HPLC content detection method for reference sample of traditional Chinese medicine preparation

Abstract

The invention relates to an HPLC (High Performance Liquid Chromatography) content detection method for a reference sample of a traditional Chinese medicine preparation, which comprises the following steps: 1) preparation of a reference substance solution: taking a kukoamine B reference substance, a mulberroside A reference substance and a liquiritin reference substance, and adding a solvent to prepare the reference substance solution; 2) preparation of a test solution: taking a Bai San reference sample, and adding a solvent to prepare the test solution; and 3) determination: taking the reference substance solution and the test solution, injecting the reference substance solution and the test solution into a high performance liquid chromatograph to obtain a chromatogram, and calculating the contents of kukoamine B, mulberroside A and liquiritin according to the chromatogram. According to the method, gradient elution is adopted, acetonitrile is taken as a mobile phase A, and a phosphoric acid solution is taken as a mobile phase B.

Description

Technical field:

[0001] The invention relates to a method for detecting effective ingredients in a traditional Chinese medicine preparation, and in particular to a HPLC content detection method for a Xiebaisan reference sample. Background technology:

[0002] Xiebaisan comes from "Xiaoer Yaozheng Zhijue" and was created by Qian Yi, a doctor in the Song Dynasty. It is composed of mulberry bark, radix rehmanniae, licorice and polished rice. It has the effects of clearing lung heat, relieving asthma and relieving cough. It is a commonly used prescription for treating lung heat and cough. It has been listed in the "Ancient Classic Prescription Catalog (First Batch)" by the State Administration of Traditional Chinese Medicine. In the prescription, mulberry bark is sweet and cold and enters the lungs, clears the lungs and relieves heat, relieves asthma and relieves cough. Although it clears the lungs, it does not hurt the lungs, so it is the main medicine; radix rehmanniae is sweet and cold in nature, clears the stagnant fire in the lungs, and has the function of nourishing yin, so it is the minister medicine; licorice and polished rice nourish the stomach and harmonize the middle, cultivate the soil to produce gold to support the lung qi, and are sweet and moderate, which can make the cold not hurt the stomach, and can reconcile all the medicines, and are adjuvant medicines. All the medicines are used together to play the effects of clearing the lungs, clearing heat, relieving cough and relieving asthma. It mainly treats cough caused by stagnant fire and lung heat in the lungs. Modern research shows that the main effects of Xiebaisan are antipyretic, anti-inflammatory, anti-pathogenic microorganism, expectorant and antitussive.

[0003] Xiebaisan is one of the ancient classic prescriptions encouraged by the country for development. Practice has shown that its effectiveness and safety are beyond doubt. As for quality controllability, the key lies in how to establish quality standards based on pharmacological effects and strictly control the quality during the preparation process. However, the ingredients selected for testing Xiebaisan by existing technologies are not representative enough.

[0004] The invention patent application with application number "201910812642.4" provides a method for determining the HPLC characteristic spectrum of the Xiebaisan reference sample, and establishes the characteristic spectrum of the Xiebaisan reference sample. Although this method can comprehensively and accurately evaluate the overall and intrinsic quality of the Xiebaisan reference sample, it does not study the content of the reference sample, especially the content of the effective active ingredients.

[0005] The invention patent application with application number "202010666660.9" provides a UPLC content determination method for Xiebaisan granules. Although this method can accurately evaluate the content of morphine and morusin A in Xiebaisan granules, licorice has not been studied. At the same time, this method uses the UPLC method. The UPLC instrument is expensive and is not widely used among manufacturers. From production cost to maintenance cost, it is relatively high and is not as widely used as HPLC instruments.

[0006] Based on this, a low-cost, efficient and simultaneous detection method for the content of multiple active ingredients in the Xiebaisan formula was developed, which is of great significance to the quality control of Xiebaisan. Summary of the invention:

[0007] The present invention has found a low-cost, high-efficiency HPLC content detection method for a reference sample of Xiebaisan, a classic Chinese medicine prescription, which can simultaneously detect the contents of multiple indicators of medicinal ingredients in the Xiebaisan prescription.

[0008] The invention provides a HPLC content detection method of cefotaxime, morusin A and liquiritin in a Xiebaisan reference sample, taking cefotaxime contained in the bark of Lycium bark, morusin A contained in the bark of Morus alba and liquiritin contained in Licorice in the Xiebaisan formula as detection objects, and provides a content detection method.

[0009] According to the monarch, minister, assistant and envoy in the Chinese medicine prescription, the relevant medicinal flavors in the prescription were analyzed in combination with the efficacy of Xiebai San. Morus alba bark is the dried root bark of the mulberry plant of the Moraceae family, which is a cough and phlegm-relieving drug. It has the effects of purging heat and relieving asthma, promoting diuresis and reducing swelling, and is used to treat lung heat cough and asthma, edema, oliguria, and facial edema. Morus alba bark is cold in nature and has the effects of clearing the liver and lowering blood pressure. Morus alba glycoside A is the main diphenylethylene glycoside active ingredient in Morus alba bark, which has a strong antitussive effect and a certain degree of asthma-relieving effect, which is consistent with the main function of Xiebai San and is the main active ingredient in Morus alba bark; (2) Lycium bark is the dried root bark of Lycium barbarum or Lycium ningxiaensis of the Solanaceae family, which is included in the 2020 edition of the Chinese Pharmacopoeia. It is cold in nature, sweet in taste, and enters the lung, liver, and kidney meridians. It has the effects of cooling blood and removing steam, clearing the lungs and reducing fire, and is mainly used for yin deficiency hot flashes, bone steaming and night sweats, lung heat cough, hemoptysis, epistaxis, and internal heat and thirst. Modern pharmacological studies have found that the bark of Chinese wolfberry also has the effects of lowering blood sugar and blood pressure and antibacterial effects. Lycopodiol is a major active ingredient in the Chinese medicine bark of Chinese wolfberry. (3) Licorice is the dried root and rhizome of the leguminous plant Licorice, which is sweet and neutral in nature. It enters the heart, lung, spleen, and stomach meridians. It has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving pain, and harmonizing various medicines. It is used for spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough with sputum, abdominal and limb cramps and pain, carbuncle and sore, and relieves drug toxicity and potency. Liquiritin is a major active ingredient in the Chinese medicine licorice.

[0010] Therefore, morusin A in Morus alba bark, lycoside B in Lycium bark, and liquiritin in Licorice were studied as content detection items, and an HPLC content detection method for Xiebaisan reference sample was provided. The detection method has the following steps:

[0011] 1) Preparation of reference solution: Take tricholoma ethopropin, mulberry glycoside A and liquiritin reference substances, add solvent and prepare reference solution.

[0012] 2) Preparation of test solution: Take Xiebaisan reference sample, add solvent, and prepare test solution.

[0013] 3) Determination: Take the reference solution and the test solution, inject them into the high performance liquid chromatograph to obtain the chromatogram, and calculate the contents of lycoside A, morusin A and liquiritin according to the chromatogram.

[0014] Wherein, the solvent is selected from acetic acid, methanol or a mixture thereof.

[0015] Preferably, the solvent is a mixture of acetic acid and methanol.

[0016] Most preferably, the solvent is a mixture of 0.25% acetic acid and 30% methanol, wherein the percentages are by weight.

[0017] Preferably, the method of the present invention adopts high performance liquid chromatography, and the chromatographic column is filled with octadecylsilane bonded silica gel ( AQ-C18, 4.6mm×250mm, 5μm)

[0018] Preferably, the method of the present invention adopts gradient elution.

[0019] Preferably, in the method of the present invention, acetonitrile is used as mobile phase A and phosphoric acid solution is used as mobile phase B.

[0020] The most preferred method of the present invention comprises the following steps:

[0021] 1) Preparation of reference solution: Take appropriate amount of cerebroside A reference substance, mulberry glycoside A reference substance and liquiritin reference substance, weigh accurately, add 30% methanol solution containing 0.25% acetic acid to prepare a mixed solution containing 50 μg of cerebroside A, 50 μg of mulberry glycoside A and 10 μg of liquiritin per 1 ml.

[0022] 2) Preparation of test solution: Take about 0.1 g of reference sample powder, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of 30% methanol solution containing 0.25% acetic acid, stopper, weigh, ultrasonically treat for 45 minutes, cool, weigh again, make up the lost weight with 30% methanol solution containing 0.25% acetic acid, shake well, filter, and take the filtrate.

[0023] 3) Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph to obtain the chromatogram, and calculate the contents of scopolamine, morusin A and liquiritin based on the chromatogram.

[0024] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler ( AQ-C18, 4.6mm×250mm, 5μm); acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; flow rate 1ml per minute; column temperature 30℃; detection wavelength 280nm. The theoretical plate number calculated based on the mulberry glycoside A peak should not be less than 2000.

[0025] Time (min) Mobile phase A (%) Mobile phase B (%) 0～9 8→10 92→90 9～15 10→13 90→87 15～23 13→33 87→67 23～35 33 67

[0026] The reagents, samples and standard reference substances used in the present invention can all be purchased on the market.

[0027] The Xiebaisan reference sample of the present invention can be prepared by the following method:

[0028] Take appropriate amounts of fried mulberry bark, roasted rehmannia root bark, and fried licorice, and grind them into the coarsest powder. Weigh 2.95g of fried mulberry bark, 2.95g of roasted rehmannia root bark, 0.30g of fried licorice, and 2.0g of polished rice slices. Add 300ml of water, cover, and soak for 30 minutes. Boil over high heat (500W) and then turn to low heat (300W), decoct for about 19 minutes, filter through a 300-mesh filter cloth while hot, and concentrate under reduced pressure at 65℃ to a solid-liquid ratio of 1:1.5-1:2. Freeze-dry and grind to obtain the product.

[0029] The HPLC content detection method of ethoxylated tricholoma, morusin A and liquiritin in a Xiebaisan reference sample claimed in the present invention adopts an HPLC detection method, and carries out necessary linear relationships, reproducibility and stability such as chromatographic conditions and system suitability tests, preparation of reference solution and preparation of test solution.

[0030] The present invention further verifies the method of the present invention, including specificity, injection precision, linearity, sample stability, repeatability, intermediate precision, and accuracy durability (including investigations of different chromatographic columns, different flow rates, different column temperatures, and different wavelengths) to ensure that the detection method is complete and effective.

[0031] The specificity of the method was verified by examining whether the blank interfered with the determination of the main component.

[0032] The precision of the instrument was investigated by continuous sampling.

[0033] The aim was to investigate whether the peak areas of tricholoma ethopropin, mulberry glycoside A and liquiritin were linearly correlated with their concentrations within the designed range.

[0034] Investigate the stability of the test solution over a certain period of time.

[0035] Six samples of the test solution were measured repeatedly to investigate the repeatability of the method.

[0036] In the same laboratory, different times, different analysts, and different equipment were used to examine the intermediate precision.

[0037] The accuracy of this method was investigated by adding a control substance of known concentration to the test substance.

[0038] Investigate the robustness of the experimental method under slight changes in measurement conditions.

[0039] Exclusive

[0040] plan

[0041] (1) Blank solvent interference test: Take a 30% methanol solution containing 0.25% acetic acid and inject it according to the proposed method.

[0042] (2) Reference substance positioning test: Take a mixed reference substance solution of lemborexanthin, morusin A and liquiritin, and inject it according to the proposed method for inspection.

[0043] (3) Test sample: Take the method validation batch sample - Xiebaisan lyophilized powder (XBS(J)-GG240101), prepare the test sample solution, and perform sampling inspection according to the proposed method.

[0044] Acceptance criteria

[0045] (1) The blank solvent should have no interference at each chromatographic peak.

[0046] (2) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0047] (3) The separation degree between the adjacent peaks of lycopodiol, morusin A and liquiritin detected in the test solution shall not be less than 1.5.

[0048] (4) The entire analytical test process (including solution preparation, instrument parameters, result calculation, etc.) must be consistent with the proposed analytical method.

[0049] Results Figure 1 Investigation on the specificity of Xiebaisan standard sample content determination

[0050] The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there was no interference from the blank solution, indicating that the method had good specificity.

[0051] Injection precision

[0052] plan

[0053] Take lemborexanthin, mulberry glycoside A and liquiritin to prepare a mixed reference solution according to the proposed method, and perform continuous sampling and determination according to the proposed analytical method.

[0054] Acceptance criteria

[0055] (1) The RSD of the peak areas of tricholoma ethyl, morusin A and liquiritin shall not be greater than 2.0%, and the RSD of the retention time shall not be greater than 1.0%.

[0056] (2) The theoretical plate numbers of the tricholoma glycoside and liquiritin peaks shall not be less than 5000, and the theoretical plate number of the mulberry glycoside A peak shall not be less than 8000.

[0057] result

[0058] Table 1. Results of the precision test of Xiebaisan standard sample content determination

[0059]

[0060]

[0061] The results showed that the RSD of retention time of lycopodipine was 0.8%, and the RSD of peak area was 0.5%; the RSD of retention time of mulberry glycoside A was 0.1%, and the RSD of peak area was 0.1%; the RSD of retention time of liquiritin was 0.1%, and the RSD of peak area was 0.1%. The RSD of retention time of each component was less than 1.0%, and the RSD of peak area was less than 1.0%. The theoretical plate number of lycopodipine and liquiritin was greater than 5000, and the theoretical plate number of mulberry glycoside A was greater than 8000. This shows that the instrument has good precision.

[0062] Linear

[0063] plan

[0064] Take appropriate amount of cerebroside ethanol, mulberry glycoside A and liquiritin reference substances, weigh them accurately, prepare them according to 200% of the concentration of the reference substance solution, dissolve them in 30% methanol solution containing 0.25% acetic acid and quantitatively dilute them step by step to prepare 100%, 50%, 25%, and 10% linear test solutions. According to the proposed analysis method, prepare 200%, 100%, 50%, 25%, and 10% linear solutions respectively, and perform linear regression calculation with the concentration (μg / ml) of cerebroside ethanol, mulberry glycoside A and liquiritin as the horizontal coordinate and the peak area as the vertical coordinate.

[0065] Acceptance criteria

[0066] (1) Draw a linear graph of concentration-peak area, and there is a linear relationship.

[0067] (2) Report the linear equation, residual sum of squares, and the linear correlation coefficient (r) must not be less than 0.999.

[0068] result

[0069] Table 2. Results of linearity test for Xiebaisan standard sample content determination

[0070]

[0071]

[0072] The standard curve for determination of the content of lycopodine in Xiebaisan reference sample is shown in Figure 2

[0073] The standard curve for the determination of mulberry glycoside A in Xiebaisan standard sample is shown in Figure 3

[0074] The standard curve for determination of liquiritin content in Xiebaisan reference sample is shown in Figure 4

[0075] The experimental results show that in the range of 5.106-102.1 μg / ml, the linear equation of tricholoma ethyl is: y=0.0613x-0.0524, the correlation coefficient r=1.0000, and the linear relationship is good; in the range of 4.708-94.13 μg / ml, the linear equation of mulberry glycoside A is: y=0.2495x+0.0182, the correlation coefficient r=0.9999, and the linear relationship is good; in the range of 0.9636-19.27 μg / ml, the linear equation of liquiritin is: y=0.2943x+0.0164, the correlation coefficient r=0.9999, and the linear relationship is good.

[0076] Sample stability

[0077] plan

[0078] Take the method validation batch sample (XBS(J)-GG240101), prepare the test solution according to the proposed method, and inject the sample at 0h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h at room temperature to examine the stability of the test solution. If the stability at room temperature does not meet the acceptance criteria, examine the stability of the sample under refrigeration (2-10°C).

[0079] Acceptance criteria

[0080] The detection of chromatographic peaks of lycopodiol, mulberry glycoside A and liquiritin in the test solution should remain stable. Compared with 0h, the peak area RAD of lycopodiol, mulberry glycoside A and liquiritin should not be greater than 2.0%.

[0081] result

[0082] Table 3. Results of the stability study of the Xiebaisan standard sample content determination samples

[0083]

[0084]

[0085] The results showed that the RAD of the peak areas of lemborexanthin, mulberry glycoside A and liquiritin within 48 hours were all less than 1.0%, indicating that the test solution was stable within 48 hours.

[0086] Repeatability

[0087] plan

[0088] Without taking the sample from the methodology validation batch (XBS(J)-GG240101), prepare 6 test sample solutions in parallel according to the proposed method, and inject and measure according to the proposed analytical method.

[0089] Acceptance criteria

[0090] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0091] (2) The detection of chromatographic peaks of lycopodiol, morusin A and liquiritin in the test solution should be consistent. The RSD of the detection amount of lycopodiol and morusin A should not be greater than 1.5%, and the RSD of the detection amount of liquiritin should not be greater than 2.0%.

[0092] result

[0093] Table 4. Results of the repeatability test of Xiebaisan standard sample content determination

[0094] Serial number Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) Repeatability -1 27.603 21.841 2.640 Repeatability -2 27.072 21.752 2.635 Repeatability - 3 27.702 21.768 2.632 Repeatability - 4 27.962 21.985 2.661 Repeatability -5 27.639 21.763 2.633 Repeatability - 6 27.013 21.692 2.633 RSD(%) 1.4 0.5 0.5

[0095] The results showed that the RSDs of the contents of tricholoma ethainoside in the six test solutions prepared from the same batch of samples were all less than 1.5%, and the RSDs of the contents of mulberry glycoside A and liquiritin were less than 1.0%, indicating that the analytical method had good repeatability.

[0096] Intermediate precision

[0097] plan

[0098] Take the method validation batch sample (XBS(J)-GG240101), prepare 6 test sample solutions in parallel according to the proposed method, and perform the analysis according to the proposed analytical method.

[0099] Acceptance criteria

[0100] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0101] (2) The detection of chromatographic peaks of lycopodiol, morusin A and liquiritin in the test solution should be consistent. The RSD of the detection amount of lycopodiol and morusin A should not be greater than 1.5%, and the RSD of the detection amount of liquiritin should not be greater than 2.0%.

[0102] result

[0103] Table 5. Results of the intermediate precision test of Xiebaisan standard sample content determination

[0104]

[0105] Remark:

[0106] Personnel 1, Instrument: High Performance Liquid Chromatograph, Thermo Vanquish, No.: JS-01-164; Experiment Date: 2024.04.03 Personnel 2, Instrument: High Performance Liquid Chromatograph, Waters Arc, No.: JS-01-170; Experiment Date: 2024.04.06

[0107] The results showed that different analysts used different chromatographs to measure samples on different dates, for a total of 12 samples, the RSDs of the contents of tricholoma ethopropene and morusin A were no more than 1.5%, and the RSD of the content of liquiritin was no more than 1.0%. The intermediate precision of this analysis method was good.

[0108] Accuracy

[0109] plan

[0110] 0.05 g of the method validation batch sample (XBS(J)-GG240101) was accurately weighed, and then a certain amount of the reference substance with a known content of the tested component was accurately added, and 6 parallels were prepared according to 100% of the measured concentration. According to the proposed analytical method, continuous sampling was performed, and the accuracy of the method was evaluated by the recovery rate of lycoside, morusin A and liquiritin.

[0111] Acceptance criteria

[0112] The recovery rates of tricholoma ethopropin and morusin A should be between 95% and 102%, and the RSD should not be greater than 1.5%. The recovery rate of liquiritin should be between 92% and 105%, and the RSD should not be greater than 2.0%.

[0113] result

[0114] Table 6. Results of the recovery rate study of Xiebaisan standard sample content determination (digoupietrin)

[0115]

[0116] Table 7. Results of the recovery rate of Xiebaisan standard sample content determination (mulberry glycoside A)

[0117]

[0118] Table 8. Results of the recovery rate study of Xiebaisan standard sample content determination (liquiritin)

[0119]

[0120] The results showed that the recovery rate of tricholoma ethyl was in the range of 95.3% to 98.9%, with an average recovery rate of 96.5% and an RSD of 1.4%; the recovery rate of mulberry glycoside A was in the range of 97.8% to 100.0%, with an average recovery rate of 99.3% and an RSD of 1.0%; the recovery rate of liquiritin was in the range of 98.2% to 100.8%, with an average recovery rate of 100.1% and an RSD of 1.0%, indicating that the method has good accuracy.

[0121] Durability

[0122] plan

[0123] Take the method validation batch sample (XBS(J)-GG240101) and prepare the test solution according to the proposed method. After fine-tuning according to the table below, inject the sample according to the proposed chromatographic conditions:

[0124] Table 9. Variation degree of each parameter in durability test

[0125]

[0126] Acceptance criteria

[0127] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0128] (2) The detection of chromatographic peaks of lycopodiol, mulberry glycoside A and liquiritin in the test solution should be consistent, and the RSD of the detection amounts of lycopodiol, mulberry glycoside A and liquiritin should not be greater than 2.0%.

[0129] result

[0130] (1) Investigation of different column temperatures

[0131] Table 10. Results of investigation at different column temperatures 1

[0132] Column temperature (℃) Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) 25 28.040 21.942 2.685 30 27.603 21.841 2.640 35 30.339 21.915 2.588 RSD(%) 5.2 0.3 1.9

[0133] The results showed that when the column temperature was 35°C, the content of lycopodiol increased, so the temperature range was reduced to 28-32°C and the experiment was repeated, and the original conditions were also re-run. The results are as follows:

[0134] Table 11. Results of investigation at different column temperatures 2

[0135] Column temperature (℃) Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) 28 27.724 23.161 2.631 30 28.001 23.175 2.635 32 29.469 23.284 2.641 RSD(%) 3.4 0.3 0.2

[0136] The results showed that the RSD value of the scutellariae content at different column temperatures (±2°C) was 3.4%, which did not meet the standard, indicating that the analytical method had poor durability within the column temperature range of ±2°C, so the column temperature needed to be fixed at 30°C during testing.

[0137] (2) Investigation of different flow rates

[0138] Compare the effects of different flow rates of 0.9ml / min, 1.0ml / min, and 1.1ml / min on the determination of the contents of lycopodiol, mulberry glycoside A, and licorice in the Xiebaisan benchmark sample. Take the "repeatability test" test solution as the test solution for durability test, set the flow rates to 0.9ml / min, 1.0ml / min, and 1.1ml / min, respectively, and the other conditions follow the proposed chromatographic conditions to determine the contents of lycopodiol, mulberry glycoside A, and licorice at different flow rates.

[0139] Table 12. Results of investigation at different flow rates

[0140] Flow rate (ml / min) Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) 0.9 27.674 21.400 2.711 1.0 27.603 21.841 2.640 1.1 27.921 21.412 2.704 RSD(%) 0.7 1.2 1.5

[0141] The results showed that the RSD values ​​of the contents of scopolamine, morusin A and liquiritin at different flow rates were all less than 1.5%, indicating that the analytical method had good durability within the range of slight changes in flow rate.

[0142] (3) Investigation at different wavelengths

[0143] Compare the effects of different wavelengths of 275nm, 280nm, and 285nm on the determination of the content of cerebroside ethyne, mulberry glycoside A, and licorice glycoside in the Xiebaisan reference sample. Take the "repeatability study" test solution as the test solution for durability study, set the wavelengths to 275nm, 280nm, and 285nm, and determine the content of cerebroside ethyne, mulberry glycoside A, and licorice glycoside at different wavelengths according to the chromatographic conditions.

[0144] Table 13. Results of investigation at different wavelengths

[0145] Wavelength (nm) Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) 275 27.887 21.790 2.626 280 27.603 21.841 2.640 285 27.929 21.785 2.643 RSD(%) 0.7 0.2 0.4

[0146] The results showed that the RSD values ​​of the contents of scoparia acetylcholine, morusin A and liquiritin at different wavelengths were all less than 1.0%, indicating that the analytical method had good durability within the range of slight wavelength changes.

[0147] (4) Investigation of different chromatographic columns

[0148] Column 1: Welch AQ-C18, 4.6 mm × 250 mm, 5 um, A-106 (lot number: 60230502488), column 2: Agilent 5TC-C18, 4.6 mm × 250 mm, 5 um, A-109 (lot number: SN 722594).

[0149] Table 14. Results of investigation on different chromatographic columns 1

[0150] Chromatographic columns Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) Column 1 28.667 21.874 2.626 Column 2 29.701 22.454 2.704 RAD(%) 1.8 1.3 1.5

[0151] The results show that after replacing the chromatographic columns of different brands, the RAD values ​​of the content of each component are all less than 2.0%. We plan to re-examine the chromatographic columns of the same brand with different batch numbers after purchasing them, and re-run the original chromatographic columns. The results are as follows:

[0152] Column 3: Welch AQ-C18, 4.6mm×250mm, 5um, A-127 (Batch number: SN602312002103).

[0153] Table 15. Results of investigation on different chromatographic columns 2

[0154] Chromatographic columns Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) Column 1 27.065 20.844 2.592 Column 3 27.039 20.862 2.565 RAD(%) 0.1 0.1 0.6

[0155] The results showed that after replacing the same brand but different batches of chromatographic columns, the RAD values ​​of the contents of each component were all less than 2.0%. Combined with the results of the investigation of different brands of chromatographic columns, the method had good durability when using chromatographic columns of the same specification from different manufacturers or different batches from the same manufacturer.

[0156] (5) Investigation of different phosphoric acid concentrations

[0157] The effects of different phosphate concentration mobile phases on the determination of the contents of lycopodiol, mulberry glycoside A and liquiritin in Xiebaisan standard samples were compared. Xiebaisan standard samples were taken, and the test solution was prepared according to the proposed method. The determination was carried out according to the proposed chromatographic conditions. Except for the use of mobile phases with different phosphate concentrations (the phosphate concentrations were 0.045%, 0.050% and 0.055%, respectively), the contents of lycopodiol, mulberry glycoside A and liquiritin were determined according to the proposed chromatographic conditions.

[0158] Table 16. Results of investigations at different phosphoric acid concentrations

[0159] Phosphoric acid concentration Dioscorea ethyl (mg / g) Mulberry glycoside A (mg / g) Licorice glycoside (mg / g) 0.045％ 29.045 23.191 2.650 0.050％ 28.001 23.175 2.635 0.055％ 28.359 23.146 2.633 RSD(%) 1.9 0.1 0.4

[0160] The results showed that the RSD values ​​of the contents of scoparia acetylcholine, morusin A and liquiritin at different phosphate concentrations were all less than 2.0%, indicating that the analytical method had good durability within a small range of phosphate concentration changes.

[0161] Summary of Methodological Validation

[0162] In the methodological verification test of the content of Xiebaisan benchmark samples of cephalothin, mulberry glycoside A, and licoricein, the blank had no interference and good specificity; the theoretical plate numbers of cephalothin and licoricein were greater than 5000, and the theoretical plate number of mulberry glycoside A was greater than 8000; cephalothin had good linearity in the concentration range of 5.106-102.1μg / ml, mulberry glycoside A had good linearity in the concentration range of 4.708-94.13μg / ml, and licoricein had good linearity in the concentration range of 0.9636-19.27μg / ml; injection Precision, repeatability and intermediate precision are good; the test solution is stable within 48 hours at room temperature; the recovery rate of cephalothin is between 95.3% and 98.9%, the recovery rate of mulberry glycoside A is between 97.8% and 100.0%, and the recovery rate of liquiritin is 98.2% to 100.8%, with high accuracy; except for the poor durability at different column temperatures (±2°C), the durability results for different flow rates (±0.1ml / min), different wavelengths (±5nm), different phosphate concentrations (±10%) and different chromatographic columns are good. After verification, this method can be accurately used for the quantitative determination of the contents of cephalothin, mulberry glycoside A and liquiritin in the Xiebaisan reference sample. The present invention is to improve the existing technology to improve the sensitivity and accuracy, and the detection range is wider. To this end, the present invention conducted the following comparative experiments:

[0163]

[0164]

[0165] Beneficial effects of the present invention

[0166] The present invention discloses an HPLC content detection method for eurycoma longifolia, morusin A and liquiritin in a Xiebaisan reference sample. The method has the beneficial effect that the present invention is studied for the purpose of developing and researching the quality standard requirements for the national classic prescription Xiebaisan. The present invention adopts an HPLC content detection method, which is simple and feasible after methodology verification and easy to operate. The method is applied to the quality evaluation of Xiebaisan reference samples, and provides a content detection method for the quality standard research of Xiebaisan reference samples, so that the method has certain quality controllability. Description of the drawings:

[0167] Figure 1 Investigation on the specificity of Xiebaisan standard sample content determination

[0168] Figure 2 Standard curve of determination of dioscorea ethyl content in Xiebaisan reference sample

[0169] Figure 3 Standard curve of determination of mulberry glycoside A content in Xiebaisan standard sample

[0170] Figure 4Standard curve of determination of liquiritin content in Xiebaisan reference sample

[0171] Figure 5 Reference solution (Peak 1: cephalothin; Peak 2: mulberry glycoside A; Peak 3: liquiritin)

[0172] Figure 6 Test solution (peak 1: lycoside; peak 2: mulberry glycoside A; peak 3: liquiritin) Specific implementation method:

[0173] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0174] Example 1

[0175] The following methods were used to test samples during the production process, and the results are as follows:

[0176] 1) Preparation of reference solution: Take appropriate amount of cerebroside A reference substance, mulberry glycoside A reference substance and liquiritin reference substance, weigh accurately, add 30% methanol solution containing 0.25% acetic acid to prepare a mixed solution containing 50 μg of cerebroside A, 50 μg of mulberry glycoside A and 10 μg of liquiritin per 1 ml.

[0177] 2) Preparation of test solution: Take about 0.1 g of reference sample powder, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of 30% methanol solution containing 0.25% acetic acid, stopper, weigh, ultrasonically treat for 45 minutes, cool, weigh again, make up the lost weight with 30% methanol solution containing 0.25% acetic acid, shake well, filter, and take the filtrate.

[0178] 3) Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph to obtain the chromatogram, and calculate the contents of scopolamine, morusin A and liquiritin based on the chromatogram.

[0179] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler ( AQ-C18, 4.6mm×250mm, 5μm); acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; flow rate 1ml per minute; column temperature 30℃; detection wavelength 280nm. The theoretical plate number calculated based on the mulberry glycoside A peak should not be less than 2000.

[0180] Time (min) Mobile phase A (%) Mobile phase B (%) 0～9 8→10 92→90 9～15 10→13 90→87 15～23 13→33 87→67 23～35 33 67

[0181] The product testing data of 3 batches during the production process are as follows:

[0182] batch number Dioscorea ethyl (mg / g) Mulberry glycoside A content (mg / g) Licorice glycoside content (mg / g) XBS(J)-GG240701 12.5 22.6 1.72 XBS(J)-GG240702 18.5 22.2 2.41 XBS(J)-GG240703 20.1 19.2 1.61

Claims

1. A HPLC content detection method for Xiebaisan reference sample, the detection method comprises the following steps: 1) Preparation of reference solution: Take the lycoside A reference substance, the morusin A reference substance, and the liquiritin reference substance, add a solvent, and prepare a reference solution; 2) Preparation of test solution: Take Xiebaisan standard sample, add solvent, and prepare test solution; 3) Determination: Take the reference solution and the test solution, inject them into the high performance liquid chromatograph to obtain the chromatogram, and calculate the contents of lycoside A, morusin A and liquiritin according to the chromatogram.

2. The detection method according to claim 1, wherein: The solvent is selected from acetic acid, methanol or a mixture thereof.

3. The detection method according to claim 2, wherein: The solvent is a mixture of acetic acid and methanol.

4. The detection method according to claim 3, wherein: The solvent is a mixture of 0.25% acetic acid and 30% methanol.

5. The detection method according to claim 1, wherein the method adopts high performance liquid chromatography, and the chromatographic column is filled with octadecylsilane bonded silica gel ( AQ-C18, 4.6mm×250mm, 5μm). The detection method according to claim 1 , wherein gradient elution is used.

7. The detection method according to claim 1, wherein acetonitrile is used as mobile phase A and phosphoric acid solution is used as mobile phase B.

8. The detection method according to claim 1, wherein the steps are as follows: 1) Preparation of reference solution: Take appropriate amount of cerebroside reference substance, mulberry glycoside A reference substance, and liquiritin reference substance, weigh accurately, add 30% methanol solution containing 0.25% acetic acid to prepare a mixed solution containing 50 μg of cerebroside, 50 μg of mulberry glycoside A, and 10 μg of liquiritin per 1 ml, and obtain: 2) Preparation of test solution: Take about 0.1g of the reference sample powder, weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 30% methanol solution containing 0.25% acetic acid, stopper it tightly, weigh it, ultrasonically treat it for 45 minutes, let it cool, weigh it again, make up the lost weight with 30% methanol solution containing 0.25% acetic acid, shake it well, filter it, and take the filtrate to obtain, 3) Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph to obtain the chromatogram, and calculate the contents of scopolamine, morusin A and liquiritin according to the chromatogram. in, The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler ( AQ-C18, 4.6mm×250mm, 5μm); acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate 1ml per minute; column temperature 30℃; detection wavelength 280nm. The theoretical plate number calculated based on the mulberry glycoside A peak should be no less than 2000,

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