Method for detecting content of effective components in Gu Tong Ling tincture

Abstract

The present application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a content detection method of effective components in Goutongling tincture. The present application aims to provide a content detection method of effective components in Goutongling tincture, which comprises the following steps: a, preparation of test sample solution: a certain amount of Goutongling tincture is loaded on an activated solid phase extraction column, eluted, washed, eluted, collected, evaporated, diluted, filtered, and then a continuous filtrate is obtained; b, preparation of control sample solution; c, chromatographic conditions: octadecylsilane-bonded silica gel is used as a filler, acetonitrile and phosphate buffered saline solution are used as mobile phases for gradient elution, the detection wavelength is 210 nm, the column temperature is 25-40 DEG C, the flow rate is 0.25-1.0 mL / min; and d, determination method: the control sample solution and the test sample solution are precisely taken respectively, injected into a high performance liquid chromatograph, and determined, and then the content detection method of effective components in Goutongling tincture is obtained. The method is simple and easy to operate, time-saving, and solves the problem of few quality control indicators.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine testing technology, specifically relating to a method for detecting the content of active ingredients in Gutongling Tincture. Background Technology

[0002] Bone Pain Relief Tincture is composed of six herbs: Artemisia selengensis, dried ginger, dragon's blood, borneol, frankincense, and myrrh. It has the effects of warming the meridians and dispelling cold, dispelling wind and promoting blood circulation, and relieving pain. It is a commonly used medicine for treating lumbar and cervical spondylosis, rheumatoid arthritis, and frozen shoulder. In Bone Pain Relief Tincture, Artemisia selengensis is the principal herb, playing a significant role in dispelling wind and dampness, promoting blood circulation, and relieving pain. Its main active ingredients are alkaloids, including artemisinin A and artemisinin B.

[0003] The 2020 edition of the Chinese Pharmacopoeia only specifies the content determination of artemisinin in Gutongling Tincture. The method is as follows: Accurately measure 25 mL of the product and place it in an evaporating dish. Evaporate it in a water bath to about 10 mL. Adjust the pH value to 2-3 with dilute hydrochloric acid. Transfer it to a separatory funnel and wash the container with an appropriate amount of 0.1 mol / L hydrochloric acid. Add the washings to the separatory funnel to make the total acid solution about 25 mL. Wash twice with chloroform by gentle shaking. Combine the chloroform solutions and wash twice with 0.1 mol / L hydrochloric acid. Combine the above acid-water solutions, add 2 g of sodium chloride, and shake gently to dissolve. Adjust the pH value to 9 with ammonia solution. 10. Extract with chloroform by shaking 5 times, combine the chloroform extracts, add an appropriate amount of anhydrous sodium sulfate to dehydrate, filter, collect the filtrate, wash the residue twice with chloroform (5 ml each time), combine the filtrates, evaporate to near dryness in a 60℃ water bath, add 1 ml of anhydrous ethanol, evaporate naturally, dissolve in methanol and transfer to a 25 ml volumetric flask, add methanol to the mark, sonicate (power 250W, frequency 33kHz) for 5 minutes, cool, add methanol to the mark, shake well, let stand, filter, and collect the filtrate; accurately pipette 10 μl each of the reference solution and the test solution, inject into the liquid chromatograph, and determine the result.

[0004] This method is complex, time-consuming, prone to errors, and has poor reproducibility. Under the given chromatographic conditions, the target peak and interfering peaks are not completely separated, leading to inaccurate results. Furthermore, this method can only detect artemisinin in Artemisia capillaris. Therefore, it is necessary to develop an accurate, stable, and easy-to-operate detection method capable of determining more alkaloid components in Artemisia capillaris to better control the quality of Gutongling Tincture. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting the content of active ingredients in bone pain relief tincture. The method is simple and easy to operate, and takes little time. It can simultaneously detect the content of artemisinin, artemisinogenin and benzoyl aconitine in Artemisia annua. It solves the problems of the existing technology, such as the preparation method of the test solution is too complicated, time-consuming and labor-intensive; the target peak cannot be completely separated, the analysis time is long; and there are few quality control indicators.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is to provide a method for detecting the content of effective ingredients in bone pain relief tincture, which includes the following steps:

[0007] a. Preparation of test solution: Measure out bone pain relief tincture, load it onto the activated solid phase extraction column, rinse, elute, collect the eluent, evaporate to dryness, make up to volume, and filter to obtain test solution;

[0008] b. Preparation of reference solution: Weigh appropriate amounts of artemisinin, artemisinin and benzoyl aconitine reference standards, add methanol to prepare a mixed reference solution containing 120 μg of artemisinin, 60 μg of artemisinin and 20 μg of benzoyl aconitine per 1 mL.

[0009] c. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile and phosphate buffer solution were used as the mobile phase for gradient elution; the detection wavelength was 210 nm, the column temperature was 25–40 °C, and the flow rate was 0.25–1.0 mL / min.

[0010] d. Determination method: Accurately pipette 2 μL of the reference solution and the test solution into the high performance liquid chromatograph and determine the result.

[0011] Further, in step a, the preparation method of the test solution is as follows: activate the mixed cation exchange solid phase extraction column with methanol and water respectively, load 1-5 mL of bone pain relief tincture onto the column, rinse the solid phase extraction column with 2% formic acid aqueous solution and methanol in sequence, discard the eluent, elute with 5% ammonia-methanol solution, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 2 mL, shake well and filter to obtain the test solution.

[0012] Furthermore, the mixed-type cation exchange solid-phase extraction column was activated with 0–6 mL of methanol and 0–6 mL of water, respectively.

[0013] Preferably, the mixed-type cation exchange solid-phase extraction column is activated with 3 mL of methanol and 3 mL of water, respectively.

[0014] Further, 2 mL of bone pain relief tincture was measured and loaded onto the sample.

[0015] Further, the solid-phase extraction column was rinsed sequentially with 3–12 mL of 2% formic acid aqueous solution and 3–9 mL of methanol.

[0016] Preferably, the solid-phase extraction column is washed sequentially with 3 mL of 2% formic acid aqueous solution and 3 mL of methanol.

[0017] Further, elute with 6–12 mL of 5% ammonia-methanol solution.

[0018] Preferably, elution is performed using 6 mL of 5% ammonia-methanol solution.

[0019] Further, in step c, the concentration of the phosphate buffer solution is 1–25 mmol / L; the pH value is 7.3.

[0020] Preferably, the phosphate buffer solution is a 5 mmol / L disodium hydrogen phosphate solution.

[0021] Further, the gradient elution is as follows:

[0022] The time is 0–12 min, the acetonitrile concentration is 20–27%, and the phosphate buffer solution concentration is 80–73%.

[0023] The time is 12–12.1 min, the acetonitrile concentration is 27–70%, and the phosphate buffer solution concentration is 73–30%.

[0024] The time was 12.1–17 min, the acetonitrile content was 70%, and the phosphate buffer solution was 30%.

[0025] The time is 17–17.1 min, the acetonitrile content is 70–20%, and the phosphate buffer solution content is 30–80%.

[0026] The time was 17.1–25 min, the acetonitrile content was 20%, and the phosphate buffer solution was 80%.

[0027] Further, in step c, the column temperature is 35°C; the flow rate is 1.0 mL / min.

[0028] Furthermore, in step c, the theoretical plate number, calculated based on the benzoyl aconitine peak, should be no less than 8000.

[0029] The inventors investigated various mobile phase gradient elution conditions and found that under the above-mentioned mobile phase gradient elution conditions, artemisinin, artemisinogenin, and benzoyl aconitine could all be completely separated.

[0030] The beneficial effects of this invention are:

[0031] This invention utilizes high-performance liquid chromatography (HPLC). By optimizing the preparation method of the test sample and the chromatographic conditions, it can simultaneously determine the contents of artemisinin, artemisinogenin, and benzoyl aconitine in Gutongling tincture. This method exhibits good linearity, repeatability, stability, and recovery, effectively controlling the quality of Gutongling tincture. The method optimizes the test sample preparation process and employs a mixed-type cation exchange solid-phase extraction column for purification. This simplifies the operation, reduces the amount of organic solvent used, and eliminates the highly toxic organic reagent chloroform, making it more environmentally friendly and reducing harm to the human body. Furthermore, the method optimizes the elution procedure of the mobile phase in the chromatographic conditions, ensuring complete separation of the three target peaks: artemisinin, artemisinogenin, and benzoyl aconitine. The entire analytical process is short, and the results are accurate and reliable. Attached Figure Description

[0032] Figure 1 The chromatograms of artemisinin, artemisinin and benzoyl aconitine in the bone pain relief tincture obtained in Example 1 are shown below (1 is artemisinin, 2 is artemisinin, and 3 is benzoyl aconitine).

[0033] Figure 2 The chromatogram of artemisinin in bone pain relief tincture as measured in Comparative Example 1 is shown.

[0034] Figure 3 Chromatograms of artemisinin and artemisinin in bone pain relief tincture separated by different mobile phases.

[0035] Figure 4 Chromatograms of artemisinin, artemisinin and benzoyl aconitine in bone pain relief tincture with different salt concentrations.

[0036] Figure 5 Chromatograms of artemisinin, artemisinin and benzoyl aconitine in bone pain relief tincture separated by different chromatographic columns.

[0037] Figure 6 Chromatograms of artemisinin, artemisinin and benzoyl aconitine in bone pain relief tincture separated at different column temperatures.

[0038] Figure 7 Chromatograms of artemisinin, artemisinin and benzoyl aconitine in bone pain relief tincture separated by different instruments.

[0039] Figure 8 To investigate the linear relationship, the standard curve of artemisinin was examined.

[0040] Figure 9 To investigate the linear relationship, the standard curve of artemisinin was examined.

[0041] Figure 10 The standard curve of benzoyl aconitine was examined to investigate the linear relationship.

[0042] Figure 11 The chromatograms obtained from the specificity test are as follows (from top to bottom: Gutongling tincture sample, benzoyl aconitine, artemisinin, artemisinin A, and Gutongling tincture negative sample). Detailed Implementation

[0043] The present invention will be further described in detail below through specific embodiments.

[0044] Example 1

[0045] Preparation of the test solution: Activate a solid-phase extraction column packed with a mixed cation exchange adsorbent using 3 mL of methanol and 3 mL of water, respectively. Accurately measure 2 mL of bone pain relief tincture and load it onto the column. Elute the solid-phase extraction column sequentially with 3 mL of 2% formic acid aqueous solution and 3 mL of methanol. Discard the eluent. Elute with 6 mL of 5% ammonia-methanol solution. Collect the eluent in an evaporating dish and evaporate to dryness in a water bath. Dissolve the residue in methanol and transfer it to a 2 mL volumetric flask. Add methanol to the mark, shake well, filter, and collect the filtrate.

[0046] Preparation of reference solution: Weigh appropriate amounts of artemisinin, artemisinin-enrichin, and benzoyl aconitine reference standards, add methanol to prepare a mixed reference solution containing 120 μg of artemisinin, 60 μg of artemisinin, and 20 μg of benzoyl aconitine per 1 mL.

[0047] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel (4.6 × 10⁵ mm, 5 μm) was used as the stationary phase; acetonitrile and 5 mmol / L phosphate buffer were used as the mobile phase, and gradient elution was performed according to Table 1. The detection wavelength was 210 nm, and the column temperature was 35 °C. The theoretical plate number, calculated based on the benzoyl aconitine peak, should not be less than 8000.

[0048] Assay: Accurately pipette 2 μL each of the reference solution and the test solution into the high-performance liquid chromatograph (HPLC) and determine the results. See below for details. Figure 1 .

[0049] Based on the external standard method, each 1 mL of Gutongling Tincture contains Artemisia capillaris as a component of Artemisia capillaris (C). 22 H 33 NO2), Artemisia annua extract (C) 22 H 31 NO3), and benzoyl aconitine (C 32 H 45 NO 10 The total amount of the three substances shall not be less than 154mg.

[0050] Table 1. Mobile phase gradient conditions used in Example 1

[0051] Time (minutes) Acetonitrile (%) 5 mmol / L phosphate buffer (%) 0～12 20→27 80→73 12～12.1 27→70 73→30 12.1～17 70 30 17～17.1 70→20 30→80 17.1～25 20 80

[0052] Comparative Example 1 was tested using the standard drug methods of the 2020 edition of the Chinese Pharmacopoeia.

[0053] Preparation of the test solution: Accurately measure 25 mL of this product and place it in an evaporating dish. Evaporate in a water bath to approximately 10 mL. Adjust the pH to 2–3 with dilute hydrochloric acid. Transfer the solution to a separatory funnel and wash the container with an appropriate amount of 0.1 mol / L hydrochloric acid. Add the washings to the separatory funnel, making the total acid solution approximately 25 mL. Gently wash twice with chloroform (10 mL, 5 mL). Combine the chloroform solutions and wash twice with 0.1 mol / L hydrochloric acid (5 mL, 3 mL). Combine the above acid-water solutions, add 2 g of sodium chloride, and gently shake to dissolve. Adjust the pH to 9–10 with ammonia solution. Extract five times with chloroform (25 mL, 15 mL, 10 mL, 10 mL, 10 mL), combine the chloroform extracts, add an appropriate amount of anhydrous sodium sulfate to dehydrate, filter, collect the filtrate, wash the residue twice with chloroform (5 mL each time), combine the filtrates, evaporate to near dryness in a 60°C water bath, add 1 mL of anhydrous ethanol, evaporate naturally, dissolve in methanol and transfer to a 25 mL volumetric flask, add methanol to the mark, sonicate (250 W, 33 kHz) for 5 minutes, cool, add methanol to the mark, shake well, let stand, filter, and collect the filtrate to obtain the final product.

[0054] Preparation of reference solution: Accurately weigh an appropriate amount of artemisinin reference standard and add methanol to prepare a solution containing 100 μg of artemisinin per 1 mL.

[0055] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; methanol-phosphate buffer solution (1.97 g disodium hydrogen phosphate and 0.22 g potassium dihydrogen phosphate, dissolved in water and diluted to 1000 mL, pH adjusted to 7.3 with 80% phosphoric acid solution) (72:28) was used as the mobile phase; the detection wavelength was 210 nm. The theoretical plate number, calculated based on the artemisinin peak, should be no less than 1500.

[0056] Accurately pipette 10 μL each of the reference solution and the test solution and inject them into the liquid chromatograph for determination. Results are shown below. Figure 2 .

[0057] Experimental Example 1: Selection of Chromatographic Conditions

[0058] The test solution prepared in Example 1 was used as the sample.

[0059] 1.1 Selection of mobile phase

[0060] Two mobile phases, methanol-5 mmol / L phosphate buffer (gradient elution according to Table 2) and acetonitrile-5 mmol / L phosphate buffer (gradient elution according to Example 1), were investigated. The results showed that both mobile phases could separate the target peak from the interfering peaks; only the elution order of artemisinin and artemisinogen was reversed. Furthermore, when acetonitrile was used as the mobile phase, the chromatographic baseline was more stable and the peak symmetry was better than with methanol. Therefore, acetonitrile-phosphate buffer was chosen as the mobile phase. (See...) Figure 3 .

[0061] Table 2. Mobile phase gradient conditions used for methanol-5 mmol / L phosphate buffer.

[0062] Time (minutes) Methanol (%) 5 mmol / L phosphate buffer (%) 0～5 60 40 5～15 60→64 40→36 15～15.1 64→90 36→10 15.1～20 90 10 20～20.1 90→60 10→40 20.1～25 60 40

[0063] 1.2 Salt Concentration Investigation

[0064] The study investigated the separation of the target peak using four mobile phases: 1 mmol / L mixed phosphate solution, 5 mmol / L mixed phosphate solution, 25 mmol / L mixed phosphate solution, and 5 mmol / L disodium hydrogen phosphate solution (all adjusted to pH 7.3). The results showed that all four mobile phases effectively separated the target peak. The 5 mmol / L disodium hydrogen phosphate solution, being a single salt, was the simplest to prepare, and was ultimately selected as the mobile phase. See [link to relevant documentation]. Figure 4 .

[0065] 1.3 Column Investigation

[0066] Based on the pH value of the mobile phase, chromatographic columns from different manufacturers and of different models were investigated for separating basic compounds. Both UPLC and HPLC columns achieved complete separation of the target peak. (See...) Figure 5 However, UPLC columns (2.1*100mm, 1.8μm) have smaller particle sizes and higher pressures, making them more susceptible to column clogging by salts. Therefore, a chromatographic column with octadecylsilane-bonded silica gel (4.6*150mm, 5μm) was selected for the experiment.

[0067] 1.4 Column Temperature Investigation

[0068] The separation of the target peak was ensured at column temperatures of 25℃, 30℃, 35℃, and 40℃, with the best separation effect observed at 30–40℃. Therefore, a column temperature of 35℃ was selected. (See...) Figure 6 .

[0069] 1.5 Instrumental Examination

[0070] The proposed chromatographic conditions were used to conduct analyses on different instruments to examine the robustness of the method. The results showed that the method could achieve complete separation of the target peak on all different instruments. See [link / reference]. Figure 7 .

[0071] Experimental Example 2: Investigation of the preparation method of the test sample solution

[0072] 2.1 Selection of purification method

[0073] Four purification methods were investigated: HPD-100 macroporous adsorption resin, basic alumina, 732 type cation exchange resin, and mixed cation exchange solid-phase extraction column. The results showed that the sample purified by the mixed cation exchange solid-phase extraction column could completely separate the three target peaks without loss of content. Therefore, the mixed cation exchange solid-phase extraction column was selected as the purification method for the finished product of Gutongling Tincture.

[0074] 2.2 Investigation on the amount of activating solvent used

[0075] Solid-phase extraction (SPE) columns were activated with 0 mL, 1 mL, 3 mL, and 6 mL of methanol and water, respectively. 3 mL of bone pain relief tincture was accurately measured and loaded onto the column (in duplicate). The column was then rinsed sequentially with 6 mL of 2% formic acid aqueous solution and 6 mL of methanol. The rinsing solutions were discarded. The SPE columns were then further eluted with 6 mL of ammonia-methanol solution. The eluent was collected, evaporated to dryness in a water bath in an evaporating dish, and the residue was dissolved in methanol and transferred to a 2 mL volumetric flask. Methanol was added to the mark, the mixture was shaken well, and the solution was filtered. The filtrate was then collected. The results are shown in Table 3.

[0076] Table 3. Investigation of Activation Solvent Dosage (n=2)

[0077]

[0078] The results showed that when the solid-phase extraction column was activated with 3 mL (methanol, water) or 6 mL (methanol, water) of activating solvent, the contents of artemisinin, artemisinin, and benzoyl aconitine were consistent and relatively high. Since the amount of organic solvent used for activation was small, the activation solvent volume of 3 mL (methanol, water) was selected.

[0079] 2.3 Sample Loading Quantity Investigation

[0080] The solid-phase extraction column was activated sequentially with 3 mL of methanol and 3 mL of water. 1 mL, 2 mL, and 3 mL of bone pain relief tincture were then accurately loaded onto the column, respectively. The column was elute sequentially with 6 mL of 2% formic acid aqueous solution and 6 mL of methanol. The eluent was discarded. The extraction column was then further eluted with 6 mL of 5% ammonia-methanol solution. The eluent was collected, evaporated to dryness in a water bath in an evaporating dish, and the residue was dissolved in methanol and transferred to a 2 mL volumetric flask. Methanol was added to the mark, shaken well, and filtered. The filtrate was then collected. The results are shown in Table 4.

[0081] Table 4. Sample loading quantity investigation (n=2)

[0082]

[0083]

[0084] The results showed that when the sample loading volume was 3 mL, the solid phase extraction column was overloaded, and when the sample loading volume was 1 mL, the concentration was too low. Therefore, a sample loading volume of 2 mL was recommended.

[0085] 2.4 Investigation on the dosage of rinsing solution

[0086] (1) 2% formic acid aqueous solution

[0087] The solid-phase extraction column was activated sequentially with 3 mL of methanol and 3 mL of water. 2 mL of bone pain relief tincture was accurately loaded onto the column, followed by rinsing with 3 mL, 6 mL, 9 mL, and 12 mL of 2% formic acid aqueous solution, and then with 9 mL of methanol. The eluent was discarded. The extraction column was then further eluted with 12 mL of 5% ammonia-methanol solution. The eluent was collected, evaporated to dryness in a water bath in an evaporating dish, and the residue was dissolved in methanol and transferred to a 2 mL volumetric flask. Methanol was added to the mark, shaken well, and filtered. The filtrate was then collected. The results are shown in Table 5.

[0088] Table 5. Investigation of the dosage of 2% formic acid aqueous solution (n=2)

[0089]

[0090] The results showed that when the rinsing volume of 2% formic acid aqueous solution was 3 mL and 6 mL, the contents of artemisinin, artemisinin and benzoyl aconitine were basically the same. When the rinsing volume was greater than or equal to 9 mL, the content of artemisinin decreased to varying degrees. Therefore, the rinsing volume of 2% formic acid aqueous solution was selected as 3 mL, which can retain the content of the target substances and achieve the purpose of removing impurities.

[0091] (2) Methanol

[0092] The solid-phase extraction column was activated sequentially with 3 mL of methanol and 3 mL of water. 2 mL of bone pain relief tincture was accurately loaded onto the column, followed by rinsing with 3 mL of 2% formic acid aqueous solution, then with 3 mL, 6 mL, and 9 mL of methanol, respectively. The eluent was discarded. The extraction column was then eluted with 12 mL of 5% ammonia-methanol solution. The eluent was collected, evaporated to dryness in a water bath in an evaporating dish, and the residue was dissolved in methanol and transferred to a 2 mL volumetric flask. Methanol was added to the mark, shaken well, and filtered. The filtrate was then collected. The results are shown in Table 6.

[0093] Table 6. Investigation of Methanol Usage (n=2)

[0094]

[0095] The results showed that the contents of artemisinin, artemisinin and benzoyl aconitine were basically the same after rinsing with three different volumes of methanol, so a methanol volume of 3 mL was sufficient.

[0096] 2.5 Analysis of Elution Buffer Dosage

[0097] The solid-phase extraction column was activated sequentially with 3 mL of methanol and 3 mL of water. 2 mL of bone pain relief tincture was accurately loaded onto the column, followed by rinsing with 3 mL of 2% formic acid aqueous solution and 3 mL of methanol. The eluents were discarded. The column was then eluted with 6 mL, 9 mL, and 12 mL of 5% ammonia-methanol solution, respectively. The eluents were collected and evaporated to dryness in a water bath in an evaporating dish. The residue was dissolved in methanol and transferred to a 2 mL volumetric flask. Methanol was added to the mark, the mixture was shaken well, and the solution was filtered. The filtrate was then collected. The results are shown in Table 7.

[0098] Table 7. Investigation of the dosage of 5% ammonia water and methanol (n=2)

[0099]

[0100] The results in the table above show that 6 mL, 9 mL, and 12 mL of 5% ammonia and methanol can all elute artemisinin, artemisinin, and benzoyl aconitine, with 6 mL being the preferred eluent volume.

[0101] Experimental Example 3: Methodological Validation

[0102] Preparation of reference solution: Weigh appropriate amounts of artemisinin, artemisinogenin, and benzoyl aconitine reference standards, and add methanol to prepare a mixed reference solution containing 120 μg of artemisinin, 60 μg of artemisinogenin, and 40 μg of benzoyl aconitine per 1 mL.

[0103] 3.1 System Suitability Test

[0104] According to the method of this invention, a mixed reference solution of aconitine, aconitine and benzoyl aconitine was injected into the liquid chromatograph, and five injections were performed consecutively. The peak areas were recorded and the RSD values ​​were calculated. The results are shown in Table 8. The injection precision was good, the theoretical plate number of benzoyl aconitine was greater than 8000, the resolution of each peak was greater than 1.5, and the symmetry factor was 0.95 to 1.05, indicating that the system has good applicability.

[0105] Table 8 Results of the injection precision test (n=5)

[0106] Component name Peak area 1 Peak area 2 Peak area 3 Peak area 4 Peak area 5 average value RSD% Artemisia capillaris on snow 387.263 377.781 388.135 379.978 383.109 383.253 1.17 Snow on a branch of artemisia annua 274.823 279.127 277.269 283.605 273.635 277.692 1.42 Benzoyl aconitine 167.473 165.189 166.265 170.476 165.373 166.955 1.30

[0107] 3.2 Linear

[0108] Preparation of reference stock solution: Take appropriate amounts of artemisinin, artemisinin-enrichin, and benzoyl aconitine reference standards, add methanol to prepare a mixed reference stock solution containing 404.6544 μg of artemisinin, 199.10904 μg of artemisinin, and 872.615 μg of benzoyl aconitine per 1 mL.

[0109] Accurately measure 1 mL, 1 mL, 1 mL, 1 mL, and 1 mL of the above mixed reference standard stock solution into 2 mL, 5 mL, 10 mL, 50 mL, and 100 mL volumetric flasks, respectively. Dilute to the mark with methanol, shake well, and filter. Inject 2 μL each of methanol (blank) and the above reference standard solution into the high-performance liquid chromatograph (HPLC), record the chromatograms, and determine the peak areas. Plot standard curves for each reference standard with the reference standard concentration as the x-axis and the peak area as the y-axis, and calculate the linear regression equation, correlation coefficient, and linear range. The results are shown in Tables 9, 10, and 11. Figure 8 , Figure 9 , Figure 10 .

[0110] Table 9. Linear Relationship of Artemisinin in Snowdrop

[0111] Concentration μg / mL 0 4.046544 8.093088 40.46544 80.93088 202.3272 404.6544 Peak area 0 35.093 74.712 399.827 783.014 1929.536 3745.179

[0112] The linear regression equation for artemisia capillaris is y = 9.2656x + 17.67, with a correlation coefficient r = 0.9997.

[0113] Table 10. Investigation of the linear relationship between Artemisia annua and Artemisia selengensis.

[0114] Concentration μg / mL 0 1.9910904 3.982181 19.910904 39.82181 99.55452 199.10904 Peak area 0 25.108 65.392 278.663 557.579 1380.653 2733.864

[0115] The linear regression equation for Artemisia annua is: y = 13.718x + 7.18, with a correlation coefficient r = 0.99996.

[0116] Table 11. Linearity of Benzoyl Aconitine

[0117] Concentration μg / mL 0 8.72615 17.4523 87.2615 174.523 436.3075 872.615 Peak area 0 76.129 159.400 761.865 1536.760 3881.155 7684.523

[0118] The linear regression equation for benzoyl aconitine is: y = 8.81907x + 2.39720, with a correlation coefficient r = 0.99997.

[0119] The experimental results showed that, within the concentration range of 0–404.6544 μg / mL, the peak area of ​​artemisinin showed a good linear relationship with the concentration when the injection volume was 2 μL; within the concentration range of 0–199.10904 μg / mL, the peak area of ​​artemisinin showed a good linear relationship with the concentration when the injection volume was 2 μL; and within the concentration range of 0–872.615 μg / mL, the peak area of ​​benzoyl aconitine showed a good linear relationship with the concentration when the injection volume was 2 μL.

[0120] 3.3 Specificity Examination

[0121] Take a negative sample of this product (lacking Artemisia selengensis), prepare a negative sample solution according to the preparation method of the test solution, and determine it. The results show no interfering peaks at the corresponding retention times of the reference standard peak, indicating good method specificity. (See attached image) Figure 11 .

[0122] 3.4 Stability of the test solution

[0123] Bone Pain Relief Tincture was prepared according to the method of this invention, and the contents and RSD values ​​of Artemisinin, Artemisinogenin, and Benzoyl Aconitine were measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 16h, 20h, 24h, 30h, 36h, 40h, and 48h, respectively. The results are shown in Table 12. This indicates that the test solution is relatively stable within 48h.

[0124] Table 12 Stability Study of Test Sample Solutions

[0125]

[0126]

[0127] 3.5 Repeatability

[0128] Six test solutions were prepared from the bone pain relief tincture according to the method of this invention. The contents and RSD values ​​of artemisinin, artemisinin, and benzoyl aconitine were determined and calculated according to the method. The results are shown in Table 13. This indicates that the method has good repeatability.

[0129] Table 13 Stability Study of Test Sample Solutions (n=6)

[0130] serial number 1 2 3 4 5 6 mean RSD% Artemisia capillaris content (μg / mL) 101 106 104 106 103 103 104 1.62 Artemisia selengenide content (μg / mL) 53 54 54 55 53 52 53 1.79 Benzoyl aconitine content (μg / mL) 49 48 50 49 49 48 49 1.90 Total amount (μg / mL) 203 208 208 210 205 203 206 1.42

[0131] 3.6 Recovery rate

[0132] Accurately measure 1 mL of bone pain relief tincture with known content (6 portions in total), add a mixed reference solution of artemisinin, artemisinogenin, and benzoyl aconitine (artemisinin 107 μg / mL, artemisinogenin 65 μg / mL, benzoyl aconitine 43 μg / mL), prepare the sample according to the method of this invention, inject 2 μL into the ultra-high performance liquid chromatograph, determine the content, and calculate the recovery rate according to the following formula. The results show that the average recovery rate of artemisinin is 98%, with an RSD of 3.04%; the average recovery rate of artemisinogenin is 106%, with an RSD of 4.09%, indicating that the method has good accuracy; the average recovery rate of benzoyl aconitine is 95%, with an RSD of 3.99%. The results are shown in Tables 14, 15, and 16.

[0133]

[0134] Table 14 Results of Artemisinin Recovery Experiment (n=6)

[0135]

[0136] Table 15 Results of Artemisinin Recovery Experiment (n=6)

[0137]

[0138] Table 16. Experimental results of the recovery rate of benzoyl aconitine (n=6)

[0139]

[0140] 3.7 Intermediate Precision Examination

[0141] The results of different analysts using different instruments at different times to measure the same batch of bone pain relief tincture were examined. The results are shown in Table 17. There was no significant difference in the content results, indicating that the intermediate precision of this method is good.

[0142] Table 17 Intermediate Precision Results (n=3)

[0143]

[0144] 3.8 Content Limit

[0145] According to the method of this invention, 12 batches of the finished product of Gutongling Tincture were tested. The results showed that the total amount of artemisinin, artemisinogenin and benzoyl aconitine ranged from 193 to 298 μg / mL, with an average value of 232 μg / mL. See Table 18.

[0146] Table 18. Results of determination of effective ingredient content of 12 batches of Gutongling Tincture (n=2) using Artemisia argyi.

[0147]

[0148] In summary, the method of this invention can simultaneously determine the content of artemisinin, artemisinogenin, and benzoyl aconitine in bone pain relief tincture. Furthermore, the method is simple to operate, requires a small amount of organic solvent, and eliminates the highly toxic organic reagent chloroform, making it more environmentally friendly and reducing harm to the human body. The method of this invention exhibits good linearity, repeatability, stability, and recovery rate, and can effectively control the quality of bone pain relief tincture.

Claims

1. A method for detecting the content of active ingredients in Bone Pain Relief Tincture, characterized in that: Includes the following steps: a. Preparation of the test solution: Activate the mixed cation exchange solid-phase extraction column with methanol and water respectively, and load 1-5 mL of bone pain relief tincture onto the column. Elute the solid-phase extraction column with 2% formic acid aqueous solution and methanol in sequence. Discard the eluent, elute with 5% ammonia-methanol solution, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 2 mL, shake well and filter. Take the filtrate as the test solution. b. Preparation of reference solution: Weigh appropriate amounts of artemisinin, artemisinin and benzoyl aconitine reference standards, add methanol to prepare a mixed reference solution containing 120 μg of artemisinin, 60 μg of artemisinin and 20 μg of benzoyl aconitine per 1 mL. c. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile and phosphate buffer solution were used as the mobile phase for gradient elution; the detection wavelength was 210 nm; the column temperature was 25–40 °C; the flow rate was 0.25–1.0 mL / min; the concentration of the phosphate buffer solution was 1–25 mmol / L; and the pH value was 7.

3. The gradient elution is as follows: The time is 0–12 min, the acetonitrile concentration is 20–27%, and the phosphate buffer solution concentration is 80–73%. The time is 12–12.1 min, the acetonitrile concentration is 27–70%, and the phosphate buffer solution concentration is 73–30%. The time was 12.1–17 min, the acetonitrile content was 70%, and the phosphate buffer solution was 30%. The time is 17–17.1 min, the acetonitrile content is 70–20%, and the phosphate buffer solution content is 30–80%. The time was 17.1–25 min, the acetonitrile concentration was 20%, and the phosphate buffer solution was 80%. d. Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the high performance liquid chromatograph, and determine the result.

2. The method for detecting the content of active ingredients in the bone pain relief tincture according to claim 1, characterized in that: Meet at least one of the following: The mixed-type cation exchange solid-phase extraction column was activated with 0-6 mL of methanol and 0-6 mL of water, respectively. Measure 2 mL of bone pain relief tincture and load it onto the sample; The solid-phase extraction column was rinsed sequentially with 3-12 mL of 2% formic acid aqueous solution and 3-9 mL of methanol. Elute with 6-12 mL of 5% ammonia-methanol solution.

3. The method for detecting the content of active ingredients in bone pain relief tincture according to claim 2, characterized in that: The mixed-type cation exchange solid-phase extraction column was activated with 3 mL of methanol and 3 mL of water, respectively.

4. The method for detecting the content of active ingredients in bone pain relief tincture according to claim 2, characterized in that: The solid-phase extraction column was rinsed sequentially with 3 mL of 2% formic acid aqueous solution and 3 mL of methanol.

5. The method for detecting the content of active ingredients in bone pain relief tincture according to claim 2, characterized in that: Elute with 6 mL of 5% ammonia-methanol solution.

6. The method for detecting the content of active ingredients in the bone pain relief tincture according to claim 1, characterized in that: The phosphate buffer solution is a 5 mmol / L disodium hydrogen phosphate solution.

7. The method for detecting the content of active ingredients in the bone pain relief tincture according to claim 1, characterized in that: In step c, the column temperature is 35°C and the flow rate is 1.0 mL / min.

8. The method for detecting the content of active ingredients in the bone pain relief tincture according to claim 1, characterized in that: In step c, the theoretical plate number, calculated based on the benzoyl aconitine peak, should be no less than 8000.

9. The method for detecting the content of active ingredients in the bone pain relief tincture according to claim 1, characterized in that: In step d, precisely pipette 2 μL each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph.

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