Specific chromatogram construction method and application of prepared kusnezoff monkshood root aqueous extract and formula granules

Abstract

The invention provides a specific chromatogram construction method and application of a prepared kusnezoff monkshood root aqueous extract and formula granules, and the specific chromatogram construction method of the prepared kusnezoff monkshood root comprises the following steps: obtaining a specific chromatogram of an object to be detected by adopting a high performance liquid chromatography; specific chromatographic conditions of the high performance liquid chromatography are provided, and octadecylsilane chemically bonded silica is used as a chromatographic column of a filler; acetonitrile is used as a mobile phase A, an aqueous solution containing 0.18%-0.22% of triethylamine and 0.18%-0.22% of glacial acetic acid is used as a mobile phase B, and elution is performed according to a specific gradient program; the detection wavelength is 238 to 242 nm in 0 to 14 minutes, the detection wavelength is 278 to 282 nm in 14 to 38.5 minutes, and the detection wavelength is 238 to 242 nm in 38.5 to 59 minutes. The material information in the characteristic chromatogram obtained by the construction method is comprehensive, and the overall quality control of the prepared kusnezoff monkshood root and the identification of easily mixed products of the prepared kusnezoff monkshood root can be realized through the related information in the characteristic chromatogram represented by the method.

Description

Technical Field

[0001] The present invention relates to the field of identification of traditional Chinese medicines, and particularly to a method for constructing a characteristic chromatogram and its application for the water extract and formula granules of processed Aconitum kusnezoffii. Background Art

[0002] Processed Aconitum kusnezoffii is the dried tuber of Aconitum kusnezoffii Reichb. of the Ranunculaceae family, and the original plant is a perennial herb.

[0003] Aconitum kusnezoffii has a wide range of uses and is applied in many fields such as traditional Chinese medicine decoction pieces, proprietary Chinese medicines, veterinary medicines, etc. Aconitum kusnezoffii is widely used clinically; there are many characteristic methods for Aconitum kusnezoffii herbs or decoction pieces in the literature or patents. And the characteristic methods for Aconitum kusnezoffii herbs and decoction pieces are basically based on the chromatographic conditions under the content determination item of "processed Aconitum kusnezoffii"; moreover, there are significant differences in the material basis between Aconitum kusnezoffii herbs or decoction pieces and the water extract of processed Aconitum kusnezoffii obtained after extraction, concentration, and drying. In the current research on the characteristic chromatogram of the water extract of processed Aconitum kusnezoffii and its preparations, the disclosed chromatographic conditions are as follows:

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

[0005] The characteristic chromatograms of the water extract of processed Aconitum kusnezoffii and its preparations disclosed in the prior art, such as Figure 1 shown, have few characteristic peaks and incomplete characterization information. From the chromatogram, a large part of the chromatographic peaks are stacked in the first 5 minutes, and the separation effect is not good, which is not conducive to the overall quality control. Summary of the Invention

[0006] Regarding the problem that the characteristic chromatograms of the water extract of processed Aconitum kusnezoffii and its preparations obtained by the characteristic chromatogram construction methods disclosed in the prior art have few characteristic peaks and are not conducive to the overall quality control, the present invention provides a method for constructing a characteristic chromatogram with more characteristic peaks and better realizing the overall quality control of the water extract of processed Aconitum kusnezoffii and its preparations; at the same time, the present invention also provides the application of the characteristic chromatogram constructed by this construction method in the overall quality control of the water extract of processed Aconitum kusnezoffii and its preparations, as well as the application of the characteristic chromatogram constructed by this construction method in the identification of easily confused varieties of Aconitum kusnezoffii.

[0007] A method for constructing a characteristic chromatogram of processed Aconitum kusnezoffii, which obtains the characteristic chromatogram of the analyte by high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are as follows:

[0008] A chromatographic column packed with octadecylsilyl 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, and elution is carried out according to the following gradient program:

[0009]

[0010] Among them, the detection wavelength is 238 - 242 nm from 0 to 14 minutes, 278 - 282 nm from 14 to 38.5 minutes, and 238 - 242 nm from 38.5 to 59 minutes.

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

[0012] and / or, the column temperature is 28 - 32 °C;

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

[0014] and / or, the number of theoretical plates calculated based on the benzoylmesaconine peak should be not less than 5000.

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

[0016] When the analyte is the water extract of Radix Aconiti Kusnezoffii preparata or the preparation of the water extract of Radix Aconiti Kusnezoffii preparata, obtain the characteristic chromatogram of the test solution; the preparation process of the test solution is as follows: take the water extract of Radix Aconiti Kusnezoffii preparata or the preparation of the water extract of Radix Aconiti Kusnezoffii preparata, weigh accurately, add solvent, weigh again, perform ultrasonic treatment, take out, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate to obtain it;

[0017] When the analyte is the accompanying decoction pieces, obtain the characteristic chromatogram of the reference solution of the accompanying decoction pieces; the preparation process of the reference solution of the accompanying decoction pieces is as follows: take the accompanying decoction pieces, add water, perform heat reflux treatment, filter, concentrate, add the corresponding solvent, cool, filter, and take the subsequent filtrate to obtain it;

[0018] When the analyte is the reference substance, obtain the characteristic chromatogram of the reference solution of the reference substance; the preparation process of the reference solution of the reference substance is as follows: take the reference substance, weigh accurately, and prepare the reference solution of the reference substance with the solvent; the reference substances include monoester - type alkaloids, preferably including benzoylmesaconine, benzoylhypaconine, and benzoylaconine.

[0019] The preparation of the processed Radix Aconiti Kusnezoffii water extract includes processed Radix Aconiti Kusnezoffii water extract paste, processed Radix Aconiti Kusnezoffii water extract dry powder, processed Radix Aconiti Kusnezoffii formula granules or processed Radix Aconiti Kusnezoffii water extract preparation;

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

[0021] And / or, the solvent in the reference substance solution is a hydrochloric acid methanol solution with a mass concentration of 0.01%;

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

[0023] The characteristic chromatogram includes characteristic peaks from peak 1 to peak 10. Among them, peak 8 is the characteristic peak corresponding to benzoylaconine, peak 9 is the characteristic peak corresponding to benzoylaconitine, and peak 10 is the characteristic peak corresponding to benzoylhypaconitine;

[0024] Taking peak 8 as the S peak, calculate the relative retention times of peaks 1 - 7. The relative retention times should be within the range of ±10% of the specified values. The specified values 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, peak 7: 0.70 in sequence.

[0025] Application of the characteristic chromatogram constructed by the above construction method in the overall quality control of the processed Radix Aconiti Kusnezoffii water extract and its preparations.

[0026] Application of the characteristic chromatogram constructed by the above construction method in differentiating the easily confused products of the processed Radix Aconiti Kusnezoffii water extract and its preparations;

[0027] In the said characteristic chromatogram, taking the characteristic peak corresponding to benzoylaconine as the S peak, the characteristic peaks with relative retention times of 0.58 ± 10% and 0.62 ± 10% to the S peak are peak 5 and peak 6 respectively;

[0028] The ratio of the relative peak areas of peak 5 and peak 6 is K 56 , when K 56 ≤2, it is the processed Radix Aconiti Kusnezoffii water extract and its preparations, and the rest are the easily confused products of the processed Radix Aconiti Kusnezoffii water extract and its preparations.

[0029] The said K 56 is 0.50 - 1.73 for the processed Radix Aconiti Kusnezoffii water extract and its preparations, and the said K 56 is 5.28 - 31.61 for the easily confused products of the processed Radix Aconiti Kusnezoffii water extract and its preparations.

[0030] The easily confused products of the processed Radix Aconiti Kusnezoffii water extract and its preparations include the water extracts and their preparations of Radix Aconiti Chuanwu, Aconiti Lateralis Praeparata Thunb. (White Prepared Lateral Root) and Aconiti Lateralis Praeparata Thunb. (Black Prepared Lateral Root).

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

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

[0033] 2. The construction method of the present invention has the advantages of simple operation, stability, high precision, good reproducibility, etc., and can quickly and accurately identify the quality of products, providing a scientific basis for comprehensively establishing the quality control standards of processed Radix Aconiti Kusnezoffii formula granules.

[0034] 3. In the application of the present invention, the substance information in the characteristic spectrum obtained by using the construction method of the present invention is more comprehensive, and it can be more effectively applied to the overall quality control of the water extract of processed Radix Aconiti Kusnezoffii and its preparations.

[0035] 4. In the application of the present invention, in the characteristic spectrum obtained by using the construction method of the present invention, through K 56 It can effectively realize the identification of processed Radix Aconiti Kusnezoffii and its easily confused varieties, effectively distinguish processed Radix Aconiti Kusnezoffii from processed Radix Aconiti Chuanwu, white prepared lateral root of aconite and black prepared lateral root of aconite, and can achieve more strict control over the overall quality of processed Radix Aconiti Kusnezoffii. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required to be used in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 It is the characteristic spectrum of the processed Radix Aconiti Kusnezoffii formula granules in Comparative Example 1 of the present invention.

[0038] Figure 2 It is the control characteristic spectrum of the test solution and the reference substance solution of the reference in Example 1 of the present invention.

[0039] Figure 3 It is the control characteristic spectrum of the test solution and the accompanying sliced herb reference solution in Example 1 of the present invention.

[0040] Figure 4 It is the characteristic spectrum of the processed Radix Aconiti Kusnezoffii formula granules in Example 1 of the present invention.

[0041] Figure 5 It is the control characteristic chromatogram of the water extracts of Radix Aconiti Lateralis Preparata and Radix Aconiti Kusnezoffii Preparata in Example 2 of the present invention.

[0042] Figure 6 It is the control characteristic chromatogram of the water extracts of Radix Aconiti Lateralis Praeparata Alba and Radix Aconiti Kusnezoffii Preparata in Example 2 of the present invention.

[0043] Figure 7 It is the control characteristic chromatogram of the water extracts of Radix Aconiti Lateralis Praeparata Nigra and Radix Aconiti Kusnezoffii Preparata in Example 2 of the present invention.

[0044] Figure 8 It is the chromatogram of the delayed test in Example 3 of the present invention.

[0045] Figure 9 It is the characteristic chromatogram of different chromatographic columns in Example 5 of the present invention. Detailed implementation manners

[0046] For those not specifying specific experimental steps or conditions in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0047] Instruments: XPR26 / A micro analytical balance (METTLER TOLEDO); JM-A5002 analytical balance (Yuyao Beiqi Measuring and Weighing Equipment 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 chromatographic workstation; ML204T electronic balance (METTLER TOLEDO); MSA6.6S-0CE-DI electronic balance (Sartorius); JJ500 electronic balance (Changshu Shuangjie Testing Instrument Factory); KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

[0048] Test drugs: Freeze-dried powder of the standard decoction of Radix Aconiti Kusnezoffii Preparata (15 batches): K466BJ01, K466BJ02, K466BJ03, K466BJ04, K466BJ05, K466BJ06, K466BJ07, K466BJ08, K466BJ09, K466BJ10, K466BJ11, K466BJ12, K466BJ13, K466BJ14, K466BJ15.

[0049] Formula granules of Radix Aconiti Kusnezoffii Preparata: K466CP11, K466CP12, K466CP15;

[0050] Accompanying pieces of Radix Aconiti Kusnezoffii Preparata: K466YP11;

[0051] Reference substances: Benzoylaconine (National Institutes for Food and Drug Control, batch number 111794 - 202307, purity: 98.0%); Benzoylhypaconine (National Institutes for Food and Drug Control, batch number 111796 - 202207, purity: 96.4%); Benzoylmesaconine (National Institutes for Food and Drug Control, batch number 111795 - 202106, purity: 96.3%); Reference extract of aconite diester alkaloids (National Institutes for Food and Drug Control, batch number 112029 - 202302, purity: mesaconitine 32.9%, hypaconitine 30.9%, aconitine 32.7%).

[0052] Reagents: Acetonitrile (Merck chromatographic grade), Triethylamine (Fisher chromatographic grade), Glacial acetic acid (Fisher chromatographic grade); Water is distilled water (Watsons); Other reagents are all of analytical grade.

[0053] Example 1

[0054] A method for constructing the characteristic fingerprint of processed Kusnezoff Monkshood Root is as follows:

[0055] 1. Preparation of test samples:

[0056] Preparation of freeze - dried powder of standard decoction of processed Kusnezoff Monkshood Root: Take the slices of processed Kusnezoff Monkshood Root, place them in a sand 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 for 60 minutes, filter while it is hot, and set aside; for the second decoction, add 10 times the amount of water, bring to a boil over high heat, then simmer for 40 minutes, filter while it is hot; Combine the filtrates, concentrate (concentration temperature 65°C), concentrate to a clear paste with a density of 1.05 - 1.07 g / ml, and obtain the freeze - dried powder of the standard decoction of processed Kusnezoff Monkshood Root by freeze - drying the clear paste.

[0057] Preparation of processed Kusnezoff Monkshood Root formula granules: Take 5000 g of the slices of processed Kusnezoff Monkshood Root, extract twice. For the first extraction, add 14 times the amount of water, extract at boiling (100°C) for 1.5 hours; for the second extraction, add 10 times the amount of water, extract at boiling (100°C) for 1.5 hours; Filter the medicinal liquid while it is hot with a 150 - mesh filter cloth, concentrate under reduced pressure below 70°C to a relative density of

[0058] 1.05 - 1.07 (60°C); Spray - dry, add an appropriate amount of excipients, the inlet air temperature is 170°C ± 5°C; Add an appropriate amount of excipients, mix evenly, and granulate by dry granulation to make 1000 g.

[0059] 2. Construction of the characteristic fingerprint of test samples

[0060] 2.1. Solution preparation

[0061] Preparation of the test solution: Take an appropriate amount of the test sample (processed Kusnezoff Monkshood Formula Granules K466CP11), grind it finely, take about 1.0 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of 0.01% hydrochloric acid solution, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 0.01% hydrochloric acid solution, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0062] Preparation of the reference solution of the accompanying cut crude drug: Take about 10 g of the accompanying cut crude drug of processed Kusnezoff Monkshood (K466YP01), reflux it by heating twice, add 100 ml of water each time, for 60 minutes each time, filter, combine the filtrates, concentrate them under reduced pressure to nearly dry at 65 °C, dissolve the residue in 20 ml of 0.01% hydrochloric acid solution, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the reference solution of the accompanying cut crude drug.

[0063] Reference solution of the reference substance: Prepare a solution of the mixed reference substances of monoester-type alkaloids as the reference solution of the reference substance, and the solvent in the reference solution of the reference substance is a hydrochloric acid methanol solution with a mass concentration of 0.01%.

[0064] 2.2 Chromatographic method

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

[0066] Waters XSelect HSS T3 (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); use acetonitrile as mobile phase A and water (containing 0.2% triethylamine and 0.2% glacial acetic acid) as mobile phase B, and perform gradient elution according to the regulations in Table 1 below; the flow rate is 1.0 ml / min; the column temperature is 30 °C; the detection wavelength: 240 nm from 0 to 14 minutes, 280 nm from 14 to 38.5 minutes, 240 nm from 38.5 to 59 minutes. The number of theoretical plates calculated based on the benzoylmesaconine peak should be not less than 5000.

[0067] Table 1

[0068]

[0069] Accurately pipette 20 μl each of the reference solution of the accompanying cut crude drug and the reference solution of the reference substance, and 10 μl of the test solution, inject them into the liquid chromatograph, and determine to obtain the results.

[0070] The characteristic chromatograms of the above reference solution of the accompanying cut crude drug, reference solution of the reference substance, and test solution are as Figures 2 - 3 shown, and the results of the test solution are shown in Table 2 below.

[0071] Table 2

[0072]

[0073] From the above Figure 2 It can be seen that the characteristic spectrum shows 10 peaks, of which peak 8 is benzoyl mesaconitine, peak 9 is benzoyl mesaconitine, and peak 10 is benzoyl mesaconitine, and there is no interference from the negative excipients; from the results in Table 2, it can be seen that the characteristic spectrum construction method of the present invention is used to detect the prepared Radix Aconiti Kusnezoffii formula granules, and the peak shape, separation degree, and theoretical plate number of each characteristic peak meet the requirements. Figure 3 It can be seen that through chromatographic comparison, the 10 characteristic peaks of the processed Radix Aconiti Kusnezoffii formula granules and the processed Radix Aconiti Kusnezoffii slices are consistent, indicating that the substances corresponding to the characteristic peaks are completely transferred from the processed Radix Aconiti Kusnezoffii slices to the formula granules, and the two are well correlated.

[0074] Fifteen batches of lyophilized powder of standard decoction of Aconitum kusnezoffii and three batches of granules of Aconitum kusnezoffii were tested to obtain characteristic spectra. The similarity of the characteristic spectra of the 15 batches of lyophilized powder of Aconitum kusnezoffii and the three batches of granules of Aconitum kusnezoffii was calculated. The similarity range was between 0.902 and 0.988, which was highly correlated and met the quality control requirements. At the same time, the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012.1 version) was used, with the S1 liquid phase spectrum as the reference spectrum, and the median was calculated to obtain the control characteristic spectrum of the Aconitum kusnezoffii granules, and the common peaks were identified. The results are shown in Tables 3-6 and Figure 4 shown.

[0075] Table 3 Retention time and relative retention time of 15 batches of freeze-dried powder of standard decoction of Aconitum kusnezoffii

[0076]

[0077]

[0078] Table 4 Peak area and relative peak area of 15 batches of freeze-dried powder of standard decoction of Aconitum kusnezoffii

[0079]

[0080]

[0081] Table 5 Retention time and relative retention time table of three batches of Radix Aconiti Kusnezoffii granules

[0082]

[0083] Table 6 Peak area and relative peak area of 3 batches of prepared Aconiti Kusnezoffii granules

[0084]

[0085] Tables 3 and 4 and Figure 4It can be seen that there should be 10 characteristic peaks in the chromatogram of the test sample, and the relative retention times of the 10 characteristic peaks in the chromatogram of the accompanying medicinal material reference material should correspond to each other. Among them, peak 8, peak 9, and peak 10 should correspond to the retention times of the benzoylmeaconitine reference material, the benzoylmeaconitine reference material, and the benzoylmeaconitine reference material reference material, respectively. The peak corresponding to the benzoylmeaconitine reference material reference material is the S peak. The relative retention times of the characteristic peaks of peaks 1 to 7 and the S peak are calculated. The relative retention times should be within the range of ±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] Application of a method for constructing a characteristic spectrum of processed Aconitum kusnezoffii preparations in determining whether processed Aconitum kusnezoffii is easily confused with other products.

[0088] Research has revealed that Aconite, Chuanwu, and Kusnezoffia are very similar, often leading to confusion and even the mixing of Aconite dyes with Kusnezoffia. Aconite, Chuanwu, and Kusnezoffia are all plants of the Ranunculaceae family, but from different genera. Aconite is a processed product of the rootlets of Aconitum carmichaeli Debx; Chuanwu is the dried rootlet of Aconitum carmichaeli Debx; and Kusnezoffia is the dried root of Aconitum kusnezoffi Reichb. Because these three medicinal herbs originate from the same family and are closely related, they share significant similarities in chemical composition and pharmacological effects.

[0089] The present invention mainly studies the differences between processed Radix Aconiti Kusnezoffii mixed products (processed Radix Aconiti Kusnezoffii, white aconite root, and Heishun Pian) and processed Radix Aconiti Kusnezoffii standard decoction. According to the processing method of processed Radix Aconiti Kusnezoffii, processed Radix Aconiti Kusnezoffii medicinal materials are processed to obtain processed Radix Aconiti Kusnezoffii decoction slice samples; white aconite root and Heishun Pian are already in sheet form and do not need to be processed. The processed Radix Aconiti Kusnezoffii, processed Radix Aconiti Kusnezoffii, Heishun Pian, and white aconite decoction slices are decocted using the decoction conditions of processed Radix Aconiti Kusnezoffii standard decoction (soaking for 30 minutes, decocting twice, adding 14 times the amount of decoction slices in water for the first decoction and decocting for 60 minutes, and adding 10 times the amount of decoction slices in water for the second decoction and decocting for 40 minutes) to obtain the standard decoction. The standard decoction is concentrated and freeze-dried to obtain processed Radix Aconiti Kusnezoffii standard decoction freeze-dried powder, processed Radix Aconiti Kusnezoffii standard decoction freeze-dried powder, Heishun Pian standard decoction freeze-dried powder, and white aconite standard decoction freeze-dried powder, respectively.

[0090] Use the construction method of characteristic chromatogram to detect the characteristics of freeze-dried powder of standard decoction of processed Aconitum kusnezoffii Reichb., freeze-dried powder of standard decoction of processed Aconitum carmichaelii Debx., freeze-dried powder of standard decoction of Radix Aconiti Lateralis Preparata, and freeze-dried powder of standard decoction of Radix Aconiti Lateralis Preparata Praeparata. The results show that there are differences in the characteristic chromatograms of each counterfeit product. In the characteristic chromatograms of the test samples obtained by using the same construction method as in Example 1, there are obvious differences between processed Aconitum carmichaelii Debx., Radix Aconiti Lateralis Preparata Praeparata, and Radix Aconiti Lateralis Preparata and the standard decoction of processed Aconitum kusnezoffii Reichb. at characteristic peaks 5 and 6, as Figures 5 - 7 shown. At the same time, the ratio of the peak areas of peaks 5 and 6 in the characteristic chromatogram is K 56 , and the results are shown in Tables 7 and 8.

[0091] Table 7

[0092]

[0093] Table 8

[0094]

[0095]

[0096] From the research results of the peak areas shown in the above Tables 7-8, it can be seen that the ratio K 56 of the peak areas of peaks 5 and 6 in 15 batches of standard decoctions of processed Aconitum kusnezoffii Reichb. is 0.50-1.73, and the average value is 0.91; the ratio K 56 of the peak areas of peaks 5 and 6 in 3 batches of formula granules of processed Aconitum kusnezoffii Reichb. shown in the above Table 6 is 1.06-1.11. In the above Table 8, the ratio K 56 of the peak areas of peaks 5 and 6 in the freeze-dried powders of the standard decoctions of the easily confused processed Aconitum carmichaelii Debx., Radix Aconiti Lateralis Preparata Praeparata, and Radix Aconiti Lateralis Preparata ranges from 5.28 to 31.61. It can be seen that there are significant data differences between the standard decoction of processed Aconitum kusnezoffii Reichb. and the standard decoctions of the three easily confused products, and there are no cross data. It can be distinguished between the standard decoction of processed Aconitum kusnezoffii Reichb. and the standard decoctions of the easily confused processed Aconitum carmichaelii Debx., Radix Aconiti Lateralis Preparata Praeparata, and Radix Aconiti Lateralis Preparata by the ratio K 56 of the peak areas of peaks 5 and 6 not exceeding 2.0 (rounded off the average value + 3SD).

[0097] Example 3

[0098] This example is used to verify the methodology of the chromatographic conditions in Example 1.

[0099] 1. Specificity study

[0100] Take about 1.0 g of each of the formula granules of processed Aconitum kusnezoffii Reichb. and maltodextrin, and prepare the test solution of the formula granules of processed Aconitum kusnezoffii Reichb. and the negative control solution of the excipient respectively according to the method of Example 1. The results show that the negative solution has no interference on the characteristic peaks, and the method has good specificity.

[0101] 2. Delayed test

[0102] Take the test solution of the processed Kusnezoff Monkshood Formula Granules, inject the sample according to the chromatographic conditions in Example 1, and record the chromatogram at 2 times the retention time of the mobile phase. The results are shown in Figure 8 . Through Figure 8 The results show that there are no obvious trailing peaks after 60 minutes, indicating that this chromatographic method has good delay performance.

[0103] 3. Repeatability investigation

[0104] Take 6 portions of the processed Kusnezoff Monkshood Formula Granules (batch number: K466CP12), grind them finely, and determine them according to the method in Example 1 to obtain their characteristic chromatograms. Using the 8th peak as the reference peak S, calculate the relative retention time and relative peak area of the 1st - 7th characteristic peaks; and calculate the RSD. The results are shown in Table 9 - Table 10.

[0105] Table 9 Retention Time and Relative Retention Time Table for Repeatability Investigation of Characteristic Chromatograms

[0106]

[0107] Table 10 Peak Area and Relative Peak Area Table for Repeatability Investigation of Characteristic Chromatograms

[0108]

[0109] According to the results of the repeatability investigation, the RSD of the relative retention time of each characteristic peak is in the range of 0.00% - 0.06%, and the RSD of the relative peak area of each characteristic peak is in the range of 0.22% - 0.59%. The ratio of the peak areas of peak 5 and peak 6 is not greater than 2.0. It shows that the repeatability of the retention time of the characteristic peaks of this characteristic chromatogram is good.

[0110] 4. Intermediate precision investigation

[0111] Take the processed Kusnezoff Monkshood Formula Granules (batch number: K466CP12), grind them finely, prepare the samples according to the method in Example 1, and conduct intermediate precision tests by different analysts at different times using another high - performance liquid chromatograph. Determine and obtain their characteristic chromatograms. Using the 8th peak as the reference peak S, calculate the relative retention time and relative peak area of the 1st - 7th characteristic peaks; calculate the RSD value with the results of the repeatability investigation. The results are shown in Table 11 and Table 12.

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

[0113]

[0114] Table 12 Peak Area and Relative Peak Area Table for Intermediate Precision Investigation

[0115]

[0116]

[0117] The relative retention time RSD of each characteristic peak is in the range of 0.00% to 0.14%, and the RSD range of the relative retention time between different instruments is 0.05% to 1.07%; the RSD of the relative peak area is in the range of 0.39% to 3.15%, and the RSD% range of the relative peak area between different instruments is 0.49% to 7.16%. The ratio of the peak areas of peak 5 and peak 6 is not greater than 2.0. From the results of the intermediate precision investigation, it shows that the intermediate precision of this characteristic chromatogram is good.

[0118] 5. Stability investigation

[0119] Take the same granule 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 the 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 table

[0124]

[0125] From the results of the stability experiment, it can be seen 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%, and the RSD of the relative peak area is in the range of 0.34% to 1.65%. The ratio of the peak areas of peak 5 and peak 6 is not greater than 2.0.

[0126] Example 4

[0127] A method for constructing the characteristic chromatogram of a prepared kusnezoff monkshood preparation, which is different from Example 1 in that the preparation of the test solution is different.

[0128] Take the prepared kusnezoff monkshood formula granules (batch number: K466CP12), grind them finely, weigh 1.0g, 1.5g, and 2.0g respectively, place them in a stoppered conical flask, add 25 ml of 0.01% hydrochloric acid water respectively, tightly stopper, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, take out, let cool, shake well, and filter to obtain the test solution. Precisely pipette 10 μl of each test solution, inject it into a high performance liquid chromatograph, and measure the peak area of each peak.

[0129] Table 15 Investigation of characteristic chromatograms at different extraction concentrations

[0130]

[0131]

[0132] The analysis results show that under different extraction concentrations, the number and peak area of each characteristic peak do not differ much. After comprehensive consideration, an extraction concentration of 1.0 g / 25 ml is selected.

[0133] Example 5

[0134] A method for constructing the characteristic chromatogram of the prepared Kusnezoff Monkshood preparation, which is different from Example 1 in that the chromatographic conditions are as follows:

[0135] (1) Investigation of different column temperatures

[0136] Take the prepared Kusnezoff Monkshood formula granules (batch number: K466CP12), grind them finely, prepare the test solution according to the test solution preparation method in Example 1, and measure the characteristic chromatogram at different column temperatures (28 °C, 30 °C, and 32 °C) according to the chromatographic conditions in Example 1. Using peak 8 as the reference peak S, calculate the relative retention times of characteristic peaks 1-7, and calculate the RSD. The results are shown in Table 16.

[0137] Table 16 Retention times and relative retention times for the investigation of different column temperatures

[0138]

[0139] Summary: By investigating different column temperatures (28 °C, 30 °C, and 32 °C) respectively, the RSD of the relative retention times of the characteristic peaks is in the range of 0.72% - 4.36%, and the relative retention times of each characteristic peak are within ±10% of the specified value, indicating that the relative retention times of the characteristic peaks under this method have good durability with respect to the column temperature.

[0140] (2) Investigation of different flow rates

[0141] Take the prepared Kusnezoff Monkshood formula granules (batch number: K466CP12), grind them finely, prepare the test solution according to the test solution preparation method in Example 1, and measure the characteristic chromatogram 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 8 as the reference peak S, calculate the relative retention times of characteristic peaks 1-7, and calculate the RSD. The results are shown in Table 17.

[0142] Table 17 Retention times and relative retention times for the investigation of different flow rates

[0143]

[0144] At different flow rates (0.9 ml / min, 1.0 ml / min, and 1.1 ml / min), the relative retention times of the characteristic peaks had an RSD in the range of 0.51% - 7.49%. 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 had good robustness to the flow rate.

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

[0146] Take the prepared Kusnezoff Monkshood formula granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the preparation method of the test solution in Example 1. Measure the characteristic chromatograms under different acetic acid 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) according to the chromatographic conditions in Example 1. Take the 8th peak as the reference peak S, calculate the relative retention times of the 1st - 7th characteristic peaks, and calculate the RSD. The results are shown in Table 18.

[0147] Table 18 Retention times and relative retention times for the investigation of different glacial acetic acid concentrations

[0148]

[0149]

[0150] At different glacial acetic acid 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), the relative retention times of the characteristic peaks had an RSD in the range of 0.14% - 0.70%. 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 had good robustness to different glacial acetic acid concentrations.

[0151] (4) Investigation of different triethylamine concentrations

[0152] Take the prepared Kusnezoff Monkshood formula granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the preparation method of the test solution in Example 1. Measure the characteristic chromatograms 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. Take the 8th peak as the reference peak S, calculate the relative retention times of the 1st - 7th characteristic peaks, and calculate the RSD. The results are shown in Table 19.

[0153] Table 19 Retention times and relative retention times for the investigation of 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 time RSD of each characteristic peak was in the range of 0.01% - 1.00%, 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 peak under this method had good durability for different triethylamine concentrations.

[0157] (5) Investigation of different chromatographic columns

[0158] Take the freeze-dried powder of the standard decoction of Radix Aconiti Kusnezoffii Praeparata (batch number: K466CP12), prepare the test solution according to the preparation method of the test solution in Example 1, and determine it according to the chromatographic conditions in Example 1 to investigate the durability of the chromatographic method for different chromatographic columns. The results are as Figure 9 shown in Table 20.

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

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

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

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

[0163]

[0164] It can be seen from the data and chromatogram results that due to the differences in the fillers of different chromatographic columns, there are significant differences in the elution positions and peak shapes of each characteristic peak. Therefore, to ensure the durability of the chromatographic method, the present invention preferably fixedly uses one of the chromatographic columns, and preferably uses chromatographic column Xselect HSS T3 (4.6*250mm, 5μm).

[0165] Example 6

[0166] A method for constructing the characteristic chromatogram of a preparation of Radix Aconiti Kusnezoffii Praeparata, which is different from Example 1 in that the chromatographic detection wavelength is different, specifically as follows:

[0167] Take the prepared Kusnezoff Monkshood Formula Granules (batch number: K466CP12), grind them finely, and prepare the test solution according to the preparation method of the test solution in Example 1. Measure the characteristic spectrum at different detection wavelengths (switch from 238 to 278 and then back to 238, switch from 240 to 280 and then back to 240, switch from 242 to 282 and then back to 242) according to the chromatographic conditions in Example 1. Take the 8th peak as the reference peak S, calculate the relative retention time of the 1st to 7th characteristic peaks, and calculate the RSD. The results are shown in Table 21.

[0168] Table 21 Retention Time and Relative Retention Time at Different Detection Wavelengths

[0169]

[0170] At different detection wavelengths (switch from 238 to 278 and then back to 238, switch from 240 to 280 and then back to 240, switch from 242 to 282 and then back to 242), the RSD of the relative retention time of each characteristic peak is in the range of 0.00% - 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 peaks under this method has good durability for different detection wavelengths.

[0171] Example 7

[0172] A method for constructing the characteristic spectrum of a prepared Kusnezoff Monkshood preparation, which is different from Example 1 in that during the preparation of the reference substance solution, hydrochloric acid aqueous solutions with different mass concentrations are used as solvents, specifically as follows:

[0173] Take the prepared Kusnezoff Monkshood Formula Granules (batch number: K466CP12), grind them finely, accurately add 25 ml of the corresponding solvents (0.01% hydrochloric acid aqueous solution, 0.02% hydrochloric acid aqueous solution, 0.03% hydrochloric acid aqueous solution) respectively, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, let it cool, weigh again, make up the lost weight with the corresponding solvents respectively, shake well, filter, and take the continuous filtrate as the test solution. Measure the characteristic spectrum according to the chromatographic conditions in Example 1. Take the 8th peak as the reference peak S, calculate the relative retention time of the 1st to 7th characteristic peaks, and calculate the RSD. The results are shown in Table 22.

[0174] Table 22 Investigation of Different Concentrations of Hydrochloric Acid in Water

[0175]

[0176] There is no obvious difference in the peak area of each characteristic peak at different concentrations of hydrochloric acid aqueous solutions (0.01% hydrochloric acid aqueous solution, 0.02% hydrochloric acid aqueous solution, 0.03% hydrochloric acid aqueous solution). Therefore, in this study, a hydrochloric acid in water concentration of 0.01% can be selected.

[0177] Comparative Example 1

[0178] A method for constructing a characteristic chromatogram of a prepared kusnezoff monkshood preparation, which is different from Example 1 in that the high performance liquid chromatography method is different, specifically as follows:

[0179] Chromatographic column: Waters ACQUITY HSS T3 (2.1 mm × 100 mm, 1.8 μm); flow rate 0.3 ml / min; column temperature 30 °C; detection wavelength 240 nm; mobile phase: acetonitrile - 0.1% phosphoric acid solution is subjected to gradient elution according to the regulations in Table 23 below.

[0180] Table 23

[0181]

[0182] The chromatogram obtained under this condition is as Figure 1 shown. The characteristic chromatogram method of the water extract of prepared kusnezoff monkshood and its preparation has few characteristic peaks and incomplete characterization information. From the chromatogram, a large part of the chromatographic peaks are piled up in the first 5 minutes.

[0183] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of processed Kusnezoff Monkshood, characterized in that, Obtain the characteristic chromatogram of the analyte by high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are as follows: A chromatographic column filled with octadecylsilyl-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, and perform elution according to the following gradient program: Among them, the detection wavelength from 0 to 14 minutes is 238-242 nm, the detection wavelength from 14 to 38.5 minutes is 278-282 nm, and the detection wavelength from 38.5 to 59 minutes is 238-242 nm.

2. The construction method according to claim 1, characterized in that In the chromatographic conditions of the high performance liquid chromatography, the column length of the chromatographic column is 250 mm, the column inner diameter is 4.6 mm, and the particle size is 5 μm; and / or, the column temperature is 28-32 °C; and / or, the flow rate is 0.9-1.1 ml / min; and / or, the theoretical plate number calculated based on the benzoylaconine peak should be not less than 5000.

3. The construction method according to claim 1, characterized in that In the described high performance liquid chromatography method, the chromatographic column is Waters Xselect HSS T3, phenomenex Gemini C18 or SHIMADZU Shim-pack GISTC18-AQ.

4. The construction method according to any one of claims 1-3, characterized in that When the analyte is the aqueous extract of processed Aconitum kusnezoffii Reichb. or the preparation of the aqueous extract of processed Aconitum kusnezoffii Reichb., obtain the characteristic chromatogram of the test solution; the preparation process of the test solution is as follows: take the aqueous extract of processed Aconitum kusnezoffii Reichb. or the preparation of the aqueous extract of processed Aconitum kusnezoffii Reichb., weigh accurately, add solvent, weigh, perform ultrasonic treatment, take out, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate to obtain. When the analyte is the accompanying cut crude drug, obtain the characteristic chromatogram of the reference solution of the accompanying cut crude drug; the preparation process of the reference solution of the accompanying cut crude drug is as follows: take the accompanying cut crude drug, add water, perform heat reflux treatment, filter, concentrate, add the corresponding solvent, cool, filter, and take the subsequent filtrate to obtain. When the analyte is the reference substance, obtain the characteristic chromatogram of the reference substance solution; the preparation process of the reference substance solution is as follows: take the reference substance, weigh accurately, and prepare the reference substance solution with the solvent; the reference substance includes monoester-type alkaloids, preferably including benzoylaconine, benzoylaconitine, and benzoylhypaconitine.

5. The construction method according to claim 4, characterized in that, The preparation of the aqueous extract of processed Aconitum kusnezoffii Reichb. includes the aqueous extract of processed Aconitum kusnezoffii Reichb., the dried powder of the aqueous extract of processed Aconitum kusnezoffii Reichb., the formula granules of processed Aconitum kusnezoffii Reichb., or the aqueous preparation of processed Aconitum kusnezoffii Reichb.; and / or, the solvent in the test solution is an aqueous hydrochloric acid solution with a mass concentration of 0.01%-0.03%; and / or, the addition amount of the solvent in the test solution is (1-2) g / 25 ml.

6. The construction method according to any one of claims 1 to 3, characterized in that, The characteristic chromatogram includes characteristic peaks from peak 1 to peak 10. Among them, peak 8 is the characteristic peak corresponding to benzoylaconine, peak 9 is the characteristic peak corresponding to benzoylaconitine, and peak 10 is the characteristic peak corresponding to benzoylhypaconitine; Taking peak 8 as the S peak, calculate the relative retention time of peaks 1-7 compared with the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values 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 in sequence.

7. Application of the characteristic chromatogram constructed by the construction method according to any one of claims 1-6 in the overall quality control of the aqueous extract of processed Aconitum kusnezoffii Reichb. and its preparations.

8. Use of the characteristic fingerprint constructed by the construction method according to any one of claims 1-6 in identifying easily confused products of the water extract of Aconitum kusnezoffii Reichb. preparata and its preparations; In the said characteristic fingerprint, taking the characteristic peak corresponding to benzoylaconine as the S peak, the characteristic peaks with relative retention times of 0.58±10% and 0.62±10% relative to the S peak are peak 5 and peak 6 respectively; The ratio of the peak area of peak 5 to that of peak 6 is K 56 , when K 56 ≤ 2, it is the water extract of Aconitum kusnezoffii Reichb. and its preparations, and the rest are the easily confused products of the water extract of Aconitum kusnezoffii Reichb. and its preparations.

9. The application according to claim 8, characterized in that, The said K 56 When it is 0.50 to 1.73, it is the water extract of processed Aconitum kusnezoffii Reichb. and its preparations. The said K 56 When it is 5.28 to 31.61, it is the easily confused product of the water extract of processed Aconitum kusnezoffii Reichb. and its preparations.

10. The application according to claim 8 or 9, characterized in that, The easily confused products of the water extract of Aconitum kusnezoffii Reichb. preparata and its preparations include the water extracts of Aconitum carmichaeli Debx. preparata, Radix Aconiti Lateralis Preparata (processed with white skin removed) and Radix Aconiti Lateralis Preparata (processed with black skin removed) and their preparations.

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