Method for detecting characteristic spectrum of four seasons green formula granules and quality control method

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, and relates to a detection method and quality control method for characteristic chromatogram of four seasons green formula granules, which is detected by ultra-high performance liquid chromatography, and chromatographic conditions are as follows: octadecylsilane bonded silica is used as filler, acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, and a gradient elution program includes: 0-15 min, the volume ratio of mobile phase A and mobile phase B is 8%:92%; 15-30 min, the volume ratio of mobile phase A and mobile phase B is 8%:92% to 18%:82%; 30-50 min, the volume ratio of mobile phase A and mobile phase B is 18%:82% to 22%:78%; 50-60 min, the volume ratio of mobile phase A and mobile phase B is 22%:78%; the characteristic chromatogram of four seasons green formula granules detected by the above method is stable in baseline, balanced in distribution, good in peak type, and good in peak separation degree.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and particularly relates to a detection method and quality control method for characteristic chromatogram of Ilex chinensis granules. BACKGROUND

[0002] Ilex chinensis is the dried leaves of Ilex chinensis Sims in Aquifoliaceae, which has the effects of clearing heat and resolving toxins, removing blood stasis and reducing swelling, and is used for treating lung heat cough, sore throat, dysentery, hypochondriac pain, hot stranguria, and external treatment of burns and scalds, skin ulcers, etc. Ilex chinensis has a long history of medicinal use, and has been recorded in Tang Dynasty's Bencao Shiyi since the Tang Dynasty. It is widely used in modern clinical practice, and is often used to treat various inflammatory diseases, burns and scalds, ulcers, etc.

[0003] The quality control of Ilex chinensis in Chinese Pharmacopoeia 2020 edition includes original plant varieties, decoction piece processing, decoction piece appearance, physicochemical identification and other items. The literature also describes the chemical components of Ilex chinensis, including organic acids, flavonoids and other components. However, on the one hand, the content determination or identification of Ilex chinensis granules by the above single component cannot detect and control the quality of Ilex chinensis granules as a whole; on the other hand, the content determination of single component combined with the identification of other components of Ilex chinensis granules is time-consuming and laborious, and is difficult to be widely applied in production practice.

[0004] At present, the reports on the quality control of Ilex chinensis by characteristic chromatogram are mostly for Ilex chinensis decoction pieces, not for Ilex chinensis granules. Although Ilex chinensis granules are a type of preparation processed from Ilex chinensis decoction pieces, the physicochemical properties of Ilex chinensis granules processed by production and processing have changed greatly compared with Ilex chinensis decoction pieces, so that the current detection method for Ilex chinensis decoction pieces cannot be applied to the detection of Ilex chinensis granules. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a detection method and quality control method for characteristic chromatogram of Ilex chinensis granules, which can be more specific and targeted for Ilex chinensis granules, play a standardized, standardized and objective description and evaluation, and can comprehensively and quickly detect Ilex chinensis granules, which has important significance for the comprehensive quality detection and overall quality control of Ilex chinensis granules.

[0006] To this end, the present application provides the following technical solution:

[0007] A detection method for characteristic chromatogram of Ilex chinensis granules, which is detected by ultra-high performance liquid chromatography, and the chromatographic conditions are as follows:

[0008] The chromatographic column uses octadecylsilane-bonded silica gel as the filler, acetonitrile as the mobile phase A, and 0.1% v / v phosphoric acid solution as the mobile phase B, and the gradient elution program includes:

[0009] From 0 to 15 min, the volume ratio of mobile phase A to mobile phase B was 8%:92%.

[0010] Over 15-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 8%:92% to 18%:82%.

[0011] Over 30-50 minutes, the volume ratio of mobile phase A to mobile phase B changed from 18%:82% to 22%:78%.

[0012] The volume ratio of mobile phase A to mobile phase B is 22%:78% for 50-60 minutes.

[0013] Optionally, the chromatographic conditions include at least one of the following:

[0014] 1) Detection wavelength: 254nm;

[0015] 2) The flow rate is 0.18-0.22 mL / min;

[0016] 3) Column temperature is 23-27℃;

[0017] 4) The chromatographic column is Waters ACQUITY UPLC HSS T3, Shim-pack GIST C18-Aq, Phenomenex Luna Omega C18 or Dikma Endeavorsil C18; optionally, the chromatographic column is Waters ACQUITY UPLC HSS T3, with specifications of 150mm × 2.1mm and 1.8μm.

[0018] 5) The injection volume is 1.8-2.2 μL, with 2 μL being an option.

[0019] Optionally, it also includes a method for preparing the test solution, including:

[0020] Take the sample, add solvent to extract, filter, and collect the filtrate to obtain the final product.

[0021] Optionally, it also includes a method for preparing the reference solution, including:

[0022] Preparation of reference solution for medicinal materials: Take the *Ilex chinensis* reference medicinal material, add solvent to extract, filter, and take the filtrate to obtain the solution;

[0023] And / or, preparation of reference solutions: Take protocatechuic acid and 4,5-O-dicaffeoylquinic acid reference standards and add them to a solvent to prepare a mixed solution containing 50 μg of each reference standard per 1 ml, which is used as the reference solution.

[0024] Optionally, the reference solution for the control medicinal material is prepared using water or methanol as the solvent.

[0025] And / or, in the extraction step, first add water and heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 50 v / v% methanol aqueous solution to the residue and ultrasonically extract for 30 minutes, filter, and collect the filtrate;

[0026] And / or, the mass-to-volume ratio of the Four Seasons Green reference medicinal material to water is 1:20, with a ratio of g / ml;

[0027] And / or, the mass-to-volume ratio of the *Ilex chinensis* reference herb and the 50 v / v% methanol aqueous solution is 1:20, with a ratio of g / ml;

[0028] Optional, preparation of reference solution for control medicinal material: Take 1g of Sijiqing reference medicinal material, place it in a stoppered conical flask, add 20ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 20ml of 50v / v% methanol aqueous solution to the residue, sonicate at 250W power and 40kHz frequency for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution;

[0029] Optionally, the preparation of the reference solution: Take appropriate amounts of protocatechuic acid and 4,5-O-dicaffeoylquinic acid reference standards, accurately weigh them, and add 50 v / v% methanol aqueous solution to prepare a mixed solution containing 50 μg of each per 1 ml, which is used as the reference solution.

[0030] A method for determining the content of *Lysimachia christinae* formula granules, comprising:

[0031] Take the test solution and the reference solution, and perform ultra-high performance liquid chromatography (UHPLC) in the detection method of the characteristic spectrum of the Sijiqing formula granules.

[0032] The application of the detection method of the characteristic spectrum of the Sijiqing formula granules or the content determination method of the Sijiqing formula granules in the quality testing of Sijiqing formula granules.

[0033] A method for quality testing of Sijiqing formula granules includes the steps of obtaining a characteristic spectrum of the test sample according to the method for detecting the characteristic spectrum of Sijiqing formula granules, and comparing the characteristic spectrum with a control characteristic spectrum.

[0034] The control feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of Sijiqing formula granules as a standard according to the detection method of the feature spectrum of Sijiqing formula granules.

[0035] Optionally, the reference characteristic spectrum includes 16 characteristic peaks, with peak 4 being protocatechuic acid and peak 16 being 4,5-O-dicaffeoylquinic acid; peak 4 is used as the first reference peak S1, and the relative retention times of peaks 1-3, peaks 5-9 and peak S1 are within the range of ±10% of a specified value, with the specified value for peak 1 being 0.46, peak 2 being 0.74, peak 3 being 0.85, peak 5 being 1.63, and peak 6 being 1. Peak 7 has a specified value of 2.27, peak 8 has a specified value of 2.53, and peak 9 has a specified value of 2.92. Peak 16 is used as the second reference peak S2. The relative retention times of peaks 10-15 with peak S2 are within ±10% of the specified values. The specified values ​​for peak 10 are 0.63, peak 11 is 0.67, peak 12 is 0.70, peak 13 is 0.75, peak 14 is 0.92, and peak 15 is 0.97.

[0036] Optionally, peak 4 (S1 peak) corresponds to protocatechuic acid; peak 5 corresponds to protocatechuic aldehyde; peak 7 corresponds to syringin; peak 8 corresponds to chlorogenic acid; peak 9 corresponds to caffeic acid; peak 10 corresponds to 1,3-O-dicaffeoylquinic acid; peak 12 corresponds to quercetin-3-O-mori disaccharide glycoside; peak 14 corresponds to 3,5-O-dicaffeoylquinic acid; and peak 16 (S2) corresponds to 4,5-O-dicaffeoylquinic acid.

[0037] The technical solution of this invention has the following advantages:

[0038] 1. This invention provides a method for detecting the characteristic chromatogram of Sijiqing (a type of herbal medicine) formula granules. The method employs ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions are as follows: the column is packed with octadecylsilane-bonded silica gel; acetonitrile is used as mobile phase A; and 0.1 v / v% phosphoric acid solution is used as mobile phase B. The gradient elution program includes: 0-15 min, mobile phase A and mobile phase B volume ratio of 8%:92%; 15-30 min, mobile phase A and mobile phase B volume ratio of 8%:92% → 18%:82%; 30-50 min, mobile phase A and mobile phase B volume ratio of 18%:82% → 22%:78%; 50 min... -60 min, the volume ratio of mobile phase A to mobile phase B is 22%:78%; the above method was used to detect the characteristic chromatogram of Sijiqing formula granules. The chromatographic conditions were stable, the baseline of the chromatogram was stable, the distribution of common chromatographic peaks was even, the peak shape was good, the peak resolution was good, and there were 9 known components among the characteristic peaks. The content of protocatechuic acid, protocatechuic aldehyde, syringin, chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, quercetin-3-O-mori disaccharide glycoside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid can be determined simultaneously. It can comprehensively and rapidly detect the effective components and their content in Sijiqing formula granules.

[0039] Furthermore, the above method is designed based on Sijiqing formula granules, which is more specific and targeted for the quality control of Sijiqing formula granules. It can play a standardized, normalized, and objective role in description and evaluation. Therefore, the above method can be used to conduct comprehensive quality testing and overall quality control of Sijiqing formula granules, which helps to improve the safety and stability of the drug. Attached Figure Description

[0040] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 These are the characteristic chromatograms of 18 batches of Sijiqing formula granules in Example 1 of this invention;

[0042] Figure 2 This is a comparative characteristic spectrum of the Sijiqing formula granules in Example 1 of the present invention;

[0043] Figure 3 This is a characteristic chromatogram of the *Ilex chinensis* reference medicinal material in Example 1 of this invention;

[0044] Figure 4 This is the location feature map of the Four Seasons Green Formula Granules Reference Standard in Example 1 of the present invention;

[0045] Figure 5 This is a characteristic chromatogram of the Four Seasons Green Formula Granules with a flow rate of 0.2 ml / min in Example 2 of the present invention;

[0046] Figure 6 This is a characteristic chromatogram of the Sijiqing formula granules with a flow rate of 0.18 ml / min in Example 2 of the present invention;

[0047] Figure 7 This is a characteristic chromatogram of the Four Seasons Green Formula Granules with a flow rate of 0.22 ml / min in Example 2 of the present invention;

[0048] Figure 8 This is a characteristic spectrum of the *Ilex cornuta* formula granules with a column temperature of 23°C in Example 3 of this invention;

[0049] Figure 9 This is a characteristic spectrum of the Four Seasons Green Formula Granules with a column temperature of 25°C in Example 3 of the present invention;

[0050] Figure 10 This is a characteristic spectrum of the *Ilex cornuta* formula granules with a column temperature of 27°C in Example 3 of this invention;

[0051] Figure 11 This is a characteristic chromatogram of the *Ilex cornuta* formulation particles on chromatographic column 1 in Example 4 of the present invention;

[0052] Figure 12 This is a characteristic chromatogram of the *Ilex cornuta* formulation particles in chromatographic column 2 of Example 4 of the present invention;

[0053] Figure 13 This is a characteristic chromatogram of the *Ilex cornuta* formulation particles in chromatographic column 3 of Example 4 of the present invention;

[0054] Figure 14 This is a characteristic chromatogram of the *Ilex cornuta* formulation particles on chromatographic column 4 in Example 4 of the present invention;

[0055] Figure 15 This is a characteristic chromatogram of the *Ilex chinensis* formulation granules obtained by chromatograph 2 in Embodiment 5 of the present invention;

[0056] Figure 16 This is a characteristic chromatogram of the Sijiqing formula granules obtained by chromatograph 1 in Embodiment 5 of the present invention;

[0057] Figure 17 This is a characteristic chromatogram of the Sijiqing formula granules obtained by chromatograph 3 in Embodiment 5 of the present invention. Detailed Implementation

[0058] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0059] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0060] The pharmaceutical preparation of *Ilex cornuta* described in this invention is prepared by the following method:

[0061] Take *Ilex chinensis* and extract it under reflux at least once, adding 6-12 times its weight of water each time for at least 0.5 hours. Filter, combine the filtrates, and concentrate the filtrate to a relative density of 1.05-1.10 g / mL at 60°C. Add conventional excipients and follow conventional processes to prepare clinically acceptable tablets, capsules, pills, granules, honey-processed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0062] The test sample used in this embodiment is Sijiqing (Ilex chinensis) formula granules. The specific preparation method of Sijiqing formula granules is as follows: Sijiqing is taken and extracted twice by heating and reflux. For the first extraction, 10 times the weight of water is added and soaked for 30 minutes, then heated and refluxed for 0.5 hours and filtered. For the second extraction, 8 times the weight of water is added and extracted for 0.5 hours and filtered. The filtrates are combined and concentrated to a relative density of 1.05 g / mL at 60°C. The filtrates are then spray-dried. The dry powder is mixed with the excipient maltodextrin, and then dry-granulated to produce granules.

[0063] The instruments, reagents, and excipients involved in the examples are as follows:

[0064] Instruments and equipment:

[0065] Chromatograph 1: Waters Hclass chromatography system, including Quaternary Solvent Manager quaternary pump, FTN-H autosampler, PDA diode array detector, and Empower chromatography management system;

[0066] Chromatograph 2: Thermo Fisher Vanquish chromatography system, including VF-P20-A quaternary pump, VF-A10-A autosampler, VF-D11-A DAD detector, and Chromeleon chromatography workstation;

[0067] Chromatograph 3: Agilent 1290 chromatography system, including G7104A quaternary pump, G7129B autosampler, G7117A1290DAD detector, and Agilent liquid chromatography workstation;

[0068] Other instruments: High-power digitally controlled ultrasonic instrument (KQ-400KDB, Kunshan Ultrasonic Instrument Co., Ltd.), 0.001 g electronic balance (Shimadz μAY120, Shimadzu Corporation, Japan), 0.001 g electronic balance (Sartoriμs SQP SECMRA225D-1CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.), digital display constant temperature water bath (HH-S6, Jiangsu Jinyi Instrument Technology Co., Ltd.).

[0069] Chromatographic column:

[0070] Waters ACQUITY UPLC HSS T3 2.1mm×150mm, 1.8μm

[0071] Agilent InfinityLab Poroshell 120SB-C18, 2.1×150mm, 1.9μm

[0072] Shimadzu Shim-pack GIST_HP C18-Aq 2.1mm×150mm, 1.9μm;

[0073] Reagents:

[0074] Methanol and acetonitrile were chromatographic grade (Merck); phosphoric acid (chromatographic grade, 85-90%, Aladdin); glacial acetic acid (HPLC, >99.9%, Aladdin); formic acid (chromatographic grade, ≥98%, Aladdin); water (Milli-Q).

[0075] Drug trials:

[0076] Four Seasons Ivy (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 380066-202107).

[0077] Protocatechuic acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 110809-202207, purity 97.5%);

[0078] 4,5-O-dicaffeoylquinic acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 111894-202205, purity 94.9%);

[0079] 3,5-O-dicaffeoylquinic acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 111782-202208, purity 95.9%);

[0080] Protocatechuic aldehyde reference standard (purchased from the National Institutes for Food and Drug Control, batch number 110810-202210, purity 99.9%);

[0081] 1,3-O-dicaffeoylquinic acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 111717-201402, purity 94.5%);

[0082] Chlorogenic acid (purchased from the National Institutes for Food and Drug Control, batch number 10753-202119, purity 96.3%)

[0083] Caffeic acid (purchased from the National Institutes for Food and Drug Control, batch number 110885-201703, purity 99.7%)

[0084] Syringin (purchased from the National Institutes for Food and Drug Control, batch number 111574-202106, purity 94.3%)

[0085] Sijiqing Formula Granules (Batch Nos.: 1802002W, 1804002S, 1905001S, 1907001W, 2003001W, 2005002S, 2007003S, 2012001W, 2102001S, 2104001S, 2110002W, 2112001S, 2203002S, 2204001S, 2206002S, 2208001W, 2301003S, 2307001S).

[0086] Example 1

[0087] This embodiment provides a method for detecting the characteristic chromatograms of Sijiqing formula granules, using ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions are as follows:

[0088] The chromatographic column was packed with octadecylsilane-bonded silica gel (Waters ACQUITY UPLC HSS T3 column, 150 mm × 2.1 mm, 1.8 μm). Acetonitrile was used as mobile phase A, and 0.1% v / v phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below. The detection wavelength was 254 nm; the column temperature was 25 °C; and the flow rate was 0.2 mL / min. The theoretical plate number, calculated based on the protocatechuic acid peak, should be no less than 5000.

[0089] Table 1. Gradient elution program

[0090] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～15 8 92 15～30 8→18 92→82 30～50 18→22 82→78 50～60 22 78

[0091] Preparation of reference solution: Take 1g of *Ilex chinensis* reference material, place it in a stoppered conical flask, add 20ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 20ml of 50v / v% methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of protocatechuic acid and 4,5-O-dicaffeoylquinic acid reference standards, accurately weigh them, and add 50v / v% methanol to prepare a mixed solution containing 50μg of each per ml, as the reference solution.

[0092] Preparation of the test solution: Take an appropriate amount of Sijiqing formula granules, grind them finely, take 0.3g, accurately weigh it, place it in a stoppered conical flask, add 20ml of 50v / v% methanol, stopper tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, take it out, cool it, shake it well, filter it, and take the filtrate to obtain the test solution.

[0093] Determination method: Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0094] In this embodiment, 18 batches of Sijiqing (a type of herb) formula granules were used to formulate a reference characteristic chromatogram for Sijiqing formula granules. The determination method was carried out by ultra-high performance liquid chromatography (UHPLC) as described above. Eighteen characteristic chromatograms were obtained as follows: Figure 1 As shown, Figure 1 The product batches S1 to S18 correspond to are 1802002W, 1804002S, 1905001S, 1907001W, 2003001W, 2005002S, 2007003S, 2012001W, 2102001S, 2104001S, 2110002W, 2112001S, 2203002S, 2204001S, 2206002S, 2208001W, 2301003S, and 2307001S, respectively. The results show that the relative retention times of each characteristic peak of the 18 batches of Sijiqing formula granules are all within ±10% of the specified values, as shown in the table below.

[0095] Multiple batches of data were imported into the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission for data matching. Using chromatogram S1 as the reference chromatogram, a control characteristic chromatogram was generated based on the median. Sixteen common characteristic peaks with verified response values, good resolution, and high purity were obtained and identified. The resulting control characteristic chromatogram is shown below. Figure 2 As shown.

[0096] Table 2. Relative retention times of 18 batches of Sijiqing formula granules

[0097]

[0098]

[0099]

[0100] Table 3. Similarity of 18 batches of Sijiqing formula granules

[0101] Batch number Similarity 1802002W 0.998 1804002S 0.999 1905001S 0.997 1907001W 0.998 2003001W 0.999 2005002S 1.000 2007003S 0.999 2012001W 0.999 2102001S 0.997 2104001S 0.998 2110002W 0.999 2112001S 1.000 2203002S 0.999 2204001S 0.999 2206002S 0.998 2208001W 0.983 2301003S 0.983 2307001S 0.983

[0102] The results of identifying and locating the common characteristic peaks mentioned above, combined with the characteristic spectrum of *Ilex chinensis* (a type of medicinal herb), localization using reference standards, and relevant literature, are shown in the table below. Figure 3 and Figure 4 As can be seen from the characteristic spectrum 3 of *Ilex chinensis* (Sijiqing) as a reference medicinal material, its main characteristic components are phenolic acids. The results, obtained through reference standard localization, are as follows: Figure 4 As shown in the figure (S1: Characteristic chromatogram of Sijiqing formula granules; S2: 1,3-O-dicaffeoylquinic acid; S3: Quercetin-3-O-morum disaccharide glycoside; S4: Caffeic acid; S5: Chlorogenic acid; S6: Syringin; S7: Protocatechuic acid; S8: Protocatechuic aldehyde; S9: 3,5-O-dicaffeoylquinic acid; S10: 4,5-O-dicaffeoylquinic acid), the characteristic chromatogram of Sijiqing formula granules shows a total of 16 chromatographic peaks, of which 9 peaks (peaks 4, 5, 7, 8, 9, 10, 12, 13, and 15) are known components. Based on literature review, protocatechuic acid and 4,5-O-dicaffeoylquinic acid are the main active components in Sijiqing. Therefore, the peak corresponding to the protocatechuic acid reference standard was selected as peak S1, and the peak corresponding to the 4,5-O-dicaffeoylquinic acid reference standard was selected as peak S2.

[0103] Table 4. Relative retention time of characteristic spectra of Sijiqing formula granules

[0104]

[0105]

[0106] Table 5. Relative peak areas of characteristic spectra of Sijiqing formula granules

[0107]

[0108] Table 6. Relative retention times of fingerprint spectra of *Ilex chinensis* (a type of medicinal herb) as a control.

[0109]

[0110] Table 7. Relative peak areas of fingerprint spectra of *Ilex chinensis* (a type of medicinal herb) as a control.

[0111]

[0112] The results above indicate that the chromatogram of the test sample should show 16 characteristic peaks, corresponding to the retention times of the 16 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 4 and 16 should correspond to the retention times of the reference peaks of protocatechuic acid and 4,5-O-dicaffeoylquinic acid, respectively. The peak corresponding to the protocatechuic acid reference peak is peak S1. The relative retention times of peaks 1-3 and 5-9 should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.46 (peak 1), 0.74 (peak 2), 0.85 (peak 3), 1.63 (peak 5), 1.87 (peak 6), 2.27 (peak 7), 2.53 (peak 8), and 2.92 (peak 9). The peak corresponding to the 4,5-O-dicaffeoylquinic acid reference peak is peak S2. The relative retention times of peaks 10-15 should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.63 (peak 10), 0.67 (peak 11), 0.70 (peak 12), 0.75 (peak 13), 0.92 (peak 14), and 0.97 (peak 15). Peak 4 (S1 peak) corresponds to protocatechuic acid; peak 5 corresponds to protocatechuic aldehyde; peak 7 corresponds to syringin; peak 8 corresponds to chlorogenic acid; peak 9 corresponds to caffeic acid; peak 10 corresponds to 1,3-O-dicaffeoylquinic acid; peak 12 corresponds to quercetin-3-O-mori disaccharide glycoside; peak 14 corresponds to 3,5-O-dicaffeoylquinic acid; and peak 16 (S2) corresponds to 4,5-O-dicaffeoylquinic acid.

[0113] Furthermore, when using the characteristic chromatogram detection method of the Sijiqing formula granules in this embodiment for quality control, the test sample of Sijiqing formula granules is tested according to the aforementioned characteristic chromatogram of Sijiqing formula granules. The chromatogram of the test sample is compared with the reference characteristic chromatogram. If at least 16 characteristic peaks identical to those in the reference characteristic chromatogram appear in the chromatogram of the test sample, the peak corresponding to the protocatechuic acid reference peak is designated as peak S1. The relative retention times of peaks 1-3 and peaks 5-9 are calculated, and their relative retention times should be within ±10% of the specified value. The peak corresponding to the 4,5-O-dicaffeoylquinic acid reference peak is designated as peak S2. The relative retention times of peaks 10-15 are calculated, and their relative retention times should be within ±10% of the specified value. Then, the quality of the test sample of Sijiqing formula granules is qualified; otherwise, it is unqualified. The above quality control detection method is more specific and targeted for Sijiqing formula granules, and plays a standardized, normalized, and objective role in description and evaluation.

[0114] Example 2: Investigation of Flow Rate

[0115] The difference between this embodiment and Example 1 is that the characteristic chromatograms of Sijiqing formula granules (2307001S) were detected at different flow rates in the chromatographic conditions. The flow rates were set to 0.2 ml / min, 0.22 ml / min, and 0.18 ml / min, respectively. The detection results are as follows:Figure 5 to Figure 7 As shown in the table below, the separation effect of each chromatographic peak is good and the system adaptability parameters are relatively similar with slight fluctuations in flow rate. However, the relative retention time of each characteristic peak shows significant fluctuations. Considering the relative retention time of each characteristic peak specified in the characteristic chromatogram, the optimal flow rate is 0.2 ml / min.

[0116] Table 8. Results of relative retention time for different flow velocities

[0117]

[0118] Table 9. Results of relative peak area at different flow velocities

[0119]

[0120]

[0121] Example 3: Investigation of column temperature

[0122] The difference between this embodiment and Example 1 is that the characteristic chromatograms of the Sijiqing formula granules (2307001S) were detected at different column temperatures in the chromatographic conditions. The column temperatures were set to 23℃, 25℃, and 27℃, respectively. The detection results are as follows: Figure 8 to Figure 10 The results are shown in the table below. The results indicate that with fluctuations in column temperature, the separation of each chromatographic peak was good and the system adaptability parameters were relatively similar. However, the relative retention times of each characteristic peak showed significant fluctuations. Considering the relative retention times specified for each characteristic peak in fingerprint chromatograms, the optimal column temperature was 25℃.

[0123] Table 10. Results of relative retention times at different column temperatures

[0124]

[0125]

[0126] Table 11. Relative peak areas at different column temperatures

[0127]

[0128] Example 4: Investigation of the chromatographic column

[0129] The difference between this embodiment and Example 1 is that different chromatographic columns were used to detect the characteristic chromatograms of Sijiqing formula granules (2307001S). The chromatographic columns were set as follows: Column 1: Waters HSS T3; Column 2: Shim-pack GIST C18-Aq; Column 3: Phenomenex Luna Omega C18; Column 4: Dikma Endeavorsil C18. The detection results are as follows. Figure 11 to Figure 14The results, as shown in the table below, indicate that the chromatographic information presented by different brands of high-performance liquid chromatography (HPLC) is relatively complete, and none of the characteristic peaks are missing, suggesting that different models of HPLC columns have good instrument durability for this method.

[0130] Table 12. Comparison of relative retention times for different brands of chromatographic columns

[0131]

[0132]

[0133] Table 13. Comparison of relative peak areas of different brands of chromatographic columns

[0134]

[0135]

[0136] Example 5: Investigation of the Chromatograph

[0137] The difference between this embodiment and Embodiment 1 is that different chromatograms were used to detect the characteristic chromatograms of Sijiqing formula granules (2307001S). The chromatograms were set as follows: Chromatograph 1: Waters; Chromatograph 2: Thermo Fisher Scientific; Chromatograph 3: Agilent. The detection results are as follows: Figure 15 to Figure 17 As shown in the table below. Comparing the fingerprint spectra obtained by high-performance liquid chromatography (HPLC), it can be found that the chromatographic information presented by different brands of HPLC is relatively complete, and none of the characteristic peaks are missing, indicating that different models of HPLC have good instrument durability for this method.

[0138] Table 14. Relative Retention Time for Instrument Durability Testing

[0139]

[0140] Table 15. Relative Peak Area in Instrument Durability Test

[0141]

[0142]

[0143] Experimental Methodology Validation

[0144] 1. Instrument precision

[0145] The same Sijiqing formula granules (2307001S) test solution was injected 6 times according to the method in Example 1. The RSD of the relative retention time of the characteristic peak should be less than 2.0%. The results show that the instrument precision is good.

[0146] Table 16. Relative Retention Time Results of Precision Test

[0147]

[0148]

[0149] Table 17. Results of relative peak area in precision test

[0150]

[0151] 2. Repeatability test

[0152] Six test solutions were prepared in parallel according to the method in Example 1, and the RSD of the relative retention time of the characteristic peak was calculated to be less than 2.0%. The results showed that the method had good repeatability.

[0153] Table 18. Relative Retention Time Results of Repeatability Tests

[0154]

[0155]

[0156] Table 19. Results of Relative Peak Area in Repeatability Tests

[0157]

[0158]

[0159] 3. Stability test

[0160] The same Sijiqing formula granules (2307001S) test solution was taken and measured at 0h, 2h, 4h, 8h, 12h and 24h according to the method under the fingerprint spectrum section. The RSD of the relative retention time of the characteristic peak should be less than 2.0%. The results show that the test solution is stable within 24h and meets the measurement requirements.

[0161] Table 20 Results of relative retention times in stability tests

[0162]

[0163]

[0164] Table 21. Results of relative peak area in stability test

[0165]

[0166] 4. Exclusivity

[0167] Accurately pipette 2 μl each of the test solution and the negative control solution (excluding the excipients and reagents of Sijiqing) and inject them into the high performance liquid chromatograph. Perform the test according to the method in Example 1. The result was negative and there was no interference.

[0168] 5. Intermediate precision (personnel)

[0169] Three researchers prepared and measured the Sijiqing formula granules (2307001S) test solution according to the method in Example 1. The RSD of the relative retention time of the characteristic peak was calculated to be less than 2.0%, and the results showed that the intermediate precision was good.

[0170] Table 22. Intermediate Precision (Personnel) Relative Retention Time

[0171]

[0172] Table 23. Relative Peak Area of ​​Intermediate Precision (Personnel)

[0173]

[0174]

[0175] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting the characteristic chromatogram of Four Seasons Green Formula Granules, characterized in that, Ultra-high performance liquid chromatography (UHPLC) was used for detection, and the chromatographic conditions were as follows: Using acetonitrile as mobile phase A and 0.1% v / v phosphoric acid solution as mobile phase B, the gradient elution program includes: From 0 to 15 min, the volume ratio of mobile phase A to mobile phase B was 8% : 92%. Over 15-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 8%:92% to 18%:82%. Over 30-50 minutes, the volume ratio of mobile phase A to mobile phase B changed from 18%:82% to 22%:78%. For 50-60 minutes, the volume ratio of mobile phase A to mobile phase B is 22%:78%. The detection wavelength was 254 nm; the chromatographic columns were Waters ACQUITY UPLC HSST3 (150 mm × 2.1 mm, 1.8 µm), Shim-pack GIST C18-Aq (150 mm × 2.1 mm, 1.9 µm), and Phenomenex Luna Omega C18 (150 mm × 2.1 mm, 1.9 µm). The method for preparing the test solution includes: taking the test sample, adding 50% methanol for extraction, filtering, and taking the filtrate to obtain the solution; Preparation of reference solutions: 1,3-O-dicaffeoylquinic acid, quercetin-3-O-morum disaccharide glycoside, caffeic acid, chlorogenic acid, syringin, protocatechuic acid, protocatechuic aldehyde, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid reference standards were prepared into reference solutions.

2. The method for detecting the characteristic spectrum of the Four Seasons Green Formula Granules according to claim 1, characterized in that, The chromatographic conditions include at least one of the following: 1) The flow rate is 0.18-0.22 mL / min; 2) Column temperature is 23-27℃; 3) The chromatographic column was a Waters ACQUITY UPLC HSS T3; 4) The injection volume is 1.8-2.2µL.

3. The method for detecting the characteristic spectrum of the Four Seasons Green Formula Granules according to claim 1, characterized in that, The chromatographic conditions include an injection volume of 2 µL.

4. The method for detecting the characteristic spectrum of the Four Seasons Green Formula Granules according to any one of claims 1-3, characterized in that, It also includes methods for preparing reference solutions, including: Preparation of reference solution for control medicinal materials: Take the control medicinal material of Sijiqing, add solvent to extract, filter, and take the filtrate to obtain the solution.

5. The method for detecting the characteristic spectrum of the Four Seasons Green Formula Granules according to claim 4, characterized in that, Preparation of reference solution for medicinal materials: The solvent is water or methanol solution; And / or, in the extraction step, first add water and heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 50 v / v% methanol aqueous solution to the residue and ultrasonically extract for 30 minutes, filter, and collect the filtrate; And / or, the mass-to-volume ratio of the Four Seasons Green reference medicinal material to water is 1:20, with a ratio of g / ml; And / or, the mass-to-volume ratio of the *Ilex chinensis* reference herb and the 50 v / v% methanol aqueous solution is 1:20, with a ratio of g / ml.

6. The method for detecting the characteristic spectrum of the Four Seasons Green Formula Granules according to claim 5, characterized in that, Preparation of reference solution for control medicinal materials: Take 1g of Sijiqing reference medicinal material, add 20ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 20ml of 50 v / v % methanol aqueous solution to the residue, sonicate at 250W power and 40kHz frequency for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution. And / or, preparation of reference solution: Take appropriate amounts of protocatechuic acid and 4,5-O-dicaffeoylquinic acid reference standards, accurately weigh them, and add 50 v / v% methanol aqueous solution to prepare a mixed solution containing 50 μg of each per 1 ml, as the reference solution.

7. A method for determining the content of *Lysimachia christinae* formula granules, characterized in that, include: Take the test solution and the reference solution, and perform ultra-high performance liquid chromatography (UHPLC) according to the detection method of the characteristic spectrum of Sijiqing formula granules as described in any one of claims 1-6.

8. The method for detecting the characteristic spectrum of Sijiqing formula granules according to any one of claims 1-6 or the method for determining the content of Sijiqing formula granules according to claim 7, applied to the quality testing of Sijiqing formula granules.

9. A method for quality testing of Four Seasons Green Formula Granules, characterized in that, The method includes the steps of obtaining the characteristic spectrum of the test sample by the detection method of the characteristic spectrum of Sijiqing formula granules according to any one of claims 1-6, and comparing the characteristic spectrum with the reference characteristic spectrum; The control feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of Sijiqing formula granules as a standard according to the detection method of feature spectrum of Sijiqing formula granules as described in any one of claims 1-6.

10. The method for quality testing of *Clerodendrum trichotomum* formula granules according to claim 9, characterized in that, The reference characteristic spectrum includes 16 characteristic peaks, with peak 4 being protocatechuic acid and peak 16 being 4,5-O-dicaffeoylquinic acid. Peak 4 is used as the first reference peak S1. The relative retention times of peaks 1-3 and peaks 5-9 with peak S1 are within ±10% of the specified values. The specified values ​​for peak 1 are 0.46, peak 2 is 0.74, peak 3 is 0.85, peak 5 is 1.63, peak 6 is 1.87, peak 7 is 2.27, peak 8 is 2.53, and peak 9 is 2.

92. Peak 16 is used as the second reference peak S2. The relative retention times of peaks 10-15 with peak S2 are within ±10% of the specified values. The specified values ​​for peak 10 are 0.63, peak 11 is 0.67, peak 12 is 0.70, peak 13 is 0.75, peak 14 is 0.92, and peak 15 is 0.

97.

11. The quality testing method for Sijiqing formula granules according to claim 10, characterized in that, Peak 4 corresponds to protocatechuic acid; peak 5 corresponds to protocatechuic aldehyde; peak 7 corresponds to syringin; peak 8 corresponds to chlorogenic acid; peak 9 corresponds to caffeic acid; peak 10 corresponds to 1,3-O-dicaffeoylquinic acid; peak 12 corresponds to quercetin-3-O-morum disaccharide glycoside; peak 14 corresponds to 3,5-O-dicaffeoylquinic acid; peak 16 corresponds to 4,5-O-dicaffeoylquinic acid.

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