090] Prepare the test solution and negative control solution according to the proposed method and conduct the test. Figure 4 . Figure 4 This is a graph of specific results.

[0091] The results showed that the chromatogram of negative solution had no interference with the determination of the peak to be tested, indicating that the method had good specificity.

[0092] 3.2 Precision investigation

[0093] The reference solution was injected 6 times continuously, the peak areas of rosmarinic acid and 4-caffeoylquinic acid were recorded, and the RSD values were calculated. The results are shown in Table 3.

[0094] Table 3 Precision inspection results

[0095]

[0096] The results showed that the RSD value of the peak area of rosmarinic acid was 0.3%, and the RSD value of the peak area of 4-caffeoylquinic acid was 0.5%, indicating that the injection precision of this method was good.

[0097] 3.3 Linear Relationship

[0098] Take rosmarinic acid and 4-caffeoylquinic acid reference substances and prepare a mixed standard mother solution of rosmarinic acid solution (51.95605μg / ml, purity 90.5%) and 4-caffeoylquinic acid (45.54296μg / ml, purity 99.7%). Take 1, 2, 5, 10, 15, and 25 ml respectively and place them in a 25 ml volumetric flask. Dilute to the mark with 70%, shake well, and filter. Accurately draw 10μl and inject it into the liquid chromatograph. Analyze and obtain the peak area. Draw the response curve with the rosmarinic acid and 4-caffeoylquinic acid content (X) as the horizontal axis and the peak area (Y) as the vertical axis. The results are shown in Table 4. Figure 5 、 6 . Figure 5 Standard curve of rosmarinic acid; Figure 6 Standard curve of 4-caffeoylquinic acid.

[0099] Table 4 Analysis results of standard curves of rosmarinic acid and 4-caffeoylquinic acid

[0100]

[0101] The results showed that the linear relationship for rosmarinic acid was y = 3,553.4968x - 6,136.5358, R² = 0.9999, over the concentration range of rosmarinic acid (20.78242-519.5605 μg / ml). The linear relationship for 4-caffeoylquinic acid was y = 3,648.4793x - 5,517.6844, R² = 0.9999, over the concentration range of 18.217184-455.4296 μg / ml. Both rosmarinic acid and 4-caffeoylquinic acid exhibited good linear relationships.

[0102] 3.4 Repeatability

[0103] Six portions of the same test sample (batch number: 010094-1708001) were taken, 0.5 g of each portion was accurately weighed, and the test sample solution was prepared by the same operator according to the proposed method. The total content of rosmarinic acid and 4-caffeoylquinic acid in the six portions of the test sample was calculated. The results are shown in Table 5.

[0104] Table 5 Repeatability test results

[0105]

[0106] The results showed that the RSD value of the total content of rosmarinic acid and 4-caffeoylquinic acid was 1.8%, indicating that the method had good reproducibility.

[0107] 3.5 Sample recovery

[0108] About 0.25 g of a test sample with a known content (batch number: 010094-1708001, rosmarinic acid content 0.33%, 4-caffeoylquinic acid content 0.048%) was taken, and a total of 6 portions were accurately weighed. A certain amount of rosmarinic acid reference substance was accurately added to each portion. The test solution was prepared and measured according to the proposed method. The recovery rate was calculated. The results are shown in Table 6. The calculation formula is as follows:

[0109]

[0110] Table 6 Rosmarinic acid sample recovery experimental results

[0111]

[0112] Table 7 4-caffeoylquinic acid sample recovery test results

[0113]

[0114] The results showed that the average recovery rate of rosmarinic acid was 104.95% with an RSD of 1.7%, and the average recovery rate of 4-caffeoylquinic acid was 99.2% with an RSD of 2.4%, indicating that the method has good accuracy.

[0115] 3.6 Intermediate precision

[0116] 3.6.1 Investigation with different instruments

[0117] Based on the experimental conditions proposed above, two portions of Dafeijincao powder (batch number: 010094-1708001) were precisely weighed to prepare test solutions. The solutions were then analyzed on Shimadzu, Agilent 1260, and Waters 2998-e2695 high performance liquid chromatographs (all using Agilent 5TC-C18(2) 250×4.6mm columns). The rosmarinic acid content was calculated, and the results are shown in Table 8.

[0118] Table 8 Experimental results of different instruments

[0119]

[0120] The results showed that the RSD values of rosmarinic acid and 4-caffeoylquinic acid content determination results were 1.9% and 1.6%, respectively, indicating that the intermediate precision of this method was good.

[0121] 3.6.2 Inspection by different personnel and time

[0122] Based on the experimental conditions proposed above, different individuals (A and B) accurately weighed Dafeijincao powder (batch number: 010094-1708001) at different times (T1 and T2) to prepare test samples for determination. The calculated rosmarinic acid content is shown in Table 9.

[0123] Table 9 Results of investigations of different personnel and time

[0124]

[0125] The results showed that the RSD values of rosmarinic acid and 4-caffeoylquinic acid were 1.2% and 1.9%, respectively, indicating that the method had good intermediate precision.

[0126] 3.7 Durability inspection

[0127] 3.7.1 Column durability assessment

[0128] Based on the experimental conditions proposed above, the chromatographic columns were Agilent 5TC-C18(2), KromasilC18, Agilent ZORBAX Eclipse XDB C 18 See Table 10.

[0129] Table 10 Durability inspection results

[0130]

[0131] The results showed that the chromatographic indicators of the different columns were good. The RSD values of rosmarinic acid and 4-caffeoylquinic acid were 0.1% and 1.9% respectively. The chromatographic columns of this method were very durable.

[0132] 3.7.2 Stability test

[0133] The same test solution (batch number: 010094-1708001) was taken and the peak areas of rosmarinic acid and 4-caffeoylquinic acid were determined at 0, 2, 4, 8, 16, and 24 hours, respectively. The results are shown in Table 11.

[0134] Table 11 Rosmarinic acid stability test results

[0135]

[0136] The results showed that under the experimental conditions, the RSD value of rosmarinic acid was 0.9%, and the RSD value of 4-caffeoylquinic acid was 2.0%, indicating that the test solution had good stability within 24 hours.

[0137] Example 4 Verification of Radix Psoraleae Radix and Herba Decoction Pieces and Establishment of HPLC Content Detection Method

[0138] 4.1 The verification results of 21 batches of Dafeijincao medicinal materials and decoction pieces are shown in Tables 12 and 13.

[0139] Table 12 Verification of content of 21 batches of Dafeijincao herbs

[0140]

[0141]

[0142] Table 13 Verification of content of 21 batches of Dafeijincao decoction pieces

[0143]

[0144]

[0145] Conclusion: The content determination method of Dafeijincao medicinal materials and decoction pieces can effectively detect Dafeijincao medicinal materials and decoction pieces, which proves that the method is feasible.

[0146] Example 5 Determination of the content of Dafeijincao herb

[0147] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, and gradient elution as specified in the table below; the column temperature was 30°C; the detection wavelength was 330 nm, and the number of theoretical plates calculated based on the rosmarinic acid peak should be no less than 4000.

[0148]

[0149] Preparation of reference solution: Take appropriate amount of rosmarinic acid and 4-caffeoylquinic acid reference substances, weigh accurately, and add 70% methanol to prepare a mixed standard solution containing 20 μg of rosmarinic acid and 20 μg of 4-caffeoylquinic acid per 1 ml.

[0150] Preparation of test solution: Take about 0.5 g of the product (passed through No. 3 sieve), weigh accurately, place in a stoppered conical flask, add 50 ml of 70% methanol accurately, weigh the weight, ultrasonically treat for 30 minutes, let cool, weigh again, make up the lost weight with 70% methanol, shake well, and obtain.

[0151] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0152] Example 6 Standard decoction HPLC content determination method:

[0153] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, and gradient elution as specified in Table 1 below; the column temperature was 30°C; the detection wavelength was 330 nm, and the number of theoretical plates calculated based on the rosmarinic acid peak should be no less than 4000.

[0154] Preparation of reference solution: Take appropriate amount of rosmarinic acid and 4-caffeoylquinic acid reference substances, weigh accurately, and add 70% methanol to prepare a mixed standard solution containing 20 μg of rosmarinic acid and 20 μg of 4-caffeoylquinic acid per 1 ml.

[0155] Preparation of test solution: Take about 0.1 g of the product, weigh it accurately, place it in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh it, treat it with ultrasound for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, and the solution is ready.

[0156] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0157] Example 7 Preparation of test solution

[0158] 7.1 Extraction solvent investigation

[0159] Approximately 0.1 g of the standard decoction (batch number: DFJC-BT-210901) was accurately weighed and placed in a stoppered conical flask. The sample was tested using methanol, 50% methanol, 70% methanol, ethanol, 30% ethanol, and 25 ml of water as the extraction solvent. The flask was sealed and weighed. Ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. The flask was allowed to cool and weighed again. The loss was made up with the extraction solvent, shaken well, filtered, and the filtrate was collected. 10 μl of each filtrate was injected into a chromatograph to analyze and calculate the rosmarinic acid and 4-caffeoylquinic acid contents under different extraction methods. The results are shown in Table 14.

[0160] Table 14 Analysis results of different extraction solvents

[0161]

[0162] The results showed that 70% methanol had the highest extraction efficiency and was used as the extraction solvent.

[0163] 7.2 Investigation of extraction methods

[0164] Approximately 0.1 g of the standard decoction (batch number: DFJC-BT-210901) was accurately weighed and placed in a stoppered conical flask. 25 ml of 70% methanol was accurately added and weighed. The decoction was then refluxed and sonicated for 30 minutes, respectively. The decoction was cooled and weighed again. The remaining weight was made up with 70% methanol and shaken well. The results are shown in Table 15.

[0165] Table 15 Analysis results of different extraction methods

[0166]

[0167] The results showed that there was little difference in the extraction efficiency between reflux and ultrasonic extraction, and ultrasonic extraction was preferred due to its simplicity.

[0168] 7.3 Extraction time investigation

[0169] Approximately 0.1 g of the standard decoction (batch number: DFJC-BT-210901) was accurately weighed and placed in a stoppered conical flask. 25 ml of 70% methanol was accurately added, the flask was sealed, and the weight was determined. Ultrasonic treatment (power 600 W, frequency 40 kHz) was then performed. The sample was extracted for 20 minutes, 30 minutes, and 40 minutes, respectively. The sample was allowed to cool, and the weight was determined again. The weight loss was supplemented with 70% methanol, shaken well, filtered, and the filtrate was collected. The rosmarinic acid and 4-caffeoylquinic acid contents at different extraction times were analyzed and calculated. The results are shown in Table 16.

[0170] Table 16 Analysis results of different extraction times

[0171]

[0172] The results showed that the total content of rosmarinic acid and 4-caffeoylquinic acid was higher when the extraction time was 30 minutes. Taking all factors into consideration, the extraction time of the test sample was determined to be 30 minutes.

[0173] 7.4 Investigation of solvent addition amount

[0174] Approximately 0.1 g of a standard decoction (batch number: DFJC-BT-210901) was accurately weighed and placed in a stoppered conical flask. 15 ml, 25 ml, and 50 ml of 70% methanol were accurately added, respectively. The flask was stoppered and weighed. Ultrasonic treatment (power 600 W, frequency 40 kHz) was performed. The sample was extracted for 30 minutes for observation. The sample was allowed to cool, and the weight was reweighed. The weight loss was supplemented with 70% methanol, shaken well, filtered, and the filtrate was collected. The rosmarinic acid and 4-caffeoylquinic acid contents at different extraction times were analyzed and calculated. The results are shown in Table 17.

[0175] Table 17 Analysis results of different extraction times

[0176]

[0177] The results showed that the amount of solvent added had little effect on the contents of rosmarinic acid and 4-caffeoylquinic acid in the test sample. Taking all factors into consideration, the amount of solvent added to the test sample was determined to be 25 ml.

[0178] In summary: Through the investigation of the extraction method, solvent, extraction time, and the amount of solvent added, the preparation method of the test solution is determined as follows: take about 0.1g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, and you have it.

[0179] Example 8 Methodological Investigation

[0180] 8.1 Specificity Experiment

[0181] Prepare the test solution and negative control solution according to the proposed method and conduct the test. Figure 7 , Figure 7 The results show that the negative solution chromatogram has no interference with the determination of the peak to be tested, indicating that the method has good specificity.

[0182] 8.2 Precision Investigation

[0183] The reference solution was sampled 6 times continuously, the peak areas of rosmarinic acid and 4-caffeoylquinic acid were recorded, and the RSD values were calculated. The results are shown in Table 18.

[0184] Table 18 Precision inspection results

[0185]

[0186] The results showed that the RSD value of the peak area of rosmarinic acid was 0.7%, and the RSD value of the peak area of 4-caffeoylquinic acid was 1.2%. The injection precision of this method was good.

[0187] 8.3 Linear Relationship

[0188] Take rosmarinic acid and 4-caffeoylquinic acid reference substances and prepare a mixed standard mother solution of rosmarinic acid solution (51.95605μg / ml, purity 90.5%) and 4-caffeoylquinic acid (45.54296μg / ml, purity 99.7%). Take 1, 2, 5, 10, and 25 ml respectively and place them in a 25 ml volumetric flask. Dilute to the mark with 70%, shake well, and filter. Accurately draw 10μl and inject it into the liquid chromatograph. Analyze and obtain the peak area. Draw the response curve with the rosmarinic acid and 4-caffeoylquinic acid content (X) as the horizontal axis and the peak area (Y) as the vertical axis. The results are shown in Table 6. Figure 8 、 9 . Figure 8 Standard curve of rosmarinic acid;

[0189] Figure 9 This is the standard curve of 4-caffeoylquinic acid.

[0190] Table 19 Standard curve analysis results

[0191]

[0192] The results showed that the concentration range of rosmarinic acid was 20.78242~519.5605μg / ml, with a linear relationship of y=3,553.4968x-6,136.5358, R2=0.9999; the concentration range of 4-caffeoylquinic acid was 18.217184~455.4296μg / ml, with a linear relationship of y=3,648.4793x-5,517.6844, R2=0.9999.

[0193] 8.4 Repeatability

[0194] Six portions of the same test sample (batch number: DFJC-BT-210901) were taken, 0.1 g of each was accurately weighed, and the test sample solution was prepared by the same operator according to the proposed method. The contents of rosmarinic acid and 4-caffeoylquinic acid in the six portions of the test sample were calculated. The results are shown in Table 20.

[0195] Table 20 Repeatability test results

[0196]

[0197] The results showed that the RSD value of rosmarinic acid content was 0.9%, and the RSD value of 4-caffeoylquinic acid content was 1.2%, indicating that the method has good reproducibility.

[0198] 8.5 Sample recovery

[0199] Take about 0.05 g of the test sample with known content (batch number: DFJC-BT-210901, rosmarinic acid content 5.9 mg / g, 4-caffeoylquinic acid content 3.2 mg / g), a total of 6 portions, accurately weighed, and accurately added a certain amount of rosmarinic acid reference substance to each. Prepare the test solution and determine it according to the proposed method. Calculate the recovery rate. The results are shown in Tables 21 and 22. The calculation formula is as follows:

[0200]

[0201] Table 21 Rosmarinic acid sample recovery experimental results

[0202]

[0203] Table 22 4-caffeoylquinic acid sample recovery test results

[0204]

[0205] The results showed that the average recovery rate of rosmarinic acid was 101% with an RSD of 2.0%; the average recovery rate of 4-caffeoylquinic acid was 99.8% with an RSD of 1.8%, indicating that the method had good accuracy.

[0206] 8.6 Intermediate precision

[0207] 8.6.1 Inspection of different instruments

[0208] Based on the experimental conditions proposed above, two portions of the standard decoction of Dafeijincao (batch number: DFJC-BT-210901) were accurately weighed to prepare test solutions. The solutions were then analyzed on Shimadzu, Agilent 1260, and Waters 2998-e2695 high performance liquid chromatographs (all using Agilent 5TC-C18(2) 250×4.6mm columns). The contents of rosmarinic acid and 4-caffeoylquinic acid were calculated. The results are shown in Table 10.

[0209] Table 23 Experimental results of different instruments

[0210]

[0211] The results showed that the RSD values of the determination results of rosmarinic acid and 4-caffeoylquinic acid were 1.9% and 1.9%, respectively, indicating that the intermediate precision of this method was good.

[0212] 8.6.2 Inspection by different personnel and time

[0213] Based on the experimental conditions proposed above, different individuals (A and B) accurately weighed a standard decoction of Dafeijincao (Batch No.: DFJC-BT-210901) at different times (T1 and T2) to prepare test samples for determination. The calculated rosmarinic acid content is shown in Table 24.

[0214] Table 24 Results of investigations of different personnel and time

[0215]

[0216] The results showed that the RSD value of the determination results of rosmarinic acid was 1.8%, and the RSD value of the determination results of 4-caffeoylquinic acid was 2.8%. The intermediate precision of this method was good.

[0217] 8.7 Durability Assessment

[0218] 8.7.1 Column Durability Assessment

[0219] Based on the experimental conditions proposed above, the chromatographic columns were Agilent 5TC-C18(2), KromasilC18, Agilent ZORBAX Eclipse XDB C 18 See Table 12.

[0220] Table 25 Durability test results

[0221]

[0222]

[0223] The results showed that the analytical chromatographic indicators of different chromatographic columns were good. Among the six measurement results, the RSD value of rosmarinic acid was 0.9%, and the RSD value of 4-caffeoylquinic acid was 2.3%. The chromatographic column of this method had good durability.

[0224] 8.7.2 Stability test

[0225] The same test solution (batch number: DFJC-BT-210901) was taken and the rosmarinic acid peak area was measured at 0, 2, 4, 8, 16, and 24 hours, respectively. The results are shown in Table 26.

[0226] Table 26 Rosmarinic acid stability test results

[0227]

[0228] The results showed that under the experimental conditions, the RSD value of the rosmarinic acid peak area was 0.3%, and the RSD value of the 4-caffeoylquinic acid peak area was 2.2%, indicating that the test solution had good stability within 24 hours.

[0229] Example 9 The verification results of 21 batches of standard decoction of Radix Pseudostellariae are shown in Table 27.

[0230] Table 27 Content verification of 21 batches of Dafeijincao standard decoction

[0231]

[0232]

[0233] Conclusion: The content determination method of Dafeijincao standard decoction can effectively detect Dafeijincao standard decoction, which proves that the method is feasible.

[0234] Example 10 HPLC content determination method of the formulated granules:

[0235] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, and gradient elution as specified in Table 1; the column temperature was 30°C; the detection wavelength was 330 nm, and the number of theoretical plates calculated based on the rosmarinic acid peak should be no less than 4000.

[0236] Preparation of reference solution: Take appropriate amount of rosmarinic acid and 4-caffeoylquinic acid reference substances, weigh accurately, and add 70% methanol to prepare a mixed standard solution containing 20 μg of rosmarinic acid and 20 μg of 4-caffeoylquinic acid per 1 ml.

[0237] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, about 0.1g, weigh it accurately, put it into a stoppered conical flask, add 25ml of 70% methanol accurately, weigh it, treat it with ultrasound for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, and the solution is ready.

[0238] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0239] Example 11 Preparation of test solution

[0240] 11.1 Extraction Solvent Investigation

[0241] Approximately 0.1 g of the formulated granules (batch number: 1902061) were accurately weighed and placed in a stoppered conical flask. The sample was extracted using methanol, 50% methanol, 70% methanol, ethanol, 30% ethanol, and 25 ml of water. The flask was sealed and weighed. Ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. The flask was allowed to cool and weighed again. The weight loss was made up with the extraction solvent. The filtrate was shaken well, filtered, and the filtrate was collected. 10 μl of each extraction solution was injected into a chromatograph to analyze and calculate the rosmarinic acid and 4-caffeoylquinic acid contents of the different extraction methods. The results are shown in Table 28.

[0242] Table 28 Analysis results of different extraction solvents

[0243]

[0244] The results showed that 70% methanol had the highest extraction efficiency and was used as the extraction solvent.

[0245] 11.2 Investigation of extraction methods

[0246] Accurately weigh approximately 0.1 g of the formulated granules (batch number: 1902061) and place in a stoppered conical flask. Accurately add 25 ml of 70% methanol and weigh the mixture. Reflux and sonicate for 30 minutes, respectively. Allow to cool, weigh again, and make up the lost weight with 70% methanol. Shake well to obtain the final product. Results are shown in Table 29.

[0247] Table 29 Analysis results of different extraction methods

[0248]

[0249] The results showed that there was little difference in the extraction efficiency between reflux and ultrasonic extraction, and ultrasonic extraction was preferred due to its simplicity.

[0250] 11.3 Extraction time investigation

[0251] Approximately 0.1 g of the formulated granules (batch number: 1902061) were accurately weighed and placed in a stoppered conical flask. 25 ml of 70% methanol was accurately added, the flask was sealed, and the weight was determined. Ultrasonic treatment (power 600 W, frequency 40 kHz) was then performed. The sample was extracted for 20 minutes, 30 minutes, and 40 minutes. The sample was allowed to cool, and the weight was determined again. The weight loss was made up with 70% methanol, shaken well, filtered, and the filtrate was collected. The rosmarinic acid and 4-caffeoylquinic acid contents at different extraction times were analyzed and calculated. The results are shown in Table 30.

[0252] Table 30 Analysis results of different extraction times

[0253]

[0254] The results showed that the content of rosmarinic acid was higher when the extraction time was above 30 minutes. Taking all factors into consideration, the extraction time of the test sample was determined to be 30 minutes.

[0255] 11.4 Investigation of solvent addition amount

[0256] Approximately 0.1 g of the formulated granules (batch number: 1902061) were accurately weighed and placed in a stoppered conical flask. 15 ml, 25 ml, and 50 ml of 70% methanol were accurately added, respectively. The flask was sealed and weighed. Ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. The sample was allowed to cool, and the weight was reweighed. The weight loss was made up with 70% methanol, shaken well, filtered, and the filtrate was collected. The rosmarinic acid and 4-caffeoylquinic acid contents at different extraction times were analyzed and calculated. The results are shown in Table 31.

[0257] Table 31 Analysis results of different extraction times

[0258]

[0259] The results showed that the amount of solvent added had little effect on the content of rosmarinic acid in the test sample. Taking all factors into consideration, the amount of solvent added to the test sample was determined to be 25 ml.

[0260] In summary: Through the investigation of the extraction method, solvent, extraction time, and the amount of solvent added, the preparation method of the test solution is determined as follows: take an appropriate amount of this product, grind it into powder, about 0.1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, and you have it.

[0261] Example 12 Methodological Investigation

[0262] 12.1 Specificity Experiment

[0263] Prepare the test solution and negative control solution according to the proposed method and conduct the test. Figure 10 . Figure 10 The specificity result graph is shown in Figure 2. The results show that the negative solution chromatogram has no interference with the determination of the peak to be tested, indicating that the method has good specificity.

[0264] 12.2 Precision Investigation

[0265] The reference solution was sampled six times continuously, the peak areas of rosmarinic acid and 4-caffeoylquinic acid were recorded, and the RSD values were calculated. The results are shown in Table 32.

[0266] Table 32 Precision inspection results

[0267]

[0268] The results showed that the RSD value of the peak area of rosmarinic acid was 0.7%, and the RSD value of the peak area of 4-caffeoylquinic acid was 2.7%. The injection precision of this method was good.

[0269] 12.3 Linear Relationship

[0270] Take rosmarinic acid and 4-caffeoylquinic acid reference substances and prepare a mixed standard mother solution of rosmarinic acid solution (51.95605μg / ml, purity 90.5%) and 4-caffeoylquinic acid (45.54296μg / ml, purity 99.7%). Take 1, 2, 5, 10, 15, and 25ml respectively and place them in a 25ml volumetric flask. Dilute to the mark with 70%, shake well, and filter. Accurately pipette 10μl and inject it into the liquid chromatograph. Analyze to obtain the peak area. Plot the response curve with the rosmarinic acid concentration (X) and the 4-caffeoylquinic acid concentration as the horizontal axis and the peak area (Y) as the vertical axis. The results are shown in Table 33. Figure 11 、 12 .

[0271] Table 33 Standard curve analysis results

[0272]

[0273] The results showed that the linear relationship of rosmarinic acid in the concentration range of 20.78242~519.5605μg / ml was y=3,553.4968x-6,136.5358, R2=0.9999; the linear relationship of 4-caffeoylquinic acid in the concentration range of 18.217184~455.4296μg / ml was y=3,648.4793x-5,517.6844, R2=0.9999, showing a good linear relationship.

[0274] 12.4 Repeatability

[0275] Six portions of the same test sample (batch number: 1902061) were taken, 0.1 g of each was accurately weighed, and the test sample solution was prepared by the same operator according to the proposed method. The rosmarinic acid and 4-caffeoylquinic acid contents of the six portions of the test sample were calculated. The results are shown in Table 34.

[0276] Table 34 Repeatability test results

[0277]

[0278] The results showed that the RSD values of rosmarinic acid and 4-caffeoylquinic acid were 0.4% and 0.7%, respectively, indicating that the method had good repeatability.

[0279] 12.5 Accuracy

[0280] About 0.05 g of a test sample (batch number: 1902061, rosmarinic acid content 5.4 mg / g) of known content was taken in 6 portions and accurately weighed. A certain amount of rosmarinic acid reference substance was accurately added to each portion. The test solution was prepared and measured according to the proposed method. The recovery rate was calculated. The results are shown in Tables 35 and 36. The calculation formula is as follows:

[0281]

[0282] Table 35 Rosmarinic acid sample recovery experimental results

[0283]

[0284] Table 36 4-caffeoylquinic acid sample recovery test results

[0285]

[0286] The results showed that the average recovery rate of rosmarinic acid was 102%, and the RSD value was 1.1%; the average recovery rate of 4-caffeoylquinic acid was 98.9%, and the RSD value was 2.7%, indicating that the method had good accuracy.

[0287] 12.6 Intermediate Precision

[0288] 12.6.1 Inspection of Different Instruments

[0289] Based on the experimental conditions proposed above, two portions of Dafeijincao formula granules (batch number: 1902061) were precisely weighed to prepare test solutions. The solutions were then analyzed on Shimadzu, Agilent 1260, and Waters 2998-e2695 high performance liquid chromatographs (all using Agilent 5TC-C18(2) 250×4.6mm columns). The contents of rosmarinic acid and 4-caffeoylquinic acid were calculated. The results are shown in Table 37.

[0290] Table 37 Experimental results of different instruments

[0291]

[0292] The results showed that the RSD value of the determination results of rosmarinic acid was 1.2%, and the RSD value of the determination results of 4-caffeoylquinic acid was 3.1%, indicating that the intermediate precision of this method was good.

[0293] 12.6.2 Inspection by different personnel and time

[0294] Based on the above experimental conditions, different individuals (A and B) accurately weighed Dafeijincao granules (Batch No. 1902061) at different times (T1 and T2) to prepare test samples for determination. The calculated rosmarinic acid and 4-caffeoylquinic acid contents are shown in Table 38.

[0295] Table 38 Results of investigations of different personnel and time

[0296]

[0297] The results showed that the RSD value of the determination results of rosmarinic acid was 1.1%, and the RSD value of the determination results of 4-caffeoylquinic acid was 2.6%. The intermediate precision of this method was good.

[0298] 12.7 Durability Assessment

[0299] 12.7.1 Column Durability Assessment

[0300] Based on the experimental conditions proposed above, the chromatographic columns were Agilent 5TC-C18(2), KromasilC18, Agilent ZORBAX Eclipse XDB C 18 See Table 39.

[0301] Table 39 Durability test results

[0302]

[0303] The results showed that the analytical chromatographic indicators of different chromatographic columns were good. Among the six measurement results, the RSD value of rosmarinic acid was 1.5%, and the RSD value of 4-caffeoylquinic acid was 2.7%. The chromatographic column of this method had good durability.

[0304] 12.7.2 Stability Test

[0305] The same test solution (batch number: 1902061) was taken and the peak areas of rosmarinic acid and 4-caffeoylquinic acid were determined at 0, 2, 4, 8, 16, and 24 hours, respectively. The results are shown in Table 40.

[0306] Table 40 Rosmarinic acid stability test results

[0307]

[0308] The results showed that under the experimental conditions, the RSD value of the rosmarinic acid peak area was 0.2%, and the RSD value of the 4-caffeoylquinic acid peak area was 2.0%. The test solution had good stability within 24 hours.

[0309] Example 13 The verification results of three batches of Radix Psoraleae formula granules are shown in Table 41.

[0310] Table 41 Content verification of 3 batches of Dafeijincao formula granules

[0311]

[0312] Conclusion: The content determination method of Dafeijincao formula granules can be used to effectively detect Dafeijincao formula granules, proving that this method is feasible.

[0313] Example 14 Identification of Fake Large Lung Tendon Grass and Lung Meridian Grass

[0314] Lungwort: The dried whole herb of the Umbelliferae plant Sanicμla lamelligera Hance.

[0315] Lung meridian grass: the dried whole herb of Aletris spicata (Thμnb.) Franch., a plant of the Liliaceae family.

[0316] Lung meridian herb is a common counterfeit of large lung tendon herb. Because of their similar names and properties, confusion can easily occur during use. This method uses high-performance liquid chromatography to distinguish the two.

[0317] The chromatographic conditions and test sample preparation conditions are as follows:

[0318] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, and gradient elution as specified in Table 1; the column temperature was 30°C; the detection wavelength was 330 nm, and the number of theoretical plates calculated based on the rosmarinic acid peak should be no less than 4000.

[0319] Preparation of reference solution: Take appropriate amount of rosmarinic acid and 4-caffeoylquinic acid reference substances, weigh accurately, and add 70% methanol to prepare a mixed standard solution containing 20 μg of rosmarinic acid and 20 μg of 4-caffeoylquinic acid per 1 ml.

[0320] Preparation of the test solution: Take about 1.0 g of Dafeijincao and lung meridian herbal medicine powder (passed through No. 3 sieve), accurately weigh, place in a stoppered conical flask, accurately add 50 ml of 30% methanol, weigh the weight, ultrasonically treat for 30 minutes, let cool, weigh again, make up the lost weight with 30% methanol, shake well, and obtain.

[0321] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. Figure 13 .Depend on Figure 13 It can be seen that under the same chromatographic conditions and relatively good separation effect, the fake lung meridian grass did not produce any peaks when used in combination with 4-caffeoylquinic acid and rosmarinic acid, indicating that the two components 4-caffeoylquinic acid and rosmarinic acid do not exist in the fake lung meridian grass.

[0322] Conclusion: The HPLC method for content determination of Dafeijincao can be used to identify counterfeit products of D.

[0323] Comparative Example 1 Chromatographic conditions using caffeic acid as reference substance:

[0324] The chromatographic conditions and system suitability test were performed with octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and isocratic elution was performed with methanol:0.1% phosphoric acid (20:80); the column temperature was 30°C; the detection wavelength was 323 nm, and the theoretical plate number calculated based on the 4-caffeoylquinic acid peak should be no less than 4000.

[0325] Preparation of reference solution: Take an appropriate amount of caffeic acid reference, accurately weigh it, and add 70% methanol to make a solution containing 20 μg per 1 ml.

[0326] Preparation of test solution: Take about 0.5 g of the product (passed through No. 3 sieve), weigh accurately, place in a stoppered conical flask, add 50 ml of water accurately, weigh the weight, ultrasonically treat for 30 minutes, let cool, weigh again, make up the lost weight with water, shake well, and obtain.

[0327] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0328] 1. Investigation of different solvents

[0329] Approximately 0.5 g of Dafeijincao herb (batch number: 010094-1708001, passed through a No. 3 sieve) was accurately weighed and placed in a stoppered conical flask. The sample was extracted using 50 ml of methanol, 50% methanol, 70% methanol, ethanol, 30% ethanol, and water. The flask was sealed and weighed. Ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. The flask was allowed to cool and weighed again. The weight loss was made up with the extraction solvent, shaken well, filtered, and the filtrate was collected. 10 μl of each extraction solution was injected into a chromatograph and analyzed for caffeic acid content under different extraction methods. The results are shown in Table 42.

[0330] Table 42 Analysis results of different extraction solvents

[0331]

[0332] The results show that when the extraction solvent is water, the caffeic acid content of the herb is the highest, so water is tentatively selected as the extraction solvent.

[0333] 2. Stability inspection

[0334] Using the above-mentioned chromatographic conditions, the medicinal material test sample solution (batch number: 010094-1708001) was taken and the caffeic acid peak area was measured at 0, 2, 4, 8, 16, and 24 hours. The results are shown in Table 43.

[0335] Table 43 Caffeic acid stability test results

[0336]

[0337] The results showed that under the experimental conditions, the RSD value of the caffeic acid peak area was 70.9%, and the test solution was extremely unstable within 24 hours. Therefore, caffeic acid was not selected as a reference substance.

[0338] Comparative Example 2: Same mobile phase gradient, different organic phases

[0339] Based on the above selected chromatographic conditions, methanol was replaced by acetonitrile, and the other conditions were the same. The relevant investigations were carried out and the results are shown in Figure 14 , Figure 14 It is the chromatogram of Comparative Example 2;

[0340] The results showed that under the same gradient conditions, the chromatogram showed poor peak separation after methanol was replaced by acetonitrile.

[0341] Comparative Example 3: Same mobile phase gradient, different mobile phases

[0342] Based on the above selected chromatographic conditions, methanol: 0.1% phosphoric acid was replaced with acetonitrile: water, and the other conditions were the same. The relevant investigations were carried out, and the results are shown in Figure 15 , Figure 15It is the chromatogram of Comparative Example 3;

[0343] The results showed that under the same gradient conditions, after the mobile phase was replaced from methanol-0.1% phosphoric acid to acetonitrile-water, the chromatogram showed that the method basically had no peaks.

[0344] Comparative Example 4: Same mobile phase, different mobile phase gradients

[0345] Based on the mobile phase selected above, the mobile phase elution gradient was changed. The gradient is shown in Table 44. The results are shown in Figure 16 It is the chromatogram of Comparative Example 4.

[0346] Table 44

[0347]

[0348] The results showed that under the same mobile phase conditions, different mobile phase gradients resulted in poor peak separation in the chromatograms.

[0349] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for determining the content of Dafeijincao medicinal pieces, standard decoctions, and formula granules by HPLC, comprising: A) Dissolve the test sample in a solvent and extract to obtain a test solution; the solvent is methanol, 50% methanol, 70% methanol, or water; the extraction is performed by heating reflux extraction or ultrasonic extraction; The invention also includes preparing a reference solution: taking rosmarinic acid and 4-caffeoylquinic acid respectively, dissolving them in 70% methanol to obtain a reference solution; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the dafeijincao herbal medicine slices, standard decoction, and formula granules are qualitatively analyzed based on the chromatogram of the reference; B) measuring the test solution by high performance liquid chromatography to obtain HPLC characteristic spectra of the Dafeijincao herbal medicine slices, standard decoction, and formula granules; The HPLC conditions are as follows: a C18 column; mobile phase A is a methanol solution; mobile phase B is a 0.1% phosphoric acid aqueous solution; gradient elution; detection wavelength is 330 nm; The gradient elution is specifically as follows: 0-5min, phase A: 15%-40%, phase B: 85-60%; 5-35min, phase A: 40%-65%, phase B: 60%-35%.

2. The method according to claim 1, characterized in that The concentrations of the reference solution are specifically as follows: rosmarinic acid is 21-520 μg / mL, and 4-caffeoylquinic acid is 18-455 μg / mL.

3. The method according to claim 1, characterized in that The chromatographic column is Agilent 5 TC-C 18 、KromasilC 18 , Agilent ZORBAX Eclipse XDB C 18 250×4.6mm 5μm; column temperature 25~35℃; the theoretical plate number calculated based on the rosmarinic acid peak should be no less than 4000.

4. The method according to claim 1, wherein The flow rate of the mobile phase is 0.8-1.2 mL / min; the injection volume is 10 μL.

5. The method according to claim 1, wherein In step A), the heating and reflux extraction time is 30 minutes; the ultrasonic power is 600W, the frequency is 40kHz; and the ultrasonic time is 30-40 minutes.

6. The method according to claim 1, characterized in that The ratio of the mass g of the test sample raw material to the volume mL of the solvent is 0.5:50; The raw materials of the test sample are one or more of the following: large lung tendon herb materials, large lung tendon herb slices, large lung tendon herb standard decoction or large lung tendon herb formula granules.

7. A method for identifying characteristic patterns of Dafeijincao medicinal pieces, standard decoctions, and formula granules, characterized in that: The method according to any one of claims 1 to 6 is used for detection, and the detection results are analyzed.

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