Method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component simultaneous determination method

Abstract

The present invention discloses a method for determining the contents of six components in Qingwei Bao'an Pills by the method of multi-component quantification by single marker, belonging to the technical field of traditional Chinese medicine detection. The method for determining the contents of six components in Qingwei Bao'an Pills by the method of multi-component quantification by single marker provided by the present invention uses neohesperidin as an internal reference substance. According to the relative correction factors obtained between neohesperidin and liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate, the contents of six main pharmacologically active components in Qingwei Bao'an Pills can be quickly detected, the detection efficiency can be improved, a basis is provided for the quality control standard of Qingwei Bao'an Pills products, and it is convenient for the production and detection of Qingwei Bao'an Pills.

Description

Technical Field

[0001] The invention relates to a method for determining the contents of six components in Qingwei Baoan Pills by adopting a one-test-multiple-evaluation method, and belongs to the technical field of traditional Chinese medicine detection. Background Art

[0002] There are 14 herbs in the prescription of Qingwei Baoan Pills, namely, bran-fried Atractylodes macrocephala, Liushenqu (fried with bran), dried tangerine peel, Poria cocos, Amomum villosum, vinegar green peel, ginger Magnolia bark, fried malt, licorice, betel nut, bran-fried Citrus aurantium, Citrus aurantium, white wine koji, and fried hawthorn. It has the effects of digesting food, removing stagnation, and soothing the stomach and stopping vomiting. It is often used clinically for gastrointestinal stagnation caused by food stagnation, with symptoms such as children's food stagnation, milk stagnation, abdominal distension, vomiting, irritability, and thirst. In the prescription, bran-fried Atractylodes macrocephala and Poria cocos are the main herbs, which invigorate the spleen and dry dampness; dried tangerine peel, Amomum villosum, vinegar green peel, ginger Magnolia bark, bran-fried Citrus aurantium, and Citrus aurantium are the assistant herbs, which regulate qi and remove dampness, dredge qi, and eliminate distension; six-shenqu (fried with bran), fried malt, fried hawthorn, and white wine koji are used to digest food and remove stagnation, betel nut kills insects and eliminates accumulation; roasted licorice harmonizes the herbs. The combination of these medicines has the effect of strengthening the spleen and stomach, and digesting food and relieving stagnation.

[0003] The 14 traditional Chinese medicines in the Qingwei Baoan Pills have complex chemical compositions, including saponins, furans, alkaloids, flavonoids, polysaccharides, lactones and other compounds. The content determination indicators specified in the 2020 edition of the "Chinese Pharmacopoeia" under the Qingwei Baoan Pills content determination item are magnolol and honokiol. Literature research and analysis found that it is difficult to comprehensively evaluate the quality of Qingwei Baoan Pills based solely on the content of magnolol and honokiol. With the deepening of clinical application and research, it is of great significance to establish a scientific, reasonable, convenient and feasible quality control method to control the quality of Qingwei Baoan Pills and ensure clinical efficacy.

[0004] It should be noted that the above contents belong to the technical knowledge of the inventor and do not necessarily constitute prior art. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for determining the contents of six components in Qingwei Baoan Pills by using a one-test-multiple-evaluation method, which can quickly detect the contents of the six components in Qingwei Baoan Pills that mainly play a pharmacological role, and improve the detection efficiency.

[0006] The present invention achieves the above-mentioned purpose by adopting the following technical solutions:

[0007] The method for determining the contents of six components in Qingwei Baoan Pills by using the one-test-multiple-evaluation method comprises the following steps:

[0008] (1) preparing mixed reference substance solutions of different concentrations, wherein the mixed reference substance solution is a mixed solution of six single reference substances: liquiritin, naringin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate;

[0009] (2) Determine the chromatographic conditions for high performance liquid chromatography analysis:

[0010] Use octadecylsilane chemically bonded silica as the filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B for gradient elution. The detection wavelength is 275 nm from 0 to 55 min and 237 nm from 55 to 80 min. The flow rate is 1 ml / min and the column temperature is 30 °C;

[0011] (3) Take mixed reference substance solutions with different concentrations, inject them respectively, and perform high performance liquid chromatography analysis. Use neohesperidin as the internal reference substance to determine the relative correction factors of liquiritin, rutacarpin, naringin, hesperidin, and ammonium glycyrrhizinate;

[0012] (4) Determination of the content of the components to be measured in the test solution:

[0013] Prepare the test solution of Qingwei Bao'an Pills and the reference substance solution of neohesperidin. Perform high performance liquid chromatography analysis according to the chromatographic conditions in step (2). Locate the chromatographic peaks of each component to be measured according to the relative retention time. Using the concentration and peak area of the reference substance solution of neohesperidin as the reference, calculate the contents of liquiritin, rutacarpin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate in the test solution respectively according to the relative correction factors determined in step (3).

[0014] Furthermore, step (1) is specifically as follows:

[0015] Use methanol as the solvent to prepare single reference substance solutions of six reference substances, namely liquiritin, rutacarpin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate. Take the six single reference substance solutions and mix them to prepare a stock solution of the mixed reference substance solution;

[0016] Dilute the stock solution of the mixed reference substance solution by the multiple dilution method to obtain mixed reference substance solutions with different concentrations.

[0017] Even further, in step (1), the preparation methods of the six single reference substance solutions of liquiritin, rutacarpin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate are as follows:

[0018] Take six reference substances, namely liquiritin, rutacarpin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate, dissolve them in methanol respectively to prepare six single reference substance solutions with concentrations of 1.59784 mg / ml, 2.04918 mg / ml, 2.14864 mg / ml, 2.01186 mg / ml, 1.69517 mg / ml, and 1.23360 mg / ml respectively.

[0019] Furthermore, the specific conditions for gradient elution in step (2) are as follows:

[0020] 0 - 48 min, mobile phase A: 12% - 23%, mobile phase B: 88% - 77%;

[0021] 48 - 49 min, mobile phase A: 23% - 41%, mobile phase B: 77% - 59%;

[0022] 49 - 60 min, mobile phase A: 41%, mobile phase B: 59%;

[0023] 60 - 61 min, mobile phase A: 41% - 60%, mobile phase B: 59% - 40%;

[0024] 61 - 70 min, mobile phase A: 60%, mobile phase B: 40%;

[0025] 70 - 80 min, mobile phase A: 60% - 12%, mobile phase B: 40% - 88%.

[0026] Furthermore, in step (3), the relative retention time of neohesperidin is 1.00, and the chromatographic peak relative retention times of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate relative to neohesperidin are 0.63, 0.80, 0.87, 0.92, and 1.32, respectively.

[0027] Furthermore, in step (3), the correction factor of neohesperidin is 1.00, and the relative correction factors of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate relative to neohesperidin are 0.80, 1.21, 1.02, 1.01, and 2.12, respectively.

[0028] Furthermore, in step (3), the calculation formula for the relative correction factor is shown in formula (Ⅰ):

[0029]

[0030] Wherein, W s is the concentration of the neohesperidin reference solution, A s is the peak area of the neohesperidin reference solution, W k is the concentration of the test component, A k is the peak area of the test component; s is neohesperidin, k is the test component, f s is the correction factor of neohesperidin, f k is the correction factor of the test component.

[0031] Furthermore, in step (4), the preparation method of the test solution is as follows:

[0032] Cut Qingwei Bao'an Pills into pieces, accurately weigh 1.5 g, place them in a stoppered conical flask, add 30 ml of methanol, weigh, heat under reflux for 60 min, cool, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0033] Further, in step (4), the preparation method of the neohesperidin reference solution is: dissolve neohesperidin in methanol to prepare a neohesperidin reference solution with a concentration of 251.48 μg / ml.

[0034] The beneficial effects of the present invention include but are not limited to:

[0035] The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component simultaneous determination method using one reference substance provided by the present invention takes neohesperidin as the internal reference substance. According to the relative correction factors obtained between neohesperidin and liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate, it can quickly detect the contents of the six main pharmacologically active components in Qingwei Bao'an Pills, improve the detection efficiency, provide a basis for the quality control standard of Qingwei Bao'an Pills products, and facilitate the production and detection of Qingwei Bao'an Pills.

[0036] The detection conditions of the high performance liquid chromatography (HPLC) used in the present invention have good stability, repeatability, precision, and reliability, and the detection results are accurate, which is suitable for the large-scale detection of Qingwei Bao'an Pills. Description of the Drawings

[0037] The drawings described herein are used to provide a further understanding of the present invention and form a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0038] Figure 1 It is the chromatogram of the test solution obtained by the reflux and ultrasonic extraction methods.

[0039] Figure 2 It is the chromatogram of each sample in the specificity verification.

[0040] Figure 3 It is the chromatogram measured using different chromatographic columns. Detailed Embodiments

[0041] The present invention will be further described in detail below. However, it should be noted that the following specific embodiments only give specific operation examples of the present invention in an exemplary manner, but the protection scope of the present invention is not limited thereto. The protection scope of the present invention is only defined by the claims. Those skilled in the art can obviously think that various other improvements and replacements can be made to the embodiments described in the present invention within the protection scope defined by the claims of the present invention, and still be able to achieve the same technical effects and reach the ultimate technical purpose of the present invention.

[0042] Unless otherwise specified, each raw material in this specification is purchased through commercial channels. The raw materials and instruments used are as follows:

[0043] I. Instruments and reagents

[0044] Instruments:

[0045] Agilent 1260 high performance liquid chromatograph, BSA224S-CW electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), XS105 electronic balance (METTLER TOLEDO), KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), KMD adjustable temperature electric heating mantle, HH-8 constant temperature water bath (Jiangsu Zhongda Instrument Technology Co., Ltd.).

[0046] Chromatographic column:

[0047]

[0048] Drugs:

[0049] Glabridin reference substance (batch number: 111610-202209, National Institutes for Food and Drug Control, purity, 95.2%);

[0050] Narirutin reference substance (batch number: 103394, Jiangsu Yongjian Medical Technology Co., Ltd., purity, 98.0%);

[0051] Naringin reference substance (batch number: 110722-202116, National Institutes for Food and Drug Control, purity, 93.5%);

[0052] Neohesperidin reference substance (batch number: 111857-202305, National Institutes for Food and Drug Control, purity, 99.6%);

[0053] Hesperidin reference substance (batch number: 110721-202220, National Institutes for Food and Drug Control, purity, purity, 97.2%);

[0054] Ammonium glycyrrhizinate reference substance (batch number: 110731-202122, National Institutes for Food and Drug Control, purity, 94.4%).

[0055] The preparation method of Qingwei Bao'an Pills is as follows: Weigh 90 g of stir-fried Atractylodes macrocephala, 90 g of six-ingredient medicated leaven (stir-fried with bran), 90 g of tangerine peel, 90 g of Poria cocos, 90 g of amomum villosum, 90 g of green tangerine peel (processed with vinegar), 90 g of magnolia officinalis (processed with ginger), 90 g of stir-fried malt, 90 g of roasted licorice root, 90 g of areca nut, 90 g of stir-fried Fructus aurantii, 90 g of Fructus aurantii immaturus, 180 g of fermented rice for making liquor, and 360 g of stir-fried hawthorn fruit, a total of 14 kinds of decoction pieces. Crush them into fine powder, sieve, and mix evenly. Add 120 - 130 g of refined honey to every 100 g of the powder to make big honey pills or small honey pills, then you get it. Produce 3 batches of small honey pill samples of Qingwei Bao'an Pills by the same process, and the batch numbers are 2205001, 2206001, and 2206002 respectively.

[0056] Reagents:

[0057] Acetonitrile (Fisher Chemical) and phosphoric acid (Fisher Scientific) are of chromatographic purity, methanol is of analytical purity, and water is purified water of Watson.

[0058] II. Determination method

[0059] The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component quantification method provided by the present invention includes the following steps:

[0060] (1) Prepare mixed reference substance solutions with different concentrations, where the mixed reference substance solution is a mixed solution of six single reference substances including liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate.

[0061] During actual operation, first, use methanol as the solvent to prepare single reference substance solutions of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate respectively. Take the six single reference substance solutions and mix them to prepare a stock solution of the mixed reference substance solution; then, use the multiple dilution method to dilute the stock solution of the mixed reference substance solution to obtain mixed reference substance solutions with different concentrations.

[0062] In one embodiment, the specific preparation method of the six single reference substance solutions is as follows: Weigh appropriate amounts of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate reference substances accurately, dissolve them in methanol respectively to obtain a liquiritin reference substance solution with a concentration of 1.59784 mg / ml, a narirutin reference substance solution with a concentration of 2.04918 mg / ml, a naringin reference substance solution with a concentration of 2.14864 mg / ml, a neohesperidin reference substance solution with a concentration of 2.01186 mg / ml, a hesperidin reference substance solution with a concentration of 1.69517 mg / ml, and an ammonium glycyrrhizinate reference substance solution with a concentration of 1.23360 mg / ml.

[0063] The specific preparation method of the mixed reference substance solution stock solution is as follows: precisely pipette 1 ml of liquiritin reference substance solution, 2 ml of narirutin reference substance solution, 5 ml of naringin reference substance solution, 5 ml of neohesperidin reference substance solution, 5 ml of hesperidin reference substance solution, and 2 ml of ammonium glycyrrhizinate reference substance solution into a 20-ml volumetric flask respectively, mix well to prepare a mixed reference substance solution stock solution with concentrations of 79.89 μg / ml, 204.92 μg / ml, 537.16 μg / ml, 502.97 μg / ml, 423.79 μg / ml, and 123.36 μg / ml respectively, denoted as mixed reference substance solution ①.

[0064] Precisely pipette 1 ml of mixed reference substance solution ① into a 2-ml volumetric flask, add methanol to the scale, shake well, and denote it as mixed reference substance solution ②;

[0065] Precisely pipette 1 ml of mixed reference substance solution ② into a 2-ml volumetric flask, add methanol to the scale, shake well, and denote it as mixed reference substance solution ③;

[0066] Precisely pipette 1 ml of mixed reference substance solution ③ into a 2-ml volumetric flask, add methanol to the scale, shake well, and denote it as mixed reference substance solution ④;

[0067] Precisely pipette 1 ml of mixed reference substance solution ④ into a 2-ml volumetric flask, add methanol to the scale, shake well, and denote it as mixed reference substance solution ⑤.

[0068] (2) Determine the chromatographic conditions of the high-performance liquid chromatography analysis method:

[0069] Using octadecylsilane chemically bonded silica as the filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution. The detection wavelength is 275 nm from 0 to 55 min and 237 nm from 55 to 80 min, the flow rate is 1 ml / min, and the column temperature is 30 °C;

[0070] The specific conditions of the gradient elution are as follows:

[0071] From 0 to 48 min, mobile phase A: 12% - 23%, mobile phase B: 88% - 77%;

[0072] From 48 to 49 min, mobile phase A: 23% - 41%, mobile phase B: 77% - 59%;

[0073] From 49 to 60 min, mobile phase A: 41%, mobile phase B: 59%;

[0074] From 60 to 61 min, mobile phase A: 41% - 60%, mobile phase B: 59% - 40%;

[0075] 61 - 70 min, mobile phase A: 60%, mobile phase B: 40%;

[0076] 70 - 80 min, mobile phase A: 60% - 12%, mobile phase B: 40% - 88%.

[0077] (3)Precisely pipette 10 μl of each of the mixed reference substance solutions ① - ⑤ with different concentrations, inject them separately, and perform high - performance liquid chromatography analysis. Using the concentration of each reference substance as the abscissa and the corresponding chromatographic peak area as the ordinate, plot the standard curve to obtain the regression equations for the six components. The results are shown in Table 1; according to the regression equations, using neohesperidin as the internal reference substance, determine the relative correction factors of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate.

[0078] Table 1 Regression equations, correlation coefficients, and linear ranges of the six components

[0079] Component Regression equation Correlation coefficient r Linear range (μg / ml) Glabroside y = 20.476x + 6.0563 0.9998 4.9～79.89 Narirutin y = 13.281x + 27.548 0.9995 12.81～204.92 Naringin y = 15.935x + 42.973 0.9999 33.57～537.16 Neohesperidin y = 16.204x + 57.059 0.9999 31.44～502.97 Hesperidin y = 16.367x + 12.344 0.9999 26.49～423.79 Ammonium glycyrrhizinate y = 7.6928x + 6.7608 0.9998 7.71～123.36

[0080] The calculation formula for the relative correction factor is shown in formula (Ⅰ):

[0081]

[0082] Among them, W s is the concentration of the neohesperidin reference substance solution, A s is the peak area of the neohesperidin reference substance solution, W k is the concentration of the component to be measured, A k is the peak area of the component to be measured; s is neohesperidin, k is the component to be measured, f s is the correction factor of neohesperidin, f k is the correction factor of the component to be measured.

[0083] Among them, the correction factor of neohesperidin is 1.00. The relative correction factors of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate relative to neohesperidin are shown in Table 2.

[0084] Table 2 Relative correction factors

[0085]

[0086] The relative retention time of neohesperidin is 1.00. The relative retention times of the chromatographic peaks of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate relative to neohesperidin are 0.63, 0.80, 0.87, 0.92, and 1.32, respectively.

[0087] (4)Determination of the content of the component to be measured in the test solution:

[0088] Prepare the test solution of Qingwei Bao'an Pills and the reference solution of neohesperidin. Perform high-performance liquid chromatography analysis according to the chromatographic conditions in step (2). Locate the chromatographic peaks of each component to be measured based on the relative retention time. Using the concentration and peak area of the reference solution of neohesperidin as a reference, calculate the contents of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate in the test solution respectively according to the relative correction factor determined in step (3).

[0089] Among them, the preparation method of the test solution is as follows:

[0090] Crush Qingwei Bao'an Pills, accurately weigh 1.5 g, place them in a stoppered conical flask, add 30 ml of methanol, weigh, heat under reflux for 60 min, cool, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution. The effects of small honeyed pills and large honeyed pills of Qingwei Bao'an Pills are the same. The following only takes the small honeyed pills of Qingwei Bao'an Pills as an example for research.

[0091] The preparation method of the reference solution of neohesperidin is as follows: Dissolve neohesperidin in methanol to prepare a reference solution of neohesperidin with a concentration of 251.48 μg / ml.

[0092] III. Investigation on the preparation method of the test solution

[0093] 3.1 Investigation on different extraction solvents

[0094] Crush Qingwei Bao'an Pills (batch number 2205001), accurately weigh 1.5 g, place them in a stoppered conical flask, and add 30 ml of water, 20% methanol, 50% methanol, 80% methanol, methanol, and ethanol respectively. Heat under reflux for 60 min to prepare the test solution. Measure the peak area of the test solution and calculate the contents of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate. The results are shown in Table 3. Different extraction solvents have a great influence on each component. Through comprehensive analysis of the contents of each component to be measured, the extraction of each component to be measured is relatively more complete when using methanol for extraction.

[0095] Table 3 Determination results of the contents of six components under different extraction solvents

[0096]

[0097]

[0098] 3.2 Investigation on different extraction methods

[0099] The Qingwei Bao'an Pills (batch number 2205001) were minced, and 1.5 g was accurately weighed and placed in a stoppered conical flask. 30 ml of methanol was added, and two parallel samples were prepared. The test solutions were prepared by heating under reflux and ultrasonic treatment (power 250 W, frequency 40 kHz) for 60 min respectively, and then injected into the liquid chromatograph for determination. The contents of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate were calculated, and the results are shown in Table 4 and Figure 1 . Figure 1 The upper chromatogram in the middle is the chromatogram obtained by detecting the test solution extracted by heating under reflux, and the lower chromatogram is the chromatogram obtained by detecting the test solution extracted by ultrasonic extraction. It can be seen that the contents of each component to be detected are higher when extracted by heating under reflux.

[0100] Table 4 Determination results of the contents of six components under different extraction methods

[0101]

[0102] IV. Methodology investigation

[0103] 4.1 Specificity verification

[0104] The other 13 herbs except roasted licorice root were pulverized into fine powder, and according to the powder-honey ratio of 1∶1.2 - 1.3, refined honey was added to prepare a negative sample without liquiritin and ammonium glycyrrhizinate;

[0105] The other 10 herbs except green tangerine peel, dried tangerine peel, stir-fried immature bitter orange, and stir-fried bitter orange shell were pulverized into fine powder, and according to the powder-honey ratio of 1∶1.2 - 1.3, refined honey was added to prepare a negative sample without narirutin, naringin, neohesperidin, and hesperidin;

[0106] According to the preparation method of the test solution, a negative control solution was prepared; a mixed reference substance solution was used as a control; and methanol solvent was used as a blank solvent.

[0107] The test solution, negative control solution, mixed reference substance solution, and blank solvent were subjected to HPLC detection to obtain chromatograms, and the results are shown in Figure 2 . It can be seen that there are no chromatographic peaks in the negative control solution and the blank solvent at the retention times of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate, proving that the HPLC detection method of the present invention has good specificity.

[0108] 4.2 Repeatability test

[0109] Six test solution samples were prepared in parallel and detected by HPLC. Using the mixed reference solution as a control, the RSD was calculated, and the results are shown in Table 5. The RSD of liquiritin content was 1.70%, the RSD of narirutin content was 1.41%, the RSD of naringin content was 1.45%, the RSD of neohesperidin content was 1.43%, the RSD of hesperidin content was 2.14%, and the RSD of ammonium glycyrrhizinate content was 1.95%. It was proved that the repeatability of the HPLC detection conditions of the present invention was good and met the analysis requirements.

[0110] Table 5 Results of Repeatability Test

[0111]

[0112] 4.3 Intermediate Precision

[0113] Six test solution samples were prepared in parallel. Different analysts used another high-performance liquid chromatograph at different times to conduct a repeatability test. Using the mixed reference solution as a control, the RSD was calculated, and the results are shown in Table 6. The RSD of liquiritin content determined by different instruments in Qingwei Bao'an Pills was 4.40%, the RSD of narirutin content was 2.08%, the RSD of naringin content was 1.79%, the RSD of neohesperidin content was 1.54%, the RSD of hesperidin content was 1.97%, and the RSD of ammonium glycyrrhizinate content was 2.00%, meeting the analysis requirements.

[0114] Table 6 Results of Intermediate Precision Test

[0115]

[0116]

[0117] 4.4 Recovery

[0118] Six portions of Qingwei Bao'an Pills with known content were taken, shredded, and accurately weighed 1.5 g. They were placed in a stoppered conical flask, and appropriate amounts of reference solutions of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate were accurately added respectively. The test solution was prepared according to the preparation method of the test solution. Using the mixed reference solution as a control, the results are shown in Tables 7 - 12. The recovery rate of liquiritin was between 94.76% and 101.88%, the recovery rate of narirutin was between 94.54% and 101.46%, the recovery rate of naringin was between 93.14% and 98.51%, the recovery rate of neohesperidin was between 96.51% and 101.56%, the recovery rate of hesperidin was between 94.69% and 100.77%, and the recovery rate of ammonium glycyrrhizinate was between 93.42% and 99.46%. It was proved that the HPLC detection conditions of the present invention met the analysis requirements.

[0119] The calculation formula for the recovery rate is as follows:

[0120] Recovery rate (%) = [(Measured value - Amount of the analyte contained in the test sample) / Amount of the reference substance added] × 100%

[0121] Table 7 Recovery of liquiritin added

[0122]

[0123] Table 8 Recovery of narirutin added

[0124]

[0125]

[0126] Table 9 Recovery of naringin added

[0127]

[0128] Table 10 Recovery of neohesperidin added

[0129]

[0130] Table 11 Recovery of hesperidin added

[0131]

[0132] Table 12 Recovery of ammonium glycyrrhizinate added

[0133]

[0134]

[0135] 4.5 Stability

[0136] The prepared test sample solution was injected once at 0 h, 2 h, 6 h, 12 h, and 24 h respectively. Using the mixed reference substance solution as a control, the content of the analyte in the sample was determined, and the RSD was calculated. The results are shown in Table 13. Within 24 hours, the RSD of the liquiritin content was 1.74%, the RSD of the narirutin content was 1.54%, the RSD of the naringin content was 0.88%, the RSD of the neohesperidin content was 0.54%, the RSD of the hesperidin content was 0.94%, and the RSD of the ammonium glycyrrhizinate content was 1.43%. It was proved that the test sample solution had good stability and met the analysis requirements.

[0137] Table 13 Results of the stability test

[0138]

[0139] V. Calculation of relative correction factor and investigation of robustness

[0140] 5.1 Influence of Different Instruments and Columns on Relative Correction Factors

[0141] The relative correction factors were determined using different instruments and different columns respectively. A mixed reference solution was used as the control, and the RSD was calculated. The results are shown in Table 14. The RSD values of the measured relative correction factors were between 1.00% and 2.31%, indicating good reproducibility of the relative correction factors of the components to be measured among different instruments and different columns. Figure 3 Figure 5 shows the chromatograms obtained using different columns.

[0142] Table 14 Influence of Different Instruments and Different Columns on Relative Correction Factors of Components to be Measured

[0143]

[0144]

[0145] 5.2 Different Flow Rates

[0146] The relative correction factors were determined using different flow rates respectively. The other detection conditions of HPLC were the same as those under "II. Determination Method". A mixed reference solution was used as the control, and the RSD was calculated. The results are shown in Table 15. The RSD values of the measured relative correction factors were between 0.64% and 1.21%, indicating good reproducibility of the relative correction factors of the components to be measured among different flow rates.

[0147] Table 15 Influence of Different Flow Rates on Relative Correction Factors of Components to be Measured

[0148]

[0149] 5.3 Different Column Temperatures

[0150] The relative correction factors were determined using different column temperatures respectively. The other detection conditions of HPLC were the same as those under "II. Determination Method". A mixed reference solution was used as the control, and the RSD was calculated. The results are shown in Table 16. The RSD values of the measured relative correction factors were between 0.51% and 1.51%, indicating good reproducibility of the relative correction factors of the components to be measured among different column temperatures.

[0151] Table 16 Influence of Different Column Temperatures on Relative Correction Factors of Components to be Measured

[0152]

[0153] VI. Location of Chromatographic Peaks of Components to be Measured:

[0154] Calculate the relative retention time (r) and retention time difference (ΔtR) of liquiritin, narirutin, naringin, hesperidin, ammonium glycyrrhizinate reference substance solutions and the internal reference neohesperidin. The HPLC detection conditions are the same as those under "II. Determination Method", and investigate their reproducibility between different instruments and different chromatographic columns, calculate the RSD, and the results are shown in Tables 17 - 18. The relative retention time fluctuations of each component to be measured are small between different chromatographic columns and different instruments, and the RSD values are all <2%. Finally, select the relative retention times of each component to be measured in different instruments and different chromatographic columns as the peak positioning basis.

[0155] Table 17 Relative retention times of each component to be measured in different instruments and different chromatographic columns

[0156]

[0157]

[0158] Table 18 Retention time differences of each component to be measured in different instruments and different chromatographic columns

[0159]

[0160] VII. Comparison of the results of multi - component determination by single - point calibration and external standard method for content determination

[0161] Respectively take the small honey pills of Qingwei Bao'an Pills 2205001, 2206001, and 2206002 as test samples, prepare test sample solutions, and according to the HPLC test conditions under "II. Determination Method".

[0162] Multi - component determination by single - point calibration (QAMS): Prepare a reference substance solution of neohesperidin with a concentration of 251.48 μg / ml, and calculate the contents of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate according to the average value of the relative correction factors calculated in Table 2.

[0163] External standard method (ESM): Use a mixed reference substance solution as a control, inject 10 μl of the mixed reference substance solution and the test sample solution respectively, and calculate the contents of liquiritin, narirutin, naringin, hesperidin, ammonium glycyrrhizinate and neohesperidin.

[0164] The contents of the six components to be measured in the test sample solutions detected by the above two methods are shown in Table 19. After t - test comparison of the contents calculated by the external standard method and the multi - component determination by single - point calibration method, P is much greater than 0.05, indicating that there is no significant difference in the contents measured by the two methods, and the relative error between the two groups of contents is <3%, indicating that the multi - component determination by single - point calibration method established in the present invention has good accuracy and can be used for the content determination of Qingwei Bao'an Pills.

[0165] Table 19 Contents of six components to be measured determined by multi - component determination by single - point calibration and external standard method

[0166]

[0167] RE represents the relative error.

[0168] The above specific embodiments shall not be construed as limiting the scope of the present invention. For those skilled in the art of this technology, any alternative improvements or transformations made to the embodiments of the present invention fall within the scope of protection of the present invention.

[0169] Those not detailed in the present invention are all well-known technologies to those skilled in the art of this technology.

Claims

1. A method for determining the contents of six components in Qingwei Bao'an Pills by the method of multi-component determination with single marker, characterized in that, It includes the following steps: (1) Prepare mixed reference substance solutions with different concentrations, where the mixed reference substance solution is a mixed solution of six single reference substances, namely liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate; (2) Determine the chromatographic conditions for high performance liquid chromatography analysis: Using octadecylsilane chemically bonded silica as the filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution. The detection wavelength is 275 nm for 0 - 55 min and 237 nm for 55 - 80 min, the flow rate is 1 ml / min, and the column temperature is 30 °C; The specific conditions for gradient elution are: 0 - 48 min, mobile phase A: 12% - 23%, mobile phase B: 88% - 77%; 48 - 49 min, mobile phase A: 23% - 41%, mobile phase B: 77% - 59%; 49 - 60 min, mobile phase A: 41%, mobile phase B: 59%; 60 - 61 min, mobile phase A: 41% - 60%, mobile phase B: 59% - 40%; 61 - 70 min, mobile phase A: 60%, mobile phase B: 40%; 70 - 80 min, mobile phase A: 60% - 12%, mobile phase B: 40% - 88%; (3) Take mixed reference substance solutions with different concentrations, inject samples respectively, and perform high performance liquid chromatography analysis. Using neohesperidin as the internal reference substance, determine the relative correction factors of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate; (4) Determination of the content of the components to be measured in the test solution: Prepare the test solution of Qingwei Bao'an Pills and the reference solution of neohesperidin, perform high performance liquid chromatography analysis according to the chromatographic conditions in step (2), locate the chromatographic peaks of each component to be measured according to the relative retention time, take the concentration and peak area of the reference solution of neohesperidin as the reference, and calculate the contents of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate in the test solution respectively according to the relative correction factors determined in step (3); The preparation method of the test solution is: Crush Qingwei Bao'an Pills, accurately weigh 1.5 g, place it in a stoppered conical flask, add 30 ml of methanol, weigh, heat under reflux for 60 min, cool, make up the lost weight with methanol, shake well, filter, and take the continuous filtrate to obtain the test solution.

2. The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component determination with single marker method according to claim 1, wherein, (1) Specifically: Using methanol as the solvent, prepare single reference substance solutions of six reference substances, namely liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate respectively. Take the six single reference substance solutions and mix them to prepare a stock solution of the mixed reference substance solution; Dilute the stock solution of the mixed reference substance solution by the multiple dilution method to obtain mixed reference substance solutions with different concentrations.

3. The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component assay with single marker according to claim 2, wherein In step (1), the preparation methods of the six single reference substance solutions of liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate are: Six reference substances, namely liquiritin, narirutin, naringin, hesperidin, neohesperidin, and ammonium glycyrrhizinate, were separately dissolved in methanol to prepare six single-reference substance solutions with concentrations of 1.59784 mg / ml, 2.04918 mg / ml, 2.14864 mg / ml, 2.01186 mg / ml, 1.69517 mg / ml, and 1.23360 mg / ml, respectively.

4. The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component assay with single marker according to claim 1, wherein, In step (3), the relative retention time of neohesperidin was 1.00, and the relative retention times of the chromatographic peaks of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate relative to neohesperidin were 0.63, 0.80, 0.87, 0.92, and 1.32, respectively.

5. The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component simultaneous determination method according to claim 1, wherein, In step (3), the correction factor of neohesperidin was 1.00, and the relative correction factors of liquiritin, narirutin, naringin, hesperidin, and ammonium glycyrrhizinate relative to neohesperidin were 0.80, 1.21, 1.02, 1.01, and 2.12, respectively.

6. The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component simultaneous determination method according to claim 1, wherein In step (3), the calculation formula for the relative correction factor is shown in formula (Ⅰ): Among them, W s is the concentration of the neohesperidin reference solution, and A s is the peak area of the neohesperidin reference solution. W k is the concentration of the component to be measured, and A k is the peak area of the component to be measured; s is neohesperidin, k is the component to be measured, and f s is the correction factor of neohesperidin, and f k is the correction factor of the component to be measured.

7. The method for determining the contents of six components in Qingwei Bao'an Pills by the multi-component determination with single marker method according to claim 1, wherein In step (4), the preparation method of the neohesperidin reference substance solution was as follows: Neohesperidin was dissolved in methanol to prepare a neohesperidin reference substance solution with a concentration of 251.48 μg / ml.

Images