CN115097031B - Method for establishing HPLC (high Performance liquid chromatography) characteristic spectrum of tonifying middle-jiao and Yi oral liquid - Google Patents
Method for establishing HPLC (high Performance liquid chromatography) characteristic spectrum of tonifying middle-jiao and Yi oral liquid Download PDFInfo
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Abstract
The invention discloses a method for establishing an HPLC (high performance liquid chromatography) characteristic spectrum of a tonifying middle-jiao and Yi oral liquid, belonging to the field of analysis of traditional Chinese medicine preparations. The method optimizes the characteristic spectrum detection conditions, screens the marked characteristic peaks based on the chemical pattern recognition technology, and finally determines the HPLC characteristic spectrum analysis method through methodology investigation. By Kromasil 100-5-C 18 Chromatographic column (4.6X250 mm,5 μm); acetonitrile (A) -0.2% formic acid water solution (B) is taken as a mobile phase; gradient elution, flow rate 0.80mL/min; and (3) detecting the wavelength of 260nm, and performing high performance liquid chromatography analysis to obtain the characteristic spectrum of the tonifying middle-jiao and Yi oral liquid. The method has the advantages of good repeatability, high stability and precision, simple operation and the like, and the characteristic spectrum of the oral liquid for tonifying middle-jiao and Qi, which is measured by the method, fully shows the chemical component characteristics of the oral liquid, and provides reference for the subsequent quality evaluation research of the oral liquid for tonifying middle-jiao and Qi.
Description
Field of the art
The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly relates to a method for establishing a characteristic spectrum of a Chinese medicinal oral liquid for tonifying middle-jiao and Qi.
(II) background art
The Chinese medicine compound preparation is prepared from ten medicinal materials of radix astragali preparata, dangshen, stir-fried largehead atractylodes rhizome, honey-fried licorice root, chinese angelica, dried orange peel, cimicifuga foetida, bupleurum root, ginger and Chinese date, and the original prescription is created by the east wall of the famous doctor of the Jinyuan period, and is first carried in the internal and external injury distinguished confusion theory, and has the effects of tonifying middle-jiao and Qi and raising yang and trapping. In the formula, astragalus root can be used for patients with weakness of spleen and stomach and pathogenic factors of lung qi, and can avoid spontaneous perspiration from damaging primordial qi; licorice is sweet in taste, can purge heart fire and remove spleen heat, and neutralize spleen and stomach primordial qi, and the processed product has stronger effect; bai Zhu can relieve stomach heat; cimicifugae rhizoma and bupleuri radix can promote the ascending of qi due to the sweet and warm nature of qi-tonifying herbs; dried orange peel regulates qi and helps yang qi rise to dissipate stagnant qi. The research shows that the oral liquid for strengthening the middle-jiao and replenishing qi has the pharmacological effects of regulating stomach and intestine, enhancing immunity, resisting inflammation, resisting fatigue and the like; has certain curative effect on digestive system diseases, respiratory system diseases, urinary system diseases, gynecological diseases and other diseases.
The Chinese medicine and compound characteristic map is an important means for controlling the quality of medicinal materials and compound preparations, and is also a key for ensuring clinical curative effect. The characteristic map has the characteristics of integrity and ambiguity, well accords with the characteristics of complexity and diversity of traditional Chinese medicine components, and can evaluate and control the quality of traditional Chinese medicine and compound. The composition for invigorating middle-warmer and replenishing qi has various dosage forms, such as mixture, pill, granule, oral liquid, etc., wherein the mixture, pill, granule are recorded in the 2020 edition of Chinese pharmacopoeia, and the oral liquid for invigorating middle-warmer and replenishing qi has no definite quality standard. Aiming at the situation, the invention adopts an HPLC method to study the characteristic spectrum of the middle-jiao tonifying qi-tonifying oral liquid, strictly controls the quality of the middle-jiao tonifying qi-tonifying oral liquid, comprehensively and intuitively reflects the types and the amounts of the internal active components of the middle-jiao tonifying qi-tonifying oral liquid, and provides a reference for the establishment of a quality evaluation system of the subsequent middle-jiao tonifying qi-tonifying oral liquid.
(III) summary of the invention
The invention aims to solve the defects of the prior art, provides a characteristic spectrum detection method of the tonifying middle-jiao and Yi oral liquid, provides a new technical method for controlling the quality of the preparation, and aims to solve the problems of the prior quality control technology of the tonifying middle-jiao and Yi oral liquid.
In order to achieve the above object, the present invention provides the following technical solutions: the evaluation method of the oral liquid for tonifying middle-jiao and Qi based on the characteristic spectrum specifically comprises the following steps:
(1) Preparation of test sample and mixed reference substance solution
Precisely measuring 1mL of the tonifying middle-jiao and Yi oral liquid in a volumetric flask, fixing the volume to 5mL by using methanol, performing ultrasonic treatment for 3min, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution. Taking appropriate amount of calycosin-7-O-glucoside, glycyrrhizin, ferulic acid, hesperidin and ammonium glycyrrhizinate reference substances, precisely weighing, and adding methanol to obtain mixed reference substance solutions containing 20 μg, 50 μg, 5 μg, 170 μg, 10 μg and 200 μg respectively per 1 mL.
(2) Optimization of feature maps
The chromatographic conditions (mobile phase aqueous phase acid, wavelength, column temperature, flow speed and chromatographic column) in the characteristic spectrum construction are optimized, and the optimal high performance liquid chromatographic conditions of the oral liquid for tonifying middle-jiao and Qi are established.
(3) Selection of characteristic peaks
Different from the common characteristic peaks which are selected according to the height of the chromatographic peaks, the method utilizes a chemical pattern recognition technology (CA, PCA, PLS-DA) to analyze the chromatographic peaks of the oral liquid for supplementing the middle-jiao and replenishing the qi in different batches, and screens out the chromatographic peaks with larger influence on the quality of samples. And determining characteristic peaks of the chromatogram by taking the screening result as a reference.
(4) Methodology investigation of feature maps
The method for establishing the characteristic spectrum is used for examining precision, repeatability and stability, and the analysis method is proved to be suitable for analyzing the characteristic spectrum of the tonifying middle-jiao and Yi oral liquid.
(5) Establishing a characteristic spectrum of the oral liquid for strengthening middle-jiao and replenishing qi
The conditions of the high performance liquid chromatography are as follows: chromatographic column: kromasil 100-5-C 18 (4.6X250 mm,5 μm); mobile phase: acetonitrile (a) -0.2% formic acid in water (B); the detection wavelength is 260nm; column temperature is 35 ℃; the flow rate is 0.80mL/min; the sample injection amount is 10 mu L; the gradient elution procedure was as follows:
compared with the prior art, the invention has the beneficial effects that:
(1) When characteristic peaks in the HPLC characteristic spectrum of the tonifying middle-jiao and Yi oral liquid are selected, the chemical pattern recognition technology is utilized to screen out components with larger mass difference, and the components are sequentially selected as reference characteristic peaks.
(2) The HPLC characteristic spectrum of the tonifying middle-jiao and Yi oral liquid established by the invention is calibrated with 14 characteristic peaks, and the chromatographic peak in the constructed HPLC characteristic spectrum has better separation degree, rich characteristic spectrum information, and the established method is simple, convenient and reliable, high in stability and precision and good in repeatability.
(3) The HPLC characteristic spectrum of the oral liquid for tonifying middle-jiao and Qi constructed by the invention fills the defect of the quality control technology of the oral liquid formulation of the prescription, and provides guarantee for improving the stability, reliability and safety of the product quality.
(IV) description of the drawings
FIG. 1 is a chromatogram of the mobile phase aqueous acid investigation result in example 1.
FIG. 2 is a chromatogram of the wavelength investigation result in example 1
FIG. 3 is a chromatogram of the results of the column temperature investigation in example 1.
FIG. 4 is a chromatogram of the flow rate measurement result in example 1.
FIG. 5 is a chromatogram of the results of the durability test of the column in example 1.
FIG. 6 is a pattern of common peaks for the tonic and beneficial oral liquid markers in example 2.
Fig. 7 is a superposition chromatogram of various batches of the middle-jiao and Qi-tonifying oral liquid in example 2.
FIG. 8 is a graph showing the clustering analysis (A) and principal component analysis score in example 2.
Fig. 9 shows partial least squares discriminant analysis score map (a) and VIP map (B) in example 2.
FIG. 10 is an HPLC characteristic spectrum of the tonifying middle-jiao and Yi oral liquid in example 2.
(fifth) detailed description of the invention
The invention will be further illustrated by the following specific examples, although the scope of the invention is not limited thereto.
In the following examples, specific batches of the related middle-jiao and Qi-tonifying oral liquid are shown in the following table 1:
table 1 sample information of oral liquid for invigorating middle warmer and replenishing Qi
| Numbering device | Batch of | Numbering device | Batch of |
| S1 | 150302 | S12 | 200701 |
| S2 | 150905 | S13 | 200905 |
| S3 | 180302 | S14 | 201108 |
| S4 | 180502 | S15 | 201215 |
| S5 | 180604 | S16 | 210101 |
| S6 | 181010 | S17 | 210102 |
| S7 | 181215 | S18 | 210103 |
| S8 | 190406 | S19 | 210112 |
| S9 | 190902 | S20 | 210115 |
| S10 | 200101 | S21 | 210301 |
| S11 | 200605 | S22 | 210401 |
Example 1
In this example, chromatographic conditions (mobile phase aqueous phase acid, wavelength, column temperature, flow rate, chromatographic column) in the construction of the HPLC characteristic spectrum are examined, and the technical scheme is further described.
(1) Mobile phase aqueous acid investigation
The effect on chromatographic peaks when the aqueous phase was water, 0.1% phosphoric acid water, 0.1% formic acid, and 0.2% formic acid water was examined, and the results are shown in fig. 1.
The results show that: when the aqueous phase acid of the mobile phase is 0.2% of formic acid water, the whole baseline is stable, and the separation degree between chromatographic peaks is good, so that the aqueous phase of the mobile phase is 0.2% of formic acid water.
(2) Wavelength investigation
Sample solutions were sampled and chromatograms at 237nm, 260nm, 283nm, 316nm wavelengths were extracted, and the results were shown in FIG. 2.
The results show that: when the detection wavelength is 260nm, the information amount of the chromatographic peak is large, the base line of the chromatographic peak is stable, and the separation degree between the chromatographic peaks is good, so that the detection wavelength is determined to be 260nm.
(3) Column temperature investigation
The column temperatures were set at 25℃and 30℃and 35℃respectively, and the results are shown in FIG. 3.
The results show that: when the column temperature is 35 ℃, the chromatographic peak type is symmetrical, and the separation degree is moderate, so the column temperature is set to be 35 ℃.
(4) Flow rate investigation
The flow rates were set to 0.60ml/min, 0.80ml/min, and 1.00ml/min, respectively, and the results are shown in FIG. 4.
The results show that: when the flow rate is 0.80ml/min, each characteristic peak-to-peak type is good, and the separation degree is moderate, so the flow rate is determined to be 0.80ml/min.
(5) Chromatographic column durability investigation
Based on the construction method of the characteristic spectrum of the tonifying middle-jiao Yi oral liquid, 4 chromatographic columns of Agilent ZORBAX SB-C18 (4.6 mm multiplied by 250mm,5.0 mu m), ACE Excel 5C 18-AR (4.6 mm multiplied by 250mm,5.0 mu m), kromasil 100-5-C18 (4.6 mm multiplied by 250mm,5.0 mu m) and Diamond 5 mu m C (4.6 mm multiplied by 250mm,5.0 mu m) are respectively examined, and the obtained results are shown in figure 5.
The results show that: the chromatographic patterns of different chromatographic columns have larger difference, wherein compared with the chromatographic columns, the Kromasil 100-5-C18 chromatographic column has more chromatographic peak information and better separation degree of each peak, so the Kromasil 100-5-C18 chromatographic column is fixed in the liquid phase condition to be used as an analysis column of the characteristic spectrum of the middle-jiao tonifying oral liquid.
Example 2
In this example, the chemical pattern recognition analysis is performed on the common peak in the HPLC profile construction, and the present technical scheme is further described.
(1) Similarity evaluation
And (3) taking 20 common peaks of 22 batches of middle-jiao and qi-tonifying oral liquid sample solutions as a reference, marking the common peak chromatograms as shown in fig. 6, evaluating the similarity, and overlapping chromatograms of middle-jiao and qi-tonifying oral liquid in different batches as shown in fig. 7, wherein the result shows that the similarity is 0.950-0.994. The overall characteristic similarity of the chemical components is high, the quality of the oral liquid is relatively stable, but the RSD value of the relative peak area is larger, which indicates that the content of the chemical components is different.
(2) Clustering and principal component analysis
Taking 20 common peak areas of 22 batches of middle-tonifying and benefit oral liquid as variables, introducing SPSS 19.0 and SIMCA13.0 software, performing systematic clustering and principal component analysis on samples, wherein the obtained results show that PCA and CA results are consistent, and the classification results are as follows: 15 year batch, 18 year and 19 year batch, 20 year and 21 year batch, as shown in fig. 8. In the principal component analysis, the characteristic value of > 1 is taken as an extraction standard, the accumulated variance contribution rate of the first 5 principal components is 84.676 percent and is larger than 80 percent, and the first 5 principal components can explain most of information of the common peaks of the fingerprint spectrums of the 20 middle-jiao and Qi tonifying oral liquids, so that the middle-jiao and Qi tonifying oral liquids can be evaluated by using the 5 principal components, and the characteristic value and the variance contribution rate of each principal component are shown in the following table 2.
TABLE 2 sample principal component eigenvalues and cumulative variance contribution rates
(3) Partial least squares discriminant analysis
Based on the principal component analysis result, the PLS-DA model is adopted to carry out supervised discriminant analysis, and after the model is automatically fitted, the interpretation rate R of the X matrix is calculated 2 X=0.611, model stability parameter R 2 Y=0.719, predictive power parameter Q 2 And (0.589) is larger than 0.5, which indicates that the established PLS-DA model has good stability and strong prediction capability. Through minimum deviationAnd (3) performing square discriminant analysis to obtain a score graph and a variable importance projection value graph (VIP graph), wherein the score graph and the variable importance projection value graph are shown in FIG. 9.
From the above chemical pattern recognition analysis, peak 7, peak 17, peak 4, peak 15, peak 12, peak 20, peak 8, peak 18, peak 11, peak 13, peak 16 were selected. The 11 chemical components are proved to have obvious influence on the quality difference of the samples, and are main potential marking components for causing the quality difference of the oral liquid. Based on the screening result, 14 characteristic chromatographic peaks are selected according to the height of the chromatographic peak, and 12 characteristic peaks are identified according to the comparison of the mixed reference substance and the HPLC-QTOF-MSMS analysis, see figure 10.
Example 3
Based on examples 1 and 2, the methodology of this example was examined, and specifically includes:
(1) Precision investigation
According to the preparation method of the sample solution in the third invention, the sample solution of the oral liquid (batch: 210101) for supplementing middle-jiao and replenishing qi is prepared, sample injection is carried out for 3 times according to different testers and different time, the relative retention time and the relative peak area of each characteristic peak are calculated by taking the No. 9 hesperidin chromatographic peak as a reference peak (S), and the obtained results are shown in tables 3-4 below.
TABLE 3 precision investigation results (relative retention time)
| Numbering device | Peak 1 | Peak 2 | Peak 3 | Peak 4 | Peak 5 | Peak 6 | Peak 7 | Peak 8 | Peak 9 (S) | Peak 10 | Peak 11 | Peak 12 | Peak 13 | Peak 14 |
| Personnel 1 | 0.167 | 0.365 | 0.800 | 0.816 | 0.834 | 0.858 | 0.903 | 0.917 | 1.00 | 1.04 | 1.16 | 1.29 | 1.49 | 1.55 |
| Personnel 2 | 0.173 | 0.372 | 0.799 | 0.815 | 0.833 | 0.857 | 0.902 | 0.917 | 1.00 | 1.04 | 1.16 | 1.29 | 1.49 | 1.55 |
| At different times | 0.167 | 0.368 | 0.799 | 0.815 | 0.832 | 0.855 | 0.901 | 0.916 | 1.00 | 1.04 | 1.16 | 1.30 | 1.49 | 1.55 |
| RSD% | 1.87 | 1.79 | 0.95 | 1.01 | 0.05 | 0.07 | 0.10 | 0.16 | 0.16 | 0.06 | 0.00 | 0.07 | 0.04 | 0.14 |
Table 4 results of precision investigation (relative peak area)
| Numbering device | Peak 1 | Peak 2 | Peak 3 | Peak 4 | Peak 5 | Peak 6 | Peak 7 | Peak 8 | Peak 9 (S) | Peak 10 | Peak 11 | Peak 12 | Peak 13 | Peak 14 |
| Personnel 1 | 0.326 | 0.439 | 0.643 | 0.398 | 0.587 | 0.093 | 0.462 | 0.300 | 1.00 | 1.58 | 0.39 | 0.20 | 0.20 | 1.09 |
| Personnel 2 | 0.336 | 0.444 | 0.650 | 0.399 | 0.585 | 0.094 | 0.477 | 0.306 | 1.00 | 1.58 | 0.39 | 0.19 | 0.20 | 1.07 |
| At different times | 0.336 | 0.443 | 0.617 | 0.391 | 0.573 | 0.096 | 0.488 | 0.315 | 1.00 | 1.58 | 0.38 | 0.20 | 0.21 | 1.11 |
| RSD% | 1.68 | 0.57 | 2.73 | 0.99 | 1.24 | 1.30 | 2.77 | 2.56 | 0.00 | 0.19 | 0.83 | 0.96 | 2.53 | 1.61 |
The results show that: the relative retention time RSD of the characteristic peak and the relative peak area RSD of the characteristic peak are not more than 5%, and the method has good precision.
(2) Repeatability investigation
According to the preparation method of the sample solution in the third invention, 6 parts of sample solution of the oral liquid (batch: 210101) for tonifying middle-jiao and Qi is prepared, the relative retention time and the relative peak area of each characteristic peak are calculated by taking the No. 9 hesperidin chromatographic peak as a reference peak (S), and the obtained results are shown in tables 5-6 below.
TABLE 5 repeatability test results (relative retention time)
| Numbering device | Peak 1 | Peak 2 | Peak 3 | Peak 4 | Peak 5 | Peak 6 | Peak 7 | Peak 8 | Peak 9 (S) | Peak 10 | Peak 11 | Peak 12 | Peak 13 | Peak 14 |
| 1 | 0.172 | 0.371 | 0.800 | 0.815 | 0.832 | 0.855 | 0.900 | 0.915 | 1.00 | 1.04 | 1.16 | 1.29 | 1.49 | 1.56 |
| 2 | 0.175 | 0.372 | 0.797 | 0.815 | 0.834 | 0.859 | 0.905 | 0.919 | 1.00 | 1.04 | 1.16 | 1.29 | 1.48 | 1.54 |
| 3 | 0.176 | 0.372 | 0.797 | 0.815 | 0.834 | 0.859 | 0.905 | 0.919 | 1.00 | 1.04 | 1.16 | 1.29 | 1.48 | 1.54 |
| 4 | 0.176 | 0.372 | 0.796 | 0.815 | 0.834 | 0.859 | 0.905 | 0.919 | 1.00 | 1.04 | 1.16 | 1.29 | 1.48 | 1.54 |
| 5 | 0.176 | 0.372 | 0.797 | 0.815 | 0.834 | 0.859 | 0.905 | 0.919 | 1.00 | 1.04 | 1.16 | 1.29 | 1.48 | 1.54 |
| 6 | 0.175 | 0.372 | 0.798 | 0.816 | 0.835 | 0.859 | 0.905 | 0.919 | 1.00 | 1.04 | 1.16 | 1.29 | 1.48 | 1.54 |
| RSD% | 0.80 | 0.14 | 0.15 | 0.04 | 0.12 | 0.20 | 0.24 | 0.16 | 0.00 | 0.22 | 0.07 | 0.05 | 0.49 | 0.50 |
TABLE 6 repeatability test results (relative peak area)
| Numbering device | Peak 1 | Peak 2 | Peak 3 | Peak 4 | Peak 5 | Peak 6 | Peak 7 | Peak 8 | Peak 9 (S) | Peak 10 | Peak 11 | Peak 12 | Peak 13 | Peak 14 |
| 1 | 0.344 | 0.439 | 0.628 | 0.393 | 0.573 | 0.086 | 0.475 | 0.301 | 1.00 | 1.54 | 0.39 | 0.23 | 0.19 | 1.11 |
| 2 | 0.323 | 0.446 | 0.650 | 0.392 | 0.577 | 0.090 | 0.417 | 0.268 | 1.00 | 1.56 | 0.42 | 0.21 | 0.18 | 1.07 |
| 3 | 0.324 | 0.451 | 0.657 | 0.397 | 0.581 | 0.089 | 0.426 | 0.276 | 1.00 | 1.58 | 0.44 | 0.22 | 0.19 | 1.10 |
| 4 | 0.332 | 0.457 | 0.657 | 0.397 | 0.583 | 0.090 | 0.424 | 0.275 | 1.00 | 1.58 | 0.43 | 0.21 | 0.19 | 1.10 |
| 5 | 0.327 | 0.436 | 0.650 | 0.394 | 0.574 | 0.089 | 0.420 | 0.270 | 1.00 | 1.56 | 0.43 | 0.22 | 0.19 | 1.08 |
| 6 | 0.332 | 0.445 | 0.648 | 0.391 | 0.575 | 0.091 | 0.417 | 0.266 | 1.00 | 1.56 | 0.41 | 0.20 | 0.19 | 1.06 |
| RSD% | 2.33 | 1.69 | 1.62 | 0.64 | 0.72 | 2.10 | 5.22 | 4.68 | 0.00 | 0.84 | 4.23 | 5.28 | 2.07 | 1.60 |
The results show that: the relative retention time RSD and relative peak area RSD of the characteristic peak of the method are not more than 5%, and the method has good repeatability.
(3) Stability investigation
According to the preparation method of the sample solution in the third invention, 1 part of the sample solution of the middle-jiao and Qi tonifying oral liquid (batch: 210101) is prepared, sample injection measurement is carried out at 0h, 2h, 4h, 6h, 12h and 24h respectively, the relative retention time and the relative peak area of each characteristic peak are calculated by taking the No. 9 hesperidin chromatographic peak as a reference peak (S), and the obtained results are shown in the following tables 7-8.
TABLE 7 stability investigation results (relative retention time)
| Numbering device | Peak 1 | Peak 2 | Peak 3 | Peak 4 | Peak 5 | Peak 6 | Peak 7 | Peak 8 | Peak 9 (S) | Peak 10 | Peak 11 | Peak 12 | Peak 13 | Peak 14 |
| 0h | 0.173 | 0.371 | 0.799 | 0.815 | 0.832 | 0.856 | 0.902 | 0.916 | 1.00 | 1.04 | 1.16 | 1.30 | 1.49 | 1.55 |
| 2h | 0.172 | 0.372 | 0.800 | 0.816 | 0.833 | 0.857 | 0.902 | 0.917 | 1.00 | 1.04 | 1.16 | 1.29 | 1.49 | 1.55 |
| 4h | 0.171 | 0.369 | 0.798 | 0.814 | 0.832 | 0.855 | 0.901 | 0.917 | 1.00 | 1.04 | 1.16 | 1.29 | 1.49 | 1.55 |
| 6h | 0.174 | 0.372 | 0.799 | 0.814 | 0.831 | 0.855 | 0.900 | 0.916 | 1.00 | 1.04 | 1.16 | 1.30 | 1.49 | 1.55 |
| 12h | 0.172 | 0.370 | 0.798 | 0.814 | 0.832 | 0.855 | 0.901 | 0.917 | 1.00 | 1.04 | 1.16 | 1.29 | 1.49 | 1.55 |
| 24h | 0.173 | 0.371 | 0.798 | 0.815 | 0.832 | 0.857 | 0.903 | 0.917 | 1.00 | 1.04 | 1.16 | 1.29 | 1.48 | 1.54 |
| RSD% | 0.53 | 0.33 | 0.08 | 0.08 | 0.09 | 0.12 | 0.11 | 0.05 | 0.00 | 0.14 | 0.06 | 0.09 | 0.12 | 0.11 |
TABLE 8 stability investigation results (relative peak area)
| Numbering device | Peak 1 | Peak 2 | Peak 3 | Peak 4 | Peak 5 | Peak 6 | Peak 7 | Peak 8 | Peak 9 (S) | Peak 10 | Peak 11 | Peak 12 | Peak 13 | Peak 14 |
| 0h | 0.334 | 0.449 | 0.594 | 0.398 | 0.583 | 0.093 | 0.479 | 0.312 | 1.00 | 1.58 | 0.377 | 0.200 | 0.205 | 1.08 |
| 2h | 0.334 | 0.453 | 0.593 | 0.394 | 0.585 | 0.095 | 0.477 | 0.307 | 1.00 | 1.59 | 0.388 | 0.189 | 0.209 | 1.09 |
| 4h | 0.335 | 0.441 | 0.590 | 0.391 | 0.578 | 0.100 | 0.493 | 0.324 | 1.00 | 1.58 | 0.379 | 0.200 | 0.211 | 1.10 |
| 6h | 0.332 | 0.448 | 0.587 | 0.398 | 0.579 | 0.092 | 0.490 | 0.318 | 1.00 | 1.59 | 0.389 | 0.197 | 0.208 | 1.08 |
| 12h | 0.336 | 0.451 | 0.585 | 0.394 | 0.583 | 0.099 | 0.489 | 0.323 | 1.00 | 1.59 | 0.384 | 0.197 | 0.212 | 1.10 |
| 24h | 0.335 | 0.446 | 0.596 | 0.398 | 0.587 | 0.096 | 0.461 | 0.301 | 1.00 | 1.57 | 0.369 | 0.200 | 0.201 | 1.05 |
| RSD% | 0.44 | 0.93 | 0.72 | 0.76 | 0.61 | 3.40 | 2.46 | 2.90 | 0.00 | 0.43 | 1.98 | 2.11 | 1.93 | 1.47 |
The results show that: the relative retention time RSD of the characteristic peak and the relative peak area RSD of the characteristic peak are smaller than 5%, and the sample solution is stable within 24 hours.
Example 4
The method for establishing the HPLC characteristic spectrum based on the embodiments 1, 2 and 3 has good precision, repeatability and stability, and is suitable for the technical means of detecting the characteristic spectrum of the tonifying middle-jiao and Yi oral liquid. Specific HPLC characterization methods are shown below.
Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (Kromasil 100-5C) 18 Chromatographic column, 4.6X250 mm,5 μm); acetonitrile is taken as a mobile phase A, 0.2% formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the following table;the flow rate is 0.8mL/min; column temperature is 35 ℃; the detection wavelength was 260nm.
Preparing reference solution, namely preparing mixed reference solutions containing 10 mug, 25 mug, 2.5 mug, 85 mug and 100 mug of calycosin-7-O-glucoside reference, glycyrrhizin reference, ferulic acid reference, hesperidin reference, ammonium glycyrrhizate reference and a proper amount of reference solution, precisely weighing, and adding methanol into each 1 mL.
And (3) preparing a sample solution, namely taking 1mL of oral liquid for tonifying middle-jiao and Qi, and fixing the volume to 5mL by using methanol.
The measurement method comprises respectively precisely sucking 10 μl of mixed reference solution and sample solution, and injecting into liquid chromatograph for measurement
Finally, the following steps are provided: the characteristic spectrum of the middle-jiao tonifying and qi-tonifying oral liquid comprises 14 characteristic peaks, wherein the corresponding chromatographic peaks of hesperidin are taken as S peaks, and the relative retention time of the peak 9 (S) and the peak 1, the peak 2, the peak 3, the peak 4, the peak 5, the peak 6, the peak 7, the peak 8, the peak 10, the peak 11, the peak 12, the peak 13 and the peak 14 is calculated, wherein the relative retention time is within +/-10% of a specified value, and the specified value is 0.175 (peak 1), 0.372 (peak 2), 0.797 (peak 3), 0.815 (peak 4), 0.834 (peak 5), 0.858 (peak 6), 0.904 (peak 7), 0.918 (peak 8), 1.040 (peak 10), 1.156 (peak 11), 1.294 (peak 12), 1.480 (peak 13) and 1.541 (peak 14).
Claims (4)
1. The method for establishing the HPLC characteristic spectrum of the tonifying middle-jiao and Yi oral liquid is characterized by comprising the following steps:
(1) Preparation of test sample and mixed reference substance solution
Precisely measuring 1mL of the tonifying middle-jiao and Yi oral liquid in a volumetric flask, fixing the volume to 5mL by using methanol, performing ultrasonic treatment for 3min, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution; taking appropriate amount of calycosin-7-O-glucoside, glycyrrhizin, ferulic acid, hesperidin and ammonium glycyrrhizinate reference substances, precisely weighing, and adding methanol to obtain mixed reference substance solutions containing 20 μg, 50 μg, 5 μg, 170 μg, 10 μg and 200 μg respectively per 1 mL;
(2) Optimization of feature maps
Chromatographic conditions in the feature map construction: optimizing mobile phase aqueous phase acid, wavelength, column temperature, flow rate and chromatographic column, and establishing optimal high performance liquid chromatography conditions of the oral liquid for supplementing middle-jiao and replenishing qi;
(3) Selection of characteristic peaks
Different from the common characteristic peak, the characteristic peak is selected according to a single standard of chromatographic peak height, and a chemical pattern recognition technology is utilized: CA. PCA and PLS-DA analyze chromatographic peaks of the oral liquid for supplementing middle-jiao and replenishing qi in different batches, and screen chromatographic peaks with great influence on sample quality; taking the screening result as a reference, determining a characteristic peak of the chromatogram;
(4) Methodology investigation of feature maps
The precision, repeatability and stability of the method for establishing the characteristic spectrum are inspected, and the analysis method is proved to be applicable to the analysis of the characteristic spectrum of the tonifying middle-jiao and Yi oral liquid;
(5) Establishing a characteristic spectrum of the oral liquid for strengthening middle-jiao and replenishing qi
The conditions of the high performance liquid chromatography are as follows: chromatographic column: kromasil 100-5-C 18 : 4.6X250 mm,5 μm; mobile phase: acetonitrile (a) -0.2% formic acid in water (B); the detection wavelength is 260nm; column temperature is 35 ℃; the flow rate is 0.80mL/min; the sample injection amount is 10 mu L; the gradient elution procedure was as follows:
。
2. The method for establishing an HPLC profile of a fluid for middle-jiao and Yi oral administration of claim 1, wherein in the step (1), the power of the ultrasonic treatment is 250W and the frequency is 40kHz.
3. The method for establishing an HPLC characteristic spectrum of a liquid for tonifying middle-jiao and Yi oral administration according to claim 1, wherein 14 characteristic peaks are included in the characteristic spectrum, wherein No. 3 peak, no. 4 peak, no. 5 peak, no. 6 peak, no. 7 peak, no. 8 peak, no. 9 peak, no. 10 peak, no. 11 peak, no. 12 peak, no. 13 peak, and No. 14 peak are respectively calycosin-7-O-glucoside, apigenin, glycyrrhizin, ferulic acid, isoferulic acid, rutin, hesperidin, sodium benzoate, formononetin, calycosin, glycyrrhizin G2, and glycyrrhizic acid.
4. The method for establishing an HPLC profile of a middle-warmer and beneficial oral liquid according to claim 1, wherein in the step (3), the middle-warmer and beneficial oral liquid profile includes 14 characteristic peaks, peak 9 and peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 10, peak 11, peak 12, peak 13, peak 14 are calculated by taking the peak corresponding to hesperidin as S peak, the relative retention time is within ±10% of a specified value, and the specified value is peak 1:0.175, peak 2:0.372, peak 3:0.797, peak 4:0.815, peak 5:0.834, peak 6:0.858, peak 7:0.904, peak 8:0.918, peak 10:1.040, peak 11:1.156, peak 12:1.294, peak 13:1.480, peak 14:1.541.
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