A detection method for Yuye Jiedu Granules and its application

Abstract

The present application relates to the technical field of quality control and detection of traditional Chinese medicine, and in particular to a detection method and application of Yuye Jiedu Granules. The method is a YCM Meteoric Core C18 (150×4.6mm, 2.7μm) chromatographic column, with a flow rate of 1.0mL / min, a column temperature of 30°C, a wavelength of 254nm, acetonitrile as mobile phase A, and 0.05% phosphoric acid aqueous solution as mobile phase B, with gradient elution; the method is applied to the construction of the fingerprint spectrum of Yuye Jiedu Granules and the content determination of 12 components. The methodological verification shows that the method of the present invention realizes the effective detection of 19 characteristic peaks of Yuye Jiedu Granules, and the effective content determination of 12 identified peaks is carried out. The method can be well used for the quality control of Yuye Jiedu Granules.

Description

Technical Field

[0001] This application relates to the technical field of quality control and detection of traditional Chinese medicines, and particularly to a detection method for Yuye Jiedu Granules and its application. Background Art

[0002] Yuye Jiedu Granules is a traditional Chinese medicine compound preparation composed of seven herbs, namely Mussaenda pubescens, Lonicera hypoglauca, Ilex asprella, Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica. Controlling single components or several components of single herbs in traditional Chinese medicine compound preparations can no longer meet the requirements for overall quality control of traditional Chinese medicine compound preparations. Fingerprint / characteristic spectrum can reflect the chemical composition, distribution characteristics, distribution ratio, content characteristics, potential physiological activity, and quantitative characteristics of drug efficacy of traditional Chinese medicine, which is consistent with the integrity, characteristic, multi-target, and non-linear characteristics that should be reflected in the quality control of traditional Chinese medicine. Currently, there is no content determination index in the quality standard of Yuye Jiedu Granules. It is necessary to develop a detection method that can comprehensively control its quality and apply it to the construction of the fingerprint spectrum and the content determination of multiple indicators of Yuye Jiedu Granules. Summary of the Invention

[0003] To solve the above technical problems, the object of the present invention is to provide a detection method for Yuye Jiedu Granules. The method uses high performance liquid chromatography to simultaneously detect 19 components in Yuye Jiedu Granules, and 12 of these components are: neochlorogenic acid, methylshanzhiside, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside;

[0004] The chromatographic conditions of the detection method are: a C18 chromatographic column, a detection wavelength of 254 nm, acetonitrile as mobile phase A, and a 0.05%-0.1% phosphoric acid aqueous solution as mobile phase B, with gradient elution;

[0005] The gradient elution program is: 0 - 3 min, 3% A; 3 - 25 min, 3 - 16% A; 25 - 30 min, 16% A; 30 - 50 min, 16 - 20% A; 50 - 70 min, 25 - 35% A;

[0006] Take an appropriate amount of Yuye Jiedu Granules sample with a 50%-70% methanol solution, extract it by ultrasonic or reflux, filter, and take the subsequent filtrate to obtain the test solution; precisely absorb the test solution and inject it into the liquid chromatograph for determination according to the above method, record the chromatogram, and that's it.

[0007] Preferably, a 0.05% phosphoric acid aqueous solution is used as mobile phase B. The model of the C18 chromatographic column is YCM MeteoricCore C18, with a specification of 150×4.6 mm, 2.7 μm. The flow rate is 1.0 mL / min, and the column temperature is 30°C.

[0008] Preferably, a sample of Yuye Jiedu Granules is taken with an appropriate amount of 50%-70% methanol solution, ultrasonically extracted for 10 minutes-30 minutes, filtered, and the filtrate is taken to obtain the test solution.

[0009] Preferably, take a sample of Yuye Jiedu Granules and add an appropriate amount of 50% methanol solution, ultrasonically extract for 10 minutes, filter, and take the filtrate to obtain the test solution; the ultrasonic extraction power is 300W, 40kHz.

[0010] The above method is used in the construction of the fingerprint spectrum of Yuye Jiedu Granules. The construction method of the fingerprint spectrum of Yuye Jiedu Granules is as follows:

[0011] (1) Take the test solution, reference solution and blank solvent of Yuye Jiedu Granules, and perform the test according to the above method, and record the chromatogram;

[0012] (2) By comparing the peak elution time, the spectra of the reference substance and the test substance, the characteristic peaks are calibrated, and the chromatographic peaks are identified and labeled;

[0013] (3) Set the reference peak, calculate the relative retention time and relative peak area of other characteristic peaks, and establish the fingerprint of Yuye Detoxification Granules based on the relative retention time.

[0014] A total of 19 characteristic peaks were calibrated in the fingerprint spectrum of Yuye Jiedu Granules, and the reference peak was set as isochlorogenic acid B;

[0015] The relative Poly times of the 19 characteristic peaks relative to the reference peaks are: peak 1 is 0.3397, peak 2 is 0.3688, peak 3 is 0.4471, peak 4 is 0.4686, peak 5 is 0.5066, peak 6 is 0.5897, peak 7 is 0.6552, peak 8 is 0.6751, peak 9 is 0.8600, peak 10 is 0.8840, peak 11 is 1.0000, peak 12 is 1.0466, peak 13 is 1.0666, peak 14 is 1.2108, peak 15 is 1.3182, peak 16 is 1.5807, peak 17 is 1.6166, peak 18 is 1.6363, peak 19 is 1.7059;

[0016] The relative peak areas of the 19 characteristic peaks with respect to the reference peak are as follows: the relative peak area of peak No. 1 is 0.6483, that of peak No. 2 is 0.0443, that of peak No. 3 is 0.0516, that of peak No. 4 is 0.9365, that of peak No. 5 is 0.7678, that of peak No. 6 is 0.0831, that of peak No. 7 is 0.1857, that of peak No. 8 is 0.1669, that of peak No. 9 is 0.3707, that of peak No. 10 is 0.1810, that of peak No. 11 is 1.0000, that of peak No. 12 is 0.5307, that of peak No. 13 is 0.1819, that of peak No. 14 is 1.1556, that of peak No. 15 is 0.0453, that of peak No. 16 is 0.0349, that of peak No. 17 is 0.5827, that of peak No. 18 is 0.0679, and that of peak No. 19 is 0.1213;

[0017] Identify and label 12 chromatographic peaks, which are: peak No. 1 is neochlorogenic acid, peak No. 3 is gardenoside methyl ester, peak No. 4 is chlorogenic acid, peak No. 5 is cryptochlorogenic acid, peak No. 6 is loganin, peak No. 8 is secologanin, peak No. 9 is luteoloside, peak No. 11 is isochlorogenic acid B, peak No. 12 is isochlorogenic acid A, peak No. 14 is isochlorogenic acid C, peak No. 16 is luteolin, and peak No. 17 is buddleoside.

[0018] The reference substance solution includes the reference substances corresponding to the above 12 labeled peaks.

[0019] The application of the above detection method or the fingerprint of Yuye Jiedu Granules in the determination of the contents of 12 components in Yuye Jiedu Granules. The 12 components are neochlorogenic acid, gardenoside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside. The content determination method is as follows: Take the sample of Yuye Jiedu Granules, extract it by ultrasonic extraction with 50% methanol solution, filter, and take the subsequent filtrate to obtain the test solution. Take the test solution and determine it according to the above method, and calculate the contents of the 12 components through the relative retention time and relative peak area.

[0020] The numbers of the 12 components are: peak No. 1 is neochlorogenic acid, peak No. 3 is gardenoside methyl ester, peak No. 4 is chlorogenic acid, peak No. 5 is cryptochlorogenic acid, peak No. 6 is loganin, peak No. 8 is secologanin, peak No. 9 is luteoloside, peak No. 11 is isochlorogenic acid B, peak No. 12 is isochlorogenic acid A, peak No. 14 is isochlorogenic acid C, peak No. 16 is luteolin, and peak No. 17 is buddleoside. The relative retention times and relative peak areas of the 12 components are as described above.

[0021] The linear ranges of the content determination of the 12 components are as follows: neochlorogenic acid is 19.796-158.368 μg / ml, shanzhioside methyl ester is 0.8176-40.8811 μg / ml, chlorogenic acid is 19.9534-159.6269 μg / ml, cryptochlorogenic acid is 20.4232-163.3856 μg / ml, loganin is 0.8825-44.1242 μg / ml, deoxylognain is 5.7977-57.9768 μg / ml, luteolin is 0.8648-43.2425 μg / ml, isochlorogenic acid B is 20.9328-167.4624 μg / ml, isochlorogenic acid A is 8.1379-81.3792 μg / ml, isochlorogenic acid C is 11.0544~154.7616 μg / ml, luteolin is 0.2018~20.1845 μg / ml, and montanol is 8.0978~80.9776 μg / ml:

[0022] The detection method of the present invention has been verified by methodology, and has achieved effective separation and detection of 19 characteristic peaks of Yuye Jiedu Granules. Compared with the separation and detection of multiple components of a single medicinal material, it can achieve a more comprehensive quality control study of Yuye Jiedu Granules; through the detection of a large number of batches of Yuye Jiedu Granule samples by this method, a fingerprint of Yuye Jiedu Granules is constructed, which can more completely characterize the quality attributes of Yuye Jiedu Granule preparations, and at the same time, 12 identified peaks are studied for content determination. According to methodology verification, this method can be well used for the content determination and comprehensive quality control of 12 key indicator components of Yuye Jiedu Granules. This method has simple sample processing, does not require expensive experimental instruments, does not require the purchase of a large number of reference substances for external standard method quantification, and can achieve the content determination of 19 characteristic peaks of the key components of each medicinal material of Yuye Jiedu Granules, achieving a better characterization of the quality attributes of Yuye Jiedu Granules, and has important value in production quality control and quality control research. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1-1 Fingerprints at different detection wavelengths (220nm, 254nm, 280nm);

[0025] Figure 1-2Fingerprint spectra at different detection wavelengths (310 nm, 340 nm, 370 nm, 400 nm);

[0026] Figure 2 Fingerprint spectra under different mobile phases;

[0027] Figure 3 Fingerprint spectra peak identification result diagram of Yuye Jiedu Granules;

[0028] Figure 4-1 Peak attribution result of fingerprint spectra of Yuye Jiedu Granules of the present invention (solvent blank);

[0029] Figure 4-2 Peak attribution result of fingerprint spectra of Yuye Jiedu Granules of the present invention (test solution, Mussaenda pubescens, Lonicera japonica, Ilex asprella);

[0030] Figure 4-3 Peak attribution result of fingerprint spectra of Yuye Jiedu Granules of the present invention (Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum, Centella asiatica);

[0031] Figure 5-1 Specificity HPLC diagram for the determination of the contents of 12 components in Yuye Jiedu Granules of the present invention (in the figure, 1 is neochlorogenic acid; 2 is methyl gardenoside; 3 is chlorogenic acid; 4 is cryptochlorogenic acid; 5 is loganin; 6 is secologanin; 7 is luteoloside; 8 is isochlorogenic acid B; 9 is isochlorogenic acid A; 10 is isochlorogenic acid C; 11 is luteolin; 12 is buddleoside);

[0032] Figure 5-2 Specificity HPLC diagram for the determination of the contents of 12 components in Yuye Jiedu Granules of the present invention. Detailed implementation manners

[0033] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0034] Example 1: Development of the detection method for Yuye Jiedu Granules and its application in the construction of fingerprint spectra

[0035] 1.1 Instruments and reagents

[0036] 1.1.1 Instruments

[0037] Thermo Ultimate 3000, high performance liquid chromatograph (DAD detector); SB-5200D ultrasonic cleaner (Ningbo Xinzhi Biotech Co., Ltd.); HH-8 digital display constant temperature water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); ES-502HA electronic balance (Changsha Xiangping Technology Development Co., Ltd.); SQP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); XSR205DU / A electronic analytical balance (METTLER).

[0038] 1.1.2 Test drugs

[0039] Yuye Jiedu Granules, produced by Sanjin Group Hunan Sanjin Pharmaceutical Co., Ltd., a total of 30 batches; acetonitrile, methanol, and phosphoric acid are chromatographically pure, water is pure water, and other reagents are all analytically pure.

[0040] 1.2 Method establishment

[0041] 1.2.1 Chromatographic condition establishment

[0042] ① Investigation of elution gradient

[0043] Comprehensively considering factors such as baseline, number of peaks, and resolution of each chromatographic peak, after gradient exploration, the preliminary method for preparing the test solution is as follows: Take an appropriate amount of this product, grind it finely, take about 5 g, weigh it precisely, place it in a stoppered conical flask, precisely add 25 ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 10 minutes, take it out and let it cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate, that is, obtain it.

[0044] The proposed chromatographic conditions are: YCM Meteoric Core C18 (150×4.6 mm, 2.7 μm) chromatographic column, flow rate 1.0 mL / min, column temperature 30 °C, wavelength 254 nm, injection volume 5 μl, using acetonitrile as mobile phase A and 0.05% phosphoric acid aqueous solution as mobile phase B, and perform gradient elution according to the regulations in Table 1.

[0045] Table 1 Fingerprint gradient elution program

[0046]

[0047] ② Wavelength selection

[0048] Take the test solution of this product and compare the chromatograms at wavelengths of 220 nm, 254 nm, 280 nm, 310 nm, 340 nm, 370 nm, and 400 nm. The results are shown in Figure 1-1 and Figure 1-2The results showed that a full scan was performed at wavelengths of 190 - 400 nm using a diode array detector (DAD). According to factors such as the number of chromatographic peaks, peak response values and their proportions, peak resolution, tailing factor, and baseline stability, the fingerprint of Yuye Jiedu Granules was determined at a wavelength of 254 nm.

[0049] ③ Selection of mobile phase types

[0050] The mobile phase types investigated were acetonitrile - water, acetonitrile - 0.1% formic acid aqueous solution, acetonitrile - 0.1% phosphoric acid aqueous solution, and acetonitrile - 0.05% phosphoric acid aqueous solution. The results are shown in Figure 2 . The results showed that when acetonitrile - aqueous solution was used as the mobile phase, the number of peaks was small and the baseline was unstable; when acetonitrile - 0.1% formic acid aqueous solution was used as the mobile phase, the separation of each chromatographic peak was poor in the time periods of 15 - 20 min and 30 - 35 min; when acetonitrile - 0.1% phosphoric acid aqueous solution and acetonitrile - 0.05% phosphoric acid aqueous solution were used as the mobile phase, the separation effect and peak shape of each chromatographic peak were relatively good. Therefore, acetonitrile - 0.05% phosphoric acid aqueous solution with a lower acid concentration was selected as the mobile phase.

[0051] 1.2.2 Investigation of the preparation method of the test solution

[0052] (1) Extraction method

[0053] Take an appropriate amount of this product, grind it finely, take about 5 g, a total of 2 portions, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 70% methanol, and treat it by ultrasonic (power 300 W, frequency 40 kHz) and heating under reflux (80 °C) for 10 minutes respectively. Take it out and let it cool, then weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0054] Determine the fingerprint according to the established chromatographic conditions. The results are shown in Table 2.

[0055] The results showed that in the fingerprint of the test solution extracted by ultrasonic method, the asymmetry of the chromatographic peak at a retention time of 12.330 min was 1.10, that at 16.957 min was 1.09, that at 18.353 min was 1.09, and that at 21.313 min was 1.00;

[0056] while the corresponding asymmetry of the peaks extracted by reflux was 1.13, 1.21, 1.15, and 1.11 in sequence, indicating that the peak shape of the fingerprint of the test solution extracted by ultrasonic method was better than that extracted by reflux in the time period of 5 - 25 min. Considering the convenience of operation, ultrasonic extraction was selected as the extraction method for this product.

[0057] Table 2 Results of the investigation of extraction methods

[0058]

[0059] (2) Extraction solvent

[0060] Take an appropriate amount of this product, grind it finely, take about 5 g, a total of 9 portions, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of anhydrous ethanol, 70% ethanol, water, 10% methanol, 30% methanol, 50% methanol, 70% methanol, 90% methanol, and methanol respectively, ultrasonically treat (power 300 W, frequency 40 kHz) for 10 minutes, take out and let cool, weigh again, make up the lost weight with each extraction solvent, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0061] Determine the fingerprint according to the established chromatographic conditions, and the results are shown in Table 3.

[0062] Table 3 Results of the investigation of extraction solvents

[0063]

[0064] The results show that when treating the test sample, for the test sample treated with water, it cannot pass through the microporous filter head; for the test samples treated with 10% methanol and 30% methanol, it is relatively difficult to pass through the microporous filter head; for the test samples treated with anhydrous ethanol and 70% ethanol, the overall peak shape is not good, with serious tailing or fronting; for the samples treated with 90% methanol and methanol, the peak shape of the chromatographic peak with a retention time of 12.330 min is not good, and the peak asymmetry is only 0.7 - 0.8; the overall peak shapes of 50% methanol and 70% methanol are better, and the overall peak area of the sample treated with 50% methanol is slightly larger than that of the sample treated with 70% methanol. Therefore, 50% methanol is selected as the extraction solvent.

[0065] (3) Extraction time

[0066] Take an appropriate amount of this product, grind it finely, take about 5 g, a total of 3 portions, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 50% methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 10 minutes, 20 minutes, and 30 minutes respectively, take out and let cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0067] Determine the fingerprint according to the established chromatographic conditions, and the results are shown in Table 4.

[0068] Table 4 Results of the investigation of extraction time

[0069]

[0070] The results show that ultrasonic treatment for 10 minutes has basically completed the extraction. Considering time and energy saving, the ultrasonic time is selected as 10 minutes.

[0071] (4) Sampling amount (see Table 5)

[0072] Table 5 Results of the investigation of sampling amount

[0073]

[0074] Take an appropriate amount of this product, grind it finely, take about 2.5 g, 5 g and 10 g, a total of 3 portions, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 50% methanol, treat with ultrasound (power 300 W, frequency 40 kHz) for 10 minutes, take out and let cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0075] Inject 10 μl of the 2.5 g sample, 5 μl of the 5 g sample, and 2.5 μl of the 10 g sample for injection. For the rest, determine the fingerprint according to the established chromatographic conditions. The results are shown in Table 5.

[0076] The results show that when the sampling amount is 2.5 g, the extraction is more complete compared to 5 g and 10 g, and the filtration operation during the sample treatment process is more convenient. Therefore, the sampling amount is selected as 2.5 g and the injection volume is 10 μl.

[0077] 1.2.3 Determination of the analysis method of the present invention

[0078] After comprehensive investigation and research, the analysis method for Yuye Jiedu Granules is determined as follows:

[0079] Chromatographic conditions: YCM Meteoric Core C18 (150×4.6 mm, 2.7 μm) chromatographic column, flow rate 1.0 mL / min, column temperature 30 °C, wavelength 254 nm. Use acetonitrile as mobile phase A and 0.05% phosphoric acid aqueous solution as mobile phase B, and perform gradient elution according to the regulations in Table 6, and rinse and pre-equilibrate for 30 min.

[0080] Table 6 Fingerprint gradient elution program

[0081]

[0082] Preparation of the test solution: Take an appropriate amount of this product, grind it finely, take about 2.5 g, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of 50% methanol, stopper tightly, weigh, treat with ultrasound for 10 minutes (300 W, 40 kHz), take out and let cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0083] Determination method: Accurately pipette 10 μl of the test solution, inject it into the liquid chromatograph, determine, and record the chromatogram to obtain the chromatogram.

[0084] 1.3 Application and verification of the detection method for Yuye Jiedu Granules in the construction of fingerprint

[0085] 1.3.1 Identification of chromatographic peaks

[0086] According to the chromatographic method determined above, the test solution, reference solution (loganin, isorhamnetin, quercetin, kaempferol, rutin, luteolin, chlorogenic acid, buddleoside, asperosaponin VI, L-phenylalanine, D-phenylalanine, madecassoside, madecassic acid, sophocarpine, shanzhiside methyl ester, ilicifolioside A, asiatic acid, secologanin, macranthoidin A, acacetin, 1,5-dicaffeoylquinic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, methyl mussaenoside, 8-O-acetylshanzhiside methyl ester) and solvent blank were detected in sequence. The results are shown in Figure 3 .

[0087] By comparing the peak elution times and the spectrograms of the reference and test substances, it was determined that peak 1 marked in the figure is neochlorogenic acid, peak 3 is shanzhiside methyl ester, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is loganin, peak 8 is secologanin, peak 9 is luteoloside, peak 11 is isochlorogenic acid B, peak 12 is isochlorogenic acid A, peak 14 is isochlorogenic acid C, peak 16 is luteolin, and peak 17 is buddleoside.

[0088] 1.3.2 Peak attribution

[0089] Respectively weigh Mussaenda pubescens, Lonicera confusa, Ilex asprella, Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum, Centella asiatica according to the prescription ratio, add 25 ml of water to each, decoct for 30 minutes, and evaporate the filtrate of each medicinal liquid to dryness. Subsequently, follow the preparation method of the test solution in "1.2.3" to prepare the test substances of each single herb in sequence. According to the determined chromatographic conditions, inject samples for detection respectively. The results are shown in Figure 4-1 , Figure 4-2 , Figure 4-3 , Table 7.

[0090] Table 7 Peak attribution results of the fingerprint of Yuye Jiedu Granules

[0091]

[0092] Peak attribution results of 12 identified chromatographic peaks: Peak 1 (neochlorogenic acid) and peak 4 (chlorogenic acid) are common peaks of 7 herbs; peak 3 (shanzhiside methyl ester) is a characteristic peak of Mussaenda pubescens; peak 5 (cryptochlorogenic acid), peak 11 (isochlorogenic acid B), peak 12 (isochlorogenic acid A), and peak 14 (isochlorogenic acid C) are common peaks of Mussaenda pubescens, Lonicera confusa, Ilex asprella, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; peak 6 (loganin) is a common peak of Mussaenda pubescens and Lonicera confusa; peak 8 (secologanin) is a common peak of Lonicera confusa and Ilex asprella; peak 9 (luteoloside) is a common peak of Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; peak 16 (luteolin) and peak 17 (buddleoside) are common peaks of Chrysanthemum morifolium and Chrysanthemum indicum.

[0093] Peak attribution results of other unassigned chromatographic peaks: Peak 2 is a common peak of Lonicera confusa, Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; Peak 7 is a common peak of Lonicera confusa, Ilex asprella, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; Peaks 15 and 18 are attributed to Chrysanthemum indicum; Peaks 10, 13, and 19 are common peaks of Chrysanthemum morifolium and Chrysanthemum indicum; Peak 19 is a common peak of Ilex asprella and Helicteres angustifolia.

[0094] 1.3.3 Repeatability test

[0095] Prepare 6 samples of the test solution according to the established analytical method, inject them successively for detection, and examine the repeatability. Calibrate 19 characteristic peaks, using Peak 11 (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results show that the RSD of the relative retention time of the 19 common peaks does not exceed 1.0%, and the RSD of the relative peak area does not exceed 5.0%, indicating that the method has good repeatability.

[0096] 1.3.4 Solution stability test

[0097] Prepare 1 sample of the test solution according to the established analytical method, and measure the fingerprint at 0h, 10h, 20h, 40h, and 50h respectively to examine the stability. Calibrate 19 characteristic peaks, using Peak 11 (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results show that the RSD of the relative retention time of the 19 common peaks does not exceed 1.0%, and the RSD of the relative peak area does not exceed 5.0%, indicating that the solution has good stability within 50 hours.

[0098] 1.3.5 Precision test

[0099] Prepare 1 sample of the test solution according to the established analytical method, inject it continuously for 6 times, and examine the precision. Calibrate 19 characteristic peaks, using Peak 11 (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results are shown in Table 8-9.

[0100] Table 8 Results of relative retention time of fingerprint precision

[0101]

[0102] The results of relative peak area are shown in Table 9.

[0103] Table 9 Results of relative peak area of fingerprint precision

[0104]

[0105] The results show that the RSD of the relative retention time of the 19 common peaks does not exceed 1.0%, and the RSD of the relative peak area does not exceed 5.0%, indicating that the instrument precision is good.

[0106] 1.4 Fingerprint Analysis of Yuye Jiedu Granules

[0107] Determine the fingerprints of 30 batches of Yuye Jiedu Granules according to the method described in 1.2.3, calibrate 19 common characteristic peaks, use the 11th peak (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results are shown in Tables 10-1, 10-2, 11-1, and 11-2.

[0108] Table 10-1 Results of Relative Retention Time of Fingerprints of 30 Batches of Samples

[0109]

[0110] The results show that the RSD of the relative retention time of 19 common peaks does not exceed 2.0%, indicating that the relative retention time of Yuye Jiedu Granules is relatively stable and will not vary significantly due to batches. The fingerprint of this product can be established based on the relative retention time.

[0111] Table 10-2 Results of Relative Retention Time of Fingerprints of 30 Batches of Samples (Continued Table)

[0112]

[0113] Table 11-1 Results of Relative Peak Area of Fingerprints of 30 Batches of Samples

[0114]

[0115] Table 11-2 Results of Relative Peak Area of Fingerprints of 30 Batches of Samples (Continued Table)

[0116]

[0117] Example 2: Method for Determining the Contents of 12 Components in Yuye Jiedu Granules

[0118] Yuye Jiedu Granules is a traditional Chinese medicine compound preparation composed of seven herbs, namely Mussaenda pubescens, Lonicera confusa, Ilex asprella, Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica. There are currently no content determination indicators in the quality standard. Based on the above fingerprint research, multi-index content determination studies are to be carried out on 12 components, namely neochlorogenic acid, methylshanzhiside, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside.

[0119] 2.1 Instruments and Reagents

[0120] 2.1.1 Instruments

[0121] Thermo Ultimate 3000, high performance liquid chromatograph (DAD detector); SB-5200D ultrasonic cleaner (Ningbo Xinzhi Biotech Co., Ltd.); HH-8 digital display constant temperature water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); ES-502HA electronic balance (Changsha Xiangping Technology Development Co., Ltd.); SQP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); XPE105 electronic analytical balance (METTLER).

[0122] 2.1.2 Test drugs

[0123] Yuye Jiedu Granules, produced by Sanjin Group Hunan Sanjin Pharmaceutical Co., Ltd., a total of 30 batches. Acetonitrile, methanol, and phosphoric acid are chromatographically pure, water is purified water, and other reagents are all analytically pure.

[0124] 2.2 Chromatographic conditions

[0125] Same as the detection method under "1.2.3" of Example 1.

[0126] 2.3 Methodology verification

[0127] 2.3.1 Specificity test

[0128] Preparation of individual reference substance solutions: Weigh appropriate amounts of reference substances of neochlorogenic acid, gardenoside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside accurately, and dissolve them in methanol respectively to prepare individual reference substance stock solutions with concentrations of 197.96, 204.41, 199.53, 204.23, 220.62, 193.26, 216.21, 209.33, 203.45, 221.09, 201.84, and 80.98 μg / mL respectively. Pipette appropriate amounts of the reference substance stock solutions respectively, add 50% methanol, and dilute to obtain individual reference substance solutions with concentrations of 83.14, 20.44, 83.80, 93.95, 15.44, 32.85, 17.30, 83.73, 32.55, 68.54, 2.02, and 44.54 μg / mL respectively.

[0129] Prepare a test sample solution, an individual reference substance solution, and a solvent blank (50% methanol) respectively according to the determined method. Pipette 10 μl accurately from each, inject them into the high performance liquid chromatograph, and determine according to the determined chromatographic conditions. The results are shown in Figure 5-1 、 Figure 5-2 。

[0130] The results show that there are no corresponding chromatographic peaks at the positions corresponding to the reference substance chromatographic peaks and the test sample chromatographic peaks in the solvent blank, indicating no interference and good specificity of this method.

[0131] 2.3.2 Precision Test

[0132] Prepare 1 sample solution according to the determined method, and repeat the determination 6 times, recording the peak areas of 12 components.

[0133] The results show that the RSDs of the average peak areas of the 12 components are between 0.09% and 1.44%, indicating that the instrument precision is good and meets the requirements of quantitative analysis.

[0134] 2.3.3 Solution Stability

[0135] Prepare 1 sample solution according to the determined method, and measure it at 0h, 6h, 12h, 18h, 24h, 30h, 36h, and 42h respectively, recording the peak areas of the target substances. The results show that the RSDs of the average peak areas of the 12 components are between 0.14% and 1.51%, indicating that the sample solution has good stability within 42h.

[0136] 2.3.4 Repeatability Test

[0137] Prepare 6 sample solutions in parallel according to the determined method, measure the peak areas, and calculate the contents of 12 components.

[0138] The results show that: for the new chlorogenic acid in this batch of samples, the average content is 0.5670 mg / g and the RSD is 1.85%; for methyl gardenoside, the average content is 0.1277 mg / g and the RSD is 1.29%; for chlorogenic acid, the average content is 0.5348 mg / g and the RSD is 2.09%; for cryptochlorogenic acid, the average content is 0.5784 mg / g and the RSD is 2.07%; for loganin, the average content is 0.1046 mg / g and the RSD is 2.66%; for secologanin, the average content is 0.1824 mg / g and the RSD is 2.46%; for luteoloside, the average content is 0.1416 mg / g and the RSD is 2.46%; for isochlorogenic acid B, the average content is 0.6257 mg / g and the RSD is 0.67%; for isochlorogenic acid A, the average content is 0.2635 mg / g and the RSD is 0.61%; for isochlorogenic acid C, the average content is 0.5351 mg / g and the RSD is 0.97%; for luteolin, the average content is 0.0096 mg / g and the RSD is 1.54%; for buddleoside, the average content is 0.3803 mg / g and the RSD is 0.53%. According to the requirements of the General Principles 9101 "Guidelines for the Validation of Analytical Methods for Drug Quality Standards" in the Fourth Part of the Chinese Pharmacopoeia 2020 Edition, when the content of the component to be measured in the sample is 1 mg / g, the RSD% of repeatability should be less than 3%. The test results all meet the requirements, indicating that this method has good repeatability.

[0139] 2.3.5 Linear Range

[0140] Inject the prepared series of reference substance solutions under the chromatographic conditions described in item "2.2", and record the peak areas of the reference substance solutions of each component and their corresponding dilution solutions. Taking the concentration as the abscissa (X) and the peak area as the ordinate (Y), draw the standard curves in sequence, and the results are shown in Tables 12-1 and 12-2.

[0141] Table 12-1 Results of the linear range investigation of 12 reference substances

[0142]

[0143] Table 12-2 Results of the linear range investigation of 12 reference substances (continued table)

[0144]

[0145] The results show that the linear relationships of the 12 components are all good within their respective mass concentration ranges.

[0146] 2.3.6 Sample addition recovery rate (accuracy)

[0147] Precisely measure 3.584, 0.852, 3.454, 3.544, 0.683, 1.231, 0.787, 3.84, 1.681, 3.029, 0.065, and 5.876 mL of the reference substance stock solutions of neochlorogenic acid, methyl gardenoside, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside respectively, and place them in stoppered conical flasks. Evaporate to dryness in a low-temperature water bath at 50-60 °C. There are a total of 6 portions, which are labeled as test samples 1-6 in sequence.

[0148] Take Yuye Jiedu Granules (with known contents of neochlorogenic acid 0.5670 mg / g, methyl gardenoside 0.1277 mg / g, chlorogenic acid 0.5348 mg / g, cryptochlorogenic acid 0.5784 mg / g, loganin 0.1046 mg / g, secologanin 0.1824 mg / g, luteoloside 0.1416 mg / g, isochlorogenic acid B 0.6257 mg / g, isochlorogenic acid A 0.2635 mg / g, isochlorogenic acid C 0.5351 mg / g, luteolin 0.0096 mg / g, and buddleoside 0.3803 mg / g), grind them finely, take about 1.25 g, weigh accurately, and add them to test samples 1-6 in sequence. Then precisely add 25 mL of 50% methanol, stopper tightly, weigh, ultrasonically treat for 10 minutes (300 W, 40 kHz), take out and let it cool, weigh again, make up for the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate as the test solution.

[0149] Determine the content according to the established chromatographic conditions, and calculate the recovery rate by the following formula. The results show that the recovery rates of the 12 components are between 97.81% and 104.72%, and the RSDs are between 0.52% and 1.81%. According to the requirements of the General Principles 9101 "Guidelines for the Validation of Analytical Methods for Drug Quality Standards" in the Fourth Part of the Chinese Pharmacopoeia 2020 Edition, when the content of the component to be measured in the sample is 1 mg / g, the recovery rate limit is 90% - 108%. The detection results of the 12 components all meet the requirements, indicating that the accuracy of this method is good.

[0150] 2.3.7 Content Determination Results of Multiple Batches of Yuye Jiedu Granules

[0151] Determine the contents of 12 components in 30 batches of Yuye Jiedu Granules according to the detection method under "1.2.3" in Example 1. The results are shown in Tables 13-1 and 13-2. The results show that among the 12 components, the relatively higher contents are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid C, buddleoside, while the contents of methyl gardenoside, loganin, and luteolin are relatively lower.

[0152] Table 13-1 Content Determination Results of 12 Components in 30 Batches of Yuye Jiedu Granules (mg / g)

[0153]

[0154] Table 13-2 Content Determination Results of 12 Components in 30 Batches of Yuye Jiedu Granules (mg / g) (Continued Table)

[0155]

[0156] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A detection method for Yuye Jiedu Granules, wherein the Yuye Jiedu Granules are composed of seven traditional Chinese medicines, namely Mussaenda pubescens, Lonicera confusa, Ilex asprella, Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum and Centella asiatica, and is characterized in that: Simultaneously determine 12 components in Yuye Jiedu Granules by high performance liquid chromatography. The 12 components are: neochlorogenic acid, gardenoside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin and buddleoside; The chromatographic conditions of the detection method are: C18 chromatographic column, detection wavelength 254 nm, using acetonitrile as mobile phase A and 0.05%-0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution; The gradient elution program is: 0 - 3 min, 3% A; 3 - 25 min, 3 - 16% A; 25 - 30 min, 16% A; 30 - 50 min, 16 - 20% A; 50 - 60 min, 20 - 25% A; 60 - 70 min, 25 - 35% A; Take an appropriate amount of Yuye Jiedu Granules sample and extract it with 50%-70% methanol solution by ultrasonic or reflux method, filter it, and take the continued filtrate to obtain the test solution; precisely absorb the test solution and inject it into the liquid chromatograph for determination according to the above method, and record the chromatogram.

2. The method according to claim 1, wherein: Use 0.05% phosphoric acid aqueous solution as mobile phase B.

3. The method according to claim 1, characterized in that: The model of the C18 chromatographic column is YCM Meteoric CoreC18, with a specification of 150×4.6 mm, 2.7 μm.

4. The method according to claim 1, characterized in that: Take an appropriate amount of Yuye Jiedu Granules sample and extract it with 50%-70% methanol solution by ultrasonic extraction for 10 - 30 minutes, filter it, and take the continued filtrate to obtain the test solution.

5. The method according to claim 4, characterized in that: Take an appropriate amount of Yuye Jiedu Granules sample and extract it with 50% methanol solution by ultrasonic extraction for 10 minutes, filter it, and take the continued filtrate to obtain the test solution; the ultrasonic extraction power is 300 W, 40 kHz.

6. The method according to claim 1, characterized in that: Flow rate 1.0 mL / min, column temperature 30 °C.

7. The method according to any one of claims 1-6, characterized in that: Calculate the contents of the 12 components by relative retention time and relative peak area; The 12 components: peak 1 is neochlorogenic acid, peak 3 is gardenoside methyl ester, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is loganin, peak 8 is secologanin, peak 9 is luteoloside, peak 11 is isochlorogenic acid B, peak 12 is isochlorogenic acid A, peak 14 is isochlorogenic acid C, peak 16 is luteolin, peak 17 is buddleoside; Taking isochlorogenic acid B as the reference peak, the relative retention times of the 12 components relative to the reference peak are: peak 1 is 0.3397, peak 3 is 0.4471, peak 4 is 0.4686, peak 5 is 0.5066, peak 6 is 0.5897, peak 8 is 0.6751, peak 9 is 0.8600, peak 11 is 1.0000, peak 12 is 1.0466, peak 14 is 1.2108, peak 16 is 1.5807, peak 17 is 1.6166; The relative peak areas of the 12 components with respect to the reference peak are as follows: the peak No. 1 is 0.6483, the peak No. 3 is 0.0516, the peak No. 4 is 0.9365, the peak No. 5 is 0.7678, the peak No. 6 is 0.0831, the peak No. 8 is 0.1669, the peak No. 9 is 0.3707, the peak No. 11 is 1.0000, the peak No. 12 is 0.5307, the peak No. 14 is 1.1556, the peak No. 16 is 0.0349, and the peak No. 17 is 0.5827.

8. The method according to claim 7, wherein The linear ranges for the determination of the contents of the 12 components are as follows: For neochlorogenic acid, it is 19.796 - 158.368 μg / ml; for gardenoside methyl ester, it is 0.8176 - 40.8811 μg / ml; for chlorogenic acid, it is 19.9534 - 159.6269 μg / ml; for cryptochlorogenic acid, it is 20.4232 - 163.3856 μg / ml; for loganin, it is 0.8825 - 44.1242 μg / ml; for secologanin, it is 5.7977 - 57.9768 μg / ml; for luteoloside, it is 0.8648 - 43.2425 μg / ml; for isochlorogenic acid B, it is 20.9328 - 167.4624 μg / ml; for isochlorogenic acid A, it is 8.1379 - 81.3792 μg / ml; for isochlorogenic acid C, it is 11.0544 - 154.7616 μg / ml; for luteolin, it is 0.2018 - 20.1845 μg / ml; for buddleoside, it is 8.0978 - 80.9776 μg / ml.

9. The application of the method according to any one of claims 1 - 6 in the construction of the fingerprint of Yuye Jiedu Granules, and the method for constructing the fingerprint of Yuye Jiedu Granules is as follows: (1) Take the test solution, reference solution and blank solvent of Yuye Jiedu Granules, and perform detection according to the method described in any one of claims 1 - 6, and record the chromatogram; (2) Calibrate the characteristic peaks by comparing the peak emergence times, and the spectra of the reference substance and the test substance, identify and label the chromatographic peaks; (3) Set the reference peak, calculate the relative retention times and relative peak areas of other characteristic peaks, and establish the fingerprint of Yuye Jiedu Granules based on the relative retention times.

10. The application according to claim 9, wherein: A total of 19 characteristic peaks are calibrated in the fingerprint of Yuye Jiedu Granules, and the reference peak is set as isochlorogenic acid B; The relative retention times of the 19 characteristic peaks with respect to the reference peak are as follows: the peak No. 1 is 0.3397, the peak No. 2 is 0.3688, the peak No. 3 is 0.4471, the peak No. 4 is 0.4686, the peak No. 5 is 0.5066, the peak No. 6 is 0.5897, the peak No. 7 is 0.6552, the peak No. 8 is 0.6751, the peak No. 9 is 0.8600, the peak No. 10 is 0.8840, the peak No. 11 is 1.0000, the peak No. 12 is 1.0466, the peak No. 13 is 1.0666, the peak No. 14 is 1.2108, the peak No. 15 is 1.3182, the peak No. 16 is 1.5807, the peak No. 17 is 1.6166, the peak No. 18 is 1.6363, and the peak No. 19 is 1.7059; The relative peak areas of 19 characteristic peaks with respect to the reference peak are as follows: the relative peak area of peak 1 is 0.6483, that of peak 2 is 0.0443, that of peak 3 is 0.0516, that of peak 4 is 0.9365, that of peak 5 is 0.7678, that of peak 6 is 0.0831, that of peak 7 is 0.1857, that of peak 8 is 0.1669, that of peak 9 is 0.3707, that of peak 10 is 0.1810, that of peak 11 is 1.0000, that of peak 12 is 0.5307, that of peak 13 is 0.1819, that of peak 14 is 1.1556, that of peak 15 is 0.0453, that of peak 16 is 0.0349, that of peak 17 is 0.5827, that of peak 18 is 0.0679, and that of peak 19 is 0.1213; Identify and label 12 chromatographic peaks: peak 1 is neochlorogenic acid, peak 3 is gardenoside methyl ester, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is loganin, peak 8 is secologanin, peak 9 is luteoloside, peak 11 is isochlorogenic acid B, peak 12 is isochlorogenic acid A, peak 14 is isochlorogenic acid C, peak 16 is luteolin, and peak 17 is buddleoside.

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