CN116165301B - A method for simultaneously determining the contents of six components in Jinlian Qingre Granules - Google Patents
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Abstract
The invention discloses a method for simultaneously measuring the contents of 6 components in lotus heat-clearing particles, which respectively selects 3 substances as internal references, calculates correction factors under each internal reference, and can simultaneously calculate the contents of other 5 substances according to the correction factors and the HPLC chromatographic elution peak area of an object to be measured. According to the invention, a one-measurement-multiple-evaluation (QAMS) method for simultaneously measuring the contents of 6 components in the golden lotus heat-clearing particles is established by taking any 1 of three components orientin-2' -O-beta-galactoside, orientin and vitexin which are clear in pharmacological action and high in content in golden lotus heat-clearing particles as internal references, and the results are compared and confirmed with the measurement results of an external standard method. The method disclosed by the invention uses less reference substances, can simplify the detection process, improve the detection efficiency and reduce the detection cost, and provides support for the comprehensive evaluation of the quality of the preparation and the improvement of the quality standard.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine component measurement, in particular to a method for simultaneously measuring the contents of 6 components in lotus heat-clearing particles.
Background
The lotus flower heat-clearing granule takes trollius chinensis and dyers woad leaf as principal drugs, heat-clearing and detoxifying, rhizoma anemarrhenae and gypsum as ministerial drugs, auxiliary principal drugs for clearing away qi-flowing and heat so as to reduce high fever rapidly, radix scrophulariae and radix rehmanniae as adjuvant drugs for nourishing yin and clearing heat, and increasing liquid so as to moisten dryness, promote body fluid to relieve restlessness, clear nutrient and cool blood and detoxify, and is particularly suitable for people with impairment of body fluid and fluid robbing, and bitter almond as conductant drugs for dispersing lung qi and relieving cough and reducing sputum.
At present, the content of vitexin is only used as a quality control index of the lotus heat-clearing particles, and the lotus heat-clearing particles consist of 7 traditional Chinese medicines, have complex components, and are difficult to comprehensively and accurately evaluate the internal quality by using a single component as a detection index. In recent years, multi-index component detection analysis has become the development direction of traditional Chinese medicine quality control, but the separation difficulty of partial traditional Chinese medicine chemical reference substances is high, and the multi-index quality control tends to increase the detection cost, so that the application of a multi-index quality control mode in production, scientific research and medicine supervision is limited. A multi-component simultaneous measurement is realized by using an effective component as an internal reference substance in a multi-evaluation (QAMS) method, constructing a Relative Correction Factor (RCF) between each component and the internal reference substance, and measuring the content of one effective component. However, the traditional Chinese medicine QAMS has the phenomenon of repeated research of RCF, namely, the analysis method is the same, the components to be detected are the same, and RCF is repeatedly built by adopting different internal references, so that resources are wasted, and the initial purpose of QAMS building is violated.
Therefore, how to provide a method for simultaneously measuring the contents of various components in the lotus heat-clearing particles based on one measurement and multiple evaluation methods of different internal references is a problem to be solved by the skilled in the art.
Disclosure of Invention
In view of the above, the invention establishes a QAMS method which takes any 1 of three components orientin-2' -O-beta-galactoside, orientin and vitexin with definite pharmacological action and higher content in monarch drug trollius chinensis as internal references, can simultaneously measure the content of 6 components in the trollius chinensis heat-clearing particles, and compares and confirms with the measurement result of an external standard method, thereby providing support for the overall evaluation of the quality of the preparation and the improvement of quality standard.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A method for simultaneously measuring the contents of 6 components in lotus heat-clearing particles comprises the steps of respectively selecting 3 substances as internal references, calculating correction factors under each internal reference, and simultaneously calculating the contents of 6 substances according to any correction factor and the HPLC chromatographic elution peak area of the object to be measured.
Preferably, the correction factor RCFi/s=fi/fs= (Wi/Ai)/(Ws/As) = (wi×as)/(ws×ai) under each internal reference is calculated, wherein fi and fs are absolute correction factors of the component to be measured and the internal reference, ai and Wi are peak areas and masses of the component to be measured, and As and Ws are peak areas and masses of the internal reference, respectively.
Preferably, the three internal references are orientin-2' -O-beta-galactoside, orientin and vitexin respectively.
Preferably, the HPLC chromatographic elution condition is that the mobile phase is acetonitrile (A) to 0.1 percent of H 3PO4 aqueous solution (B), and the gradient elution is ：0～5min,B：97％;5～15min,B：97％～95％;15～25min,B：95％～90％;25～91min,B：90％～68％;92～95min,B：68％～97％;96～100min,B：97％;, and the sample injection amount is 10 mu L.
According to the technical scheme, the traditional Chinese medicine has the effects of multiple components and multiple effects, the internal quality of the traditional Chinese medicine is difficult to comprehensively and accurately evaluate by taking a single component as a detection index, and the detection analysis of the components of the multiple indexes becomes the development direction of the quality control of the traditional Chinese medicine. The QAMS method can realize multi-component quality control and overcome the difficulties of shortage of reference substances and high detection cost. The research establishes a QAMS method which takes any 1 of three components orientin-2' -O-beta-galactoside, orientin and vitexin with definite pharmacological action and higher content in trollius chinensis serving as monarch drug of the trollius chinensis bunge heat-clearing particles as internal references, can simultaneously measure 6 components in the trollius chinensis bunge heat-clearing particles, constructs RCFs between 3 internal references and components to be measured, and systematically examines the durability of the RCFs. The method has the advantages that the method is adopted to measure 6 components in 21 batches of lotus heat-clearing particles by the QAMS method, and the difference percentage between the measured results by the external standard method is less than 3.0%, so that the constructed QAMS method is accurate and reliable, the content of the 6 components in the lotus heat-clearing particles can be measured simultaneously under the condition that any one of three internal references is used as a reference, the resource waste caused by repeatedly establishing RCF is avoided, and the support is provided for the comprehensive evaluation of the quality of the preparation and the improvement of the quality standard.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing HPLC chromatograms of a mixed control solution, sample solution and negative sample solution;
FIG. 2 is a graph of the spectra of a photodiode array detector (PDA) for the corresponding chromatographic peaks in the 6 component controls and samples.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The apparatus and reagents used in the examples
Instrument for measuring and controlling the intensity of light
Prominenece LC-20A high performance liquid chromatography system was (Shimadzu corporation), agilent 1260 high performance liquid chromatograph (Agilent corporation, U.S.), KQ5200B type ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.), XS105DU type analytical balance (Mettler-Toledo, switzerland), TGL-16G high speed centrifuge (Shanghai Anting scientific instruments Co.).
Reagent
Reference substances including mangiferin (lot number: 111607-201704), orientin-2' -O-beta-galactoside (lot number: P01O11F 126428), orientin (lot number: S19GB 161663), veratric acid (lot number: DN1127BA 13), vitexin (lot number: D29GB 172870) are all purchased from Shanghai source leaf Biotechnology Co., ltd., harba russia (lot number: PS 000404), and are all purchased from Chengdu PS Biotechnology Co., ltd, and the purity of the reference substances is not less than 98%. Chromatographic pure acetonitrile was purchased from Fisher corporation and chromatographic water was purchased from haha group limited, wa, hangzhou.
21 Batches of lotus heat-clearing particles are provided by Ningxia Qiyuan pharmaceutical industry Co.
In the prior art, vitexin is only used as a quality evaluation index of the lotus heat-clearing particles, and it is difficult to comprehensively and accurately evaluate the internal quality of the lotus heat-clearing particles, while examples 1-8 provide technical permissions for determining the content of 6 components by a later one-test-multiple-evaluation method for verifying whether the content of 6 components in the lotus heat-clearing particles can be determined simultaneously.
Example 1 preparation of sample solutions for samples
This example examined different extraction solvents (water, methanol, ethanol) and showed the highest extraction of each component when methanol was used as the solvent. And further comparing the concentration of methanol (30%, 50%, 70%), the extraction time (20, 30, 40, 50 min) and the extraction solvent dosage (10, 25, 50, 100 mL), optimizing by taking the extraction efficiency of 6 components to be detected in the lotus heat-clearing particles as an index, and finally determining the preparation method of the lotus heat-clearing particle sample solution, wherein the preparation method comprises the steps of adding 25mL of 50% methanol solution and carrying out ultrasonic extraction for 30min.
The specific process comprises the steps of taking a sample of lotus heat-clearing particles, grinding, precisely weighing about 0.5g, placing the sample into a conical flask with a plug, precisely adding 25ml of 50% methanol solution, sealing and weighing. Ultrasonic extraction (power 250w, frequency 33 KHz) for 30 mm, taking out, cooling, adding solvent to the weight, and shaking. Taking a proper amount, centrifuging (10000 r.min -1) for 10min, and taking supernatant as a sample solution.
EXAMPLE 2 preparation of Mixed control solution
Respectively precisely weighing appropriate amounts of mangiferin, orientin-2' -O-beta-galactoside, orientin, veratric acid, vitexin and harpagoside reference substances, adding methanol to prepare reference substance stock solutions with the concentrations of 1.00, 0.420, 0.350, 0.232 and 1.00 mg/mL -1 respectively, precisely sucking the reference substance stock solutions respectively into the same 5mL volumetric flask, adding methanol to fix the volume, shaking uniformly to prepare mixed reference substance solutions ① with the concentrations of the 6 reference substances of 24.0, 160, 210, 35.0, 46.4 and 16.0 mug/mL -1 respectively, and diluting the mixed reference substance solutions ① times to obtain mixed reference substance solutions ②、③、④、⑤、⑥ respectively.
EXAMPLE 3 preparation of negative sample solution
According to the prescription proportion of the trollius chinensis heat-clearing granules and the technological conditions of extraction, drying, forming and the like, negative samples of trollius chinensis, rhizoma anemarrhenae and radix scrophulariae are respectively prepared, and test solutions of the negative samples are prepared according to the method of the example 1.
EXAMPLE 4 linear relationship investigation
Taking the mixed reference substance solution ①～⑥ in the embodiment 2, sampling under the detection condition of HPLC chromatography, and taking the chromatographic condition into consideration, testing and examining the separation effect of gradient elution of different mobile phase compositions (methanol-water, acetonitrile-water, methanol-formic acid, glacial acetic acid, phosphoric acid aqueous solution, acetonitrile-formic acid, glacial acetic acid and phosphoric acid aqueous solution), wherein the result shows that the acetonitrile-phosphoric acid aqueous solution is better as a mobile phase chromatographic peak for separation. Meanwhile, different phosphoric acid water concentrations (0.05%, 0.1%, 0.2% and 0.5%) are examined, and the result shows that acetonitrile-0.1% phosphoric acid water solution is taken as a mobile phase, and each chromatographic peak can be well separated.
At the same time, the effect of changes in column temperature (25, 30, 35 ℃) and flow rate (0.9, 1.0, 1.1mL.min -1) on chromatographic peak separation, relative retention time and RCF of each component tested was examined. The results show that the variation of these two factors has no significant effect on the separation of the individual chromatographic peaks, the relative retention time and the RCF.
Finally, the chromatographic conditions determined were:
The chromatographic column was AGILENT ECLIPSE Plus C 18 (250 mm. Times.4.6 mm,5 μm), the flow rate was 1.0 mL. Min -1, the column temperature was 30℃and the detection wavelength was 272nm. The mobile phase is acetonitrile (A) to 0.1 percent of H 3PO4 aqueous solution (B), and the sample injection amount of the gradient elution ：0～5min,B：97％;5～15min,B：97％～95％;15～25min,B：95％～90％;25～91min,B：90％～68％;92～95min,B：68％～97％;96～100min,B：97％; is 10 mu L. The chromatograms of the mixed control solution, sample solution and negative sample solution under the above conditions are shown in figure 1.
The chromatographic peaks of 6 components to be measured are identified by comparing the retention time of the chromatographic peaks of the control and the sample with the spectrum of the photodiode array detector (PD A), see FIG. 2.
Taking mixed reference substance solution ①～⑥, detecting according to the chromatographic sampling condition and the elution condition, and recording peak areas of mangiferin, orientin-2' -O-beta-L-galactoside, orientin, veratric acid, vitexin and harpagoside. The mass concentration of each control was plotted on the abscissa (X) and the peak area on the ordinate (Y), and the results are shown in Table 1.
Table 16 linear relationship of compounds
EXAMPLE 5 precision test
Taking a mixed reference substance solution ①, sampling for 6 times according to the chromatographic conditions, and recording peak areas of mangiferin, orientin-2' -O-beta-galactoside, orientin, veratric acid, vitexin and harpagoside, wherein as a result, RSD (RELATIVE STANDARD displacement) of 6 component peak areas PA (peak area) is respectively 0.35%, 0.24%, 0.26%, 0.33%, 0.53%, and RSD of retention time RT (retention tim e) is between 0.04 and 0.15%, which indicates that the precision of the instrument is within a specified range.
Example 6 repeatability test
The same part of lotus heat-clearing granule sample is precisely weighed, 6 parts of sample solution is prepared in parallel according to the method of the embodiment 1, and the sample solution is sampled and measured according to the chromatographic analysis conditions, so that the average contents of mangiferin, orientin-2-O-galactoside, orientin, veratric acid, vitexin and harpagoside are respectively 0.34, 2.03, 2.25, 0.19, 0.68 and 0.12 mg.g -1, and RSD is respectively 1.25%, 1.80%, 1.65%, 1.16%, 1.38% and 0.79%, which indicates that the method is good in repeatability and meets the analysis requirements.
Example 7 stability test
1 Part of the sample solution for the repeatability test of example 6 was sampled at 0, 2, 4, 8, 12 and 24 hours, and the chromatograms were recorded to calculate the RSD of the peak areas of the components. Within 24 hours, RSD of peak areas of mangiferin, orientin-2' -O-beta-galactoside, orientin, veratric acid, vitexin and harpagoside are respectively 0.43%, 0.54%, 0.79%, 0.66% and 0.55%, and the solution has good stability within 24 hours.
Example 8 sample recovery test
Taking 6 parts of the same batch of lotus heat-clearing granule samples with calculated content, placing about 0.5g of each part into a conical flask with a plug, adding a reference substance which is 100% of the content of each component in the sample, preparing a sample solution according to the method of example 1, carrying out sample injection measurement under the chromatographic conditions, and calculating the sample recovery rate of 6 components to be detected, wherein the average recovery rates of the peaks of the mangiferin, orientin-2' -O-beta-galactoside, orientin, veratric acid, vitexin and harpagoside are respectively 99.7%, 96.4%, 94.6%, 101.2%, 96.0%, 98.1%, RSD is respectively 3.0%, 0.69%, 1.2%, 3.3%, 1.9% and 2.5%, which indicates that the method is good in accuracy.
EXAMPLE 9 establishment of Relative Correction Factor (RCF) of test element and internal reference
Calculation of RCF
The experiment adopts a multipoint correction method to calculate RCF, namely taking sample injection amounts and measured peak areas of 6 mass points of orientin-2 '-O-beta-galactoside, orientin and vitexin 3 reference substances in a mixed reference substance solution ①～⑥ under the linear relation investigation item of the embodiment 4, and calculating RCF of orientin-2' -O-beta-galactoside, orientin and vitexin of other 5 components to be detected and internal reference substances according to a formula RCFi/s=fi/fs= (Wi/Ai)/(Ws/As) = (wi×As)/(Ws×ai). RCFi/s in the formula are relative correction factors, fi and fs of the component to be measured and the internal reference substance, absolute correction factors of the component to be measured and the internal reference substance, ai and Wi are peak areas and masses of the component to be measured, and As and Ws are peak areas and masses of the internal reference substance. The results are expressed as "mean (RSD)" (n=6), see table 2.
TABLE 2 calculation of relative correction factor RCF i/s
The results show that when 3 components are used as internal reference substances respectively, RSD of RCF of 6 mass points of the other 5 components to be detected and the internal reference substances is less than 2.0%.
RCF reproducibility investigation
The mixed reference solution ① of example 2 was analyzed by using AGILENT ECLIPSE Plus C18 column (250 mm. Times.4.6 mm,5 μm), shimadzu InertSustain C column (250 mm. Times.4.6 mm,5 μm) and Agilent Zorbax SB-C18 column (250 mm. Times.4.6 mm,5 μm) under the condition of chromatographic detection, respectively, and by using different types of HPLC (SHIMADZU Prominenece LC-20A HPLC system, agilent 1260 HPLC system), the RCF of the other 5 components to be measured and the reference were calculated, respectively, and the results were expressed as "mean value (RSD)" (n=6) as shown in Table 3.
TABLE 3 influence of different chromatography columns and chromatography systems on RCF
The results show that when different chromatographic columns and chromatographic systems are adopted, 3 components of orientin-2' -O-beta-galactoside, orientin and vitexin are respectively used as internal references, RSD of RCF values of the other 5 components to be detected and the internal references are less than 5.0%, and the reproducibility is good.
Positioning of chromatographic peaks of components to be measured
Recording the retention time of each component chromatographic peak measured by using 2 high performance liquid chromatography systems and 3 chromatographic columns, and calculating the relative retention time of the other 5 components to be measured and the internal reference when 3 components of orientin-2' -O-beta-galactoside, orientin and vitexin are respectively used as the internal reference. Calculation formula Relative Retention Time (RRT) =rt test/Rt internal reference, the results are expressed as "mean (RSD)" (n=6), see table 4
TABLE 4 influence of different chromatography columns and chromatography systems on relative retention time
The result shows that when 3 components of orientin-2' -O-beta-galactoside, orientin and vitexin are respectively used as internal references, RSD of the other 5 components to be detected and the internal references relative retention time is less than 3.0%, and RRT fluctuation of each component is small and no significant difference exists when different chromatographic columns and chromatographic systems are adopted.
Example 10 comparison of QAMS method with External Standard Method (ESM) content measurement results
21 Batches of lotus heat-clearing granule samples are taken, sample solutions are prepared according to the method of example 1, sample injection measurement is carried out according to the chromatographic analysis conditions, the peak areas of 6 components to be measured are recorded, the peak areas are brought into the respective linear regression equation, and the content is calculated by an ESM method. Orientin-2' -O-beta-galactoside, orientin and vitexin are respectively used As internal references, and the contents of the other 5 components to be detected are calculated according to the formula wi=RCF i/s XAiXWs/As and the QAMS method. The results obtained by the 2 methods were compared by calculating the percent difference (pd= |qams-esm|×2/[ (qams+esm)) ×100%) and are shown in tables 5 to 7.
TABLE 5 ESM and QAMS determination of 6 component content in JINLIAN QING RE granule (mg. G -1)
TABLE 6
TABLE 7
The result shows that when orientin-2' -O-beta-galactoside, orientin and vitexin are respectively used as internal references, the RSD of the calculation results of the QAMS method of 6 ingredients in 21 batches of the lotus heat-clearing particles is less than 2.0%, and the difference percentages of the measurement results of the QAMS method and the ESM method are less than or equal to 3.0%, so that the QAMS method constructed by the experiment is accurate and reliable, and the content of 6 ingredients in the lotus heat-clearing particles can be measured simultaneously under the condition that any one of three internal references is only used as a reference.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (1)
1. A method for simultaneously measuring the contents of 6 components in lotus heat-clearing particles is characterized by respectively selecting 3 substances as internal references, calculating correction factors under each internal reference, and calculating the contents of the other 5 substances according to any correction factor and HPLC chromatographic elution peak area of a sample solution, wherein the 6 components are mangiferin, orientin-2' -O-beta-galactoside, orientin, veratric acid, vitexin and harpagoside;
calculating correction factors RCFi/s=fi/fs= (Wi/Ai)/(Ws/As) = (wi×as)/(ws×ai) under each internal reference, wherein fi and fs are absolute correction factors of the component to be measured and the internal reference respectively, ai and Wi are peak areas and masses of the component to be measured respectively, and As and Ws are peak areas and masses of the internal reference respectively;
The three internal references are orientin-2' -O-beta-galactoside, orientin and vitexin respectively;
The HPLC chromatographic elution condition is that the mobile phase is acetonitrile A-0.1% H 3PO4 water solution B, and the sample injection amount of gradient elution ：0~5 min,B：97％;5~15 min,B：97％~95％;15~25 min,B：95％~90％;25~91 min,B：90％~68％;92~95 min,B：68％~97％;96~100 min,B：97％; is 10 mu L;
AGILENT ECLIPSE Plus C18 column, 250 mm ×4.6 mm,5 μm;
The preparation process of the sample solution comprises the steps of taking a lotus heat-clearing granule sample, grinding, precisely weighing about 0.5 g, placing in a conical bottle with a plug, precisely adding 25ml of 50% methanol solution, sealing, weighing, ultrasonic extracting with power of 250w, frequency of 33KHz and 30 mm, taking out, cooling, supplementing the lost weight with solvent, shaking uniformly, taking a proper amount, centrifuging 10 min in 10000 r min -1, and taking supernatant as the sample solution.
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| SG114468A1 (en) * | 2000-06-23 | 2005-09-28 | Univ Singapore | Separation materials for chromatography and electrophoresis applications comprising cyclodextrins cross-linked and chemically bonded to a support via urethane linkages |
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