Method for measuring morphine content in cough-relieving and phlegm-reducing pill

Abstract

The invention discloses a method for measuring morphine content in a cough-relieving and phlegm-reducing pill, and aims to improve the quality standard of the cough-relieving and phlegm-reducing pill and guarantee the medication safety of a patient. The method adopts a high performance liquid chromatography and comprises the following steps: precisely weighing a cough-relieving and phlegm-reducing pill sample, grinding, and carrying out ultrasonic extraction by taking 20% methanol containing 5% glacial acetic acid as an extraction solvent to prepare a test solution; preparing a morphine reference substance solution; according to the method, octadecylsilane chemically bonded silica is taken as a filling agent, acetonitrile and a specific mixed solution are taken as a mobile phase for gradient elution, and determination is carried out under the conditions that the column temperature is 30 DEG C and the detection wavelength is 210 nm. The method is verified by specificity, accuracy, repeatability and the like, the result is reliable, the linear relation is good, a low-toxicity organic reagent is used, the method is environment-friendly, the method can be used as a quality control means of Chinese patent medicines containing poppy shells, and the blank of morphine content determination in the existing standard is filled.

Description

A method for determining the morphine content in cough-relieving and expectorant pills Technical Field

[0001] This invention relates to a method for determining morphine content, and in particular to a method for determining the morphine content in a cough-relieving and expectorant pill. Background Technology

[0002] The Cough-Relieving and Phlegm-Resolving Pill is included in Part I of the 2020 edition of the Chinese Pharmacopoeia. This product clears the lungs, resolves phlegm, relieves cough, and calms asthma. It is used for phlegm-heat obstructing the lungs, chronic cough, hemoptysis, wheezing, and insomnia. The prescription for the Cough-Relieving and Phlegm-Resolving Pill consists of twenty-five herbs: poppy husk, platycodon root, anemarrhena rhizome, angelica root, tangerine peel, rhubarb (processed), prepared licorice root, fritillaria bulb, gypsum, bitter almond, perilla leaf, lepidium seed, coltsfoot flower (processed), stemona root (processed), scrophularia root, ophiopogon root, buddleja flower, asparagus root, schisandra fruit (processed), bitter orange peel (fried), trichosanthes seed, pinellia tuber (processed with ginger), costus root, aristolochic acid (processed), and mulberry leaf. The preparation method is to grind the above twenty-five herbs into a fine powder, sift, mix evenly, make into pills with water, dry, and polish.

[0003] The pharmacopoeia standard for Zhike Huatan Pills only includes microscopic identification of rhubarb, licorice, and schisandra, and thin-layer chromatography identification of poppy shell and rhubarb. Based on this, we have added a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the detection of aristolochic acid I, which we have established. Among the ingredients in the Zhike Huatan Pills prescription, only poppy shell may contain morphine. The original standard did not establish a morphine content determination method. To improve the quality standard, facilitate the control and evaluation of the overall intrinsic quality of the drug, and ensure patient safety, we have now established an HPLC method for morphine content detection and rationally set a total morphine limit for Zhike Huatan Pills. Summary of the Invention

[0004] The technical problem solved by this invention is that the existing standards for cough-relieving and expectorant pills do not include a method for determining morphine content. This invention explores a method for detecting morphine content in cough-relieving and expectorant pills.

[0005] The technical solution of the present invention: A method for determining the morphine content in cough-relieving and phlegm-reducing pills, which is carried out according to the following steps: (1) Preparation of test solution: Take a sample of cough-relieving and phlegm-reducing pills, grind it finely, accurately weigh 0.1-2g, place it in a stoppered conical flask, accurately add 15-50mL of 10-30% methanol solution containing 1-10% glacial acetic acid, stopper tightly, weigh, sonicate for 15-45min, the power of the sonication is 400-600W, the frequency is 12-38kHz, take it out, cool it and weigh it again, replenish the lost weight with 10-30% methanol solution containing 1-10% glacial acetic acid, shake well, filter. (1) Filter the filtrate through a 0.1-0.8 μm filter membrane to obtain the test solution for later use; (2) Preparation of reference stock solution: Accurately weigh the morphine reference standard, add 10-30% methanol solution containing 1-10% glacial acetic acid to prepare a morphine reference stock solution containing 350-450 ug per 1 mL for later use; (3) Preparation of reference solution: Accurately measure the morphine reference stock solution, add 10-30% methanol solution containing 1-10% glacial acetic acid to prepare 1.15-4.55 ug / mL morphine reference solution, 5.51-10.33 ug / mL morphine reference solution, and 10.17- 20.47 ug / mL morphine reference solution, 31.56-55.89 ug / mL morphine reference solution and 70.45-96.37 ug / mL morphine reference solution, for later use; (4) Determination method: Accurately pipette 10 μL of 1.15-4.55 ug / mL morphine reference solution, 5.51-10.33 ug / mL morphine reference solution, 10.17-20.47 ug / mL morphine reference solution, 31.56-55.89 ug / mL morphine reference solution and 70.45-96.37 ug / mL morphine reference solution respectively and 10 μL of the test solution was injected into the liquid chromatograph, and the determination was performed according to the chromatographic conditions. A standard curve was prepared with peak area as the ordinate and reference concentration as the abscissa. The amount of morphine equivalent in the test solution was read from the standard curve and calculated. Chromatographic conditions: octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A; an equal volume mixture of 0.01 mol / L heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate was used as mobile phase B; the column temperature was 30℃; the detection wavelength was 210 nm; the flow rate was 1.0 mL / min; and the injection volume was 10 μL. Gradient elution was performed according to the table below: Preparation of the test solution in step (1) above: Take the cough-relieving and phlegm-reducing pill sample, grind it into a fine powder, accurately weigh 0.6-1.4g, place it in a stoppered conical flask, accurately add 21-32mL of 16-24% methanol solution containing 3-8% glacial acetic acid, stopper tightly, weigh it, sonicate for 26-38min, the power of the sonication is 460-530W, the frequency is 19-29kHz, take it out, cool it and weigh it again, use 16-24% methanol solution containing 3-8% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter it through a 0.22-0.5μm filter membrane to obtain the test solution.

[0006] Specifically, in step (1) above, the preparation of the test solution is as follows: Take the sample of Zhike Huatan Pill, grind it into a fine powder, accurately weigh 1g, place it in a stoppered conical flask, accurately add 25mL of 20% methanol solution containing 5% glacial acetic acid, seal tightly, weigh, sonicate for 30min, the power of the sonication is 500W, the frequency is 25kHz, take it out, cool it and weigh it again, use 20% methanol solution containing 5% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter it through a 0.45μm filter membrane to obtain the test solution.

[0007] Preparation of the reference stock solution in step (2) above: Accurately weigh the morphine reference standard and add 16-24% methanol solution containing 3-8% glacial acetic acid to prepare a morphine reference stock solution containing 380-420ug per 1mL.

[0008] Specifically, in step (2) above, the preparation of the reference stock solution is as follows: accurately weigh the morphine reference standard and add 20% methanol solution containing 5% glacial acetic acid to prepare a stock solution containing 411.28ug per 1mL.

[0009] Preparation of reference solutions in step (3) above: Accurately measure the morphine reference stock solution and add 16-24% methanol solution containing 3-8% glacial acetic acid to prepare morphine reference solutions of 2.86-3.74 ug / mL, 6.96-9.12 ug / mL, 15.23-17.84 ug / mL, 37.25-49.26 ug / mL, and 77.39-88.94 ug / mL.

[0010] Specifically, in step (3) above, the preparation of the reference solution is as follows: accurately measure the morphine reference stock solution and add 20% methanol solution containing 5% glacial acetic acid to prepare 3.29ug / mL morphine reference solution, 8.23ug / mL morphine reference solution, 16.45ug / mL morphine reference solution, 41.13ug / mL morphine reference solution and 82.26ug / mL morphine reference solution.

[0011] The determination method in step (4) above: accurately pipette 10 μL of morphine reference solution with concentrations of 2.86-3.74 ug / mL, 6.96-9.12 ug / mL, 15.23-17.84 ug / mL, 37.25-49.26 ug / mL, and 77.39-88.94 ug / mL, and inject 10 μL of the test solution into the liquid chromatograph. Determine the concentration according to the chromatographic conditions. Prepare a standard curve with peak area as the ordinate and reference concentration as the abscissa. Read the amount of morphine equivalent in the test solution from the standard curve and calculate the result.

[0012] Specifically, the determination method in step (4) above is as follows: accurately pipette 10 μL of 3.29 ug / mL morphine reference solution, 8.23 ​​ug / mL morphine reference solution, 16.45 ug / mL morphine reference solution, 41.13 ug / mL morphine reference solution and 82.26 ug / mL morphine reference solution respectively, and inject them into the liquid chromatograph. Perform the determination according to the chromatographic conditions. Prepare a standard curve with peak area as the ordinate and reference concentration as the abscissa. Read the amount of morphine equivalent in the test solution from the standard curve and calculate the result.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the method for detecting morphine content in cough-relieving and expectorant pills developed by the present invention is scientific, stable and reliable. At the same time, the experiment uses low-toxicity organic reagents, which not only does not pollute the environment, but the obtained detection method can also be used as a quality control means for traditional Chinese medicine containing poppy shells, which greatly improves the safety of patients. Attached Figure Description

[0014] Figure 1: Chromatogram 1 for specificity test; Figure 2: Chromatogram 2 for specificity test; Figure 3: Chromatogram 3 for specificity test; Figure 4: Chromatogram 1 for sample accuracy test; Figure 5: Chromatogram 2 for sample accuracy test; Figure 6: Chromatogram 3 for sample accuracy test; Figure 7: Chromatogram 4 for sample accuracy test; Figure 8: Chromatogram 5 for sample accuracy test; Figure 9: Chromatogram 6 for sample accuracy test; Figure 10: Chromatogram 1 for sample accuracy test; Figure 11: Chromatogram 2 for sample accuracy test; Figure 12: Chromatogram 3 for sample accuracy test; Figure 13: Chromatogram 4 for sample accuracy test; Figure 14: Chromatogram 5 for sample accuracy test; Figure 15: Chromatogram 6 for sample accuracy test; Figure 16: Chromatogram 7 of the sample accuracy test; Figure 17: Chromatogram 1 of the sample repeatability test; Figure 18: Chromatogram 2 of the sample repeatability test; Figure 19: Chromatogram 3 of the sample repeatability test; Figure 20: Chromatogram 4 of the sample repeatability test; Figure 21: Chromatogram 5 of the sample repeatability test; Figure 22: Chromatogram 6 of the sample repeatability test; Figure 23: Chromatogram 7 of the sample repeatability test; Figure 24: Chromatogram 8 of the sample repeatability test; Figure 25: Chromatogram 9 of the sample repeatability test; Figure 26: Chromatogram 10 of the sample repeatability test; Figure 27: Chromatogram 11 of the sample repeatability test; Figure 28: Chromatogram 12 of the sample repeatability test; Figure 29: Chromatogram 13 of the sample repeatability test. Detailed Implementation

[0015] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically described in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0016] Example 1: Chromatographic conditions for the determination of morphine content in Zhike Huatan Pills: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A; an equal volume mixture of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate was used as mobile phase B; the column temperature was 30℃; the detection wavelength was 210 nm; the flow rate was 1.0 mL / min; and the injection volume was 10 μL. Gradient elution was performed according to the table below: The chromatographic conditions used in Examples 2-6 below are the same as those in Example 1.

[0017] Example 2:Method for determining morphine content in Zhike Huatan Pills (1) Preparation of test solution: Take Zhike Huatan Pills sample, grind finely, accurately weigh 1g, place in a stoppered conical flask, accurately add 25mL of 20% methanol solution containing 5% glacial acetic acid, seal tightly, weigh, sonicate for 30min, the power of the sonication is 500W, the frequency is 25kHz, take out, cool and weigh again, use 20% methanol solution containing 5% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter through a 0.45μm filter membrane to obtain the test solution for later use; (2) Preparation of reference stock solution: Accurately weigh morphine reference standard, add 20% methanol solution containing 5% glacial acetic acid to prepare a stock solution containing 411.28ug per 1mL for later use; (3) Preparation of reference solution: Accurately measure morphine reference stock solution, add 20% methanol solution containing 5% glacial acetic acid. Prepare 3.29ug / mL morphine reference solution, 8.23ug / mL morphine reference solution, 16.45ug / mL morphine reference solution, 41.13ug / mL morphine reference solution and 82.26ug / mL morphine reference solution for later use; (4) Determination method: Accurately pipette 10μL of 3.29ug / mL morphine reference solution, 8.23ug / mL morphine reference solution, 16.45ug / mL morphine reference solution, 41.13ug / mL morphine reference solution and 82.26ug / mL morphine reference solution and 10μL of test solution, inject them into the liquid chromatograph, and determine according to the chromatographic conditions. Prepare a standard curve with peak area as the ordinate and reference concentration as the abscissa. Read the amount of morphine in the test solution from the standard curve and calculate it.

[0018] Example 3:Method for determining morphine content in Zhike Huatan Pills (1) Preparation of test solution: Take a sample of Zhike Huatan Pills, grind it finely, accurately weigh 0.1g, place it in a stoppered conical flask, accurately add 15mL of 10% methanol solution containing 1% glacial acetic acid, stopper tightly, weigh, sonicate for 15min, the power of the sonication is 400W, the frequency is 12kHz, take it out, cool it and weigh it again, and make up the loss with 10% methanol solution containing 1% glacial acetic acid. (1) Weight, shake well, filter, and filter the filtrate through a 0.1 μm filter membrane to obtain the test solution for later use; (2) Preparation of reference stock solution: accurately weigh the morphine reference standard, add 10% methanol solution containing 1% glacial acetic acid to prepare a morphine reference stock solution containing 350 ug per 1 mL for later use; (3) Preparation of reference solution: accurately measure the morphine reference stock solution, add 10% methanol solution containing 1% glacial acetic acid to prepare a solution containing 1.15 ug / mL. Morphine reference solutions, 5.51ug / mL morphine reference solutions, 10.17ug / mL morphine reference solutions, 31.56ug / mL morphine reference solutions and 70.45ug / mL morphine reference solutions, are prepared for use; (4) Determination method: Accurately pipette 10μL of 1.15-4.55ug / mL morphine reference solutions, 5.51-10.33ug / mL morphine reference solutions, 10.17-20.47ug / mL morphine reference solutions, 31.56-55.89ug / mL morphine reference solutions and 70.45-96.37ug / mL morphine reference solutions and 10μL of test solution, respectively, and inject them into the liquid chromatograph. Determine according to the chromatographic conditions. Prepare a standard curve with peak area as the ordinate and reference concentration as the abscissa. Read the amount of morphine in the test solution from the standard curve and calculate it.

[0019] Example 4:Method for determining morphine content in Zhike Huatan Pills (1) Preparation of test solution: Take Zhike Huatan Pills sample, grind finely, accurately weigh 0.6g, place in a stoppered conical flask, accurately add 21mL of 16% methanol solution containing 3% glacial acetic acid, stopper tightly, weigh, sonicate for 26min, the power of the sonication is 460W, the frequency is 19kHz, take out, cool and weigh again, use 16% methanol solution containing 3% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter through a 0.22μm filter membrane to obtain the test solution, for use; (2) Preparation of reference stock solution: Accurately weigh morphine reference standard, add 16% methanol solution containing 3% glacial acetic acid to prepare morphine reference stock solution containing 380ug per 1mL, for use; (3) Preparation of reference solution: Accurately measure morphine reference stock solution, add 16% methanol solution containing 3% glacial acetic acid Methanol solution was used to prepare 2.86 ug / mL morphine reference solution, 6.96 ug / mL morphine reference solution, 15.23 ug / mL morphine reference solution, 37.25 ug / mL morphine reference solution and 77.39 ug / mL morphine reference solution for later use; (4) Determination method: 10 μL of 2.86 ug / mL morphine reference solution, 6.96 ug / mL morphine reference solution, 15.23 ug / mL morphine reference solution, 37.25 ug / mL morphine reference solution and 77.39 ug / mL morphine reference solution respectively and 10 μL of test solution were injected into the liquid chromatograph and determined according to the chromatographic conditions. A standard curve was prepared with peak area as the ordinate and reference concentration as the abscissa. The amount of morphine in the test solution was read from the standard curve and calculated.

[0020] Example 5: Method for determining morphine content in Zhike Huatan Pills (1) Preparation of test solution: Take Zhike Huatan Pills sample, grind finely, accurately weigh 1.4g, place in a stoppered conical flask, accurately add 32mL of 24% methanol solution containing 8% glacial acetic acid, stopper tightly, weigh, sonicate for 38min, the power of the sonication is 530W, the frequency is 29kHz, take out, cool and weigh again, use 24% methanol solution containing 8% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter through a 0.5μm filter membrane to obtain the test solution.

[0021] (2) Preparation of reference stock solution: Accurately weigh the morphine reference standard and add 24% methanol solution containing 8% glacial acetic acid to prepare a morphine reference stock solution containing 420ug per mL for later use; (3) Preparation of reference solution: Accurately measure the morphine reference stock solution and add 24% methanol solution containing 8% glacial acetic acid to prepare 3.74ug / mL morphine reference solution, 9.12ug / mL morphine reference solution, 17.84ug / mL morphine reference solution, 49.26ug / mL morphine reference solution and 88.94ug / mL morphine reference solution. Solution, for later use; (4) Determination method: Accurately pipette 10 μL of 3.74 ug / mL morphine reference solution, 9.12 ug / mL morphine reference solution, 17.84 ug / mL morphine reference solution, 49.26 ug / mL morphine reference solution and 88.94 ug / mL morphine reference solution and inject 10 μL of test solution into the liquid chromatograph, and determine according to the chromatographic conditions. Prepare a standard curve with peak area as the ordinate and reference concentration as the abscissa. Read the amount of morphine in the test solution from the standard curve and calculate it.

[0022] Example 6: Methods for determining morphine content in Zhike Huatan Pills (1) Preparation of test solution: Take Zhike Huatan Pill sample, grind finely, accurately weigh 2g, place in a stoppered conical flask, accurately add 50mL of 30% methanol solution containing 10% glacial acetic acid, stopper tightly, weigh, sonicate for 45min, the power of the sonication is 600W, the frequency is 38kHz, take out, cool and weigh again, use 30% methanol solution containing 10% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter through a 0.8μm filter membrane to obtain the test solution for later use; (2) Preparation of reference stock solution: Accurately weigh morphine reference standard, add 30% methanol solution containing 10% glacial acetic acid to prepare morphine reference stock solution containing 450ug per 1mL for later use; (3) Preparation of reference solution: Accurately measure morphine reference stock solution, add 30% methanol solution containing 10% glacial acetic acid to prepare morphine reference stock solution containing 450ug per 1mL for later use; (3) Preparation of reference solution: Accurately measure morphine reference stock solution, add 30% methanol solution containing 10% glacial acetic acid to prepare morphine reference stock solution containing 450ug per 1mL. Prepare 4.55ug / mL morphine reference solution, 10.33ug / mL morphine reference solution, 20.47ug / mL morphine reference solution, 55.89ug / mL morphine reference solution and 96.37ug / mL morphine reference solution with alcohol solution for later use; (4) Determination method: accurately pipette 10μL of 4.55ug / mL morphine reference solution, 10.33ug / mL morphine reference solution, 20.47ug / mL morphine reference solution, 55.89ug / mL morphine reference solution and 96.37ug / mL morphine reference solution respectively and inject 10μL of test solution into liquid chromatograph, determine according to chromatographic conditions, prepare standard curve with peak area as ordinate and reference concentration as abscissa, read the amount of morphine in test solution from standard curve and calculate.

[0023] To verify the feasibility of the method of this invention, a large number of experimental studies were conducted. The results of the experimental studies are as follows: 1. Extraction solvent: Take the product with different filling amounts, grind it finely, accurately weigh about 1g, and place it in a stoppered conical flask. Accurately add 25mL of the extraction solvent containing 5% glacial acetic acid (20% methanol, 20% methanol, and methanol solution), respectively. Seal tightly, weigh, and sonicate (power 500W, frequency 25kHz) for 30min. Remove, cool, and weigh again. Make up the lost weight with 20% methanol, 20% methanol, and methanol solutions containing 5% glacial acetic acid, respectively. Shake well, filter, and take the filtrate. Filter through a 0.45μm microporous membrane to obtain the product. Accurately pipette 10μL each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the results.

[0024] Results: Using 5% glacial acetic acid in 20% methanol, 20% methanol, and methanol as extraction solvents, the highest morphine content was obtained when using 5% glacial acetic acid in 20% methanol as the extraction solvent. Therefore, 5% glacial acetic acid in 20% methanol was selected as the extraction solvent. Because the target peak and impurity peaks were not completely separated when methanol was used as the extraction solvent, their content could not be calculated. See Table 1 for detailed results.

[0025] Table 1 Selection of Extraction Solvent 2. Extraction method: Take approximately 1g of the sample (based on the difference in fill weight), grind it finely, and place it in a stoppered conical flask. Accurately add 25mL of 20% methanol solution containing 5% glacial acetic acid as the extraction solvent. Seal the flask tightly and weigh. Extract by ultrasonic treatment (500W, 25kHz) for 30 minutes and by reflux for 30 minutes. Remove the flask, cool it, and weigh it again. Make up the weight loss with 20% methanol solution containing 5% glacial acetic acid. Shake well, filter, and filter the filtrate through a 0.45μm microporous membrane. Accurately inject 10μL each of the reference solution and the test solution into the liquid chromatograph for determination.

[0026] Results: Since the results of the two different extraction methods were not significantly different, and considering that ultrasonic extraction was simpler, ultrasonic extraction was chosen as the extraction method. The results are detailed in Table 2.

[0027] Table 2 Selection of Extraction Method 3. Extraction Time: Take approximately 1g of the sample (based on the difference in fill weight), grind it finely, and place it in separate stoppered conical flasks. Accurately add 25mL of 20% methanol solution containing 5% glacial acetic acid as the extraction solvent. Seal tightly, weigh, and sonicate (500W, 25kHz) for 15, 30, and 45 minutes respectively. Remove, cool, and weigh again. Make up the weight loss with 20% methanol solution containing 5% glacial acetic acid, shake well, filter, and filter the filtrate through a 0.45μm microporous membrane. Accurately inject 10μL each of the reference solution and the test solution into the liquid chromatograph for determination.

[0028] Results: There was almost no difference in morphine content before and after ultrasonic extraction at different times, indicating that the extraction was complete at 15 minutes. Therefore, the ultrasonic extraction time was set at 15 minutes. See Table 3 for details.

[0029] Table 3 Selection of Extraction Time 4. Investigation of Extraction Solvent Volume: Take approximately 1g of the sample from the different fill weights, grind it finely, and place it in separate stoppered conical flasks. Add 15, 25, and 50 mL of 20% methanol solution containing 5% glacial acetic acid to the flasks respectively. Seal the flasks tightly, weigh them, and sonicate them (500W, 25kHz) for 30 minutes. Remove the flasks, cool them, and weigh them again. Make up the weight loss with 20% methanol solution containing 5% glacial acetic acid, shake well, filter, and filter the filtrate through a 0.45μm microporous membrane. Accurately inject 10μL each of the reference solution and the test solution into the liquid chromatograph for determination.

[0030] Results: The morphine content increased with the increase of the extraction solvent volume. Since the difference in morphine content between adding 50 mL and 25 mL of solvent was not significant, the extraction solvent volume was set at 25 mL to save solvent. The results are detailed in Table 4.

[0031] Table 4 Selection of Extraction Solvent Amount 5. Specificity: Prepare sample solutions and negative sample solutions for determination, and examine whether the chromatograms of the sample solutions and negative sample solutions have corresponding chromatographic peaks at the corresponding positions in the chromatograms of the reference solution, and whether there is any interference from the negative sample solution.

[0032] Results: In the chromatogram of the negative sample solution, no chromatographic peak corresponding to the retention time of the reference standard was observed. In the chromatogram of the test sample solution, a chromatographic peak corresponding to the retention time of the reference standard was observed. The verification results meet the requirements, as detailed in Figures 1, 2, and 3.

[0033] 6. Accuracy: Take 6 portions of the same batch of cough-relieving and expectorant pills, accurately weigh them, and add 5 mL of morphine reference solution of different concentrations to each portion. Prepare the test solution according to the above method. Perform the determination according to the method, calculate the spiked recovery rate and RSD, and record the chromatograms. The results are detailed in Table 5, and Figures 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16.

[0034] Table 5 Accuracy Test 7. Repeatability: Take 6 samples of the same batch of cough-relieving and phlegm-reducing pills, accurately weigh them, and prepare the test solution according to the above method. Perform the determination according to the assay method, calculate the content and RSD, and record the chromatogram. The results are detailed in Table 6, and Figures 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.

[0035] Table 6 Repeatability Tests 8. Intermediate precision: Another analyst processed the samples and reference standards on different days and recorded the chromatograms. The assay was performed according to the prescribed method, the content was calculated, and the chromatograms were recorded. The results are detailed in Table 7.

[0036] Table 7 Intermediate Precision Test 9. Linearity and Range: Accurately weigh an appropriate amount of morphine reference standard and add it to a 20% methanol solution containing 5% glacial acetic acid to prepare a stock solution containing 411.28 μg per mL. Accurately measure appropriate amounts of the stock solution and add it to a 20% methanol solution containing 5% glacial acetic acid to prepare solutions containing 3.29 μg, 8.23 ​​μg, 16.45 μg, 41.13 μg, and 82.26 μg of morphine per mL, respectively. Determine the concentration of morphine under chromatographic conditions. Perform linear fitting with peak area Y as the ordinate and reference standard concentration X as the abscissa to obtain the linear equation.

[0037] Results: Linear regression was performed with the standard concentration X as the abscissa and the peak area Y as the ordinate. A working curve was plotted, yielding the linear regression equation: y = 0.8187X + 0.1509, R². 2 =1. The results are shown in Table 8. In the range of 3.29-82.26 ug / ml, the injection amount of the reference standard showed a good linear relationship with the peak area. The results are detailed in Table 8.

[0038] Table 8. Linear Relationship of Morphine Reference Standards 10. Limit of Detection (LOD) and Limit of Quantification (LOQ): Signal-to-Noise Ratio (SNR) Method: The LOD was determined using a SNR of 3, and the LOQ was determined using a SNR of 10. See Table 9 for detailed results.

[0039] Table 9 Results of Limit of Detection and Limit of Quantification 11. Robustness: This test examines the ability of the morphine content detection method in Zhike Huatan Pills to withstand minor variations in certain parameters (column temperature, column manufacturer). Change one parameter while keeping others constant, and prepare the test solution as described above. Perform the determination according to the method, calculate the content and RSD, and record the chromatogram. Results are detailed in Table 10.

[0040] Table 10. Determination results of chromatographic columns of different temperatures and brands. 12. Stability: Accurately weigh an appropriate amount of morphine reference standard, add 20% methanol solution containing 5% glacial acetic acid to prepare a solution containing 15 μg per mL. Take samples of the same batch of cough-relieving and expectorant pills and prepare them according to the method for preparing the test solution. Under the above chromatographic conditions, determine the peak area values ​​of the same test solution and the same reference solution at 0h, 24h, 48h, and 72h, respectively, and investigate the peak area change of aristolochic acid component and calculate the RSD. The results are detailed in Table 11.

[0041] Table 11 Sample stability test The method for detecting the morphine limit in Zhike Huatan Pills developed based on the above experiments is scientific, stable, and reliable. At the same time, the use of low-toxicity organic reagents during the experiment not only avoids environmental pollution, but the obtained detection method can also be used as a quality control means for traditional Chinese medicines containing poppy shells, greatly improving patient safety.

Claims

1. A method for determining the morphine content in a cough-relieving and expectorant pill, characterized in that: The method for determining morphine content is carried out according to the following steps: (1) Preparation of test solution: Take the sample of Zhike Huatan Pill, grind it finely, accurately weigh 0.1-2g, place it in a stoppered conical flask, accurately add 15-50mL of 10-30% methanol solution containing 1-10% glacial acetic acid, stopper tightly, weigh, sonicate for 15-45min, the power of the sonication is 400-600W, the frequency is 12-38kHz, take it out, cool it and weigh it again, make up the lost weight with 10-30% methanol solution containing 1-10% glacial acetic acid, shake well, filter, and take the filtrate through 0.1-0.8μm. (1) Filter membrane to obtain the test solution, for later use; (2) Preparation of reference stock solution: Accurately weigh morphine reference standard, add 10-30% methanol solution containing 1-10% glacial acetic acid to prepare morphine reference stock solution containing 350-450ug per 1mL, for later use; (3) Preparation of reference solution: Accurately measure morphine reference stock solution, add 10-30% methanol solution containing 1-10% glacial acetic acid to prepare 1.15-4.55ug / mL morphine reference solution, 5.51-10.33ug / mL morphine reference solution, 10.17-20.47ug / mL morphine reference solution. Morphine reference solutions, 31.56-55.89 ug / mL morphine reference solutions and 70.45-96.37 ug / mL morphine reference solutions, are prepared for use; (4) Determination method: Accurately pipette 10 μL of each of the following morphine reference solutions: 1.15-4.55 ug / mL morphine reference solutions, 5.51-10.33 ug / mL morphine reference solutions, 10.17-20.47 ug / mL morphine reference solutions, 31.56-55.89 ug / mL morphine reference solutions and 70.45-96.37 ug / mL morphine reference solutions, and prepare 10 μL of the test solution. μL, injected into the liquid chromatograph, and determined according to the chromatographic conditions. A standard curve was prepared with the peak area as the ordinate and the mass concentration of the reference standard as the abscissa. The amount of morphine equivalent in the test solution was read from the standard curve and calculated. (5) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material, acetonitrile was used as the mobile phase A, and an equal volume mixture of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate was used as the mobile phase B. The column temperature was 30℃, the detection wavelength was 210 nm, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. Gradient elution was performed according to the following table:

2. The method for determining the morphine content in the cough-relieving and phlegm-reducing pills according to claim 1, characterized in that: Preparation of the test solution in step (1): Take the cough-relieving and phlegm-reducing pill sample, grind it finely, accurately weigh 0.6-1.4g, place it in a stoppered conical flask, accurately add 21-32mL of 16-24% methanol solution containing 3-8% glacial acetic acid, stopper tightly, weigh it, sonicate for 26-38min, the power of the sonication is 460-530W, the frequency is 19-29kHz, take it out, cool it and weigh it again, use 16-24% methanol solution containing 3-8% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter it through a 0.22-0.5μm filter membrane to obtain the test solution.

3. The method for determining the morphine content in the cough-relieving and phlegm-reducing pills according to claim 2, characterized in that: Preparation of the test solution in step (1): Take the cough-relieving and expectorant pill sample, grind it into a fine powder, accurately weigh 1g, place it in a stoppered conical flask, accurately add 25mL of 20% methanol solution containing 5% glacial acetic acid, seal tightly, weigh, sonicate for 30min, the power of the sonication is 500W, the frequency is 25kHz, take it out, cool it and weigh it again, use 20% methanol solution containing 5% glacial acetic acid to make up the lost weight, shake well, filter, take the filtrate and filter it through a 0.45μm filter membrane to obtain the test solution.

4. The method for determining the morphine content in the cough-relieving and expectorant pills according to claim 1, characterized in that: Preparation of the reference stock solution in step (2): Accurately weigh the morphine reference standard and add 16-24% methanol solution containing 3-8% glacial acetic acid to prepare a morphine reference stock solution containing 380-420ug per 1mL.

5. The method for determining the morphine content in the cough-relieving and phlegm-reducing pills according to claim 4, characterized in that: Preparation of the reference stock solution in step (2): Accurately weigh the morphine reference standard and add 20% methanol solution containing 5% glacial acetic acid to prepare a stock solution containing 411.28ug per 1mL.

6. The method for determining the morphine content in the cough-relieving and phlegm-reducing pills according to claim 1, characterized in that: Preparation of reference solutions in step (3): Accurately measure the morphine reference stock solution and add 16-24% methanol solution containing 3-8% glacial acetic acid to prepare morphine reference solutions of 2.86-3.74 ug / mL, 6.96-9.12 ug / mL, 15.23-17.84 ug / mL, 37.25-49.26 ug / mL, and 77.39-88.94 ug / mL.

7. The method for determining the morphine content in the cough-relieving and phlegm-reducing pills according to claim 6, characterized in that: In step (3), the preparation of the reference solution is as follows: the morphine reference stock solution is accurately measured and added to a 20% methanol solution containing 5% glacial acetic acid to prepare 3.29 ug / mL morphine reference solution, 8.23 ​​ug / mL morphine reference solution, 16.45 ug / mL morphine reference solution, 41.13 ug / mL morphine reference solution and 82.26 ug / mL morphine reference solution.

8. The method for determining the morphine content in the cough-relieving and phlegm-reducing pills according to claim 1, characterized in that: The determination method in step (4) is as follows: accurately pipette 10 μL of morphine reference solution with concentrations of 2.86-3.74 ug / mL, 6.96-9.12 ug / mL, 15.23-17.84 ug / mL, 37.25-49.26 ug / mL, and 77.39-88.94 ug / mL, and inject 10 μL of the test solution into the liquid chromatograph. Perform the determination according to the chromatographic conditions. Prepare a standard curve with peak area as the ordinate and reference concentration as the abscissa. Read the amount of morphine equivalent in the test solution from the standard curve and calculate the result.

9. The method for determining the morphine content in the cough-relieving and phlegm-reducing pills according to claim 8, characterized in that: The determination method in step (4) is as follows: accurately pipette 10 μL of each of the following morphine reference solutions: 3.29 ug / mL morphine reference solution, 8.23 ​​ug / mL morphine reference solution, 16.45 ug / mL morphine reference solution, 41.13 ug / mL morphine reference solution, and 82.26 ug / mL morphine reference solution, and inject them into the liquid chromatograph. Perform the determination according to the chromatographic conditions. Prepare a standard curve with peak area as the ordinate and reference concentration as the abscissa. Read the amount of morphine equivalent in the test solution from the standard curve and calculate the result.

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