A method for establishing HPLC fingerprint of a kidney-nourishing and liver-nourishing mixture and a method for determining multi-index component content

Abstract

The present application belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a method for establishing HPLC fingerprint of Yishen Yanggan mixture and a method for determining multi-index component content. The method for establishing fingerprint comprises the following steps: mixing Yishen Yanggan mixture and methanol solution to perform ultrasonic treatment, performing HPLC detection on the obtained test sample solution and mixed control sample solution, introducing the obtained chromatogram into fingerprint software to obtain HPLC fingerprint of Yishen Yanggan mixture. The pre-treatment method is simple, and the characteristic components are reserved completely. The present application adopts high performance liquid chromatography, and has high precision, good reproducibility and good stability. The obtained fingerprint has 15 common peaks, which can effectively monitor the quality of Yishen Yanggan mixture, and provides guarantee for comprehensive and effective control of the quality of Yishen Yanggan mixture.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, specifically relating to a method for establishing an HPLC fingerprint of a kidney-tonifying and liver-nourishing compound and a method for determining the content of multiple components. Background Technology

[0002] The Kidney-Nourishing and Liver-Tonifying Compound is composed of multiple traditional Chinese medicines, including Astragalus membranaceus, Cornus officinalis, Epimedium brevicornu, and Cnidium monnieri. It has the effects of tonifying Qi and strengthening the spleen, nourishing the kidneys and liver, and strengthening muscles and bones. Clinically, it is used for various diseases and chronic strain caused by liver and kidney deficiency, blood deficiency, and spermatorrhea, with symptoms such as dizziness, blurred vision, fatigue, swelling and pain in the lower back and limbs, dry mouth and eyes, weakness and soreness in the limbs, irregular menstruation, inability to lift heavy objects, or hemiplegia. It has significant therapeutic effects and no adverse reactions.

[0003] Currently, the main active ingredient, astragaloside A, in Yishen Yanggan Heji (a traditional Chinese medicine preparation) is determined using a dual-wavelength thin-layer scanning method. However, due to the complexity of the components in traditional Chinese medicine preparations, the determination of the content of a single indicator component cannot fully guarantee the quality of Yishen Yanggan Heji. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for establishing an HPLC fingerprint of a kidney-tonifying and liver-nourishing compound and a method for determining the content of multiple components. This invention uses HPLC to detect the kidney-tonifying and liver-nourishing compound and obtain a fingerprint for quality evaluation and control, providing a guarantee for the comprehensive and effective quality control of the kidney-tonifying and liver-nourishing compound.

[0005] This invention provides a method for establishing the HPLC fingerprint of a kidney-tonifying and liver-nourishing compound, comprising the following steps:

[0006] The kidney-tonifying and liver-nourishing compound was mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution;

[0007] A mixed reference solution is provided, wherein the references in the mixed reference solution include mononoside, loganin, verbascoside, icariin and osthol;

[0008] The test solution and the mixed reference solution were analyzed by HPLC. The obtained chromatograms were imported into fingerprinting software to identify common peaks and obtain the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound.

[0009] The conditions for the HPLC detection include:

[0010] Chromatographic column: C18 column; Mobile phase: Phase A is 0.05–0.2% (v / v) aqueous phosphoric acid solution, Phase B is acetonitrile; Gradient elution, elution program: 0–10 min, Phase B volume fraction increases from 5% to 10%; 10–30 min, Phase B volume fraction increases from 10% to 18%; 30–50 min, Phase B volume fraction increases from 18% to 30%; 50–70 min, Phase B volume fraction increases from 30% to 50%; 70–85 min, Phase B volume fraction increases from 50% to 100%; Flow rate: 0.6–1 mL / min; Column temperature: 25–35℃; Detection wavelength: 240–260 nm.

[0011] Preferably, the volume fraction of methanol in the methanol-water solution is 40% to 60%.

[0012] Preferably, the volume ratio of the kidney-tonifying and liver-nourishing compound to the methanol aqueous solution is 1:4 to 6.

[0013] Preferably, the chromatographic column is a ZORBAX Eclipse Plus C18 column.

[0014] Preferably, phase A is a 0.1% (v / v) aqueous solution of phosphoric acid, the flow rate is 0.8 mL / min, and the column temperature is 30 °C.

[0015] Preferably, the detection wavelength is 250 nm.

[0016] Preferably, the injection volume for HPLC detection is 5–20 μL.

[0017] Preferably, the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound includes 15 common peaks, and the relative retention times with peak 7 are as follows: peak 1: 0.22, peak 2: 0.46, peak 3: 0.51, peak 4: 0.65, peak 5: 0.71, peak 6: 0.72, peak 7: 1.00, peak 8: 1.04, peak 9: 1.22, peak 10: 1.36-1.37, peak 11: 1.52-1.53, peak 12: 1.56, peak 13: 1.77, peak 14: 2.14-2.15, peak 15: 2.27-2.28; wherein peak 3 is mononoside, peak 5 is loganin, peak 7 is verbascoside, peak 12 is icariin, and peak 15 is osthol.

[0018] This invention also provides a method for determining the content of multiple indicators in a kidney-tonifying and liver-nourishing compound, wherein the multiple indicators include two or more of mononoside, loganin, verbascoside, icariin, and osthol, and the determination method includes the following steps:

[0019] The test kidney-tonifying and liver-nourishing compound was mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution.

[0020] The test solution was subjected to HPLC analysis, and the content of multiple index components in the kidney-tonifying and liver-nourishing compound was obtained based on the obtained chromatographic peak area and the predetermined standard curve; the predetermined standard curve is a linear curve between the concentration of each of the multiple index components and the corresponding peak area.

[0021] The conditions for the HPLC detection include:

[0022] Chromatographic column: C18 column; Mobile phase: Phase A is 0.05–0.2% (v / v) aqueous phosphoric acid solution, Phase B is acetonitrile; Gradient elution, elution program: 0–10 min, Phase B volume fraction increases from 5% to 10%; 10–30 min, Phase B volume fraction increases from 10% to 18%; 30–50 min, Phase B volume fraction increases from 18% to 30%; 50–70 min, Phase B volume fraction increases from 30% to 50%; 70–85 min, Phase B volume fraction increases from 50% to 100%; Flow rate: 0.6–1 mL / min; Column temperature: 25–35℃; Detection wavelength: 240–330 nm.

[0023] Preferably, the detection wavelengths of mononoside and loganin are 240 nm, the detection wavelengths of verrucoside and isoflavone glucoside are 260 nm, the detection wavelengths of icariin are 270 nm, and the detection wavelengths of osthol are 322 nm.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a method for establishing an HPLC fingerprint of a kidney-tonifying and liver-nourishing compound, comprising the following steps: mixing the kidney-tonifying and liver-nourishing compound with a methanol-water solution and subjecting it to ultrasonic treatment to obtain a test solution; providing a mixed reference solution, wherein the reference standards in the mixed reference solution include mononoside, loganin, verrucoside, icariin, and osthol; performing HPLC detection on the test solution and the mixed reference solution, importing the obtained chromatogram into fingerprinting software, identifying common peaks, and obtaining the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound. The pretreatment method of this invention is simple, and the characteristic components are completely preserved. This invention uses high-performance liquid chromatography (HPLC), which has high precision, good reproducibility, and good stability. The fingerprint obtained by this invention identifies 15 common peaks, which can effectively monitor the quality of the kidney-tonifying and liver-nourishing compound. This invention uses HPLC to detect the kidney-tonifying and liver-nourishing compound and obtain a fingerprint for quality evaluation and control, providing a guarantee for the comprehensive and effective control of the quality of the kidney-tonifying and liver-nourishing compound.

[0026] This invention also provides a method for determining the content of multiple indicators in a kidney-tonifying and liver-nourishing compound, which can simultaneously determine the content of five indicator components (including mononoside, loganin, verbascoside, icariin, and osthol). This method plays an important role in effectively controlling the batch-to-batch stability and efficacy of the product, and lays the foundation for improving the quality standards of the kidney-tonifying and liver-nourishing compound. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The superimposed chromatograms and control chromatograms of 10 batches of the Kidney-Nourishing and Liver-Brewing Compound in Example 1 are shown.

[0029] Figure 2 The chromatogram is of the mixed reference solution in Example 1, where 3 is mononoside, 5 is loganin, 7 is romaine isoflavone glucoside, 12 is icariin, and 15 is osthol.

[0030] Figure 3 The chromatograms are of the mixed reference solution, test solution, and blank solution in Example 2; where A is the mixed reference solution, B is the test solution, and C is the blank solution. Detailed Implementation

[0031] This invention provides a method for establishing the HPLC fingerprint of a kidney-tonifying and liver-nourishing compound, comprising the following steps:

[0032] The kidney-tonifying and liver-nourishing compound was mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution;

[0033] A mixed reference solution is provided, wherein the references in the mixed reference solution include mononoside, loganin, verbascoside, icariin and osthol;

[0034] The test solution and the mixed reference solution were analyzed by HPLC. The obtained chromatograms were imported into fingerprinting software to identify common peaks and obtain the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound.

[0035] The conditions for the HPLC detection include:

[0036] Chromatographic column: C18 column; Mobile phase: Phase A is 0.05–0.2% (v / v) aqueous phosphoric acid solution, Phase B is acetonitrile; Gradient elution, elution program: 0–10 min, Phase B volume fraction increases from 5% to 10%; 10–30 min, Phase B volume fraction increases from 10% to 18%; 30–50 min, Phase B volume fraction increases from 18% to 30%; 50–70 min, Phase B volume fraction increases from 30% to 50%; 70–85 min, Phase B volume fraction increases from 50% to 100%; Flow rate: 0.6–1 mL / min; Column temperature: 25–35℃; Detection wavelength: 240–260 nm.

[0037] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0038] This invention involves mixing a kidney-tonifying and liver-nourishing compound with a methanol-water solution and then subjecting the mixture to ultrasonic treatment to obtain a test solution.

[0039] In this invention, the kidney-tonifying and liver-nourishing compound is derived from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine.

[0040] In this invention, the volume fraction of methanol in the methanol aqueous solution is preferably 40% to 60%, more preferably 50%.

[0041] In this invention, the volume ratio of the kidney-tonifying and liver-nourishing compound to the methanol aqueous solution is preferably 1:4 to 6, more preferably 1:5.

[0042] In this invention, the ultrasonic treatment time is preferably 0.5 to 1.5 hours, more preferably 1 hour, the power is preferably 250 to 350 W, more preferably 300 W, and the frequency is preferably 35 to 45 kHz, more preferably 40 kHz.

[0043] The present invention provides a mixed reference solution, wherein the reference standards in the mixed reference solution include monoglobulin, loganin, verbascoside, icariin and osthol.

[0044] In this invention, the concentrations of mononoside, loganin, verbascoside, icariin, and osthol in the mixed reference solution are preferably 50 μg / mL.

[0045] The present invention performs HPLC detection on the test solution and the mixed reference solution, imports the obtained chromatogram into fingerprinting software, identifies common peaks, and obtains the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound.

[0046] The conditions for the HPLC detection include:

[0047] Chromatographic column: C18 column; Mobile phase: Phase A is 0.05–0.2% (v / v) aqueous phosphoric acid solution, Phase B is acetonitrile; Gradient elution, elution program: 0–10 min, Phase B volume fraction increases from 5% to 10%; 10–30 min, Phase B volume fraction increases from 10% to 18%; 30–50 min, Phase B volume fraction increases from 18% to 30%; 50–70 min, Phase B volume fraction increases from 30% to 50%; 70–85 min, Phase B volume fraction increases from 50% to 100%; Flow rate: 0.6–1 mL / min; Column temperature: 25–35℃; Detection wavelength: 240–260 nm.

[0048] In this invention, the chromatographic column is preferably a ZORBAX Eclipse Plus C18 column, with a preferred specification of 4.6 mm × 250 mm and 5 μm.

[0049] In this invention, phase A is preferably a phosphoric acid aqueous solution with a volume fraction of 0.1%.

[0050] In this invention, the flow rate is preferably 0.8 mL / min.

[0051] In this invention, the column temperature is preferably 30°C.

[0052] In this invention, the injection volume for HPLC detection is preferably 5 to 20 μL, more preferably 10 μL.

[0053] In this invention, the detection wavelength is preferably 250 nm.

[0054] In this invention, the fingerprinting software is a "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," preferably version 130723, published by the National Pharmacopoeia Commission in 2012. The process of importing the fingerprinting software includes: after determining the reference chromatogram, generating a control chromatogram using the averaging method, employing multi-point correction Mark peak matching, and establishing superimposed chromatograms and control chromatograms for multiple batches of the kidney-tonifying and liver-nourishing compound.

[0055] In this invention, the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound includes 15 common peaks, with the following relative retention times to peak 7: peak 1: 0.22, peak 2: 0.46, peak 3: 0.51, peak 4: 0.65, peak 5: 0.71, peak 6: 0.72, peak 7: 1.00, peak 8: 1.04, peak 9: 1.22, peak 10: 1.36–1.37, peak 11: 1.52–1.53, peak 12: 1.56, peak 13: 1.77, peak 14: 2.14–2.15, peak 15: 2.27–2.28; among which peak 3 is mononoside, peak 5 is loganin, peak 7 is verrucoside glucoside, peak 12 is icariin, and peak 15 is osthol. The fingerprint established by this invention has high similarity.

[0056] This invention also provides a method for determining the content of multiple indicators in a kidney-tonifying and liver-nourishing compound, wherein the multiple indicators include two or more of mononoside, loganin, verbascoside, icariin, and osthol, and the determination method includes the following steps:

[0057] The test kidney-tonifying and liver-nourishing compound was mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution.

[0058] The test solution was subjected to HPLC analysis, and the content of multiple index components in the kidney-tonifying and liver-nourishing compound was obtained based on the obtained chromatographic peak area and the predetermined standard curve; the predetermined standard curve is a linear curve between the concentration of each of the multiple index components and the corresponding peak area.

[0059] The conditions for the HPLC detection include:

[0060] Chromatographic column: C18 column; Mobile phase: Phase A is 0.05–0.2% (v / v) aqueous phosphoric acid solution, Phase B is acetonitrile; Gradient elution, elution program: 0–10 min, Phase B volume fraction increases from 5% to 10%; 10–30 min, Phase B volume fraction increases from 10% to 18%; 30–50 min, Phase B volume fraction increases from 18% to 30%; 50–70 min, Phase B volume fraction increases from 30% to 50%; 70–85 min, Phase B volume fraction increases from 50% to 100%; Flow rate: 0.6–1 mL / min; Column temperature: 25–35℃; Detection wavelength: 240–330 nm.

[0061] In this invention, the preparation of the test solution is the same as that in the method for establishing the fingerprint spectrum, and will not be described again here.

[0062] In this invention, except for the detection wavelength, the conditions for HPLC detection are the same as those in the fingerprint spectrum establishment method, and will not be repeated here.

[0063] In this invention, the detection wavelength of mononoside and loganin is preferably 240 nm, the detection wavelength of verrucoside is preferably 260 nm, the detection wavelength of icariin is preferably 270 nm, and the detection wavelength of osthol is preferably 322 nm.

[0064] In this invention, the linear range of the predetermined standard curve is preferably 15.6–1000 μg / mL for mononoside, 9.4–600 μg / mL for loganin, 1.9–120 μg / mL for verbascoside, 2.3–150 μg / mL for icariin, and 0.3–20 μg / mL for osthol.

[0065] The multi-index component content determination method provided by this invention has good linearity, good precision, good repeatability, good stability, and the recovery rate meets the requirements.

[0066] To further illustrate the present invention, the method for establishing the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound and the method for determining the content of multiple components provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0067] In this embodiment of the invention, the instruments and materials used include:

[0068] 1. Instruments: High-performance liquid chromatograph (Agilent Technologies, Inc., USA, Agilent 1260); G3KT18273 vortex mixer (Thermo Fisher Scientific); AS60 / 220.R2 0.0001 ppm balance (Radiwag, Poland); IQ7005 ultrapure water preparation system (Millipore); CTUW-X15-400 ultrasonic cleaner (Qingdao Shuihe Chuangxin Electronic Technology Co., Ltd.).

[0069] 2. Materials: Methanol, acetonitrile (chromatographic grade, Fisher Scientific, USA); formic acid (ROE, USA); phosphoric acid (Thermo Fisher Scientific (China) Co., Ltd.); standard reference standards: monoglobulin, 111998-2022050, purity 97.8%; loganin, 11640-202309, purity 99.2%; verrucoside, 11920-202308, purity 96.9%; icariin, 110737-202017, purity 98.1%; osthol, 110822-202412, purity 99.9%; all standard reference standards were purchased from the China National Institutes for Food and Drug Control. Ultrapure water was prepared using a Milli-Q ultrapure water preparation system. The batch number information for the Kidney-Nourishing and Liver-Brightening Compound is shown in Table 1, all originating from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine.

[0070] Table 1 Sample Batch Number Information

[0071] S1 230601 S6 240702 S2 230602 S7 240901 S3 230603 S8 240902 S4 231201 S9 240401 S5 240701 S10 240402

[0072] Example 1: Establishment of fingerprint map

[0073] 1. Chromatographic conditions

[0074] Chromatographic column: ZORBAX Eclipse Plus C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase: Phase A was 0.1% (v / v) aqueous phosphoric acid solution, and Phase B was acetonitrile; Gradient elution, elution gradient: 0–10 min, 5%–10% B; 10–30 min, 10%–18% B; 30–50 min, 18%–30% B; 50–70 min, 30%–50% B; 70–85 min, 50%–100% B; Flow rate: 0.8 mL / min; Column temperature: 30℃; Injection volume: 10.0 μL; Wavelength: 250 nm.

[0075] 2. Prepare the reference standard stock solution

[0076] Five standard reference standards were accurately weighed using a balance with a volume fraction of 0.0001. The standard reference standards were dissolved in a 50% methanol aqueous solution to prepare a 10 mg / mL solution, which was then stored at 4°C.

[0077] 3. Preparation of the test solution

[0078] Accurately measure 2 mL of the kidney-tonifying and liver-nourishing compound sample and place it in a 10 mL volumetric flask. Add 50% methanol aqueous solution to dilute to the mark of the volumetric flask, sonicate for 1 h (300 W, 40 kHz), cool, make up the weight loss, shake well, and filter through a microporous membrane (0.22 μm) to obtain the test solution.

[0079] 4. Establishment and similarity evaluation of HPLC fingerprint of the kidney-tonifying and liver-nourishing compound.

[0080] Samples of Yishen Yanggan Compound (S1-S10) were taken and test solutions were prepared according to method 3. The samples were then analyzed under the chromatographic conditions described in method 1. The chromatographic data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 130723, National Pharmacopoeia Commission, 2012)". Using chromatogram S7 as the reference chromatogram, a control chromatogram was generated using the mean method. Multi-point correction Mark peak matching was employed to establish superimposed chromatograms and control chromatograms (R) for 10 batches of Yishen Yanggan Compound. (See attached table). Figure 1 A total of 15 common peaks were identified, and their values ​​were compared with those of a mixed standard solution with a concentration of 50 μg / mL. Figure 2The comparison identified five components: peak 3 (monoside), peak 5 (loganin), peak 7 (verrucaside), peak 12 (icariin), and peak 15 (ostrichin). The similarity analysis results of the 10 batches of the kidney-tonifying and liver-nourishing compound are shown in Table 2. All similarity scores were above 0.995, indicating a high degree of similarity. This preliminarily suggests that the chemical composition of the 10 batches of samples is highly consistent, and their quality is uniform and stable.

[0081] Table 210 Similarity Analysis of Kidney-Nourishing and Liver-Breeding Compound

[0082] S1 1 S6 0.995 S2 1 S7 0.995 S3 1 S8 0.996 S4 0.999 S9 0.998 S5 0.995 S10 0.998

[0083] 1) Precision test

[0084] Take the 7th batch of Yishen Yanggan Mixture, prepare the test solution, and inject it 6 times consecutively. Use the chromatographic peak of the 7th peak with the higher peak area (S) as the reference peak and calculate the relative standard deviation (RSD) of the relative retention time of each common peak. Rt (See Table 3, RSD) Rt The results were all less than 0.74%, and the relative standard deviation (RSD) of the relative peak area was less than 0.74%. S (See Table 4, RSD) S The results were all less than 4.66%, indicating that the instrument had good precision.

[0085] Table 3. Relative retention time precision results of the fingerprint chromatogram of Yishen Yanggan Compound (n=6)

[0086] 1 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.74 2 0.46 0.46 0.46 0.47 0.47 0.46 0.46 0.66 3 0.51 0.51 0.51 0.52 0.52 0.51 0.51 0.58 4 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.31 5 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.17 6 0.72 0.72 0.72 0.72 0.73 0.73 0.72 0.21 7 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 8 1.04 1.04 1.03 1.05 1.05 1.05 1.04 0.65 9 1.22 1.22 1.22 1.22 1.22 1.22 1.22 0.09 10 1.36 1.37 1.36 1.37 1.37 1.36 1.37 0.17 11 1.53 1.53 1.53 1.52 1.52 1.51 1.52 0.30 12 1.56 1.56 1.56 1.56 1.56 1.55 1.56 0.31 13 1.77 1.77 1.77 1.78 1.78 1.77 1.77 0.27 14 2.14 2.15 2.14 2.15 2.15 2.15 2.15 0.26 15 2.27 2.28 2.27 2.29 2.29 2.29 2.28 0.39

[0087] Table 4. Relative peak area precision results of the fingerprint spectrum of Yishen Yanggan Compound (n=6)

[0088]

[0089]

[0090] 2) Repeatability test

[0091] Six parallel test solutions were prepared from the 7th batch of Yishen Yanggan Compound, and the solutions were injected for determination. The chromatographic peak of verbascoside (S) was used as the reference peak. The results are shown in Tables 5 and 6. RSD Rt Less than 1.26%, RSD S The result of less than 4.75% indicates that the method has good repeatability.

[0092] Table 5. Relative retention time repeatability results of fingerprint chromatograms of Yishen Yanggan Compound (n=6)

[0093]

[0094]

[0095] Table 6. Repeatability results of relative peak areas in the fingerprint chromatogram of Yishen Yanggan Compound (n=6)

[0096] 1 0.97 0.96 1.00 1.00 0.97 0.94 0.98 2.47 2 0.58 0.61 0.64 0.67 0.61 0.61 0.62 4.75 3 5.86 5.86 6.08 6.08 5.93 5.94 5.96 1.70 4 0.20 0.20 0.21 0.21 0.21 0.20 0.20 3.65 5 2.14 2.14 2.19 2.23 2.16 2.16 2.17 1.63 6 0.36 0.36 0.36 0.37 0.36 0.36 0.36 1.03 7 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 8 0.02 0.02 0.02 0.03 0.02 0.02 0.02 4.33 9 0.64 0.62 0.65 0.66 0.63 0.66 0.64 2.33 10 0.59 0.58 0.62 0.62 0.60 0.59 0.60 2.82 11 0.73 0.75 0.76 0.76 0.74 0.74 0.75 1.62 12 0.62 0.64 0.66 0.66 0.64 0.64 0.64 2.11 13 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.82 14 0.09 0.09 0.10 0.10 0.09 0.09 0.09 1.71 15 0.04 0.04 0.04 0.04 0.04 0.04 0.04 3.07

[0097] 3) Stability test

[0098] The 7th batch of Yishen Yanggan Compound was used to prepare a test solution. The solution was injected at 0, 2, 4, 8, 12, and 24 hours, with the chromatographic peak of the 7th peak (S) of verbascoside isoflavone. The results are shown in Tables 7 and 8. RSD Rt Less than 0.96%, RSD S A value less than 3.88% indicates that the test solution has good stability within 24 hours.

[0099] Table 7. Relative retention time stability results of the fingerprint chromatograms of the Kidney-Nourishing and Liver-Brightening Compound (n=6)

[0100]

[0101]

[0102] Table 8. Stability results of relative peak area in the fingerprint chromatogram of the Kidney-Nourishing and Liver-Brightening Compound (n=6)

[0103] 1 0.90 0.94 0.89 0.90 0.94 0.86 0.91 3.51 2 0.63 0.61 0.62 0.60 0.63 0.57 0.61 3.88 3 5.88 5.86 5.96 5.86 5.96 5.85 5.90 0.82 4 0.25 0.25 0.25 0.26 0.25 0.25 0.25 1.05 5 2.14 2.14 2.18 2.14 2.18 2.14 2.15 0.91 6 0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.59 7 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 8 0.02 0.02 0.02 0.03 0.02 0.02 0.02 3.83 9 0.64 0.63 0.63 0.63 0.63 0.64 0.63 1.16 10 0.58 0.60 0.61 0.58 0.61 0.58 0.59 2.13 11 0.73 0.73 0.74 0.77 0.74 0.73 0.74 1.84 12 0.63 0.63 0.64 0.65 0.64 0.63 0.64 1.84 13 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.78 14 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.77 15 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.90

[0104] Example 2 Content Determination

[0105] 1. Chromatographic conditions

[0106] Chromatographic column: ZORBAX Eclipse Plus C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase: Phase A was 0.1% (v / v) phosphoric acid aqueous solution, and Phase B was acetonitrile; Gradient elution, elution gradient: 0–10 min, 5%–10% B; 10–30 min, 10%–18% B; 30–50 min, 18%–30% B; 50–70 min, 30%–50% B; 70–85 min, 50%–100% B; Flow rate: 0.8 mL / min; Column temperature: 30℃; Injection volume: 10.0 μL; Wavelengths: 240 nm (monoside, loganin), 260 nm (verrucaside), 270 nm (icariin), 322 nm (ostrichin).

[0107] The preparation methods for the reference stock solution and the test solution are the same as in Example 1.

[0108] 2. Methodological Examination

[0109] 1) Specificity examination

[0110] The chromatograms of the mixed reference solution (each reference standard concentration was 50 μg / mL), the test solution, and the blank solution were compared. The results are shown below. Figure 3 In the chromatographic solution, A is a mixed reference solution, B is the test solution, and C is a blank solution (1. mononoside; 2. loganin; 3. verbascoside; 4. icariin; 5. osthol). It can be seen that the peaks of each analyte are well separated, and the solvent has no effect on the chromatographic peaks, indicating that the method has good specificity.

[0111] 2) Examination of linear relationships

[0112] Accurately measure each reference standard stock solution and dilute it with 50% (v / v) methanol aqueous solution to obtain a mixed reference standard solution with a concentration of 1000 μg / mL for mononoside, 600 μg / mL for loganin, 120 μg / mL for verrucoside, 150 μg / mL for icariin, and 20 μg / mL for osthol. This solution is then serially diluted with 50% (v / v) methanol aqueous solution by 2, 2, 2, 2, 2, and 2 times to obtain the mixed reference standard solutions of each concentration in the standard curve. Inject each of the above solutions for analysis, record the corresponding peak areas, and plot the content determination standard curve with peak area as the ordinate (Y) and mass concentration as the abscissa (X). Calculate the linear regression equation; the results are shown in Table 9. The limit of quantitation (LLOQ) is the reference standard concentration corresponding to a signal-to-noise ratio (S / N) of 10; the results are shown in Table 9.

[0113] Table 9. Results of linear relationship analysis of five components in the Kidney-Nourishing and Liver-Brewing Compound.

[0114]

[0115] 3) Precision test

[0116] The 7th batch of Yishen Yanggan Compound was used to prepare test solutions, which were injected six times consecutively. The results are shown in Table 10. The calculated RSD of the peak areas of the five analytes was less than 0.70%, indicating good intra-day precision of the instrument. Injections were repeated twice daily for three consecutive days. The results are shown in Table 11. The RSD of the peak areas of the five analytes was less than 3.13%, indicating good inter-day precision of the instrument.

[0117] In Tables 10 to 15 below, C1: mononoside, C2: loganin, C3: verbascoside, C4: icariin, and C5: osthol.

[0118] Table 10 Intra-day precision results of five test components in the Kidney-Nourishing and Liver-Brightening Compound (n=6)

[0119] C1 8190.80 8220.70 8219.50 8276.30 8320.80 8330.20 8259.72 0.70 C2 3525.80 3527.50 3544.90 3549.40 3559.20 3567.80 3545.77 0.47 C3 1091.10 1093.80 1096.80 1098.60 1102.90 1103.60 1097.80 0.45 C4 1238.70 1237.90 1236.20 1249.80 1253.00 1255.30 1245.15 0.68 C5 162.30 161.50 162.10 161.80 161.70 162.60 162.00 0.25

[0120] Table 11. Daytime precision results of five tested components in the Kidney-Nourishing and Liver-Brightening Compound (n=6)

[0121] C1 8359.00 8371.00 8389.20 8463.60 8385.10 8786.00 8458.98 1.94 C2 3576.40 3588.20 3593.80 3614.10 3628.20 3813.30 3635.67 2.45 C3 1103.20 1109.20 1114.80 1127.20 1121.70 1176.70 1125.47 2.36 C4 1261.30 1260.40 1270.10 1278.90 1276.10 1334.40 1280.20 2.16 C5 164.60 162.90 153.50 153.90 154.00 159.20 158.02 3.13

[0122] 4) Repeatability test

[0123] Six test solutions were prepared in parallel from the 7th batch of Yishen Yanggan Compound, and the results were shown in Table 12. The RSD of the contents of the five test components were all less than 1.40%, indicating that the method has good repeatability.

[0124] Table 12. Repeatability results of five tested components in the Kidney-Nourishing and Liver-Brightening Compound (mg / g, n=6)

[0125] C1 1.88 1.92 1.93 1.95 1.94 1.94 1.93 1.17 C2 1.10 1.12 1.13 1.14 1.14 1.15 1.13 1.40 C3 0.15 0.15 0.15 0.15 0.15 0.15 0.15 1.34 C4 0.24 0.25 0.25 0.25 0.25 0.25 0.25 1.20 C5 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.63

[0126] 5) Stability test

[0127] The 7th batch of Yishen Yanggan Compound was used to prepare test solutions. The solutions were injected at 0, 2, 4, 8, 12 and 24 h. The results are shown in Table 13. The peak area RSD of the five test components was less than 4.83%, indicating that the test solutions had good stability within 24 h.

[0128] Table 13. Stability results of five tested components in the Kidney-Nourishing and Liver-Brightening Compound (n=6)

[0129] C1 8190.80 8219.50 8359.00 8463.60 8443.30 8536.50 8368.78 1.66 C2 3525.80 3544.90 3576.40 3614.10 3611.50 3622.90 3582.60 1.13 C3 1091.10 1096.80 1103.20 1127.20 1123.30 1161.90 1117.25 2.34 C4 1238.70 1236.20 1261.30 1278.90 1123.30 1287.70 1237.68 4.83 C5 162.30 162.10 164.60 153.90 153.30 153.80 158.33 3.28

[0130] 6) Recovery rate experiment

[0131] Six portions (2 mL each) of the 7th batch of Yishen Yanggan Mixture were accurately measured. Appropriate amounts of each reference standard were added to each portion, and six parallel test solutions were prepared. The solutions were injected and analyzed, and the peak areas were recorded. The average recoveries of monoglobulin, loganin, verrucoside, icariin, and osthol were calculated. The results are shown in Table 14, and were 105.53%, 113.62%, 89.08%, 90.78%, and 96.17%, respectively. The RSD values ​​were all less than 2.82%, indicating that the recovery rate of this method met the requirements.

[0132] Table 14. Recovery rates of five test components in the Kidney-Nourishing and Liver-Brightening Compound (n=6)

[0133]

[0134] 7) Content determination

[0135] Test solutions were prepared from each batch of Yishen Yanggan Compound, and the samples were injected for analysis. The content of each component was calculated according to the external standard method. The results are shown in Table 15.

[0136] Table 15. Results of content determination of different batches of Yishen Yanggan Mixture

[0137]

[0138]

[0139] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for establishing an HPLC fingerprint of a kidney-tonifying and liver-nourishing compound, characterized by comprising the following steps: The kidney-tonifying and liver-nourishing compound was mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution. A mixed reference solution is provided, wherein the reference standards in the mixed reference solution include mononoside, loganin, verbascoside, icariin and osthol; The test solution and the mixed reference solution were analyzed by HPLC. The obtained chromatograms were imported into fingerprinting software to identify common peaks and obtain the HPLC fingerprint of the kidney-tonifying and liver-nourishing compound. The conditions for the HPLC detection include: Chromatographic column: C18 column; Mobile phase: Phase A is 0.05-0.2% (v / v) aqueous phosphoric acid solution, Phase B is acetonitrile; Gradient elution, elution program: 0-10 min, Phase B volume fraction increases from 5% to 10%; 10-30 min, Phase B volume fraction increases from 10% to 18%; 30-50 min, Phase B volume fraction increases from 18% to 30%; 50-70 min, Phase B volume fraction increases from 30% to 50%; 70-85 min, Phase B volume fraction increases from 50% to 100%; Flow rate: 0.6-1 mL / min; Column temperature: 25-35℃; Detection wavelength: 240-260 nm; Phase A is a 0.1% (v / v) aqueous solution of phosphoric acid; the flow rate is 0.8 mL / min; and the column temperature is 30 °C. The HPLC fingerprint of the kidney-tonifying and liver-nourishing compound includes 15 common peaks, with the following relative retention times to peak 7: Peak 1: 0.22, Peak 2: 0.46, Peak 3: 0.51, Peak 4: 0.65, Peak 5: 0.71, Peak 6: 0.72, Peak 7: 1.00, Peak 8: 1.04, Peak 9: 1.22, Peak 10: 1.36~1.37, Peak 11: 1.52~1.53, Peak 12: 1.56, Peak 13: 1.77, peak 14: 2.14~2.15, peak 15: 2.27~2.28; among which peak 3 is mononoside, peak 5 is loganin, peak 7 is romaine isoflavone glucoside, peak 12 is icariin, and peak 15 is osthol.

2. The method for establishing according to claim 1, characterized in that, The volume fraction of methanol in the methanol-water solution is 40% to 60%.

3. The method for establishing according to claim 2, characterized in that, The volume ratio of the kidney-tonifying and liver-nourishing compound to the methanol aqueous solution is 1:4~6.

4. The method for establishing according to claim 1, characterized in that, The chromatographic column used was a ZORBAX Eclipse Plus C18 column.

5. The method for establishing according to claim 1, characterized in that, The detection wavelength is 250nm.

6. The method for establishing according to claim 1, characterized in that, The injection volume for HPLC detection is 5~20 μL.

7. A method for determining the content of multiple indicators in a kidney-tonifying and liver-nourishing compound, characterized in that, The multi-index components include two or more of mononoside, loganin, verbascoside, icariin, and osthol, and the determination method includes the following steps: The test solution was obtained by mixing the kidney-tonifying and liver-nourishing compound with a methanol-water solution and then ultrasonically treating it. The test solution was subjected to HPLC analysis, and the content of multiple index components in the kidney-tonifying and liver-nourishing compound was obtained based on the obtained chromatographic peak area and the predetermined standard curve; the predetermined standard curve is a linear curve between the concentration of each of the multiple index components and the corresponding peak area. The conditions for the HPLC detection include: Chromatographic column: C18 column; Mobile phase: Phase A is 0.05-0.2% (v / v) aqueous phosphoric acid solution, Phase B is acetonitrile; Gradient elution, elution program: 0-10 min, Phase B volume fraction increases from 5% to 10%; 10-30 min, Phase B volume fraction increases from 10% to 18%; 30-50 min, Phase B volume fraction increases from 18% to 30%; 50-70 min, Phase B volume fraction increases from 30% to 50%; 70-85 min, Phase B volume fraction increases from 50% to 100%; Flow rate: 0.6-1 mL / min; Column temperature: 25-35℃; Detection wavelength: 240-330 nm; Phase A is a 0.1% (v / v) aqueous solution of phosphoric acid, the flow rate is 0.8 mL / min, and the column temperature is 30 °C.

8. The method for determining the content of multiple components according to claim 7, characterized in that, The detection wavelengths for mononoside and loganin are 240 nm, for verbascoside isoflavone glucoside is 260 nm, for icariin is 270 nm, and for osthol is 322 nm.

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