CN111007169B - Construction method and quality evaluation method of fingerprint of strong loquat syrup - Google Patents
Construction method and quality evaluation method of fingerprint of strong loquat syrup Download PDFInfo
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- CN111007169B CN111007169B CN201911274545.0A CN201911274545A CN111007169B CN 111007169 B CN111007169 B CN 111007169B CN 201911274545 A CN201911274545 A CN 201911274545A CN 111007169 B CN111007169 B CN 111007169B
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Abstract
The invention relates to the field of medicines, and particularly discloses a construction method and quality of fingerprint of strong loquat syrupAnd (4) an evaluation method. The construction method of the fingerprint of the strong loquat dew comprises the following steps: preparing reference substance solution and test solution by using 30% methanol, and adopting Halo under the chromatographic conditionR5C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle size 5 μm), mobile phase 3-40: 97-60 parts of mixed solution; the flow rate is 0.5-1.5 mL/min; the detection wavelength is 254-325 nm; the column temperature is 30-40 ℃, and the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peaks; and respectively and precisely sucking 5-20 mu L of reference substance solution and sample solution, injecting the solutions into a liquid chromatograph, and recording chromatographic peaks for 4-30 min to obtain the strong loquat fingerprint chromatogram. The fingerprint and the construction method thereof provided by the invention can avoid the conditions of filling, reducing the amount of material feeding, changing the processing, production process and the like which are not strict, thereby cutting off the possibility of producing inferior medicines.
Description
Technical Field
The invention relates to the field of medicines, in particular to a construction method and a quality evaluation method of a fingerprint of strong loquat syrup.
Background
In the prior art, a certain component of the strong loquat dew is often subjected to qualitative or quantitative research, such as identification or content of menthol, identification or content of poppy shell, identification of loquat leaf and identification of stemona root. Generally, 1-2 medicinal materials are selected to establish a standard, illegal manufacturers may adopt certain means for the standard, such as illegal addition, and the like, so that the condition that the medicine is qualified in inspection but actually is inferior can occur.
When qualitative or quantitative research is carried out on various components, the problems of mutual interference, easy overlapping and unclear spectrogram and the like exist.
Therefore, it is urgently needed to develop a method for monitoring and evaluating the overall quality of the strong loquat distillate product and construct a clear and non-interference fingerprint spectrum which can qualitatively and/or quantitatively detect various important active ingredients in the strong loquat distillate and serve as an objective basis for monitoring and evaluating the quality of the product.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a construction method and a quality evaluation method of a fingerprint of strong loquat dew.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in a first aspect, the invention provides a method for constructing fingerprint of strong loquat dew, which comprises the following steps:
(1) preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
(2) preparation of a test solution: placing a 3mL strong loquat dew sample on a C18(6cc) small column, adding 12mL water for elution, then using 12mL 30% methanol solution for elution, collecting water eluate and 30% methanol eluate, combining, evaporating to dryness, adding a proper amount of 30% methanol into residue for dissolving, quantitatively transferring to a 2mL volumetric flask, adding 30% methanol for constant volume, shaking up, filtering, and taking a subsequent filtrate to obtain the loquat dew;
(3) chromatographic conditions are as follows: by HaloR5C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm), octadecylsilane chemically bonded silica as filler;
the mobile phase is 3-40% of acetonitrile-0.4% phosphoric acid solution: 97-60 parts of mixed solution; the flow rate is 0.5-1.5 mL/min; the detection wavelength is 254-325 nm; the column temperature is 30-40 ℃, and the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peaks;
(4) and (3) determination: precisely absorbing 5-20 mu L of reference substance solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain strong loquat fingerprint chromatogram.
Further, acetonitrile is taken as a mobile phase A, 0.4% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; equilibration time 10 minutes; the flow rate was 1.0mL per minute; the detection wavelength is a gradient wavelength; the column temperature is 35 ℃;
further, the filtrate was filtered through a filter paper to obtain a subsequent filtrate.
In the second aspect, the standard fingerprint of strong loquat dew is characterized in that samples of strong loquat dew with qualified quality and excellent quality in different batches are selected as test samples, the fingerprint is manufactured according to the construction method, and a map representing the average state is generated through software, namely the standard fingerprint of strong loquat dew.
Different batches of strong loquat dew samples with qualified quality and excellent quality are taken as standard test samples, and the standard test samples are selected from 100 representative batches with superior quality in more than 6000 batches produced in the last two years.
Further, the standard fingerprint spectrum of the strong loquat dew is composed of 15 characteristic peaks, wherein in the characteristic peaks, the peak 1 is a common peak, the peak 2 is a stemona root peak, the peak 3 is a common peak, the peak 4 is a loquat leaf peak, the peak 5 is a common peak, the peak 6 is a common peak, the peak 7 is a stemona root peak, the peak 8 is a common peak, the peak 9 is a common peak, the peak 10 is a chlorogenic acid peak, the peak 11 is a mulberry bark peak, the peak 12 is a common peak, the peak 13 is a common peak, the peak 14 is a common peak, and the peak 15 is a common peak.
In a third aspect, the invention provides a quality evaluation method of strong loquat syrup, which is characterized in that a sample to be tested is taken as a test sample, the construction method is adopted to obtain a fingerprint of the sample to be tested, traditional Chinese medicine chromatogram fingerprint similarity evaluation software is utilized to analyze the fingerprint of the sample to be tested and the standard fingerprint of the strong loquat syrup, and the qualified product is obtained when the similarity is more than 0.90.
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The invention has the beneficial effects that:
the method optimizes the reference solution, the test solution, the chromatographic conditions and the like, so that the strong loquat fingerprint spectrum constructing method provided by the invention can construct the strong loquat fingerprint spectrum with clear characteristic peaks and no interference. The standard fingerprint spectrum manufactured by the construction method can simultaneously display 15 clear characteristic peaks which are not overlapped and have no interference, the characteristic peaks are respectively from loquat leaves, stemona roots, white mulberry barks and the like, the existence and the relative position of each characteristic peak can be used for qualitative analysis, and the size of the characteristic peak can be used for quantitative analysis.
If the species, the producing area, the processing technology and the extraction method of the medicinal materials deviate, the quantity, the position and the size of the characteristic peaks can be changed, and the variation is compared with a standard map through special software, and the medicinal materials can be judged to be unqualified when exceeding the standard. Therefore, multiple dimensions such as medicinal material originality, producing area, processing technology, production technology and the like are controlled simultaneously in a fingerprint mode, and the situation that the processing and production technology are not strict with secondary filling, material feeding reduction, material feeding change and the like is avoided, so that the possibility of producing inferior medicines is cut off.
Drawings
FIG. 1 shows a standard fingerprint of strong loquat dew constructed by the construction method of the invention.
FIG. 2 shows the results of tests conducted on 15 lots of the product in example 3 of the present invention.
FIG. 3 is a fingerprint of the Eriobotrya japonica Lindl constructed by the construction method of comparative example 1.
FIG. 4 is a fingerprint of the Eriobotrya japonica Lindl constructed by the construction method of comparative example 2.
FIG. 5 is a fingerprint of the Eriobotrya japonica Lindl constructed by the construction method of comparative example 3.
FIG. 6 is a fingerprint of the Eriobotrya japonica Lindl constructed by the construction method of comparative example 4.
FIG. 7 is a fingerprint of the Eriobotrya japonica Lindl constructed by the construction method of comparative example 5.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 construction of fingerprint
1. Preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
2. preparing a test solution: placing a 3mL strong loquat dew sample on a C18(6cc) small column, adding 12mL of water for elution, then using 12mL of 30% methanol solution for elution, collecting water eluent and 30% methanol eluent, combining, drying by distillation, adding a proper amount of 30% methanol into residue for dissolution, quantitatively transferring into a 2mL volumetric flask, adding 30% methanol for constant volume, shaking up, filtering, and taking subsequent filtrate to obtain the loquat dew;
3. chromatographic conditions are as follows: by HaloR5C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm), octadecylsilane chemically bonded silica as filler;
acetonitrile is taken as a mobile phase A, 0.4 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; equilibration time 10 minutes; the flow rate was 1.0mL per minute; the detection wavelength is a gradient wavelength; the column temperature is 35 ℃; the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peak;
4. and (3) determination: precisely absorbing 5-20 mu L of reference substance solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain strong loquat fingerprint chromatogram.
Example 2 construction of Standard fingerprints
Selecting 100 representative batches of products with superior quality from 6000 batches of strong loquat dew products produced in the last two years as standard test samples, making a fingerprint spectrum according to the method described in the embodiment 1, and generating a spectrum representing an average state through software, namely the strong loquat dew standard fingerprint spectrum (as shown in figure 1).
The standard fingerprint spectrum of the strong loquat dew is composed of 15 characteristic peaks, wherein in the characteristic peaks, a peak 1 is a common peak, a peak 2 is a stemona root peak, a peak 3 is a common peak, a peak 4 is a loquat leaf peak, a peak 5 is a common peak, a peak 6 is a common peak, a peak 7 is a stemona root peak, a peak 8 is a common peak, a peak 9 is a common peak, a peak 10 is a chlorogenic acid peak, a peak 11 is a cortex mori radicis peak, a peak 12 is a common peak, a peak 13 is a common peak, a peak 14 is a common peak, and a peak 15 is a common peak.
Example 3 verification of Standard fingerprints
Selecting 15 batches of products with qualified representative quality, constructing a fingerprint spectrum according to the method in the embodiment 1, and analyzing the fingerprint spectrum of the obtained sample and the standard fingerprint spectrum obtained in the embodiment 2 by using traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation software.
The software screenshot is shown in FIG. 2, and the data in the table shows that the similarity between 15 batches of samples is 0.864-0.997, and the similarity with the generated standard atlas is 0.962-0.997.
Comparative example 1
This comparative example differs from example 1 in that a chromatography column consisting of (Halo)R5C18250mm X4.6 mm, 5 μm) to (150mm X4.6 mm, 4 μm), and a fingerprint constructed using the sample of example 2 is shown in FIG. 3, and it can be seen that the spectrum shows 12 sample peaks, and the peak area and retention time of the chromatographic peak are inconsistent with those of the standard spectrum.
Comparative example 2
This comparative example differs from example 1 in that the gradient mobile phase of example 1 was changed to a constant ratio (5: 95 acetonitrile: 0.4% phosphoric acid solution) run, and the remaining steps and operating parameters were the same as in example 1. The fingerprint constructed by the sample of example 2 is shown in fig. 4, and it can be seen that only 9 chromatographic peaks are displayed, and the peak area and retention time of the chromatographic peaks are inconsistent with those of the standard spectrum.
Comparative example 3
This comparative example differs from example 1 in that the elution solvent was changed in the treatment of the test article in example 1: the 30% methanol was changed to 10% methanol and the remaining steps and operating parameters were the same as in example 1. The fingerprint constructed by using the sample of example 2 is shown in fig. 5, and it can be seen that 4 chromatographic peaks are lacked, and the peak area and retention time of the chromatographic peaks are inconsistent with those of the standard spectrum.
Comparative example 4
This comparative example differs from example 1 in that the detection wavelength in example 1 is changed:
0-5.5 minutes: the wavelength is changed from 310nm to 254nm,
5.5-9.5 minutes: the wavelength is changed from 258nm to 310nm,
9.5-11.5 minutes: the wavelength is changed from 325nm to 254nm,
11.5-14.0 min: the wavelength is changed from 254nm to 310nm,
the remaining steps and operating parameters were the same as in example 1. Fig. 6 shows that the fingerprint constructed by using the sample of example 2 lacks 9 chromatographic peaks, and the peak area and retention time of the chromatographic peaks are inconsistent with those of the standard chromatogram.
Comparative example 5
This comparative example differs from example 1 in that the elution solvent was changed in the treatment of the test article in example 1: the 30% methanol was changed to 100% methanol and the remaining steps and operating parameters were the same as in example 1. The fingerprint constructed by using the sample of example 2 is shown in fig. 7, and it can be seen that 3 chromatographic peaks are lacked, and the peak area and retention time of the chromatographic peak are inconsistent with those of the standard spectrum.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. The construction method of the fingerprint of the strong loquat dew is characterized by comprising the following steps:
(1) preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
(2) preparation of a test solution: placing a 3mL strong loquat dew sample on a C18 small column, eluting with 6cc water by 12mL, eluting with 30% methanol solution by 12mL, collecting water eluate and 30% methanol eluate, mixing, evaporating to dryness, dissolving residue with a proper amount of 30% methanol, quantitatively transferring to a 2mL volumetric flask, adding 30% methanol to a constant volume, shaking up, filtering, and taking a subsequent filtrate to obtain the loquat dew;
(3) chromatographic conditions are as follows: by HaloR5C18 chromatographic column, octadecylsilane chemically bonded silica is filler; acetonitrile is taken as a mobile phase A, 0.4 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; equilibration time 10 minutes; the flow rate was 1.0mL per minute; the detection wavelength is a gradient wavelength; the column temperature is 35 ℃;
the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peak;
(4) and (3) determination: precisely absorbing 5-20 mu L of reference substance solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain strong loquat fingerprint chromatogram.
2. The method of claim 1, wherein the filtrate is obtained by filtration through a filter paper.
3. A quality evaluation method of strong loquat dew is characterized in that a sample to be tested is used as a test sample, the fingerprint of the sample to be tested is obtained by the steps (1) to (4) of the construction method according to claim 1 or 2, the fingerprint of the sample to be tested and the fingerprint of the strong loquat dew according to claim 1 or 2 are analyzed by traditional Chinese medicine chromatography fingerprint similarity evaluation software, and the strong loquat dew with the similarity of more than 0.90 is a qualified product.
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| CN104634888B (en) * | 2014-11-25 | 2016-08-17 | 杭州胡庆余堂药业有限公司 | A kind of intensified loquet distillate fingerprint pattern quality control method |
| CN105372350B (en) * | 2015-12-07 | 2017-10-31 | 合肥今越制药有限公司 | A kind of low-sugar type intensified loquet distillate fingerprint control method |
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