Evaluation method of waist-strengthening and kidney-invigorating tablets based on liquid chromatography

Abstract

The invention provides an evaluation method of a waist-strengthening kidney-invigorating tablet based on liquid chromatography, which comprises the following steps: establishing a contrast characteristic spectrum of the waist-strengthening kidney-invigorating tablet through high performance liquid chromatography to obtain a characteristic peak of the contrast characteristic spectrum and a specified value of the relative retention time of the characteristic peak, and evaluating that the quality of the waist-strengthening kidney-invigorating tablet meets the specification. The accuracy of existing quality control indexes can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to an evaluation method for Zhuangyao Jianshen tablets based on liquid chromatography. Background Art

[0002] Zhuangyao Jianshen Tablets are provided by Jingzhongshan National Medicine (Tangshan) Co., Ltd. The prescription consists of 9 kinds of Chinese medicinal materials, including Cibotium barometz, Black Tiger, Millettia reticulata, Rhizoma Cynomorii, Fructus Corni, Viscum album (steamed), Rhizoma Cibotii, Fructus Ligustri Lucidi (steamed), and Fructus Cuscutae (prepared with salt water).

[0003] The current quality standard for Zhuangyao Jianshen Tablets is "Volume 13 of the Ministry of Health's Drug Standards for Chinese Medicine Formulas and Preparations", which only stipulates properties, identification (1 microscopic identification, 3 colorimetric physical and chemical identifications, and 1 mistletoe thin-layer chromatography identification) and inspection (universal inspection under tablets). However, this product is a large compound of 9 herbs with complex ingredients, but the existing quality control standards are relatively simple.

[0004] Therefore, it is necessary to improve the quality control evaluation method of Zhuangyao Jianshen Tablets. Summary of the invention

[0005] The invention provides an evaluation method for Zhuangyao Jianshen tablets based on liquid chromatography, which is used to solve the problem of simple quality control standards for Zhuangyao Jianshen tablets with complex ingredients in the prior art.

[0006] The present invention provides an evaluation method for Zhuangyao Jianshen tablets based on liquid chromatography, comprising the following steps:

[0007] (1) Preparation of test solution: Take Zhuangyao Jianshen tablets from different batches, remove the sugar coating, grind into powder and place in a stoppered conical flask, add 10 ml of 70% ethanol, weigh and then perform ultrasonic treatment, cool to room temperature, weigh again, make up the lost weight with 70% ethanol, shake well, take the filtrate, and obtain test solution from different batches;

[0008] (2) Preparation of reference solution: Take the reference substance, weigh it accurately, and add ethanol to make a solution containing 80 μg per 1 ml to obtain the reference solution;

[0009] (3) Characteristic spectrum determination: Accurately pipette the reference solution and different batches of test solution respectively, perform high performance liquid chromatography test, and obtain the characteristic spectra of different batches;

[0010] (4) Generation of characteristic peaks: The characteristic spectra of different batches were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the National Pharmacopoeia Committee in AIA format for data processing, and the characteristic spectra were MARK peak matched using the multi-point correction method to generate characteristic peaks of different batches;

[0011] (5) Establishment of reference characteristic spectrum: Determine the common peaks and reference peaks of the characteristic spectrum of different batches according to the peak areas and retention times of the characteristic peaks of different batches;

[0012] According to the relative peak areas and relative retention times of the common peaks and reference peaks of the characteristic spectra of different batches, the characteristic peaks of the control characteristic spectra and the prescribed values ​​of the relative retention times of the characteristic peaks are determined to obtain the control characteristic spectra;

[0013] (6) Evaluation of Zhuangyao Jianshen Tablets: When the characteristic peaks of Zhuangyao Jianshen Tablets include the characteristic peaks of the reference characteristic spectrum, and the relative retention time of the characteristic peaks of Zhuangyao Jianshen Tablets and the reference peak is within 5% of the specified value of the characteristic peak of the reference characteristic spectrum, it is confirmed that the quality of Zhuangyao Jianshen Tablets meets the requirements.

[0014] Compared with the prior art, the advantages of the present invention are as follows: a control characteristic spectrum of Zhuangyao Jianshen Tablets is established by high performance liquid chromatography, and characteristic peaks of the control characteristic spectrum and specified values ​​of the relative retention times of the characteristic peaks are obtained, which are used to evaluate whether the quality of Zhuangyao Jianshen Tablets meets the regulations, and can improve the accuracy of existing quality control indicators.

[0015] Furthermore, in step (5), the control characteristic spectrum includes 16 characteristic peaks, and the specified values ​​of the relative retention times of the 16 characteristic peaks are: peak 1: 0.71, peak 2: 0.77, peak 3: 1.00, peak 4: 1.35, peak 5: 1.63, peak 6: 1.94, peak 7: 2.05, peak 8: 2.49, peak 9: 2.72, peak 10: 3.61, peak 11: 3.81, peak 12: 4.28, peak 13: 4.77, peak 14: 5.07, peak 15: 5.46, peak 16: 6.01.

[0016] Further, the reference substance is 5-hydroxymethylfurfural; and / or,

[0017] The reference peak is 5-hydroxymethylfurfural peak.

[0018] Furthermore, in step (3), the chromatographic column used in the HPLC test is ACE Excel C 18 or WatersSymmetry C 18 .

[0019] Furthermore, in step (3), the flow rate of the sample in the HPLC test is 1.0 to 1.05 ml / min.

[0020] Furthermore, in step (3), the mobile phase A of the HPLC test is acetonitrile, and the mobile phase B is 0.2% phosphoric acid solution; the gradient elution program is:

[0021] 0-17min: Fluidity A increases from 5% to 12% at a constant rate, and fluidity B decreases from 95% to 88% at a constant rate;

[0022] 17-25min: Fluidity A increases from 12% to 20% at a constant rate, and fluidity B decreases from 88% to 80% at a constant rate;

[0023] 25-30min: Fluidity A increases from 20% to 23% at a constant rate, and fluidity B decreases from 80% to 77% at a constant rate;

[0024] 30-45min: Fluidity A increases from 23% to 40% at a constant rate, and fluidity B decreases from 77% to 60% at a constant rate;

[0025] 45-55min: Fluidity A increases from 40% to 60% at a constant rate, and fluidity B decreases from 60% to 40% at a constant rate;

[0026] 55-70min: Fluidity A increases from 60% to 70% at a constant rate, and fluidity B decreases from 40% to 30% at a constant rate;

[0027] 70-72min: Fluidity A increases uniformly from 70% to 90%, and fluidity B decreases uniformly from 30% to 10%.

[0028] Furthermore, in step (3), the column temperature of the HPLC test is 30-40°C.

[0029] Furthermore, in step (3), the detection wavelength of the high performance liquid chromatography test is 255 to 265 nm.

[0030] Furthermore, in step (3), the theoretical plate number of the high performance liquid chromatography test is not less than 5000 calculated based on the reference peak.

[0031] Furthermore, in step (1), the power of ultrasonic treatment is 250 W, the frequency is 40 kHz, and the time is 10 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparative diagram of the extraction solvent in the embodiment of the present invention;

[0033] Figure 2 This is a comparison chart of the extraction time investigation in the embodiment of the present invention;

[0034] Figure 3 This is a graph showing the results of the characteristic spectrum measurement of the blank solvent in the embodiment of the present invention;

[0035] Figure 4 It is a characteristic spectrum-precision investigation diagram in the embodiment of the present invention;

[0036] Figure 5It is a characteristic spectrum-stability investigation diagram in the embodiment of the present invention;

[0037] Figure 6 It is a characteristic spectrum-repeatability investigation diagram in the embodiment of the present invention;

[0038] Figure 7 The characteristic spectra of 17 batches of Zhuangyao Jianshen Tablets in the embodiment of the present invention;

[0039] Figure 8 This is a comparison chart of 7 batches of Zhuangyao Jianshen Tablets in the examples of the present invention;

[0040] Fig. 9 This is a characteristic peak identification diagram of gallic acid, protocatechuic acid, 5-hydroxymethylfurfural, and protocatechuic aldehyde in the embodiments of the present invention;

[0041] Fig.10 This is a characteristic peak identification diagram of formononetin, genistein, daidzein, and ellagic acid in the embodiments of the present invention;

[0042] Fig.11 It is a comparison spectrum of eight characteristic peaks of gallic acid, protocatechuic aldehyde, protocatechuic acid, 5-hydroxymethylfurfural, ellagic acid, daidzein, genistein, and formononetin in the embodiment of the present invention;

[0043] Fig.12 The chromatograms of the characteristic spectra of different column temperatures in the embodiments of the present invention;

[0044] Fig.13 It is a chromatogram of characteristic patterns of different flow rates in the embodiment of the present invention;

[0045] Fig.14 It is a chromatogram of characteristic spectra of different wavelengths in the embodiment of the present invention;

[0046] Fig.15 The chromatograms are characteristic spectra of different chromatographic columns in the embodiments of the present invention;

[0047] Fig.16 It is a chromatogram of characteristic spectra of different instruments in the embodiments of the present invention;

[0048] Fig.17 It is a comparison chart of Zhuangyao Jianshen Tablets and a comparison chart of the characteristic chart of the Chinese medicine of Gouji in the embodiment of the present invention;

[0049] Fig.18 It is a comparison chart of Zhuangyao Jianshen Tablets and Black Tiger Medicinal Material Characteristic Chart in the embodiments of the present invention;

[0050] Fig.19 It is a comparison chart of Zhuangyao Jianshen Tablets and Millettia Spatholobi medicinal material characteristic chart in the embodiments of the present invention;

[0051] Fig. 20It is a comparison chart of Zhuangyao Jianshen Tablets and Niu Dali medicinal material characteristic chart in the embodiment of the present invention;

[0052] Fig.21 It is a comparison chart of Zhuangyao Jianshen Tablets and the characteristic chart of Rosa Laevigata in the embodiments of the present invention;

[0053] Fig. 22 It is a comparison chart of Zhuangyao Jianshen Tablets and characteristic chart of Herba Mori in the embodiments of the present invention;

[0054] Fig.23 It is a comparison chart of Zhuangyao Jianshen Tablets and Qianjinba medicinal material characteristic chart in the embodiments of the present invention;

[0055] Fig.24 It is a comparison chart of Zhuangyao Jianshen Tablets and characteristic chart of Ligustrum lucidum medicinal material in the embodiment of the present invention;

[0056] Fig.25 It is a comparison chart of Zhuangyao Jianshen Tablets and Cuscuta australis medicinal material characteristic chart in the embodiments of the present invention;

[0057] Fig.26 Characteristic spectra of 5 batches of Zhuangyao Jianshen Tablet intermediates in the examples of the present invention;

[0058] Fig. 27 This is a comparison chart of 5 batches of Zhuangyao Jianshen Tablet intermediates in the examples of the present invention;

[0059] Fig.28 This is a comparison chart of dry powder and tablets in the examples of the present invention;

[0060] Fig.29 It is the reference characteristic spectrum in the examples of the present invention. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] The materials, instruments and reagents used in the evaluation method of the present invention are as follows:

[0063] Instrument: High performance liquid chromatograph (LC-15C pump, SPD-15C UV detector, SIL-10AF autosampler, CTO-15C column oven, LC chromatography workstation, Shimadzu Corporation, Japan)

[0064] Chromatographic column: Waters Symmetry C 18 (4.6×250mm, 5μm)

[0065] Electronic analytical balance: TB-215D, BSA224S-CW (Beijing Sartorius)

[0066] KQ-250 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., power 250W, frequency 40kHz)

[0067] Acetonitrile: chromatographic grade (Fisher, USA)

[0068] Phosphoric acid: chromatographically pure (Fisher, USA)

[0069] Ethanol: analytical grade (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.)

[0070] Ultrapure water: UPR-II-20L ultrapure water system (Sichuan UPR Ultrapure Technology Co., Ltd.)

[0071] 5-Hydroxymethylfurfural (98.4%, 111626-202316), gallic acid (89.9%, 110831-201204), protocatechuic aldehyde (98.2%, 110810-201007), protocatechuic acid (97.5%, 110809-202207), ellagic acid (88.8%, 111959-201903), daidzein (99.3%, 111502-202304), genistein (99.1%, 111704-201302), and formononetin (99.0%, 111703-202401) were all purchased from China Food and Drug Identification Institute.

[0072] Zhuangyao Jianshen Tablets are provided by Jingzhongshan National Pharmaceutical Group, with batch numbers 211201, 211202, 211203, 211204, 211205, 220101, 220102, 220103, 220301, 220302, 220303, 220401, 220402, 220403, 220501, 230602, and 240101, numbered S1 to S17 respectively.

[0073] Wherein, the determination of chromatographic conditions comprises the following steps:

[0074] (1) Investigation of mobile phase

[0075] By consulting the literature, acetonitrile-0.1% phosphoric acid, acetonitrile-0.05% phosphoric acid, acetonitrile-0.2% phosphoric acid and methanol-0.2% phosphoric acid were selected as mobile phases to explore the characteristic chromatographic conditions. By comparing the chromatograms under different mobile phases, acetonitrile-0.2% phosphoric acid was finally selected as the mobile phase for gradient elution.

[0076] (2) Determination of the optimal absorption wavelength

[0077] The DAD detector was used to scan the characteristic spectrum at all wavelengths, and it was found that the number of chromatographic peaks was large and the separation was relatively high under the detection wavelength of 260nm, so the detection wavelength of the characteristic spectrum was determined to be 260nm.

[0078] (3) Determination of chromatographic conditions

[0079] Chromatographic column: Waters Symmetry C 18 (4.6×250mm, 5μm); acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; flow rate was 1.0ml / min; detection wavelength was 260nm, and column temperature was 35℃.

[0080]

[0081] The preparation of the reference solution comprises the following steps:

[0082] Take an appropriate amount of 5-hydroxymethylfurfural reference substance, weigh it accurately, and add ethanol to make a solution containing 80 μg per 1 ml.

[0083] The preparation method of the test solution includes the following steps:

[0084] (1) Investigation of extraction solvent

[0085] Take the same batch of samples, remove the sugar coating, weigh accurately, grind finely, take about 0.5g of each, put it in a stoppered conical bottle, accurately add pure water, 50% methanol, 70% methanol, methanol, 50% ethanol, 70% ethanol, 10ml of ethanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 20 minutes, cool, make up the loss weight with the corresponding solvent, mix well, filter, and obtain. See the results. Figure 1 ; As can be seen from the above figure, the extraction effect is better when the solvent is 70% methanol and 70% ethanol, and the number of chromatographic peaks is larger, but the chromatographic peak baseline obtained by 70% ethanol extraction is relatively stable, so 70% ethanol is finally selected as the extraction solvent.

[0086] (2) Investigation of ultrasound time

[0087] Take the same batch of samples, remove the sugar coating, weigh accurately, grind finely, take about 0.5g of each, put it in a stoppered conical bottle, accurately add 10ml of 70% ethanol, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 10, 20, and 30 minutes respectively, weigh it again, make up the lost weight with 70% ethanol, shake well, filter, and you are done. Figure 2 ,Depend on Figure 2 It was found that the extraction effects of the three different ultrasonic times were basically the same. In order to save time, 10 minutes was selected as the ultrasonic time for the test solution.

[0088] (3) Determination of the test sample preparation method

[0089] Take 10 tablets of this product, remove the sugar coating, accurately weigh, grind, take about 0.5g, accurately weigh, put in a stoppered conical flask, accurately add 10ml of 70% ethanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 10 minutes, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter, and take the filtrate to obtain.

[0090] The methodological investigation includes the following steps:

[0091] (1) Exclusivity

[0092] Take the blank solvent as the negative sample solution, draw the reference solution, negative sample solution and test solution, and test according to the characteristic spectrum determination conditions. The blank solvent has no interference with the characteristic spectrum determination. The results are shown in Figure 3 .

[0093] (2) Precision

[0094] Take the same sample, prepare it according to the test sample preparation method, and inject it continuously 6 times according to the known chromatographic conditions, with an injection volume of 10μl. Taking the chromatographic peak of 5-hydroxymethylfurfural as the reference peak, the RSD value of the relative retention time of the 16 characteristic peaks is calculated to be 0.00%~0.07%, and the RSD value of the relative peak area is 0.00%~2.78%, indicating that the instrument has good precision. The results are shown in Figure 4 , Tables 1~2.

[0095] Table 1 Relative retention time ratio-precision investigation

[0096]

[0097]

[0098] Table 2 Relative peak area ratio - precision investigation

[0099]

[0100] (3) Stability

[0101] The sample under precision was injected at 0h, 6h, 12h, 18h, 12h, and 24h under known conditions, and the chromatographic peak of 5-hydroxymethylfurfural was used as the reference peak. The RSD values ​​of the relative retention time of the 16 characteristic peaks were calculated to be 0.00% to 0.05%, and the RSD values ​​of the relative peak area were 0.00% to 2.80%. This shows that the sample has good stability within 24h. The results are shown in Figure 5 , Tables 3~4.

[0102] Table 3 Relative retention time ratio-stability study

[0103]

[0104]

[0105] Table 4 Relative peak area ratio-stability study

[0106]

[0107] (4) Repeatability study

[0108] Take the same batch of samples and prepare 6 test solutions in parallel. According to the known conditions, 10 μl of the sample was injected in sequence for detection. The chromatographic peak of 5-hydroxymethylfurfural was used as the reference peak. The RSD value of the relative retention time of the common peak was calculated to be 0.00% to 0.07%, and the RSD value of the relative peak area was 0.00% to 2.68%, indicating that the method has good repeatability. The results are shown in Figure 6 , Tables 5~6.

[0109] Table 5 Relative retention time ratio - repeatability study

[0110]

[0111]

[0112] Table 6 Relative peak area ratio - repeatability study

[0113]

[0114] The establishment of characteristic spectra of 17 batches of Zhuangyao Jianshen Tablets includes the following steps:

[0115] 17 batches of Zhuangyao Jianshen tablets were taken respectively, and the test solution was prepared according to the confirmed test solution preparation method, and the characteristic spectrum of 17 batches of Zhuangyao Jianshen tablets was established. The obtained spectrum was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software of the National Pharmacopoeia Committee in AIA format for data processing, and the chromatographic peaks of 17 batches of Zhuangyao Jianshen tablets were MARK matched using the multi-point correction method to generate characteristic peaks of multiple batches of Zhuangyao Jianshen tablets. The results are shown in Figures 7-8 .

[0116] According to the relevant parameters given by the characteristic spectra of 17 batches of Zhuangyao Jianshen Tablets, the spectra of 17 batches of test samples were compared, and it was found that 16 peaks were common to the 17 batches of test samples, with relatively high response values ​​and relatively stable relative retention times, so these 16 peaks were determined to be common characteristic peaks. Among them, the chromatographic peak area of ​​characteristic peak No. 3 was large and the separation was good, so the common peak No. 3 was used as the reference peak S, and the relative retention time and relative peak area of ​​16 characteristic peaks in 17 batches of Zhuangyao Jianshen Tablets were calculated, as shown in Tables 7 and 8.

[0117] Table 7 Relative retention time of 17 batches of Zhuangyao Jianshen tablets

[0118]

[0119]

[0120] Table 8 Relative peak areas of 17 batches of Zhuangyao Jianshen tablets

[0121]

[0122]

[0123] From the data in the table, it can be seen that the relative retention time RSD of the 16 common characteristic peaks of 17 batches of Zhuangyao Jianshen Tablets is between 0.00% and 0.62%, indicating that the relative retention time of each characteristic peak of the characteristic spectrum is relatively stable. Taking the average relative retention time of 17 batches of Zhuangyao Jianshen Tablets as the standard, considering that different instruments, chromatographic columns, personnel and other factors will cause certain differences in the results, the relative retention time should be within ±5% of the specified value. Taking Peak 3 (5-hydroxymethylfurfural) as the reference peak, the relative retention times of other characteristic peaks were calculated, and the specified relative retention times of the characteristic spectra of 17 batches of Zhuangyao Jianshen Tablets were determined to be 0.71 (peak 1), 0.77 (peak 2), 1.00 (peak 3), 1.35 (peak 4), 1.63 (peak 5), 1.94 (peak 6), 2.05 (peak 7), 2.49 (peak 8), 2.72 (peak 9), 3.61 (peak 10), 3.81 (peak 11), 4.28 (peak 12), 4.77 (peak 13), 5.07 (peak 14), 5.46 (peak 15), and 6.01 (peak 16).

[0124] The similarity between the characteristic spectra of 17 batches of Zhuangyao Jianshen Tablets and the control is shown in Table 9. The similarity between each batch of Zhuangyao Jianshen Tablets and the control spectra is higher than 0.97, which is a high similarity, proving that the quality of the 17 batches of samples is relatively stable and the chemical composition is consistent.

[0125] Table 9 Similarity of 17 batches of Zhuangyao Jianshen Tablets Zhuangyao Jianshen Tablets

[0126]

[0127]

[0128] Characteristic peak identification includes the following steps:

[0129] Zhuangyao Jianshen Tablets are composed of 9 kinds of Chinese medicinal materials, including Cibotium barometz, Black Tiger, Millettia reticulata, Rhizoma Cynoglossi, Fructus Rosae Laevigatae, Viscum album (steamed), Rhizoma Coptidis, Fructus Ligustri Lucidi (steamed), and Semen Cuscutae (prepared with salt water). After consulting the relevant literature on the chemical composition of each medicinal flavor, the characteristic peaks were identified by comparing the retention time with that of the reference substance.

[0130] According to the chromatographic conditions determined by the characteristic spectrum of Zhuangyao Jianshen Tablets, gallic acid, protocatechuic aldehyde, protocatechuic acid, 5-hydroxymethylfurfural, ellagic acid, daidzein, genistein, formononetin and other reference substances were injected and tested. The experimental results are as follows: Fig. 9 , Fig.10 shown.

[0131] From the experimental results, it can be seen that in the characteristic spectrum of Zhuangyao Jianshen Tablets, characteristic peaks with the same retention time as those of gallic acid, protocatechuic aldehyde, protocatechuic acid, 5-hydroxymethylfurfural, ellagic acid, daidzein, genistein, and formononetin reference substances can be found, and they can be reflected in all batches of samples. Therefore, among the common peaks determined in the characteristic spectrum of Zhuangyao Jianshen Tablets, 8 characteristic peaks of gallic acid, protocatechuic aldehyde, protocatechuic acid, 5-hydroxymethylfurfural, ellagic acid, daidzein, genistein, and formononetin were identified. Fig.11 .

[0132] Durability inspection includes the following steps:

[0133] (1) Different column temperatures

[0134] Take the same sample solution, fix other chromatographic conditions, set the column temperature to 30℃, 35℃, and 40℃ respectively, and investigate the effect of column temperature change on the relative retention time of each characteristic peak. Fig.12 The relative retention time of each characteristic peak is shown in Table 10.

[0135] Table 10 Relative retention time of characteristic spectra at different column temperatures

[0136]

[0137] The results showed that 16 characteristic peaks were present in the chromatogram of the test sample at different column temperatures. The chromatographic peak of 5-hydroxymethylfurfural was taken as the S peak, and the relative retention times of the remaining chromatographic peaks were calculated to be within the specified range of relative retention time. The column temperature varied between 30-40℃, which had little effect on the characteristic spectrum.

[0138] (2) Different flow rates

[0139] Take the same sample solution, fix other chromatographic conditions, set the flow rate to 0.95ml / min, 1.0ml / min, and 1.05ml / min, respectively, and investigate the effect of flow rate change on the relative retention time of each characteristic peak. The chromatograms at different flow rates are shown in Fig.13 The relative retention time of each characteristic peak is shown in Table 11.

[0140] Table 11 Relative retention time of characteristic spectra at different flow rates

[0141]

[0142] The results showed that there were 16 characteristic peaks in the chromatogram of the test sample at different flow rates. The 5-hydroxymethylfurfural chromatographic peak was taken as the S peak, and the relative retention times of the remaining chromatographic peaks were calculated. When the flow rate was 1.0ml / min and 1.05ml / min, the relative retention times were within the specified range; but when the flow rate was 0.95ml / min, the relative retention time of chromatographic peak No. 12 was not within the specified range, so the specified flow rate was 1.0ml / min or 1.05ml / min.

[0143] (3) Different wavelengths

[0144] Take the same sample solution, fix other chromatographic conditions, set the wavelength to 255nm, 260nm, and 265nm respectively, and investigate the effect of wavelength change on the relative retention time of each characteristic peak. Fig.14 The relative retention time of each characteristic peak is shown in Table 12.

[0145] Table 12 Relative retention time of characteristic spectra at different wavelengths

[0146]

[0147] The results showed that there were 16 characteristic peaks in the chromatogram of the test sample at different wavelengths. The chromatographic peak of 5-hydroxymethylfurfural was taken as the S peak, and the relative retention times of the remaining chromatographic peaks were calculated to be within the specified range of relative retention time. The wavelength varied between 260±5nm, which had little effect on the characteristic spectrum.

[0148] (4) Different chromatographic columns

[0149] Take the same sample solution, fix other chromatographic conditions, and use Pheromones ACE Excel C 18 , 4.6×250mm, 5μm (column 1); Agilent Eclipse XDB-C 18 , 4.6×250 mm, 5 μm (chromatographic column 2); Waters Symmetry C 18 , 4.6×250mm, 5μm (chromatographic column 3) to investigate the effects of different chromatographic columns on the relative retention time of each characteristic peak. The chromatograms of different chromatographic columns are shown in Fig.15 The relative retention time of each characteristic peak is shown in Table 13.

[0150] Table 13 Relative retention time of characteristic spectra of different chromatographic columns

[0151]

[0152] The chromatogram shows that the separation effect of the Agilent column is not good. Both the Pheromone and Waters columns show 16 characteristic peaks in the chromatogram of the test sample. The 5-hydroxymethylfurfural chromatographic peak is the S peak, and the relative retention times of the remaining chromatographic peaks are calculated, all within the specified range of relative retention time. Therefore, the ACE Excel C18 (4.6×250mm, 5μm) and Waters Symmetry C18 (4.6×250mm, 5μm) columns are fixedly selected for the characteristic spectrum.

[0153] (5) Investigation of different chromatographs

[0154] In order to investigate the durability of the chromatograph, the same sample solution was taken and measured using chromatographs of different brands (Shimadzu, Agilent 1260II, Waters e2695) to calculate the relative retention time of each characteristic peak. The chromatograms of different instruments are shown in Fig.16 The relative retention time of each characteristic peak is shown in Table 14.

[0155] Table 14 Relative retention time of characteristic spectra of different instruments

[0156]

[0157] The results showed that 16 characteristic peaks were present in the chromatograms of the test samples under different instruments. The chromatographic peak of 5-hydroxymethylfurfural was taken as the S peak. The relative retention times of the remaining chromatographic peaks were all within the specified range of relative retention time. The use of different instruments had little effect on the determination of characteristic spectra.

[0158] Correlation analysis includes the following steps:

[0159] 1. Correlation analysis between medicinal materials and tablets

[0160] Take about 0.5g of each medicinal powder (passed through No. 3 sieve), weigh accurately, put it in a round-bottom flask, add 50ml of water, heat and reflux for 1h, filter, take 3ml of the filtrate, add 7ml of anhydrous ethanol, filter, and take the filtrate to obtain the test solution of each medicinal flavor.

[0161] According to the determined characteristic spectrum chromatographic conditions and detection methods, the 9 medicinal materials were sampled and tested respectively, and the chromatograms were recorded and compared with the control spectrum of Zhuangyao Jianshen Tablets. The 16 characteristic peaks were attributed to the prescription medicinal flavors. The results are shown in Table 15. Figures 17 to 25 .

[0162] Table 15 The results of attribution of the common peaks in the characteristic spectrum of Zhuangyao Jianshen Tablets to the Chinese medicine prescription

[0163]

[0164]

[0165] Intermediate-tablet correlation analysis includes the following steps:

[0166] Five batches of Zhuangyao Jianshen Tablets intermediates (batch numbers QT240203, QT240204, QT240205, QT240301, QT240302) were taken respectively, and the test solution was prepared according to the confirmed test solution preparation method, and the characteristic spectra of the five batches of Zhuangyao Jianshen Tablets intermediates were determined and established. The obtained spectra were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software of the National Pharmacopoeia Committee in AIA format for data processing, and the chromatographic peaks of the five batches of Zhuangyao Jianshen Tablets intermediates were MARK matched using the multi-point correction method. The characteristic spectra and reference spectra of the five batches of Zhuangyao Jianshen Tablets intermediates are shown in Figures 26-27 The relative retention times of the common peaks are shown in Table 16.

[0167] Table 16 Relative retention time of 5 batches of Zhuangyao Jianshen Tablets intermediates

[0168]

[0169]

[0170] It can be seen from the data in the table that the RSD values ​​of the relative retention times of 16 common characteristic peaks of 5 batches of Zhuangyao Jianshen Tablets intermediates are between 0.00% and 0.04%, indicating that the relative retention time of each characteristic peak of the characteristic spectrum is relatively stable. The average relative retention times of the characteristic spectra of 5 batches of Zhuangyao Jianshen Tablets intermediates are 0.71 (peak 1), 0.77 (peak 2), 1.00 (peak 3), 1.34 (peak 4), 1.63 (peak 5), 1.95 (peak 6), 2.05 (peak 7), 2.49 (peak 8), 2.72 (peak 9), 3.62 (peak 10), 3.81 (peak 11), 4.28 (peak 12), 4.77 (peak 13), 5.08 (peak 14), 5.47 (peak 15), and 6.01 (peak 16), which are all within the specified value range of the relative retention time of Zhuangyao Jianshen Tablets.

[0171] The reference spectra of the intermediate and tablets of Zhuangyao Jianshen Tablets were imported into the similarity evaluation software, and the reference spectra of the tablets were used as the reference spectra to compare the intermediate and tablets. It was found that the main chromatographic peaks in the intermediate could be attributed to those in the tablets (such as Fig.28 ), the similarity between the two is 0.961, which proves that there is no significant change in the main chemical components of the tablets and intermediates.

[0172] The draft standard for the characteristic spectrum of Zhuangyao Jianshen Tablets is as follows:

[0173] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0174] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate 1.0 ml per minute; detection wavelength 260 nm. The theoretical plate number calculated based on the 5-hydroxymethylfurfural peak should be no less than 5000.

[0175]

[0176] Preparation of reference solution: Take an appropriate amount of 5-hydroxymethylfurfural reference substance, weigh it accurately, and add ethanol to make a solution containing 80 μg per 1 ml.

[0177] Preparation of test solution: Take 10 tablets of this product, remove the sugar coating, accurately weigh, grind, take about 0.5g, accurately weigh, put in a stoppered conical flask, accurately add 10ml of 70% ethanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 10 minutes, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter, and take the filtrate.

[0178] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, record the chromatogram, and obtain the result.

[0179] The characteristic spectrum of the test sample should show 16 characteristic peaks. The peak corresponding to the reference peak is the S peak. The relative retention time of the remaining characteristic peaks and the S peak is calculated. The relative retention time should be within the range of ±5% of the specified value. The specified value is: 0.71 (peak 1), 0.77 (peak 2), 1.00 (peak 3), 1.35 (peak 4), 1.63 (peak 5), 1.94 (peak 6), 2.05 (peak 7), 2.49 (peak 8), 2.72 (peak 9), 3.61 (peak 10), 3.81 (peak 11), 4.28 (peak 12), 4.77 (peak 13), 5.07 (peak 14), 5.46 (peak 15), 6.01 (peak 16). The reference characteristic spectrum is as follows: Fig.29 shown.

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein by equivalents. If these modifications and variations fall within the scope of the claims of the present invention and their equivalent technologies, they should be considered to be within the protection scope of the present invention.

Claims

1. A method for evaluating Zhuangyao Jianshen Tablets based on liquid chromatography, characterized in that: The following steps are involved: (1) Preparation of test solution: Take Zhuangyao Jianshen tablets from different batches, remove the sugar coating, grind into powder and place in a stoppered conical flask, add 10 ml of 70% ethanol, weigh and then perform ultrasonic treatment, cool to room temperature, weigh again, make up the lost weight with 70% ethanol, shake well, take the filtrate, and obtain different batches of test solution; (2) Preparation of reference solution: Take the reference substance, weigh it accurately, and add ethanol to make a solution containing 80 μg per 1 ml to obtain the reference solution; (3) Characteristic spectrum determination: Accurately pipette the reference solution and different batches of test solution respectively, perform high performance liquid chromatography test, and obtain the characteristic spectra of different batches; (4) Generation of characteristic peaks: The characteristic spectra of different batches were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the National Pharmacopoeia Committee in AIA format for data processing, and the characteristic spectra were MARK matched using the multi-point correction method to generate characteristic peaks of different batches; (5) Establishment of reference characteristic spectrum: Determine the common peaks and reference peaks of the characteristic spectrum of different batches according to the peak areas and retention times of the characteristic peaks of different batches; According to the relative peak areas and relative retention times of the common peaks and reference peaks of the characteristic spectra of different batches, the characteristic peaks of the control characteristic spectra and the prescribed values ​​of the relative retention times of the characteristic peaks are determined to obtain the control characteristic spectra; (6) Evaluation of Zhuangyao Jianshen Tablets: When the characteristic peaks of Zhuangyao Jianshen Tablets include the characteristic peaks of the reference characteristic spectrum, and the relative retention time of the characteristic peaks of Zhuangyao Jianshen Tablets and the reference peak is within 5% of the specified value of the characteristic peak of the reference characteristic spectrum, it is confirmed that the quality of Zhuangyao Jianshen Tablets meets the requirements.

2. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to claim 1, characterized in that: In step (5), the reference characteristic spectrum includes 16 characteristic peaks, and the prescribed values ​​of the relative retention times of the 16 characteristic peaks are: peak 1: 0.71, peak 2: 0.77, peak 3: 1.00, peak 4: 1.35, Peak 5: 1.63, Peak 6: 1.94, Peak 7: 2.05, Peak 8: 2.49, Peak 9: 2.72, Peak 10: 3.61, Peak 11: 3.81, Peak 12: 4.28, Peak 13: 4.77, Peak 14: 5.07, Peak 15: 5.46, Peak 16: 6.

01.

3. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to claim 2, characterized in that: The reference substance is 5-hydroxymethylfurfural; and / or, The reference peak is 5-hydroxymethylfurfural peak.

4. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to any one of claims 1 to 3, characterized in that: In step (3), the chromatographic column used for the HPLC test is ACE Excel C 18 or Waters Symmetry C 18 .

5. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to any one of claims 1 to 3, characterized in that: In step (3), the flow rate of the sample for HPLC test is 1.0 to 1.05 ml / min.

6. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to any one of claims 1 to 3, characterized in that: In step (3), the mobile phase A of the HPLC test is acetonitrile, and the mobile phase B is 0.2% phosphoric acid solution; the gradient elution program is: 0-17min: Fluidity A increases from 5% to 12% at a constant rate, and fluidity B decreases from 95% to 88% at a constant rate; 17-25min: Fluidity A increases from 12% to 20% at a constant rate, and fluidity B decreases from 88% to 80% at a constant rate; 25-30min: Fluidity A increases from 20% to 23% at a constant rate, and fluidity B decreases from 80% to 77% at a constant rate; 30-45min: Fluidity A increases from 23% to 40% at a constant rate, and fluidity B decreases from 77% to 60% at a constant rate; 45-55min: Fluidity A increases from 40% to 60% at a constant rate, and fluidity B decreases from 60% to 40% at a constant rate; 55-70min: Fluidity A increases from 60% to 70% at a constant rate, and fluidity B decreases from 40% to 30% at a constant rate; 70-72min: Fluidity A increases uniformly from 70% to 90%, and fluidity B decreases uniformly from 30% to 10%.

7. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to any one of claims 1 to 3, characterized in that: In step (3), the column temperature of the HPLC test is 30-40°C.

8. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to any one of claims 1 to 3, characterized in that: In step (3), the detection wavelength of the high performance liquid chromatography test is 255 to 265 nm.

9. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to any one of claims 1 to 3, characterized in that: In step (3), the theoretical plate number of the high performance liquid chromatography test is not less than 5000 calculated based on the reference peak.

10. The evaluation method of Zhuangyao Jianshen Tablets based on liquid chromatography according to claim 1, characterized in that: In step (1), the power of ultrasonic treatment is 250 W, the frequency is 40 kHz, and the time is 10 min.

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