Method for measuring contents of various chemical components in kidney-tonifying and liver-nourishing prescription

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a method for determining the content of various chemical components in a kidney-tonifying and liver-nourishing prescription. The determination method comprises the following steps: mixing a to-be-detected kidney-tonifying and liver-nourishing prescription sample with an aqueous solution of alcohol, carrying out ultrasonic treatment, and carrying out HPLC-MS / MS detection on the obtained test solution to obtain the content of each chemical component. According to the invention, a sample to be detected is subjected to a simple pretreatment process and then is detected by HPLC-MS / MS; the method can be used for simultaneously measuring the contents of 20 chemical components, namely xanthoxylin, bergamolactone, cnidium lactone, imperatorin, calycosin, calycosin glucoside, siloside-B, icariin, epimedin-B, epimedin-C, apigenin, quercetin, swertiamarin, loganin, morroniside, isoquercitrin, baohuoside-I, icariside-I, rutin and astragaloside. The method can reflect the overall characteristics of the prescription and can be used for quality control.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a method for determining the contents of multiple chemical components in a kidney-tonifying and liver-nourishing prescription. Background Art

[0002] The kidney-tonifying and liver-nourishing prescription is composed of multiple traditional Chinese medicines such as Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao, Cornus officinalis Sieb. et Zucc., Epimedium brevicornu Maxim., and Cnidium monnieri (L.) Cuss., and has the effects of invigorating qi and strengthening the spleen, tonifying the kidney and nourishing the liver, and strengthening tendons and bones. Clinically, it is used for various diseases and chronic strains caused by deficiency of the liver and kidney and insufficiency of blood and essence. This prescription has been clinically used for many years with remarkable curative effects and no adverse reactions found.

[0003] The kidney-tonifying and liver-nourishing prescription contains various chemical components such as polysaccharides, flavonoids, saponins, iridoids and their glycosides, triterpenes, lignans, organic acids, coumarins, etc. At present, for the quality evaluation of the kidney-tonifying and liver-nourishing prescription, thin-layer chromatography is mainly used for qualitative identification of astragaloside IV and ursolic acid and determination of the content of astragaloside IV. Traditional Chinese medicine compound prescriptions have the characteristics of multiple components, multiple targets, and multiple pathways. Only determining a single index component cannot well show the overall picture of traditional Chinese medicine compound prescriptions. Therefore, there is an urgent need to develop a detection method that can reflect the overall characteristics of the kidney-tonifying and liver-nourishing prescription. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for determining the contents of multiple chemical components in a kidney-tonifying and liver-nourishing prescription. The determination method of the present invention can simultaneously determine the contents of 20 chemical components in the kidney-tonifying and liver-nourishing prescription and can reflect the overall characteristics of the kidney-tonifying and liver-nourishing prescription.

[0005] The present invention provides a method for determining the contents of multiple chemical components in a kidney-tonifying and liver-nourishing prescription, wherein the kidney-tonifying and liver-nourishing prescription includes Epimedium brevicornu Maxim., Cnidium monnieri (L.) Cuss., Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao, and Cornus officinalis Sieb. et Zucc., and the determination method includes the following steps:

[0006] Mixing a sample of the kidney-tonifying and liver-nourishing prescription to be tested with an aqueous solution of alcohol and performing ultrasonic treatment to obtain a test solution;

[0007] Performing HPLC-MS / MS detection on the test solution, and obtaining the contents of the chemical components in the kidney-tonifying and liver-nourishing prescription according to the obtained mass spectrometry peak areas and a predetermined standard curve; the predetermined standard curve is a linear curve between the respective concentrations of the chemical components and the corresponding mass spectrometry peak areas;

[0008] The chemical components include at least two of xanthotoxin, bergapten, osthole, imperatorin, calycosin, calycosin-7-O-β-D-glucoside, icariside-B, icariin, epimedin-B, epimedin-C, apigenin, quercetin, swertiamarin, loganin, morroniside, isoquercitrin, baohuoside-I, icarisidin-I, rutin, and astragaloside IV;

[0009] The chromatographic conditions for the HPLC-MS / MS detection include:

[0010] Chromatographic column: C18 column; Mobile phase: Phase A is an aqueous formic acid solution with a volume fraction of 0.05 - 0.2%, and Phase B is acetonitrile; Gradient elution, elution program: 0 - 1 min, the volume fraction of Phase B increases from 5% to 15%; 1 - 5 min, the volume fraction of Phase B increases from 15% to 35%; 5 - 6 min, the volume fraction of Phase B increases from 35% to 36%; 6 - 10 min, the volume fraction of Phase B increases from 36% to 70%; 10 - 13 min, the volume fraction of Phase B increases from 70% to 80%; Flow rate: 0.3 - 0.6 mL / min; Column temperature: 25 - 35 °C;

[0011] The mass spectrometric conditions for the HPLC-MS / MS detection include:

[0012] Ion source: Electrospray ionization source; Detection mode: Multiple reaction monitoring; Drying gas temperature: 280 - 320 °C; Drying gas flow rate: 9 - 13 L / min; Nebulizer pressure: 25 - 35 psi.

[0013] Preferably, the aqueous solution of alcohol includes an aqueous methanol solution or an aqueous ethanol solution; the volume fraction of alcohol in the aqueous solution of alcohol is 40% - 60%.

[0014] Preferably, the dosage ratio of the kidney-tonifying and liver-nourishing prescription to the aqueous solution of alcohol is 0.05 g : (4 - 6) mL.

[0015] Preferably, the time for ultrasonic treatment is 50 - 70 min, the power is 250 - 350 W, and the frequency is 35 - 45 kHz.

[0016] Preferably, the chromatographic column is C18 column.

[0017] Preferably, the mobile phase A is an aqueous formic acid solution with a volume fraction of 0.1%.

[0018] Preferably, the flow rate is 0.4 mL / min.

[0019] Preferably, the drying gas temperature is 300 °C; the drying gas flow rate is 11 L / min; the nebulizer pressure is 30 psi.

[0020] Preferably, the parent ions detected by HPLC-MS / MS include: xanthotoxin 217.2, bergapten 217.2, osthole 245.3, imperatorin 271.2, calycosin 285.2, calycosin-7-O-β-D-glucoside 447.4, icariin-B 647.4, icariin 677.0, epimedin B 809.5, epimedin C 823.5, apigenin 269.0, quercetin 301.1, swertiamarin 403.2, loganin 435.3, morroniside 451.2, isoquercitrin 463.2, baohuoside-I 513.1, icariside II 529.3, rutin 609.2, astragaloside IV 829.6; Allowable deviation: ±0.2 DA.

[0021] Preferably, the daughter ions detected by HPLC-MS / MS include: xanthotoxin 174.2, bergapten 202.1, osthole 131.2, imperatorin 147.1, calycosin 270.3, calycosin-7-O-β-D-glucoside 285.2, icariin-B 369.3, icariin 369.3, epimedin B 369.4, epimedin C 369.4, apigenin 117.1, quercetin 151.1, swertiamarin 125.1, loganin 227.2, morroniside 140.9, isoquercitrin 300.1, baohuoside-I 366.0, icariside II 367.2, rutin 301.0, astragaloside IV 783.5; Allowable deviation: ±0.2 DA.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a method for determining the contents of various chemical components in a kidney-tonifying and liver-nourishing prescription. The kidney-tonifying and liver-nourishing prescription includes epimedium, cnidium fruit, roasted astragalus, and fructus corni. The determination method includes the following steps: mixing a sample of the kidney-tonifying and liver-nourishing prescription to be tested with an aqueous solution of alcohol and performing ultrasonic treatment to obtain a test solution; performing HPLC-MS / MS detection on the test solution, and obtaining the contents of the chemical components in the kidney-tonifying and liver-nourishing prescription according to the obtained mass spectrometry peak areas and a predetermined standard curve; the predetermined standard curve is a linear curve between the respective concentrations of the chemical components and the corresponding mass spectrometry peak areas.

[0024] After simple pretreatment of the sample to be tested, the present invention uses HPLC-MS / MS for detection, and can simultaneously determine the contents of multiple chemical components in the kidney-tonifying and liver-nourishing formula, including xanthotoxin, bergapten, osthole, imperatorin, calycosin, calycosin-7-O-β-D-glucoside, icariside B, icariin, epimedin B, epimedin C, apigenin, quercetin, swertiamarin, loganin, morroniside, isoquercitrin, baohuoside I, icarisidin I, rutin, and astragaloside IV, up to 20 kinds, which can reflect the overall characteristics of the traditional Chinese medicine compound and can be used for the quality control of the kidney-tonifying and liver-nourishing formula.

[0025] The data of the examples show that the determination method of the present invention has strong specificity, high sensitivity, high precision, and good accuracy. The HPLC-MS / MS method established by the present invention can efficiently and accurately monitor the contents of 20 characteristic components in the kidney-tonifying and liver-nourishing formula, providing a reference for the quality research of the kidney-tonifying and liver-nourishing formula. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 MRM chromatograms of the standard reference solution at 2 μg / mL (A) and the S1 sample (B), where 1. xanthotoxin; 2. bergapten; 3. osthole; 4. imperatorin; 5. calycosin; 6. calycosin-7-O-β-D-glucoside; 7. icariside B; 8. icariin; 9. epimedin B; 10. epimedin C; 11. apigenin; 12. quercetin; 13. swertiamarin; 14. loganin; 15. morroniside; 16. isoquercitrin; 17. baohuoside I; 18. icarisidin I; 19. rutin; 20. astragaloside IV;

[0028] Figure 2 Total ion chromatograms of the standard reference solution at 2 μg / mL in the positive ion (A) and negative ion (B) modes with different mobile phases. Detailed Embodiments

[0029] The present invention provides a method for determining the contents of multiple chemical components in a kidney-tonifying and liver-nourishing formula, the kidney-tonifying and liver-nourishing formula includes epimedium, cnidium fruit, astragalus membranaceus, and cornel, and the determination method includes the following steps:

[0030] Mix the sample of the kidney-tonifying and liver-nourishing formula to be tested with an aqueous solution of alcohol and perform ultrasonic treatment to obtain a test solution;

[0031] Perform HPLC-MS / MS detection on the test sample solution, and obtain the contents of the chemical components in the Yishen Yanggan formula according to the obtained mass spectrometry peak areas and the predetermined standard curves; the predetermined standard curves are linear curves between the concentrations of the chemical components and their corresponding mass spectrometry peak areas.

[0032] The chemical components include at least two of xanthotoxin, bergapten, osthole, imperatorin, calycosin, calycosin-7-O-β-D-glucoside, icariin-B, icariin, epimedin-B, epimedin-C, apigenin, quercetin, swertiamarin, loganin, morroniside, isoquercitrin, baohuoside-I, icariside-II, rutin, and astragaloside IV.

[0033] The chromatographic conditions for the HPLC-MS / MS detection include:

[0034] Chromatographic column: C18 column; Mobile phase: Phase A is an aqueous formic acid solution with a volume fraction of 0.05 - 0.2%, and Phase B is acetonitrile; Gradient elution, elution program: 0 - 1 min, the volume fraction of Phase B increases from 5% to 15%; 1 - 5 min, the volume fraction of Phase B increases from 15% to 35%; 5 - 6 min, the volume fraction of Phase B increases from 35% to 36%; 6 - 10 min, the volume fraction of Phase B increases from 36% to 70%; 10 - 13 min, the volume fraction of Phase B increases from 70% to 80%; Flow rate: 0.3 - 0.6 mL / min; Column temperature: 25 - 35 °C.

[0035] The mass spectrometry conditions for the HPLC-MS / MS detection include:

[0036] Ion source: Electrospray ionization source; Detection mode: Multiple reaction monitoring; Dry gas temperature: 280 - 320 °C; Dry gas flow rate: 9 - 13 L / min; Nebulizer pressure: 25 - 35 psi.

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

[0038] In the present invention, the test Yishen Yanggan formula sample is mixed with an aqueous solution of alcohol and subjected to ultrasonic treatment to obtain the test sample solution.

[0039] In the present invention, roasted epimedium, cnidium monnieri, roasted astragalus membranaceus, and fructus corni in the Yishen Yanggan formula are preferably used in the form of powders, and the powders are preferably powders passing through a 60-mesh sieve.

[0040] In the present invention, the mass ratio of roasted epimedium, cnidium monnieri, roasted astragalus membranaceus, and fructus corni in the Yishen Yanggan formula is preferably (1 - 4):(1 - 4):(3 - 12):(1 - 4).

[0041] In the present invention, the aqueous solution of alcohol preferably comprises an aqueous solution of methanol or ethanol, more preferably an aqueous solution of ethanol. The volume fraction of alcohol in the aqueous solution of alcohol is preferably 40% - 60%, more preferably 50%.

[0042] In the present invention, the dosage ratio of the kidney-tonifying and liver-nourishing formula to the aqueous solution of alcohol is preferably 0.05 g : (4 - 6) mL, more preferably 0.05 g : 5 mL.

[0043] In the present invention, the time of ultrasonic treatment is preferably 50 - 70 min, more preferably 60 min (1 h), the power is preferably 250 - 350 W, more preferably 300 W, and the frequency is preferably 35 - 45 kHz, more preferably 40 kHz.

[0044] In the present invention, after ultrasonic treatment, it preferably further includes filtration, dilution, and re-filtration. Both the filtration and re-filtration preferably use a 0.22 μm microporous filter membrane; the dilution multiple is preferably 10 times, and the dilution preferably uses an aqueous solution of ethanol with a volume fraction of 50%.

[0045] After obtaining the test solution, in the present invention, the test solution is subjected to HPLC-MS / MS detection. According to the obtained mass spectrometry peak area and a predetermined standard curve, the content of chemical components in the kidney-tonifying and liver-nourishing formula is obtained; the predetermined standard curve is a linear curve between the concentration of each chemical component and the corresponding mass spectrometry peak area.

[0046] In the present invention, the chromatographic column is preferably a C18 column with a specification of 2.1×100 mm, 3.5 μm.

[0047] In the present invention, the mobile phase A is preferably an aqueous solution of formic acid with a volume fraction of 0.1%.

[0048] In the present invention, the flow rate is preferably 0.4 mL / min.

[0049] In the present invention, the injection volume for HPLC-MS / MS detection is preferably 1 - 10 μL, more preferably 5 μL.

[0050] In the present invention, the column temperature is preferably 30 °C

[0051] In the present invention, the drying gas temperature is preferably 300 °C; the drying gas flow rate is preferably 11 L / min; the nebulizer pressure is preferably 30 psi.

[0052] In the present invention, the mass spectrometry parameters of the chemical components are preferably as shown in Table 1:

[0053] Table 1 Ion pairs and mass spectrometry parameters of substances to be measured

[0054]

[0055]

[0056] In the present invention, the determination method of the chemical component content preferably uses external standard method for quantification.

[0057] In the present invention, the linear range of the predetermined standard curve is preferably: xanthotoxin 0.8 - 320 ng / mL, bergapten 1.5 - 600 ng / mL, osthole 10 - 4000 ng / mL, imperatorin 4 - 1600 ng / mL, calycosin 0.5 - 200 ng / mL, calycosin-7-O-β-D-glucoside 0.8 - 320 ng / mL, acanthoside-B 0.3 - 120 ng / mL, icariin 2.5 - 1000 ng / mL, epimedin B 2.5 - 1000 ng / mL, epimedin C 8 - 3200 ng / mL, apigenin 0.4 - 160 ng / mL, quercetin 0.4 - 160 ng / mL, swertiamarin 1 - 400 ng / mL, loganin 12 - 4800 ng / mL, morroniside 20 - 8000 ng / mL, isoquercitrin 1.5 - 600 ng / mL, baohuoside-I 0.5 - 200 ng / mL, icariside II 0.4 - 160 ng / mL, rutin 1 - 400 ng / mL, astragaloside IV 12 - 4800 ng / mL.

[0058] The determination method of the present invention has a simple pretreatment process, good linear relationship, and good results in precision, repeatability, stability and standard addition recovery test. It can simultaneously determine the contents of 20 chemical components in the kidney-tonifying and liver-nourishing prescription, and can provide a reference for the quality evaluation of the kidney-tonifying and liver-nourishing prescription.

[0059] In order to further illustrate the present invention, the determination method of the contents of various chemical components in the kidney-tonifying and liver-nourishing prescription provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0060] In the embodiments of the present invention, the instruments used include:

[0061] 1290 ultra-high performance liquid chromatograph, 6470 triple quadrupole tandem mass spectrometer (Agilent Technologies, USA); Milli-Q IQ 7005 ultrapure water preparation instrument (Millipore Corporation); vortex mixer (Thermo Fisher Scientific); high-speed temperature-controlled centrifuge (Eppendorf); ultrasonic cleaner (Tianjin Zhizhu Technology Co., Ltd.); one over one hundred thousand balance (Radwag, Poland);

[0062] The test drugs include:

[0063] Xanthotoxin (DST200510-062), bergapten (DST200813-012), imperatorin (DST200624-003), calycosin (DST220226-012), calycosin-7-O-β-D-glucoside (DSTDM001301), astragaloside B (DST190411-163), icariin (DST180730-091), quercetin (DST180521-028), baohuoside I (DSTDY009201), icariside I (DST190411-094), rutin (DST210520-017) were purchased from Chengdu Deste Biotechnology Co., Ltd.; apigenin (MUST-17022605), isoquercitrin (MUST-18051005) were purchased from Chengdu Manit Biotechnology Co., Ltd.; epimedin C (111780-201905), osthole (110822-202412), loganin (111640-202309), morroniside (111998-202205), astragaloside IV (110781-202219), epimedin B (000142-202309), swertiamarin (000929-202301) were purchased from National Institutes for Food and Drug Control; methanol and acetonitrile were purchased from Fisher Company (USA); ethanol was purchased from Tianjin Concord Technology Co., Ltd.; formic acid was purchased from ROE Company (USA).

[0064] The medicinal materials include:

[0065] In an Excel spreadsheet, the RAND function was used to randomly sort different batches of each medicinal material to obtain 16 random combinations, resulting in a kidney-tonifying and liver-nourishing formula. The numbering information is shown in Table 2:

[0066] Table 2 Sample source information

[0067]

[0068] Example 1

[0069] 1. Instrument conditions

[0070] 1) Chromatographic conditions

[0071] Chromatographic column: C18 column (2.1×100 mm, 3.5 μm); mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B) by volume; gradient elution, elution gradient: 0 - 1 min, 5 - 15% B; 1 - 5 min, 15 - 35% B; 5 - 6 min, 35 - 36% B; 6 - 10 min, 36 - 70% B; 10 - 13 min, 70 - 80% B; flow rate: 0.4 mL / min; injection volume: 5 μL; column temperature: 30 °C.

[0072] 2) Mass spectrometry conditions

[0073] Ion source: electrospray ionization source (ESI); detection mode: multiple reaction monitoring (MRM); drying gas temperature: 300 °C; drying gas flow rate: 11 L / min; nebulizer pressure: 30 psi. The ion pairs and mass spectrometry parameters of 20 substances to be measured are shown in Table 1.

[0074] 2. Preparation of solutions

[0075] 1) Preparation of reference substance solution

[0076] Appropriately weigh the standard reference substances of xanthotoxin, bergapten, osthole, imperatorin, calycosin, calycosin-7-O-β-D-glucoside, gymnemic acid-B, icariin, epimedin-B, epimedin-C, apigenin, quercetin, swertiamarin, loganin, morroniside, isoquercitrin, baohuoside-I, icariside-II, rutin, and astragaloside IV, dissolve them in a methanol aqueous solution with a volume fraction of 80%, and prepare a standard reference substance stock solution with a concentration of 1 mg / mL. Place it in a refrigerator at 4 °C for standby.

[0077] 2) Preparation of test solution

[0078] Precisely weigh 50 mg of the roasted epimedium powder, cnidium fruit powder, roasted astragalus root powder, and dogwood fruit powder (sieved through 60 mesh) according to the prescription ratio, place them in a 5 mL volumetric flask, add an ethanol aqueous solution with a volume fraction of 50% to the scale line, ultrasonically treat for 1 h (300 W, 40 kHz), cool, make up for the weight loss, shake well, filter through a 0.22 μm microporous filter membrane, precisely measure 1 mL of the subsequent filtrate, place it in a 10 mL volumetric flask, add an ethanol aqueous solution with a volume fraction of 50% to the scale, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0079] Methodology investigation of Example 2

[0080] 1. Standard curve regression equation

[0081] Precisely measure the stock solution of the standard reference substance, and dilute it with an aqueous methanol solution with a volume fraction of 80% to obtain a mixed reference substance solution with the concentration of imperatorin being 1600 ng / mL, the concentrations of xanthotoxin and calycosin-7-O-β-D-glucoside being 320 ng / mL, the concentrations of bergapten and isoquercitrin being 600 ng / mL, the concentration of osthole being 4000 ng / mL, the concentrations of calycosin and baohuoside-I being 200 ng / mL, the concentration of jianhuoside-B being 120 ng / mL, the concentrations of icariin and epimedin B being 1000 ng / mL, the concentration of epimedin C being 3200 ng / mL, the concentrations of apigenin, quercetin and icariside II being 160 ng / mL, the concentrations of swertiamarin and rutin being 400 ng / mL, the concentrations of loganin and astragaloside IV being 4800 ng / mL, and the concentration of morroniside being 8000 ng / mL. Dilute it successively by 2, 2, 2, 2.5, 2, 2.5, 2, 2 times with an aqueous methanol solution with a volume fraction of 80% to obtain mixed reference substance solutions with different concentrations, and perform injection analysis. Use the weighted least squares method for regression calculation. The abscissa (X) is the concentration of the analyte, the ordinate (Y) is the peak area of the analyte, and the weighting coefficient is 1 / X to obtain the linear regression equation of each component. Calculate the lowest limit of quantitation (LLOQ) with the reference substance concentration when the signal-to-noise ratio (S / N) is 10. The results are shown in Table 3.

[0082] Table 3 Regression equations, linear ranges, correlation coefficients and lowest limits of quantitation of 20 chemical components

[0083]

[0084] Figure 1 MRM chromatograms of the standard reference substance solution with the concentration of each component being 2 μg / mL (A) and the MRM chromatogram of sample S1 (B), where 1. xanthotoxin; 2. bergapten; 3. osthole; 4. imperatorin; 5. calycosin; 6. calycosin-7-O-β-D-glucoside; 7. jianhuoside-B; 8. icariin; 9. epimedin B; 10. epimedin C; 11. apigenin; 12. quercetin; 13. swertiamarin; 14. loganin; 15. morroniside; 16. isoquercitrin; 17. baohuoside-I; 18. icariside II; 19. rutin; 20. astragaloside IV. It can be seen that the peak shapes of each compound are good and there is no interference with each other.

[0085] 2. Precision test

[0086] Prepare the test solution according to the method of Example 1, inject continuously for 6 times, and calculate the RSD values of the peak areas of each compound. The intra-day precision is shown in Table 4.

[0087] Prepare the test solution according to the method of Example 1, inject repeatedly for 2 times, inject continuously for 3 days, and calculate the RSD values of the peak areas of each compound. The inter-day precision results are shown in Table 5.

[0088] Intraday precision results of 20 chemical components (n = 6)

[0089]

[0090] Interday precision results of 20 chemical components (n = 6)

[0091]

[0092] 3. Repeatability test

[0093] The test solution was prepared according to the method of Example 1, and 6 parallel samples were prepared and injected respectively. The RSD values of the contents of each compound were calculated. The results are shown in Table 6, indicating that the method has good repeatability.

[0094] Repeatability results of 20 chemical components (n = 6, μg / g)

[0095]

[0096]

[0097] 4. Stability test

[0098] The test solution was prepared according to the method of Example 1. The prepared test solution was injected at 0, 2, 4, 8, 12, and 24 h respectively, and the RSD values of the peak areas of each compound were calculated. The results are shown in Table 7, indicating that each compound has good stability.

[0099] Stability results of 20 chemical components (n = 6)

[0100]

[0101]

[0102] 5. Spiked recovery test

[0103] Precisely weigh 50 mg of Epimedii Folium powder, Fructus Cnidii powder, Astragali Radix powder, and Cornus officinalis powder (sieved through 60-mesh sieve) according to the prescription ratio, weigh 6 parallel samples, add a certain amount of mixed reference solution respectively, prepare in parallel according to the preparation method of the test solution in Example 1, inject respectively, and calculate the spiked recoveries of each compound. The results are shown in Table 8.

[0104] Spiked recovery results of 20 chemical components (n = 6)

[0105]

[0106]

[0107] 6. Content determination results

[0108] The test solution was prepared according to the method of Example 1 for determination. The concentrations of 20 compounds were recorded and the contents were calculated. The results are shown in Table 9. The results indicate that there are differences in the contents of each component among different batches. The components with higher contents in all batches are loganin, morroniside, and astragaloside IV, while the contents of apigenin, quercetin, and icariside II are lower. The above results can provide a reference for the quality evaluation of Yishen Yanggan Prescription.

[0109] Table 9 Contents of 20 chemical components in 16 batches of Yishen Yanggan Prescription (μg / g, n = 3)

[0110]

[0111]

[0112]

[0113] Note: “-” in the table indicates that the content is lower than the lowest quantitation limit and is not detected.

[0114] Comparative Example 1

[0115] The difference from Example 1 is that mobile phase B was replaced with methanol, and the other conditions were the same.

[0116] Figure 2 Total ion chromatograms of 2 μg / mL standard reference solution in positive ion (A) and negative ion (B) modes for different mobile phases; among them, ME / 0.1% FA-H 2 O is the mobile phase of Comparative Example 1, ACN / 0.1% FA-H 2 O is the mobile phase of Example 1. The abscissa is time, unit min, and the ordinate is response. Elution gradient: 0 - 20 min, the volume fraction of phase B increases from 5% to 100%.

[0117] It can be seen that in Example 1, with acetonitrile - 0.1% formic acid aqueous solution as the mobile phase, each component was effectively separated, and the overall peak elution time was earlier and the elution efficiency was higher. Therefore, the present invention uses acetonitrile - 0.1% formic acid aqueous solution as the optimal mobile phase.

[0118] Comparative Example 2

[0119] The difference from Example 1 is that the mobile phase elution gradient program is different, and the other conditions are the same. Gradient conditions I and Gradient conditions II were used respectively, as shown in Table 10 and Table 11 below:

[0120] Table 10 Gradient conditions I

[0121] Time (min) Acetonitrile (%) Aqueous solution of formic acid with a volume fraction of 0.1% (%) 0～1 5→20 95→80 1～4 20→40 80→60 4～6 40 60 6～15 40→80 60→20

[0122] Table 11 Gradient Condition II

[0123] Time (min) Acetonitrile (%) Aqueous solution of formic acid with a volume fraction of 0.1% (%) 0～1 5→20 95→80 1～4.5 20→41 80→59 4.5～5.5 41→41.5 59→58.5 5.5～15 41.5→80 58.5→20

[0124] The results show that in Gradient Conditions I and II, the resolution of rutin is low and there are consecutive peaks, while the separation effect of the gradient condition of Example 1 is the best and the time is shorter. Therefore, the gradient condition of Example 1 is selected as the mobile phase elution gradient in the present invention.

[0125] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to the embodiments of the present invention without creative labor, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for determining the contents of multiple chemical components in a prescription for nourishing the kidney and liver, wherein the prescription comprises roasted epimedium, fructus cnidium, roasted astragalus and fructus corni, characterized in that: The determination method comprises the following steps: The sample of the kidney-tonifying and liver-nourishing prescription to be tested is mixed with an aqueous solution of alcohol and subjected to ultrasonic treatment to obtain a test solution; The test solution is subjected to HPLC-MS / MS detection, and the content of the chemical components in the kidney-tonifying and liver-nourishing prescription is obtained according to the obtained mass spectrum peak area and a predetermined standard curve; the predetermined standard curve is a linear curve between the concentration of each chemical component and the corresponding mass spectrum peak area; The chemical components include at least two of the following: zanthoxylum toxin, bergamot lactone, osthole, imperatorin, calycosin, calycosin glucoside, argentein-B, icariin, chamoside-B, chamoside-C, apigenin, quercetin, dangoside, loganin, morroniside, isoquercetin, bauroside-I, icariin-I, rutin and astragaloside IV; The chromatographic conditions for the HPLC-MS / MS detection include: Chromatographic column: C18 column; mobile phase: phase A is a 0.05-0.2% formic acid aqueous solution, phase B is acetonitrile; gradient elution, elution program: 0-1min, the volume fraction of phase B increases from 5% to 15%; 1-5min, the volume fraction of phase B increases from 15% to 35%; 5-6min, the volume fraction of phase B increases from 35% to 36%; 6-10min, the volume fraction of phase B increases from 36% to 70%; 10-13min, the volume fraction of phase B increases from 70% to 80%; flow rate: 0.3-0.6mL / min; column temperature: 25-35℃; The mass spectrometry conditions for the HPLC-MS / MS detection include: Ion source: electrospray ion source; detection mode: multiple reaction ion monitoring; drying gas temperature: 280-320°C; drying gas flow rate: 9-13 L / min; nebulizer pressure: 25-35 psi.

2. The measuring method according to claim 1, characterized in that The alcohol aqueous solution includes methanol aqueous solution or ethanol aqueous solution; the volume fraction of alcohol in the alcohol aqueous solution is 40% to 60%.

3. The measuring method according to claim 2, characterized in that The dosage ratio of the kidney-tonifying and liver-nourishing prescription to the alcohol aqueous solution is 0.05 g: (4-6) mL.

4. The measuring method according to claim 1, characterized in that The ultrasonic treatment lasts for 50 to 70 minutes, the power is 250 to 350 W, and the frequency is 35 to 45 kHz.

5. The measuring method according to claim 1, characterized in that The chromatographic column is C18 column.

6. The measuring method according to claim 1, characterized in that The mobile phase A is a formic acid aqueous solution with a volume fraction of 0.1%.

7. The measuring method according to claim 6, characterized in that The flow rate was 0.4 mL / min.

8. The measuring method according to claim 1, characterized in that The drying gas temperature is 300° C.; the drying gas flow rate is 11 L / min; and the atomizer pressure is 30 psi.

9. The measuring method according to claim 1, characterized in that The parent ions detected by the HPLC-MS / MS include: xanthoxylum toxin 217.2, bergamot lactone 217.2, osthole 245.3, imperatorin 271.2, calycosin 285.2, calycosin glucoside 447.4, scutellarin-B 647.4, icariin 677.0, chondrin-B 809.5, chondrin-C 823.5, apigenin 269.0, quercetin 301.1, danganin 403.2, loganin 435.3, morroniside 451.2, isoquercitrin 463.2, baohuoside-I 513.1, icariin-I 529.3, rutin 609.2, and astragaloside IV 829.6; the allowable deviation is ±0.2DA.

10. The measuring method according to claim 1 or 9, characterized in that: The product ions detected by the HPLC-MS / MS include: xanthoxylum toxin 174.2, bergamot lactone 202.1, osthole 131.2, imperatorin 147.1, calycosin 270.3, calycosin glucoside 285.2, scutellaria glycoside-B 369.3, icariin 369.3, chondrin-B 369.4, chondrin-C 369.4, apigenin 117.1, quercetin 151.1, dangioside 125.1, loganin 227.2, morroniside 140.9, isoquercitrin 300.1, baohuoside-I 366.0, icariin-I 367.2, rutin 301.0, and astragaloside IV 783.5; the allowable deviation is ±0.2DA.

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