A method for simultaneously determining the contents of six components in Jinlian Qingre Granules

Abstract

The present invention discloses a method for simultaneously determining the contents of 6 components in Jinlian Qingre Granules. 3 substances are selected as internal reference substances respectively, and the correction factor under each internal reference substance is calculated. The contents of the other 5 substances can be simultaneously calculated according to the correction factor and the HPLC chromatographic elution peak area of ​​the substance to be measured. The present invention establishes a one-measurement-multiple-evaluations (QAMS) method using any one of the three components of Jinlian Qingre Granule, orientin-2”-O-β-galactoside, orientin, and vitexin, which have clear pharmacological effects and high contents in Jinlian Qingre Granules as an internal reference substance, and can simultaneously determine the contents of 6 components in Jinlian Qingre Granules, and compare and confirm with the determination results of the external standard method. The method disclosed in the present invention uses fewer reference substances, can simplify the detection process, improve detection efficiency, and reduce detection costs, and provides support for the comprehensive evaluation of the quality of the preparation and the improvement of quality standards.

Description

Technical Field

[0001] The invention relates to the technical field of determination of components of traditional Chinese medicines, and more specifically to a method for simultaneously determining the contents of six components in Jinlian Qingre Granules. Background Art

[0002] Jinlian Qingre Granules uses golden lotus and isatis indigotica as the main medicines to clear away heat and detoxify; Anemarrhena asphodeloides and Gypsum are the assistant medicines to help the main medicines clear away the heat in the qi and quickly reduce high fever; Scrophularia ningpoensis and Rehmannia glutinosa are the adjuvant medicines to nourish yin and clear away heat, increase fluid to moisten dryness, produce body fluid to relieve restlessness, clear the blood and cool the blood to detoxify, and are especially suitable for those with damaged body fluids; Bitter almond is the guiding medicine to clear the lungs, relieve cough and resolve phlegm.

[0003] At present, only the content of vitexin is used as the quality control index of Jinlian Qingre Granules. However, this product is composed of 7 Chinese medicines with complex ingredients. It is difficult to fully and accurately evaluate its intrinsic quality by using only a single ingredient as a detection index. In recent years, multi-index component detection and analysis has become the development direction of Chinese medicine quality control, but some Chinese medicine chemical reference substances are difficult to separate, and multi-index quality control is bound to increase the detection cost, thus limiting the application of multi-index quality control mode in production, scientific research, and drug supervision. The one-measurement-multiple-evaluation (QAMS) method uses one active ingredient as an internal reference to construct the relative correction factor (RCF) between each component and the internal reference. By determining the content of one active ingredient, the simultaneous determination of multiple components is achieved. However, there is a phenomenon of repeated RCF research in the current Chinese medicine QAMS, that is, the same analysis method and the same component to be measured are used to repeatedly establish RCF using different internal references, which not only wastes resources but also violates the original intention of establishing QAMS.

[0004] Therefore, how to provide a method for simultaneously determining the contents of multiple components in Jinlian Qingre Granules by a one-measurement-multiple-evaluation method based on different internal reference materials is an urgent problem that technicians in this field need to solve. Summary of the invention

[0005] In view of this, the present invention establishes a QAMS method for simultaneously determining the contents of six components in Jinlian Qingre Granules by using any one of the three components with clear pharmacological effects and high contents in Jinlian Lotus, namely orientin-2"-O-β-galactoside, orientin and vitexin, as an internal reference, and compares and confirms the results with the determination results of the external standard method, so as to provide support for the comprehensive evaluation of the quality of the preparation and the improvement of quality standards.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for simultaneously determining the contents of 6 components in Jinlian Qingre Granules, wherein 3 substances are selected as internal reference substances, and correction factors under each internal reference substance are calculated. The contents of the 6 substances can be simultaneously calculated according to any correction factor and the HPLC chromatographic elution peak area of ​​the substance to be tested.

[0008] Preferably, the correction factor RCFi / s=fi / fs=(Wi / Ai) / (Ws / As)=(Wi×As) / (Ws×Ai) for each internal reference is calculated; wherein fi and fs are the absolute correction factors of the component to be measured and the internal reference, Ai and Wi are the peak area and mass of the component to be measured, and As and Ws are the peak area and mass of the internal reference, respectively.

[0009] Preferably, the three internal reference substances are orientin-2"-O-β-galactoside, orientin and vitexin.

[0010] Preferably, the HPLC chromatographic elution conditions are: mobile phase: acetonitrile (A) ~ 0.1% H3PO4 aqueous solution (B), 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%; injection volume 10μL.

[0011] Through the above technical solutions, we can know that Chinese medicine has the characteristics of multiple components and multiple functions. It is difficult to fully and accurately evaluate its intrinsic quality by using only a single component as a detection indicator. Multi-index component detection and analysis has become the development direction of Chinese medicine quality control. The QAMS method can not only achieve multi-component quality control, but also overcome the difficulties of shortage of reference materials and high detection costs. In this study, a QAMS method was established, which can simultaneously determine the six components in Jinlian Qingre Granules by using any one of the three components of Jinlian Qingre Granules, namely orientin-2"-O-β-galactoside, orientin and vitexin, with clear pharmacological effects and high contents in Jinlian Qingre Granules as internal reference substances. The RCF between the three internal reference substances and the components to be measured was constructed, and its durability was systematically investigated. The six components in 21 batches of Jinlian Qingre Granules were determined by QAMS method using the technical parameters established by the present invention. The percentage differences from the actual results measured by the external standard method were all <3.0%, indicating that the constructed QAMS method is accurate and reliable, and can simultaneously determine the contents of the six components in Jinlian Qingre Granules when only any one of the three internal reference substances is used as a reference substance, avoiding the waste of resources caused by repeated establishment of RCF, and providing support for the comprehensive evaluation of the quality of the preparation and the improvement of quality standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0013] Figure 1 The accompanying drawings are HPLC chromatograms of mixed reference solution, sample solution and negative sample solution;

[0014] Figure 2 The attached figure is a photodiode array detector (PDA) spectrum diagram of the corresponding chromatographic peaks in the six component reference substances and samples. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Instruments and reagents used in the examples

[0017] instrument

[0018] Prominenece LC-20A high performance liquid chromatography system (Shimadzu Corporation, Japan); Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); KQ5200B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); XS105DU analytical balance (Mettler-Toledo, Switzerland); TGL-16G high speed centrifuge (Anting Scientific Instrument Co., Ltd., Shanghai).

[0019] Drug testing

[0020] Reference substances: mangiferin (batch number: 111607-201704), purchased from China Food and Drug Inspection Institute; orientin-2”-O-β-galactoside (batch number: P01O11F126428), orientin (batch number: S19GB161663), veratric acid (batch number: DN1127BA13), vitexin (batch number: D29GB172870), all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; harpagoside (batch number: PS000404), purchased from Chengdu Pusi Biotechnology Co., Ltd., the purity of the reference substances was ≥98%. Chromatographic grade acetonitrile was purchased from Fisher, and water for chromatographic analysis was purchased from Hangzhou Wahaha Group Co., Ltd.

[0021] Batch 21 of Jinlian Qingre Granules was provided by Ningxia Qiyuan Pharmaceutical Co., Ltd.

[0022] In the prior art, only vitexin is used as a quality evaluation index for Jinlian Qingre Granules, which makes it difficult to comprehensively and accurately evaluate its intrinsic quality. Examples 1-8 are used to verify whether the contents of the six components in Jinlian Qingre Granules can be determined simultaneously, so as to provide technical permission for the subsequent one-test-multiple-evaluation method to determine the contents of the six components.

[0023] Example 1 Preparation of test sample solution

[0024] This example investigates different extraction solvents (water, methanol, ethanol), and the results show that the extraction rate of each component is the highest when methanol is used as the solvent. The methanol concentration (30%, 50%, 70%), extraction time (20, 30, 40, 50min) and extraction solvent dosage (10, 25, 50, 100ml) are further compared, and the extraction efficiency of the six components to be tested in Jinlian Qingre Granules is used as an index for optimization, and the preparation method of the Jinlian Qingre Granules test solution is finally determined as follows: add 25mL of 50% methanol solution and ultrasonically extract for 30min.

[0025] The specific process is as follows: Take a sample of Jinlian Qingre Granules, grind it into powder, accurately weigh about 0.5g, put it in a stoppered conical bottle, accurately add 25ml of 50% methanol solution, seal it, and weigh it. Perform ultrasonic extraction (power 250w, frequency 33KHz) for 30min, take it out, cool it, use solvent to make up the lost weight, and shake it well. Take an appropriate amount, centrifuge (10000r·min -1 ) for 10 min, and the supernatant was used as the test sample solution.

[0026] Example 2 Preparation of mixed reference solution

[0027] Accurately weigh appropriate amounts of mangiferin, orientin-2"-O-β-galactoside, orientin, veratric acid, vitexin, and harpaoside reference substances, and add methanol to make the concentrations of 1.00, 1.00, 0.420, 0.350, 0.232, and 1.00 mg·mL, respectively. -1 Accurately pipette appropriate amount of the above reference substance stock solution into the same 5mL volumetric flask, add methanol to make up to volume, shake well, and make the above 6 reference substances with concentrations of 24.0, 160, 210, 35.0, 46.4, and 16.0μg·mL -1 The mixed reference solution ① is diluted in multiple ratios to obtain mixed reference solutions ②, ③, ④, ⑤, and ⑥, respectively.

[0028] Example 3 Preparation of negative sample solution

[0029] According to the prescription ratio of Jinlian Qingre Granules and the process conditions of extraction, drying, molding, etc., negative samples lacking Jinlianhua, lacking Zhimu, and lacking Scrophularia were prepared respectively, and the test solutions of each negative sample were prepared according to the method of Example 1.

[0030] Example 4 Linear Relationship Investigation

[0031] Take the mixed reference solution ①~⑥ in Example 2, and inject under HPLC chromatographic detection conditions. When considering the chromatographic conditions, the separation effect of gradient elution with different mobile phase compositions (methanol-water, acetonitrile-water, methanol-formic acid, glacial acetic acid, phosphoric acid aqueous solution, acetonitrile-formic acid, glacial acetic acid, phosphoric acid aqueous solution) was tested. The results showed that acetonitrile-phosphoric acid aqueous solution was used as the mobile phase for better separation of chromatographic peaks. At the same time, different phosphoric acid aqueous concentrations (0.05%, 0.1%, 0.2%, 0.5%) were investigated, and the results showed that acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase, and each chromatographic peak could be better separated.

[0032] At the same time, the column temperature (25, 30, 35°C) and flow rate (0.9, 1.0, 1.1 mL.min -1 The results showed that the changes of these two factors had no significant effect on the separation, relative retention time and RCF of each chromatographic peak.

[0033] Finally, the chromatographic conditions were determined as follows:

[0034] Column: Agilent EClipse Plus C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm); flow rate 1.0 mL min -1 , column temperature 30℃, detection wavelength: 272nm. Mobile phase: acetonitrile (A) ~ 0.1% H3PO4 aqueous solution (B), 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%; injection volume 10μL. The chromatograms of the mixed reference solution, sample solution and negative sample solution under the above conditions are shown in Figure 1 .

[0035] By comparing the retention time of the chromatographic peaks of the reference and samples and the photodiode array detector (PDA) spectra, the chromatographic peaks of the six components to be tested were identified. Figure 2 .

[0036] Take mixed reference solution ①~⑥, detect according to the above chromatographic injection and elution conditions, and record the peak areas of mangiferin, orientin-2"-O-β-L-galactoside, orientin, veratric acid, vitexin and harpaoside. Linear regression was performed with the mass concentration of each reference substance as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 1.

[0037] Table 1 Linear relationships of 16 compounds

[0038]

[0039] Example 5 Precision Test

[0040] Take the mixed reference solution ① and inject it 6 times according to the above chromatographic conditions. Record the peak areas of mangiferin, orientin-2"-O-β-galactoside, orientin, veratric acid, vitexin and harpaoside. The RSDs (relative standard deviations) of the peak areas PA (peak area) of the six components are 0.35%, 0.24%, 0.24%, 0.26%, 0.33% and 0.53%, respectively. The RSD of the retention time RT (retention time) is between 0.04 and 0.15%, indicating that the precision of the instrument is within the specified range.

[0041] Example 6 Repeatability Test

[0042] The same Jinlian Qingre Granule sample was accurately weighed, and 6 sample solutions were prepared in parallel according to the method of Example 1. The samples were injected and measured according to the above chromatographic analysis conditions. The results showed that the average contents of mangiferin, orientin-2-O-galactoside, orientin, veratric acid, vitexin, and harpagoside were 0.34, 2.03, 2.25, 0.19, 0.68, and 0.12 mg·g, respectively. -1 , RSDs were 1.25%, 1.80%, 1.65%, 1.16%, 1.38% and 0.79%, respectively, indicating that the method had good repeatability and met the analysis requirements.

[0043] Example 7 Stability Test

[0044] Take one portion of the test solution of the repeatability test in Example 6, inject once at 0, 2, 4, 8, 12, and 24 hours, record the chromatogram, and calculate the RSD of the peak area of ​​each component. Within 24 hours, the RSDs of the peak areas of mangiferin, orientin-2"-O-β-galactoside, orientin, veratric acid, vitexin, and harpagoside were 0.43%, 0.54%, 0.79%, 0.66%, and 0.55%, respectively, and the solution had good stability within 24 hours.

[0045] Example 8 Sample recovery experiment

[0046] Take 6 samples of Jinlian Qingre Granules from the same batch with calculated content, each weighing about 0.5 g, and place them in a stoppered conical flask, add a reference substance equivalent to 100% of the content of each component in the sample, prepare the test sample solution according to the method in Example 1, inject and measure under the above-mentioned chromatographic conditions, and calculate the recovery rates of the 6 components to be tested. The results show that the average recoveries of mangiferin, orientin-2"-O-β-galactoside, orientin, veratric acid, vitexin and harpagoside peaks are 99.7%, 96.4%, 94.6%, 101.2%, 96.0% and 98.1%, respectively, and the RSDs are 3.0%, 0.69%, 1.2%, 3.3%, 1.9% and 2.5%, respectively, indicating that the method has good accuracy.

[0047] Example 9 Establishment of the relative correction factor (RCF) between the component to be measured and the internal reference

[0048] Calculation of RCF

[0049] In this experiment, the multi-point calibration method was used to calculate the RCF, that is, the injection amount and measured peak area of ​​6 mass points of the three reference substances, orientin-2"-O-β-galactoside, orientin, and vitexin in the mixed reference solution ①~⑥ under the linear relationship investigation item of Example 4 were taken, and the RCF of the other five components to be tested and the internal reference substances when orientin-2"-O-β-galactoside, orientin, and vitexin were used as internal reference substances were calculated according to the formula RCFi / s=fi / fs=(Wi / Ai) / (Ws / As)=(Wi×As) / (Ws×Ai). In the formula, RCFi / s is the relative correction factor of the component to be tested and the internal reference substance, fi and fs are the absolute correction factors of the component to be tested and the internal reference substance, Ai and Wi are the peak area and mass of the component to be tested, respectively, and As and Ws are the peak area and mass of the internal reference substance, respectively. The results are expressed as "mean (RSD)" (n=6), see Table 2.

[0050] Table 2 Relative correction factor RCF i / s Calculation results

[0051]

[0052] The results showed that when three components were used as internal references, the RSDs of the RCFs of the other five components to be tested and the six mass points of the internal references were all less than 2.0%.

[0053] RCF reproducibility study

[0054] Take the mixed reference solution of Example 2 ①, according to the chromatographic detection conditions, adopt Agilent Eclipse Plus C18 column (250mm×4.6mm, 5μm), Shimadzu InertSustain C18 column (250mm×4.6mm, 5μm), Agilent Zorbax SB-C18 column (250mm×4.6mm, 5μm), respectively, analyze on high performance liquid chromatographs of different models (SHIMADZU Prominenece LC-20A high performance liquid chromatography system, Agilent 1260 high performance liquid chromatography system), calculate when taking 3 components as internal reference substances, the RCF of other 5 components to be tested and internal reference substance, the results are expressed as "mean (RSD)" (n=6), see Table 3.

[0055] Table 3 Effects of different chromatographic columns and chromatographic systems on RCF

[0056]

[0057] The results showed that when different chromatographic columns and chromatographic systems were used, when orientin-2"-O-β-galactoside, orientin and vitexin were used as internal reference substances, the RSDs of the RCF values ​​of the other five components to be tested and the internal reference substances were all less than 5.0%, indicating good reproducibility.

[0058] Positioning of chromatographic peaks of components to be measured

[0059] The retention time of the chromatographic peaks of each component measured by the above two HPLC systems and three chromatographic columns was recorded, and the relative retention time of the other five components to be tested and the internal reference was calculated when the three components of orientin-2"-O-β-galactoside, orientin and vitexin were used as internal references. The calculation formula is: relative retention time (RRT) = Rt test substance / Rt internal reference substance. The results are expressed as "mean (RSD)" (n = 6), see Table 4

[0060] Table 4 Effects of different chromatographic columns and chromatographic systems on relative retention time

[0061]

[0062] The results showed that when orientin-2"-O-β-galactoside, orientin and vitexin were used as internal reference substances, the RSDs of the relative retention times of the other five components to be tested and the internal reference substances were all <3.0%. When different chromatographic columns and chromatographic systems were used, the RRT fluctuations of each component were small and there was no significant difference.

[0063] Example 10 Comparison of the results of content determination by QAMS method and external standard method (ESM)

[0064] Take 21 batches of Jinlian Qingre Granules, prepare the test sample solutions according to the method in Example 1, inject and measure according to the above chromatographic analysis conditions, record the peak areas of the 6 components to be tested, and insert them into their respective linear regression equations to calculate the content by ESM method. Orientin-2"-O-β-galactoside, orientin, and vitexin are used as internal reference substances, respectively, according to the formula Wi = RCF i / s × Ai × Ws / As, QAMS method was used to calculate the contents of the other five components to be tested. The results obtained by the two methods were compared by calculating the difference percentage (PD = |QAMS-ESM| × 2 / [(QAMS+ESM))] × 100%), as shown in Tables 5 to 7.

[0065] Table 5 Results of determination of the contents of six components in Jinlian Qingre Granules by ESM and QAMS (mg·g -1 )

[0066]

[0067]

[0068] Table 6

[0069]

[0070]

[0071] Table 7

[0072]

[0073]

[0074] The results showed that when orientin-2"-O-β-galactoside, orientin and vitexin were used as internal reference substances, the RSDs of the QAMS calculation results of the six components in 21 batches of Jinlian Qingre Granules were all <2.0%, and the percentage differences between the results determined by the QAMS method and the ESM method were all ≤3.0%, indicating that the QAMS method constructed in this experiment is accurate and reliable, and can simultaneously determine the contents of the six components in Jinlian Qingre Granules with only one of the three internal reference substances.

[0075] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0076] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously determining the contents of six components in Jinlian Qingre Granules, characterized in that: Three substances were selected as internal reference substances, and the correction factor of each internal reference substance was calculated. The contents of the other five substances were calculated according to any correction factor and the HPLC chromatographic elution peak area of ​​the test sample solution; the six components were mangiferin, orientin-2''-O-β-galactoside, orientin, veratric acid, vitexin and harpagoside; Calculate the correction factor RCFi / s = fi / fs = (Wi / Ai) / (Ws / As) = (Wi×As) / (Ws×Ai) for each internal reference; where fi and fs are the absolute correction factors of the component to be measured and the internal reference, Ai and Wi are the peak area and mass of the component to be measured, and As and Ws are the peak area and mass of the internal reference, respectively. The three internal reference substances were orientin-2''-O-β-galactoside, orientin, and vitexin; HPLC elution conditions were as follows: mobile phase: acetonitrile A~0.1% H3PO4 aqueous solution B, 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%; injection volume 10 μL; Chromatographic column: Agilent EClipse Plus C18 column, specification: 250 mm × 4.6 mm, 5 μm; The preparation process of the test sample solution is as follows: take the Jinlian Qingre Granule sample, grind it into powder, accurately weigh about 0.5 g, put it in a stoppered conical flask, accurately add 25 ml of 50% methanol solution, seal it tightly, and weigh it; ultrasonic extraction, power 250w, frequency 33KHz, 30min, take it out, cool it, make up the lost weight with solvent, shake it well, take an appropriate amount, 10000 r·min -1 Centrifuge for 10 min and use the supernatant as the test sample solution.

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