A method for constructing a characteristic chromatogram of a water extract of grass wu and a formula granule and application thereof

Abstract

This invention provides a method for constructing characteristic chromatograms of processed Aconitum carmichaelii water extract and its formulation granules, and its application. The method for constructing characteristic chromatograms of processed Aconitum carmichaelii involves using high-performance liquid chromatography (HPLC) to obtain the characteristic chromatograms of the analyte. Specific HPLC conditions are provided, including a column packed with octadecylsilane-bonded silica gel; acetonitrile as mobile phase A; and an aqueous solution containing 0.18%-0.22% triethylamine and 0.18%-0.22% glacial acetic acid as mobile phase B, eluted according to a specific gradient program. The detection wavelengths are 238-242 nm for 0-14 minutes, 278-282 nm for 14-38.5 minutes, and 238-242 nm for 38.5-59 minutes. The characteristic chromatograms obtained by this invention provide comprehensive material information. The relevant information in the characteristic chromatograms characterized by this method can be used to achieve overall quality control of processed Aconitum carmichaelii and identification of easily confused processed Aconitum carmichaelii products.

Description

Technical Field

[0001] This invention relates to the field of identification of traditional Chinese medicine, specifically to a method and application for constructing characteristic spectra of processed Aconitum carmichaelii water extract and its formulation granules. Background Technology

[0002] Prepared aconite is the dried tuberous root of Aconitum kusnezoffii Reichb., a plant belonging to the Ranunculaceae family. The original plant is a perennial herb.

[0003] Aconitum carmichaelii has a wide range of uses, applied in many fields such as traditional Chinese medicine decoction pieces, prepared Chinese medicines, and veterinary drugs. Its clinical applications are extensive; numerous literature and patents contain characteristic methods for Aconitum carmichaelii raw materials or decoction pieces. Furthermore, the characteristic methods for Aconitum carmichaelii raw materials and decoction pieces are primarily based on the chromatographic conditions under the "processed Aconitum carmichaelii" assay. Moreover, the material basis of raw Aconitum carmichaelii raw materials or decoction pieces differs significantly from that of the processed Aconitum carmichaelii aqueous extract obtained through extraction, concentration, and drying. Currently, the publicly disclosed chromatographic conditions for the characteristic chromatograms applicable to processed Aconitum carmichaelii aqueous extracts and their preparations are as follows:

[0004] The chromatographic column was a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm); the flow rate was 0.3 ml / min; the column temperature was 30 °C; the detection wavelength was 240 nm; the mobile phase was acetonitrile-0.1% phosphoric acid solution for gradient elution, as described in Comparative Example 1.

[0005] The existing technology discloses characteristic spectra of aconite root water extract and its preparations, such as Figure 1 As shown, it has few characteristic peaks and incomplete characterization information. From the chromatogram, a large portion of the chromatographic peaks are accumulated in the first 5 minutes, resulting in poor separation and hindering overall quality control. Summary of the Invention

[0006] Existing methods for constructing characteristic spectra yield characteristic spectra of processed aconite water extract and its preparations with fewer characteristic peaks, which is detrimental to overall quality control. This invention provides a method for constructing characteristic spectra with more characteristic peaks, which can better achieve overall quality control of processed aconite water extract and its preparations. Furthermore, this invention also provides applications of the characteristic spectra constructed by this method in the overall quality control of processed aconite water extract and its preparations, as well as applications of the characteristic spectra constructed by this method in the identification of easily confused processed aconite products.

[0007] A method for constructing characteristic chromatograms of processed aconite, employing high-performance liquid chromatography (HPLC) to obtain the characteristic chromatograms of the analyte; the chromatographic conditions for the HPLC method are as follows:

[0008] A chromatographic column packed with octadecylsilane-bonded silica gel was used; acetonitrile was used as mobile phase A, and an aqueous solution containing 0.18%-0.22% triethylamine and 0.18%-0.22% glacial acetic acid was used as mobile phase B. Elution was performed according to the following gradient program:

[0009]

[0010] The detection wavelengths for 0–14 minutes are 238–242 nm, for 14–38.5 minutes are 278–282 nm, and for 38.5–59 minutes are 238–242 nm.

[0011] In the chromatographic conditions of the high performance liquid chromatography method, the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

[0012] And / or, the column temperature is 28-32℃;

[0013] And / or, the flow rate is 0.9-1.1 ml / min;

[0014] And / or, the theoretical plate number calculated based on the benzoyl neoaconitine peak should be no less than 5000.

[0015] In the high-performance liquid chromatography method described above, the chromatographic column is Waters Xselect HSS T3 or phenomenex Gemini C18. Or SHIMADZU Shim-pack GIST C18-AQ.

[0016] When the analyte is a water extract of Aconitum carmichaelii or a preparation of Aconitum carmichaelii water extract, a characteristic chromatogram of the test solution is obtained; the preparation process of the test solution is as follows: take the water extract of Aconitum carmichaelii or the preparation of Aconitum carmichaelii water extract, accurately weigh it, add solvent, weigh it, sonicate it, take it out, cool it, weigh it again, replenish the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain the test solution;

[0017] When the analyte is a piece of medicinal herb, a characteristic spectrum of the reference solution of the medicinal herb is obtained; the preparation process of the reference solution of the medicinal herb is as follows: take the medicinal herb, add water, reflux, filter, concentrate, add the corresponding solvent, cool, filter, and take the filtrate to obtain the solution;

[0018] When the analyte is a reference standard, a characteristic chromatogram of the reference standard solution is obtained; the preparation process of the reference standard solution is as follows: take the reference standard, accurately weigh it, and add solvent to prepare the reference standard solution; the reference standard includes monoester-type alkaloids, preferably including benzoylnephatonine, benzoylhypoaconitine, and benzoylnephatonine.

[0019] The preparations of aconite water extract include aconite water extract paste, aconite water extract dried powder, aconite formula granules, or aconite water extract preparations.

[0020] And / or, the solvent in the test solution is an aqueous hydrochloric acid solution with a mass concentration of 0.01% to 0.03%;

[0021] And / or, the solvent in the reference solution is a 0.01% (w / w) hydrochloric acid methanol solution;

[0022] And / or, the amount of solvent added to the test solution is (1-2) g / 25 ml.

[0023] The characteristic spectrum includes characteristic peaks 1-10, wherein peak 8 is the characteristic peak corresponding to benzoyl aconitine, peak 9 is the characteristic peak corresponding to benzoyl aconitine, and peak 10 is the characteristic peak corresponding to benzoyl hypoaconitine.

[0024] Taking peak 8 as the S peak, calculate the relative retention times of peaks 1 to 7. The relative retention times should be within ±10% of the specified values, which are peak 1: 0.14, peak 2: 0.23, peak 3: 0.27, peak 4: 0.43, peak 5: 0.58, peak 6: 0.62, and peak 7: 0.70, respectively.

[0025] The application of the characteristic chromatograms constructed by the above method in the overall quality control of Aconitum carmichaelii water extract and preparations.

[0026] Application of the characteristic chromatograms constructed by the above method in identifying easily confused products of processed Aconitum carmichaelii water extract and its preparations;

[0027] In the characteristic spectrum, the characteristic peak corresponding to benzoyl neoaconitine is the S peak, and the characteristic peaks with relative retention times of 0.58±10% and 0.62±10% with the S peak are peak 5 and peak 6, respectively.

[0028] The ratio of the relative peak areas of peak 5 to peak 6 is K. 56 When K 56 When the concentration is ≤2, it is processed aconite water extract and its preparations; the rest are easily confused products of processed aconite water extract and its preparations.

[0029] The K 56 When the concentration is 0.50–1.73, it is used for the preparation of Aconitum carmichaelii water extract and its preparations. 56 When the concentration is 5.28–31.61, it is an easily confused substance with aconite water extract and its preparations.

[0030] The easily confused products of the prepared aconite water extract and its preparations include the water extracts and preparations of prepared aconite root, prepared white aconite root slices, and prepared black aconite root slices.

[0031] The technical solution of this invention has the following advantages:

[0032] 1. This invention provides a method for constructing a characteristic spectrum of processed aconite root. The characteristic spectrum obtained by this method has more characteristic peaks and more comprehensive material information, which more fully characterizes the chemical components in the aqueous extract of processed aconite root and its preparations, and can better achieve overall quality control of the aqueous extract of processed aconite root and its preparations. At the same time, the characteristic spectrum of the aqueous extract of processed aconite root and its preparations obtained by the construction method of this invention also contains characteristic components that can be used to distinguish processed aconite root and its easily confused products, providing an effective guarantee for the identification of processed aconite root and its easily confused products.

[0033] 2. The construction method of the present invention has the advantages of being simple, stable, highly precise, and reproducible. It can quickly and accurately identify the quality of the product and provide a scientific basis for the comprehensive establishment of quality control standards for Aconitum carmichaelii formula granules.

[0034] 3. In the application of the present invention, the material information in the feature spectrum obtained by the construction method of the present invention is more comprehensive and can be more effectively applied to the overall quality control of Aconitum carmichaelii water extract and its preparations.

[0035] 4. In the application of this invention, the feature map obtained by the construction method of this invention, through K... 56 It can effectively identify processed aconite and its easily confused products, and effectively distinguish processed aconite from processed Sichuan aconite, white aconite slices and black aconite slices, and can achieve more stringent control over the overall quality of processed aconite. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is a characteristic chromatogram of the prepared Aconitum carmichaelii formula granules in Comparative Example 1 of the present invention.

[0038] Figure 2 These are comparative characteristic chromatograms of the test solution and the reference solution in Example 1 of this invention.

[0039] Figure 3 These are comparative characteristic chromatograms of the test sample solution and the accompanying medicinal slices reference solution in Example 1 of the present invention.

[0040] Figure 4 This is a characteristic chromatogram of the prepared Aconitum carmichaelii formula granules in Example 1 of the present invention.

[0041] Figure 5 This is a comparative characteristic spectrum of the aqueous extracts of processed Aconitum carmichaelii and processed Aconitum kusnezoffii in Example 2 of the present invention.

[0042] Figure 6 This is a comparative characteristic spectrum of Aconitum carmichaelii root slices and processed Aconitum carmichaelii water extract in Example 2 of the present invention.

[0043] Figure 7 This is a comparative characteristic spectrum of black aconite slices and processed Aconitum carmichaelii water extract in Example 2 of the present invention.

[0044] Figure 8 This is a graph of the delayed test in Embodiment 3 of the present invention.

[0045] Figure 9 These are characteristic chromatograms of different chromatographic columns in Example 5 of the present invention. Detailed Implementation

[0046] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0047] Instruments: XPR26 / A micro-analytical balance (Mettler Toledo); JM-A5002 analytical balance (Yuyao Beiqi Metrology & Weighing Instrument Co., Ltd.); ML204T / 02 analytical balance (Mettler Toledo); SB-5200DT ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); High-performance liquid chromatograph (Waters e2695 series, with Waters 2998PDA detector); Empower 3 chromatography workstation; ML204T electronic balance (Mettler Toledo); MSA6.6S-0CE-DI electronic balance (Sartorius); JJ500 electronic balance (Changshu Shuangjie Test Instrument Factory); KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0048] Test drug: Freeze-dried powder of prepared aconite root decoction (15 batches): K466BJ01, K466BJ02, K466BJ03, K466BJ04, K466BJ05, K466BJ06, K466BJ07, K466BJ08, K466BJ09, K466BJ10, K466BJ11, K466BJ12, K466BJ13, K466BJ14, K466BJ15.

[0049] Prepared Aconitum carmichaelii granules: K466CP11, K466CP12, K466CP15;

[0050] Prepared Aconitum carmichaelii slices: K466YP11;

[0051] Reference standards: Benzoyl aconitine (National Institutes for Food and Drug Control, batch number 111794-202307, purity: 98.0%); Benzoyl hypoaconitine (National Institutes for Food and Drug Control, batch number 111796-202207, purity: 96.4%); Benzoyl neoaconitine (National Institutes for Food and Drug Control, batch number 111795-202106, purity: 96.3%); Aconitine diester alkaloid reference extract (National Institutes for Food and Drug Control, batch number 112029-202302, purity: neoaconitine 32.9%, hypoaconitine 30.9%, aconitine 32.7%).

[0052] Reagents: Acetonitrile (Merck chromatographic grade), triethylamine (Fisher chromatographic grade), glacial acetic acid (Fisher chromatographic grade); water was distilled water (Watson's); all other reagents were of analytical grade.

[0053] Example 1

[0054] A method for constructing a characteristic map of processed aconite, the specific process of which is as follows:

[0055] 1. Preparation of the test sample:

[0056] Preparation of freeze-dried powder of prepared aconite root standard decoction: Take prepared aconite root slices, place them in a clay pot, soak for 30 minutes, add 14 times the amount of water for the first decoction, bring to a boil over high heat, then simmer over low heat for 60 minutes, filter while hot and set aside; add 10 times the amount of water for the second decoction, bring to a boil over high heat, then simmer over low heat for 40 minutes, filter while hot; combine the filtrates, concentrate (concentration temperature 65℃), concentrate to a density of 1.05~1.07g / ml clear extract, freeze-dry the clear extract to obtain freeze-dried powder of prepared aconite root standard decoction.

[0057] Preparation of processed aconite granules: Take 5000g of processed aconite slices, extract twice. For the first decoction, add 14 times the amount of water and extract at boiling (100℃) for 1.5 hours; for the second decoction, add 10 times the amount of water and extract at boiling (100℃) for 1.5 hours. Filter the liquid while hot through a 150-mesh filter cloth, and concentrate under reduced pressure below 70℃ until the relative density is [value missing].

[0058] 1.05~1.07 (60℃); spray drying, add appropriate amount of excipients, air inlet temperature is 170℃±5℃; add appropriate amount of excipients, mix well, dry granulation to make 1000g.

[0059] 2. Construction of the characteristic spectrum of the test sample

[0060] 2.1 Solution Preparation

[0061] Preparation of the test solution: Take an appropriate amount of the test sample (prepared Aconitum carmichaelii granules K466CP11), grind it into a fine powder, take about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 0.01% hydrochloric acid solution, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 0.01% hydrochloric acid solution, shake it well, filter it, and take the filtrate to obtain the test solution.

[0062] Preparation of reference solution for prepared aconite root slices: Take about 10g of prepared aconite root slices (K466YP01), heat under reflux twice, adding 100ml of water each time, for 60 minutes each time, filter, combine the filtrates and concentrate under reduced pressure at 65℃ to near dryness, add 20ml of 0.01% hydrochloric acid water to dissolve the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the reference solution for prepared aconite root slices.

[0063] Reference solution: Prepare a solution of monoester-type alkaloid mixed reference standard as the reference solution. The solvent in the reference solution is a 0.01% (w / w) hydrochloric acid methanol solution.

[0064] 2.2 Chromatographic Methods

[0065] The following chromatographic conditions were used for detection:

[0066] Waters XSelect HSS T3 column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile as mobile phase A, water (containing 0.2% triethylamine and 0.2% glacial acetic acid) as mobile phase B, gradient elution according to the specifications in Table 1; flow rate 1.0 ml / min; column temperature 30℃; detection wavelength: 240 nm for 0–14 min, 280 nm for 14–38.5 min, and 240 nm for 38.5–59 min. The theoretical plate number, calculated based on the benzoyl neoaconitine peak, should be no less than 5000.

[0067] Table 1

[0068]

[0069] Accurately pipette 20 μl each of the accompanying medicinal slices reference solution and the control standard reference solution, and 10 μl of the test solution, inject them into the liquid chromatograph, and determine the result.

[0070] The characteristic chromatograms of the above-mentioned accompanying medicinal slices reference solution, control standard reference solution, and test solution are as follows: Figures 2-3 As shown in Table 2, the results of the test solution are as follows.

[0071] Table 2

[0072]

[0073] From the above Figure 2 As can be seen, the characteristic spectrum shows 10 peaks, of which peak 8 is benzoylneoprothiolane, peak 9 is benzoylneoprothiolane, and peak 10 is benzoylhypoaconitine, with no interference from the negative excipient. Table 2 shows that the characteristic spectrum construction method of this invention, when used to detect the processed aconite granules, ensures that the peak shape, resolution, and theoretical plate number of each characteristic peak meet the requirements. Figure 3 It can be seen that, through spectral comparison, the 10 characteristic peaks of processed aconite granules and processed aconite slices are consistent, indicating that the substances corresponding to the characteristic peaks are completely transferred from processed aconite slices to granules, and the two have a good correlation.

[0074] Fifteen batches of freeze-dried powder of processed aconite root standard decoction and three batches of processed aconite root formula granules were tested to obtain characteristic chromatograms. Similarity calculations were performed on the characteristic chromatograms of the 15 batches of freeze-dried powder and the 3 batches of processed aconite root formula granules. The similarity ranged from 0.902 to 0.988, indicating a high correlation, which meets the quality control requirements. Simultaneously, using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (version 2012.1), with the S1 liquid chromatography chromatogram as the reference chromatogram, the median was used to calculate the characteristic chromatogram of the processed aconite root formula granules, and common peaks were identified. The results are shown in Tables 3-6 and 3-6. Figure 4 As shown.

[0075] Table 3. Retention Time and Relative Retention Time of 15 Batches of Freeze-dried Powder of Standard Aconitum carmichaelii Decoction

[0076]

[0077]

[0078] Table 4. Peak area and relative peak area of ​​freeze-dried powder of 15 batches of prepared aconite root standard decoction

[0079]

[0080]

[0081] Table 5. Retention Time and Relative Retention Time of Three Batches of Processed Aconitum carmichaelii Formula Granules

[0082]

[0083] Table 6. Peak area and relative peak area of ​​granules in three batches of processed Aconitum carmichaelii formula.

[0084]

[0085] Through Tables 3 and 4, and Figure 4It is known that the chromatogram of the test sample should show 10 characteristic peaks, and the relative retention times should correspond to the 10 characteristic peaks in the chromatogram of the accompanying reference materials. Among them, peaks 8, 9, and 10 should correspond to the retention times of the reference standards for benzoyl aconitine, benzoyl aconitine, and benzoyl hypoaconitine, respectively. The peak corresponding to the reference standard for benzoyl aconitine is designated as the S peak. The relative retention times of characteristic peaks 1 to 7 and the S peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.14 (peak 1), 0.23 (peak 2), 0.27 (peak 3), 0.43 (peak 4), 0.58 (peak 5), 0.62 (peak 6), and 0.70 (peak 7).

[0086] Example 2

[0087] The application of a method for constructing a characteristic spectrum of processed aconite preparations in identifying easily confused standard decoctions of processed aconite.

[0088] Research shows that Aconitum carmichaelii, Aconitum kusnezoffii, and Aconitum kusnezoffii are very similar, often leading to confusion, and even instances of dyed Aconitum carmichaelii being mixed into Aconitum kusnezoffii. All three are Ranunculaceae plants, but belong to different genera. Aconitum carmichaelii is a processed product of the lateral root of Aconitum carmichaeli Debx; Aconitum kusnezoffii is the dried mother root of Aconitum carmichaeli Debx; and Aconitum kusnezoffii is the dried root of Aconitum kusnezoffire Reichb. Because these three drugs originate from the same family and are closely related, they share significant similarities in chemical composition and pharmacological effects.

[0089] This invention primarily investigates the differences between adulterated processed aconite (processed Sichuan aconite, white aconite root slices, and black aconite root slices) and standard decoction of processed aconite. Processed Sichuan aconite was processed according to the processing method for processed aconite to obtain processed aconite root slices. White aconite and black aconite root slices were already in slice form and required no further processing. The processed aconite, processed Sichuan aconite, black aconite, and white aconite root slices were decocted using the standard decoction conditions for processed aconite (soaking for 30 minutes, decocting twice; the first decoction added 14 times the amount of water and decocted for 60 minutes; the second decoction added 10 times the amount of water and decocted for 40 minutes) to obtain a standard decoction. This standard decoction was then concentrated and freeze-dried to obtain freeze-dried powders of the following standard decoctions: processed aconite, processed Sichuan aconite, black aconite, and white aconite.

[0090] The characteristic spectra of the following products were analyzed using a method for constructing characteristic spectra: freeze-dried powder of prepared Aconitum carmichaelii decoction, freeze-dried powder of prepared Aconitum kusnezoffii decoction, freeze-dried powder of prepared Aconitum carmichaelii slices, and freeze-dried powder of prepared Aconitum carmichaelii slices. The results showed that each adulterant product differed in its characteristic spectra. Using the same construction method as in Example 1, the characteristic spectra of the tested samples obtained showed significant differences between prepared Aconitum kusnezoffii, prepared Aconitum carmichaelii slices, and prepared Aconitum carmichaelii slices at characteristic peaks 5 and 6 compared to the standard decoction of prepared Aconitum carmichaelii. Figures 5-7 As shown. Simultaneously, the ratio of the peak areas of peaks 5 and 6 in the feature map is obtained as K. 56 The results are shown in Tables 7 and 8.

[0091] Table 7

[0092]

[0093] Table 8

[0094]

[0095]

[0096] As can be seen from the peak area research results shown in Tables 7 and 8 above, the peak area ratio K of peak 5 to peak 6 in 15 batches of prepared aconite root standard decoction is... 56 The values ​​ranged from 0.50 to 1.73, with an average of 0.91. The peak area ratio K of peak 5 to peak 6 in the three batches of processed aconite granules shown in Table 6 above is also relevant. 56 The value ranges from 1.06 to 1.11. In Table 8 above, the peak area ratio K of peak 5 to peak 6 in the freeze-dried powders of easily confused products such as Aconitum carmichaelii, Aconitum carmichaelii root slices, and Aconitum carmichaelii root slices is... 56 The range is 5.28–31.61. It is evident that the peak 5 / peak 6 ratios of the prepared aconite root standard decoction and the three easily confused standard decoctions show significant differences and no overlap. This can be addressed by analyzing the peak area ratio K of peak 5 to peak 6. 56 The value must not exceed 2.0 (mean + 3SD rounded down) to distinguish processed aconite from easily confused processed aconite root, white aconite root slices, and black aconite root slices in standard decoctions.

[0097] Example 3

[0098] This embodiment is used to validate the chromatographic conditions in Example 1.

[0099] 1. Specificity research

[0100] Approximately 1.0 g each of prepared aconite granules and maltodextrin were taken and prepared into a test solution of prepared aconite granules and a negative control solution of excipients according to the method in Example 1. The results showed that the negative control solution did not interfere with the characteristic peaks, indicating that the method has good specificity.

[0101] 2. Delayed testing

[0102] Take the test solution of prepared Aconitum carmichaelii granules and inject it according to the chromatographic conditions in Example 1. Record the chromatogram of retention time of the mobile phase at two times the value. The results are shown in the figure. Figure 8 .pass Figure 8 The results showed that no obvious hysteresis peak appeared after 60 minutes, indicating that this chromatographic method has good delay properties.

[0103] 3. Repeatability of examinations

[0104] Six portions of prepared Aconitum carmichaelii granules (batch number: K466CP12) were taken, ground into a fine powder, and their characteristic spectra were determined according to the method in Example 1. Using peak 8 as the reference peak S, the relative retention times and relative peak areas of characteristic peaks 1 to 7 were calculated, and the RSD was calculated. The results are shown in Tables 9 and 10.

[0105] Table 9 Retention time and relative retention time for repeatability testing of characteristic maps

[0106]

[0107] Table 10: Peak area and relative peak area for repeatability testing of characteristic spectra.

[0108]

[0109] Based on the repeatability test results, the relative retention time RSD of each characteristic peak is in the range of 0.00% to 0.06%, and the relative peak area RSD of each characteristic peak is in the range of 0.22% to 0.59%. The peak area ratio of peak 5 to peak 6 is not greater than 2.0. This indicates that the retention time repeatability of the characteristic peaks in this characteristic spectrum is good.

[0110] 4. Intermediate precision test

[0111] Take the prepared Aconitum carmichaelii formula granules (batch number: K466CP12), grind them finely, and prepare the sample according to the method in Example 1. Different analysts used another high performance liquid chromatograph at different times to conduct intermediate precision tests and obtain its characteristic chromatograms. Using peak No. 8 as the reference peak S, the relative retention time and relative peak area of ​​characteristic peaks No. 1 to No. 7 were calculated. The results and the results of the repeatability test were used to calculate the RSD value. The results are shown in Tables 11 and 12.

[0112] Table 11 Retention Time and Relative Retention Schedule for Intermediate Precision Examination

[0113]

[0114] Table 12: Intermediate Precision Examination Peak Area and Relative Peak Area

[0115]

[0116]

[0117] The relative retention time (RSD) of each characteristic peak ranged from 0.00% to 0.14%, with the RSD between different instruments ranging from 0.05% to 1.07%. The RSD of the relative peak area ranged from 0.39% to 3.15%, with the RSD% between different instruments ranging from 0.49% to 7.16%. The peak area ratio of peak 5 to peak 6 was no greater than 2.0. The intermediate precision results indicate that the intermediate precision of this characteristic spectrum is good.

[0118] 5. Stability test

[0119] Take the same particle test solution and measure it at 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h respectively. Calculate the relative retention time and relative peak area of ​​characteristic peaks 1 to 7 and peak 8, and calculate RSD. The results are shown in Tables 13 and 14.

[0120] Table 13 Stability retention time and relative retention time table

[0121]

[0122]

[0123] Table 14 Stability Peak Area and Relative Peak Area

[0124]

[0125] The stability test results show that the characteristic components in the sample solution are stable within 24 hours. The relative retention time RSD of each characteristic peak is in the range of 0.00% to 0.11%, the relative peak area RSD is in the range of 0.34% to 1.65%, and the peak area ratio of peak 5 to peak 6 is not greater than 2.0.

[0126] Example 4

[0127] A method for constructing a characteristic spectrum of aconite preparations differs from Example 1 in that the preparation of the test solution is different.

[0128] Take the prepared Aconitum carmichaelii granules (batch number: K466CP12), grind them finely, and weigh out 1.0g, 1.5g, and 2.0g respectively. Place them in stoppered conical flasks, add 25ml of 0.01% hydrochloric acid solution to each, seal tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Remove, cool, shake well, and filter to obtain the test samples. Accurately pipette 10μl of each test sample and inject it into the high-performance liquid chromatograph to determine the peak area of ​​each peak.

[0129] Table 15. Characteristic chromatograms of different extraction concentrations.

[0130]

[0131]

[0132] The analysis results show that the number and area of ​​each characteristic peak are not significantly different under different extraction concentrations. After comprehensive consideration, the extraction concentration of 1.0g / 25ml was selected.

[0133] Example 5

[0134] A method for constructing a characteristic chromatogram of processed aconite root preparation differs from Example 1 in that the chromatographic conditions are different, as detailed below:

[0135] (1) Investigation at different column temperatures

[0136] Take the prepared Aconitum carmichaelii formula granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the test solution preparation method in Example 1. The characteristic chromatograms were obtained by measuring them at different column temperatures (28℃, 30℃ and 32℃) according to the chromatographic conditions of Example 1. Using peak No. 8 as the reference peak S, the relative retention times of characteristic peaks No. 1 to No. 7 were calculated, and the RSD was calculated. The results are shown in Table 16.

[0137] Table 16 Retention times and relative retention times at different column temperatures

[0138]

[0139] Summary: The relative retention times (RSD) of characteristic peaks were examined at different column temperatures (28℃, 30℃, and 32℃). The RSD of the characteristic peaks ranged from 0.72% to 4.36%, and the relative retention times of each characteristic peak were within ±10% of the specified value. This indicates that the relative retention times of characteristic peaks under this method have good robustness to column temperature.

[0140] (2) Investigation of different flow velocities

[0141] Take the prepared Aconitum carmichaelii formula granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the test solution preparation method in Example 1. The characteristic chromatograms were obtained by measuring them at different flow rates (0.9 ml / min, 1.0 ml / min and 1.1 ml / min) according to the chromatographic conditions in Example 1. Using peak No. 8 as the reference peak S, the relative retention times of characteristic peaks No. 1 to No. 7 were calculated, and the RSD was calculated. The results are shown in Table 17.

[0142] Table 17 Retention times and relative retention times at different flow velocities

[0143]

[0144] At different flow rates (0.9 ml / min, 1.0 ml / min and 1.1 ml / min), the relative retention time RSD of the characteristic peaks ranged from 0.51% to 7.49%, and the relative retention time of each characteristic peak was within ±10% of the specified value, indicating that the relative retention time of the characteristic peaks under this method has good robustness to flow rate.

[0145] (3) Investigation of different glacial acetic acid concentrations

[0146] Take the prepared Aconitum carmichaelii formula granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the test solution preparation method in Example 1. The characteristic chromatograms were obtained by measuring the acetic acid at different concentrations (0.18% glacial acetic acid + 0.20% triethylamine, 0.20% glacial acetic acid + 0.20% triethylamine, 0.22% glacial acetic acid + 0.20% triethylamine) under the chromatographic conditions of Example 1. Using peak 8 as the reference peak S, the relative retention times of characteristic peaks 1 to 7 were calculated, and the RSD was calculated. The results are shown in Table 18.

[0147] Table 18 Retention times and relative retention times at different glacial acetic acid concentrations

[0148]

[0149]

[0150] At different concentrations of glacial acetic acid (0.18% glacial acetic acid + 0.20% triethylamine, 0.20% glacial acetic acid + 0.20% triethylamine, 0.22% glacial acetic acid + 0.20% triethylamine), the relative retention times (RSD) of each characteristic peak were in the range of 0.14% to 0.70%, and the relative retention times of each characteristic peak were within ±10% of the specified value, indicating that the relative retention times of the characteristic peaks under this method have good robustness to different concentrations of glacial acetic acid.

[0151] (4) Investigation of different triethylamine concentrations

[0152] Take the prepared Aconitum carmichaelii formula granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the test solution preparation method in Example 1. The test solution was determined under different triethylamine concentrations (0.20% glacial acetic acid + 0.18% triethylamine, 0.20% glacial acetic acid + 0.20% triethylamine, 0.20% glacial acetic acid + 0.22% triethylamine) according to the chromatographic conditions in Example 1 to obtain its characteristic chromatogram. Using peak 8 as the reference peak S, the relative retention times of characteristic peaks 1 to 7 were calculated, and the RSD was calculated. The results are shown in Table 19.

[0153] Table 19 Retention times and relative retention times at different triethylamine concentrations

[0154]

[0155]

[0156] At different triethylamine concentrations (0.20% glacial acetic acid + 0.18% triethylamine, 0.20% glacial acetic acid + 0.20% triethylamine, 0.20% glacial acetic acid + 0.22% triethylamine), the relative retention times (RSD) of each characteristic peak were in the range of 0.01% to 1.00%, and the relative retention times of each characteristic peak were within ±10% of the specified value, indicating that the relative retention times of the characteristic peaks under this method have good robustness to different triethylamine concentrations.

[0157] (5) Investigation of different chromatographic columns

[0158] The freeze-dried powder of Aconitum carmichaelii (batch number: K466CP12) was used to prepare a test solution according to the preparation method of the test solution in Example 1. The chromatographic conditions of Example 1 were used to determine the robustness of the chromatographic method to different chromatographic columns. The results are as follows: Figure 9 As shown in Table 20.

[0159] Column 1: SHIMADZU Shim-pack GIST C18-AQ (4.6*250mm, 5μm),

[0160] Column 2: Waters Xselect HSS T3 (4.6*250mm, 5μm),

[0161] Column 3: phenomenex Gemini C18 (4.6*250mm, 5μm);

[0162] Table 20 Relative retention time tables under different chromatographic columns

[0163]

[0164] As can be seen from the data and chromatographic results, due to differences in different chromatographic column packing materials, the elution positions and peak shapes of each characteristic peak vary considerably. Therefore, in order to ensure the robustness of the chromatographic method, this invention preferably uses a fixed type of chromatographic column, preferably the Xselect HSS T3 column (4.6*250mm, 5μm).

[0165] Example 6

[0166] A method for constructing a characteristic chromatogram of aconite preparations differs from Example 1 in that the chromatographic detection wavelength is different, as detailed below:

[0167] Take the prepared Aconitum carmichaelii formula granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the test solution preparation method in Example 1. The test solution was measured at different detection wavelengths (238 switched to 278 and then back to 238, 240 switched to 280 and then back to 240, 242 switched to 282 and then back to 242) according to the chromatographic conditions in Example 1 to obtain its characteristic chromatogram. With peak 8 as the reference peak S, the relative retention times of characteristic peaks 1 to 7 were calculated, and the RSD was calculated. The results are shown in Table 21.

[0168] Table 21 Retention time and relative retention time for different detection wavelengths

[0169]

[0170] Under different detection wavelengths (238 switching to 278 and then back to 238, 240 switching to 280 and then back to 240, 242 switching to 282 and then back to 242), the relative retention time RSD of each characteristic peak is in the range of 0.00% to 0.16%, and the relative retention time of each characteristic peak is within ±10% of the specified value, indicating that the relative retention time of the characteristic peak under this method has good robustness to different detection wavelengths.

[0171] Example 7

[0172] A method for constructing a characteristic spectrum of aconite preparations differs from Example 1 in that, in the preparation of the reference solution, hydrochloric acid aqueous solutions with different mass concentrations are used as the solvent, as detailed below:

[0173] Take the prepared Aconitum carmichaelii formula granules (batch number: K466CP12), grind them finely, and accurately add 25 ml of the corresponding solvents (0.01% hydrochloric acid aqueous solution, 0.02% hydrochloric acid aqueous solution, and 0.03% hydrochloric acid aqueous solution), weigh them, and sonicate them (power 250W, frequency 40kHz) for 30 minutes. Take them out, let them cool, weigh them again, and make up the weight loss with the corresponding solvents. Shake well, filter, and take the filtrate as the test solution. Determine its characteristic chromatogram according to the chromatographic conditions of Example 1. Using peak 8 as the reference peak S, calculate the relative retention time of characteristic peaks 1 to 7, and calculate the RSD. The results are shown in Table 22.

[0174] Table 22 Investigation of different hydrochloric acid concentrations

[0175]

[0176] There was no significant difference in the peak area of ​​each characteristic peak under different concentrations of hydrochloric acid aqueous solution (0.01% hydrochloric acid aqueous solution, 0.02% hydrochloric acid aqueous solution, 0.03% hydrochloric acid aqueous solution), so a concentration of 0.01% was selected for this study.

[0177] Comparative Example 1

[0178] A method for constructing a characteristic chromatogram of processed aconite root preparations differs from Example 1 in that the high-performance liquid chromatography method is different, as detailed below:

[0179] Waters ACQUITY Column HSS T3 (2.1 mm × 100 mm, 1.8 μm); flow rate 0.3 ml / min; column temperature 30 ℃; detection wavelength 240 nm; mobile phase was acetonitrile-0.1% phosphoric acid solution, and gradient elution was performed according to the specifications in Table 23 below.

[0180] Table 23

[0181]

[0182] The spectrum obtained under these conditions is as follows Figure 1 As shown, the characteristic chromatograms of aconite water extract and its preparations have few characteristic peaks and incomplete characterization information. From the chromatogram, a large portion of the chromatographic peaks are concentrated in the first 5 minutes.

[0183] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of aconite root water extract and its formulation granules, characterized in that, The characteristic chromatogram of the test sample solution was obtained by high performance liquid chromatography (HPLC); the chromatographic conditions for the HPLC method were as follows: The chromatographic column was a Waters Xselect HSS T3; acetonitrile was used as mobile phase A, and an aqueous solution containing 0.18%-0.22% triethylamine and 0.18%-0.22% glacial acetic acid was used as mobile phase B, eluted according to the following gradient program: The detection wavelengths for 0-14 minutes are 238-242 nm, for 14-38.5 minutes are 278-282 nm, and for 38.5-59 minutes are 238-242 nm. The preparation process of the test solution is as follows: take the aqueous extract of Aconitum carmichaelii or the formula granules of Aconitum carmichaelii, weigh them accurately, add solvent, weigh them, sonicate, take them out, cool them, weigh them again, replenish the lost weight with the corresponding solvent, shake well, filter, and take the filtrate to obtain the test solution; the solvent is a hydrochloric acid aqueous solution with a mass concentration of 0.01%-0.03%; The characteristic spectrum includes characteristic peaks 1-10, wherein peak 8 is the characteristic peak corresponding to benzoyl aconitine, peak 9 is the characteristic peak corresponding to benzoyl aconitine, and peak 10 is the characteristic peak corresponding to benzoyl hypoaconitine. Taking peak 8 as peak S, calculate the relative retention times of peaks 1 to 7 compared to peak S. The relative retention times should be within ±10% of the specified values, which are peak 1: 0.14, peak 2: 0.23, peak 3: 0.27, peak 4: 0.43, peak 5: 0.58, peak 6: 0.62, and peak 7: 0.

70.

2. The construction method according to claim 1, characterized in that, In the chromatographic conditions of the high performance liquid chromatography method, the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm. And / or, the column temperature is 28-32℃; And / or, the flow rate is 0.9-1.1 ml / min; And / or, the theoretical plate number calculated based on the benzoyl neoaconitine peak should be no less than 5000.

3. The construction method according to claim 1 or 2, characterized in that, The detection includes the use of reference solutions, the preparation process of which is as follows: take the reference standard, accurately weigh it, and add a solvent to prepare the reference solution; the reference standard includes benzoylneopioid, benzoylaconitine, and benzoylhypoconitine.

4. The construction method according to claim 3, characterized in that, The amount of solvent added to the test solution is (1-2) g / 25 ml.

5. The application of the characteristic spectrum constructed by the construction method according to any one of claims 1-4 in the overall quality control of processed Aconitum carmichaelii water extract and formulation granules.

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