Establishment method of HPLC (High Performance Liquid Chromatography) fingerprint spectrum of kidney-tonifying and liver-nourishing mixture and multi-index component content determination method

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to an establishment method of an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of a kidney-tonifying and liver-nourishing mixture and a multi-index component content determination method. The establishment method of the fingerprint spectrum comprises the following steps: mixing the kidney-tonifying and liver-nourishing mixture and a methanol aqueous solution, carrying out ultrasonic treatment, carrying out HPLC detection on an obtained test solution and a mixed reference substance solution, and introducing an obtained chromatogram into fingerprint spectrum software for treatment, so as to obtain the HPLC fingerprint spectrum of the kidney-tonifying and liver-nourishing mixture. The pretreatment method disclosed by the invention is simple, the characteristic components are completely reserved, and the high performance liquid chromatography is adopted, so that the precision is high, the reproducibility is good, and the stability is good; according to the fingerprint spectrum obtained by the invention, 15 common peaks are determined, the quality of the kidney-tonifying and liver-nourishing mixture can be effectively monitored, and a guarantee is provided for comprehensive and effective control of the quality of the kidney-tonifying and liver-nourishing mixture.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control of traditional Chinese medicines, and specifically relates to a method for establishing an HPLC fingerprint spectrum of a kidney-tonifying and liver-nourishing mixture and a method for determining the content of multiple index components. Background Art

[0002] The Yishen Yanggan mixture is composed of many Chinese medicinal herbs such as Astragalus, Cornus officinalis, Epimedium, Cnidium monnieri, etc. It has the effects of invigorating Qi and strengthening the spleen, nourishing the kidney and liver, and strengthening tendons and bones. It is clinically used for various diseases caused by liver and kidney deficiency, blood deficiency and essence deficiency, and chronic strain, with symptoms such as dizziness, blurred vision, fatigue, swelling and pain in the waist, back and limbs, dry mouth and eyes, soreness and weakness in limbs, irregular menstruation, inability to hold weight or hemiplegia, etc. It has significant efficacy and no adverse reactions.

[0003] At present, dual-wavelength thin-layer scanning is mainly used to determine the content of astragaloside Ⅳ, the main active ingredient in Yishen Yanggan mixture. However, due to the complex composition of traditional Chinese medicine preparations, the content determination of a single indicator component cannot fully guarantee the quality of Yishen Yanggan mixture. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for establishing an HPLC fingerprint spectrum of Yishen Yanggan mixture and a method for determining the content of multiple index components. The present invention uses HPLC to detect Yishen Yanggan mixture to obtain a fingerprint spectrum for quality evaluation and control, providing a guarantee for the comprehensive and effective control of the quality of Yishen Yanggan mixture.

[0005] The present invention provides a method for establishing an HPLC fingerprint of a Yishen Yanggan mixture, comprising the following steps:

[0006] The Yishen Yanggan mixture and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution;

[0007] Providing a mixed reference solution, wherein the reference substances in the mixed reference solution include morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole;

[0008] The test solution and the mixed reference solution are subjected to HPLC detection, and the obtained chromatogram is imported into the fingerprint software to identify the common peaks to obtain the HPLC fingerprint of Yishen Yanggan mixture;

[0009] The conditions for the HPLC detection include:

[0010] Chromatographic column: C18 column; mobile phase: phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-10 min, the volume fraction of phase B increases from 5% to 10%; 10-30 min, the volume fraction of phase B increases from 10% to 18%; 30-50 min, the volume fraction of phase B increases from 18% to 30%; 50-70 min, the volume fraction of phase B increases from 30% to 50%; 70-85 min, the volume fraction of phase B increases from 50% to 100%; flow rate: 0.6-1 mL / min; column temperature: 25-35° C.; detection wavelength 240-260 nm.

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

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

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

[0014] Preferably, the phase A is a phosphoric acid aqueous solution with a volume fraction of 0.1%, 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 to 20 μL.

[0017] Preferably, the HPLC fingerprint of the Yishen Yanggan mixture 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 morroniside, peak 5 is loganin, peak 7 is calycosin glucoside, peak 12 is icariin, and peak 15 is osthole.

[0018] The present invention also provides a method for determining the content of multiple index components in a kidney-tonifying and liver-nourishing mixture, wherein the multiple index components include two or more of morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole, and the determination method comprises the following steps:

[0019] The Yishen Yanggan mixture to be tested and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution;

[0020] The test solution is subjected to HPLC detection, and the content of multiple index components in the Yishen Yanggan mixture is obtained according to the obtained chromatographic peak area and a 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 a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-10 min, the volume fraction of phase B increases from 5% to 10%; 10-30 min, the volume fraction of phase B increases from 10% to 18%; 30-50 min, the volume fraction of phase B increases from 18% to 30%; 50-70 min, the volume fraction of phase B increases from 30% to 50%; 70-85 min, the volume fraction of phase B increases from 50% to 100%; flow rate: 0.6-1 mL / min; column temperature: 25-35° C.; detection wavelength 240-330 nm.

[0023] Preferably, the detection wavelength of morroniside and loganin is 240 nm, the detection wavelength of calycosin glucoside is 260 nm, the detection wavelength of icariin is 270 nm, and the detection wavelength of osthole is 322 nm.

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

[0025] The present invention provides a method for establishing an HPLC fingerprint of a Yishen Yanggan mixture, comprising the following steps: mixing the Yishen Yanggan mixture and a methanol aqueous solution for ultrasonic treatment to obtain a test solution; providing a mixed reference solution, wherein the reference substances in the mixed reference solution include morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole; performing HPLC detection on the test solution and the mixed reference solution, importing the obtained chromatogram into a fingerprint software, identifying common peaks, and obtaining an HPLC fingerprint of the Yishen Yanggan mixture. The pretreatment method of the present invention is simple, and the characteristic components are retained intact. The present invention adopts high performance liquid chromatography (HPLC), which has high precision, good reproducibility and good stability; the fingerprint obtained by the present invention determines 15 common peaks in total, and can effectively monitor the quality of the Yishen Yanggan mixture. The present invention adopts HPLC to detect the Yishen Yanggan mixture to obtain a fingerprint for quality evaluation and control, providing a guarantee for the comprehensive and effective control of the quality of the Yishen Yanggan mixture.

[0026] The present invention also provides a method for determining the content of multiple index components in the Yishen Yanggan mixture, which can simultaneously determine the contents of five index components (including morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole), plays an important role in effectively controlling the stability and effectiveness of product batches, and lays a foundation for improving the quality standard of the Yishen Yanggan mixture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The superimposed spectra and the reference spectra of 10 batches of Yishen Yanggan mixture in Example 1;

[0029] Figure 2 is a chromatogram of the mixed reference solution in Example 1, wherein 3 is morroniside, 5 is loganin, 7 is calycosin isoflavone glucoside, 12 is icariin, and 15 is osthole;

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

[0031] The present invention provides a method for establishing an HPLC fingerprint of a Yishen Yanggan mixture, comprising the following steps:

[0032] The Yishen Yanggan mixture and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution;

[0033] Providing a mixed reference solution, wherein the reference substances in the mixed reference solution include morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole;

[0034] The test solution and the mixed reference solution are subjected to HPLC detection, and the obtained chromatogram is imported into the fingerprint software to identify the common peaks to obtain the HPLC fingerprint of Yishen Yanggan mixture;

[0035] The conditions of the HPLC detection include:

[0036] Chromatographic column: C18 column; mobile phase: phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-10 min, the volume fraction of phase B increases from 5% to 10%; 10-30 min, the volume fraction of phase B increases from 10% to 18%; 30-50 min, the volume fraction of phase B increases from 18% to 30%; 50-70 min, the volume fraction of phase B increases from 30% to 50%; 70-85 min, the volume fraction of phase B increases from 50% to 100%; flow rate: 0.6-1 mL / min; column temperature: 25-35° C.; detection wavelength 240-260 nm.

[0037] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0038] The invention mixes the kidney-tonifying and liver-nourishing mixture with a methanol aqueous solution and performs ultrasonic treatment to obtain a test solution.

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

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

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

[0042] In the present invention, the ultrasonic treatment time is preferably 0.5 to 1.5 h, more preferably 1 h, 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 invention provides a mixed reference substance solution, wherein the reference substances in the mixed reference substance solution include morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole.

[0044] In the present invention, the concentrations of morroniside, loganin, calycosin glucoside, icariin and osthole 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 fingerprint software, identifies the common peaks, and obtains the HPLC fingerprint of Yishen Yanggan mixture;

[0046] The conditions for the HPLC detection include:

[0047] Chromatographic column: C18 column; mobile phase: phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-10 min, the volume fraction of phase B increases from 5% to 10%; 10-30 min, the volume fraction of phase B increases from 10% to 18%; 30-50 min, the volume fraction of phase B increases from 18% to 30%; 50-70 min, the volume fraction of phase B increases from 30% to 50%; 70-85 min, the volume fraction of phase B increases from 50% to 100%; flow rate: 0.6-1 mL / min; column temperature: 25-35° C.; detection wavelength 240-260 nm.

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

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

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

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

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

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

[0054] In the present invention, the fingerprint software is a "Chinese medicine chromatographic fingerprint similarity evaluation system", and the "Chinese medicine chromatographic fingerprint similarity evaluation system" is preferably version 130723, State Pharmacopoeia Committee, 2012. The introduction of the fingerprint software includes: after determining the reference spectrum, generating a reference spectrum by the average method, using multi-point correction Mark peak matching, and establishing superimposed spectra and reference spectra of multiple batches of Yishen Yanggan mixture.

[0055] In the present invention, the HPLC fingerprint of the Yishen Yanggan mixture includes 15 common peaks, and the relative retention time with peak 7 is 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 morroniside, peak 5 is loganin, peak 7 is calycosin isoflavone glucoside, peak 12 is icariin, and peak 15 is osthole. The fingerprint established by the present invention has high similarity.

[0056] The present invention also provides a method for determining the content of multiple index components in a kidney-tonifying and liver-nourishing mixture, wherein the multiple index components include two or more of morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole, and the determination method comprises the following steps:

[0057] The Yishen Yanggan mixture to be tested and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution;

[0058] The test solution is subjected to HPLC detection, and the content of multiple index components in the Yishen Yanggan mixture is obtained according to the obtained chromatographic peak area and a 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 of the HPLC detection include:

[0060] Chromatographic column: C18 column; mobile phase: phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-10 min, the volume fraction of phase B increases from 5% to 10%; 10-30 min, the volume fraction of phase B increases from 10% to 18%; 30-50 min, the volume fraction of phase B increases from 18% to 30%; 50-70 min, the volume fraction of phase B increases from 30% to 50%; 70-85 min, the volume fraction of phase B increases from 50% to 100%; flow rate: 0.6-1 mL / min; column temperature: 25-35° C.; detection wavelength 240-330 nm.

[0061] In the present invention, the preparation of the test solution is the same as that in the fingerprint establishment method, which will not be repeated here.

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

[0063] In the present invention, the detection wavelength of morroniside and loganin is preferably 240 nm, the detection wavelength of calycosin isoflavone glucoside is preferably 260 nm, the detection wavelength of icariin is preferably 270 nm, and the detection wavelength of osthole is preferably 322 nm.

[0064] In the present invention, the linear range of the predetermined standard curve is: morroniside is preferably 15.6-1000 μg / mL, loganin is preferably 9.4-600 μg / mL, calycosin is preferably 1.9-120 μg / mL, icariin is preferably 2.3-150 μg / mL, and osthole is preferably 0.3-20 μg / mL.

[0065] The multi-index component content determination method provided by the present invention has good linear relationship, 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 Yishen Yanggan mixture and the method for determining the content of multiple index components provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0067] In the embodiments of the present invention, the instruments and materials used include:

[0068] 1. Instruments: High performance liquid chromatograph (Agilent 1260, USA); G3KT18273 vortex mixer (Thermo Fisher Scientific); AS60 / 220.R2 1 / 100,000 balance (Radwag, Poland); IQ7005 ultrapure water preparation instrument (Millipore); CTUW-X15-400 ultrasonic cleaning machine (Qingdao Shuihe Chuangxin Electronic Technology Co., Ltd.).

[0069] 2. Materials: methanol, acetonitrile (chromatographic grade, Fisher, USA); formic acid (ROE, USA); phosphoric acid (Thermo Fisher Scientific (China) Co., Ltd.); standard reference substances morroniside, 111998-2022050, purity 97.8%; loganin, 11640-202309, purity 99.2%; calycosin glucoside, 11920-202308, purity 96.9%; icariin, 110737-202017, purity 98.1%; osthole, 110822-202412, purity 99.9%; the above standard reference substances were purchased from the China Food and Drug Inspection Institute. Ultrapure water was prepared by Milli-Q ultrapure water preparation instrument. The batch number information of Yishen Yanggan Mixture is shown in Table 1, all of which are from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine.

[0070] Table 1 Sample batch number information table

[0071] serial number batch number serial number batch number 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

[0073] 1. Chromatographic conditions

[0074] Chromatographic column: ZORBAX Eclipse Plus C18 chromatographic column (4.6mm×250mm, 5μm); mobile phase: phase A is 0.1% by volume phosphoric acid aqueous solution, phase B is acetonitrile; gradient elution, elution gradient: 0-10min, 5%-10% B; 10-30min, 10%-18% B; 30-50min, 18%-30% B; 50-70min, 30%-50% B; 70-85min, 50%-100% B; flow rate: 0.8mL / min; column temperature: 30℃; injection volume: 10.0μL; wavelength 250nm.

[0075] 2. Prepare reference stock solution

[0076] Take 5 kinds of standard reference substances respectively, accurately weigh them with a 1 / 100,000 balance, dissolve the weighed standard substances in a 50% by volume methanol aqueous solution, and finally prepare a 10 mg / mL solution, which is stored in a refrigerator at 4°C.

[0077] 3. Prepare the test solution

[0078] Accurately measure 2 mL of Yishen Yanggan mixture sample, place it in a 10 mL volumetric flask, add 50% by volume methanol aqueous solution to the volumetric flask scale line, ultrasonically treat for 1 h (300 W, 40 kHz), cool, make up for weight loss, shake well, and pass through a microporous filter membrane (0.22 μm) to obtain the test solution.

[0079] 4. Establishment and similarity evaluation of HPLC fingerprint of Yishen Yanggan mixture

[0080] Take samples of Yishen Yanggan mixture (S1-S10), prepare the test solution according to the method in 3, and analyze the sample according to the chromatographic conditions in 1. Import the chromatographic data into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (130723 Edition, State Pharmacopoeia Committee, 2012)", use chromatogram S7 as a reference spectrum, generate a reference spectrum by the average method, and use multi-point correction Mark peak matching to establish the superimposed spectrum and reference spectrum (R) of 10 batches of Yishen Yanggan mixture, see Figure 1 A total of 15 common peaks were identified, and the chromatograms of mixed reference solutions with a concentration of 50 μg / mL for each reference substance were compared ( Figure 2) were compared, and 5 components were identified, namely, peak 3 morroniside, peak 5 loganin, peak 7 calycosin isoflavone glucoside, peak 12 icariin and peak 15 osthole. The similarity analysis results of 10 batches of Yishen Yanggan mixture are shown in Table 2. The similarity results are all above 0.995, with a high similarity, which preliminarily shows that the chemical composition of the 10 batches of samples is highly consistent and the quality is uniform and stable.

[0081] Table 2 Similarity analysis of 10 batches of Yishen Yanggan mixture

[0082] serial number Similarity serial number Similarity 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] The 7th batch of Yishen Yanggan mixture was used to prepare the test solution, and the sample was injected 6 times continuously. The chromatographic peak of No. 7 calycosin glucoside with a higher peak area was used as the reference chromatographic peak (S), and the relative standard deviation (RSD) of the relative retention time of each common peak was calculated. Rt ), see Table 3, RSD Rt The results were all less than 0.74%, and the relative standard deviation (RSD 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 Yishen Yanggan mixture fingerprint (n=6)

[0086] Peak 1 2 3 4 5 6 average value RSD(%) 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 Yishen Yanggan mixture fingerprint (n=6)

[0088]

[0089]

[0090] 2) Repeatability test

[0091] Take the 7th batch of Yishen Yanggan mixture, prepare 6 test sample solutions in parallel, inject and measure, take the 7th chromatographic peak of calycosin isoflavone glucoside as the reference peak (S), the results are shown in Tables 5 and 6, RSD Rt Less than 1.26%, RSD S It is less than 4.75%, indicating that the method has good repeatability.

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

[0093]

[0094]

[0095] Table 6 Relative peak area repeatability results of Yishen Yanggan mixture fingerprint (n=6)

[0096] Peak 1 2 3 4 5 6 average value RSD(%) 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 mixture was used to prepare the test solution, and the samples were injected at 0, 2, 4, 8, 12, and 24 h respectively. The chromatographic peak of No. 7 calycosin isoflavone glucoside was used as the reference peak (S). The results are shown in Tables 7 and 8. RSD Rt Less than 0.96%, RSD S Less than 3.88%, indicating that the test solution has good stability within 24 hours.

[0099] Table 7 Relative retention time stability results of Yishen Yanggan mixture fingerprint (n=6)

[0100]

[0101]

[0102] Table 8 Relative peak area stability results of Yishen Yanggan mixture fingerprint (n=6)

[0103] Peak 0h 2h 4h 8h 12h 24h average value RSD(%) 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 chromatographic column (4.6mm×250mm, 5μm); mobile phase: phase A is a 0.1% volume fraction of phosphoric acid aqueous solution, and phase B is acetonitrile; gradient elution, elution gradient: 0-10min, 5%-10% B; 10-30min, 10%-18% B; 30-50min, 18%-30% B; 50-70min, 30%-50% B; 70-85min, 50%-100% B; flow rate: 0.8mL / min; column temperature: 30℃; injection volume: 10.0μL; wavelength 240nm (morroniside, loganin), 260nm (calycosin isoflavone glucoside), 270nm (icariin), 322nm (osthole).

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

[0108] 2. Methodology

[0109] 1) Specificity inspection

[0110] Compare the chromatograms of the mixed reference solution (each reference solution has a concentration of 50 μg / mL), the test solution, and the blank solution. Figure 3 , where A is the mixed reference solution, B is the test solution, and C is the blank solution (1. Morroniside; 2. Loganin; 3. Calycosin glucoside; 4. Icariin; 5. Osthole). It can be seen that the peak separation of each component to be tested is good, and the solvent has no effect on the chromatographic peak, indicating that the method has good specificity.

[0111] 2) Linear relationship investigation

[0112] Accurately measure each reference substance stock solution, dilute with 50% methanol aqueous solution to obtain a mixed reference substance standard solution with a concentration of 1000 μg / mL of morroniside, 600 μg / mL of loganin, 120 μg / mL of calycosin glucoside, 150 μg / mL of icariin, and 20 μg / mL of osthole, and dilute stepwise with 50% methanol aqueous solution by 2, 2, 2, 2, 2, 2 times to obtain mixed reference substance standard solutions of each concentration in the standard curve. Take each of the above solutions for sampling and analysis, record the corresponding peak area, use the chromatographic peak area as the ordinate (Y), and the mass concentration as the abscissa (X), draw the standard curve of the content determination, calculate the linear regression equation, and the results are shown in Table 9. The reference substance concentration corresponding to the signal-to-noise ratio (S / N) of 10 is the lowest limit of quantification (LLOQ), and the results are shown in Table 9.

[0113] Table 9 Results of linear relationship investigation of the five components in Yishen Yanggan mixture

[0114]

[0115] 3) Precision test

[0116] The 7th batch of Yishen Yanggan mixture was used to prepare the test solution, and the samples were injected 6 times continuously. The results are shown in Table 10. The calculated peak area RSDs of the 5 components to be tested were all less than 0.70%, indicating that the intra-day precision of the instrument was good. The injections were repeated 2 times a day for 3 consecutive days. The results are shown in Table 11. The peak area RSDs of the 5 components to be tested were all less than 3.13%, indicating that the inter-day precision of the instrument was good.

[0117] In the following Tables 10 to 15, C1: morroniside, C2: loganin, C3: calycosin glucoside, C4: icariin, and C5: osthole.

[0118] Table 10 Intra-day precision results of the five tested components of Yishen Yanggan mixture (n=6)

[0119] Element 1 2 3 4 5 6 average value RSD(%) 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 Inter-day precision results of the five tested components of Yishen Yanggan mixture (n=6)

[0121] Element 1 2 3 4 5 6 average value RSD(%) 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] The 7th batch of Yishen Yanggan mixture was used to prepare 6 test solutions in parallel, and the samples were injected for determination. The results are shown in Table 12. The RSDs of the contents of the five components to be tested were all less than 1.40%, indicating that the method had good repeatability.

[0124] Table 12 Repeatability results of the five tested components of Yishen Yanggan mixture (mg / g, n=6)

[0125] Element 1 2 3 4 5 6 average value RSD(%) 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 mixture was used to prepare the test solution, which was injected at 0, 2, 4, 8, 12, and 24 h. The results are shown in Table 13. The RSDs of the peak areas of the five components to be tested were all less than 4.83%, indicating that the test solution had good stability within 24 h.

[0128] Table 13 Stability results of 5 tested components of Yishen Yanggan mixture (n=6)

[0129] Element 0h 2h 4h 8h 12h 24h average value RSD(%) 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) Sample recovery experiment

[0131] Accurately measure 6 portions of the 7th batch of Yishen Yanggan mixture, 2 mL each, add appropriate amount of each reference substance, prepare 6 test sample solutions in parallel, inject and measure, record the peak area, calculate the average recovery of morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole, and the results are shown in Table 14, which are 105.53%, 113.62%, 89.08%, 90.78% and 96.17%, respectively, and the RSD values ​​are all less than 2.82%, indicating that the recovery of this method meets the requirements.

[0132] Table 14 Recovery results of five components of Yishen Yanggan mixture (n=6)

[0133]

[0134] 7) Content determination

[0135] Take each batch of Yishen Yanggan mixture to prepare the test solution, inject and analyze, and calculate the content of each component according to the external standard method. The results are shown in Table 15.

[0136] Table 15 Content determination results of different batches of Yishen Yanggan mixture

[0137]

[0138]

[0139] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for establishing an HPLC fingerprint of a Yishen Yanggan mixture, characterized in that: The following steps are involved: The Yishen Yanggan mixture and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution; Providing a mixed reference solution, wherein the reference substances in the mixed reference solution include morroniside, loganin, calycosin isoflavone glucoside, icariin and osthole; The test solution and the mixed reference solution are subjected to HPLC detection, and the obtained chromatogram is imported into the fingerprint software to identify the common peaks to obtain the HPLC fingerprint of Yishen Yanggan mixture; The conditions of the HPLC detection include: Chromatographic column: C18 column; mobile phase: phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-10 min, the volume fraction of phase B increases from 5% to 10%; 10-30 min, the volume fraction of phase B increases from 10% to 18%; 30-50 min, the volume fraction of phase B increases from 18% to 30%; 50-70 min, the volume fraction of phase B increases from 30% to 50%; 70-85 min, the volume fraction of phase B increases from 50% to 100%; flow rate: 0.6-1 mL / min; column temperature: 25-35° C.; detection wavelength 240-260 nm.

2. The establishment method according to claim 1, characterized in that: The volume fraction of methanol in the methanol aqueous solution is 40% to 60%.

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

4. The establishment method according to claim 1, characterized in that: The chromatographic column is a ZORBAX Eclipse Plus C18 chromatographic column.

5. The establishment method according to claim 1 or 4, characterized in that: The phase A is a phosphoric acid aqueous solution with a volume fraction of 0.1%, the flow rate is 0.8 mL / min, and the column temperature is 30°C.

6. The establishment method according to claim 1, characterized in that: The detection wavelength is 250 nm.

7. The establishment method according to claim 1, characterized in that: The injection volume of the HPLC detection is 5 to 20 μL.

8. The establishment method according to claim 1, characterized in that: The HPLC fingerprint of the Yishen Yanggan mixture 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; peak 3 is morroniside, peak 5 is loganin, peak 7 is calycosin glucoside, peak 12 is icariin, and peak 15 is osthole.

9. A method for determining the content of multiple index components in a kidney-tonifying and liver-nourishing mixture, characterized in that: The multi-index components include two or more of morroniside, loganin, calycosin glucoside, icariin and osthole, and the determination method includes the following steps: The Yishen Yanggan mixture to be tested and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution; The test solution is subjected to HPLC detection, and the content of multiple index components in the Yishen Yanggan mixture is obtained according to the obtained chromatographic peak area and a 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 of the HPLC detection include: Chromatographic column: C18 column; mobile phase: phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-10 min, the volume fraction of phase B increases from 5% to 10%; 10-30 min, the volume fraction of phase B increases from 10% to 18%; 30-50 min, the volume fraction of phase B increases from 18% to 30%; 50-70 min, the volume fraction of phase B increases from 30% to 50%; 70-85 min, the volume fraction of phase B increases from 50% to 100%; flow rate: 0.6-1 mL / min; column temperature: 25-35° C.; detection wavelength 240-330 nm.

10. The method for determining the content of multiple index components according to claim 9, characterized in that: The detection wavelengths of morroniside and loganin are 240 nm, the detection wavelength of calycosin glucoside is 260 nm, the detection wavelength of icariin is 270 nm, and the detection wavelength of osthole is 322 nm.

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