CN112557546B

CN112557546B - Method for detecting UPLC fingerprint spectrum and multi-component content of exocarpium citri grandis pear syrup

Info

Publication number: CN112557546B
Application number: CN202011507715.8A
Authority: CN
Inventors: 杨丽莹; 王艳慧; 黄洁文; 吴舒楹; 秦群英; 黄绮韵
Original assignee: Guangdong Huazhou Traditional Chinese Medicine Factory Pharmaceutical Co ltd; GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Current assignee: Guangdong Huazhou Traditional Chinese Medicine Factory Pharmaceutical Co ltd; GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2022-12-20
Anticipated expiration: 2040-12-18
Also published as: CN112557546A

Abstract

The invention relates to a method for detecting UPLC fingerprint spectrum and multi-component content of pummelo peel pear syrup. The detection method of the UPLC fingerprint comprises the following steps: accurately weighing exocarpium Citri rubrum pear syrup, adding organic solvent, filtering, collecting filtrate, and preparing sample solution; precisely absorbing the test solution, and performing ultra-high performance liquid chromatography to obtain the UPLC fingerprint of the exocarpium citri grandis paste; wherein, the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, and the mobile phase B is water; gradient elution mode is adopted. The invention also provides a detection method capable of simultaneously determining the multi-component content of the chrysoidine pear syrup. The method is accurate and reliable, has strong specificity and good repeatability, and can quickly, comprehensively, effectively and efficiently control the product quality.

Description

Method for detecting UPLC fingerprint spectrum and multi-component content of exocarpium citri grandis pear syrup

Technical Field

The invention relates to the technical field of medicines, in particular to a method for detecting UPLC fingerprint spectrum and multi-component content of pummelo peel pear syrup.

Background

The exocarpium citri grandis paste is a traditional Chinese medicine preparation, contains pear, pummelo peel, loquat leaf, bitter apricot seed, chinese magnoliavine fruit, unibract fritillary bulb, asparagus fern and dwarf lilyturf tuber, has the effects of nourishing yin, clearing away the lung-heat, relieving cough and reducing sputum, and is used for treating lung-stomach yin deficiency, dry mouth and throat and less long-term expectoration.

At present, the drug standard (traditional Chinese medicine ingredient preparation WS 3-B-0666-91) issued by the ministry of execution of the chrysoidine pear syrup only requires the relative density, insoluble substances, the filling amount and the microbial limit of the chrysoidine pear syrup, does not relate to the inspection of medicinal materials in a prescription, and cannot effectively control the product quality. In the currently published quality research of the chrysoidine pear paste, thin-layer chromatography (TLC) identification methods of medicinal materials are adopted. 3238 Zxft 3238 is used for thin-layer identification of exocarpium Citri Grandis, semen Armeniacae amarum and fructus Schisandrae chinensis in exocarpium Citri Grandis fructus Pyri paste, and the method requires preparation of three samples, and adopts three different thin-layer development conditions, and also provides a method for determining naringin single component content in exocarpium Citri Grandis (3262 Zxft 3262. Quality standard research of exocarpium Citri Grandis Pyri paste [ J ] Chinese medicinal materials, 2003 (05): 363-365.). However, the orange pear syrup is rich in internal substances, the difference of the properties of each component is large, and the traditional single component content measurement is not enough to indicate the overall quality of the product. At present, a rapid detection method capable of simultaneously identifying multiple medicinal materials in the chrysoidine pear syrup or simultaneously determining the content of multiple components in the chrysoidine pear syrup is lacked, and the quality and the clinical curative effect of a product are difficult to control effectively.

Disclosure of Invention

Based on the above, a UPLC fingerprint detection method for the chrysoidine pear syrup is needed, which can be used for quickly identifying multiple raw medicinal materials in the chrysoidine pear syrup, and can be used for measuring the content of multiple index components, so that the quality of the chrysoidine pear syrup can be quickly, comprehensively, effectively and efficiently controlled.

The invention is realized by the following technical scheme:

a method for detecting a UPLC fingerprint of exocarpium citri grandis paste comprises the following steps:

accurately weighing exocarpium Citri rubrum pear syrup, adding organic solvent, filtering, collecting filtrate, and preparing sample solution;

precisely absorbing the test solution, and performing ultra-high performance liquid chromatography to obtain the UPLC fingerprint of the exocarpium citri grandis paste;

wherein, the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, and the mobile phase B is water; adopting a gradient elution mode;

the organic solvent adding method comprises the following steps: firstly adding a solvent A, and then adding a solvent B;

the volume ratio of the solvent A to the test solution is 1 (2-5);

the solvent A is a methanol water solution with the volume fraction of 20-50% or an ethanol water solution with the volume fraction of 20-50%;

the solvent B is methanol or ethanol.

In one embodiment, the mobile phase a is acetonitrile containing acid; and/or the mobile phase B is water containing acid; wherein the volume fraction of the acid in the mobile phase A and/or the mobile phase B is 0.01-4%;

the acid is acetic acid or formic acid.

In one embodiment, the volume fraction of the acid in the mobile phase a and/or the mobile phase B is 0.01 to 0.5%;

in one embodiment, the gradient elution mode is: the volume percentage of the mobile phase A is changed from 5 percent to 90 percent in 0-24 min.

In one embodiment, the gradient elution mode is: the volume percent of the mobile phase A is changed from 5 percent to 20 percent within 0-11 min; the volume percentage of the mobile phase A is changed from 20 percent to 90 percent within 11-24 min.

In one embodiment, the conditions of the ultra performance liquid chromatography assay are: the chromatographic column is C ₁₈ A chromatographic column; the flow rate of the mobile phase is 0.2-0.4 ml/min; the column temperature is 20-45 ℃; the detection wavelength is 200-220 nm, 244-264 nm or 310-330 nm.

In one embodiment, the detection wavelength is 205-215 nm, 250-260 nm, or 315-325 nm.

The invention also provides a method for detecting the multi-component content of the chrysoidine pear syrup, which comprises the following steps:

accurately weighing a reference substance, adding methanol for dissolving, and preparing a reference substance solution; the reference substance is at least one of naringin, rhoifolin and schizandrol A;

precisely sucking the reference substance solution and the test solution, and performing ultra-high performance liquid chromatography measurement;

wherein the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, and the mobile phase B is water; gradient elution mode is adopted.

In one embodiment, the adding the organic solvent comprises the following steps: adding a solvent A and then adding a solvent B;

the volume ratio of the solvent A to the test solution is 1 (2-5);

the solvent B is methanol or ethanol.

In one embodiment, the mobile phase a is acetonitrile containing acid; and/or

The mobile phase B is water containing acid;

wherein the volume fraction of the acid in the mobile phase A and/or the mobile phase B is 0.01-4%;

the acid is acetic acid or formic acid.

In one embodiment, the conditions of the ultra performance liquid chromatography assay are: the chromatographic column is C ₁₈ A chromatographic column; the flow rate of the mobile phase is 0.2-0.4 ml/min; the column temperature is 20-45 ℃; the detection wavelength is 244-264 nm or 310-330 nm.

In one embodiment, the detection wavelength is 250-260 nm or 315-325 nm.

Compared with the prior art, the invention has the following beneficial effects:

the UPLC fingerprint of the tangerine peel pear syrup is constructed for the first time, the chemical component characteristics of multiple medicinal materials of the tangerine peel pear syrup are fully displayed, the multiple medicinal materials in the tangerine peel pear syrup can be identified simultaneously, the content of multiple index components can be measured simultaneously, the product quality can be effectively controlled, the detection time and the detection cost are saved to a great extent, and compared with a high performance liquid chromatography, the detection time is further shortened by the ultra performance liquid chromatography.

Drawings

FIG. 1 is a UPLC spectrum of a sample solution obtained by different sample preparation methods;

FIG. 2 is a UPLC spectrum at 320nm wavelength obtained for different mobile phases;

FIG. 3 is a UPLC spectrum at 254nm wavelength obtained for different mobile phases;

FIG. 4 is a UPLC spectrum at a wavelength of 210nm obtained from different mobile phases;

FIG. 5 is a UPLC fingerprint spectrum of pummelo peel pear syrup at a wavelength of 320nm;

FIG. 6 is a UPLC fingerprint spectrum of pummelo peel pear syrup at 254nm wavelength;

FIG. 7 is UPLC fingerprint spectrum of pummelo peel pear syrup at 210nm wavelength.

Detailed Description

The UPLC fingerprint spectrum and the method for detecting the content of multiple components of the exocarpium citri grandis paste of the present invention are further described in detail with reference to the specific examples below.

precisely absorbing a test solution, and performing ultra-high performance liquid chromatography to obtain an UPLC fingerprint of the exocarpium citri grandis paste;

wherein, the mobile phase A adopted by the ultra-performance liquid chromatography is acetonitrile, and the mobile phase B is water; gradient elution mode is adopted.

The mobile phase is an acetonitrile-water system, so that the peak-off time is short, and more peak information can be kept.

In one specific example, the addition of the organic solvent comprises the steps of: adding a solvent A and then adding a solvent B;

the volume ratio of the solvent A to the test solution is 1 (2-5);

the solvent B is methanol or ethanol.

A small amount of 20-50% methanol or ethanol is used for diluting and dissolving a sample, and then the methanol or ethanol is added to a certain concentration, so that impurities can be effectively removed, detection equipment can be protected, medicinal material component information of the chrysoidine pear syrup can be kept to the maximum extent, component characteristic peaks of loquat leaves, pummelo peel, schisandra chinensis and bitter almonds can be detected at the same time, and the method has good repeatability. In addition, compared with liquid-liquid extraction, the method reduces the use of organic reagents, is beneficial to environmental protection and saves the detection cost.

In one specific example, the solvent a is a methanol aqueous solution with a volume fraction of 20 to 50%. Specifically, the volume fraction of the aqueous methanol solution (solvent a) includes, but is not limited to, the following percentages: 20%, 30%, 40%, 45%, 47%, 48%, 49%, 50%.

In one specific example, the volume ratio of the solvent A to the test solution is 1 (2-5). Specifically, the volume ratio of solvent a to the test solution includes, but is not limited to 1:2, 1:3, 1.

In one specific example, the mobile phase a is an acid-containing acetonitrile; and/or

The mobile phase B is water containing acid;

wherein the volume fraction of the acid in the mobile phase A and/or the mobile phase B is 0.01-4%.

The inventor of the patent tries to add a certain proportion of acid into the mobile phase, finds that the peak shape is obviously improved, and the UPLC fingerprint spectrum under the condition of 310-330 nm of the red tangerine peel pear syrup can be obtained when the volume fraction of the acid in the mobile phase is 0.01-4%.

In one specific example, the acid is added into the mobile phase A or/and the mobile phase B, so that the volume fraction of the acid in the mobile phase is 0.01-0.5%. Specifically, the volume fraction of acid in the mobile phase includes, but is not limited to, the following percentages: 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.3%, 0.5%.

The inventor of the patent further improves the base line and related chromatographic peaks of the chromatogram by adjusting the volume fraction of the mobile phase, and finds that the optimal acid concentration is 0.01-0.5%.

In one specific example, the acid is acetic acid or formic acid.

The inventor of the patent screens out the mobile phase added with formic acid or acetic acid, and can achieve the effects of improving the peak pattern and increasing the separation degree.

In one particular example, the acid is acetic acid.

In one specific example, the gradient elution mode is: the volume percentage of the mobile phase A is changed from 5 percent to 95 percent in 0-24 min.

In one specific example, the gradient elution mode is: the volume percent of the mobile phase A is changed from 5 percent to 20 percent within 0-11 min; the volume percentage of the mobile phase A is changed from 20 percent to 90 percent within 11-24 min.

In one specific example, the conditions for the ultra performance liquid chromatography assay are: the chromatographic column is C ₁₈ A chromatographic column; the flow rate of the mobile phase is 0.2-0.4 ml/min; the column temperature is 20-45 ℃; the detection wavelength is 200-220 nm, 244-264 nm or 310-330 nm.

When the flow rate of the mobile phase is 0.1ml/min, the peak time is very slow, the separation effect is poor, and when the flow rate is 0.5ml/min, the system pressure is very high, so that the instrument is easily damaged, and the use and maintenance of the instrument are not facilitated. The inventor of the present patent studied to obtain an optimum flow rate of 0.2 to 0.4ml/min.

In one specific example, the flow rate is 0.3 to 0.4ml/min. Specifically, flow rates of the mobile phase include, but are not limited to, 0.3ml/min, 0.34ml/min, 0.35ml/min, 0.36ml/min, 0.4ml/min.

A diode array detector (PDA) is adopted for full-wavelength scanning, UPLC spectrums obtained by 190-400 nm are compared, and as a result, when wavelengths of 200-220 nm, 244-264 nm and 310-330 nm are detected, more chromatographic peaks are presented in the spectrums, and meanwhile, the spectrum peak separation degree is good, the peak number is moderate, and the peak shape is good.

In one specific example, the detection wavelength is 205 to 215nm, 250 to 260nm, or 315 to 325nm.

Further, the detection wavelength is 210nm, 254nm or 320nm.

In one specific example, the column temperature is 20 ℃, 30 ℃, 35 ℃, 40 ℃ or 45 ℃.

The inventor of the patent examines the separation condition of chromatographic peaks under different column temperatures, and the result shows that the chromatographic peaks under different column temperatures are slightly different, but the detection effect can be achieved.

In one specific example, the chromatography column is an ACQUIT UPLC HSS T3 column, an ACQUIT UPLC HSS C18 column, an ACQUIT UPLC CSH C18 column, a CORTECS UPLC C18 column, or a CORTECS UPLC C18+ column.

In one specific example, the chromatography column is an ACQUIT UPLC HSS T3 column, a CORTECS UPLC C18 column, or a CORTECS UPLC C18+ column.

The ACQUIT UPLC HSS T3 column and either CORTECS UPLC C18 column or CORTECS UPLC C18+ column provide better separation of the various spectral peaks and better peak shapes.

precisely weighing a reference substance, adding methanol for dissolving, and preparing a reference substance solution; the reference substance is at least one of naringin, rhoifolin and schizandrol A;

precisely sucking the reference solution and the test solution, and performing ultra-high performance liquid chromatography;

in one specific example, the addition of the organic solvent comprises the steps of: firstly adding a solvent A, and then adding a solvent B;

the volume ratio of the solvent A to the test solution is 1 (2-5);

the solvent B is methanol or ethanol.

In one specific example, the solvent A is 20-50% methanol aqueous solution by volume fraction. Specifically, the volume fraction of the aqueous methanol solution (solvent a) includes, but is not limited to, the following percentages: 20%, 30%, 40%, 45%, 47%, 48%, 49%, 50%.

The mobile phase B is water containing acid;

In one specific example, the volume fraction of acid in mobile phase a and/or mobile phase B is 0.01 to 0.5%. Specifically, the volume fraction of acid in the mobile phase includes, but is not limited to, the following percentages: 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.3%, 0.5%.

In one specific example, the acid is acetic acid or formic acid.

In one particular example, the acid is acetic acid.

In one specific example, the gradient elution mode is: the volume percentage of the mobile phase A is changed from 5 percent to 90 percent in 0-24 min.

In one specific example, the conditions for the ultra performance liquid chromatography assay are: the chromatographic column is C ₁₈ A chromatographic column; the flow rate of the mobile phase is 0.2-0.4 ml/min; the column temperature is 20-45 ℃; the detection wavelength is 244-264 nm or 310-330 nm.

In one specific example, the detection wavelength is 250 to 260nm or 315 to 325nm.

In one specific example, the detection wavelength is 254nm or 320nm.

The following are specific examples, and the starting materials used in the examples are all commercially available products unless otherwise specified.

Example 1 detection method of UPLC fingerprint of exocarpium Citri rubrum pear syrup

The embodiment provides a method for detecting a UPLC fingerprint spectrum of pummelo peel pear syrup.

1 instruments and reagents

1.1 instruments

The ultra-high performance liquid chromatograph is a Waters Acquity H-Class UPLC system, and the chromatographic column is C ₁₈ (2.1 mm. Times.100mm, 1.8 μm) column.

1.2 reagents

Acetonitrile, methanol, formic acid and acetic acid are chromatographically pure; the water is ultrapure water; other reagents were analytically pure.

1.3 reagent

The 10 batches of chrysoidine pear syrup were produced by Huazhou Chinese medicine factories, qinzhou, inc. of Xiangxue pharmacy, guangzhou.

2 methods and results

2.1 preparation of test solutions

Precisely weighing 2.5g of exocarpium Citri rubrum pear syrup, adding 2.5ml of 50% methanol, shaking to fully dissolve exocarpium Citri rubrum pear syrup, gradually adding methanol, diluting to 10ml, shaking, filtering, and collecting filtrate to obtain the sample solution.

2.2 methods of measurement

Precisely absorbing the test solution, and injecting the test solution into an ultra-high performance liquid chromatograph for determination to obtain the UPLC fingerprint of the exocarpium citri grandis paste;

the chromatographic conditions are as follows: the chromatographic column is ACQUIT UPLC HSS T3 column (2.1 mm × 100mm,1.8 μm); mobile phase: acetonitrile is used as a mobile phase A, acetic acid water solution with the volume fraction of 0.05 percent is used as a mobile phase B, elution is carried out according to the elution mode of the table 1, and the flow rate is 0.35ml/min; the detection wavelength is 210nm, 254nm or 320nm; the column temperature was 30 ℃.

TABLE 1 mobile phase gradient elution mode

Note: the fingerprint pattern has all peaks in 0-24 min, and the purpose of 24-26 min is to return the mobile phase to the initial proportion, so that the next sample can be conveniently injected.

In addition, this example screens the experimental conditions:

(1) Optimization of preparation method of test solution

Because the exocarpium citri rubrum pear syrup has high sugar content and very viscous samples, target components of the exocarpium citri rubrum pear syrup can be dissolved in an organic solvent, but the exocarpium citri rubrum pear syrup sample cannot be dissolved in the organic solvent, and if the exocarpium citri rubrum pear syrup is directly extracted by the organic solvent, the sample can be adhered to the wall of a bottle, so that the problems of incomplete dissolution and poor experimental repeatability are caused. If the water or the low-concentration organic solvent is used for dissolving, the problem of poor experimental repeatability can be solved, but the problems of insufficient purification of a sample, pollution to instruments and equipment and a chromatographic column and influence on the service life of the instruments and the chromatographic column can be caused.

On the other hand, the components of the medicinal materials such as loquat leaves, pummelo peel, schisandra chinensis, bitter apricot seeds and the like in the chrysoidine pear syrup have large differences, and if the sample is not processed properly, the components are lost and cannot be detected.

Through a large number of experiments, the inventor researches and examines preparation methods of various samples to be tested, such as an organic solvent extraction method (ethyl acetate, trichloromethane and water saturated n-butanol), a macroporous resin adsorption method, an organic solvent dissolution method (methanol and ethanol) and the like, wherein UPLC spectrograms of sample solutions prepared by different methods are shown in figure 1. The result shows that a small amount of 20-50% methanol or ethanol is firstly used for diluting and dissolving a sample, and then the methanol or ethanol is added to a certain concentration, so that not only can the impurities be effectively removed and the detection equipment be protected, but also the medicinal material component information of the chrysoidine pear syrup can be retained to the maximum extent, the component characteristic peaks of the loquat leaves, the pummelo peel, the schisandra chinensis and the bitter almonds can be detected at the same time, and the method has good repeatability.

Furthermore, the component information of the orange pear syrup sample obtained by dissolving the orange pear syrup by the methanol is most abundant, and all components are not interfered by the solvent.

(2) Selection of mobile phase

Firstly, the influence of a methanol-water system and an acetonitrile-water system on component separation is investigated, the peak time of the acetonitrile-water system is shorter than that of the methanol-water system, and more peak information can be kept, so that the acetonitrile-water system is selected for further condition optimization.

In order to improve the peak shape and increase the degree of separation, the inventors of the present invention tried to add a certain proportion of acid to the aqueous phase, and found that the peak shape was significantly improved. As shown in figure 2, the kind and the addition amount of the acid have small influence on the UPLC fingerprint spectrum under 320nm, and when the addition amount of the mobile phase acid is 0.01-4% of the volume fraction, the UPLC fingerprint spectrum under 320nm of the tangerine peel pear syrup can be obtained; the addition of the acid slightly affects the spectrum baseline under 254nm, as shown in fig. 3, when the addition of the acid is more than 0.5%, the spectrum baseline is unstable, but the detection effect can be achieved; the acid addition has an effect on the spectrum at 210nm, and as shown in fig. 4, when the acid addition is greater than 0.5%, the baseline noise at 210nm is large, and the detection effect is poor. Therefore, the optimal acid concentration is 0.01-0.5%. After screening, the formic acid and the acetic acid can achieve the effect of improving separation within the range of 0.01 to 0.5 percent.

(3) Selection of elution gradient

Searching the UPLC elution gradient to obtain the optimal separation gradient, so that the UPLC chromatogram of the tangerine peel pear paste has rich peak information, good peak separation degree and good peak shape.

(4) Selection of flow rate

In the UPLC research, the flow rate of 0.1ml/min shows that the peak time is very slow, the separation effect is poor, and when the flow rate of 0.5ml/min shows that the system pressure is very high, the instrument is easy to damage, and the use and maintenance of the instrument are not facilitated. Therefore, the optimum flow rate is 0.2 to 0.4ml/min.

(5) Selection of detection wavelength

A diode array detector (PDA) is adopted for full-wavelength scanning, UPLC spectrums obtained from 190-400 nm are compared, and as a result, when the wavelength of 210nm, 254nm or 320nm is detected, more chromatographic peaks are presented in the spectrums, and meanwhile, the resolution of each chromatographic peak is better, the number of peaks is moderate, the peak shape is better, so that the wavelength at 210nm, 254nm or 320nm is determined as the fingerprint spectrum detection wavelength.

(6) Investigation of chromatographic columns

5 different types of columns ACQUIT UPLC HSS T3 (2.1 mm. Times.100mm, 1.8 μm), ACQUIT UPLC HSS C18 (2.1 mm. Times.100mm, 1.8 μm), ACQUIT UPLC CSH C18 (2.1 mm. Times.100mm, 1.7 μm), CORTECS UPLC C18 (2.1 mm. Times.100mm, 1.6 μm), CORTECS UPLC C18+ (2.1 mm. Times.100mm, 1.6 μm) were examined, in which the ACQUIT UPLC HSS T3 (2.1 mm. Times.100mm, 1.8 μm), CORTECS UPLC C18 (2.1 mm. Times.100mm, 1.6 μm) and CORTECS UPLC C18+ (2.1 mm. Times.100mm, 1.6 μm) columns exhibited better resolution of the respective spectral peaks and the shapes were better.

(7) Selection of column temperature

The separation condition of each spectrum peak under the column temperature conditions of 20 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ is investigated in the experiment, and the result shows that the spectrum difference is small under different column temperatures, the peak-out time is slightly different, but the detection effect can be achieved.

The UPLC fingerprint is established by adopting the method, the fingerprint measurement data of 10 batches of the chrysoidine pear syrup is introduced into a Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) compiled by the national pharmacopoeia committee, a fingerprint common mode is generated by using an average method, and the common mode is taken as the chrysoidine pear syrup standard fingerprint.

The fingerprint spectrum of exocarpium Citri rubrum fructus Pyri paste adopts three wavelengths, wherein 8 common characteristic peaks (shown in figure 5) exist at 320nm, 8 common characteristic peaks (shown in figure 6) exist at 254nm, and 6 characteristic peaks (shown in figure 7) exist at 210 nm.

The 8 common peaks were calibrated at 320nm, and the ratio of the non-common peak area to the total area was less than 10%, as shown in table 2:

TABLE 2 common peaks of Citrus reticulata Blanco extract at 320nm

Numbering	Relative retention of time to peak
		1	0.375
2	0.398
		3	0.433
4	0.611
		5 (naringin)	1.000
6 (rhoifolin)	1.026
		7	1.198
8	1.612

The 8 common peaks are calibrated under 254nm, the ratio of the non-common peak area to the total area is less than 10%, and specifically, as shown in table 3, two chromatographic peaks of 10.8min and 18.0min are preservative auxiliary material peaks and are not included in the total peak area.

TABLE 3 common peaks of fingerprint spectrum 254nm of exocarpium Citri rubrum and fructus Pyri paste

The 6 common peaks are calibrated under 210nm, the ratio of the non-common peak area to the total area is less than 10%, and specifically, as shown in table 4, two chromatographic peaks of 10.8min and 18.0min are preservative auxiliary material peaks and are not included in the total peak area.

TABLE 4 common peak of fingerprint of exocarpium Citri rubrum and fructus Pyri paste at 210nm

Numbering	Relative retention of time to peak
		1	0.463
2	0.471
		3 (naringin)	1.000
4 (rhoifolin)	1.026
		5	1.198
6	1.263

2.3 method verification of fingerprint

The precision, stability and repeatability of the fingerprint detection method are inspected, and the result proves that the precision, stability and repeatability are good, and the requirement of the fingerprint is met.

(1) Precision test

The same sample solution is taken, sample introduction is carried out for 6 times continuously to obtain a fingerprint, evaluation is carried out by a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2012 edition, the result is shown in table 5, the similarity of the sample solution under 320nm, 254nm and 210nm is equal to 1.000, and the precision of the instrument is good.

TABLE 5 fingerprint precision test results

S1	S2	S3	S4	S5	S6	Comparison fingerprint
							1.000	1.000	1.000	1.000	1.000	1.000	1.000
1.000	1.000	1.000	1.000	1.000	1.000	1.000
							1.000	1.000	1.000	1.000	1.000	1.000	1.000
1.000	1.000	1.000	1.000	1.000	1.000	1.000
							1.000	1.000	1.000	1.000	1.000	1.000	1.000
1.000	1.000	1.000	1.000	1.000	1.000	1.000

(2) Stability test

The same sample solution was sampled at 0h,3h,6h,12h,24h and 48h to obtain finger-prints, and the results of evaluation by the traditional Chinese medicine chromatography finger-print similarity evaluation system 2012 edition are shown in table 6, wherein the similarity of the sample solution at 320nm, 254nm and 210nm is equal to 1.000, and the stability of the sample solution in 48h is good.

TABLE 6 stability test results of finger print

(3) Repeatability test

Taking samples of the same batch number, preparing 6 samples according to a preparation method of the samples, detecting the samples to obtain fingerprints, and evaluating the samples by a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2012 version, wherein the results are shown in table 7, the similarity of the sample solution at 320nm, 254nm and 210nm is equal to 1.000, and the method has good repeatability.

TABLE 7 results of the fingerprint repeatability test

Example 2 method for detecting content of multiple components in exocarpium citri grandis paste

The embodiment provides a method for detecting the content of multiple components in exocarpium citri grandis pear syrup, which specifically comprises the following steps:

1 instruments and reagents

1.1 instruments

1.2 reagents

Acetonitrile, methanol, formic acid and acetic acid are used as chromatographic purities; the water is ultrapure water; other reagents were analytically pure.

1.3 reagent

Comparison products: naringin, rhoifolin, schizandrin A reference substances were purchased from the Chinese food and drug testing research institute.

Sample preparation: the 10 batches of chrysoidine pear syrup were produced by the national institute of Huazhou, douken, national institute of pharmacy, guangzhou, xiangxue, inc.

2 methods and results

2.1 preparation of control solutions

Precisely weighing naringin, rhoifolin and schizandrol A reference substances, adding methanol for dissolving, and preparing reference substance solution.

2.2 preparation of test solutions

2.3 measurement method

Precisely sucking the reference solution and the test solution, and injecting into an ultra-high performance liquid chromatograph for determination;

the chromatographic conditions are as follows: the chromatographic column is ACQUIT UPLC HSS T3 column (2.1 mm × 100mm,1.8 μm); mobile phase: taking acetonitrile acetate with the volume fraction of 0.05% as a mobile phase A and acetic acid aqueous solution with the volume fraction of 0.05% as a mobile phase B, and eluting according to the elution mode shown in the table 1, wherein the flow rate is 0.35ml/min; the detection wavelengths are 254nm and 320nm; the column temperature was 30 ℃.

2.4 methodological validation of assay

The method for detecting the content measurement is verified by methodology, and the result proves that the method has good accuracy, repeatability, intermediate precision, specificity, durability and linearity and meets the requirement of content measurement.

(1) Accuracy test

Precisely weighing the same batch of pummelo peel pear paste with known content, paralleling 6 parts, respectively and precisely adding a certain amount of mixed reference substance solution, preparing the test solution according to the method under the item of preparation of the test solution, measuring and calculating the sample adding recovery rate and RSD of each component, wherein the results are shown in Table 8, the sample adding recovery rates of naringin, rhoifolin and schizandrol A are respectively 95.0-105.0%, and the RSD is respectively less than 1.0%, which indicates that the method has good accuracy.

TABLE 8 assay accuracy test results

(2) Repeatability test

Taking the same batch of samples, preparing 6 parts of samples according to the preparation method of the samples, measuring according to the detection method, recording the retention time and peak area of each peak, and the result is shown in table 9, wherein the retention time and content RSD value of each peak are both less than 2.0 percent, and the method has good repeatability.

TABLE 9 repeatability test results for the assay

(3) Intermediate precision test

The intermediate precision of different analysis time, different analysts and different analysis instruments is examined, and the result shows that the intermediate precision of the method is good.

Taking the same batch of samples, selecting three different analysis times, preparing a sample solution according to the sample solution preparation method, measuring according to the detection method, recording the retention time and peak area of each peak, and recording the retention time and content RSD value of each peak at different times to be less than 2.0 percent as shown in Table 10.

TABLE 10 results of intermediate precision test of different assay times for content determination

Taking the same lot of samples, preparing a sample solution according to a sample solution preparation method by three analysts, measuring according to a detection method, recording the retention time and peak area of each peak, and obtaining results shown in table 11, wherein the retention time and content RSD value of each peak of the samples prepared by different analysts are less than 2.0%.

TABLE 11 results of intermediate precision tests by various persons in content measurement

The same batch of samples are taken, the samples are prepared according to the sample preparation method, the measurement is carried out on three different instruments and equipment according to the detection method, the retention time and the peak area of each peak are recorded, the results are shown in table 12, and the retention time and the content RSD value of each peak in different equipment are both less than 2.0%.

TABLE 12 results of intermediate precision tests of different instruments for content determination

(4) Specificity test

And (3) preparing corresponding negative control solutions by taking the exocarpium citri grandis-deficient negative sample and the fructus schizandrae-deficient negative sample according to a test article preparation method, injecting the exocarpium citri grandis paste test article solution and the negative control solution into a liquid chromatograph to obtain a liquid chromatogram, wherein the result shows that the exocarpium citri grandis paste test article solution has corresponding chromatographic peaks at corresponding positions of rhoifolin, naringin and schizandrol A and the negative control is not interfered.

(5) Linearity and range

Precisely sucking 1 mu l of each mixed standard solution, injecting into an ultra-high performance liquid chromatograph, and processing the peak area by concentration to obtain a standard curve shown in a table 13, wherein the linear relation in the concentration range of each reference substance is good.

TABLE 13 linearity and Range of assay results

Substance(s)	Curve	r ² Value of	Range (μ g/ml)
				Naringin	y＝954.29x+1403.5	1.0000	29.872～1493.610
Rhoifolin	y＝5967x+3.812	1.0000	1.006～50.300
				Schisandrin A	y＝5925.8x+1192.4	0.9999	1.022～51.086

(6) Stability test

The same sample solution was measured at 0h,3h,6h,12h,24h and 48h respectively by the detection method, and the retention time and peak area of each peak were recorded, and the results are shown in Table 14, where the retention time and content RSD of each peak were less than 2.0%, and the method was stable.

TABLE 14 test results of solution stability (durability) of assay

2.5 Multi-batch assay results

Taking 10 batches of the chrysoidine pear syrup, preparing a test solution according to a test solution preparation method, measuring according to a detection method, and calculating the content of various components in the sample by using an external standard method, wherein the average content of the 10 batches of the chrysoidine pear syrup is shown in table 15.

TABLE 15 measurement results of multiple batches of exocarpium citri grandis paste

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A detection method of a UPLC fingerprint of exocarpium citri grandis paste is characterized by comprising the following steps:

accurately weighing exocarpium Citri rubrum pear syrup, adding organic solvent, filtering, and collecting filtrate to obtain sample solution;

wherein, the mobile phase A adopted by the ultra-high performance liquid chromatography is acid-containing acetonitrile, and the mobile phase B is acid-containing water; the volume fraction of the acid in the mobile phase A is 0.01 to 0.5 percent; the volume fraction of the acid in the mobile phase B is 0.01 to 0.5 percent; the acid is acetic acid or formic acid; adopting a gradient elution mode; the gradient elution mode is as follows: the volume percent of the mobile phase A is changed from 95 percent to 83 percent within 0 to 8 min; 8-11min, wherein the volume percentage of the mobile phase A is changed from 83% to 80%; 11-13min, the volume percentage of the mobile phase A is changed from 80% to 79.6%; 13-1695in, the volume percentage of the mobile phase A is changed from 79.6% to 73.6%;16 to 19min, and the volume percentage of the mobile phase A is changed from 73.6 percent to 61.6 percent; 19 to 23.5min, wherein the volume percentage of the mobile phase A is changed from 61.6 percent to 10 percent; 23.5 to 24min, and keeping the volume percentage of the mobile phase A to be 10 percent; the volume percentage of the mobile phase A is changed from 10% to 95% within 24-26min;

the chromatographic column adopted for the ultra-high performance liquid chromatography is C ₁₈ A chromatographic column; the flow rate of the mobile phase is 0.2 to 0.4ml/min;

the volume ratio of the solvent A to the test solution is 1 (2~5);

the solvent A is a methanol aqueous solution with the volume fraction of 20 to 50 percent or an ethanol aqueous solution with the volume fraction of 20 to 50 percent;

the solvent B is methanol or ethanol.

2. The detection method of the UPLC fingerprint of tangerine peel pear syrup according to claim 1, wherein the conditions of the ultra high performance liquid chromatography are as follows: the column temperature is 20 to 45 ℃; the detection wavelength is 200 to 220nm, 244 to 264nm or 310 to 330nm.

3. The detection method of the UPLC fingerprint of tangerine peel pear syrup according to claim 1, wherein the volume fraction of the acid in the mobile phase A is 0.01 to 0.3%.

4. The detection method of the UPLC fingerprint of tangerine peel pear syrup according to claim 1, wherein the volume fraction of the acid in the mobile phase B is 0.01 to 0.3%.

5. The detection method of the UPLC fingerprint of tangerine peel pear syrup according to claim 1, wherein the volume ratio of the solvent A to the test solution is 1 (2~4).

6. A method for detecting the content of multiple components in exocarpium citri grandis paste is characterized by comprising the following steps:

accurately weighing a reference substance, adding methanol for dissolving, and preparing a reference substance solution; the reference substances are naringin, rhoifolin and schizandrol A reference substances;

precisely sucking the reference substance solution and the test solution to perform ultra-high performance liquid chromatography;

wherein, the mobile phase A adopted by the ultra-high performance liquid chromatography determination is acid-containing acetonitrile, and the mobile phase B is acid-containing water; the volume fraction of the acid in the mobile phase A is 0.01 to 0.5 percent; the volume fraction of the acid in the mobile phase B is 0.01 to 0.5 percent; the acid is acetic acid or formic acid; adopting a gradient elution mode; the gradient elution mode is as follows: the volume percent of the mobile phase A is changed from 95 percent to 83 percent within 0 to 8 min; 8-11min, wherein the volume percentage of the mobile phase A is changed from 83% to 80%; 11-13min, the volume percentage of the mobile phase A is changed from 80% to 79.6%; 13-1695in, the volume percentage of the mobile phase A is changed from 79.6% to 73.6%;16 to 19min, and the volume percentage of the mobile phase A is changed from 73.6 percent to 61.6 percent; 19 to 23.5min, wherein the volume percent of the mobile phase A is changed from 61.6 percent to 10 percent; 23.5 to 24min, and keeping the volume percentage of the mobile phase A to be 10 percent; changing the volume percentage of the mobile phase A from 10% to 95% under 24-26min;

the organic solvent adding method comprises the following steps: adding a solvent A and then adding a solvent B;

the volume ratio of the solvent A to the test solution is 1 (2~5);

the solvent B is methanol or ethanol.

7. The method for detecting the content of the multiple components in the chrysoidine pear syrup according to claim 6, wherein the conditions of the ultra-high performance liquid chromatography are as follows: the column temperature is 20 to 45 ℃; the detection wavelength is 244 to 264nm or 310 to 330nm.

8. The method for detecting the content of the components in the orange pear syrup according to claim 6, wherein the volume fraction of the acid in the mobile phase A is 0.01 to 0.3%.

9. The method for detecting the content of the components in the orange pear syrup according to claim 6, wherein the volume fraction of the acid in the mobile phase B is 0.01 to 0.3%.

10. The method for detecting the content of multiple components in the exocarpium citri grandis pear syrup according to claim 6, wherein the volume ratio of the solvent A to the test solution is 1 (2~4).