CN118275595A

CN118275595A - Fingerprint spectrum and multi-component content determination method of nasopharynx toxicity-removing preparation

Info

Publication number: CN118275595A
Application number: CN202410678217.1A
Authority: CN
Inventors: 董福越; 严曾豪; 李鹏; 李楚源
Original assignee: Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co Ltd
Current assignee: Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co Ltd
Priority date: 2024-05-29
Filing date: 2024-05-29
Publication date: 2024-07-02
Anticipated expiration: 2044-05-29
Also published as: CN118275595B

Abstract

The invention belongs to the field of medicine detection, and particularly discloses a method for determining the content of multiple components in a nasopharynx toxicity-removing preparation by combining a fingerprint spectrum with the content of multiple components, wherein the method is used for detecting by an HPLC-DVD method. Under the cooperation of specific sample solution and chromatographic conditions, the invention not only obtains the fingerprint capable of reflecting 17 characteristic peaks of chemical components of most of medicinal materials of the nasopharynx toxicity-removing preparation formula such as wild chrysanthemum, cocklebur fruit, selfheal, radix zanthoxyli, rubus parvifolius root and the like, but also can accurately measure the content of representative components of 4 nasopharynx toxicity-removing preparations such as chlorogenic acid, gentiopicroside, rosmarinic acid and linarin based on the spectrograms under the dual wavelengths of 280nm and 334 nm.

Description

Fingerprint spectrum and multi-component content determination method of nasopharynx toxicity-removing preparation

Technical Field

The invention belongs to the field of medicine detection, and particularly relates to a method for determining the content of multiple components in a fingerprint spectrum combination of a nasopharynx toxicity-removing preparation.

Background

The nasopharynx toxin-removing preparation is a Chinese medicinal compound preparation which consists of 8 Chinese medicaments including wild chrysanthemum, fructus xanthil, rhizoma paridis, radix rubra, radix zanthoxyli, selfheal, gentian and codonopsis pilosula, and comprises pharmaceutically acceptable dosage forms such as granules, oral liquid, decoction and the like. The nasopharyngeal detoxification preparation has the effects of clearing heat and detoxicating, resolving phlegm and resolving masses, has long clinical application history, can be used for treating chronic inflammation of nasopharynx caused by accumulation of phlegm-heat toxin and blood stasis, and has definite curative effect on symptoms of secretion increase after nasopharyngeal carcinoma radiotherapy. Modern pharmacological studies show that the nasopharyngeal detoxification granule can inhibit proliferation of nasopharyngeal carcinoma CNE-2 cells, and has radiotherapy sensitization effect.

The nasopharynx toxin-removing preparation is used as a modern preparation of a traditional Chinese medicine compound, has complex chemical composition, less research on substance basis and action mechanism, and lower quality control level. The quality standard of the nasopharynx toxicity-removing granule in the 2020 edition of Chinese pharmacopoeia mainly comprises the steps of measuring the content of gentiopicroside which is a single representative component of gentian by adopting an HPLC method, and carrying out thin-layer identification on wild chrysanthemum and radix zanthoxyli, so that the quality of the nasopharynx toxicity-removing granule cannot be comprehensively evaluated. In the existing quality research, researchers mainly adopt an HPLC external standard method to measure the content of single or multiple chemical components such as gentiopicroside, rosmarinic acid, parietal saponin I, linarin and the like in the nasopharynx toxicity-removing preparation at the same time, so that the quality control level of the nasopharynx toxicity-removing preparation is improved to a certain extent, but the curative effect of the traditional Chinese medicine compound is an overall synergistic result, and the quality control of the traditional Chinese medicine is carried out by using comprehensive chemical components, so that the traditional Chinese medicine compound is accurate and reliable.

The traditional Chinese medicine fingerprint is a spectrogram or a chromatogram of chemical components of the traditional Chinese medicine obtained by means of a spectrum or chromatographic technique, has the characteristics of integral, macroscopic and fuzzy analysis and the like, and can achieve the purpose of integral quality control through the description of integral characteristics of the traditional Chinese medicine. In addition, an ideal fingerprint can achieve the purpose of qualitative identification, wherein part of the medicine spectrum is required to achieve baseline separation, so that quantitative analysis is realized, the qualitative identification is satisfied, and meanwhile, the absolute content of the representative components is controlled, so that the quality control level of the traditional Chinese medicine and the compound preparation thereof can be greatly improved. However, the method for determining the content of the multiple components by combining the fingerprint spectrum of the traditional Chinese medicine still has technical difficulties in the aspects of preparation method selection of a test sample, liquid chromatography condition fumbling, fingerprint spectrum sharing mode establishment, chromatographic peak selection, identification and the like in the application of the traditional Chinese medicine, and no report and application of quality control of the nasopharynx toxicity-removing preparation by combining the fingerprint spectrum or the fingerprint spectrum with the content determination of the multiple components are yet seen.

Disclosure of Invention

The invention aims to establish a fingerprint spectrum combined with multi-component content measurement method to comprehensively and accurately control the quality of a nasopharynx toxicity-removing preparation and ensure the curative effect of the nasopharynx toxicity-removing preparation.

The invention provides a method for measuring the content of multiple components in a nasopharynx toxicity-removing preparation by combining a fingerprint spectrum, which adopts an HPLC-DVD method to detect, and specifically comprises the following steps:

1) Preparing a reference substance solution: dissolving reference substance in methanol;

2) Sample solution preparation: extracting the nasopharynx toxicity-removing preparation to be detected with methanol solution, filtering, and collecting the filtrate;

3) Respectively sucking the reference substance solution and the sample solution, and injecting into a high performance liquid chromatograph; the chromatographic conditions were as follows:

Chromatographic column: octadecylsilane chemically bonded silica is used as a filler; mobile phase: taking formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; gradient elution procedure 0~3 min,2% B→8% B;3~15 min,8% B→13% B;15~20 min,13% B;20~25 min,13% B→20% B;25~35 min,20% B→21% B;35~43 min,21% B→28% B;43~50 min,28% B;50~60 min,28% B→50% B.

Further, the concentration of each reference substance in the reference substance solution in the step 1) is 0.001-0.05 mg.ml < -1 >.

Further, the reference substance is chlorogenic acid, gentiopicroside, rosmarinic acid and/or linarin reference substance.

Further, the mass-volume ratio of the nasopharynx toxicity removing preparation to be detected in the step 2) to the methanol solution is 1-5 g/25 ml.

Further, the concentration of the methanol solution is 10% -50%, v/v, preferably 30%, v/v.

Further, the step 2) is ultrasonic extraction, wherein the temperature of ultrasonic extraction is 30-40 ℃, the power is 250W and 40Hz, and the time is 20-40 min.

Further, step 3) the chromatographic column in the chromatographic conditions: a C18 chromatographic column, 250 mm X4.6 mm, a 5 [ mu ] m, preferably AGILENT ECLIPSE XDB-C18 chromatographic column, a column temperature of 30-35 ℃, a sample injection amount of 10 mu L, a flow rate of 1 mg.min-1 and detection wavelengths of 334 nm and 280nm.

Further, the nasopharynx toxicity-removing preparation to be detected should present 17 characteristic peaks in the chromatogram under the detection wavelength 334 nm, wherein 3 peaks should correspond to the retention time of the corresponding reference peak of the reference substance, the peak corresponding to the reference peak of the linarin is the S peak, the relative retention time of the rest peaks and the S peak is calculated, the relative retention time should be within + -5% of the specified value, and the specified value is: peak 1:0.069, peak 2:0.157, peak 3:0.209, peak 4:0.246, peak 5:0.263, peak 7:0.363, peak 8:0.415, peak 9:0.512, peak 10:0.709, peak 11:0.749, peak 12:0.812, peak 14:0.860, peak 15:0.985, peak 17:1.075.

Furthermore, the content of chlorogenic acid, rosmarinic acid and linarin in the nasopharynx toxicity-removing preparation to be detected is calculated according to an external standard method by detecting the peak area in a chromatogram at a wavelength of 334 nm, and the content of gentiopicroside is calculated according to an external standard method by detecting the peak area in a chromatogram at a wavelength of 280 nm.

Further, the nasopharynx toxicity-removing preparation to be detected is a nasopharynx toxicity-removing granule.

The HPLC-DVD method refers to a liquid chromatography detection method based on a wavelength switching detector.

According to the fingerprint spectrum combined with multi-component content measurement method of the nasopharynx toxicity-removing preparation, under the cooperation of a specific sample solution and chromatographic conditions, the fingerprint spectrum of 17 chemical component characteristic peaks of most of the medicinal materials of the nasopharynx toxicity-removing preparation, such as wild chrysanthemum flower, cocklebur fruit, selfheal, radix zanthoxyli, rubus parvifolius root and the like, can be obtained, the characteristic components of the nasopharynx toxicity-removing preparation are used as a reference on the basis of the spectrograms at the dual wavelengths of 280nm and 334nm, the accurate content measurement is carried out on the characteristic components of 4 nasopharynx toxicity-removing preparations, such as chlorogenic acid, gentiopicroside, rosmarinic acid and linarin, the problem that the gentiopicroside response in the fingerprint spectrum at 334nm is lower is solved, and the comprehensive and accurate control of the nasopharynx toxicity-removing preparation quality is realized. The method of the invention takes 60 minutes, greatly reduces the time cost, is simple and feasible, is rapid and accurate, and greatly supplements and improves the quality control level of the existing nasopharynx toxicity-removing preparation.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is an HPLC overlay fingerprint and a control fingerprint (R) of 14 samples of nasopharyngeal darcinogenic particles;

FIG. 2 HPLC plot (fingerprint) of nasopharyngeal detoxification particle samples;

FIG. 3 HPLC of negative samples of each herb (A-flos Chrysanthemi Indici, B-fructus Xanthii, C-rhizoma paridis, D-radix Rubi Parvifolii, E-radix Zanthoxyli, F-Spica Prunellae, G-radix Gentianae, H-radix Codonopsis);

FIG. 4 HPLC of nasopharyngeal detoxification sample (A) and mixed control (B) -280nm (1-chlorogenic acid; 2-gentiopicroside; 3-rosmarinic acid; 4-montanin);

FIG. 5 HPLC of nasopharyngeal detoxification sample (A) and mixed control (B) -334nm (1-chlorogenic acid; 2-gentiopicroside; 3-rosmarinic acid; 4-montanin);

FIG. 6 is an HPLC diagram of sample solutions obtained from different extraction solvents;

FIG. 7 is a HPLC chart obtained from various elution procedures;

FIG. 8 HPLC plot obtained at different column temperatures.

Detailed Description

The reagents, and equipment used in the embodiments of the invention are all known products and are obtained by purchasing commercially available products.

Example 1: establishment of quality control method for nasopharynx toxicity-removing preparation by combining fingerprint spectrum with multi-component measurement

1. Experimental materials

1.1 Reagents and medicaments

Chlorogenic acid reference (lot number: 110753-202119, purity: 96.03%), gentiopicroside reference (lot number: 110770-202219, purity: 98.10%), rosmarinic acid reference (lot number: 111871-202007, purity: 98.10%), linarin reference (lot number: 111528-202213, purity: 94.60%), all of which were purchased from China food and drug inspection institute. Nasopharyngeal detoxification granule 14 batches (lot number S1-S14, respectively, provided by chinese medicine limited, ganzhou white cloud mountain and cambodia).

Acetonitrile (chromatographic purity, merck, usa), methanol (analytical purity, guangzhou chemical reagent factory), formic acid (chromatographic purity, guangzhou chemical reagent factory), and water as ultrapure water.

1.2 Instrument for measuring and controlling the intensity of light

Agilent 1290 (Agilent, usa); column Agilent ZORBAX-C ₁₈ (250 mm x 4.6mm, 5 μm); chromatographic column AGILENT ECLIPSE XDB-C ₁₈ (250 mm X4.6 mm, 5 μm); ESA224S-CW balance (saidos (beijing) instrument science limited), CP225D balance (saidos (beijing) instrument science limited); SB25-12DTD ultrasonic cleaner (Ningbo Xinzhi biotechnology Co., ltd.).

2. Experimental method

2.1 Chromatographic conditions

A AGILENT ECLIPSE XDB-C ₁₈ column (250 mm ×4.6 mm, 5 μm) was used; mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, gradient elution is carried out at ：0~3 min,B 2%~8%;3~15 min,B 8%~13%;15~20 min,B 13%;20~25 min,B 13%~20%;25~35 min,B 20%~21%;35~43 min,B 21%~28%;43~50 min,B 28%;50~60 min,B 28%~50%; flow rate of 1 mg.min ^-1, detection wavelength is 334 nm and 280nm (HPLC-DVD mode), column temperature is 30 ℃, and sample injection amount is 10 μl.

2.2 Preparation of mixed control solution

Precisely weighing appropriate amounts of chlorogenic acid, gentiopicroside, rosmarinic acid and linarin reference substances, adding methanol to dissolve and dilute to scale, shaking uniformly, preparing mixed reference substance solutions with chlorogenic acid concentration of 0.0038, 0.0075, 0.0188, 0.03011, 0.0376 mg/ml ^-1, gentiopicroside concentration of 0.0041, 0.0082, 0.0204, 0.0327, 0.0409mg/ml, rosmarinic acid concentration of 0.0033, 0.0066, 0.0164, 0.0262, 0.0328 mg/ml and linarin concentration of 0.0031, 0.0063, 0.0156, 0.0240 and 0.0313mg/ml respectively, and storing in a refrigerator at 4 ℃ in dark place for standby.

2.3 Preparation of test solutions

Taking 1g of nasopharynx virus-clearing particles, precisely weighing, placing into a 25mL volumetric flask, adding 30% methanol to about 20mL, performing ultrasonic extraction (30-40 ℃ and 250W40 Hz) for 30min, standing to room temperature, fixing the volume to the scale with 30% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution.

3. Establishment of fingerprint spectrum of nasopharynx toxin-removing granule preparation

3.1 Precision test

About 1g of nasopharynx virus-clearing particle sample (S1) is precisely weighed, a sample solution is prepared according to the method under the item "2.3", the sample solution is measured according to the chromatographic condition under the item "2.1", the sample is continuously injected for 6 times, a 334nm chromatogram is recorded, and 4 peaks are selected as targets. At 280 nm, the relative retention time of gentiopicroside peak was calculated to be 0.07%, the relative peak area was calculated to be 0.18%, and at 334nm, the relative retention times of chlorogenic acid, rosmarinic acid and linarin were calculated to be 0.11%, 0.07% and 0.04%, respectively, indicating good instrument precision.

3.2 Repeatability test

Precisely weighing about 1g parts of the same nasopharynx virus-clearing granule (S1), preparing 6 parts of test solution in parallel according to the method under the item "2.3", measuring according to the chromatographic condition under the item "2.1", selecting 4 peaks as targets, and calculating to obtain RSDs of the relative retention time of the 4 common peaks of 0.13%, 008%, 0.28% and 0.06%, wherein the RSDs of the relative peak areas are 0.85%, 0.75%, 2.17% and 0.73%, respectively, which indicates that the method repeatability is good.

3.3 Stability test

Taking the same nasopharynx virus-clearing granule sample solution, measuring according to the chromatographic condition under the item of 2.1, continuously sampling at 0, 4, 8, 16, 32 and 48 h for 6 times for analysis, selecting 4 peaks as targets, and calculating to obtain the relative retention time RSD of the 4 common peaks which are respectively smaller than 0.58%, wherein the RSD of the relative peak areas is smaller than 0.45%, which indicates that the sample solution has good stability in 48 h.

3.4 Fingerprint establishment and similarity evaluation

1G of each 14 batches of nasopharynx virus-clearing particles is precisely weighed, a test solution is prepared according to the method under the item "2.3", then sample injection measurement is carried out according to the chromatographic conditions under the item "2.1", and a 334nm chromatogram is recorded. Introducing HPLC (high performance liquid chromatography) of 14 batches of nasopharynx virus-clearing granule samples into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.1 edition), taking a chromatogram of an S1 sample as a reference chromatogram, generating a control chromatogram (R) by a median method with a time window width of 0.3min, and performing chromatographic peak matching after multi-point correction to generate a sample superposition fingerprint (figure 1); and then taking the control fingerprint as a reference, and calculating the similarity by an average method. As a result, 17 common peaks were co-calibrated in 14 batches of nasopharyngeal darcinogenic granule samples; the similarity between each sample pattern and the reference fingerprint pattern is 0.913-0.997, which shows that 14 batches of nasopharyngeal virus-clearing granule samples have high similarity, and the similarity evaluation results are shown in table 1.

TABLE 1 HPLC fingerprint similarity evaluation results of 14 batches of nasopharyngeal darcinoma granule samples

Comparing 17 common peaks in the fingerprint spectrum with the retention time of the chromatographic peaks of the reference substance and ultraviolet absorption spectrum information, and identifying 3 of the peaks: chlorogenic acid (No. 6 peak), rosmarinic acid (No. 13 peak), linarin (No. 16 peak) with larger peak area, good peak shape and better separation degree are selected as reference peak (S) according to the retention time, the specified value is 1.000, the relative retention time of the other common characteristic peaks and the reference peak is calculated, and the relative retention time is within +/-5% of the specified value; the specified value is: peak 1:0.069, peak 2:0.157, peak 3:0.209, peak 4:0.246, peak 5:0.263, peak 7:0.363, peak 8:0.415, peak 9:0.512, peak 10:0.709, peak 11:0.749, peak 12:0.812, peak 14:0.860, peak 15:0.985, peak 17:1.075.

3.5 Medicinal material negative test

Taking the powder of each medicinal material (sieving with a 60-mesh sieve): wild chrysanthemum flower 0.390g, fructus xanthil 0.390g, rhizoma paridis 0.390g, rubus parvifolius root 0.390g, radix zanthoxyli 0.195g, selfheal 0.195g, gentian 0.117g and radix codonopsis pilosulae 0.117g, respectively and precisely weighing, and preparing a test solution according to the method under item 2.3. Then, the chromatogram was recorded as determined by the chromatographic method under item "2.1" (fig. 2 and 3), and the comparison with the fingerprint in "3.4" revealed that: 4. peaks 6, 7, 8, 15 and 16 may be derived from wild chrysanthemum, peaks 5, 6, 8, 11 and 14 may be derived from fructus Xanthii, peak 6 may be derived from radix Rubi Parvifolii, peak 4, 6 and 7 may be derived from radix Zanthoxyli, and peaks 5, 8, 13 and 14 may be derived from Prunellae Spica. Under the chromatographic conditions, signal peaks derived from rhizoma paridis, radix gentianae and radix codonopsis can not be judged.

3.6 Research knot

In the study, 14 batches of nasopharynx virus-clearing granule samples (including Baiyunshan brand and other commercial brand products) are selected, and are characterized by an HPLC-DVD fingerprint pattern, and the result shows that the similarity of 14 samples is between 0.913 and 0.997, and 17 common peaks are determined. Through a medicinal material negative test, the common peaks can be presumed to be mostly derived from wild chrysanthemum, cocklebur fruit, selfheal, radix zanthoxyli and rubus parvifolius root; the common peaks of gentian, paris polyphylla and codonopsis pilosula sources could not be identified. The method can obtain the fingerprint spectrum of 17 characteristic peaks of chemical components of most of medicinal materials of the nasopharynx toxicity-removing preparation formula, such as the wild chrysanthemum, the cocklebur fruit, the selfheal, the radix zanthoxyli, the rubus parvifolius and the like.

4. Content determination of multi-index components of nasopharyngeal detoxification preparation

4.1 HPLC chromatogram and System adaptation experiment

The same nasopharynx virus-clearing granule (S1) is precisely weighed to be about 1 g, 6 parts of test solution are prepared in parallel according to the method under the item "2.3", and the chromatograms of 334nm and 280nm are respectively recorded according to the chromatographic condition under the item "2.1", and the results are shown in figures 4 and 5.HPLC (high performance liquid chromatography) graphs show that 4 index component signal peaks of chlorogenic acid, gentiopicroside, rosmarinic acid and linarin are well separated, and the gentiopicroside response is higher at 280nm, so that the method is suitable for measuring the content of gentiopicroside; chlorogenic acid, rosmarinic acid and linarin have higher response at 334nm, and are suitable for content determination of chlorogenic acid, rosmarinic acid and linarin.

4.2 Linear relationship investigation

Precisely sucking 5 series of mixed reference solutions, respectively, injecting 10 μl each into high performance liquid chromatograph, and measuring under 2.1 chromatographic conditions to determine peak area of each reference. Regression treatment is carried out on the peak area integral value (Y) according to the mass concentration (X, mug.mL ^-1) of each solution to obtain a linear equation and a correlation coefficient R ² of chlorogenic acid, gentiopicroside, rosmarinic acid and linarin. The results showed that the sample loading and peak area of each control were well linear (Table 2).

Table 2 results of linear relationship investigation

4.3 Precision test

And precisely sucking 10 mu L of the mixed reference substance solution under the item "2.2", and continuously sampling for 6 times according to the condition under the item "2.1". RSD of gentiopicroside peak area at 280 nm was 0.18%; RSD of chlorogenic acid, rosmarinic acid and linarin at 334 nm were 1.19%, 0.73% and 0.07%, respectively. The precision of the instrument is good.

4.4 Repeatability test

Taking nasopharynx toxicity-removing particles with batch number S4, preparing 6 parts of test solution in parallel according to a method of 2.3, and determining that the RSD of gentiopicroside is 0.85% under 280 nm according to the condition of 2.1; RSD of chlorogenic acid, rosmarinic acid and linarin at 334 nm were 0.75%, 2.17% and 0.73%, respectively. The reproducibility of the method was demonstrated to be good.

4.5 Stability test

Taking nasopharynx virus-clearing particles with batch number S4, preparing a sample solution according to the method under the item of 2.3, standing at room temperature, sampling under the conditions of 0, 4, 8, 16, 32 and 48 hours according to the condition of 2.1, and measuring the RSD of gentiopicroside under the condition of 280nm to be 0.33%; RSD of chlorogenic acid, rosmarinic acid and linarin at 334 nm were 0.76%, 0.91% and 0.45%, respectively.

4.6 Sample recovery test

9 Parts of a measured sample (batch number is S3) are precisely weighed, mixed reference substance solutions which are 80%, 100% and 120% of the content of the components to be measured in the sample are precisely added respectively, the sample solution is prepared according to the method under the item of 2.3, the sample is sampled and measured according to the chromatographic condition under the item of 2.1, and the average sample recovery rates of chlorogenic acid, gentiopicroside, rosmarinic acid and linarin are calculated to be 95.15%, 96.32%, 104.39% and 98.13% respectively, and RSD is 1.14%, 1.42%, 2.29% and 0.98% respectively. The results are shown in Table 3

TABLE 3 sample recovery test results for 4 components in the samples

4.7 Sample content determination

Taking nasopharynx toxin-removing granule samples, preparing test solution according to the method under item 2.3, and measuring the content of chlorogenic acid, gentiopicroside, rosmarinic acid and linarin in each sample according to the chromatographic condition under item 2.1. The results are shown in Table 4.

TABLE 4 determination of the index Components of the granule for clearing nasopharynx

4.8 Research knot

The present study established a method for determining the content of the index ingredient in the nasopharyngeal detoxification granules. The method has good repeatability, accuracy and stability, can simultaneously measure the content of a plurality of index components, and can more scientifically and comprehensively evaluate the quality of the nasopharyngeal darcinoma granules by combining a fingerprint spectrum method.

The advantageous effects of the present invention are further illustrated by test examples below.

Test example 1: influence of sample preparation method on fingerprint

The traditional Chinese medicine compound preparation has complex components, and different extraction solvents can influence the extraction effect of active ingredients. The prescription for clearing the toxin of the nasopharynx consists of 8 medicinal materials, the polarity and other characteristics of the components are greatly different, and how to extract more components to the maximum extent and show peaks in an HPLC chart is a difficult point of the technology.

Four different methods of A, B, C, D were used to prepare the test solutions, each as follows:

a: taking 1g of nasopharynx virus-clearing particles, precisely weighing, placing into a 25mL volumetric flask, adding 10% methanol about 20mL, performing ultrasonic extraction (30-40 ℃ and 250W40 Hz) for 30min, standing to room temperature, fixing the volume to the scale with 10% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution.

B: taking 1g of nasopharynx virus-clearing particles, precisely weighing, placing into a 25mL volumetric flask, adding 30% methanol to about 20mL, performing ultrasonic extraction (30-40 ℃ and 250W40 Hz) for 30min, standing to room temperature, fixing the volume to the scale with 30% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution.

C: taking 1g of nasopharynx virus-clearing particles, precisely weighing, placing into a 25mL volumetric flask, adding 50% methanol about 20mL, performing ultrasonic extraction (30-40 ℃ and 250W40 Hz) for 30min, standing to room temperature, fixing the volume to the scale with 50% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution.

D: taking 1g of nasopharynx virus-clearing particles, precisely weighing, placing into a 25mL volumetric flask, adding 80% methanol about 20mL, performing ultrasonic extraction (30-40 ℃ and 250W40 Hz) for 30min, standing to room temperature, fixing the volume to the scale with 80% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and passing through a 0.22 mu m microporous filter membrane to obtain a sample solution.

The test solutions prepared by the four methods are respectively detected according to the chromatographic conditions of the embodiment 1, and the results are shown in fig. 6, and as can be seen from fig. 6, the signal peaks of the chromatogram of the test solution prepared by the method D are fewer and the peak shape is not ideal within 8-20 min, while the chromatogram of the test solution prepared by the method A, B, C has more peaks, good peak shape, stable base line, good peak and larger peak area, and is the optimal method selected by the invention.

Test example 2: influence of chromatographic conditions on finger print

The components of the nasopharyngeal detoxification granule are complex, isocratic elution and gradient elution (other conditions are the same as in example 1) are examined, and the following different chromatographic conditions are adopted to establish a fingerprint.

1. Selection of elution procedure

The mobile phase was chosen to be acetonitrile (a) -0.1% formic acid solution (B). Optimal chromatographic conditions were obtained by the following fumbling procedure.

Chromatographic condition 1: isocratic elution: 0-60 min,70% A;

Chromatographic condition 2: gradient elution: 0 to 27min,35 to 55 percent of A; 27-36 min,55% A; 36-43 min, 55-65% A; 43-53 min, 65-85% A; 53-60 min, 85-35% A; other conditions were the same as above.

Chromatographic condition 3: other conditions for gradient elution ：0～3min,2～10％A;3～9min,10～15％A;9～20min,15％A;20～25min,15～30％A;25～30min,30～43％A;30～40min,43～70％A;40～60min,70～100％A; were as above.

Chromatographic condition 4: other conditions for gradient elution ：0～3min,2～8％A;3～10min,8～15％A;10～18min,15％A;18～25min,15～23％A;25～40min,23～55％A;40～41min,55～70％A;41～65min,70～100％A;65～75min,100～2％A; were as above.

Chromatographic condition 5: other conditions for gradient elution ：0～3min,2～8％A;3～15min,8～13％A;15～20min,13％A;20～25min,13～20％A;25～35min,20～21％A;35～43min,21～28％A;43～50min,28％A;50～60min,28～50％A; were as above.

The results obtained for the above five elution procedures are shown in fig. 7, and can be seen from fig. 7: chromatographic conditions 1 and 2, because the initial acetonitrile concentration of the mobile phase is too high, each component gives a peak within 5min, the component signal peaks are piled up together and have a small number, and the chromatographic conditions are not suitable as analysis conditions; chromatographic conditions 3 and 4, too concentrated signal peaks also occur; in contrast, in the chromatogram of chromatographic condition 5, the component signal peaks are more, the peak separation and peak shape are good, the baseline is stable, and the fingerprint analysis is suitable.

2. Column temperature selection

The number of chromatographic peaks and the degree of separation were examined at various column temperatures (20 ℃, 25 ℃, 30 ℃, 35 ℃) under the same conditions as in example 1, and the results are shown in FIG. 8. As can be seen from FIG. 8, the number of peaks of each component was large at column temperatures of 30℃and 35℃and the degree of separation was good.

In conclusion, under the cooperation of specific sample solution and chromatographic conditions, the fingerprint spectrum of 17 chemical component characteristic peaks of most of medicinal materials of the nasopharynx toxicity-removing preparation formula such as the reactive wild chrysanthemum, the fructus xanthil, the selfheal, the radix zanthoxyli and the rubus parvifolius is obtained, and the characteristic components of the nasopharynx toxicity-removing preparation are used as a reference on the basis of the spectrograms under the dual wavelengths of 280nm and 334nm to accurately measure the content of the representative components of 4 nasopharynx toxicity-removing preparations such as chlorogenic acid, gentiopicroside, rosmarinic acid and linarin, so that the comprehensive and accurate control of the quality of the nasopharynx toxicity-removing preparation is realized.

Claims

1. A method for determining the content of a plurality of components by combining a fingerprint spectrum of a nasopharynx toxicity-removing preparation is characterized by comprising the following steps of: the method adopts an HPLC-DVD method for detection, and specifically comprises the following steps:

2. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 1, wherein the method comprises the following steps of: the concentration of each reference substance in the reference substance solution in the step 1) is 0.001-0.05 mg.ml ^-1.

3. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 2, which is characterized in that: the reference substances are chlorogenic acid, gentiopicroside, rosmarinic acid and/or linarin reference substances.

4. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 1, wherein the method comprises the following steps of: and 2) the mass volume ratio of the nasopharynx toxicity removing preparation to be tested to the methanol solution is 1-5 g/25 ml.

5. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 4, which is characterized in that: the concentration of the methanol solution is 10% -50%, v/v.

6. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 1, wherein the method comprises the following steps of: the step 2) is ultrasonic extraction, wherein the temperature of ultrasonic extraction is 30-40 ℃, the power is 250W,40Hz, and the time is 20-40 min.

7. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 1, wherein the method comprises the following steps of: the chromatographic conditions of step 3) are: chromatographic column: c ₁₈ chromatographic column, 250 mm multiplied by 4.6 mm, 5 mu m, column temperature of 30-35 ℃, sample injection amount of 10 mu L, flow rate of 1 mg.min ^-1 and/or detection wavelength of 334 nm and 280nm.

8. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 1, wherein the method comprises the following steps of: the nasopharynx toxicity-removing preparation to be detected should present 17 characteristic peaks in the chromatogram under the detection wavelength 334 nm, wherein 3 peaks should correspond to the retention time of the corresponding reference object peak of the reference substance respectively, the peak corresponding to the reference object peak of the linarin is the S peak, the relative retention time of the rest peaks and the S peak is calculated, the relative retention time is within + -5% of the specified value, and the specified value is: peak 1:0.069, peak 2:0.157, peak 3:0.209, peak 4:0.246, peak 5:0.263, peak 7:0.363, peak 8:0.415, peak 9:0.512, peak 10:0.709, peak 11:0.749, peak 12:0.812, peak 14:0.860, peak 15:0.985, peak 17:1.075.

9. The method for determining the content of the multiple components by combining the fingerprint spectrum according to claim 8, wherein the method comprises the following steps of: the content of chlorogenic acid, rosmarinic acid and linarin in the nasopharynx toxicity-removing preparation to be detected is calculated according to an external standard method by detecting the peak area in a chromatogram at the wavelength of 334 nm, and the content of gentiopicroside is calculated according to an external standard method by detecting the peak area in a chromatogram at the wavelength of 280 nm.

10. The method for determining the content of the fingerprint spectrum combined with the multiple components according to any one of claims 1 to 9, which is characterized by comprising the following steps: the nasopharynx toxicity-removing preparation to be detected is nasopharynx toxicity-removing granules.