Method for detecting content of effective components in ostealgia tincture

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for detecting the content of effective components in ostalgia tincture. The technical problem to be solved by the invention is to provide the method for detecting the content of the effective components in the ostealgia tincture, which comprises the following steps: a, preparation of a test solution: measuring the ostealgia tincture, loading the ostealgia tincture to an activated solid-phase extraction column, leaching, eluting, collecting the eluate, evaporating to dryness, fixing the volume, filtering, and taking the subsequent filtrate to obtain the test solution; b, preparing a reference substance solution; c, chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; acetonitrile and a phosphoric acid buffer salt solution are used as mobile phases for gradient elution; the detection wavelength is 210 nm, and the column temperature is 25-40 DEG C; the flow velocity is 0.25 to 1.0 mL / min; and d, determination: respectively and precisely sucking the reference substance solution and the test solution, injecting into a high performance liquid chromatograph, and determining. The method is simple and easy to operate and short in time consumption, and solves the problem of few quality control index detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for detecting the content of active ingredients in GUTONGLING TINCTURE. Background Art

[0002] GUTONGLING TINCTURE is composed of six herbs, namely ACONITUM BRACHYPODUM Diels, dried ginger, DRACONIS SANGUIS, borneol, frankincense, and myrrh, and has the effects of warming the meridians to dispel cold, expelling wind and activating blood circulation, and dredging collaterals to relieve pain. It is a commonly used medicine for treating lumbar and cervical spondylosis, rheumatoid arthritis, and scapulohumeral periarthritis. In GUTONGLING TINCTURE, ACONITUM BRACHYPODUM Diels is the monarch herb, which plays a significant role in expelling wind and dampness and promoting blood circulation to relieve pain. Its main active ingredients are alkaloids, including aconitine A and aconitine G, etc.

[0003] In the Chinese Pharmacopoeia 2020 Edition, the content detection of GUTONGLING TINCTURE only stipulates the determination of the content of aconitine A. The method is as follows: Precise measure 25 mL of this product, place it in an evaporating dish, evaporate it to about 10 mL in a water bath, adjust the pH value to 2 - 3 with dilute hydrochloric acid, transfer it to a separating funnel, wash the container with an appropriate amount of 0.1 mol / L hydrochloric acid, and incorporate the washing liquid into the separating funnel to make the total amount of acid solution about 25 mL. Gently shake and wash it twice with chloroform, combine the chloroform liquid, wash it twice with 0.1 mol / L hydrochloric acid, combine the above acid aqueous solution, add 2 g of sodium chloride, gently shake to dissolve it, adjust the pH value to 9 - 10 with ammonia test solution, shake and extract it 5 times with chloroform, combine the chloroform liquid, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, collect the filtrate, wash the residue twice with chloroform, 5 mL each time, combine the filtrate, evaporate it to nearly dry on a water bath at 60 °C, add 1 mL of absolute ethanol, naturally evaporate it to dryness, dissolve it with methanol and transfer it to a 25 mL volumetric flask, add methanol to the scale, ultrasonically treat it (power 250 W, frequency 33 kHz) for 5 minutes, let it cool, add methanol to the scale, shake well, let it stand, filter, and take the subsequent filtrate to obtain the solution; respectively precisely absorb 10 μL of the reference substance solution and the test solution, inject them into the liquid chromatograph for determination, and then obtain the result.

[0004] This method has complex operations, long time consumption, is prone to errors, and has poor reproducibility. Under this chromatographic condition, the target peak and the interference peak are not completely separated, resulting in inaccurate results; moreover, this method can only detect aconitine A. Therefore, it is necessary to develop an accurate, stable, and easy-to-operate detection method that can determine more alkaloid components in ACONITUM BRACHYPODUM Diels in order 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 GUTONGLING tincture. This method is simple, easy to operate, time-consuming, and can simultaneously detect the contents of aconitine A, aconitine G, and benzoylaconitine in Aconitum brachypodum Diels. It solves the problems existing in the prior art, such as the overly complex preparation method of the test solution, which is time-consuming and laborious; the detection target peaks cannot be completely separated, and 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 technical problems is to provide a method for detecting the content of active ingredients in GUTONGLING tincture, which includes the following steps:

[0007] a. Preparation of the test solution: Measure GUTONGLING tincture, load it onto an activated solid-phase extraction column, wash, elute, collect the eluate, evaporate to dryness, make up the volume, and filter to obtain the test solution;

[0008] b. Preparation of the reference solution: Weigh appropriate amounts of aconitine A, aconitine G, and benzoylaconitine reference substances, add methanol to make a mixed reference solution containing 120 μg of aconitine A, 60 μg of aconitine G, and 20 μg of benzoylaconitine per 1 mL;

[0009] c. Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile and phosphate buffer solution as the mobile phase for gradient elution; the detection wavelength is 210 nm, the column temperature is 25 - 40 °C; the flow rate is 0.25 - 1.0 mL / min;

[0010] d. Determination method: Precisely pipette 2 μL each of the reference solution and the test solution, inject them into a high-performance liquid chromatograph for determination, and then obtain the results.

[0011] Further, in step a, the preparation method of the test solution is as follows: Activate a mixed cation exchange solid-phase extraction column with methanol and water respectively, measure 1 - 5 mL of GUTONGLING tincture for loading, wash the solid-phase extraction column successively with 2% formic acid aqueous solution and methanol, discard the washing solution, elute with 5% ammonia methanol solution, collect the eluate, evaporate to dryness, dissolve with methanol and make up the volume to 2 mL, shake well and filter to obtain the test solution.

[0012] Further, activate the mixed cation exchange solid-phase extraction column with 0 - 6 mL of methanol and 0 - 6 mL of water respectively.

[0013] Preferably; activate the mixed cation exchange solid-phase extraction column with 3 mL of methanol and 3 mL of water respectively.

[0014] Further, measure 2 mL of GUTONGLING tincture for loading.

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

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

[0017] Furthermore, it is eluted with 6 - 12 mL of 5% ammonia methanol solution.

[0018] Preferably, it is eluted with 6 mL of 5% ammonia methanol solution.

[0019] Furthermore, 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] Furthermore, the gradient elution is as follows:

[0022] At a time of 0 - 12 min, acetonitrile is 20 - 27%, and the phosphate buffer solution is 80 - 73%;

[0023] At a time of 12 - 12.1 min, acetonitrile is 27 - 70%, and the phosphate buffer solution is 73 - 30%;

[0024] At a time of 12.1 - 17 min, acetonitrile is 70%, and the phosphate buffer solution is 30%;

[0025] At a time of 17 - 17.1 min, acetonitrile is 70 - 20%, and the phosphate buffer solution is 30 - 80%;

[0026] At a time of 17.1 - 25 min, acetonitrile is 20%, and the phosphate buffer solution is 80%.

[0027] Furthermore, 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 benzoylaconine peak should be not less than 8000.

[0029] The inventor investigated various mobile phase gradient elution conditions and found that under the above mobile phase gradient elution conditions, aconitine A, aconitine G, and benzoylaconine can all achieve complete separation.

[0030] The beneficial effects of the present invention are:

[0031] The method of the present invention uses a high performance liquid chromatograph. By optimizing the preparation method of the test sample and the detection chromatographic conditions, it can simultaneously determine the contents of hypaconitine A, hypaconitine G, and benzoylaconine in Gutingling tincture. The method of the present invention has good linearity, repeatability, stability, and recovery rate, and can well control the quality of Gutingling tincture. The method of the present invention optimizes the preparation process of the test sample, and uses a mixed cation exchange solid phase extraction column to purify and prepare the test sample solution. The operation is simple, the amount of organic solvent used is small, and the highly toxic organic reagent chloroform is removed, which is more environmentally friendly and reduces the harm to the human body. The method of the present invention optimizes the elution program of the mobile phase in the chromatographic conditions, so that the three target peaks of hypaconitine A, hypaconitine G, and benzoylaconine can be completely separated. Moreover, the entire analysis process takes a short time and the results are accurate and reliable. Description of the Drawings

[0032] Figure 1 Chromatogram of hypaconitine A, hypaconitine G, and benzoylaconine in Gutingling tincture measured in Example 1 (1 is hypaconitine A, 2 is hypaconitine G, 3 is benzoylaconine).

[0033] Figure 2 Chromatogram of hypaconitine A in Gutingling tincture measured in Comparative Example 1.

[0034] Figure 3 Chromatogram of separating hypaconitine A and hypaconitine G in Gutingling tincture with different mobile phases.

[0035] Figure 4 Chromatogram of separating hypaconitine A, hypaconitine G, and benzoylaconine in Gutingling tincture with different salt concentrations.

[0036] Figure 5 Chromatogram of separating hypaconitine A, hypaconitine G, and benzoylaconine in Gutingling tincture with different chromatographic columns.

[0037] Figure 6 Chromatogram of separating hypaconitine A, hypaconitine G, and benzoylaconine in Gutingling tincture at different column temperatures.

[0038] Figure 7 Chromatogram of separating hypaconitine A, hypaconitine G, and benzoylaconine in Gutingling tincture with different instruments.

[0039] Figure 8 Standard curve of hypaconitine A for linear relationship investigation.

[0040] Figure 9 Standard curve of hypaconitine G for linear relationship investigation.

[0041] Figure 10 The standard curve of benzoylaconitine was investigated for linear relationship.

[0042] Figure 11 Chromatograms obtained for specificity investigation (from top to bottom are Gutongling tincture sample, benzoylaconitine, artemisinin, artemisinin A, and Gutongling tincture negative sample. DETAILED DESCRIPTION

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

[0044] Example 1

[0045] Preparation of the test solution: Activate the solid phase extraction column with mixed cation exchange adsorbent as filler with 3 mL of methanol and 3 mL of water respectively, accurately measure 2 mL of Gutongling tincture and load it, elute the solid phase extraction column with 3 mL of 2% formic acid aqueous solution and 3 mL of methanol in turn, 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 scale, shake well and filter, and take the filtrate.

[0046] Preparation of reference solution: Weigh appropriate amounts of artemisinin A, artemisinin heptazone, and benzoylaconitrile reference substances, add methanol to make a mixed reference solution containing 120 μg of artemisinin A, 60 μg of artemisinin heptazone, and 20 μg of benzoylaconitrile per 1 mL.

[0047] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler (4.6×105 mm, 5 μm); acetonitrile and 5 mmol / L phosphate buffer were used as mobile phases, 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 benzoylaconitine peak should be no less than 8000.

[0048] Determination method: Accurately pipette 2 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine. Figure 1 .

[0049] According to the external standard method, the amount of artemisinin (C 22 H 33 NO2), Artemisia sutchuenensis (C 22 H 31 NO3), and benzoylaconitine (C 32 H 45 NO 10 ) The total amount of the three shall not be less than 154 mg.

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

[0051] Time (min) 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] In Comparative Example 1, the detection was carried out using the drug standard method in the Chinese Pharmacopoeia (2020 edition).

[0053] Preparation of test solution: Precisely measure 25 mL of this product, place it in an evaporating dish, evaporate it to about 10 mL in a water bath, adjust the pH value to 2 - 3 with dilute hydrochloric acid, transfer it to a separating funnel, wash the container with an appropriate amount of 0.1 mol / L hydrochloric acid, and incorporate the washing solution into the separating funnel to make the total amount of acid solution about 25 mL. Gently shake and wash with chloroform twice (10 mL, 5 mL), combine the chloroform solutions, wash with 0.1 mol / L hydrochloric acid twice (5 mL, 3 mL), combine the above acid aqueous solutions, add 2 g of sodium chloride, gently shake to dissolve, adjust the pH value to 9 - 10 with ammonia test solution, extract with chloroform by shaking 5 times (25 mL, 15 mL, 10 mL, 10 mL, 10 mL), combine the chloroform solutions, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, collect the filtrate, wash the residue with chloroform twice, 5 mL each time, combine the filtrates, evaporate to nearly dry on a water bath at 60 °C, add 1 mL of anhydrous ethanol, allow to evaporate naturally, dissolve with methanol and transfer to a 25 mL volumetric flask, add methanol to the scale, ultrasonically treat (power 250 W, frequency 33 kHz) for 5 minutes, cool, add methanol to the scale, shake well, let stand, filter, and take the subsequent filtrate, that is, obtain.

[0054] Preparation of reference solution: Precisely weigh an appropriate amount of aconitine A reference substance, and make a solution containing 100 μg of aconitine A per 1 mL with methanol.

[0055] Chromatographic conditions and system suitability test: Using octadecylsilane chemically bonded silica as the filler; using methanol - phosphate buffer solution (take 1.97 g of disodium hydrogen phosphate and 0.22 g of potassium dihydrogen phosphate, dissolve in water and dilute to 1000 mL, adjust the pH value to 7.3 with 80% phosphoric acid solution) (72:28) as the mobile phase; the detection wavelength is 210 nm. The number of theoretical plates calculated based on the aconitine A peak should be not less than 1500.

[0056] Precisely pipette 10 μL each of the reference solution and the test solution, and inject them into the liquid chromatograph for determination. The results are shown in Figure 2 .

[0057] Test Example 1 Selection of chromatographic conditions

[0058] Use the test solution prepared in Example 1 as the sample.

[0059] 1.1 Selection of mobile phase

[0060] The mobile phases of methanol-5 mmol / L phosphate buffer (gradient elution was carried out according to Table 2) and acetonitrile-5 mmol / L phosphate buffer (gradient elution was carried out according to Example 1) were investigated separately. The results showed that both mobile phases could separate the target peak from the interfering peaks. Only the elution orders of aconitine A and aconitine G were reversed. Moreover, when acetonitrile was used as the mobile phase, the chromatogram baseline was smoother and the peak symmetry was better than that with methanol. Therefore, acetonitrile-phosphate buffer was selected as the mobile phase. See Figure 3 。

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

[0062] Time (min) 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 Investigation of salt concentration

[0064] 1 mmol / L phosphate mixed salt, 5 mmol / L phosphate mixed salt, 25 mmol / L phosphate mixed salt, and 5 mmol / L disodium hydrogen phosphate solution (the pH values of the 4 mobile phases were all adjusted to 7.3) were used for investigation respectively. The results showed that all four mobile phases could separate the target peak. The 5 mmol / L disodium hydrogen phosphate solution was a single salt and was easier to prepare. Finally, 5 mmol / L disodium hydrogen phosphate solution (pH = 7.3) was selected as the mobile phase. See Figure 4 。

[0065] 1.3 Investigation of chromatographic column

[0066] According to the pH value of the mobile phase, chromatographic columns for separating basic compounds from different manufacturers and different models were investigated. Both UPLC and HPLC columns could achieve complete separation of the target peak. See Figure 5 。However, the UPLC column (2.1*100 mm, 1.8 μm) had a smaller particle size and higher pressure, and salts were likely to clog the column. Therefore, a chromatographic column filled with octadecylsilyl silica gel (4.6*150 mm, 5 μm) was selected for the experiment.

[0067] 1.4 Investigation of column temperature

[0068] Column temperatures of 25 °C, 30 °C, 35 °C, and 40 °C were investigated separately, and all could ensure the separation of the target peak. Among them, the separation effect was the best under the conditions of 30 - 40 °C, and the column temperature was selected as 35 °C. See Figure 6 。

[0069] 1.5 Investigation of instrument

[0070] Using the established chromatographic conditions, analysis was carried out on different instruments to investigate the durability of the method. The results showed that the method could achieve complete separation of the target peak on different instruments. See Figure 7 。

[0071] Investigation on the Preparation Method of Test Sample Solution

[0072] 2.1 Selection of Purification Method

[0073] Four purification methods, namely HPD-100 macroporous adsorption resin, basic alumina, 732-type cation exchange resin, and mixed-type cation exchange solid-phase extraction column, were investigated respectively. The results showed that for the test samples purified by the mixed-type cation exchange solid-phase extraction column, the three target peaks could be completely separated and the content was not lost. Therefore, the mixed-type 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 Dosage of Activation Solvent

[0075] Solid-phase extraction cartridges were activated with 0 mL, 1 mL, 3 mL, and 6 mL of methanol and water respectively. 3 mL of GUTONGLING TINCTURE was accurately measured and loaded (in duplicate). It was successively washed with 6 mL of 2% formic acid aqueous solution and methanol respectively, and the washing solutions were discarded. The solid-phase extraction cartridge was further eluted with 6 mL of ammonia-methanol, and the eluate was collected, evaporated to dryness in a water bath in an evaporating dish. The residue was dissolved with methanol and transferred to a 2 mL volumetric flask, and methanol was added to the scale, shaken well and filtered. The subsequent filtrate was obtained. The results are shown in Table 3.

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

[0077]

[0078] The results showed that when the solid-phase extraction column was activated with 3 mL (methanol, water) and 6 mL (methanol, water) of activation solvent, the contents of aconitine methyl ester, aconitine heptyl ester, and benzoylaconine were consistent and relatively high. Since the dosage of organic solvent for activation with 3 mL (methanol, water) was less, the volume of activation solvent was selected as 3 mL.

[0079] 2.3 Investigation on the Loading Volume

[0080] The solid-phase extraction column was activated with 3 mL of methanol and water respectively. 1 mL, 2 mL, and 3 mL of GUTONGLING TINCTURE were accurately measured and loaded respectively. It was successively washed with 6 mL of 2% formic acid aqueous solution and methanol respectively, and the washing solutions were discarded. The extraction cartridge was further eluted with 6 mL of 5% ammonia-methanol, and the eluate was collected, evaporated to dryness in a water bath in an evaporating dish. The residue was dissolved with methanol and transferred to a 2 mL volumetric flask. Methanol was added to the scale, shaken well and filtered. The subsequent filtrate was obtained. The results are shown in Table 4.

[0081] Table 4 Investigation on the Loading Volume (n = 2)

[0082]

[0083]

[0084] The results showed that when the sample loading volume was 3 mL, the solid-phase extraction column was overloaded. When the sample loading volume was 1 mL, the concentration was too low. Therefore, it was appropriate to choose a sample loading volume of 2 mL.

[0085] 2.4 Investigation of the amount of eluent

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

[0087] The solid-phase extraction cartridge was activated successively with 3 mL of methanol and 3 mL of water. 2 mL of GUTONGLING tincture was accurately measured and loaded onto the column. It was then eluted with 3 mL, 6 mL, 9 mL, and 12 mL of 2% formic acid aqueous solution, respectively, and 9 mL of methanol. The eluates were discarded. The extraction cartridge was further eluted with 12 mL of 5% ammonia methanol. The eluate was collected, 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 scale, shaken well and filtered. The subsequent filtrate was obtained. The results are shown in Table 5.

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

[0089]

[0090] The results showed that when the elution volume of 2% formic acid aqueous solution was 3 mL and 6 mL, the contents of bushenine A, bushenine G, and benzoylaconine were basically the same. When the elution volume was greater than or equal to 9 mL, the content of bushenine A decreased to varying degrees. Therefore, the elution volume of 2% formic acid aqueous solution was chosen as 3 mL, which could not only retain the content of the target substances but also achieve the purpose of impurity removal.

[0091] (2) Methanol

[0092] The solid-phase extraction cartridge was activated successively with 3 mL of methanol and 3 mL of water. 2 mL of GUTONGLING tincture was accurately measured and loaded onto the column. It was eluted with 3 mL of 2% formic acid aqueous solution, and then eluted with 3 mL, 6 mL, and 9 mL of methanol, respectively. The eluates were discarded. The extraction column was further eluted with 12 mL of 5% ammonia methanol. The eluate was collected, 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 scale, shaken well and filtered. The subsequent filtrate was obtained. The results are shown in Table 6.

[0093] Table 6 Investigation of the amount of methanol (n = 2)

[0094]

[0095] The results showed that after elution with three different volumes of methanol, the contents of bushenine A, bushenine G, and benzoylaconine were basically the same. Therefore, it was sufficient to choose a methanol amount of 3 mL.

[0096] 2.5 Investigation of the amount of eluent

[0097] The solid-phase extraction column was activated successively with 3 mL each of methanol and water. 2 mL of GUTONGLING tincture was precisely measured and loaded onto the column. It was then eluted successively with 3 mL each of 2% formic acid aqueous solution and methanol. The eluates were discarded. Elution was carried out separately with 6 mL, 9 mL, and 12 mL of 5% ammonia methanol. The eluates were collected, placed in an evaporating dish, and evaporated to dryness in a water bath. The residue was dissolved in methanol and transferred to a 2 mL volumetric flask. Methanol was added to the scale, shaken well, filtered, and the subsequent filtrate was obtained. The results are shown in Table 7.

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

[0099]

[0100] The results in the above table show that 6 mL, 9 mL, and 12 mL of 5% ammonia methanol can all elute bushenghao A, bushenghao G, and benzoylaconine. The preferred eluent dosage is 6 mL.

[0101] Test Example 3 Methodology verification

[0102] Preparation of reference substance solution: Appropriate amounts of bushenghao A, bushenghao G, and benzoylaconine reference substances were weighed and dissolved in methanol to prepare a mixed reference substance solution containing 120 μg of bushenghao A, 60 μg of bushenghao G, and 40 μg of benzoylaconine per 1 mL.

[0103] 3.1 System suitability test

[0104] According to the method of the present invention, a mixed reference substance solution of bushenghao A, bushenghao G, and benzoylaconine was injected into the liquid chromatograph. Five injections were made continuously, the peak areas were recorded, and the RSD value was calculated. The results are shown in Table 8. The injection precision was good. The theoretical plate number of benzoylaconine was greater than 8000, the resolution of each peak was greater than 1.5, and the symmetry factor was 0.95 - 1.05, indicating good system suitability.

[0105] Table 8 Results of investigation on injection precision (n = 5)

[0106] Component name Peak area 1 Peak area 2 Peak area 3 Peak area 4 Peak area 5 Average RSD % Bulleyaconitine A 387.263 377.781 388.135 379.978 383.109 383.253 1.17 Bulleyaconitine G 274.823 279.127 277.269 283.605 273.635 277.692 1.42 Benzoylaconine 167.473 165.189 166.265 170.476 165.373 166.955 1.30

[0107] 3.2 Linearity

[0108] Preparation of reference substance stock solution: Appropriate amounts of bushenghao A, bushenghao G, and benzoylaconine reference substances were weighed and dissolved in methanol to prepare a mixed reference substance stock solution containing 404.6544 μg of bushenghao A, 199.10904 μg of bushenghao G, and 872.615 μg of benzoylaconine per 1 mL.

[0109] Precisely measure 1 mL, 1 mL, 1 mL, 1 mL, and 1 mL of the above mixed reference stock solution respectively, place them in 2 mL, 5 mL, 10 mL, 50 mL, and 100 mL volumetric flasks, dilute to the mark with methanol, shake well, filter. Then, respectively pipette 2 μL of methanol (blank) and the above reference solution into a high performance liquid chromatograph, record the chromatogram, measure the peak areas. Using the reference concentration as the abscissa and the peak area as the ordinate, plot the standard curves of each reference substance, and calculate the linear regression equation, correlation coefficient, and linear range. The results are shown in Table 9, Table 10, Table 11, Figure 8 , Figure 9 , Figure 10 .

[0110] Table 9 Investigation on the linear relationship of aconitine A

[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 of aconitine A is y = 9.2656x + 17.67, and the correlation coefficient r = 0.9997.

[0113] Table 10 Investigation on the linear relationship of aconitine G

[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 of aconitine G is: y = 13.718x + 7.18, and the correlation coefficient r = 0.99996.

[0116] Table 11 Investigation on the linear relationship of benzoylaconine

[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 of benzoylaconine is: y = 8.81907x + 2.39720, and the correlation coefficient r = 0.99997.

[0119] The test results show that within the concentration range of 0 - 404.6544 μg / mL, when the injection volume is 2 μL, there is a good linear relationship between the peak area and concentration of aconitine A; within the range of 0 - 199.10904 μg / mL, when the injection volume is 2 μL, there is a good linear relationship between the peak area and concentration of aconitine G; within the range of 0 - 872.615 μg / mL, when the injection volume is 2 μL, there is a good linear relationship between the peak area and concentration of benzoylaconine.

[0120] 3.3 Specificity investigation

[0121] Take the negative sample of this product (lacking Bulleyan Monkshood Root), prepare the negative sample solution according to the preparation method of the test solution and determine. As a result, at the retention time position corresponding to the chromatographic peak of the reference substance, there is no interfering peak, indicating that the method has good specificity. See Figure 11 。

[0122] 3.4 Stability of the test solution

[0123] Take GUTONGLING TINCTURE, prepare the sample according to the method of the present invention, and determine at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 16h, 20h, 24h, 30h, 36h, 40h, and 48h respectively. Calculate the contents and RSD values of Bulleyaconitine A, Bulleyaconitine G, and Benzoylaconine. The results are shown in Table 12. It shows that the test solution is relatively stable within 48h.

[0124] Table 12 Investigation on the stability of the test solution

[0125]

[0126]

[0127] 3.5 Repeatability

[0128] Take GUTONGLING TINCTURE and prepare 6 portions of the test solution according to the method of the present invention. Determine according to the law, and calculate the contents and RSD values of Bulleyaconitine A, Bulleyaconitine G, and Benzoylaconine. The results are shown in Table 13. It shows that the method has good repeatability.

[0129] Table 13 Investigation on the stability of the test solution (n = 6)

[0130] Number 1 2 3 4 5 6 Mean RSD % Content of Bulleyaconitine A (μg / mL) 101 106 104 106 103 103 104 1.62 Content of Bulleyaconitine G (μg / mL) 53 54 54 55 53 52 53 1.79 Content of Benzoylaconine (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] Precisely measure 1 mL of GUTONGLING TINCTURE with known content (6 portions in total), add the mixed reference substance solution of Bulleyaconitine A, Bulleyaconitine G, and Benzoylaconine (Bulleyaconitine A 107 μg / mL, Bulleyaconitine G 65 μg / mL, Benzoylaconine 43 μg / mL), prepare the sample according to the method of the present invention, take 2 μL and inject it into the ultra-high performance liquid chromatograph to determine the content. Calculate the recovery rate according to the following formula. As a result, the average recovery rate of Bulleyaconitine A is 98%, and the RSD value is 3.04%; the average recovery rate of Bulleyaconitine G is 106%, and the RSD value is 4.09%, indicating that the method has good accuracy; the average recovery rate of Benzoylaconine is 95%, and the RSD value is 3.99%. The results are shown in Table 14, Table 15, and Table 16.

[0133]

[0135] Table 14 Results of the recovery test of snowdrop aconitine A (n = 6)

[0136]

[0137] Table 15 Results of the recovery test of snowdrop aconitine G (n = 6)

[0138]

[0139] Table 16 Results of the recovery test of benzoylaconine (n = 6)

[0140]

[0141] 3.7 Intermediate precision investigation

[0142] The same batch of Gutingling tincture was determined by different analysts at different times using different instruments. The results are shown in Table 17. There was no significant difference in the content results, indicating that the method has good intermediate precision.

[0143] Table 17 Results of intermediate precision (n = 3)

[0144]

[0145] 3.8 Content limit

[0146] According to the method of the present invention, 12 batches of Gutingling tincture finished products were determined. The results showed that the total amount of snowdrop aconitine A, snowdrop aconitine G and benzoylaconine was 193 - 298 μg / mL, with an average value of 232 μg / mL. See Table 18.

[0147] Table 18 Results of the determination of the effective components of snowdrop aconitine in 12 batches of Gutingling tincture (n = 2)

[0148]

[0149] In summary, when using the method of the present invention to determine Gutingling tincture, the contents of snowdrop aconitine A, snowdrop aconitine G and benzoylaconine in Gutingling tincture can be determined simultaneously. Moreover, the operation is simple, the amount of organic solvent used is small, and the highly toxic organic reagent chloroform is removed, which is more environmentally friendly and reduces the harm to the human body; the method of the present invention has good linearity, repeatability, stability and recovery rate, and can well control the quality of Gutingling tincture.

Claims

1. A method for detecting the content of the active ingredients in GUTONGLING TINCTURE, characterized in that: The steps include: a. Preparation of the test solution: Take a quantity of Gutongling tincture, load it onto the activated solid phase extraction column, rinse, elute, collect the eluate, evaporate to dryness, fix the volume, filter, and obtain the filtrate; b. Preparation of reference solution: weigh appropriate amounts of artemisinin, artemisinin, and benzoylaconitine reference substances, and add methanol to prepare a mixed reference solution containing 120 μg of artemisinin, 60 μg of artemisinin, and 20 μg of benzoylaconitine per 1 mL; c. Chromatographic conditions: octadecylsilane bonded silica gel as filler; acetonitrile and phosphate buffered saline as mobile phases, gradient elution; detection wavelength 210nm, column temperature 25-40℃; flow rate 0.25-1.0mL / min; d. Determination method: Accurately aspirate the reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

2. The content detection method of the active ingredient in the GUTONGLING TINCTURE according to claim 1, characterized in that: In step a, the preparation method of the test solution is: activate the mixed cation exchange solid phase extraction column with methanol and water respectively, measure 1-5 mL of Gutongling tincture and load it, elute the solid phase extraction column with 2% formic acid aqueous solution and methanol in turn, discard the eluent, elute with 5% ammonia methanol solution, collect the eluent, evaporate to dryness, dissolve it in methanol to make the volume 2 mL, shake well and filter, and take the filtrate to obtain.

3. The method for detecting the content of the active ingredient in GUTONGLING TINCTURE according to claim 2, wherein: At least one of the following is met: Activate the mixed cation exchange solid phase extraction column with 0-6 mL of methanol and 0-6 mL of water respectively; preferably, 3 mL of methanol and 3 mL of water; Measure 2 mL of Gutongling tincture and add it to the sample; The solid phase extraction column is sequentially eluted with 3-12 mL 2% formic acid aqueous solution and 3-9 mL methanol; preferably, the solid phase extraction column is sequentially eluted with 3 mL 2% formic acid aqueous solution and 3 mL methanol; The mixture is eluted with 6-12 mL of 5% ammonia methanol solution; preferably, eluted with 6 mL of 5% ammonia methanol solution.

4. The content detection method of the active ingredient in the GUTONGLING tincture according to claim 1, characterized in that: In step c, the concentration of the phosphate buffered saline solution is 1-25 mmol / L; and the pH value is 7.

3.

5. The method for detecting the content of the active ingredient in GUTONGLING TINCTURE according to claim 4, wherein: The phosphate buffered saline solution is a 5 mmol / L disodium hydrogen phosphate solution.

6. The method for detecting the content of the active ingredient in GUTONGLING tincture according to claim 1, wherein: In step c, the gradient elution is: The time is 0 to 12 minutes, the acetonitrile is 20 to 27%, and the phosphate buffered saline is 80 to 73%; The time is 12 to 12.1 min, the acetonitrile is 27 to 70%, and the phosphate buffered saline is 73 to 30%; The time is 12.1 to 17 minutes, the acetonitrile is 70%, and the phosphate buffered saline is 30%; The time is 17 to 17.1 min, the acetonitrile is 70 to 20%, and the phosphate buffered saline is 30 to 80%; The time is 17.1 to 25 minutes, the acetonitrile is 20%, and the phosphate buffered saline solution is 80%.

7. The method for detecting the content of the active ingredient in GUTONGLING TINCTURE according to claim 1, wherein: In step c, the column temperature is 35° C. and the flow rate is 1.0 mL / min.

8. The content detection method of the active ingredient in GUTONGLING TINCTURE according to claim 1, characterized in that: In step c, the number of theoretical plates calculated based on the benzoylaconitum alkali peak should be no less than 8000.

9. The content detection method of the active ingredients in GUTONGLING TINCTURE according to claim 1, characterized in that: In step d, accurately pipette 2 μL of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph.

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