CN117388397A - Quality detection method of kidney-tonifying, heat-clearing and blood-activating prescription - Google Patents

Quality detection method of kidney-tonifying, heat-clearing and blood-activating prescription Download PDF

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CN117388397A
CN117388397A CN202311350918.4A CN202311350918A CN117388397A CN 117388397 A CN117388397 A CN 117388397A CN 202311350918 A CN202311350918 A CN 202311350918A CN 117388397 A CN117388397 A CN 117388397A
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kidney
tonifying
blood
activating
clearing
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孙伟
陈翀
孙斯凡
赵静
何伟明
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Jiangsu Provincial Hospital of Chinese Medicine
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Jiangsu Provincial Hospital of Chinese Medicine
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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Abstract

The invention discloses a quality detection method of a kidney-tonifying, heat-clearing and blood-activating prescription, which comprises the steps of preparation of a sample solution, preparation of a standard curve and content measurement. The invention screens out the best preparation method of the test sample, the preparation method of the reference substance, the best chromatographic conditions and mass spectrometry analysis conditions, and the established content determination method of the kidney-tonifying, heat-clearing and blood-activating prescription can simultaneously detect the content of 11 compounds with different structural types, such as resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polygonin, astilbin, hyperin, calycosin glucoside, beta-ecdysterone and the like. The method is beneficial to comprehensively monitoring the quality of the kidney-tonifying, blood-activating and heat-clearing prescription and ensures the clinical curative effect. The method has the advantages of good stability, high accuracy, good repeatability, high precision and the like.

Description

Quality detection method of kidney-tonifying, heat-clearing and blood-activating prescription
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a quality detection method of a kidney-tonifying, heat-clearing and blood-activating prescription.
Background
The kidney-tonifying, heat-clearing and blood-activating prescription is a special intra-hospital preparation in hospitals in Jiangsu province, is taught by Sun Wei to be used for long-term clinical and basic research of Chronic Kidney Diseases (CKD), is based on inheriting the ideas of the teaching of modern Chinese medical kidney disease founds Yunxiang and the teaching of national medical university Yanqin, is innovatively developed through clinical curative effect analysis, prescription data mining, epidemiological research, expert investigation and the like, finds kidney deficiency, damp-heat, blood stasis and the like as the core pathogenesis of the CKD, creatively proposes a theory of the kidney deficiency, damp-stasis of the CKD, advocates to prevent and treat the CKD by the theory of the kidney-protecting and the delay of the kidney, and creates the kidney-tonifying, heat-clearing and blood-activating prescription on the basis. The prescription is composed of raw astragalus, angelica, giant knotweed, serissa serissoide, glabrous greenbrier rhizome, achyranthes root, pyrrosia lingua, prepared rhubarb, centella asiatica, rhizoma polygonati and flos abelmoschus manihot, has the effects of strengthening spleen and tonifying kidney, clearing damp and heat, removing blood stasis and expelling turbidity, has micro-temperature nature, is a kidney-tonifying key medicine, and can greatly tonify primordial qi. The astragalus root and the angelica are used for tonifying kidney and nourishing blood, the astragalus root and the rhizoma polygonati are used for tonifying qi and nourishing yin, and the angelica and the achyranthes root are used for activating blood and nourishing blood, so that the food is not stagnated. Centella asiatica, polygonum cuspidatum, pyrrosia lingua and flos abelmoschus manihot clear damp-heat, induce diuresis and treat stranguria; rhizoma Smilacis Glabrae, herba Artemisiae Anomalae, radix et rhizoma Rhei preparata, radix et rhizoma Rhei, and its preparation method have effects of promoting blood circulation, removing blood stasis, dredging viscera, and removing turbid pathogen. The medicines are mutually matched, and have the effects of tonifying kidney, clearing away heat, promoting blood circulation and achieving good clinical curative effect.
The kidney-tonifying, blood-activating and clearing prescription has complex components, and the quality of the prescription cannot be comprehensively and objectively detected by a single detection method. In order to fully control the clinical effectiveness and safety of the prescription, and maintain the benefit of patients, it is necessary to research and design a detection method capable of comprehensively and accurately detecting the effective components of the prescription for tonifying kidney, clearing away heat and promoting blood circulation on the basis of the prior art, and the method can comprehensively evaluate and control the quality of the prescription for tonifying kidney, clearing away heat and promoting blood circulation and provide reference basis for clinical application.
Disclosure of Invention
The invention aims to: the invention aims to overcome the defects of the prior art, and develops a quality detection method of the kidney-tonifying, clearing, benefiting and activating blood circulation prescription, and the quality of the kidney-tonifying, clearing, benefiting and activating blood circulation prescription can be objectively, correctly and effectively controlled by the method, so that the method has important guiding significance for ensuring the safety and the effectiveness of the kidney-tonifying, clearing, activating blood circulation prescription and ensuring the clinical quality.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the following technical scheme:
a quality detection method of a kidney-tonifying, heat-clearing and blood-activating prescription comprises the following steps:
step 1: preparation of kidney-tonifying, blood-activating and heat-clearing prescription sample solution
Weighing kidney-tonifying, blood-activating and heat-clearing traditional Chinese medicine, appropriately crushing, dissolving in a volumetric flask by using a methanol solution, adding methanol for volume fixation, and performing ultrasonic extraction to obtain kidney-tonifying, blood-activating and heat-clearing traditional Chinese medicine extract; precisely transferring a certain amount of extract of the kidney-tonifying, blood-activating and clearing prescription into a volumetric flask, fixing the volume of a methanol solution, shaking uniformly, centrifuging, and taking supernatant to pass through a 0.22 mu m filter membrane to obtain a sample solution of the kidney-tonifying, blood-activating and clearing prescription;
step 2: preparation of standard solution
Precisely weighing appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin, hyperoside, calycosin glucoside, beta-ecdysterone reference substance in 10mL volumetric flask, adding methanol to dissolve and dilute to scale, shaking to obtain reference stock solution with certain concentration;
precisely sucking appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside reference stock solution into the same volumetric flask, adding methanol to constant volume to scale, and shaking to obtain mixed reference working solution with certain concentration;
precisely sucking appropriate amount of calycosin glucoside and beta-ecdysterone stock solution into the same volumetric flask, adding methanol to constant volume to scale, and shaking to obtain mixed control working solution of calycosin glucoside and beta-ecdysterone at certain concentration;
step 3, preparation of standard curve
Negative ion mode: diluting the working solution of the mixed reference substances of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside prepared in the step 2 into mixed reference substance solutions with different concentrations by a multiple ratio, and taking 5 mu L of each solution for HPLC-MS/MS measurement and analysis;
positive ion mode: diluting the working solution of the calycosin glucoside and beta-ecdysterone mixed reference substance prepared in the step 2 by a multiple ratio into mixed reference substance solutions with different concentrations, and taking 5 mu L of each solution for HPLC-MS/MS determination analysis;
standard curve calculation: carrying out linear regression on the linear concentration of each standard substance combined with the detection peak area to obtain a standard curve equation, and completing calculation by using Analyst1.6;
step 4, content measurement
And (3) injecting the sample solution of the kidney-tonifying, clearing and activating blood-activating prescription prepared in the step (1) into an HPLC-MS/MS (high performance liquid chromatography-mass spectrometry) for determination and analysis under the same chromatographic and mass spectrometry conditions, substituting the detected peak area of the compound into a standard curve equation in the step (3), and calculating to obtain the content of each compound in the kidney-tonifying, clearing and activating blood-activating prescription.
As a preferred scheme, the quality detection method of the kidney-tonifying, heat-clearing and blood-activating prescription comprises the following steps of:
weighing a certain amount of traditional Chinese medicines with the functions of tonifying kidney, clearing away heat, activating blood circulation, properly crushing, dissolving in a 25mL volumetric flask by using 50% methanol, adding 50% methanol for volume fixation, and carrying out ultrasonic extraction for 20min to obtain an extract of the traditional Chinese medicines with the functions of tonifying kidney, clearing away heat, activating blood circulation; precisely transferring 5mL of extract of the kidney-tonifying, blood-activating and clearing prescription into a 100mL volumetric flask, fixing the volume by 50% methanol, shaking uniformly, centrifuging at 12000r/min for 10min, and taking supernatant to pass through a 0.22 mu m filter membrane to obtain the sample solution of the kidney-tonifying, blood-activating and clearing prescription.
As a preferred scheme, the method for detecting the quality of the kidney-tonifying, heat-clearing, blood-activating prescription comprises the following step 2: the preparation method of the standard substance solution comprises the following steps:
precisely weighing appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin, hyperoside, calycosin glucoside, and beta-ecdysterone reference substance in 10mL volumetric flask, adding methanol, dissolving and diluting to scale,shaking to obtain concentrations of 2.21, 1.07, 1.32, 1.22, 1.11, 1.29, 1.10, 1.64, 1.22, 1.12, and 1.52 mg.mL -1 The control stock solution is preserved at the temperature of minus 20 ℃ for standby;
precisely sucking appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside control stock solution into the same 10mL volumetric flask, adding methanol to constant volume to scale, shaking to obtain solutions with concentrations of 256.00, 640.00, 640.00, 640.00, 1120.00, 2080.00, 640.00, 3200.00 and 3680.00 ng.mL respectively -1 Is mixed with the working solution of the reference substance;
precisely sucking appropriate amount of calycosin glucoside and beta-ecdysterone stock solution into the same 10mL volumetric flask, adding methanol to constant volume to scale, shaking to obtain solutions with concentrations of 1600.00 and 3200.00ng·mL respectively -1 Is mixed with the working solution of the reference substance.
As a preferred scheme, the quality detection method of the kidney-tonifying, heat-clearing, blood-activating prescription comprises the following chromatographic conditions in the step 3 and the step 4: chromatographic column: watersourash C18 with a specification of 2.1mm by 150mm and 2.7 μm;
positive ion mode mobile phase: phase A0.1% formic acid/phase B acetonitrile, isocratic elution for 3min; the flow rate is 0.2mL/min; column temperature: 40 ℃; ion source: ESI; quantitative mode: MRM; ion source voltage: 5500V; ion source temperature: 400 ℃;
negative ion mode mobile phase: phase A2 mmol/L ammonium formate/phase B acetonitrile, and carrying out gradient elution; the flow rate is 0.2mL/min; column temperature: 40 ℃; ion source: ESI; quantitative mode: MRM; ion source voltage: -4000V; ion source temperature: 400 ℃.
As a preferred scheme, the mass detection method of the kidney-tonifying, heat-clearing and blood-activating prescription comprises the following mass spectrum conditions in the step 3 and the step 4:
detecting analytes in positive and negative ion modes using an API4000 tandem quadrupole mass spectrometer and electrospray ionization interface; the conversion of the precursor to the product ion is monitored by adopting a multi-reaction monitoring mode, and the set parameters are as follows: ion spray voltage: 4000V; turbine heater temperature: 500 ℃; collision activation dissociation: 10psi; air curtain gas: 25psi; specific mass spectrometry detection conditions are shown in the following table:
standard mass spectrum detection condition
Object of detection Quantitative ion pair m/z DP/V) EP/V CE/V CXP/V
Resveratrol 226.8→142.8 -100 -10 -33 -14
Rhein 238.6→182.7 -100 -10 -27 -14
Formononetin-like element 266.7→251.9 -100 -10 -30 -14
Aloe-emodin 268.7→239.8 -90 -10 -33 -14
Emodin 268.8→225.0 -110 -10 -35 -14
Chlorogenic acid 353.2→190.9 -55 -10 -20 -14
Polygonum cuspidatum glycoside 389.2→227.4 -100 -10 -20 -14
Astilbin 449.3→285.2 -110 -10 -34 -14
Hyperin 463.4→300.1 -94 -10 -34 -14
Calycosin glucoside 447.4→285.3 70 10 22 14
Beta-ecdysterone 481.4→371.3 90 10 20 14
。
As a preferred scheme, the quality detection method of the kidney-tonifying, heat-clearing and blood-activating prescription comprises the following gradient elution procedures in the step 3 and the step 4:
the positive ion elution mode is:
flow rate/mL/min A/％ B/％
0.2 60 40
The anion elution mode is:
time/min Flow rate/mL/min A/％ B/％
0 0.2 62 38
2.5 0.2 62 38
2.6 0.2 20 80
10 0.2 20 80
10.1 0.2 62 38
30 0.2 62 38
The invention relates to a quality detection method of a kidney-tonifying, blood-clearing and blood-activating prescription, which comprises the following traditional Chinese medicine components: 264g of raw astragalus, 88 parts of Chinese angelica, 132 parts of giant knotweed, 264 parts of serissa serissoides, 264 parts of glabrous greenbrier rhizome, 88 parts of achyranthes bidentata, 176 parts of pyrrosia lingua, 53 parts of prepared rhubarb, 264 parts of centella asiatica, 176 parts of prepared rhizoma polygonati and 264 parts of abelmoschus manihot flower.
Optimization of detection conditions
1. Selection of mobile phase
In the positive ion mode, pure water, 0.1% formic acid, 0.2% formic acid and 0.3% formic acid are selected as the water phase, methanol and acetonitrile are selected as the organic phase, different combinations and proportions of the water phase and the organic phase are examined, and finally 0.1% formic acid is determined: acetonitrile=60:40 as mobile phase, under this condition, each component had better chromatographic peak shape and better degree of separation.
In the negative ion mode, the water phase selects pure water and 1 mmol.L -1 Aqueous solution of methyl/ammonium acetate, 2 mmol.L -1 Aqueous solution of methyl/ammonium acetate, 5 mmol.L -1 The aqueous solution of methyl/ammonium acetate and the organic phase are selected from methanol and acetonitrile, and different combinations and proportions of the aqueous phase and the organic phase are examined to finally determine 2 mmol.L -1 The ammonium formate aqueous solution (water phase) -acetonitrile (organic phase) is eluted in a gradient way, and under the condition, the chromatographic peak shape of each component is good, and the separation degree is good.
2. Column temperature and flow rate selection
This researchThe column temperatures (25, 30, 35, 40 ℃) and the flow rates (0.2, 0.3, 0.4 mL. Min) were examined -1 ) The effect on chromatographic peaks shows that the column temperature is 40 ℃ and the flow rate is 0.2 mL-min -1 The obtained chromatographic peak has good peak shape, number and separation degree.
The beneficial effects of the invention are as follows:
(1) Because the kidney-tonifying, heat-clearing and blood-activating prescription has 11 traditional Chinese medicine compositions, the chemical components are very complex, and the prescription contains various structural compounds such as anthraquinone, flavone, aromatic acid, sterone and the like, the effective separation cannot be carried out under the conventional chromatographic conditions, and the sensitivity of detection by adopting an ultraviolet detector is not high. The invention screens out the optimal preparation method of the test sample and the preparation method of the reference substance, the optimal chromatographic separation condition and the high-sensitivity mass spectrometry analysis condition through a large number of experiments, and the established method for measuring the content of the kidney-tonifying, blood-activating and blood-activating prescription can simultaneously detect the content of 11 compounds with different structural types, such as resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin, hyperoside, calycosin glucoside, beta-ecdysterone and the like. The method can effectively characterize the quality of the kidney-tonifying, heat-clearing, blood-activating prescription, and is beneficial to comprehensively monitoring the quality of the kidney-tonifying, heat-clearing, blood-activating prescription and ensuring the clinical curative effect.
(2) The method has the advantages of good stability, high precision, high accuracy, good repeatability and the like. The quality of the kidney-tonifying, heat-clearing, blood-activating prescription can be comprehensively, objectively and accurately evaluated and controlled.
Drawings
FIG. 1 shows the molecular formulas of chlorogenic acid, polydatin, astilbin and aloe-emodin in the formula of tonifying kidney, clearing away heat and promoting blood circulation, the chromatographic peak of a standard substance and the peak Fang Sepu of tonifying kidney, clearing away heat and promoting blood circulation.
FIG. 2 shows the molecular formula of rhein, hyperin, emodin, formononetin, chromatographic peaks of standard substances and Fang Sepu peaks of kidney tonifying, blood activating and heat clearing effects.
Figure 3 shows the molecular formula of resveratrol, calycosin glucoside, beta-ecdysterone, standard substance chromatographic peak and kidney tonifying, blood circulation promoting and Fang Sepu peak. The abscissa is the peak time of the compound and the ordinate is the ionic current intensity.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, which are not to be construed as specific conditions, either as normal conditions or as recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The following examples of drugs and reagents:
resveratrol (B20044, HPLC. Gtoreq.98%), rhein (B20245, HPLC. Gtoreq.98%), formononetin (B20836, HPLC. Gtoreq.98%), aloe-emodin (B20772, HPLC. Gtoreq.98%), emodin (B20240, HPLC. Gtoreq.98%), chlorogenic acid (B20782, HPLC. Gtoreq.98%), polydatin (B20533, HPLC. Gtoreq.98%), astilbin (B20812, HPLC. Gtoreq.98%), hyperin (B20631, HPLC. Gtoreq.98%) and beta-ecdysterone (B21268, HPLC. Gtoreq.98%) were purchased from Shanghai-derived leaf biotechnology Co., ltd; calycosin glucoside (C823639, HPLC. Gtoreq.98%) was purchased from Shanghai Meilin Biotechnology Co. Methanol and acetonitrile were purchased from moesadong biotechnology company.
The main experimental instrument comprises: HPLC-MS/MS detection: agilent1200 high performance liquid chromatograph (Agilent, USA); SCIEX API4000 triple quadrupole mass spectrometer (absiex) equipped with electrospray ionization source (Electro-SprayIonization, ESI); chromatography workstation: analyst1.6; aurashellC18 column 2.1 mm. Times.150 mm,2.7 μm (Waters, USA); MICRO-17R cryocentrifuge (Thermo, USA); drict-Q5 ultra pure water machine (Millipore, USA).
Example 1
1. A quality detection method of a kidney-tonifying, heat-clearing and blood-activating prescription comprises the following steps:
step 1: preparation of kidney-tonifying, blood-activating and heat-clearing prescription sample solution
Weighing a certain amount of kidney-tonifying, heat-clearing and blood-activating prescription traditional Chinese medicines (264 parts of raw astragalus mongholicus, 88 parts of angelica sinensis, 132 parts of polygonum cuspidatum, 264 parts of serissa serissoides, 264 parts of glabrous greenbrier rhizome, 88 parts of achyranthes bidentata, 176 parts of pyrrosia lingua, 53 parts of prepared rheum officinale, 264 parts of centella asiatica, 176 parts of prepared rhizoma polygonati and 264 parts of abelmoschus manihot), appropriately crushing, dissolving in a 25mL volumetric flask with 50% methanol, adding 50% methanol for volume fixation, and carrying out ultrasonic extraction for 20min to obtain kidney-tonifying, heat-clearing and blood-activating prescription extract; precisely transferring 5mL of extract of the kidney-tonifying, blood-activating and clearing prescription into a 100mL volumetric flask, fixing the volume by 50% methanol, shaking uniformly, centrifuging at 12000r/min for 10min, and taking supernatant to pass through a 0.22 mu m filter membrane to obtain the sample solution of the kidney-tonifying, blood-activating and clearing prescription.
Step 2: preparation of standard solution
Precisely weighing appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin, hyperoside, calycosin glucoside, beta-ecdysterone reference substance in 10mL volumetric flask, dissolving with methanol, diluting to scale, shaking to obtain concentrations of 2.21, 1.07, 1.32, 1.22, 1.11, 1.29, 1.10, 1.64, 1.22, 1.12, 1.52mg·mL respectively -1 The control stock solution is preserved at the temperature of minus 20 ℃ for standby;
precisely sucking appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside control stock solution into the same 10mL volumetric flask, adding methanol to constant volume to scale, shaking to obtain solutions with concentrations of 256.00, 640.00, 640.00, 640.00, 1120.00, 2080.00, 640.00, 3200.00 and 3680.00 ng.mL respectively -1 Is mixed with the working solution of the reference substance;
precisely sucking appropriate amount of calycosin glucoside and beta-ecdysterone stock solution into the same 10mL volumetric flask, adding methanol to constant volume to scale, shaking to obtain solutions with concentrations of 1600.00 and 3200.00ng·mL respectively -1 Is mixed with the working solution of the reference substance.
Step 3, preparation of standard curve
Negative ion mode: diluting the working solution of the mixed reference substances of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside prepared in the step 2 into mixed reference substance solutions with different concentrations by a multiple ratio, and taking 5 mu L of each solution for HPLC-MS/MS measurement and analysis;
positive ion mode: diluting the working solution of the calycosin glucoside and beta-ecdysterone mixed reference substance prepared in the step 2 by a multiple ratio into mixed reference substance solutions with different concentrations, and taking 5 mu L of each solution for HPLC-MS/MS determination analysis;
standard curve calculation: carrying out linear regression on the linear concentration of each standard substance combined with the detection peak area to obtain a standard curve equation, wherein the calculation is completed by analyst1.6 software;
the standard curve equation obtained is shown in table 1 below:
TABLE 1
Object of detection Standard curve equation
Resveratrol y＝0.0482x+0.00172
Rhein y＝0.0449x+0.068
Formononetin-like element y＝0.642x+0.622
Aloe-emodin y＝0.12x+0.04
Emodin y＝0.862x+0.00368
Chlorogenic acid y＝0.353x+0.18
Polygonum cuspidatum glycoside y＝0.0317x+0.0433
Astilbin y＝0.0129x+0.123
Hyperin y＝0.0201x+0.201
Calycosin glucoside y＝0.0795x+0.132
Beta-ecdysterone y＝0.0016x+0.00242
Step 4, content measurement
And (3) injecting the sample solution of the kidney-tonifying, clearing and activating blood-activating prescription prepared in the step (1) into an HPLC-MS/MS (high performance liquid chromatography-mass spectrometry) for determination and analysis under the same chromatographic and mass spectrometry conditions, substituting the detected peak area of the compound into a standard curve equation of the step (3), and calculating to obtain the content of each compound in the kidney-tonifying, clearing and activating blood-activating prescription, as shown in the following table 2.
TABLE 2 content of Compounds (mg.g) of the recipe for tonifying Kidney, clearing away the heat-evil, promoting blood circulation -1 )
Names of Compounds Content (mg.g) -1 )
Resveratrol 0.145
Rhein 0.024
Formononetin-like element 0.014
Aloe-emodin 0.014
Emodin 0.133
Chlorogenic acid 0.250
Polygonum cuspidatum glycoside 0.515
Astilbin 2.143
Hyperin 0.543
Calycosin glucoside 0.030
Beta-ecdysterone 0.053
。
The chromatographic conditions of the step 3 and the step 4 are as follows: chromatographic column: waters AurashellC18 with a specification of 2.1mm by 150mm and 2.7 μm;
positive ion mode mobile phase: 0.1% formic acid/acetonitrile, gradient elution; the flow rate is 0.2mL/min; column temperature: 40 ℃; ion source: ESI; quantitative mode: MRM; ion source voltage: 5500V; ion source temperature: 400 ℃;
negative ion mode mobile phase: 2mmol/L ammonium formate/acetonitrile, gradient elution; the flow rate is 0.2mL/min; column temperature: 40 ℃; ion source: ESI; quantitative mode: MRM; ion source voltage: -4000V; ion source temperature: 400 ℃.
Gradient elution procedure is as follows table 3 and table 4:
TABLE 3 Positive ion elution mode
Flow rate/mL/min A/％ B/％
0.2 60 40
TABLE 4 anion elution pattern
Time/min Flow rate/mL/min A/％ B/％
0 0.2 62 38
2.5 0.2 62 38
2.6 0.2 20 80
10 0.2 20 80
10.1 0.2 62 38
30 0.2 62 38
。
The mass spectrum conditions are as follows:
detecting analytes in positive and negative ion modes using an API4000 tandem quadrupole mass spectrometer and electrospray ionization interface; the conversion of the precursor to the product ion is monitored by adopting a multi-reaction monitoring mode, and the set parameters are as follows: ion spray voltage: 4000V; turbine heater temperature: 500 ℃; collision activation dissociation: 10psi; air curtain gas: 25psi; specific mass spectrometry conditions are shown in table 4 below:
table 4 conditions for mass spectrometry detection of standard substances
Object of detection Quantitative ion pair m/z DP/V) EP/V CE/V CXP/V
Resveratrol 226.8→142.8 -100 -10 -33 -14
Rhein 238.6→182.7 -100 -10 -27 -14
Formononetin-like element 266.7→251.9 -100 -10 -30 -14
Aloe Vera(Flavin) 268.7→239.8 -90 -10 -33 -14
Emodin 268.8→225.0 -110 -10 -35 -14
Chlorogenic acid 353.2→190.9 -55 -10 -20 -14
Polygonum cuspidatum glycoside 389.2→227.4 -100 -10 -20 -14
Astilbin 449.3→285.2 -110 -10 -34 -14
Hyperin 463.4→300.1 -94 -10 -34 -14
Calycosin glucoside 447.4→285.3 70 10 22 14
Beta-ecdysterone 481.4→371.3 90 10 20 14
。
2. Methodology investigation
(1) Precision test
Taking a sample of the kidney-tonifying, blood-activating and heat-clearing prescription, preparing a sample solution according to the method in the step 1, continuously sampling and measuring for 6 times according to the chromatographic conditions, and calculating to obtain RSD (reactive power detector) of which the relative retention time is less than 1.2% (n=6) and RSD of which the relative peak area is less than 2.3% (n=6) by taking resveratrol as a reference substance, wherein the method is good in precision.
(2) Stability test
Taking samples of the kidney-tonifying, heat-clearing and blood-activating prescription, preparing test sample solutions according to the method in the step 1, respectively placing the test sample solutions for 0, 2, 4, 8, 12 and 24 hours at room temperature, carrying out sample injection measurement according to the chromatographic conditions, taking resveratrol as a reference substance, calculating RSD of the relative peak areas to be less than 1.7 percent (n=6) by taking the resveratrol as the reference substance, and calculating RSD of the relative peak areas to be less than 1.7 percent (n=6), wherein the stability of the test sample solutions at room temperature in 24 hours is good.
(3) Repeatability test
Taking 6 parts of samples of the kidney-tonifying, blood-activating and heat-clearing prescription, preparing a sample solution according to the method in the step 1, and then carrying out sample injection measurement according to the chromatographic conditions, wherein the RSD (reactive power) of the resveratrol is less than 1.1 percent (n=6) relative to the retention time, and the RSD of the calculated relative peak area is less than 2.60 percent (n=6), so that the method is good in repeatability.
The experimental results show that the detection method of the kidney-tonifying, clearing and activating blood circulation prescription has good precision, stability and repeatability, can effectively characterize the quality of the kidney-tonifying, clearing and activating blood circulation prescription, is beneficial to comprehensively monitoring the quality and ensures the clinical curative effect.

Claims (8)

1. The quality detection method of the kidney-tonifying, heat-clearing and blood-activating prescription is characterized by comprising the following steps of:
step 1: preparation of kidney-tonifying, blood-activating and heat-clearing prescription sample solution
Weighing kidney-tonifying, blood-activating and heat-clearing traditional Chinese medicine, appropriately crushing, dissolving in a volumetric flask by using a methanol solution, adding methanol for volume fixation, and performing ultrasonic extraction to obtain kidney-tonifying, blood-activating and heat-clearing traditional Chinese medicine extract; precisely transferring a certain amount of extract of the kidney-tonifying, blood-activating and clearing prescription into a volumetric flask, fixing the volume of a methanol solution, shaking uniformly, centrifuging, and taking supernatant to pass through a 0.22 mu m filter membrane to obtain a sample solution of the kidney-tonifying, blood-activating and clearing prescription;
step 2: preparation of standard solution
Precisely weighing appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin, hyperoside, calycosin glucoside, beta-ecdysterone reference substance in 10mL volumetric flask, adding methanol to dissolve and dilute to scale, shaking to obtain reference stock solution with certain concentration;
precisely sucking appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside reference stock solution into the same volumetric flask, adding methanol to constant volume to scale, and shaking to obtain mixed reference working solution with certain concentration;
precisely sucking appropriate amount of calycosin glucoside and beta-ecdysterone stock solution into the same volumetric flask, adding methanol to constant volume to scale, and shaking to obtain mixed control working solution of calycosin glucoside and beta-ecdysterone at certain concentration;
step 3, preparation of standard curve
Negative ion mode: diluting the working solution of the mixed reference substances of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside prepared in the step 2 into mixed reference substance solutions with different concentrations by a multiple ratio, and taking 5 mu L of each solution for HPLC-MS/MS measurement and analysis;
positive ion mode: diluting the working solution of the calycosin glucoside and beta-ecdysterone mixed reference substance prepared in the step 2 by a multiple ratio into mixed reference substance solutions with different concentrations, and taking 5 mu L of each solution for HPLC-MS/MS determination analysis;
standard curve calculation: performing linear regression on the linear concentration of each standard substance combined with the detection peak area to obtain a standard curve equation;
step 4, content measurement
And (3) injecting the sample solution of the kidney-tonifying, clearing and activating blood-activating prescription prepared in the step (1) into an HPLC-MS/MS (high performance liquid chromatography-mass spectrometry) for determination and analysis under the same chromatographic and mass spectrometry conditions, substituting the detected peak area of the compound into a standard curve equation in the step (3), and calculating to obtain the content of each compound in the kidney-tonifying, clearing and activating blood-activating prescription.
2. The quality detection method of the kidney-tonifying, heat-clearing and blood-activating prescription according to claim 1, which is characterized in that,
the preparation method of the sample solution of the kidney-tonifying, blood-activating prescription comprises the following steps:
weighing a certain amount of traditional Chinese medicines with the functions of tonifying kidney, clearing away heat, activating blood circulation, properly crushing, dissolving in a 25mL volumetric flask by using 50% methanol, adding 50% methanol for volume fixation, and carrying out ultrasonic extraction for 20min to obtain an extract of the traditional Chinese medicines with the functions of tonifying kidney, clearing away heat, activating blood circulation; precisely transferring 5mL of extract of the kidney-tonifying, blood-activating and clearing prescription into a 100mL volumetric flask, fixing the volume by 50% methanol, shaking uniformly, centrifuging at 12000r/min for 10min, and taking supernatant to pass through a 0.22 mu m filter membrane to obtain the sample solution of the kidney-tonifying, blood-activating and clearing prescription.
3. The quality detection method of a kidney-tonifying, heat-clearing and blood-activating prescription according to claim 1, which is characterized by comprising the following steps: the preparation method of the standard substance solution comprises the following steps:
precisely weighing appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin, hyperoside, calycosin glucoside, beta-ecdysterone reference substance in 10mL volumetric flask, dissolving with methanol, diluting to scale, shaking to obtain concentrations of 2.21, 1.07, 1.32, 1.22, 1.11, 1.29, 1.10, 1.64, 1.22, 1.12, 1.52mg·mL respectively -1 The control stock solution is preserved at the temperature of minus 20 ℃ for standby;
precisely sucking appropriate amount of resveratrol, rhein, formononetin, aloe-emodin, rhein, chlorogenic acid, polydatin, astilbin and hyperoside control stock solution into the same 10mL volumetric flask, adding methanol to constant volume to scale, shaking to obtain solutions with concentrations of 256.00, 640.00, 640.00, 640.00, 1120.00, 2080.00, 640.00, 3200.00 and 3680.00 ng.mL respectively -1 Is mixed with the working solution of the reference substance;
precisely sucking appropriate amount of calycosin glucoside and beta-ecdysterone stock solution into the same 10mL volumetric flask, adding methanol to constant volume to scale, shaking to obtain solutions with concentrations of 1600.00 and 3200.00ng·mL respectively -1 Is mixed with the working solution of the reference substance.
4. The method for detecting the quality of the kidney-tonifying, heat-clearing and blood-activating prescription according to claim 1, wherein the chromatographic conditions of the step 3 and the step 4 are as follows: chromatographic column: watersAurashell C18 with a specification of 2.1mm by 150mm and 2.7 μm;
positive ion mode mobile phase: 0.1% formic acid/acetonitrile, isocratic elution; the flow rate is 0.2mL/min; column temperature: 40 ℃; ion source: ESI; quantitative mode: MRM; ion source voltage: 5500V; ion source temperature: 400 ℃;
negative ion mode mobile phase: 2mmol/L ammonium formate/acetonitrile, gradient elution for 3min; the flow rate is 0.2mL/min; column temperature: 40 ℃; ion source: ESI; quantitative mode: MRM; ion source voltage: -4000V; ion source temperature: 400 ℃.
5. The mass detection method of the kidney-tonifying, heat-clearing and blood-activating prescription according to claim 1, wherein mass spectrometry conditions of the step 3 and the step 4 are as follows:
detecting analytes in positive and negative ion modes using an API4000 tandem quadrupole mass spectrometer and electrospray ionization interface; the conversion of the precursor to the product ion is monitored by adopting a multi-reaction monitoring mode, and the set parameters are as follows: ion spray voltage: 4000V; turbine heater temperature: 500 ℃; collision activation dissociation: 10psi; air curtain gas: 25psi; specific mass spectrometry detection conditions are shown in the following table:
standard mass spectrum detection condition
Object of detection Quantitative ion pair m/z DP/V) EP/V CE/V CXP/V Resveratrol 226.8→142.8 -100 -10 -33 -14 Rhein 238.6→182.7 -100 -10 -27 -14 Formononetin-like element 266.7→251.9 -100 -10 -30 -14 Aloe-emodin 268.7→239.8 -90 -10 -33 -14 Emodin 268.8→225.0 -110 -10 -35 -14 Chlorogenic acid 353.2→190.9 -55 -10 -20 -14 Polygonum cuspidatum glycoside 389.2→227.4 -100 -10 -20 -14 Astilbin 449.3→285.2 -110 -10 -34 -14 Hyperin 463.4→300.1 -94 -10 -34 -14 Calycosin glucoside 447.4→285.3 70 10 22 14 Beta-ecdysterone 481.4→371.3 90 10 20 14
。
6. The method for detecting the quality of a kidney-tonifying, blood-activating prescription according to claim 4, wherein the elution procedure is as follows:
the positive ion elution pattern is as follows:
flow rate/mL/min A/％ B/％ 0.2 60 40
The anion elution pattern is as follows:
time/min Flow rate/mL/min A/％ B/％ 0 0.2 62 38 2.5 0.2 62 38 2.6 0.2 20 80 10 0.2 20 80 10.1 0.2 62 38 30 0.2 62 38
。
7. The method for detecting the quality of the kidney-tonifying, blood-activating prescription according to claim 4, wherein the standard curve equation is:
object of detection Standard curve equation Resveratrol y＝0.0482x+0.00172 Rhein y＝0.0449x+0.068 Formononetin-like element y＝0.642x+0.622 Aloe-emodin y＝0.12x+0.04 Emodin y＝0.862x+0.00368 Chlorogenic acid y＝0.353x+0.18 Polygonum cuspidatum glycoside y＝0.0317x+0.0433 Astilbin y＝0.0129x+0.123 Hyperin y＝0.0201x+0.201 Calycosin glucoside y＝0.0795x+0.132 Beta-ecdysterone y＝0.0016x+0.00242
。
8. The quality detection method of the kidney-tonifying, blood-clearing and blood-activating prescription according to claim 1, wherein the traditional Chinese medicine composition of the kidney-tonifying, blood-clearing and blood-activating prescription is as follows: 264 parts of raw astragalus, 88 parts of Chinese angelica, 132 parts of giant knotweed, 264 parts of serissa serissoides, 264 parts of glabrous greenbrier rhizome, 88 parts of achyranthes bidentata, 176 parts of pyrrosia lingua, 53 parts of prepared rhubarb, 264 parts of centella asiatica, 176 parts of prepared rhizoma polygonati and 264 parts of abelmoschus manihot flower.
CN202311350918.4A 2023-10-18 2023-10-18 Quality detection method of kidney-tonifying, heat-clearing and blood-activating prescription Pending CN117388397A (en)

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