阅读说明：本技术 一种温胃舒泡腾片hplc特征图谱的检测方法 (Detection method of HPLC (high performance liquid chromatography) characteristic spectrum of WENWEISHU effervescent tablet ) 是由 方礼 郭正友 邓双炳 陈琳 刘芳芳 刘明颖 谢华萍 王章伟 简蓝 古福生 王振 于 2020-06-30 设计创作，主要内容包括：本发明公开了一种温胃舒泡腾片检测方法,利用HPLC特征图谱法进行检测,方法更加快速便捷,可用于辨别温胃舒泡腾片的真伪,提高药品质量控制。(The invention discloses a method for detecting Wenweishu effervescent tablets, which is more rapid and convenient to detect by using an HPLC characteristic map method, can be used for identifying the authenticity of the Wenweishu effervescent tablets and improving the quality control of medicines.)

1. A detection method of HPLC characteristic spectrum of WENWEISHU effervescent tablet is characterized in that,

(1) chromatographic conditions are as follows: a C18 or C8 chromatography column; taking acetonitrile as a mobile phase A and 0.02-0.2% (V/V) formic acid aqueous solution as a mobile phase B, carrying out gradient elution, and setting specific flow rate, sample introduction amount, column temperature and detection wavelength;

(2) the determination method comprises the following steps: precisely absorbing a certain amount of reference substance solution and test solution, respectively injecting into a liquid chromatograph, and recording the chromatogram for 0-48 minutes.

2. The detection method according to claim 1, wherein the preparation process of the test solution comprises:

precisely weighing the ground WENWEISHU effervescent tablet, placing in a container, adding methanol water solution, ultrasonic treating, filtering, and collecting the filtrate; preferably, the concentration of the aqueous methanol solution is 20% to 80% (V/V).

3. The detection method according to claim 1, wherein the reference solution preparation process comprises:

taking a proper amount of hesperidin, precisely weighing, and dissolving with a methanol aqueous solution for quantitative dilution;

preferably, the concentration of the methanol aqueous solution is 20 to 80% (V/V); and/or the reference substance solution is a solution containing 0.1mg of hesperidin per 1 ml.

4. The detection method according to claim 1, wherein the chromatographic column is Ultimate AQ-C18, preferably having a column length of 150mm, an inner diameter of 4.6mm, and a particle size of 3.5 μm.

5. The detection method according to claim 1,

the flow rate is 0.5-1.5 ml/min;

and/or the detection wavelength is 260-300 nm;

and/or the column temperature is 20-60 ℃;

and/or the sample injection amount is 5-20 mul.

6. The detection method according to claim 1, wherein the concentration of the aqueous formic acid solution is 0.1% (V/V).

7. The detection method according to claim 1, wherein the elution procedure of the gradient elution is as follows:

8. the detection method according to claim 1, wherein 9 characteristic peaks are required in the sample characteristic map, and the relative retention time of the 9 characteristic peaks is calculated by taking a hesperidin reference peak as an S peak, and is within +/-5% of a specified value; the specified values of the 9 characteristic peaks were 0.17 (peak 1), 0.27 (peak 2), 0.77 (peak 3), 0.80 (peak 4), 0.81 (peak 5), 0.96 (peak 6), 1.00 (peak 7), 1.20 (peak 8), and 1.22 (peak 9), respectively.

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a detection method of an HPLC (high performance liquid chromatography) characteristic spectrum of a Wenweishu effervescent tablet.

Background

The WENWEISHU is a Chinese patent medicine and is prepared into WENWEISHU tablets, WENWEISHU effervescent tablets, WENWEISHU granules or WEISHU capsules from 12 medicinal materials, wherein 2 families of WENWEISHU tablet production enterprises, 1 family of WENWEISHU effervescent tablets and WEISHU granules production enterprises respectively and 3 families of WEISHU capsules (dosage forms recorded in Chinese pharmacopoeia) production enterprises, and the WEISHU of different dosage forms has the same functions and main indications: has good curative effect and wide application in the aspects of tonifying the kidney and strengthening the spleen, warming the middle-jiao and nourishing the stomach, and promoting the circulation of qi and relieving pain; the prescription of each dosage form is consistent, and the prescription is as follows:

the quality standards of different dosage forms are different, the quality standard of the WENWEISHU Capsule is part of Chinese pharmacopoeia 2015 edition, the quality standards of the WENWEISHU tablet and the WENWEISHU effervescent tablet are registration standards approved by the State drug administration and are numbered YBZ09882006-2010Z, YBZ12172005-2010Z respectively, the quality standard of the WENWEISHU granule is a standard approved by the original Ministry of health and is numbered WS3-B-3502-98 (collected in eighteenth volume of pharmaceutical Standard Chinese medicinal prescription preparations of Ministry of health), and the main quality control indexes of each quality standard are properties, identification, inspection and content measurement; the application of the patent 'construction method of a finger print of Wenweishu granules' (application number 201810595693.1) provides a finger print of Wenweishu granules, which has a narrow application range due to the influence of factors such as the origin and the harvesting period of various medicinal materials, is only suitable for the Wenweishu granules produced by a certain production unit or even a certain period, and cannot be widely applied to the purpose of identifying authenticity; and the method has long analysis time (65 minutes is needed); the mobile phase used contained phosphoric acid and was not suitable for mass spectrometry.

Disclosure of Invention

The invention provides a detection method of HPLC characteristic spectrum of WENWEISHU effervescent tablet, which can widely identify authenticity and has short analysis time, and the mobile phase does not contain phosphoric acid, can be directly applied to a mass spectrometer, and is beneficial to identifying chemical components of various spectrum peaks.

The invention provides a method for detecting HPLC characteristic spectrum of effervescent tablets of Wenweishu effervescent tablets, which is characterized in that,

(1) chromatographic conditions are as follows: a C18 or C8 chromatography column; taking acetonitrile as a mobile phase A and 0.02-0.2% (V/V) formic acid aqueous solution as a mobile phase B, carrying out gradient elution, and setting specific flow rate, sample introduction amount, column temperature and detection wavelength;

(2) the determination method comprises the following steps: precisely absorbing a certain amount of reference substance solution and test solution, respectively injecting into a liquid chromatograph, and recording the chromatogram for 0-48 minutes.

Preferably, the preparation process of the test solution comprises:

precisely weighing the ground WENWEISHU effervescent tablet, placing in a container, adding methanol water solution, ultrasonic treating, filtering, and collecting the filtrate;

more preferably, the concentration of the methanol aqueous solution is 20% to 80% (V/V), and still more preferably 40% to 60%.

Preferably, the reference solution preparation process comprises:

taking a proper amount of hesperidin, precisely weighing, diluting with methanol water solution, dissolving and quantitatively diluting.

More preferably, the concentration of the methanol aqueous solution is 20% to 80% (V/V), and still more preferably 40% to 60%;

more preferably, the reference solution is a solution containing 0.1mg of hesperidin per 1 ml.

Preferably, the chromatographic column is an Ultimate AQ-C18 chromatographic column; preferably, the column has a length of 150mm, an inner diameter of 4.6mm and a particle diameter of 3.5. mu.m.

Preferably, the flow rate is 0.5-1.5 ml/min;

preferably, the detection wavelength is 260-300 nm;

preferably, the column temperature is 20-60 ℃;

preferably, the sample injection amount is 5-20 mul;

preferably, the concentration of the aqueous formic acid solution is 0.1% (V/V).

Preferably, the elution procedure for the gradient elution is as follows:

preferably, 9 characteristic peaks exist in the characteristic map of the test sample, a hesperidin reference peak is taken as an S peak, and the relative retention time of the 9 characteristic peaks is calculated and is within +/-5% of a specified value; the specified values of the 9 characteristic peaks were 0.17 (peak 1), 0.27 (peak 2), 0.77 (peak 3), 0.80 (peak 4), 0.81 (peak 5), 0.96 (peak 6), 1.00 (peak 7), 1.20 (peak 8), and 1.22 (peak 9).

The key points of the invention are as follows: 1. measuring by using a characteristic map method; 2. the analysis method is short in time (48 minutes) by adopting a high performance liquid chromatograph for determination; 3. identifying the authenticity of the WENWEISHU effervescent tablet by identifying 9 common peaks; 4. the adopted mobile phases are easy to volatilize components, can be directly applied to a mass spectrometer and are beneficial to identifying the chemical components of the spectral peaks.

The invention establishes a detection method capable of detecting HPLC characteristic spectrum of the Wenweishu effervescent tablet, and can be widely applied to the purpose of identifying the authenticity of the Wenweishu effervescent tablet; the method is verified to show that the effervescent tablet can be accurately and visually identified as the stomach warming and soothing effervescent tablet; the invention is suitable for detecting the characteristic spectrum of the stomach warming and relaxing effervescent tablet, is beneficial to more comprehensively reflecting the inherent multi-component characteristics of the stomach warming and relaxing effervescent tablet, and improves the quality control of the stomach warming and relaxing effervescent tablet.

Drawings

FIG. 1: HPLC contrast characteristic spectrum of WENWEISHU effervescent tablet; peak 5: echinacoside; peak 7 (S): hesperidin; peak 8: psoralen; peak 9: isopsoralen;

FIG. 2: HPLC chromatogram of test solution of WENWEISHU effervescent tablet extracted with different solvents.

Detailed Description

The present invention is illustrated below with reference to the following embodiments, but is not limited thereto.

The stomach warming and soothing effervescent tablets used in the following examples are manufactured by the applicant, and other raw materials, reagents and instruments are commercially available.

Example 1: sample preparation

1. Preparing a blank solvent: 50% (V/V) methanol aqueous solution.

2. Preparation of a test solution: weighing 3g of the finely ground WENWEISHU effervescent tablet, precisely weighing, placing into a container, adding 25ml of 50% (V/V) methanol aqueous solution, performing ultrasonic treatment for 30 minutes, cooling, filtering with a microporous membrane, and taking the subsequent filtrate.

3. Preparation of reference solutions: taking 10mg of hesperidin, precisely weighing, diluting with 50% (V/V) methanol water solution, dissolving and quantifying 100ml, namely diluting into a solution containing 0.1mg of hesperidin per 1 ml.

Example 2: method for detecting HPLC (high performance liquid chromatography) characteristic spectrum of WENWEISHU effervescent tablet

(1) Chromatographic conditions are as follows: the column was Ultimate AQ-C18(150 mm. times.4.6 mm, 3.5 μm), acetonitrile as mobile phase A, 0.1% aqueous formic acid as mobile phase B, and the gradient elution procedure was:

the flow rate is 1 ml/min; the detection wavelength is 280 nm; the column temperature is 30 ℃; the amount of sample was 10. mu.l.

(2) The determination method comprises the following steps: precisely sucking a certain amount of the reference substance solution and the test sample solution in the example 1, respectively injecting the solutions into a liquid chromatograph, and recording the chromatogram for 0-48 minutes.

(3) The judgment standard is as follows:

9 characteristic peaks exist in the characteristic map of the test sample, a hesperidin reference peak is taken as an S peak, the relative retention time of the 9 characteristic peaks is calculated, and the relative retention time is within +/-5% of a specified value; the specified values of the 9 characteristic peaks were 0.17 (peak 1), 0.27 (peak 2), 0.77 (peak 3), 0.80 (peak 4), 0.81 (peak 5), 0.96 (peak 6), 1.00 (peak 7), 1.20 (peak 8), and 1.22 (peak 9), respectively.

Example 3: methodology investigation

3.1 precision investigation: and taking the same batch of sample solution, continuously injecting samples for 6 times, detecting the characteristic spectrum, and calculating the relative retention time and RSD of each common peak, wherein the result shows that the RSD of the relative retention time of each spectrum peak is within 1.0 percent and meets the requirement.

3.2 stability investigation: and taking the same sample solution, placing at room temperature for 0h, 2h, 4h, 8h, 12h and 24h, then injecting samples, detecting a characteristic spectrum, and calculating the relative retention time and RSD of all the common peaks, wherein the result shows that the RSD is within 1.0 percent and the sample solution is stable within 24 h.

3.3 repeatability test: taking the same batch of test solution, precisely weighing 3g of finely ground WENWEISHU effervescent tablets and 6 parts of the finely ground WENWEISHU effervescent tablets, respectively preparing the test solution for analysis, detecting a characteristic spectrum, calculating the relative retention time and RSD of each common peak, and indicating that the RSD is less than 1.0 percent and the repeatability is good.

4. Results and discussion

4.1 Experimental approaches

4.1.1 screening of the column: the results of trying chromatographic columns of various types, namely Welch Ultimate XB-C18, YMC ODS-A and ACE Excel 3C18, deducting chromatographic peaks before 5 minutes and respectively obtaining the minimum separation degrees of 1.9, 0.8 and 1.4 show that the Welch Ultimate AQ-C18 chromatographic column has better separation effect.

4.1.2 selection of extraction solvent: the WENWEISHU effervescent tablet is prepared by extracting with water, 50% (V/V) methanol aqueous solution, and methanol, wherein the WENWEISHU effervescent tablet contains pregelatinized starch, has high extraction viscosity, and is difficult to filter, and the sample solution of methanol has a chromatographic peak form about 8 min, which affects the separation degree (see figure 2), so 50% (V/V) methanol aqueous solution is selected as the extraction solvent.

4.2 analysis of sample measurement results

Feature profile and common peak labeling: performing HPLC characteristic map analysis on 6 batches of WENWEISHU effervescent tablets, wherein a representative characteristic map is shown in figure 1, 9 common peaks are marked, a hesperidin reference substance peak is taken as an S peak, and the relative retention time of the characteristic peaks 1-9 is calculated and is within +/-5% of a specified value. The values to be specified were 0.17 (peak 1), 0.27 (peak 2), 0.77 (peak 3), 0.80 (peak 4), 0.81 (peak 5), 0.96 (peak 6), 1.00 (peak 7), 1.20 (peak 8), and 1.22 (peak 9). As shown in fig. 1.

4.3 conclusion

(1) The characteristic spectrum quality control mode can comprehensively reflect the internal characteristics of multiple components of the medicine, and the characteristic spectrum quality control mode is richer and more comprehensive than the information provided by measuring one or more components; the characteristic map is used for evaluating the quality through the overall 'face' of the chromatogram of the drug test sample (such as information of peak appearance time, peak number, proportion among peaks and the like).

(2) The experiment researches HPLC characteristic maps of different batches of the Wenweishu effervescent tablets, the difference of the characteristic maps of different batches is large and is related to the complexity and the uniformity of the quality of medicinal materials, if 9 characteristic peaks are quantified, the fluctuation is large, but the purpose of identifying the truth of the Wenweishu effervescent tablets can be completely achieved by taking the relative retention time of the characteristic peaks as a qualitative function, and the reliability is very high.