UPLC (Ultra Performance Liquid Chromatography) fingerprint spectrum detection method for stone-eliminating and gallbladder-benefiting traditional Chinese

Abstract

According to the UPLC fingerprint spectrum detection method for the stone-eliminating and gallbladder-benefiting traditional Chinese medicine composition, a chemical mode recognition method is combined, 12 common peaks are calibrated in total, 8 common peaks are identified through a chemical reference substance, the similarity of 15 batches of stone-eliminating and gallbladder-benefiting traditional Chinese medicine composition fingerprints is 0.932 or above, and through analysis of HCA, PCA, OPLS-DA and the like and verification of a neural network model, the similarity of the fingerprint spectrums of the stone-eliminating and gallbladder-benefiting traditional Chinese medicine composition fingerprints is determined. 15 batches of stone-eliminating and gallbladder-benefiting traditional Chinese medicine composition samples are divided into 3 classes, and 7 main chemical markers which may cause mass difference are screened out, namely chromatographic peaks 2 and 6 (baicalin), chromatographic peaks 4, chromatographic peaks 8, chromatographic peaks 7, chromatographic peaks 9 (rhein) and chromatographic peaks 5 (hesperidin). The fingerprint detection method has the advantages of simplicity, feasibility, short detection time (about 25 minutes), good stability and good repeatability, can evaluate the inherent quality of the stone-eliminating and gallbladder-benefiting traditional Chinese medicine composition, further guarantees the stability of the inherent quality of the product, and provides a reference basis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of quality detection of traditional Chinese medicine chemical components, and particularly relates to a UPLC fingerprint spectrum detection method for a Xiaoshi Lidan traditional Chinese medicine composition. BACKGROUND

[0002] The Xiaoshi Lidan traditional Chinese medicine composition is Xiaoshi Lidan capsules produced by Baoding Tianhao Pharmaceutical Co., Ltd. The medicine has the effects of soothing liver and promoting bile secretion, and promoting qi and relieving pain. The composition is prepared from 13 kinds of traditional Chinese medicines, i.e. Bupleurum chinense, Pericarpium Citri Reticulatae Viride, Radix Scrophulariae, Radix Paeoniae Alba, Radix et Rhizoma Rhei, Curcuma longa, Herba Lysimachiae, Herba Haleniae, Gekko japonicus, Herba Artemisiae Scopariae, Curcuma longa, Rhizoma Polygoni Codati, Radix Clematidis. The composition integrates the effects of soothing liver, promoting bile secretion, removing stone, dissolving stone, clearing heat and removing blood stasis, and has a proven clinical effect, and is a safe and reliable prescription for treating cholelithiasis.

[0003] The present application systematically analyzes the prior art documents of the product, and it is found that Sun Yan-tao, Simultaneous determination of paeoniflorin, hesperidin, baicalin and chrysophanol in Xiaoshi Lidan capsules by HPLC double wavelength method, China Pharmacist, publication date: 2016-04-05, the paper uses acetonitrile-0.2% phosphoric acid solution as the mobile phase, gradient elution, the detection wavelength is 230nm and 280nm, and the column temperature is 40 DEG C. The detection method is used for determining the contents of paeoniflorin, hesperidin, baicalin and chrysophanol in Xiaoshi Lidan capsules. Chen Xisheng, Hu Binxiang, Determination of baicalin in Xiaoshi Lidan capsules by HPLC method, China Modern Medicine Application, publication date: 2009-08-10, the paper uses acetonitrile-0.2% phosphoric acid (27:73) as the mobile phase, and the detection wavelength is 280nm. The content of baicalin in Xiaoshi Lidan capsules is determined. Ai Weixia, Determination of baicalin in Xiaoshi Lidan pills, Journal of Jianghan University, publication date: 2006-06-25, the content of baicalin in Xiaoshi Lidan pills is determined by using complex extraction-ultraviolet spectrophotometry with a wavelength of 262nm. Therefore, the UPLC method is used to establish the fingerprint spectrum method of Xiaoshi Lidan capsules, and combined with chemical pattern recognition analysis, an analysis and detection method reference is provided for quality control and standard improvement of the prescription preparation, so as to more comprehensively, scientifically and effectively control the quality of the prescription preparation, and ensure that it is uniformly, safely and effectively applied to clinical medicine.

[0004] Most of Chinese patent medicines are derived from ancient Chinese medicine prescriptions, which are clinically effective, and are the core application form of the overall concept of traditional Chinese medicine, syndrome differentiation and treatment and compatibility of drugs. And because most of the traditional Chinese medicine compound is decocted by more than 10 kinds of traditional Chinese medicine materials, the traditional Chinese medicine material composition contained therein is very complex. With the continuous in-depth research on the material basis, pharmacodynamic, pharmacokinetic and mechanism of traditional Chinese medicine chemical components, the analysis and detection technology of drugs has also shown a rapid development trend, and the standard of traditional Chinese medicine has made great progress. Such as the weak specificity and poor repeatability of thin layer and physicochemical identification detection method is gradually replaced by advanced and mature modern analysis technology and means such as fingerprint, characteristic spectrum, one measurement multiple evaluation, UPLC and LC-MS. Xiaoshili Dianjiaonang is collected in 'National Chinese Medicine Standard Compilation·Internal Medicine Hepatobiliary Volume', which only requires identification and inspection, and is relatively weak in controlling drug quality at the current stage. The current research on Xiaoshili Dianjiaonang is mostly focused on the clinical efficacy observation and efficacy analysis of combined medication, and there are few related researches on drug quality standard and content determination. SUMMARY

[0005] The application provides a Xiaoshili Dianjiaonang traditional Chinese medicine composition UPLC fingerprint detection method, and combines chemical pattern recognition method, 12 common peaks are calibrated, 8 common peaks are identified by chemical reference substance, the similarity of fingerprint spectra of 15 batches of Xiaoshili Dianjiaonang traditional Chinese medicine composition is above 0.932, through HCA, PCA and OPLS-DA analysis and verification of neural network model, 15 batches of Xiaoshili Dianjiaonang traditional Chinese medicine composition samples are divided into three categories, and 7 main chemical markers that may cause quality difference are screened out, which are 2, 6 (baicalin), 4, 8, 7, 9 (rhein) and 5 (hesperidin) chromatographic peaks. The fingerprint detection method has the advantages of simplicity, feasibility, short detection time (about 25 minutes), good stability and repeatability, can evaluate the internal quality of Xiaoshili Dianjiaonang traditional Chinese medicine composition, and provide a more scientific reference basis.

[0006] The technical scheme of the patent application is:

[0007] A Xiaoshili Dianjiaonang traditional Chinese medicine composition UPLC detection method, characterized in that the detection method comprises the following steps:

[0008] (1) Preparation of test solution: weigh Xiaoshili Dianjiaonang capsule content, add 100% methanol, weigh the mass, ultrasonic treatment, cool, supplement the lost mass with 100% methanol, shake and filter, and take the filtered solution;

[0009] 2. Preparation of mixed control solution: weigh each control, add methanol to prepare a mixed solution containing 10-30 ug / ml of hesperidin, 20-40 ug / ml of baicalin, 20-25 ug / ml of curcumin, 20-25 ug / ml of tazettin, 20-30 ug / ml of gallic acid, 20-30 ug / ml of chrysophanol, 20-30 ug / ml of emodin, and 15-25 ug / ml of rhein per 1 mL, and then obtain the mixed solution.

[0010] 6. Chromatographic conditions:

[0011] Chromatographic column: C 18 , mobile phase: A-acetonitrile B-0.1% formic acid aqueous solution, gradient elution: 0-3 min, 10%-13% A, 3-4 min, 13%-19% A, 4-18 min, 19%-30% A, 18-20 min, 30%-45% A, 20-25 min, 45%-100% A, 25-28 min, 100%-10% A; 28-30 min, 10%-10% A; volume flow rate: 0.1-0.5 mL / min; detection wavelength: 250-300 nm, column temperature: 25-35 °C;

[0012] 7. Fingerprint spectrum establishment

[0013] Take multiple batches of Xiaoshili Dian powder, take the test solution of step 1, and inject it into the chromatographic column under the chromatographic conditions of step 6 to determine and record the chromatogram of each sample, and then import it into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software for processing, generate the control spectrum R by the median method, and obtain the UPLC fingerprint spectrum of Xiaoshili Dian traditional Chinese medicine composition.

[0014] Preferably, the ultrasonic power in step 1 of the detection method is 700-900 W, the ultrasonic frequency is 30-50 kHz, and the ultrasonic time is 20-40 min.

[0015] Preferably, the ultrasonic power in step 1 of the detection method is 800 W, the ultrasonic frequency is 40 kHz, and the ultrasonic time is 30 min.

[0016] Preferably, in the mixed control solution of step 2 of the detection method, the concentration of hesperidin is 24.00 ug / ml, the concentration of baicalin is 29.30 ug / ml, the concentration of curcumin is 22.10 ug / ml, the concentration of tazettin is 22.80 ug / ml, the concentration of gallic acid is 25.10 ug / ml, the concentration of chrysophanol is 23.70 ug / ml, the concentration of emodin is 23.40 ug / ml, and the concentration of rhein is 21.60 ug / ml.

[0017] Preferably, in step 6 of the detection method, the C 18The model is Waters Acquity UPLC BEH, and the column specifications are 1.7μm and 2.1×100mm.

[0018] Preferably, in step (3) of the detection method, the chromatographic conditions are: flow rate: 0.3 mL / min; detection wavelength: 288 nm; column temperature: 30 °C.

[0019] Preferably, the quantity of multiple batches in step (4) is ≥10.

[0020] Preferably, the detection method is used in the quality testing, intermediate content determination, and identification of effective components of traditional Chinese medicine compositions for dissolving gallstones and promoting bile secretion.

[0021] To further illustrate the inventiveness of the detection method for the traditional Chinese medicine composition for dissolving stones and promoting bile secretion of the present invention, some of the experimental contents of the screening of the technical solution of the present invention are summarized as follows.

[0022] This study optimized and determined the extraction method, number of extractions, extraction solvent, and extraction time of the traditional Chinese medicine composition for dissolving stones and promoting bile secretion through single-factor multi-level experiments.

[0023] 1.1 Screening of extraction methods (static, ultrasonic, reflux)

[0024] Chromatographic column: Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 × 100 mm); mobile phase: A-acetonitrile, B-pure water; gradient elution (0–30 min, 10%–90% A; 30–35 min, 90%–10% A; 35–40 min, 10%–10% A); flow rate: 0.2 mL / min; detection wavelength: 254 nm; column temperature: 30 °C; injection volume: 2 μL.

[0025] 1.1.1 Let stand

[0026] Take 4.0 g of the test sample powder and place it in a stoppered conical flask. Accurately add 120 mL of methanol, tighten the screw, let stand for 24 h, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the final sample. Inject the sample according to the chromatographic conditions in section 1.1. The UPLC chromatogram is shown below. Figure 1 -S1.

[0027] 1.1.2 Ultrasound

[0028] Take 4.0 g of the test sample powder and place it in a stoppered conical flask. Accurately add 120 mL of methanol, tighten the screw, and sonicate (400 W, 40 kHz) for 1 h. Filter the solution and pass the filtrate through a 0.22 μm microporous membrane. Inject the sample according to the chromatographic conditions in section 1.1. The UPLC chromatogram is shown below. Figure 1 -S2.

[0029] 1.1.3 Reflux

[0030] Take the test sample powder 4.0 g, placed in a conical flask with a plug, precise addition of methanol 120 mL, tighten, reflux extraction 1 h, after cooling filter, take the filtrate, through 0.22 μm microporous filter membrane, get. The sample is injected under the chromatographic conditions of 1.1, the experimental results found that by comparing the UPLC chromatograms S1-S3 of standing, ultrasonic and reflux, from the baseline stability, the overall shape of the chromatographic peak, the peak position, the size of the peak and the number of peaks, etc. The baseline stability is equivalent, the peak position is basically the same, and the size of the chromatographic peak and the number of chromatographic peaks have certain differences. Among them, the number of chromatographic peaks of S2 and S3 is significantly more than that of S1, such as between the retention time 12-15 min; The size of the chromatographic peak of S2 and S3 is also significantly larger than that of S1, such as between the retention time 5-8 min. While the difference between S2 and S3 in the above indicators is not obvious, but the baseline deviation of S3 is higher than that of S2 at the retention time 7-9 min. In addition, the convenience of sample preparation of reflux (S3) is worse than that of ultrasonic (S2). In summary, the extraction method is finally determined as ultrasonic.

[0031] 1.3 Selection of extraction solvent

[0032] The chromatographic conditions are the same as those in 1.1.

[0033] 1.3.1 Pure water

[0034] Take the test sample powder 0.5 g, placed in a PVC tube with a cover, precise addition of pure water 15 mL, tighten, ultrasonic (power 400 W, frequency 40 kHz) 1 h, filter, take the filtrate, through 0.22 μm microporous filter membrane, get. The sample is injected under the chromatographic conditions of 1.1, the UPLC chromatogram is shown in the specification appendix Figure 11 -S1.

[0035] 1.3.2 Ethanol

[0036] Take the test sample powder 0.5 g, placed in a PVC tube with a cover, precise addition of ethanol 15 mL, tighten, ultrasonic (power 400 W, frequency 40 kHz) 1 h, filter, take the filtrate, through 0.22 μm microporous filter membrane, get. The sample is injected under the chromatographic conditions of 1.1, the UPLC chromatogram is shown in the specification appendix Figure 11 -S2.

[0037] 1.3.3 Acetonitrile

[0038] Take the test sample powder 0.5 g, placed in a capped PVC tube, precise add acetonitrile 15 mL, screw, ultrasonic (power 400 W, frequency 40 kHz) 1 h, filter, take the filtrate, through 0.22 μm microporous filter membrane. The sample is injected under the chromatographic conditions of 1.1, UPLC chromatogram see specification attached Figure 11 -S3.

[0039] 1.3.4 Methanol

[0040] Take the test sample powder 0.5 g, placed in a capped PVC tube, precise add acetonitrile 15 mL, screw, ultrasonic (power 400 W, frequency 40 kHz) 1 h, filter, take the filtrate, through 0.22 μm microporous filter membrane. The sample is injected under the chromatographic conditions of 1.1, UPLC chromatogram see specification attached Figure 11 -S4.

[0041] 1.3.5 90% methanol

[0042] Take the test sample powder 0.5 g, placed in a capped PVC tube, precise add acetonitrile 15 mL, screw, ultrasonic (power 400 W, frequency 40 kHz) 1 h, filter, take the filtrate, through 0.22 μm microporous filter membrane. The sample is injected under the chromatographic conditions of 1.1, UPLC chromatogram see specification attached Figure 11 -S5.

[0043] 1.3.6 70% methanol

[0044] Take the test sample powder 0.5 g, placed in a capped PVC tube, precise add acetonitrile 15 mL, screw, ultrasonic (power 400 W, frequency 40 kHz) 1 h, filter, take the filtrate, through 0.22 μm microporous filter membrane. The sample is injected under the chromatographic conditions of 1.1, UPLC chromatogram see Figure 3 -S6.

[0045] 1.3.7 50% methanol

[0046] Take the test sample powder 0.5 g, placed in a capped PVC tube, precise add acetonitrile 15 mL, screw, ultrasonic (power 400 W, frequency 40 kHz) 1 h, filter, take the filtrate, through 0.22 μm microporous filter membrane. The sample is injected under the chromatographic conditions of 1.1, UPLC chromatogram see specification attached Figure 11 -S7.

[0047] 1.3.8 30% methanol

[0048] Take the test sample powder 0.5g, placed in a PVC tube with cover, precise add 30% methanol 15mL, screw, ultrasonic (power 400W, frequency 40kHz) 1h, filter, take the filter liquid, through 0.22μm microporous filter membrane, then get. The sample is injected under the chromatographic conditions of 1.1, UPLC chromatogram is shown in the specification Figure 11 -S8.

[0049] 1.3.9 10% methanol

[0050] Take the test sample powder 0.5g, placed in a PVC tube with cover, precise add 30% methanol 15mL, screw, ultrasonic (power 400W, frequency 40kHz) 1h, filter, take the filter liquid, through 0.22μm microporous filter membrane, then get. The sample is injected under the chromatographic conditions of 1.1, UPLC chromatogram is shown in the specification Figure 11 As shown in the specification, by comparing the UPLC chromatograms S1-S9 of 9 different extraction solvents of pure water, ethanol, acetonitrile, methanol, 90% methanol, 70% methanol, 50% methanol, 30% methanol, 10% methanol, respectively, from the baseline stability, the overall shape of the chromatographic peak, the peak position, the size of the peak and the number of peaks, the baseline stability is quite, but the peak position, the size of the peak and the number of peaks have certain differences. Among them, the number of chromatographic peaks of S2 is the least, which does not meet the requirements of the development of fingerprint chromatogram; the number of chromatographic peaks of S3 is more than that of S2, but in the retention time of 7-8min, compared with S1 and S4-S9, several chromatographic peaks with large peak area are missing, which is also excluded; S1, S8 and S9 are similar, but in the retention time of 22-23min, compared with S4-S7, one chromatographic peak with good peak shape is missing, so they are all excluded; the remaining S4-S7, after comprehensive comparison, it can be seen that S4 performs well in terms of stability, number of chromatographic peaks and size of chromatographic peaks. At the same time, it can also be seen that when the extraction solvent is methanol (S4), the impurity peaks of the fingerprint chromatogram are less than those when the extraction solvent is 90% methanol (S5), 70% methanol (S6) and 50% methanol (S7). In summary, the extraction solvent is finally determined as methanol.

[0051] 1.4 extraction time screening

[0052] By comparing the UPLC chromatograms S1-S4 of 4 different extraction times of 0.5h, 1h, 1.5h, 2h, respectively, from the baseline stability, the overall shape of the chromatographic peak, the peak position, the size of the peak and the number of peaks, they are basically consistent. Therefore, considering the increase of extraction time, the detection efficiency of the fingerprint chromatogram will be reduced. Therefore, in summary, the extraction time is finally determined as 0.5h.

[0053] 1.5 ultrasonic power screening

[0054] By comparing the UPLC chromatograms S1-S4 of 800W, 400W, 200W, 100W and other four different ultrasonic powers, the baseline stability, the overall shape of the chromatographic peak, the peak position and the size of the peak were basically consistent. But the number of peaks was slightly different. S1(800W) had more peaks than S2-S4 between 6-10 min of retention time. At the same time, it was found in the experiment that lower ultrasonic power might cause the sample in the PVC tube to adhere to the bottom and not easy to shake evenly during ultrasonic process. Therefore, considering the feasibility and wide applicability of the test sample preparation method, the extraction power was finally determined as 800W.

[0055] 2. Optimization of chromatographic conditions

[0056] Test sample preparation method: take 0.5g of test sample powder, put it in a PVC tube with a lid, precisely add 15mL of methanol, tighten, ultrasonic (power 800W, frequency 40kHz) for 0.5h, filter, take the filtrate, pass through a 0.22μm microporous filter membrane to obtain.

[0057] 2.1 Selection of chromatographic column

[0058] Four commonly used UPLC chromatographic columns (C18) of different brands, models and parameters on the market were selected (Waters Acquity UPLC BEH C18(1.7μm, 2.1x100mm), chromatographic column: Agilent Zrobax RRHDSB-C18(1.8μm, 2.1x100mm), SHIMADZU Shim-pack GIST-HP C18-AQ(3μm, 2.1x100mm), Dikma Endeavorsil C18-A(1.8μm, 2.1x100mm). The influence on separation effect. Mobile phase: A-acetonitrile, B-pure water; gradient elution (0-30min, 10%-90%A; 30-35min, 90%-10%A; 35-40min, 10%-10%A); volume flow rate: 0.2mL / min; detection wavelength: 254nm; column temperature: 25℃; injection volume: 2μL.

[0059] The experimental results: by comparing the Waters BEH, Agilent RRHD, Shimadzu GIST-HP, Dikma Endeavorsil, etc. 4 common super high performance liquid chromatography column liquid chromatogram, from the baseline smooth degree, the overall shape of the chromatographic peak, the peak position, the size of the peak and the number of peaks, etc. Respectively, there are some differences. Among them, S2 in the retention time 23~26 min, the peak shape of the two main chromatographic peaks is poor, and the baseline is not flat in the retention time 8~12 min, so it is excluded first; S3 (Shimadzu GIST-HP) has the least number of peaks before the retention time 7 min, so it is also excluded; S4 (Dikma Endeavorsil) has poor peak shape in the chromatogram in the retention time 6~10 min, while S1 (Waters BEH) has significantly more peaks before the retention time 13 min, and the baseline remains stable overall. Therefore, as described above, the chromatographic column is finally determined as Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 x 100 mm).

[0060] 2.2 Selection of mobile phase

[0061] The effects of methanol-water, acetonitrile-water, methanol-0.05% phosphoric acid water, methanol-0.1% phosphoric acid water, methanol-0.05% formic acid water, methanol-0.1% formic acid water, acetonitrile-0.05% phosphoric acid water, acetonitrile-0.1% phosphoric acid water, acetonitrile-0.05% formic acid water, acetonitrile-0.1% formic acid water systems on separation effect were investigated respectively. Chromatographic column: Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 x 100 mm); volume flow rate: 0.2 mL / min; detection wavelength: 254 nm; column temperature: 25 °C; sample volume: 2 μL.

[0062] 2.2.1 Methanol-water

[0063] The samples were prepared according to the sample preparation method under item 2, and the chromatographic conditions under item 2 were used. The sample was injected under the mobile phase system and elution program, and the gradient elution program was used. The mobile phase was methanol A-water B, 0-30 min, A; methanol (10%), B water (90%), 35 min, A; methanol (100%), B water (0%), 40 min, A; methanol (10%), B water (90%). The rest of the mobile phase is: acetonitrile-water, methanol-0.05% phosphoric acid water, methanol-0.1% phosphoric acid water, methanol-0.05% formic acid water, methanol-0.1% formic acid water, acetonitrile-0.05% phosphoric acid water, acetonitrile-0.1% phosphoric acid water, acetonitrile-0.05% formic acid water, acetonitrile-0.1% formic acid water, and the proportion of the chromatographic mobile phase system is the same.

[0064] Experimental results are described in the specification Figure 12 The experimental results show that there are certain differences in terms of baseline flatness, overall shape of the chromatographic peak, peak position, peak size, and the number of peaks, etc. Among them, S2 is excluded first because of the absence of main chromatographic peaks between 16-18 min; S1 and S3-S6 have little difference in baseline flatness, overall shape of the chromatographic peak, peak position, peak size, and the number of peaks, etc., but have small peak area and no obvious chromatographic peak between 3-12 min; S7-S10 move all the chromatographic peaks forward, effectively saving time; among them, S7 and S10 have significantly more chromatographic peaks separated between 8-10 min than S8-S9. In addition, the separation degree of the chromatographic peaks of S10 is better than that of S7 between 10-12 min. In summary, the mobile phase system is finally determined as acetonitrile-0.1% formic acid water.

[0065] 2.3 Selection of detection wavelength

[0066] By comparing the UPLC chromatograms at 5 different detection wavelengths of 210 nm, 230 nm, 265 nm, 280 nm, and 288 nm, there are certain differences in terms of baseline flatness, overall shape of the chromatographic peak, peak position, peak size, and the number of peaks, etc. Among them, S1 (210 nm) and S2 (230 nm) have unstable baseline, so they are excluded first; S3 (265 nm) has low response of chromatographic peak between 7-9 min, which is excluded; S4 (280 nm) and S5 (288 nm) have basically equivalent chromatograms, but S5 (288 nm) has slightly higher response of chromatographic peak between 19-25 min. In summary, the detection wavelength is finally determined as 288 nm.

[0067] 2.4 Selection of column temperature

[0068] By comparing the UPLC chromatograms at 4 different column temperatures of 25℃, 30℃, 35℃, and 40℃, they are basically consistent in terms of baseline flatness, overall shape of the chromatographic peak, peak size, and the number of peaks, etc., only the peak position has certain differences. With the increase of column temperature, the peak position of the overall chromatographic peak moves forward. The forward movement of the peak position of the chromatographic peak will save time and save the consumption of mobile phase, but at the same time it will also reduce the service life of the chromatographic column. Considering the consumption of mobile phase, the actual use environment of the equipment, and the durability of the chromatographic column, the column temperature is finally determined as 30℃.

[0069] 2.5 Selection of flow rate

[0070] By comparing UPLC chromatograms S1-S5 at 5 different flow rates of 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, there are certain differences in terms of baseline stability, overall shape of the chromatographic peak, size of the peak, peak position and number of peaks, etc. With the increase of flow rate, the overall chromatographic peak position moves forward, and the peak area gradually decreases. At the same time, although the peak area of S1 (0.1 mL / min) is large and the response is high, the baseline is relatively uneven, the elution time is too long, which affects the efficiency, and also causes the consumption of mobile phase to increase. Therefore, considering the overall chromatogram and the consumption of mobile phase, the flow rate is finally determined to be 0.2 mL / min.

[0071] 2.6 Selection of elution conditions

[0072] Chromatographic column: Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 x 100 mm); detection wavelength: 288 nm; column temperature: 30 degrees Celsius; sample volume: 2 μL.

[0073] 2.6.1 Isocratic elution

[0074] By comparing isocratic elution UPLC chromatograms S1-S5 at 5 different mobile phase ratios of acetonitrile: 0.1% formic acid water (10:90) 40 min, acetonitrile: 0.1% formic acid water (30:70) 40 min, acetonitrile: 0.1% formic acid water (50:50) 40 min, acetonitrile: 0.1% formic acid water (70:30) 40 min, acetonitrile: 0.1% formic acid water (90:10) 40 min, there are certain differences in terms of baseline stability, overall shape of the chromatographic peak, size of the peak, peak position and number of peaks, etc. Therefore, after comprehensive consideration, it is judged that the test solution is not suitable for elution separation with isocratic elution system.

[0075] Table 1 Data of part of the screening process of the chromatographic mobile phase elution ratio conditions of the application

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] The patent technical solution of the present application has the following beneficial effects:

[0085] (1) The present application finally finds the best chromatographic conditions through a large number of experimental exploration and attempts: A-acetonitrile B-0.1% formic acid aqueous solution, gradient elution: 0-3 min, 10%-13% A, 3-4 min, 13%-19% A, 4-18 min, 19%-30% A, 18-20 min, 30%-45% A, 20-25 min, 45%-100% A, 25-28 min, 100%-10% A; 28-30 min, 10%-10% A.

[0086] (2) The present application also uses similarity evaluation, hierarchical cluster analysis (HCA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and neural network model to analyze and evaluate the fingerprints of 15 batches of Xiaoshili Dianzhong traditional Chinese medicine compositions. The HPLC fingerprint of Xiaoshili Dianzhong traditional Chinese medicine composition is calibrated with 12 common peaks, 8 of which are identified by chemical reference substances. The similarity of the fingerprints of 15 batches of Xiaoshili Dianzhong traditional Chinese medicine compositions is above 0.932. Through HCA, PCA and OPLS-DA analysis and verification of the neural network model, 15 batches of Xiaoshili Dianzhong traditional Chinese medicine composition samples are divided into 3 categories, and 7 main chemical markers that may cause quality differences are screened out, which are 2, 6 (baicalin), 4, 8, 7, 9 (rhein) and 5 (hesperidin) chromatographic peaks.

[0087] (3) Through methodological investigation, the precision test results show that the relative retention time RSD values of each common peak are 0.002%-0.158%, and the relative peak area RSD values are 0.235%-2.154%, indicating that the instrument precision is good. The stability test results show that the relative retention time RSD values of each common peak are 0.002%-0.189%, and the relative peak area RSD is 0.453%-3.014%. It shows that the test solution is stable within 24h. The repeatability test results show that the relative retention time RSD values of each common peak are 0.002%-0.165%, and the relative peak area RSD values are 0.463%-3.023%. It shows that the method has good repeatability.

[0088] (4) After establishing HPLC fingerprint chromatograms of 15 batches of Xiaoshi Lidan capsules and evaluating their similarity, this invention uses the peak areas of 12 common peaks obtained from the HPLC fingerprint chromatograms as variables. Chemical pattern recognition in chemometrics is then used to further explain the quality differences between different batches of Xiaoshi Lidan capsules. Furthermore, major biomarkers with significant influence on the quality of Xiaoshi Lidan capsules are screened to explain the main factors contributing to the quality differences. Analysis using similarity evaluation software shows that the similarity of the fingerprint chromatograms of the 15 batches of Xiaoshi Lidan traditional Chinese medicine compositions ranges from 0.932 to 1.000. A total of 12 common peaks were identified, and 8 of these common peaks were identified using reference standards, namely gallic acid, naringin, hesperidin, baicalin, rhein, curcumin, emodin, and chrysophanol. Hesperidin can relax the sphincter of Oddi, contract the gallbladder, and promote bile excretion. It can also reduce plasma and liver cholesterol, liver triglyceride levels, and the activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and acyl-CoA cholesterol transferase (ACAT) in rats, while significantly reducing the content of neutral cholesterol in animal excrement. Baicalin has pharmacological effects such as antiviral, anti-inflammatory, antibacterial, antitumor, neuroprotective, antioxidant, hemostatic, and antitumor properties. In terms of liver protection, emodin can treat carbon tetrachloride-induced liver fibrosis in rats by inhibiting the synthesis of connective tissue growth factor (CTGF) and matrix metalloproteinase inhibitor-1 (TIMP-1) and promoting the expression of matrix metalloproteinase 9 (MMP-9).

[0089] Cluster analysis using unsupervised pattern recognition was used to preliminarily evaluate the quality consistency of the choleretic and litholytic traditional Chinese medicine composition. Principal component analysis was used to compare the quality of each group, initially identifying components that influence quality differences. Orthogonal partial least squares discriminant analysis was further employed to screen components with significant impact on sample grouping. The main components contributing relatively large to quality differences were identified as peak 1 (gallic acid), peak 5 (hesperidin), peak 6 (baicalin), peak 9 (rhein), peak 12 (chrysophanol), peak 3 (narcissin), and peak 11 (emodin). This indicates that the pharmacological effect of the choleretic and litholytic capsule is the result of the combined action of multiple components, which is basically consistent with the holistic treatment concept of traditional Chinese medicine. By paying attention to the quality differences that may be caused by the above seven components and tracking the changes in the content of quality markers during the production process, the quality uniformity of the choleretic and litholytic traditional Chinese medicine composition of this invention can be better guaranteed. Attached Figure Description

[0090] Figure 1 HPLC fingerprints of 15 batches of traditional Chinese medicine compositions for dissolving stones and promoting bile secretion (S1-S15);

[0091] Figure 2Fingerprint (R) of the Chinese herbal composition for dissolving stones and promoting bile secretion; among which, chromatographic peaks 2 and 6 are baicalin, chromatographic peaks 4, 8, 7 and 9 are rhein, and chromatographic peak 5 is hesperidin;

[0092] Figure 3 HPLC fingerprint of mixed reference standards;

[0093] Figure 4 HPLC fingerprint of a traditional Chinese medicine composition for dissolving stones and promoting bile secretion;

[0094] Figure 5 Cluster analysis dendrogram of 15 batches of traditional Chinese medicine compositions for dissolving stones and promoting bile secretion;

[0095] Figure 6 Factor analysis chart of 15 batches of traditional Chinese medicine compositions for dissolving stones and promoting bile secretion;

[0096] Figure 7 PCA score chart of 15 batches of traditional Chinese medicine compositions for dissolving stones and promoting bile secretion;

[0097] Figure 8 The dispersion plot of OPLS-DA for 15 batches of traditional Chinese medicine compositions for dissolving stones and promoting bile secretion was obtained, where G1-classification group 1; G2-classification group 2; G3-classification group;

[0098] Figure 9 OPLS-DAVIP values ​​of 15 batches of traditional Chinese medicine compositions for dissolving stones and promoting bile secretion;

[0099] Figure 10 Scatter plot of OPLS-DA loadings for 15 batches of traditional Chinese medicine compositions for dissolving gallstones and promoting bile secretion;

[0100] Figure 11 Chromatograms S1-S9 represent the chromatograms of pure water (S1), ethanol (S2), acetonitrile (S3), methanol (S4), 90% methanol (S5), 70% methanol (S6), 50% methanol (S7), 30% methanol (S8), and 10% methanol (S9).

[0101] Figure 12 The UPLC chromatograms S1–S10 of 10 different mobile phase systems were compared: methanol-water, acetonitrile-water, methanol-0.05% phosphoric acid solution, methanol-0.1% phosphoric acid solution, methanol-0.05% formic acid solution, methanol-0.1% formic acid solution, acetonitrile-0.05% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.05% formic acid solution, and acetonitrile-0.1% formic acid solution. Detailed Implementation

[0102] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0103] Example 1 A detection method for UPLC fingerprint of Shuilidan Capsules

[0104] 1.1 Instruments and reagents

[0105] Waters Acquity UPLC H-class ultra-performance liquid chromatograph (Waters Corporation, USA); TE124S analytical balance (Sartorius, Germany); KQ-800KDE ultrasonic high-power digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 20 μL, 100 μL, 1000 μL, 5 mL pipette guns (Thermo, USA); Reference ultrapure water system (Merck Millipore, Germany).

[0106] Acetonitrile, methanol and formic acid were chromatographically pure and purchased from Thermo Fisher Corporation, USA. The experimental water was self-made ultrapure water. Shuilidan Capsules (0.4 g / capsule) were provided by Baoding Tianhao Pharmaceutical Co., Ltd. with 15 batches, and the batch numbers were 230403, 230404, 230601, 230602, 230602, 230603, 231001, 231002, 231101, 231102, 231103, 240403, 240404, 240601, 240602, and 240603, and the sample numbers were S1-S15, respectively. Tangeritin (batch number: 110721-202220), baicalin (batch number: 110715-202223), curcumin (batch number: 110823-202107), zephyranthine (batch number: 111997-202302), gallic acid (batch number: 110831-202407), chrysophanol (batch number: 110796-202423), emodin (batch number: 110756-202414), and rhein (batch number: 110757-202308) were purchased from China National Institute for Food and Drug Control, and the purity of each was greater than 98%.

[0107] 2 Methods and results

[0108] 2.1 Chromatographic conditions

[0109] Chromatographic column: Waters Acquity UPLC BEH C 18(1.7 pm, 2.1 x 100 mm); mobile phase: A-acetonitrile B-0.1% formic acid in water, gradient elution (0-3 min, 10%-13% A; 3-4 min, 13%-19% A; 4-18 min, 19%-30% A; 18-20 min, 30%-45% A; 20-25 min, 45%-100% A; 25-28 min, 100%-10% A; 28-30 min, 10%-10% A); volume flow rate: 0.3 mL / min; detection wavelength: 288 nm; column temperature: 30 °C; injection volume: 2 pL.

[0110] 2.2 Preparation of solutions

[0111] 2.2.1 Reference solution

[0112] Accurately weigh appropriate amount of each reference substance into a 10 mL volumetric flask, add methanol to make a mixed solution containing 24.00 pg / mL of hesperidin, 29.30 pg / mL of baicalin, 22.10 pg / mL of curcumin, 22.80 pg / mL of tazettin, 25.10 pg / mL of gallic acid, 23.70 pg / mL of chrysophanol, 23.40 pg / mL of emodin, and 21.60 pg / mL of rhein per 1 mL (see chromatogram Figure 3 ).

[0113] 2.2.2 Test solution

[0114] Accurately weigh 0.5 g of the contents of Chenshili Duan Capsules into a conical flask with a stopper, accurately add 10 mL of 100% methanol, weigh the mass, ultrasonically treat (power 800 W, frequency 40 kHz) for 30 min, cool, make up the lost mass with 100% methanol, shake well, and filter. The filtrate is the test solution.

[0115] 2.3 Methodology investigation

[0116] 2.3.1 Precision test

[0117] Weigh Chenshili Duan Capsule sample (No. 230403), prepare the test solution according to the method in item “2.2.2”, continuously inject 6 times under the conditions in item “2.1”, record the relative retention time of each common peak, and calculate the relative peak area.

[0118] The results show that the RSD values of the relative retention time of each common peak are 0.002%-0.158%, and the RSD values of the relative peak area are 0.235%-2.154%. Using the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” (2012 version) software to analyze and process the chromatograms, it is found that the similarity of the chromatograms obtained by continuous injection for 6 times is all ≥0.99 compared with the reference chromatogram, indicating that the precision of the instrument is good.

[0119] 2.3.2 Stability test

[0120] Take 6 samples of Cholestagel capsules (No. 230403), and prepare the test solution according to the method in item 2.2.2. Inject the sample under the conditions in item 2.1, and record the relative retention time of each common peak and calculate the relative peak area. The results show that the RSD of the relative retention time of each common peak is 0.002% to 0.189%, and the RSD of the relative peak area is 0.453% to 3.014%. The above experimental data show that the test solution is stable within 24 h.

[0121] 2.3.3 Reproducibility test

[0122] Take Cholestagel capsule samples (No. 230403), and prepare the test solution according to the method in item 2.2.2. Inject the sample under the conditions in item 2.1 at 0, 2, 4, 7, 12, and 24 h, respectively, and record the relative retention time of each common peak and calculate the relative peak area. The results show that the RSD of the relative retention time of each common peak is 0.002% to 0.165%, and the RSD of the relative peak area is 0.463% to 3.023%. This indicates that the method is reproducible.

[0123] 2.4 Establishment of UPLC fingerprint

[0124] Take 15 batches of Cholestagel capsule samples, and prepare the test solution according to the method in item 2.2.2. Inject the sample under the chromatographic conditions in item 2.1, record the chromatogram of each sample, and import it into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) software for processing. Take S1 (batch No. 230403) as the reference spectrum, the time window width is 0.1 min, and the chromatogram is matched by the multi-point correction method. The control spectrum (R) is generated by the median method, which can be seen in the chromatogram Figure 2 . The UPLC fingerprint of 15 batches of Cholestagel capsules is obtained (which can be seen in the chromatogram Figure 1 ), and 12 common peaks are determined.

[0125] 2.5 Identification of common peaks

[0126] Eight of the 12 common peaks are identified by comparing with the mixed reference substance using a Waters Acquity UPLC H-class ultra-high performance liquid chromatograph. The experimental results are shown in the chromatogram Figure 4 , in which peaks 1, 3, 5, 6, 9, 10, 11, and 12 are identified as gallic acid, zephyranthine, hesperidin, baicalin, rhein, curcumin, emodin, and chrysophanol, respectively.

[0127] 2.6 Similarity evaluation of the fingerprint

[0128] The similarity of 15 batches of Xiaoshili Dianjiaonang samples was calculated by analyzing the fingerprint data of the samples using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) software, and the results are shown in Table 1. The RSD values of the retention times of the common peaks were all less than 4.00%, and the similarity of the 15 batches of Xiaoshili Dianjiaonang was 0.932-1.000, indicating that the quality similarity of the different batches of Xiaoshili Dianjiaonang was high, and the chemical components contained were basically consistent, indicating that the UPLC fingerprint method established in the application was suitable for the qualitative analysis of Xiaoshili Dianjiaonang.

[0129] 2.7 Chemical pattern recognition study

[0130] 2.5.1 Cluster analysis

[0131] Hierarchical cluster analysis (HCA) was used to establish the original data matrix with the peak areas of 12 common peaks of 15 batches of Xiaoshili Dianjiaonang samples as variables, and was imported into SPSS 26.0 software, with squared Euclidean distance as the measure, to evaluate the similarities and differences of the quality of Xiaoshili Dianjiaonang between different batches, and the results are shown in the chromatogram Figure 3 . The results showed that when the classification distance was 20, the 15 batches of Xiaoshili Dianjiaonang samples could be divided into two categories, S1, S2, S5, S6, S7, S10, S11, S12, S13, S15 were in one category (G1), and S3, S4, S8, S19 were in another category (G2); when the classification distance was 15, the 15 batches of Xiaoshili Dianjiaonang samples could be divided into three categories, S1, S2, S5, S6, S7, S10, S11, S12, S13, S15 were in one category (G1), S3, S4, S8, S9 were in one category (G2), and S14 was in one category (G3), indicating that the similarity of the samples of different batches in the same year was high and the difference was small, and the different production times might be one of the important factors leading to the difference in the quality of the drug, and at the same time, it might also be related to the different growth years, picking seasons, and many other factors of the raw medicinal materials, so that there would be differences in the quality of the drug between batches.

[0132] 2.5.2 Principal component analysis

[0133] PCA as a widely used multivariate statistical analysis method, can be through the data dimensionality reduction, the presence of a certain correlation between multiple indicators into a few representative comprehensive indicators, so as to extract the main component characteristics. With 12 common peak peak area of 15 batches of Xiaoshi Lidan capsules as variables, the original data matrix was standardized by SPSS 26.0 software, and principal component (PCA) analysis was performed. Through KMO and Bartlett sphericity test in dimensionality reduction factor analysis, the results are shown in Table 2: the KMO sample adequacy number is 0.676, which is suitable for principal component analysis; Bartlett sphericity test significance p = 0.000 < 0.001, showing that the variables can provide a reasonable basis for principal component analysis. At the same time, after principal component analysis and weight calculation, the first three principal component characteristic values are greater than 1, and the cumulative contribution rate is 84.692%, as shown in Table 3; and the factor analysis of the broken stone chart also shows that there is an inflection point at the second principal component characteristic value, and then gradually tends to be flat. Based on the above analysis results, the first three components are extracted as the principal components.

[0134] With 12 common peak peak area of 15 batches of Xiaoshi Lidan capsules as variables, PCA was performed by Origin 2024 software, and the results are shown in Figure 5 . The results show that most of the sample points are within the 95% confidence interval, indicating that the 15 batches of Xiaoshi Lidan capsules samples as a whole show the characteristics of stable quality; the sum of the contribution rates of the first three principal components extracted is 88.373%, which can basically reflect the main characteristics of the 12 common peak peak areas to some extent. Figure 5 For the PCA score chart, 15 batches of samples were divided into 3 categories, the results were basically consistent with HCA, which further verified the classification results of cluster analysis.

[0135] Table 2 KMO and Bartlett sphericity test

[0136]

[0137] Table 3 Characteristic value and contribution rate of common peak of 15 batches of Xiaoshi Lidan capsules

[0138]

[0139] 2.5.3OPLS-DA analysis

[0140] Orthogonal partial least squares discriminant analysis (OPLS-DA) In order to better explain the difference contribution rate of the common peaks between different batches of Xiaoshili Dianjia Capsules, the peak areas of 12 common peaks of 15 batches of samples were introduced into SIMCA 14.10 software. The data of 3 groups of samples were subjected to supervised chemical pattern recognition analysis by OPLS-DA model which can enhance the separation of groups. The model evaluation score scatter plot, variable importance in projection (VIP) value plot and loading scatter plot were obtained, respectively, as shown in the chromatogram Figures 5 to 7 . It can be seen from Figure 8 that most of the 15 batches of Xiaoshili Dianjia Capsules fell within the 95% confidence interval, and were mainly divided into 3 groups. The OPLS-DA results were basically consistent with the PCA results; combined with the chromatogram Figure 9 , 7 relatively large contribution chromatographic peaks were determined by taking VIP>1 and error bar range in X>0 as the screening basis. The influence from large to small was peak 2, peak 6 (baicalin), peak 4, peak 8, peak 7, peak 9 (rhein), and peak 5 (hesperidin); it can be seen from the loading scatter plot of the chromatogram Figure 10 that the 7 chemical components were far away from the origin, indicating that these components had a great influence on the quality of different batches of Xiaoshili Dianjia Capsules and could be used as quality difference markers of Xiaoshili Dianjia Capsules. At the same time, it showed that the efficacy of Xiaoshili Dianjia Capsules was formed by the joint action of multiple active ingredients. It was suggested that the manufacturers of this variety should further pay attention to the above main markers in order to better guarantee the uniformity of drug quality.

[0141] 2.5.4 Similarity prediction of medicinal materials based on BP neural network

[0142] Through principal component analysis of the common peaks of HPLC chromatograms of 15 batches of Xiaoshili Dianjia Capsules, 12 common peaks were projected into 3 principal components, and the scores of the 3 principal component factors were used as input factors, and the similarity with the control chromatogram was used as output factor. DPS7.05 software was used to establish a neural network similarity fitting model of Xiaoshili Dianjia Capsules. Root mean square error (RMSE) and percent absolute error (AE) were used to evaluate the BP neural network model. The calculation formula is as follows: In the formula, Yi is the network model prediction value, Oi is the similarity measured value, and n is the sample number. After multiple fitting, the similarity neural network model of Xiaoshili Dianjia Capsules has 1 hidden layer, 3 input layer nodes, 3 hidden layer nodes, a minimum training rate of 0.1, a dynamic parameter of 0.6, a parameter SIGMOID of 0.9, an allowable error of 0.0001, a maximum iteration number of 1000, a model fitting residual of 0.01203, an RMSE of 0.0190, and an AE of 0.052% to 1.212%. Therefore, the similarity evaluation model of the Xiaoshili Dianjia Capsule fingerprint spectrum established by the BP neural network can accurately predict the similarity of the fingerprint spectrum of different Xiaoshili Dianjia Capsule samples, thereby classifying medicinal material samples with different qualities, and the classification results are consistent with the results of system clustering analysis, principal component analysis, and similarity evaluation.

[0143] Table 4: Neural network similarity fitting model of Xiaoshili Dianjia Capsules

[0144]

[0145] 4. Conclusion

[0146] Xiaoshili Dianjia Capsules are widely used in clinical practice, but their components are complex, involving multiple medicinal ingredients, and there are problems such as multiple origins and differences in production sites. Currently, there are few studies on the quality standards and content determination methods of Xiaoshili Dianjia Capsules, and the methods are relatively single. However, the overall efficacy of Xiaoshili Dianjia Capsules is achieved through the synergistic action of multiple medicinal components, and simple detection of a certain component cannot fully reflect its quality consistency and overall efficacy. Therefore, it is urgent to improve and enhance the quality control standards of Xiaoshili Dianjia Capsules from the perspective of overall medicinal efficacy components to ensure the stability and safety of its clinical efficacy.

[0147] In this experiment, UPLC fingerprint spectra of 15 batches of Xiaoshili Dianjia Capsules were established by combining chemical pattern recognition method, 12 common peaks were determined, 8 of which were identified, and the similarity of the samples was >0.943. Compared with existing research on the fingerprint spectrum of Xiaoshili Dianjia Capsules, the number of common peaks determined in this study is larger, and the identified common peaks all have important pharmacological activities. At the same time, combined with chemical pattern recognition analysis, 15 batches of Xiaoshili Dianjia Capsules can be divided into 3 categories, and 7 main markers causing quality differences among different batches of samples are found. The UPLC fingerprint spectrum established in this study is helpful for further improving the quality evaluation system of Xiaoshili Dianjia Capsules and can provide a reference for the quality control of Xiaoshili Dianjia Capsules.

Claims

1. A method for detecting UPLC fingerprint of a lithotriptic traditional Chinese medicine composition, characterized in that, The detection method comprises the following steps: (1) Preparation of test sample solution: weigh the contents of Xiaoshili Dian Capsules, add 100% methanol, weigh the mass, ultrasonic treatment, cool, supplement the lost mass with 100% methanol, shake well, filter, and take the filtrate, which is obtained; (2) Preparation of mixed control solution: weigh the control samples, add methanol to prepare a mixed solution containing 10-30 ug / ml of hesperidin, 20-40 ug / ml of baicalin, 20-25 ug / ml of curcumin, 20-25 ug / ml of tazettin, 20-30 ug / ml of gallic acid, 20-30 ug / ml of chrysophanol, 20-30 ug / ml of emodin, and 15-25 ug / ml of rhein per 1 mL, which is obtained; (3) Chromatographic conditions: Column: C 18 , mobile phase: A-acetonitrile B-0.1% formic acid in water, gradient elution: 0~3 min, 10%~13% A, 3~4 min, 13%~19% A, 4~18 min, 19%~30% A, 18~20 min, 30%~45% A, 20~25 min, 45%~100% A, 25~28 min, 100%~10% A; 28~30 min, 10%~10% A; volume flow rate: 0.1~0.5 mL / min; detection wavelength: 250~300 nm, column temperature: 25~35 °C; (4) Fingerprint spectrum establishment Take the test sample solutions of multiple batches in step (1), inject sample according to the chromatographic conditions in step (6), determine the chromatograms of each sample, and import them into the "Traditional Chinese Medicine Chromatographic Fingerprint Spectrum Similarity Evaluation System" software for processing, generate the control spectrum R by the median method, and obtain the UPLC fingerprint spectrum of Xiaoshili Dian Traditional Chinese Medicine Composition.

2. The UPLC detection method of claim 1, wherein, The ultrasonic power in step (1) of the detection method is 700-900 W, the ultrasonic frequency is 30-50 kHz, and the ultrasonic time is 20-40 min.

3. The UPLC detection method of claim 2, wherein, The ultrasonic power in step (1) of the detection method is 800 W, the ultrasonic frequency is 40 kHz, and the ultrasonic time is 30 min.

4. The quality detection method according to claim 1, wherein The concentration of hesperidin in the mixed control solution in step (2) of the detection method is 24.00 ug / ml, the concentration of baicalin is 29.30 ug / ml, the concentration of curcumin is 22.10 ug / ml, the concentration of tazettin is 22.80 ug / ml, the concentration of gallic acid is 25.10 ug / ml, the concentration of chrysophanol is 23.70 ug / ml, the concentration of emodin is 23.40 ug / ml, and the concentration of rhein is 21.60 ug / ml. The volume flow rate under the chromatographic conditions in step (6) of the detection method is 0.3 mL / min, the detection wavelength is 288 nm, and the column temperature is 30°C. The number of batches in step (7) of the detection method is greater than or equal to 10.

5. The UPLC detection method of claim 1, wherein, The detection method step ⑶C 18 Model: Waters Acquity UPLC BEH, column specifications: 1.7 μm, 2.1 x 100 mm.

6. The UPLC detection method of claim 1, wherein, The detection method is applied to the quality detection of Xiaoshili Dian Traditional Chinese Medicine Composition, the determination of intermediate content, and the identification of effective components of traditional Chinese medicine.

7. The UPLC detection method of claim 1, wherein, ​ 8. The UPLC detection method of claim 1, wherein, ​

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