A method for quality control of HPLC fingerprint of a kidney-tonifying and turbidity-draining mixture

Abstract

The application discloses a kind of kidney Yishen and turbidity releasing mixture HPLC fingerprint quality control method, including the preparation of test solution, HPLC chromatographic conditions and the establishment of fingerprint.The application carries out computer similarity evaluation to the chromatographic peak of different batches of kidney Yishen and turbidity releasing mixture, obtains the common peak of different batches of drugs, and generates HPLC control fingerprint, by comparing with the common peak and similarity in HPLC control fingerprint, the quality of kidney Yishen and turbidity releasing mixture can be comprehensively and quickly evaluated, and the chromatographic peak of each single medicine in prescription is attributed to confirm, can trace the material basis of preparation from raw material source, ensure preparation quality.Further quantitative analysis is carried out to 5 chemical components of kidney Yishen and turbidity releasing mixture, to ensure the stability and uniformity of preparation quality.

Description

Technical Field

[0001] This invention belongs to the field of detection technology for quality control of traditional Chinese medicine, specifically relating to a quality control method for HPLC fingerprint spectroscopy of Yishen Xiezhuo Compound. Background Technology

[0002] The prescription for Yishen Xiezhuo Mixture (Baoshen No. 3 Formula) is an experienced formula for treating chronic renal failure, summarized from the clinical practice of Dr. Ma Jipei, a renowned traditional Chinese medicine doctor at Wuxi Municipal Hospital of Traditional Chinese Medicine. It consists of nine Chinese herbs: Astragalus membranaceus, Cornus officinalis, Perilla frutescens leaf, Pinellia ternata, Coptis chinensis, Coix lacryma-jobi, Rheum palmatum, Prunus persica seed, and Centella asiatica. It has the effects of strengthening the spleen and kidneys, harmonizing the meridians and eliminating turbidity. Clinically, it is used for chronic renal failure (chronic kidney disease stage) and has been applied in clinical practice for many years. Clinical studies and animal efficacy experiments have shown that it has significant efficacy and no adverse reactions. However, it currently lacks a pharmaceutical preparation approval number. Therefore, it is essential to conduct pharmaceutical research, including preparation process and quality control, for its application as a medical institution preparation and for innovative traditional Chinese medicine research. Traditional Chinese medicine fingerprinting is a comprehensive and quantifiable quality control method that can effectively reflect the overall quality of traditional Chinese medicine and is widely used in traditional Chinese medicine quality control. To better control the quality of Yishen Xiezhuo Mixture, this invention establishes an HPLC fingerprinting quality control method to control the intrinsic quality of Yishen Xiezhuo Mixture. Summary of the Invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide an HPLC fingerprint quality control method for Yishen Xiezhuo Mixture, so as to provide a comprehensive and quantifiable quality control method for Yishen Xiezhuo Mixture, thereby improving the product quality of Yishen Xiezhuo Mixture and enhancing its clinical application effect.

[0004] Technical Solution: To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] A quality control method for HPLC fingerprinting of a kidney-tonifying and turbidity-eliminating compound includes the following steps:

[0006] (1) Preparation of the test solution of the kidney-tonifying and turbidity-eliminating compound:

[0007] Take the kidney-tonifying and turbidity-eliminating mixture into a volumetric flask, dilute with methanol to the mark, weigh, sonicate, cool and weigh again, make up the lost weight with methanol, shake well, filter through a microporous membrane, and take the filtrate as the test solution.

[0008] (2) Preparation of reference solution:

[0009] Weigh gallic acid, monoglucoside, loganin, verrucoside, and rhein reference standards, place them in a volumetric flask, dilute to the mark with 70% methanol solution, sonicate, cool, and then make up to the mark with 70% methanol solution. Shake well, filter through a microporous membrane, and collect the filtrate to obtain the reference solution.

[0010] (3) The test solution in step (1) and the reference solution in step (2) were detected by high performance liquid chromatography.

[0011] (4) Based on the fingerprint chromatograms of different batches of Yishen Xiezhuo Mixture test solution collected in step (3), import them into the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" to obtain the common peaks in the chromatogram of Yishen Xiezhuo Mixture and the HPLC reference fingerprint chromatogram.

[0012] (5) Take the test sample of Yishen Xiezhuo Mixture and obtain the HPLC fingerprint spectrum according to the method of steps (1) to (3). Import it into the software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" and evaluate the similarity with the HPLC reference fingerprint spectrum obtained in step (4). If the similarity is greater than 0.9, the test sample of Yishen Xiezhuo Mixture is qualified.

[0013] In step (1), 1.5 mL of the kidney-tonifying and turbidity-eliminating mixture is placed in a 10 mL volumetric flask.

[0014] In steps (1) and (2), the ultrasound conditions are as follows: 100W, 40kHz, 10min.

[0015] In step (2), the content of each component in the reference solution is as follows:

[0016] Gallic acid 49.6 μg / mL, monoglucoside 77.2 μg / mL, loganin 48.8 μg / mL, verrucoside 19.6 μg / mL, rhein 24.8 μg / mL.

[0017] In steps (1) and (2), the microporous filter membrane is a 0.22 μm microporous filter membrane.

[0018] In step (3), the detection method of high performance liquid chromatography is as follows: detection is performed using a high performance liquid chromatograph, the detection instrument is a DAD diode array detector, the detection wavelength is 254 nm, the column temperature is 30 °C, the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid water, and the flow rate is 1.0 mL·min. -1 Analysis time: 100 min; Gradient elution;

[0019] The gradient elution procedure is as follows:

[0020] 0~10min, 2%~4%A; 10~15min, 4%~6%A; 15~20min, 6%~7%A; 20~22min, 7%~10%A; 22~32min, 10%A; 32 ~40min, 10%~13%A; 40~50min, 13%A; 50~90min, 13~52%A; 90~95min, 52%A; 95~100min, 52%A~2%A.

[0021] In step (4), the HPLC reference fingerprint spectrum includes 19 common peaks, with peak 16 as the reference peak and a relative retention time of 1.000. The relative retention times of the other 18 common peaks are as follows: peak 1: 0.151±0.001, peak 2: 0.170±0.001, peak 3: 0.205±0.002, peak 4: 0.230±0.002, peak 5: 0.314±0.001, peak 6: 0.375±0.001, peak 7: 0.467±0.002. Peak 8: 0.475±0.003, Peak 9: 0.694±0.001, Peak 10: 0.887±0.001, Peak 11: 0.936±0.001, Peak 12: 0.948±0.001, Peak 13: 0.971±0.001, Peak 14: 0.976±0.001, Peak 15: 0.993±0.000, Peak 17: 1.007±0.001, Peak 18: 1.016±0.001, Peak 19: 1.192±0.003.

[0022] Among them, peak 6 is mononoside, peak 8 is loganin, and peak 19 is rhein.

[0023] Beneficial effects:

[0024] 1. This invention discloses an HPLC fingerprint quality control method for Yishen Xiezhuo Mixture. Based on the structural characteristics of the chemical components in Yishen Xiezhuo Mixture, this invention screens the optimal detection wavelength, mobile phase, mobile phase gradient elution program, column temperature, and other chromatographic analysis conditions through extensive experiments. Experimental verification shows that the HPLC fingerprint quality control method for Yishen Xiezhuo Mixture established by this invention can comprehensively, objectively, and accurately evaluate and control the quality of Yishen Xiezhuo Mixture, which is of positive significance for ensuring its clinical efficacy and safety.

[0025] 2. The HPLC fingerprint of Yishen Xiezhuo Mixture established in this invention can fully and effectively characterize the intrinsic quality of Yishen Xiezhuo Mixture, objectively reflect the sequential order and interrelationship of the intrinsic components (fingerprint characteristic peaks) of Yishen Xiezhuo Mixture, avoid the one-sidedness of using a single or a few components to distinguish the quality of Yishen Xiezhuo Mixture, and avoid the possibility of artificially adding individual components to achieve quality compliance.

[0026] 3. The HPLC fingerprint quality control method for Yishen Xiezhuo Mixture established in this invention can simultaneously detect the content of five components in Yishen Xiezhuo Mixture. The method is simple, precise, and stable. Attached Figure Description

[0027] Figure 1 The fingerprint spectra of 10 batches of Yishen Xiezhuo Mixture samples and their control fingerprint spectra R of this invention.

[0028] Figure 2 The comparative fingerprint R of the kidney-tonifying and turbidity-eliminating compound of this invention and the chromatogram of the single herb.

[0029] Figure 3 Chromatogram of mixed reference standard A (1. gallic acid 2. monoglucoside 3. loganin 4. verbascoside 5. rhein, Figure 3 (The same as BE in Chinese);

[0030] Chromatogram of the test solution of B-Yishen Xiezhuo Mixture;

[0031] Chromatogram of a negative sample solution of Cornus officinalis lacking C;

[0032] Chromatogram of a negative sample solution lacking Astragalus membranaceus (D);

[0033] Chromatogram of a negative sample solution of rhubarb lacking E. Detailed Implementation

[0034] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not and will not limit the invention as described in detail in the claims.

[0035] Example 1: A quality control method for HPLC fingerprint chromatogram of a kidney-tonifying and turbidity-eliminating compound.

[0036] 1. Instruments and Reagents

[0037] Instruments: Agilent 1200 high performance liquid chromatograph (Agilent Technologies, USA), DAD diode array detector (Agilent Technologies, USA); BSA224S-CW electronic balance (Sartorius GmbH, Germany); KQ-300E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0038] Reagents: Gallic acid reference standard (batch number: C13O9C72105, purity ≥98%), monosodium glutamate reference standard (batch number: Z12O8B45504, purity ≥97%), loganin reference standard (batch number: P22F10F81444, purity ≥98%), verbascoside reference standard (batch number: J19HB174062, purity ≥98%), and rhein reference standard (batch number: T06J10F92311, purity ≥98%) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. Acetonitrile was of chromatographic grade, water was ultrapure water, and other reagents were of analytical grade. Kidney-Nourishing and Turbidity-Clearing Compound (a self-made preparation of Wuxi Municipal Hospital of Traditional Chinese Medicine, with batch numbers of samples S1 to S10 as 20221201, 20221202, 20221203, 20221204, 20221205, 20221206, 20221207, 20221208, 20221209, and 20221210 respectively);

[0039] Table 1. Medicinal slices and batch numbers

[0040]

[0041] It was also identified by Wang Qing, deputy chief pharmacist of Wuxi Municipal Hospital of Traditional Chinese Medicine, as meeting the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0042] 2. Methods and Results

[0043] 2.1 Chromatographic conditions

[0044] Optimization of fingerprint detection conditions:

[0045] (1) Selection of detection wavelength

[0046] This invention performed a full-wavelength DAD (Diode Aperture Reduction) scan on the test solution of the Yishen Xiezhuo Compound. It was found that the sample had the most chromatographic peaks and the best peak shape at a wavelength of 254 nm. The target components gallic acid, monoglucoside, loganin, verrucoside, and rhein all showed good responses. Therefore, 254 nm was selected as the detection wavelength.

[0047] (2) Selection of mobile phase

[0048] This invention investigated different mobile phases (acetonitrile-0.1% phosphoric acid solution, methanol-0.2% phosphoric acid solution, and acetonitrile-0.2% formic acid solution). The results showed that acetonitrile-0.1% phosphoric acid solution was used as the mobile phase, and the chromatographic peaks in the Yishen Xiezhuo compound sample were better shaped. The target components gallic acid, monoglycoside, loganin, verrucoside, and rhein were all well separated, and the baseline was stable. Therefore, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase.

[0049] (3) Selection of mobile phase gradient elution program

[0050] In this invention, the components in the kidney-tonifying and turbidity-eliminating compound are mostly polar components. Based on the selection of acetonitrile-0.1% phosphoric acid water as the mobile phase, the gradient elution conditions are adjusted to improve the resolution of the chromatographic peaks of the sample. By adjusting the solvent polarity at different time points, the chromatographic peak resolution of the Yishen Xiezhuo compound sample was improved as much as possible to ensure good separation of the target components gallic acid, monoglycoside, loganin, verrucoside, and rhein. Finally, the HPLC mobile phase gradient elution program was selected as follows: Mobile phase: acetonitrile (A) - 0.1% phosphoric acid water (B) gradient elution (0-10 min, 2%-4% A; 10-15 min, 4%-6% A; 15-20 min, 6%-7% A; 20-22 min, 7%-10% A; 22-32 min, 10% A; 32-40 min, 10%-13% A; 40-50 min, 13% A; 50-90 min, 13-52% A; 90-95 min, 52% A); 95-100 min, 52% A-2% A.

[0051] (4) Selection of column temperature

[0052] This invention investigated the effects of different column temperatures (30, 35, and 40℃) on the chromatographic peak elution and the resolution of target components gallic acid, monoglycoside, loganin, verrucoside, and rhein in a sample of Yishen Xiezhuo Mixture. The results showed that at a column temperature of 30℃, the chromatographic peak shapes in the Yishen Xiezhuo Mixture sample were better, and the resolution of the target components gallic acid, monoglycoside, loganin, verrucoside, and rhein was also better. Therefore, a column temperature of 30℃ was chosen.

[0053] The final chromatographic conditions are as follows:

[0054] Chromatographic column: Eclipse XDB C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase: Acetonitrile (A) - 0.1% phosphoric acid water (B) gradient elution (0–10 min, 2%–4% A; 10–15 min, 4%–6% A; 15–20 min, 6%–7% A; 20–22 min, 7%–10% A; 22–32 min, 10% A; 32–40 min, 10%–13% A; 40–50 min, 13% A; 50–90 min, 13–52% A; 90–95 min, 52% A; 95–100 min, 52% A–2% A); Flow rate: 1.0 ml·min -1 Column temperature: 30℃; injection volume: 10μl; detection wavelength: 254nm.

[0055] This patent adjusts the detection method. Compared with the HPLC analysis method in the paper "Optimization of Preparation Process of Yishen Xiezhuo Compound by Multi-index Weighted Orthogonal Experiment" (authors Fei Qianqian et al.), the present invention detects more peaks, has better separation of each peak, and has a more stable baseline, which meets the requirements for establishing a fingerprint analysis method.

[0056] 2.2 Preparation of the test solution

[0057] Accurately pipette 1.5 ml of the kidney-tonifying and turbidity-eliminating mixture into a 10 ml volumetric flask, dilute with methanol to the mark, weigh, sonicate (100 W, 40 kHz) for 10 min, cool and weigh again, replenish the lost weight with methanol, shake well, filter through a 0.22 μm microporous membrane, and use the filtrate as the test solution.

[0058] 2.3 Preparation of Single-Herb Solution

[0059] Weigh out an appropriate amount of each herb in the prescription and prepare a single herb solution of nine herbs, including Astragalus membranaceus, Cornus officinalis, Rheum palmatum, and Coptis chinensis, according to the preparation process of Yishen Xiezhuo Mixture (12 times the amount of water, soak for half an hour, decoct twice, 1.5 hours each time). Prepare the solutions of each herb according to the method in section "2.2".

[0060] 2.4 Preparation of reference solution

[0061] Accurately weigh appropriate amounts of gallic acid, monoglobulin, loganin, verrucoside, and rhein, place them in a 10 mL volumetric flask, dilute to the mark with 70% methanol, sonicate (100 W, 40 kHz) for 10 min, cool, and then dilute to the mark with methanol. Shake well to prepare a mixed reference solution containing 49.6 μg gallic acid, 77.2 μg monoglobulin, 48.8 μg loganin, 19.6 μg verrucoside, and 24.8 μg rhein per mL. Filter the solution through a 0.22 μm microporous membrane and collect the filtrate to obtain the reference solution.

[0062] 2.5 Methodological Examination

[0063] 2.5.1 Precision Test

[0064] The test solution prepared from Yishen Guluo Compound S5 (20221205) was injected six times consecutively under the chromatographic conditions described in section "2.1", and the retention time and peak area of ​​each common peak were recorded. Using peak 16 as the reference peak, the results showed that the relative peak area RSD of each common peak was less than 2.98% (n=6), and the relative retention time RSD was less than 1.90% (n=6), indicating good instrument precision.

[0065] 2.5.2 Repeatability Test

[0066] Six test solutions were prepared in parallel using the method described in section "2.2" for the preparation of the test solution from Yishen Guluo Compound S5 (20221205). The solutions were then analyzed under the chromatographic conditions described in section "2.1". Using peak 16 as the reference peak, the RSDs of the relative retention times and relative peak areas of each common peak were <1.07% and <2.00% (n=6), respectively, indicating good repeatability of the method.

[0067] 2.5.3 Stability Test

[0068] The test solution prepared from Yishen Guluo Compound S5 (20221205) was analyzed under the chromatographic conditions described in section "2.1" at 0, 2, 4, 6, 8, 10, and 24 h after preparation. Using peak 16 as the reference peak, the RSDs of the relative retention time and relative peak area of ​​each common peak were <1.31% and <3.43 (n=7), respectively, indicating that the test solution exhibited good stability within 24 h.

[0069] 2.6 Establishment of fingerprint map

[0070] 2.6.1 Establishment of fingerprint map

[0071] Take 10 batches of Yishen Xiezhuo Mixture (S1~S10) respectively, prepare test solutions according to the method under section "2.2.2", and inject and detect according to the chromatographic conditions under section "2.1". Import the obtained chromatographic data into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software. [14-15] Automatic matching and multi-point calibration were performed on the chromatographic peaks of each fingerprint spectrum, and a total of 19 common peaks were identified. (See attached image) Figure 1 .

[0072] 2.6.2 Calibration of common peaks

[0073] According to the HPLC characteristic fingerprint of the test sample, peak 16 is symmetrical and has a large area. Therefore, it was selected as the reference peak to calculate the relative retention time and relative peak area of ​​the remaining peaks. The results are shown in Tables 2 and 3. The fingerprint of the Yishen Xiezhuo Mixture includes 19 common peaks. Peak 16 was used as the reference peak with a relative retention time of 1.000. The relative retention times of the other 18 common peaks are as follows: Peak 1 0.151±0.001, Peak 2 0.170±0.001, Peak 3 0.205±0.002, Peak 4 0.230±0.002, Peak 5 0.314±0.001, Peak 6 0.375±0.001, Peak 7 0.467±0.002, Peak 8... 0.475±0.003, Peak 9 0.694±0.001, Peak 10 0.887±0.001, Peak 11 0.936±0.001, Peak 12 0.948±0.001, Peak 13 0.971±0.001, Peak 14 0.976±0.001, Peak 15 0.993±0.000, Peak 17 1.007±0.001, Peak 18 1.016±0.001, Peak 19 1.192±0.003.

[0074] Table 2. Relative retention times of common peaks in HPLC for 10 batches of the compound preparation.

[0075]

[0076]

[0077] Table 3. Relative peak areas of common peaks in HPLC for 10 batches of the compound preparation.

[0078]

[0079]

[0080] 2.6.3 Shared Peak Attribution and Assignment

[0081] The mixed reference solution under section "2.2.1", the sample solution under section "2.2.2" (batch number: 20221205, S5), and the single herb test solution under section "2.2.3" were respectively injected and analyzed according to the chromatographic conditions under section "2.1". Nineteen common peaks were identified through full-spectrum peak matching. By comparing the chromatograms with those of the reference standards and their relative retention times, the chromatograms of the single herbs were identified as follows: peak 11 belonged to Astragalus membranaceus; peaks 1, 2, 3, 4, 5, 6, 7, and 8 belonged to Cornus officinalis (peak 6 is mononoside, and peak 8 is loganin); peaks 3 and 5 belonged to Perilla frutescens; peaks 13 and 14 belonged to Coptis chinensis; peak 5 belonged to Centella asiatica; and peaks 1, 2, 4, 5, 15, 17, and 19 belonged to Rheum palmatum (peak 19 is rhein). See [link to relevant documentation]. Figure 2 .

[0082] 2.6.4 Similarity Evaluation

[0083] Using the common pattern of Yishen Xiezhuo Compound as a control fingerprint, the similarity of 10 batches of samples was evaluated. The similarity of samples S1 to S10 were 0.933, 0.976, 0.977, 0.938, 0.956, 0.918, 0.933, 0.931, 0.971, and 0.916, respectively, indicating good similarity among the various Yishen Xiezhuo Compound samples. A similarity greater than 0.9 was used as the evaluation criterion.

[0084] Example 2: HPLC fingerprint quality control method for a kidney-tonifying and turbidity-eliminating compound (determination of the content of 5 index components)

[0085] 1. Instruments and Reagents

[0086] Instruments: Agilent 1200 high performance liquid chromatograph (Agilent Technologies, USA), DAD diode array detector (Agilent Technologies, USA); BSA224S-CW electronic balance (Sartorius GmbH, Germany); KQ-300E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0087] Reagents: Gallic acid reference standard (batch number: C13O9C72105, purity ≥98%), monosodium glutamate reference standard (batch number: Z12O8B45504, purity ≥97%), loganin reference standard (batch number: P22F10F81444, purity ≥98%), verbascoside reference standard (batch number: J19HB174062, purity ≥98%), and rhein reference standard (batch number: T06J10F92311, purity ≥98%) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. Acetonitrile was of chromatographic grade, water was ultrapure water, and other reagents were of analytical grade. Kidney-Nourishing and Turbidity-Clearing Compound (a self-made preparation of Wuxi Municipal Hospital of Traditional Chinese Medicine, with batch numbers of samples S1 to S10 as 20221201, 20221202, 20221203, 20221204, 20221205, 20221206, 20221207, 20221208, 20221209, and 20221210 respectively);

[0088] Table 4. Medicinal slices and batch numbers

[0089]

[0090] It was also identified by Wang Qing, deputy chief pharmacist of Wuxi Municipal Hospital of Traditional Chinese Medicine, as meeting the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0091] 2. Methods and Results

[0092] 2.1 Chromatographic conditions

[0093] Chromatographic column: Waters XBridge C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase: Acetonitrile (A) - 0.1% phosphoric acid water (B) gradient elution (0–10 min, 2%–4% A; 10–15 min, 4%–6% A; 15–20 min, 6%–7% A; 20–22 min, 7%–10% A; 22–32 min, 10% A; 32–40 min, 10%–13% A; 40–50 min, 13% A; 50–90 min, 13–52% A; 90–95 min, 52% A; 95–100 min, 52% A–2% A); Flow rate: 1.0 ml·min -1 Column temperature: 30℃; injection volume: 10μl; detection wavelength: 254nm.

[0094] 2.2 Preparation of the test solution

[0095] Accurately pipette 1.5 ml of the kidney-tonifying and turbidity-eliminating mixture into a 10 ml volumetric flask, dilute with methanol to the mark, weigh, sonicate (100 W, 40 kHz) for 10 min, cool and weigh again, replenish the lost weight with methanol, shake well, filter through a 0.22 μm microporous membrane, and use the filtrate as the test solution.

[0096] 2.3 Preparation of negative sample solution

[0097] According to the prescription ratio, weigh out the negative samples that are missing Astragalus membranaceus, Cornus officinalis, and Rheum palmatum in the prescription, and prepare the negative sample solutions that are missing the corresponding drugs in the prescription according to the method in section "2.2" for later use.

[0098] 2.4 Preparation of reference solution

[0099] Accurately weigh appropriate amounts of gallic acid, monoglobulin, loganin, verrucoside, and rhein, place them in a 10 mL volumetric flask, dilute to the mark with 70% methanol, sonicate (100 W, 40 kHz) for 10 min, cool, and then dilute to the mark with methanol. Shake well to prepare a mixed reference solution containing 49.6 μg gallic acid, 77.2 μg monoglobulin, 48.8 μg loganin, 19.6 μg verrucoside, and 24.8 μg rhein per mL. Filter the solution through a 0.22 μm microporous membrane and collect the filtrate to obtain the reference solution.

[0100] 2.5 Methodological Examination

[0101] 2.5.1 Specificity Examination

[0102] Take the mixed reference solution, test solution, and negative sample solution prepared under "2.2.1, 2.2.2, and 2.2.4" respectively, and inject 10 μL under the chromatographic conditions under "2.1". The baseline is stable, the peak shape is symmetrical, the resolution is good, and the peak information content is large. The negative sample solution lacking Astragalus membranaceus did not have the peak of isoflavone glycoside; the negative sample solution lacking Cornus officinalis did not have the peaks of gallic acid, monoglobin, and loganin; and the negative sample solution lacking Rheum palmatum did not have the peak of rhein. The retention time of each peak is moderate and can meet the needs of content determination. The HPLC chromatogram of the mixed reference solution is shown in [reference needed]. Figure 3 The HPLC chromatogram of the test solution of Zhong A, Yishen Xiezhuo Mixture, is shown below. Figure 3 The HPLC chromatograms of negative sample solutions lacking Cornus officinalis, Astragalus membranaceus, and Rheum palmatum are shown below. Figure 3 C, D, E.

[0103] 2.5.2 Examination of Linear Relationships

[0104] Accurately transfer an appropriate amount of the mixed reference solution of gallic acid, monoglycoside, loganin, verrucoside, and rhein from section "2.2", dilute with 70% methanol to prepare a series of solutions of different concentrations, filter through a 0.22 μm microporous membrane, and inject and determine according to the chromatographic conditions in section "2.1", recording the peak area. The concentration of each reference standard (X, μg·mL) is used as the chromatographic standard. -1 Using the x-axis as the horizontal axis and the peak area as the y-axis, a standard curve was plotted, yielding a linear regression equation. The results are shown in Table 5, indicating a good linear relationship.

[0105] Table 5. Results of the linear relationship examination of the five index components.

[0106]

[0107] 2.5.3 Precision Test

[0108] Take the mixed reference solution under section “2.2.1”, and inject it six times consecutively under the chromatographic conditions under section “2.1”. Record the peak areas of gallic acid, monoglucoside, loganin, verrucoside, and rhein. The RSDs of the results were 0.16%, 0.70%, 0.16%, 0.11%, and 0.07%, respectively, indicating that the instrument precision was good.

[0109] 2.5.4 Repeatability Test

[0110] Six test solutions were prepared in parallel using the same batch of Yishen Xiezhuo Mixture (batch number 20221205, S5) according to the method described in section “2.2.2”. The solutions were injected and the peak areas of gallic acid, monoglycoside, loganin, verrucoside, and rhein were recorded. The RSDs were 1.81%, 1.00%, 0.98%, 1.00%, and 1.01%, respectively, indicating that the method has good repeatability.

[0111] 2.5.5 Stability Test

[0112] Yishen Xiezhuo Mixture (batch number 20221205) was used to prepare a test solution according to the method described in section "2.2.2". The solution was injected at 0, 2, 4, 8, 16, and 24 hours under the chromatographic conditions described in section "2.1", and the HPLC chromatograms were recorded. The RSDs of the peak areas of gallic acid, monoglycoside, loganin, verrucoside, and rhein were 1.43%, 0.31%, 0.42%, 0.52%, and 0.52%, respectively, indicating that the test solution was stable within 24 hours.

[0113] 2.5.6 Recovery Test

[0114] Six portions of Yishen Xiezhuo Mixture (S5) with known component contents were accurately measured. A mixed reference solution containing the same content as the sample was accurately added to each portion. Six test solutions were prepared in parallel according to the method described in section "2.2.2". The solutions were then injected for analysis. The average recoveries of gallic acid, monoglycoside, loganin, verrucoside, and rhein were calculated to be 101.93%, 103.93%, 100.20%, 97.28%, and 98.77%, respectively, with RSDs of 2.72%, 2.64%, 2.84%, 2.56%, and 2.60%, respectively, indicating good accuracy of the method.

[0115] 2.5.7 Sample Content Determination

[0116] Ten batches of Yishen Xiezhuo Mixture samples were taken, and test solutions were prepared in parallel according to the method in section "2.2.2". The samples were injected according to the chromatographic conditions in section "2.1". The contents of five components in the samples, namely gallic acid, monoglucoside, loganin, verrucoside, and rhein, were calculated. The results are shown in Table 6.

[0117] Table 6. Determination of the content of five components in Yishen Xiezhuo Mixture (μg·mL) -1 )

[0118]

[0119]

Claims

1. A method for quality control of HPLC fingerprint spectroscopy of a kidney-tonifying and turbidity-eliminating compound, characterized in that, Includes the following steps: (1) Preparation of test solution of Yishen Xiezhuo Mixture: Take Yishen Xiezhuo Mixture into a volumetric flask, add methanol to dilute to the mark, weigh, sonicate, cool and weigh again, make up the lost weight with methanol, shake well, filter through a microporous membrane, and take the filtrate as the test solution. (2) Preparation of reference solution: Weigh gallic acid, monoglucoside, loganin, verrucoside and rhein reference standards, place them in volumetric flasks, dilute to the mark with 70% methanol solution, sonicate, cool and then make up to the mark with 70% methanol solution, shake well, filter through a microporous membrane, and take the filtrate to obtain the reference solution; (3) High performance liquid chromatography (HPLC) was used to detect the test solution in step (1) and the reference solution in step (2); (4) Based on the fingerprint chromatograms of different batches of Yishen Xiezhuo Mixture test solution collected in step (3), import them into the software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to obtain the common peaks in the chromatogram of Yishen Xiezhuo Mixture and the HPLC reference fingerprint chromatogram. (5) Take the sample of Yishen Xiezhuo compound to be tested, and obtain the HPLC fingerprint spectrum according to the method of steps (1) to (3). Import it into the software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and use the HPLC reference fingerprint spectrum obtained in step (4) as a reference for similarity evaluation. The detection method of the high-performance liquid chromatography (HPLC) is as follows: HPLC is used with an Eclipse XDBC18 column (250 mm × 4.6 mm, 5 μm), a DAD diode array detector, and a detection wavelength of 254 nm. Mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid water. The analysis time is 100 min. Gradient elution is performed. The gradient elution program is as follows: 0–10 min, 2%–4% A; 10–15 min, 4%–6% A; 15–20 min, 6%–7% A; 20–22 min, 7%–10% A; 22–32 min, 10% A; 32–40 min, 10%–13% A; 40–50 min, 13% A; 50–90 min, 13–52% A; 90–95 min, 52% A; 95–100 min, 52% A–2% A.

2. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 1, characterized in that, In step (1), 1.5 mL of the kidney-tonifying and turbidity-eliminating compound was placed in a 10 mL volumetric flask.

3. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 1, characterized in that, In steps (1) and (2), the ultrasound is performed under the following conditions: 100 W, 40 kHz, 10 min.

4. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 1, characterized in that, In step (2), the content of each component in the reference solution is as follows: Gallic acid 49.6 µg / mL, monoglucoside 77.2 µg / mL, loganin 48.8 µg / mL, verrucoside 19.6 µg / mL, rhein 24.8 µg / mL.

5. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 1, characterized in that, In steps (1) and (2), the microporous filter membrane is a 0.22 μm microporous filter membrane.

6. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 1, characterized in that, Column temperature: 30℃, flow rate: 1.0 mL·min -1 .

7. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 1, characterized in that, In step (4), the HPLC reference fingerprint spectrum includes 19 common peaks, with peak 16 as the reference peak and a relative retention time of 1.

000. The relative retention times of the other 18 common peaks are as follows: peak 1 0.151±0.001, peak 2 0.170±0.001, peak 3 0.205±0.002, peak 4 0.230±0.002, peak 5 0.314±0.001, peak 6 0.375±0.001, peak 7 0.467±0.002, and peak 8... 0.475±0.003, Peak 9 0.694±0.001, Peak 10 0.887±0.001, Peak 11 0.936±0.001, Peak 12 0.948±0.001, Peak 13 0.971±0.001, Peak 14 0.976±0.001, Peak 15 0.993±0.000, Peak 17 1.007±0.001, Peak 18 1.016±0.001, Peak 19 1.192±0.

003.

8. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 7, characterized in that, Peak 6 is mononoside, peak 8 is loganin, and peak 19 is rhein.

9. The HPLC fingerprint quality control method for the kidney-tonifying and turbidity-eliminating compound according to claim 1, characterized in that, In step (5), if the similarity is greater than 0.9, the sample of Yishen Xiezhuo Mixture is qualified.

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