CN107688067B - Content determination method of orifice-opening rhinitis tablets - Google Patents
Content determination method of orifice-opening rhinitis tablets Download PDFInfo
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Abstract
The invention relates to a content determination method of a resuscitation-inducing rhinitis tablet, in particular to a content determination method of active ingredients of linarin glycoside, 5-O-methylvismaidin glycoside, calycosin glycoside, magnolin, imperatorin and isoimperatorin in the resuscitation-inducing rhinitis tablet, which comprises the steps of preparation of a test solution, preparation of a reference solution and UPLC method determination. By adopting the content determination method provided by the invention, during determination, the detection baseline is stable, the separation degree is good, the detection accuracy is high, and the stability is good; the content determination time is greatly shortened, the detection efficiency is stably improved, the dosage of toxic chemical reagents is greatly reduced, and the method is more economic and environment-friendly; and simultaneously, multi-index component detection is carried out, a rapid, efficient and feasible detection method is provided for establishing a liquid-phase fingerprint atlas database of the orifice-opening rhinitis tablet, and a relatively comprehensive quantitative comparison investigation method for the stability of the effective components is easily provided for the stability investigation of the orifice-opening rhinitis tablet.
Description
Technical Field
The invention relates to content determination of Chinese patent medicines, and in particular relates to a method for determining the content of active ingredients of linarin glycoside, 5-O-methylvisammioside, calycosin glycoside, magnolin, imperatorin and isoimperatorin in an orifice-opening rhinitis tablet.
Background
The rhinitis tablet for dredging orifice is a pure Chinese medicinal compound preparation prepared by extracting six Chinese medicinal materials of fried cocklebur fruit, divaricate saposhnikovia root, astragalus, dahurian angelica root, biond magnolia flower, fried largehead atractylodes rhizome, mint and the like, and has the effects of dispelling wind, consolidating exterior, ventilating lung and dredging orifice. Can be used for treating nasal obstruction, thin or thick nasal discharge, forehead headache due to wind-heat accumulation in lung and exterior deficiency; chronic rhinitis, allergic rhinitis, sinusitis, etc. The main effective components of the orifice-opening rhinitis tablet are as follows: cimicidin, 5-O-methylvisammioside, calycosin glucoside, imperatorin, isoimperatorin, magnolin, etc.
However, the important effective components in the resuscitation inducing rhinitis do not realize more comprehensive quality detection through scientific detection.
The original standard of the orifice-opening rhinitis tablet is collected in the first part of the 2015 edition of pharmacopoeia of the people's republic of China, and only the content of imperatorin in the standard is detected. The identification and content determination methods of effective components in the orifice-opening rhinitis tablets are reported in the existing literature, but most of the identification and content determination methods are complicated in operation and long in time consumption, only one medicinal material or one component in the orifice-opening rhinitis tablets is quantitatively controlled, the orifice-opening rhinitis tablet product cannot be comprehensively qualitatively and quantitatively controlled, and simultaneous multi-component index detection cannot be realized, so that the quality of the orifice-opening rhinitis tablet product cannot be scientifically controlled.
The existing literature, namely HPLC-MS method for simultaneously determining the content of 6 components in the radix angelicae and nasopharynx granules, is modified by ChinJPharmAnal2014.34 (5) and Liu Ling from J.PharmInalysis of pharmaceutical analysis; the method is a liquid chromatography-mass spectrometry method, in actual operation, a liquid phase eluted by a mobile phase needs to be started, a mass spectrum with high alcohol nitrogen as atomizing gas needs to be started, the detection time is 30 minutes, the efficiency is low, the consumption of organic solvents and high-purity nitrogen is high, the detection cost is high, and the liquid chromatography-mass spectrometry is easy to generate a matrix effect to influence the reproducibility and the accuracy of detection.
The existing literature, HPLC method for simultaneously determining the content of 5 effective components in the four-season cold tablet, china Pharmacist 2015 3 rd part of volume 18 of 2015, vol.18No.3, zheng Zhaoxian; zhao Dantong, etc. discloses a method for simultaneously determining the content of 5 active ingredients in a cold tablet for four seasons by using an HPLC method, wherein the method simultaneously determines the content of 5 ingredients in a Chinese medicinal preparation, but the method has the advantages of long peak time, long detection time up to 50 minutes, low efficiency and large organic solvent consumption in the actual operation process. And only two components are the same as the present invention.
The existing document 5 components in the resuscitation-inducing rhinitis tablet are simultaneously determined by the RP-HPLC method, wherein the university of northwest (Nature science edition) in 2015, 4 months, 45 th volume, 2 nd stage, apr.,2015, vo1.45, no.2, zhang Qian, wang Shixiang and the like disclose a method for simultaneously determining the content of the 5 components in the resuscitation-inducing rhinitis tablet by using the RP-HPLC method, the detection time of the method is 80 minutes, the efficiency is too low, and the five components are all derived from angelica dahurica, but the effective components of other medicinal materials in the resuscitation-inducing rhinitis tablet are not quantitatively controlled, so that the optimal detection method for detecting the content of the non-resuscitation-inducing rhinitis tablet is realized.
A method for measuring the content of imperatorin in angelica dahurica in rhinitis-relieving tablets is disclosed in the first part of the 2015 edition of pharmacopoeia of the people's republic of China, and comprises the following steps: "chromatographic conditions and System suitability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-water (54; the detection wavelength was 248nm. The number of theoretical plates is not less than 5000 according to the calculation of imperatorin peak, and the preparation of a reference solution: taking a proper amount of imperatorin reference substance, precisely weighing, and adding methanol to prepare a solution containing 15 μ g per 1 mL. Preparation of a test solution: taking 20 tablets of the product, removing sugar coating of sugar-coated tablets, precisely weighing, grinding, taking about 2g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of methanol, weighing, ultrasonically treating (with the power of 250w and the frequency of 50 Hz) for 30min, cooling, weighing again, complementing the lost weight with methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. The chromatographic conditions of the method are obviously different from those of the method, and the measured components are only limited to imperatorin in angelica dahurica.
A method for detecting content of resuscitation inducing rhinitis capsule is disclosed in pharmacopoeia of the people's republic of China 2015, which has detection indexes including imperatorin in radix Angelicae Dahuricae and astragaloside IV in radix astragali. The method for measuring the content of astragaloside comprises the following steps: "chromatographic conditions and System suitability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-water (32; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 3000 calculated according to astragaloside IV, and the preparation of reference solution: taking appropriate amount of astragaloside IV reference substance, precisely weighing, and adding methanol to obtain solution containing 0.5mg per 1 mL. Preparation of a test solution: taking 30 granules of the product, pouring out the content, precisely weighing, grinding, taking about 4g, precisely weighing, placing in a soxhlet extractor, adding 50mL of methanol, heating and refluxing for 4 hours, recovering the solvent from the extracting solution and concentrating to dryness, adding 10mL of water into the residue, slightly heating to dissolve, shaking and extracting with water-saturated n-butyl alcohol for 4 times, 40mL each time, combining the n-butyl alcohol solution, fully washing with ammonia test solution for 2 times, 40mL each time, discarding the ammonia solution, recovering the solvent from the n-butyl alcohol solution until dry, adding 5mL of water into the residue to dissolve, cooling, passing through a D101 type macroporous resin column (the inner diameter is 1.5cm, the column height is 12 cm), eluting with 50mL of water, discarding the water solution, eluting with 30mL of 40% ethanol, discarding the eluent, sequentially eluting with 80mL of 70% ethanol, collecting, recovering the solvent until dry, dissolving with methanol, transferring to a 5mL volumetric flask, adding methanol until the eluent is scaled, and shaking uniformly to obtain the product. The determination method comprises the following steps: precisely sucking 5 μ L and 15 μ L of reference solution and 10 μ L of test solution, respectively, injecting into liquid chromatograph, measuring, and calculating with external standard two-point method logarithmic equation. The preparation method of the test sample is complex and takes long time. Although it controls two content detection indexes, two different detection methods are adopted, so that the cost is high, the time consumption is long, and the efficiency is low.
The above methods can not detect 6 effective components in the orifice-opening rhinitis tablet at the same time. The methods have long detection time, low efficiency and large organic solvent consumption, and can not quickly and accurately perform quantitative analysis on the components of the multi-flavor medicine of the orifice-opening rhinitis tablet.
In order to better detect the content of effective components in the orifice-opening rhinitis tablet and improve the quality of the orifice-opening rhinitis tablet, researches provide a content detection method of the orifice-opening rhinitis tablet, which has the advantages of simple operation, strong specificity, good separation degree, good stability, high efficiency and environmental protection, and can simultaneously perform multi-index component control, and is imperative.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a content determination method aiming at the active ingredients of the linarin glycoside, the 5-O-methylvismaidin glycoside, the calycosin glycoside, the magnolin, the imperatorin and the isoimperatorin in the resuscitation rhinitis tablet, and the content determination method provided by the invention has the advantages of stable detection baseline, good separation degree, high detection accuracy and good stability during detection; the content determination time is greatly shortened, the detection efficiency is stably improved, the dosage of toxic chemical reagents is greatly reduced, and the method is more economic and environment-friendly; and simultaneously, the detection of multiple index components is carried out, a rapid, efficient and feasible detection method is provided for the establishment of a liquid-phase fingerprint spectrum library of the orifice-opening rhinitis tablet, and a relatively comprehensive quantitative comparison investigation method for the stability of the effective components is easily provided for the stability investigation of the orifice-opening rhinitis tablet.
Specifically, the invention provides a method for measuring the content of active ingredients of the cimicifuga glycoside, 5-O-methylvisammioside, calycosin, magnolin, imperatorin and isoimperatorin in the resuscitation rhinitis tablet, which comprises measuring the content of the active ingredients of the cimicifuga glycoside, 5-O-methylvisammioside, calycosin, magnolin, imperatorin and isoimperatorin in the preparation; the content determination comprises the following steps: preparing a test solution, preparing a reference solution and measuring by a UPLC method.
Preferably, the chromatography conditions measured by the UPLC method are: waters BEH C18 column; the mobile phase A is acetonitrile, the mobile phase B is acetic acid with volume fraction of 0.4 per mill to 0.8 per mill, and the gradient elution method is adopted; the flow rate is 0.2-0.5 mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃.
In the chromatographic conditions, the inner diameter of the Waters BEH C18 column was 50 mm. Times.2.1mm, 1.7 μm, in order to achieve more excellent separation effect.
As a preferred embodiment, the gradient elution method is: 0-2.6min, 10-20% → 60% -80%; 2.6 to 3.5min,60 to 80% → 80 to 100% by weight A; 3.5-4.0min, 80% -100% of A;4.0 to 4.5min,80 to 100% → 10 to 20% by weight A;4.5 to 7min,10 to 20% by weight A.
In order to improve the separation degree and achieve the simultaneous detection of the contents of 6 different effective components, it is further preferred that the gradient elution method is: 0-2.6 min,18% → 70% by weight A;2.6 to 3.5min,70% → 95% A; 3.5-4.0min, 95% by weight of A;4.0 to 4.5min,95% → 18% by volume A; 4.5-7min, 18% A.
Preferably, the preparation method of the test solution comprises the following steps: taking 1-3 g of orifice-opening rhinitis tablet powder, precisely weighing, placing in a 100-200 mL triangular flask with a plug or volumetric flask, precisely adding 30-60 mL of methanol with the volume fraction of 60-80%, weighing, carrying out ultrasonic extraction at 100W and 25kHz for 30-60 min, cooling, weighing again, supplementing the weight loss by using the methanol with the volume fraction of 60-80%, filtering by using a microporous filter membrane, and taking the subsequent filtrate as a test solution.
More preferably, the preparation method of the test solution is as follows: taking 1.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing in a 100mL triangular flask with a plug or volumetric flask, precisely adding 50mL of methanol with volume fraction of 80%, weighing, ultrasonic extracting at 100W and 25kHz for 45min, cooling, weighing again, complementing the weight loss by methanol with volume fraction of 80%, filtering by a 0.22 mu m microporous membrane, and taking the subsequent filtrate as a test solution.
The reference substance solution comprises a cimicidin-glycoside reference substance solution, a 5-O-methylvisammioside reference substance solution, a calycosin-glucoside reference substance solution, an imperatorin reference substance solution, an isoimperatorin reference substance solution and a magnolin reference substance solution.
The preparation method of the cimicidin reference solution comprises the following steps: taking a cimicidin reference substance, adding methanol to dissolve until the concentration is 45-55 mug/mL for standby; the preparation method of the 5-O-methylvisammioside reference substance solution comprises the following steps: taking a 5-O-methylvisammioside reference substance, adding methanol to dissolve the reference substance until the concentration is 75-85 mug/mL for later use; the preparation method of the calycosin glucoside reference substance solution comprises the following steps: taking a calycosin glucoside reference substance, adding methanol to dissolve the calycosin glucoside reference substance until the concentration is 45-55 mug/mL for later use; the preparation method of the imperatorin reference substance solution comprises the following steps: taking an imperatorin reference substance, adding methanol to dissolve until the concentration is 55-65 mu g/mL for later use; the preparation method of the isoimperatorin reference substance solution comprises the following steps: adding methanol into an isoimperatorin reference substance to be dissolved until the concentration is 180-220 mug/mL for later use; the preparation method of the magnolin reference substance solution comprises the following steps: taking a magnolin reference substance, adding methanol to dissolve the magnolin reference substance until the concentration is 45-55 mug/mL for later use; the prepared linarin glycoside reference substance solution, 5-O-methylvisammol glycoside reference substance solution, calycosin glucoside reference substance solution, imperatorin reference substance solution, isoimperatorin reference substance solution and magnolin reference substance solution are prepared into 6-component mixed reference substance solution according to the following ratio of 2-6:5-9:2-8:2-8.
Preferably, the preparation method of the cimicidin reference solution comprises the following steps: dissolving the cimicidin reference substance with methanol to a concentration of 50 μ g/mL for use; the preparation method of the 5-O-methylvisammioside reference substance solution comprises the following steps: dissolving 5-O-methylvisammioside reference substance in methanol to a concentration of 80 μ g/mL; the preparation method of the calycosin glucoside reference solution comprises the following steps: taking a calycosin glucoside reference substance, adding methanol to dissolve until the concentration is 50 mug/mL for standby; the preparation method of the imperatorin reference substance solution comprises the following steps: dissolving imperatorin reference substance in methanol to concentration of 60 μ g/mL; the preparation method of the isoimperatorin reference substance solution comprises the following steps: dissolving isoimperatorin reference substance in methanol to concentration of 200 μ g/mL; the preparation method of the magnolin reference substance solution comprises the following steps: dissolving magnolin control in methanol to 50 μ g/mL; the prepared linarin control solution, 5-O-methylvisammol glycoside control solution, calycosin glucoside control solution, imperatorin control solution, isoimperatorin control solution and magnolin control solution were formulated into 6-component mixed control solutions in the following 5.
In a preferred embodiment of the present invention, the method for measuring the content of the active ingredients of the rhinitis-relieving tablet comprises the following steps:
(1) Preparing a test solution: taking 1-3 g of orifice-opening rhinitis tablet powder, precisely weighing, placing the powder into a 100-200 mL triangular flask or volumetric flask with a plug, precisely adding 30-60 mL of methanol with the volume fraction of 60-80%, weighing, ultrasonic extracting for 30-60 min at 100W and 25kHz, cooling, weighing again, supplementing the weight loss by using the methanol with the volume fraction of 60-80%, filtering by using a microporous filter membrane, and taking the subsequent filtrate as a test solution;
(2) Preparation of control solutions: taking a cimicidin reference substance, a 5-O-methylvisammol glycoside reference substance, a calycosin glucoside reference substance, an imperatorin reference substance, an isoimperatorin reference substance and a magnolin reference substance, adding methanol for dissolving, and respectively preparing a cimicin glycoside reference substance solution with the concentration of 45-55 mug/mL, a 5-O-methylvisammol glycoside reference substance solution with the concentration of 75-85 mug/mL, a calycosin glucoside reference substance solution with the concentration of 45-55 mug/mL, an imperatorin reference substance solution with the concentration of 55-65 mug/mL, an isoimperatorin reference substance solution with the concentration of 180-220 mug/mL and a magnolin reference substance solution with the concentration of 45-55 mug/mL for standby;
preparing a prepared linarin reference substance solution, a 5-O-methylvisammol glycoside reference substance solution, a calycosin glucoside reference substance solution, an imperatorin reference substance solution, an isoimperatorin reference substance solution and a magnolin reference substance solution into a mixed reference substance solution with 6 components according to the following ratio of 2-6:5-9:2-8:2-8;
(3) And (3) measuring by using a UPLC method: respectively and precisely sucking 1-5 μ L of each of the test solution and the mixed reference solution, injecting into an ultra-high performance liquid chromatograph, measuring, and calculating to obtain the final product;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50mm × 2.1mm,1.7 μm; the mobile phase A is acetonitrile, and the mobile phase B is acetic acid with volume fraction of 0.4-0.8 per mill; gradient elution: 0-2.6min, 10-20% → 60% -80%; 2.6-3.5min, 60% -80% → 80% -100% by weight A; 3.5-4.0min, 80% -100% of A;4.0 to 4.5min,80 to 100% → 10 to 20% by weight A; 4.5-7min, 10% -20% A; the flow rate is 0.2-0.5 mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃.
Further preferably, the method for measuring the content of the active ingredients of the rhinitis-relieving tablet comprises the following steps:
(1) Preparation of a test solution: taking 1.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing the powder into a 100mL triangular flask with a plug or volumetric flask, precisely adding 50mL of 80% methanol in volume fraction, weighing, ultrasonically extracting for 45min at 100W and 25kHz, cooling, weighing again, complementing the weight loss by 80% methanol in volume fraction, filtering by using a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate as a test solution;
(2) Preparation of control solutions: taking a cimicidin reference substance, a 5-O-methylvisammol glycoside reference substance, a calycosin glucoside reference substance, an imperatorin reference substance, an isoimperatorin reference substance and a magnolin reference substance, adding methanol for dissolving, and respectively preparing a cimicin glycoside reference substance solution with the concentration of 50 mug/mL, a 5-O-methylvisammol glycoside reference substance solution with the concentration of 80 mug/mL, a calycosin glucoside reference substance solution with the concentration of 50 mug/mL, an imperatorin reference substance solution with the concentration of 60 mug/mL, an isoimperatorin reference substance solution with the concentration of 200 mug/mL and a magnolin reference substance solution with the concentration of 50 mug/mL for standby;
preparing a 6-component mixed reference solution by mixing the prepared linarin reference solution, 5-O-methylvisammol glycoside reference solution, calycosin glucoside reference solution, imperatorin reference solution, isoimperatorin reference solution and magnolin reference solution according to the following proportion of 5;
(3) And (3) measuring by using a UPLC method: precisely sucking 1 μ L of each of the test solution and the mixed reference solution, injecting into an ultra-high performance liquid chromatograph, measuring, and calculating to obtain the final product;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50mm × 2.1mm,1.7 μm; the mobile phase A is acetonitrile, and the mobile phase B is acetic acid with volume fraction of 0.4-0.8 per mill; gradient elution: 0-2.6 min,18% → 70% by weight A;2.6 to 3.5min,70% → 95% A; 3.5-4.0min, 95 percent; 4.0 to 4.5min,95% → 18% by volume A; 4.5-7min, 18% by weight A; the flow rate is 0.2-0.5 mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃.
Through actual detection, the content of the linarin glycoside in the rhinitis-treating tablets for resuscitation is 300-500 mu g/g, the content of 5-O-methylvisammioside is 700-900 mu g/g, the content of calycosin glycoside is 150-300 mu g/g, the content of magnolin is 300-500 mu g/g, the content of imperatorin is 800-1000 mu g/g, and the content of isoimperatorin is 700-900 mu g/g.
Preferably, the content of the linarin glycoside in the orifice-opening rhinitis tablet is 413 mu g/g, the content of the 5-O-methylvisammioside is 830 mu g/g, the content of the calycosin glycoside is 254 mu g/g, the content of the magnolin is 485 mu g/g, the content of the imperatorin is 955 mu g/g, and the content of the isoimperatorin is 873 mu g/g.
The invention realizes the following beneficial effects:
1. the method has the advantages that the ultra-efficient method is established for the first time, the 6 effective components in the orifice-opening rhinitis tablet are detected simultaneously, the detection of multi-index components is realized, the rapid, efficient and feasible detection method is provided for the establishment of the liquid-phase fingerprint atlas database of the orifice-opening rhinitis tablet, the stability investigation of the orifice-opening rhinitis tablet can be easily realized, and the relatively comprehensive quantitative comparison investigation method of the stability of the effective components is provided.
2. The gradient elution method suitable for detecting the active ingredients of the resuscitation-inducing rhinitis tablets is accurately summarized, the detection time is shortened to 7min from 50-80 min of a common liquid phase, the content determination time is greatly shortened, the detection efficiency is stably improved, meanwhile, the using amount of a mobile phase is greatly reduced (from 50-80 mL to 3.5 mL), the mobile phase contains more toxic solvents such as methanol or acetonitrile, the using amount is reduced, the pollution to the environment is greatly reduced, the method is more environment-friendly, and the detection cost is also reduced.
3. Six most representative active ingredients of the four main medicinal materials in the orifice-opening rhinitis tablet, namely the linarin glycoside, the 5-O-methylvisammioside, the calycosin glycoside, the magnolin, the imperatorin and the isoimperatorin, are detected simultaneously.
4. The method has the advantages of stable detection baseline, high separation degree, high detection accuracy and good stability.
Drawings
FIG. 1 is a UPLC chromatogram of a mixed control solution of example 1;
FIG. 2 is a UPLC chromatogram of the test solution of example 1.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise specified, all the raw materials and equipment used in this example were those conventionally available in the art.
The apparatus and reagents referred to in the following examples include: ultra-high performance liquid chromatography: waters acquality UPLC (Water, usa), KQ5200DE model digital controlled ultrasonic cleaner (kunshan ultrasonic instruments ltd); electronic balances (beijing sidoris balance, ltd); an ultra-pure water machine (ABW-1001-U type Ai Kepu). Control (purchased from the chinese food and drug testing institute): cimicidine glycoside (batch No. 111710-200501); 5-O-methylvisammioside (batch No.: 111523-201509); calycosin glucoside (batch No. 111920-201505); magnolin (batch No. 110882-201206); imperatorin (batch number: 110826-201415); isoimperatorin (batch numbers: 110827-201410).
Example 1 the content of the active ingredients of linarin glycoside, 5-O-methylvisammioside, calycosin glycoside, magnolin, imperatorin, isoimperatorin in the resuscitation rhinitis tablets was determined as follows:
(1) Preparation of a test solution: taking 1.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing into a 100mL triangular flask with a plug, precisely adding 50mL of methanol with volume fraction of 80%, weighing, ultrasonic extracting at 100W and 25kHz for 45min, cooling, weighing again, complementing weight loss by methanol with volume fraction of 80%, filtering by a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate as a test solution;
(2) Preparation of control solutions: taking a cimicidin reference substance, a 5-O-methylvisammol glycoside reference substance, a calycosin glucoside reference substance, an imperatorin reference substance, an isoimperatorin reference substance and a magnolin reference substance, adding methanol for dissolving, and respectively preparing a cimicin glycoside reference substance solution with the concentration of 50 mug/mL, a 5-O-methylvisammol glycoside reference substance solution with the concentration of 80 mug/mL, a calycosin glucoside reference substance solution with the concentration of 50 mug/mL, an imperatorin reference substance solution with the concentration of 60 mug/mL, an isoimperatorin reference substance solution with the concentration of 200 mug/mL and a magnolin reference substance solution with the concentration of 50 mug/mL for standby;
preparing a 6-component mixed reference solution by mixing the prepared linarin reference solution, 5-O-methylvisammol glycoside reference solution, calycosin glucoside reference solution, imperatorin reference solution, isoimperatorin reference solution and magnolin reference solution according to the following proportion of 5;
(3) And (3) measurement by a UPLC method: precisely sucking 1 μ L of each of the test solution and the mixed reference solution, injecting into an ultra-high performance liquid chromatograph, measuring, and calculating to obtain the final product;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50mm × 2.1mm,1.7 μm; the mobile phase A is acetonitrile, and the mobile phase B is acetic acid with the volume fraction of 0.6 per mill; gradient elution: 0-2.6 min,18% → 70% by weight A;2.6 to 3.5min,70% → 95% A; 3.5-4.0min, 95% by weight of A;4.0 to 4.5min,95% → 18% by volume A; 4.5-7min, 18% A; the flow rate is 0.5mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃.
The detection result shows that the detection base line is stable, the separation degree of each target component in the sample is good, the sample peak-out time is 4-8 minutes, and the detection can be completed within 8 minutes.
Example 2 the content of the active ingredients of linarin glycoside, 5-O-methylvisammioside, calycosin glycoside, magnolin, imperatorin, isoimperatorin in the resuscitation rhinitis tablets was determined as follows:
(1) Preparation of a test solution: taking 3.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing in a 200mL volumetric flask, precisely adding 30mL of methanol with the volume fraction of 60%, weighing, ultrasonic extracting at 100W and 25kHz for 30min, cooling, weighing again, complementing the weight loss by using methanol with the volume fraction of 60%, filtering by using a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate as a test solution;
(2) Preparation of control solutions: taking a cimicidin reference substance, a 5-O-methylvisammol glycoside reference substance, a calycosin glucoside reference substance, an imperatorin reference substance, an isoimperatorin reference substance and a magnolin reference substance, adding methanol for dissolving, and respectively preparing a cimicin glycoside reference substance solution with the concentration of 45 mug/mL, a 5-O-methylvisammol glycoside reference substance solution with the concentration of 75 mug/mL, a calycosin glucoside reference substance solution with the concentration of 45 mug/mL, an imperatorin reference substance solution with the concentration of 55 mug/mL, an isoimperatorin reference substance solution with the concentration of 180 mug/mL and a magnolin reference substance solution with the concentration of 45 mug/mL for standby;
preparing a 6-component mixed reference solution by mixing the prepared linarin reference solution, 5-O-methylvisammol glycoside reference solution, calycosin glucoside reference solution, imperatorin reference solution, isoimperatorin reference solution and magnolin a ratio of 2;
(3) And (3) measuring by using a UPLC method: precisely sucking 5 μ L of each of the sample solution and the mixed reference solution, injecting into an ultra-high performance liquid chromatograph, measuring, and calculating to obtain the final product;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50 mm. Times.2.1mm, 1.7 μm; the mobile phase A is acetonitrile, and the mobile phase B is acetic acid with the volume fraction of 0.4 per mill; gradient elution: 0-2.6min, 10% → 60% A;2.6 to 3.5min,60% → 80% A; 3.5-4.0min, 80% by weight of A;4.0 to 4.5min,80% → 10% A; 4.5-7min, 10% A; the flow rate is 0.2mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃.
The detection result shows that the detection base line is stable, the separation degree of each target component in the sample is good, the effect is slightly worse than that of the embodiment 1, the sample peak-out time is 4-8 minutes, and the detection can be completed within 8 minutes.
Example 3 the content of the active ingredients of linarin glycoside, 5-O-methylvisammioside, calycosin glycoside, magnolin, imperatorin, isoimperatorin in the resuscitation rhinitis tablets was determined as follows:
(1) Preparing a test solution: taking 2.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing in a 150mL triangular flask with a plug, precisely adding 60mL of methanol with volume fraction of 70%, weighing, ultrasonic extracting for 60min at 100W and 25kHz, cooling, weighing again, complementing weight loss by methanol with volume fraction of 70%, filtering by a 0.22 mu m microporous filter membrane, and taking subsequent filtrate as a test solution;
(2) Preparation of control solutions: taking a cimicidin reference substance, a 5-O-methylvisammol glycoside reference substance, a calycosin glucoside reference substance, an imperatorin reference substance, an isoimperatorin reference substance and a magnolin reference substance, adding methanol for dissolving, and respectively preparing a cimicin glycoside reference substance solution with the concentration of 55 mug/mL, a 5-O-methylvisammol glycoside reference substance solution with the concentration of 85 mug/mL, a calycosin glucoside reference substance solution with the concentration of 55 mug/mL, an imperatorin reference substance solution with the concentration of 65 mug/mL, an isoimperatorin reference substance solution with the concentration of 220 mug/mL and a magnolin reference substance solution with the concentration of 55 mug/mL for standby;
preparing a 6-component mixed reference solution by mixing the prepared linarin reference solution, 5-O-methylvisammol glycoside reference solution, calycosin glucoside reference solution, imperatorin reference solution, isoimperatorin reference solution and magnolin reference solution according to a ratio of 8;
(3) And (3) measuring by using a UPLC method: precisely sucking 2 μ L of each of the test solution and the mixed reference solution, injecting into an ultra-high performance liquid chromatograph, measuring, and calculating to obtain;
the chromatographic conditions measured by the UPLC method are as follows: waters BEHC18 column with 50mm × 2.1mm inner diameter, 1.7 μm; the mobile phase A is acetonitrile, and the mobile phase B is acetic acid with the volume fraction of 0.8 per mill; gradient elution: 0-2.6min, 20% → 80% by weight A;2.6 to 3.5min,80% → 100% A; 3.5-4.0min, 100% by weight; 4.0 to 4.5min,100% → 20% A; 4.5-7min, 20% by weight A; the flow rate is 0.3mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃.
The detection result shows that the detection base line is stable, the separation degree of each target component in the sample is good, the effect is slightly worse than that of the embodiment 1, the sample peak-out time is 4-8 minutes, and the detection can be completed within 8 minutes.
Example 4 the content of the active ingredients of linarin glycoside, 5-O-methylvisammol glycoside, calycosin glycoside, magnolin, imperatorin, isoimperatorin in the resuscitation rhinitis tablets was determined as follows:
(1) Preparation of a test solution: taking 2.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing in a 200mL volumetric flask, precisely adding 50mL of 75% methanol in volume fraction, weighing, ultrasonic extracting for 40min at 100W and 25kHz, cooling, weighing again, complementing the weight loss by 75% methanol in volume fraction, filtering by a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate as a test solution;
(2) Preparation of control solutions: the same as example 1;
(3) And (3) measuring by using a UPLC method: the same as example 1;
the chromatographic conditions determined by the UPLC method are as follows: the same as example 1;
the detection result shows that the detection base line is stable, the separation degree of each target component in the sample is good, the effect is similar to that of the embodiment 1, the sample peak appearing time is 4-8 minutes, and the detection can be completed within 8 minutes.
Example 5 the content of the active ingredients of linarin glycoside, 5-O-methylvisammioside, calycosin glycoside, magnolin, imperatorin, isoimperatorin in the resuscitation rhinitis tablets was determined as follows:
(1) Preparation of a test solution: the same as example 1;
(2) Preparation of control solutions: the same as example 1;
(3) And (3) measuring by using a UPLC method: the same as example 1;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50mm × 2.1mm,1.7 μm; the mobile phase A is acetonitrile, and the mobile phase B is acetic acid with the volume fraction of 0.2 per mill; gradient elution: 0 to 2.6min,25% to → 55% A;2.6 to 3.5min,55% → 75% A; 3.5-4.0min, 75 percent; 4.0 to 4.5min,75% → 25% A; 4.5-7min, 25% A; the flow rate is 0.5mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃.
The detection result shows that the detection base line is stable, the separation degree of each target component in the sample is good, the effect is slightly worse than that of the embodiments 1 to 4, the sample peak-out time is 4 to 10 minutes, and the detection can be completed within 10 minutes.
Comparative example 1 the content of the effective components in the orifice-opening rhinitis tablet is measured according to the following method:
(1) Preparation of a test solution: the preparation method of the test solution is the same as that of example 1;
(2) Preparation of control solutions: the preparation method of the reference solution is the same as that of the example 1;
(3) The determination method comprises the following steps: precisely sucking 20 μ L of each of the sample solution and the mixed reference solution, injecting into a liquid chromatograph, measuring, and calculating to obtain the final product;
the chromatographic conditions of the determination method are as follows: dikma Diamonsil C18 column (4.6 mm. Times.150mm, 5 m), mobile phase A chromatographically pure methanol, mobile phase B water containing 0.05% formic acid, gradient elution (0-6 min,25% A → 75% A6-10min, 75% A → 95% A10-15min, 95% A), balanced with mobile phase initial conditions for 6min before injection, flow rate 0.8mL/min, column temperature room temperature, sample injection 20. Mu.L.
The result shows that when the comparative example method is used for detection, the spectrogram base line is not stable, the detection time is 15-30 minutes after the peak emergence time is 10 minutes, 5-O-methylvisammioside cannot be detected, the target component and the impurities cannot be completely separated, and the content measurement is unstable.
Comparative example 2 content determination of active ingredients in the resuscitation-inducing rhinitis tablets was performed according to the following method:
(1) Preparation of a test solution: the preparation method of the test solution is the same as that of example 1;
(2) Preparation of control solutions: the preparation method of the reference solution is the same as that of the example 1;
(3) The determination method comprises the following steps: precisely sucking 10 μ L of each of the sample solution and the reference solution, injecting into a liquid chromatograph, and measuring;
the chromatographic conditions of the assay are: an agent ZORBAX SB-Cl8 column (250 mm. Times.4.6 mm,5 μm); mobile phase A was acetonitrile, B was 0.1% aqueous formic acid, gradient elution (0-35min, 10% A → 35% A, 35-40min, 35% A → 90% A, 40-45min, 90% A → L0% A, 45-50min, 10% A, flow rate: 1.0mL/min, column temperature: 30 ℃, detection wavelength: 283nm, sample intake: 10. Mu.L.
The results show that when the comparative example method is used for detection, the spectrogram base line is not stable, the peak-off time is 20 minutes later, the detection time is 40-50 minutes, the calycosin glycoside, magnolin, imperatorin and isoimperatorin cannot be detected, the 6 target components cannot be accurately determined, and the requirement of quality assurance cannot be met. Comparative example 3 content determination of effective ingredients in the orifice-opening rhinitis tablet was performed according to the following method:
(1) Preparation of a test solution: the preparation method of the test solution is the same as that of example 1;
(2) Preparation of control solutions: the same preparation method as the reference solution in example 1;
(3) The determination method comprises the following steps: precisely sucking the sample solution, the reference solution and the negative reference solution by 50 μ L respectively, injecting into a liquid chromatograph, and measuring;
the chromatographic conditions of the determination method are as follows: agilent TC-C18 column (4.6 mm. Times.250mm, 55 μm), mobile phase A0.2% formic acid water, B acetonitrile, gradient elution (0-42min, 22% B43-73min, 50% B); the detection wavelength is 254nm; the volume flow is 1.0mL/min; the column temperature is 30 ℃; the amount of sample was 50. Mu.L.
The results show that when the comparative example method is used for detection, the spectrogram base line is not stable, the peak-off time is 20 minutes later, the detection time is 70-80 minutes, the linarin, the 5-O-methylvismaidin, the calycosin glycoside and the magnolin cannot be detected, the 6 target components cannot be accurately detected, and the quality guarantee requirement cannot be met.
Comparative example 4
The difference from example 1 is that the content of the rhinitis-relieving tablets was measured under the chromatographic conditions according to the method for measuring the content of the rhinitis-relieving tablets disclosed in the pharmacopoeia of china 2015 edition, first 1458.
The result shows that the detection method only can detect imperatorin and cannot simultaneously detect multi-index components, and the quantitative control requirement of the multi-index components of multiple medicinal materials cannot be met.
Comparative example 5
The difference from example 1 is that the content of the resuscitation-inducing rhinitis capsule was measured under the chromatographic conditions according to the method for measuring the content of the resuscitation-inducing rhinitis capsule disclosed in the first part, page 1459 of the "chinese pharmacopoeia 2015 edition".
The results show that the peak-off times of imperatorin and astragaloside iv were respectively 22 minutes and 20 minutes later, the detection times required 30 minutes and 35 minutes respectively, and the detection was achieved by two different test article preparation methods and chromatographic detection conditions respectively. By the two detection methods, only the imperatorin and the astragaloside can be detected respectively, multi-index components cannot be detected simultaneously, and the quantitative control requirement of the multi-components is difficult to achieve.
Example 6 investigation of linear relationship, precision test, repeatability test, stability test, recovery test and determination of sample content:
the sample solution and the control solution were treated in the same manner as in example 1, and the following tests were carried out under the same chromatographic conditions and detection methods as in example 1:
(1) And (3) linear relation investigation: the mixed control solution 0.5,1,2,4,8, 12 and 16. Mu.L samples were precisely pipetted from the bottom of example 1. Taking the peak area (Y) as an ordinate and the sample volume (X, mug) as an abscissa, drawing a standard curve to obtain regression equations of the linarin glycoside, the 5-O-methylvisammol glycoside, the calycosin glycoside, the magnolin, the imperatorin and the isoimperatorin as follows: y =3763.02X-304.96 γ =0.9999 (n = 6); y =2018.12X 22575.6 γ =0.9999 (n = 6); y =603.33X-8962.55 γ =0.999 (n = 6); y =2329.46x +68544.21 γ =0.9998 (n = 6); y =2023.62x +41224.11 γ =0.9999 (n = 6); y =700.55X-1007.56 γ =0.9999 (n = 6); the linear ranges are respectively: 0.025-0.8 mu g; 0.04-1.28 mug; 0.025-0.8 mu g; 0.03-0.96 mug; 0.03-0.96 mu g; 0.10-3.20 mug.
(2) And (3) precision test: the sample solutions prepared in example 1 were precisely extracted and continuously injected 6 times, and RSDs of peak area integral values of linarin, 5-O-methylvisammioside, calycosin, magnolin, imperatorin, and isoimperatorin were 0.5%,0.4%,0.9%,0.8%,0.3%, and 0.3%, respectively, indicating good precision of the instrument.
(3) And (3) repeatability test: taking the same batch of samples, preparing 6 parts of the sample in parallel according to the sample preparation method in the section of example 1, measuring according to the chromatographic conditions in the section of example 1, and calculating the content. The average contents of the linarin glycoside, the 5-O-methylvisammol glycoside, the calycosin glycoside, the magnolin, the imperatorin and the isoimperatorin in 6 parts of the test sample are respectively 0.37mg/g,0.72mg/g,0.31mg/g,0.37mg/g,0.79mg/g and 0.42mg/g, and the RSDs are respectively as follows: 1.0%,1.2%,1.8%,1.6%,0.8%,0.6%, indicating good reproducibility.
(4) And (3) stability test: taking the same batch of samples, preparing a test sample solution according to the test sample preparation method under the item of example 1, injecting samples at 0,2,4,6,8, 10, 12 and 24 hours after preparation, and measuring the RSD of the peak area integral values of the cimicidin, 5-O-methylvisammioside, calycosin, magnolin, imperatorin and isoimperatorin in the test sample as follows: 1.0%,1.2%,1.7%,1.5%,0.7%,0.6%, indicating that the test solution is stable within 24 h.
(5) Recovery rate test: taking 0.5g and 6 parts of a test sample, precisely weighing, precisely adding appropriate amounts of a reference substance of the primisula glycoside, the 5-O-methylvispora micrantol glycoside, the calycosin glycoside, the magnolin, the imperatorin and the isoimperatorin, preparing a test sample solution according to the preparation method of the test sample under the item of the example 1, measuring according to the chromatographic condition under the item of the example 1, and calculating the recovery rate, wherein the recovery rates (n = 9) of the primisula glycoside, the 5-O-methylvispora micrantol glycoside, the calycosin glycoside, the magnolin, the imperatorin and the isoimperatorin are respectively 97.5%,99.3%,98.0%,98.3%,99.2 and 99.5%, and the RSDs are respectively: 1.1%,1.0%,1.6%,1.3%,1.0%,0.8%.
(6) And (3) measuring the content of the sample: three batches of orifice-opening rhinitis tablets are taken, a test solution is prepared according to the method in the example 1, the contents of 6 components are measured, and the results are shown in the following table:
from the experimental results, the UPLC method is adopted to replace the traditional HPLC method for content determination, so that the time and the solvent are greatly saved, better peak type and theoretical plate number are obtained, the target component and the impurities are completely separated, the specificity is good, and the detection of various effective components can be simultaneously carried out.
The quantitative method established in the experiment is simple and easy in method, good in specificity, high in recovery rate and strong in feasibility by measuring multiple batches of samples, and is an optimal content detection method for the orifice-opening rhinitis tablets.
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to illustrate the principles of the invention, and that various changes and modifications, which will be apparent to those skilled in the art, may be made without departing from the spirit and scope of the invention and fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (9)
1. The content determination method of the orifice-opening rhinitis tablet is characterized by comprising the following steps: measuring the contents of the active ingredients of the preparation, namely the linarin glycoside, the 5-O-methylvisammol glycoside, the calycosin glycoside, the magnolin, the imperatorin and the isoimperatorin, wherein the content measurement comprises the following steps: preparing a test solution, preparing a reference solution and measuring by a UPLC method;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50mm × 2.1mm,1.7 μm; the mobile phase A is acetonitrile, the mobile phase B is acetic acid with volume fraction of 0.6 per mill, the gradient elution method; the flow rate is 0.5mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃;
the gradient elution method comprises the following steps: 0 to 2.6min,18% → 70% A;2.6 to 3.5min,70% → 95% by weight A;3.5 to 4.0min,95% by weight A;4.0 to 4.5min,95% → 18% by weight A;4.5 to 7min,18% by weight A;
the preparation method of the test solution comprises the following steps: taking 1-3 g of orifice-opening rhinitis tablet powder, precisely weighing, placing in a triangular flask with a plug or a volumetric flask with a plug of 100-200mL, precisely adding 30-60mL of methanol with volume fraction of 60-80%, weighing, 100W,25kHz, ultrasonically extracting for 30-60min, cooling, weighing again, supplementing weight loss by using methanol with volume fraction of 60-80%, filtering by using a microporous filter membrane, and taking a subsequent filtrate as a sample solution.
2. The method of claim 1, wherein: the preparation method of the test solution comprises the following steps: taking 1.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing in a 100mL triangular flask with a plug or volumetric flask, precisely adding 50mL of methanol with volume fraction of 80%, weighing, ultrasonic extracting at 100W and 25kHz for 45min, cooling, weighing again, complementing the weight loss by methanol with volume fraction of 80%, filtering by a 0.22 mu m microporous membrane, and taking the subsequent filtrate as a test solution.
3. The method of claim 1, wherein: the reference solution comprises a cimicidin glycoside reference solution, a 5-O-methylvisammioside glycoside reference solution, a calycosin glucoside reference solution, an imperatorin reference solution, an isoimperatorin reference solution and a magnolin reference solution.
4. The method of claim 3, wherein:
the preparation method of the cimicidin reference solution comprises the following steps: dissolving a cimicidin reference substance in methanol until the concentration is 45 to 55 mu g/mL for later use;
the preparation method of the 5-O-methylvisammioside reference substance solution comprises the following steps: dissolving 5-O-methylvisammioside reference substance in methanol until the concentration is 75 to 85 mu g/mL for later use;
the preparation method of the calycosin glucoside reference substance solution comprises the following steps: taking a calycosin glucoside reference substance, adding methanol to dissolve until the concentration is 45 to 55 mu g/mL for later use;
the preparation method of the imperatorin reference substance solution comprises the following steps: dissolving imperatorin reference substance in methanol until the concentration is 55 to 65 mu g/mL for later use;
the preparation method of the isoimperatorin reference substance solution comprises the following steps: adding methanol into an isoimperatorin reference substance to dissolve until the concentration is 180 to 220 mu g/mL for later use;
the preparation method of the magnolin reference substance solution comprises the following steps: taking a magnolin reference substance, and adding methanol to dissolve the magnolin reference substance until the concentration is 45-55 mu g/mL for later use;
the prepared reference solution, namely the L-ephedrine glycoside, the 5-O-methylvisammol glycoside, the calycosin glucoside, the imperatorin, the isoimperatorin and the magnolin, is prepared into a 6-component mixed reference solution according to the following ratio of 2 to 6 to 5 to 9.
5. The method of claim 4, wherein:
the preparation method of the cimicidin reference solution comprises the following steps: dissolving the cimicidin reference substance with methanol to a concentration of 50 μ g/mL for use;
the preparation method of the 5-O-methylvisammioside reference substance solution comprises the following steps: dissolving 5-O-methylvisammioside reference substance in methanol to a concentration of 80 μ g/mL;
the preparation method of the calycosin glucoside reference substance solution comprises the following steps: taking a calycosin glucoside reference substance, adding methanol to dissolve until the concentration is 50 mu g/mL for later use;
the preparation method of the imperatorin reference substance solution comprises the following steps: dissolving imperatorin reference substance in methanol to concentration of 60 μ g/mL;
the preparation method of the isoimperatorin reference substance solution comprises the following steps: adding methanol into isoimperatorin reference substance to dissolve to 200 μ g/mL for use;
the preparation method of the magnolin reference substance solution comprises the following steps: dissolving magnolin control in methanol to 50 μ g/mL;
the prepared reference solutions, i.e., the linalooside, the 5-O-methylvisammioside, the calycosin glucoside, the imperatorin, the isoimperatorin and the magnolin, were prepared into 6-component mixed reference solutions in a ratio of 5.
6. The method for measuring the content of the orifice-opening rhinitis tablet according to claim 1, which is characterized in that: the method comprises the following steps:
(1) Preparation of a test solution: taking 1 to 3g of orifice-opening rhinitis tablet powder, precisely weighing, placing the powder into a triangular flask with a stopper or a volumetric flask with a stopper of 100 to 200mL, precisely adding 30 to 60mL of methanol with volume fraction of 60 to 80%, weighing, 100W and 25kHz, ultrasonically extracting for 30 to 60min, cooling, weighing again, supplementing weight loss by using methanol with volume fraction of 60 to 80%, filtering by using a microporous filter membrane, and taking a subsequent filtrate as a test solution;
(2) Preparation of control solutions: taking a cimicidin reference substance, a 5-O-methylvisammol glycoside reference substance, a calycosin glucoside reference substance, an imperatorin reference substance, an isoimperatorin reference substance and a magnolin reference substance, adding methanol for dissolving, and respectively preparing a cimicin glycoside reference substance solution with the concentration of 45 to 55 mu g/mL, a 5-O-methylvisammol glycoside reference substance solution with the concentration of 75 to 85 mu g/mL, a calycosin glucoside reference substance solution with the concentration of 45 to 55 mu g/mL, an imperatorin reference substance solution with the concentration of 55 to 55 mu g/mL, an isoimperatorin reference substance solution with the concentration of 180 to 220 mu g/mL and a magnolin reference substance solution with the concentration of 45 to 55 mu g/mL for later use;
preparing a prepared linarin control solution, a 5-O-methylvismaioside control solution, a calycosin glucoside control solution, an imperatorin control solution, an isoimperatorin control solution and a magnolin control solution into a 6-component mixed control solution according to the following ratio of 2 to 6;
(3) And (3) measurement by a UPLC method: precisely sucking 1~5 μ L of the test solution and the mixed reference solution respectively, injecting into an ultra high performance liquid chromatograph, measuring, and calculating to obtain the final product;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50mm × 2.1mm,1.7 μm; the mobile phase A is acetonitrile, the mobile phase B is acetic acid with volume fraction of 0.6 per mill, the gradient elution method; the flow rate is 0.5mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃;
the gradient elution method comprises the following steps: 0 to 2.6min,18% → 70% A;2.6 to 3.5min,70% → 95% by weight A;3.5 to 4.0min,95% by weight A;4.0 to 4.5min,95% → 18% by weight A;4.5 to 7min,18% A.
7. The method for measuring the content of the orifice-opening rhinitis tablet according to claim 6, wherein the method comprises the following steps: the method comprises the following steps:
(1) Preparation of a test solution: taking 1.0g of orifice-opening rhinitis tablet powder, precisely weighing, placing into a 100mL triangular flask with a plug or a volumetric flask, precisely adding 50mL of methanol with volume fraction of 80%, weighing, ultrasonic extracting at 100W and 25kHz for 45min, cooling, weighing again, complementing the weight loss by using methanol with volume fraction of 80%, filtering by using a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate as a test solution;
(2) Preparation of control solutions: taking a cimicidin reference substance, a 5-O-methylvisammol glycoside reference substance, a calycosin glucoside reference substance, an imperatorin reference substance, an isoimperatorin reference substance and a magnolin reference substance, adding methanol for dissolving, and respectively preparing a cimicin glycoside reference substance solution with the concentration of 50 mug/mL, a 5-O-methylvisammol glycoside reference substance solution with the concentration of 80 mug/mL, a calycosin glucoside reference substance solution with the concentration of 50 mug/mL, an imperatorin reference substance solution with the concentration of 60 mug/mL, an isoimperatorin reference substance solution with the concentration of 200 mug/mL and a magnolin reference substance solution with the concentration of 50 mug/mL for standby;
preparing a prepared linarin glycoside reference substance solution, a 5-O-methylvisammol glycoside reference substance solution, a calycosin glucoside reference substance solution, an imperatorin reference substance solution, an isoimperatorin reference substance solution and a magnolin reference substance solution into a 6-component mixed reference substance solution according to the following ratio of 5;
(3) And (3) measuring by using a UPLC method: precisely sucking 1 μ L of each of the test solution and the mixed reference solution, injecting into an ultra high performance liquid chromatograph, measuring, and calculating to obtain;
the chromatographic conditions measured by the UPLC method are as follows: a Waters BEH C18 column with an internal diameter of 50mm × 2.1mm,1.7 μm; the mobile phase A is acetonitrile, the mobile phase B is acetic acid with the volume fraction of 0.6 per mill, the gradient elution method; the flow rate is 0.5mL/min, the detection wavelength is 254nm, and the column temperature is 35 ℃;
the gradient elution method comprises the following steps: 0 to 2.6min,18% → 70% by weight A;2.6 to 3.5min,70% → 95% by weight A;3.5 to 4.0min,95% by weight A;4.0 to 4.5min,95% → 18% by weight A;4.5 to 7min,18% by weight of A.
8. The content determination method according to any one of claims 1 to 7, wherein the content of the linarin glycoside in the resuscitation rhinitis tablet is 300 to 500 μ g/g, the content of the 5-O-methylvisammoin glycoside is 700 to 900 μ g/g, the content of the calycosin glycoside is 150 to 300 μ g/g, the content of the magnolin is 300 to 500 μ g/g, the content of the imperatorin is 800 to 1000 μ g/g, and the content of the isoimperatorin is 700 to 900 μ g/g.
9. The method for measuring the content of claim 8, wherein the orostachydis tablets have a cimicidin content of 413 μ g/g, a 5-O-methylvisammioside content of 830 μ g/g, a calycosin content of 254 μ g/g, a magnolin content of 485 μ g/g, an imperatorin content of 955 μ g/g, and an isoimperatorin content of 873 μ g/g.
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| CN113203808A (en) * | 2021-04-27 | 2021-08-03 | 西南民族大学 | Detection method of fire therapy liniment |
| CN114544833B (en) * | 2022-02-28 | 2024-04-05 | 辽宁中医药大学 | Method for constructing snuff-in-mouth orifice-opening granule multi-information drug effect prediction characteristic map |
| CN115166079B (en) * | 2022-07-04 | 2024-05-03 | 广州白云山中一药业有限公司 | Multi-component content determination method for magnolia flower rhinitis preparation |
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