Method for determining content of aconite alkaloids in traditional Chinese medicine composition containing ramulus cinnamomi and application thereof

Abstract

The application discloses a method for determining the content of aconite alkaloids in a traditional Chinese medicine composition containing Ramulus Cinnamomi, which comprises the following steps: detecting test sample solution and control sample solution of the traditional Chinese medicine composition, and obtaining the content information of the control sample of the traditional Chinese medicine composition according to the detection result; wherein the traditional Chinese medicine composition comprises Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Glycyrrhizae, Ephedra, Rhizoma Zingiberis Recens, Rhizoma Atractylodis Macrocephalae, Rhizoma Anemarrhenae, Radix Saposhnikoviae and Heishunpian, and the control sample is benzoyl neoorientaline, benzoyl orientaline, benzoyl hypoorientaline, neoorientaline, hypoorientaline and aconitine. The application carries out the content monitoring research on the monoester type alkaloids of Heishunpian medicinal flavor in the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi, and carries out the limited control research on the diester type alkaloids, and the research on the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi is carried out from the multiple dimensions of safety and effectiveness, and has important quality control significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a content determination method of aconite alkaloids in a traditional Chinese medicine composition containing Ramulus Cinnamomi and application thereof. BACKGROUND

[0002] The main active and toxic components in He Shun Pian are aconite alkaloids, which are divided into diester type aconite alkaloids (aconitine, mesaconitine, hypaconitine), monoester type aconite alkaloids (benzoyl aconine, benzoyl hypaconine, benzoyl mesaconine, etc.) and alcohol amine type alkaloids (aconine, mesaconine, hypaconine, etc.) according to chemical structure. Since there are few relevant literature reports on alcohol amine type aconite alkaloids, they have no absorption under ultraviolet, and the ultraviolet detector of high performance liquid chromatography cannot determine them, so the alcohol amine type alkaloids are not studied.

[0003] Monoester type aconite alkaloids have smaller toxicity, only 1 / 50-1 / 500 of aconitine, and have good biological activity, and are the main pharmacodynamic material basis in clinical application; alcohol amine type alkaloids have lower toxicity, only 1 / 2000-1 / 4000 of aconitine, but also have weaker efficacy.

[0004] Diester type aconite alkaloids have strong toxicity, and the toxic dose is close to the therapeutic dose, among which aconitine has extremely strong cardiotoxicity and neurotoxicity, is the main toxic component of aconite drugs, and can cause poisoning at 0.2mg orally, and can cause death at 2-5mg. Aconitine is easily absorbed from the digestive tract, has low protein binding rate, and extremely rapid poisoning occurs, and the poisoning symptoms can occur within a few minutes after accidental or excessive ingestion, and death occurs within 3-4 hours after poisoning, and autopsy is mostly suffocation death without special pathological changes. Related studies have shown that diester type alkaloids first affect the central nervous system, and then cause damage to heart function from many aspects, and when the dosage is 0.030-0.038mg / kg, arrhythmia symptoms are induced, and when the dosage is greater than 0.100mg / kg, the quality of life of patients is affected, the therapeutic window is narrow, and poisoning is easily caused.

[0005] There is no related report on the content determination method of aconite alkaloids in a traditional Chinese medicine composition containing Ramulus Cinnamomi and application thereof in the prior art. SUMMARY

[0006] Based on this, the present application provides a content determination method of aconite alkaloids in a traditional Chinese medicine composition containing Ramulus Cinnamomi, which comprises the following steps:

[0007] The traditional Chinese medicine composition test sample solution and the control sample solution are detected,

[0008] The chromatographic conditions of the detection are as follows: a chromatographic column with octadecylsilane-bonded silica gel as the filler, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffer salt aqueous solution, the gradient elution program is as follows: 0-25 min, 85% B→80% B; 25-40 min, 80% B→79% B; 40-42 min, 79% B→74% B; 42-60 min, 74% B→63% B; the flow rate is 0.5-1.5 ml / min, the column temperature is 40-42 ℃, the detection wavelength is 180-360 nm, and the injection amount is 10-30 μl;

[0009] According to the detection result, the content information of the reference substance of the traditional Chinese medicine composition is obtained;

[0010] The traditional Chinese medicine composition comprises cassia twig, white peony root, licorice, ephedra, fresh ginger, atractylodes, anemarrhena, wind-preventing and black smooth tablets, and the reference substance is benzoyl neo-ligustrazine, benzoyl protoaconitine, benzoyl hypaconitine, neo-ligustrazine, hypaconitine and aconitine.

[0011] Further, the information is the content of the reference substance in the traditional Chinese medicine composition calculated according to the corresponding peak areas in the chromatogram of the test solution of the traditional Chinese medicine composition and the chromatogram of the reference substance solution according to the external standard method.

[0012] Further, the linear equation of the benzoyl neo-ligustrazine is y=34.492x-9.3909, R 2 =0.9999.

[0013] Further, the linear equation of the benzoyl protoaconitine is y=33.75x+0.8969, R 2 =1.0000.

[0014] Further, the linear equation of the benzoyl hypaconitine is y=35.495x-7.1817, R 2 =0.9997.

[0015] Further, the linear equation of the neo-ligustrazine is y=29.6145x-14.7747, R 2 =0.9990.

[0016] Further, the linear equation of the hypaconitine is y=32.032x-5.855, R 2 =0.9998.

[0017] Further, the linear equation of the aconitine is y=29.8871x-15.1308, R 2 =0.9990.

[0018] Further, the preparation method of the test solution of the traditional Chinese medicine composition comprises: taking an appropriate amount of traditional Chinese medicine composition decoction containing cassia twig, placing it in a container, adding a first solvent, weighing, ultrasonic treatment for a period of time, cooling, weighing again, supplementing the lost weight with the first solvent, shaking, centrifuging, filtering, taking an appropriate amount of filtrate, adsorbing the filtrate with a solid phase extraction column, then eluting, collecting the eluate, reducing pressure recovery, drying to obtain a residue, adding a second solvent to the residue to dissolve and dilute, shaking, filtering to obtain the test solution of the traditional Chinese medicine composition.

[0019] Further, the container is a volumetric flask.

[0020] Further, the first solvent is hydrochloric acid.

[0021] Further, the concentration of the hydrochloric acid is 0.4-0.6 mol / L, for example, about 0.5 mol / L.

[0022] Further, the volume ratio between the traditional Chinese medicine composition decoction and the first solvent is 2-6, for example, about 4.

[0023] Further, the volume of the traditional Chinese medicine composition decoction is 60-100 ml, for example, about 80 ml.

[0024] Further, the power of the ultrasonic treatment is 300-500 W, for example, about 400 W.

[0025] Further, the frequency of the ultrasonic treatment is 30-50 kHz, for example, about 40 kHz.

[0026] Further, the time of the ultrasonic treatment is 20-60 min, for example, about 40 min.

[0027] Further, the speed of the centrifugation is 4000-6000 rpm / min, for example, about 5000 rpm / min.

[0028] Further, the time of the centrifugation is 5-15 min, for example, about 10 min.

[0029] Further, the volume ratio between the filtrate and the traditional Chinese medicine composition decoction is 0.5-1, for example, about 0.875.

[0030] Further, the volume of the filtrate is 60-80 ml, for example, about 70 ml.

[0031] Further, the temperature of the reducing pressure recovery is not higher than 40℃.

[0032] Further, the second solvent is a mixed solution of acetonitrile and ammonium acetate.

[0033] Further, the concentration of the ammonium acetate is 0.05-0.2 mol / L, for example, about 0.1 mol / L.

[0034] Further, in the second solvent, the volume ratio of the acetonitrile to the ammonium acetate is acetonitrile:ammonium acetate = about 20:about 80.

[0035] Further, the preparation method of the solid phase extraction column comprises: using a mixed cation exchange reversed phase adsorbent as a filler, and sequentially using acetonitrile and water for elution.

[0036] Further, the mass of the mixed cation exchange reversed phase adsorbent is 400-600 mg, for example, about 500 mg.

[0037] Further, the capacity of the solid phase extraction column is 4-8 ml, for example, 6 ml.

[0038] Further, the volume of the acetonitrile is 10-15 ml, for example, about 12 ml.

[0039] Further, the volume of the water is 10-15 ml, for example, about 12 ml.

[0040] Further, in the preparation method of the traditional Chinese medicine composition test sample solution, the elution method comprises: sequentially using first water, an ammonia solution (5→100), second water, methanol, acetonitrile for elution, placing for a period of time after liquid flow is exhausted, using a third solvent for elution, and collecting the eluent.

[0041] Further, the volume of the first water is 4-8 ml, for example, about 6 ml.

[0042] Further, the volume of the ammonia solution is 5-15 ml, for example, about 10 ml.

[0043] Further, the volume of the second water is 5-15 ml, for example, about 10 ml.

[0044] Further, the volume of the methanol is 5-15 ml, for example, about 10 ml.

[0045] Further, the volume of the acetonitrile is 5-15 ml, for example, about 10 ml.

[0046] Further, the placing time is 4-6 min, for example, about 5 min.

[0047] Further, the third solvent is a mixed solution of acetonitrile and ammonia water.

[0048] Further, in the third solvent, the volume ratio of the acetonitrile to the concentrated ammonia test solution is acetonitrile:concentrated ammonia test solution = about 90:about 10.

[0049] Further, the volume of the third solvent is 10-30 ml, for example, about 20 ml.

[0050] Further, the preparation method of the control solution comprises: weighing appropriate amounts of benzoyl new aconine control, benzoyl aconine control, benzoyl hypaconine control and aconitum double-ester alkaloid control extract, and adding the second solvent to prepare the control solution with the concentrations of benzoyl new aconine, benzoyl aconine, benzoyl hypaconine, new aconine, hypaconine and aconine being 1-50 μg / ml respectively.

[0051] Further, the aconitum double-ester alkaloid control extract contains new aconine, hypaconine and aconine.

[0052] Further, the concentration of benzoyl new aconine in the control solution is about 20 μg / ml.

[0053] Further, the concentration of benzoyl aconine in the control solution is about 3 μg / ml.

[0054] Further, the concentration of benzoyl hypaconine in the control solution is about 3 μg / ml.

[0055] Further, the concentration of new aconine in the control solution is about 5 μg / ml.

[0056] Further, the concentration of hypaconine in the control solution is about 5 μg / ml.

[0057] Further, the concentration of aconine in the control solution is about 5 μg / ml.

[0058] Further, the second solvent is a mixed solution of acetonitrile and ammonium acetate.

[0059] Further, the concentration of ammonium acetate is 0.05-0.2 mol / L, for example, about 0.1 mol / L.

[0060] Further, in the second solvent, the volume ratio of acetonitrile to ammonium acetate is acetonitrile:ammonium acetate=about 20:about 80.

[0061] Further, the preparation method of the decoction of the traditional Chinese medicine composition containing cassia twig comprises: weighing appropriate amounts of cassia twig, white peony root, licorice, ephedra, ginger, atractylodes, anemarrhena, windproof and black smooth tablets, placing them in a container, adding water for soaking, boiling under strong fire, simmering for a period of time under weak fire, filtering, collecting the filtrate, and obtaining the decoction of the traditional Chinese medicine composition containing cassia twig.

[0062] Further, the container is a full-automatic ceramic medicine decocting pot.

[0063] Further, the water has a volume of 1000-2000 ml, for example about 1400 ml.

[0064] Further, the ratio of the sum of the mass of the Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza, Ephedra, Rhizoma Zingiberis Recens, Radix Paeoniae Alba, Radix Anemarrhenae, Radix Saposhnikoviae and Heishunpian to the mass / volume of the water (g / ml) is 0.1-0.5, for example about 0.31.

[0065] Further, the time of the simmering is 30-90 min, for example about 60 min.

[0066] Further, the time of the simmering is 30-90 min, for example about 60 min.

[0067] Further, the filtering is 200 mesh nylon cloth filtering.

[0068] Further, the ratio of the mass between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Radix Saposhnikoviae, the Ephedra, the Radix Paeoniae Alba, the Radix Anemarrhenae and the Glycyrrhiza is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3).

[0069] Further, the ratio of the mass between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Radix Saposhnikoviae, the Ephedra, the Radix Paeoniae Alba, the Radix Anemarrhenae and the Glycyrrhiza is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5).

[0070] Further, the ratio of the mass between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Radix Saposhnikoviae, the Ephedra, the Radix Paeoniae Alba, the Radix Anemarrhenae and the Glycyrrhiza is about 4:about 2:about 5:about 4:about 2:about 5:about 3:about 4:about 2.

[0071] Further, the mass of the Ramulus Cinnamomi is in the range of 40-70 g, for example about 55.20 g.

[0072] Further, the mass of the Heishunpian is in the range of 20-40 g, for example about 27.60 g.

[0073] Further, the mass of the Rhizoma Zingiberis Recens is in the range of 50-90 g, for example about 69.00 g.

[0074] Further, the mass of the Radix Saposhnikoviae is in the range of 40-70 g, for example about 55.20 g.

[0075] Further, the mass of the Ephedra is in the range of 20-40 g, for example about 27.60 g.

[0076] Further, the mass of the Atractylodes Macrocephala Koidz. is in the range from 50 to 90 g, for example about 69.00 g.

[0077] Further, the mass of the Atractylodes Macrocephala Koidz. is in the range from 50 to 90 g, for example about 69.00 g.

[0078] Further, the mass of the Atractylodes Macrocephala Koidz. is in the range from 50 to 90 g, for example about 69.00 g.

[0079] Further, the mass of the Atractylodes Macrocephala Koidz. is in the range from 50 to 90 g, for example about 69.00 g.

[0080] Further, the flow rate is in the range from 0.6 to 1.0 ml / min, for example in the range from 0.75 to 0.85 ml / min, for example about 0.8 ml / min.

[0081] Further, the detection wavelength is in the range from 200 to 300 nm, for example about 235 nm.

[0082] Further, the injection volume is in the range from 15 to 25 μl, for example about 20 μl.

[0083] Further, the theoretical plate number of the chromatographic peak corresponding to benzoylmesaconine is not less than 3000.

[0084] Further, the chromatographic column is a Waters T3 chromatographic column or an Agilent InfintyLab Poroshell 120Aq-C18 chromatographic column or a YMC C18 chromatographic column.

[0085] Further, the chromatographic column is an Agilent InfintyLab Poroshell 120Aq-C18 chromatographic column.

[0086] Further, the specification of the chromatographic column is: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

[0087] Further, the mobile phase A is a mixed solution of acetonitrile and tetrahydrofuran.

[0088] Further, in the mobile phase A, the volume ratio of acetonitrile to tetrahydrofuran is acetonitrile: tetrahydrofuran = about 98: about 2.

[0089] Further, the acid aqueous solution, the alkaline aqueous solution and / or the buffer salt aqueous solution are selected from one or more of weak acids and their salts, weak bases and their salts with different concentrations.

[0090] Further, the acid aqueous solution, the base aqueous solution and / or the buffer salt aqueous solution is selected from formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0091] Further, the buffer salt aqueous solution is an acetic acid salt aqueous solution and / or an acetic acid salt aqueous solution.

[0092] Further, the buffer salt aqueous solution is an ammonium acetate solution and about 0.5 ml of glacial acetic acid is added to every 1000 ml of the ammonium acetate solution.

[0093] Further, the concentration of the ammonium acetate solution is 0.05-0.2 mol / L, for example, about 0.1 mol / L.

[0094] Further, the pH value of the buffer salt aqueous solution is not more than 7.0.

[0095] Further, the detection limit of benzoylmesaconitine is about 48.2 ng / ml.

[0096] Further, the detection limit of benzoylaconitine is about 111.7 ng / ml.

[0097] Further, the detection limit of benzoylhypaconitine is about 108.2 ng / ml.

[0098] Further, the detection limit of mesaconitine is about 139.7 ng / ml.

[0099] Further, the detection limit of hypaconitine is about 131.2 ng / ml.

[0100] Further, the detection limit of aconitine is about 138.8 ng / ml.

[0101] Further, the quantification limit of benzoylmesaconitine is about 0.1927 μg / ml.

[0102] Further, the quantification limit of benzoylaconitine is about 0.1955 μg / ml.

[0103] Further, the quantification limit of benzoylhypaconitine is about 0.2705 μg / ml.

[0104] Further, the quantification limit of mesaconitine is about 0.2234 μg / ml.

[0105] Further, the quantification limit of hypaconitine is about 0.2099 μg / ml.

[0106] Further, the quantitative limit of aconitine is about 0.2221 μg / ml.

[0107] According to another aspect of the present application, there is provided a use of the above-mentioned determination method in the quality detection and / or quality evaluation and / or quality control of aconitum alkaloids in traditional Chinese medicine compositions containing Ramulus Cinnamomi.

[0108] Advantages of the present application:

[0109] The present application monitors the content of monoester-type alkaloids of Heishunpian medicinal flavor in the reference sample of traditional Chinese medicine compositions containing Ramulus Cinnamomi, and controls the limit of diester-type alkaloids, which has important quality control significance for the reference sample of traditional Chinese medicine compositions containing Ramulus Cinnamomi from the aspects of safety and effectiveness. BRIEF DESCRIPTION OF DRAWINGS

[0110] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art according to these drawings without exceeding the scope of the present application.

[0111] Figure 1 It is an HPLC chromatogram under "2.2, initial conditions". Among them, A is a monoester-type alkaloid reference solution, B is a aconite diester-type alkaloid reference extract solution; 1: benzoyl mesaconitine, 2: benzoyl aconine and benzoyl hypaconine, 3: mesaconitine, 4: hypaconitine, 5: aconitine.

[0112] Figure 2 It is an HPLC chromatogram under "2.3, mobile phase gradient adjustment". Among them, A is a mixed aconite alkaloid reference solution, B is a test solution; 1: benzoyl mesaconitine, 2: benzoyl aconine, 3: benzoyl hypaconine, 4: mesaconitine, 5: hypaconitine, 6: aconitine.

[0113] Figure 3 It is an HPLC chromatogram under "2.4, preparation method optimization of test solution". Among them, A is a mixed aconite alkaloid reference solution, B is a test solution; 1: benzoyl mesaconitine, 2: benzoyl aconine, 3: benzoyl hypaconine, 4: mesaconitine, 5: hypaconitine, 6: aconitine.

[0114] Figure 4HPLC chromatogram under "2.5, adjustment of organic phase ratio". Wherein, A is the mixed control solution of aconite alkaloids, B is the test solution; 1: benzoyl neoconhydrine, 2: benzoylaconine, 3: benzoylhypaconine, 4: neoaconitine, 5: hypaconitine, 6: aconitine.

[0115] Figure 5 HPLC chromatogram under "2.6, further optimization of chromatographic conditions". Wherein, A is the mixed control solution of aconite alkaloids, B is the test solution; 1: benzoyl neoconhydrine, 2: benzoylaconine, 3: benzoylhypaconine, 4: neoaconitine, 5: hypaconitine, 6: aconitine.

[0116] Figure 6 HPLC chromatogram of the spiked test solution under "2.9, optimization of chromatographic conditions again". Wherein, 1: benzoyl neoconhydrine, 2: benzoylaconine, 3: benzoylhypaconine, 4: neoaconitine, 5: hypaconitine, 6: aconitine. DETAILED DESCRIPTION

[0117] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0118] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application, or that are given in the specification. Although any methods, conditions, materials, or articles similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods, conditions, materials, or articles are described herein.

[0119] The present application is intended to embrace all options, variations, and equivalents that can be contained in the existing art as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present application. The present application is in no way limited to the methods and materials described.

[0120] As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0121] In the present application, the term "comprising" is synonymous with "including," "containing," or "comprehending." As used herein, the terms "comprising," "including," "containing," "having" or any other similar

[0122] As described in the section of background art, there is no related report on the content determination method of aconite alkaloids in the traditional Chinese medicine composition containing Ramulus Cinnamomi and its application in the prior art. In order to solve the above problems, the present application provides a content determination method of aconite alkaloids in a traditional Chinese medicine composition containing Ramulus Cinnamomi, which comprises the following steps:

[0123] The test is performed on the test sample solution and the control sample solution of the traditional Chinese medicine composition,

[0124] The chromatographic conditions of the test are as follows: a chromatographic column with octadecylsilane bonded silica as the filler is used, the mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, the mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffer salt aqueous solution, the gradient elution program is as follows: 0-25 min, 85% B→80% B; 25-40 min, 80% B→79% B; 40-42 min, 79% B→74% B; 42-60 min, 74% B→63% B; the flow rate is 0.5-1.5 ml / min, the column temperature is 40-42℃, the detection wavelength is 180-360 nm, and the injection amount is 10-30 μl.

[0125] In the present application, when a time, concentration, ratio, volume, power, frequency, rotational speed, mass, wavelength, flow rate, injection amount, temperature, or other value or parameter is expressed in a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairings of an upper range limit or preferred value with a lower range limit or preferred value are specifically disclosed, regardless of whether the range is disclosed individually. For example, when the range "10-30" is disclosed, the described range should be interpreted as including the ranges "10-30", "10-25", "10-20", "10-15", "15-30", "15-25", "15-20", "20-30", "20-25", "25-30", etc. When numerical ranges are described herein, the range is intended to include the end values and all integers and fractions within that range, unless otherwise stated.

[0126] According to the test results, the content information of the control sample of the traditional Chinese medicine composition is obtained;

[0127] The traditional Chinese medicine composition comprises Guizhi, Baishao, Gancao, Mahuang, Shengjiang, Baizhu, Zhimu, Fangfeng and Heishunpian, and the reference substance is benzoyl neo-lappaconitine, benzoyl lappaconitine, benzoyl hypo-lappaconitine, neo-lappaconitine, hypo-lappaconitine and lappaconitine.

[0128] In a preferred embodiment, the information is used to calculate the content of the reference substance in the traditional Chinese medicine composition according to the external standard method according to the corresponding peak areas in the chromatogram of the test solution of the traditional Chinese medicine composition and the chromatogram of the reference substance solution.

[0129] In a preferred embodiment, the linear equation of the benzoyl neo-lappaconitine is y=34.492x-9.3909, R 2 =0.9999.

[0130] In a preferred embodiment, the linear equation of the benzoyl lappaconitine is y=33.75x+0.8969, R 2 =1.0000.

[0131] In a preferred embodiment, the linear equation of the benzoyl hypo-lappaconitine is y=35.495x-7.1817, R 2 =0.9997.

[0132] In a preferred embodiment, the linear equation of the neo-lappaconitine is y=29.6145x-14.7747, R 2 =0.9990.

[0133] In a preferred embodiment, the linear equation of the hypo-lappaconitine is y=32.032x-5.855, R 2 =0.9998.

[0134] In a preferred embodiment, the linear equation of the lappaconitine is y=29.8871x-15.1308, R 2 =0.9990.

[0135] In a preferred embodiment, the preparation method of the test solution of the traditional Chinese medicine composition comprises: taking a proper amount of decoction of traditional Chinese medicine composition containing Guizhi into a container, adding a first solvent, weighing, ultrasonic treating for a period of time, cooling, weighing again, supplementing the lost weight with the first solvent, shaking, centrifuging, filtering, taking a proper amount of the filtrate, adsorbing the filtrate with a solid phase extraction column, eluting, collecting the eluate, recovering under reduced pressure, drying to obtain a residue, dissolving and diluting the residue with a second solvent, shaking, filtering to obtain the test solution of the traditional Chinese medicine composition.

[0136] In a preferred embodiment, the container is a volumetric flask.

[0137] In a preferred embodiment, the first solvent is hydrochloric acid.

[0138] In a preferred embodiment, the concentration of the hydrochloric acid is 0.4-0.6 mol / L, such as about 0.5 mol / L.

[0139] In the present application, "about" means a value within ±5% of a specified value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0140] In a preferred embodiment, the ratio of the volume of the decoction of the traditional Chinese medicine composition to the first solvent is 2-6, such as about 4.

[0141] In the present application, "about" means a value within ±5% of a specified value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0142] In a preferred embodiment, the volume of the decoction of the traditional Chinese medicine composition is 60-100 ml, such as about 80 ml.

[0143] In the present application, "about" means a value within ±5% of a specified value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0144] In a preferred embodiment, the power of the ultrasonic treatment is 300-500 W, such as about 400 W.

[0145] In the present application, "about" means a value within ±5% of a specified value. For example, "about 400" includes ±5% of 400, or from 380 to 420.

[0146] In a preferred embodiment, the frequency of the ultrasonic treatment is 30-50 kHz, such as about 40 kHz.

[0147] In the present application, "about" means a value within ±5% of a specified value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0148] In a preferred embodiment, the time of the ultrasonic treatment is 20-60 min, such as about 40 min.

[0149] In the present application, "about" means a value within ±5% of a specified value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0150] In a preferred embodiment, the speed of the centrifugation is 4000-6000 rpm / min, such as about 5000 rpm / min.

[0151] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 5000" includes ±5% of 5000, or from 4750 to 5250.

[0152] In a preferred embodiment, the centrifugation time is 5-15 min, for example about 10 min.

[0153] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0154] In a preferred embodiment, the volume ratio of the filtrate to the decoction of the Chinese medicine composition is 0.5-1, for example about 0.875.

[0155] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.875" includes ±5% of 0.875, or from 0.83125 to 0.91875.

[0156] In a preferred embodiment, the volume of the filtrate is 60-80 ml, for example about 70 ml.

[0157] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 70" includes ±5% of 70, or from 66.5 to 73.5.

[0158] In a preferred embodiment, the temperature of the reduced pressure recovery is not higher than 40℃.

[0159] In a preferred embodiment, the second solvent is a mixed solution of acetonitrile and ammonium acetate.

[0160] In a preferred embodiment, the concentration of ammonium acetate is 0.05-0.2 mol / L, for example about 0.1 mol / L.

[0161] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0162] In a preferred embodiment, in the second solvent, the volume ratio of acetonitrile to ammonium acetate is acetonitrile:ammonium acetate = about 20:about 80.

[0163] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 20" includes ±5% of 20, or from 19 to 21; "about 80" includes ±5% of 80, or from 76 to 84.

[0164] In a preferred embodiment, the method of preparing the solid phase extraction column comprises using a mixed-mode cation exchange reverse-phase adsorbent as the packing material, and sequentially eluting with acetonitrile and water.

[0165] In a preferred embodiment, the mixed-mode cation exchange reverse-phase adsorbent has a mass of 400-600 mg, such as about 500 mg.

[0166] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0167] In a preferred embodiment, the solid phase extraction column has a capacity of 4-8 ml, such as 6 ml.

[0168] In a preferred embodiment, the acetonitrile has a volume of 10-15 ml, such as about 12 ml.

[0169] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0170] In a preferred embodiment, the water has a volume of 10-15 ml, such as about 12 ml.

[0171] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0172] In a preferred embodiment, in the method of preparing the test solution of the traditional Chinese medicine composition, the method of elution comprises sequentially eluting with first water, an ammonia solution (5→100), second water, methanol, acetonitrile, and then eluting with a third solvent after the liquid flow is complete and a period of time has elapsed, and collecting the eluate.

[0173] In the present application, "ammonia solution (5→100)" means a 5% ammonia solution, which is prepared by diluting 5 ml of ammonia water to 100 ml with water.

[0174] In a preferred embodiment, the first water has a volume of 4-8 ml, such as about 6 ml.

[0175] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0176] In a preferred embodiment, the ammonia solution has a volume of 5-15 ml, such as about 10 ml.

[0177] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0178] In a preferred embodiment, the volume of the second water is 5 to 15 ml, for example, about 10 ml.

[0179] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0180] In a preferred embodiment, the volume of methanol is 5 to 15 ml, for example, about 10 ml.

[0181] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0182] In a preferred embodiment, the volume of the acetonitrile is 5 to 15 ml, for example, about 10 ml.

[0183] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0184] In a preferred embodiment, the placement time is 4 to 6 minutes, for example, about 5 minutes.

[0185] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0186] In a preferred embodiment, the third solvent is a mixed solution of acetonitrile and ammonia.

[0187] In a preferred embodiment, the volume ratio of acetonitrile to concentrated ammonia solution in the third solvent is approximately 90:10.

[0188] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 90" includes ±5% of 90, or from 85.5 to 94.5; "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0189] In a preferred embodiment, the volume of the third solvent is 10 to 30 ml, for example, about 20 ml.

[0190] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0191] In a preferred embodiment, the method of preparing the control solution comprises weighing an appropriate amount of benzoylmesaconitine control, benzoylaconitine control, benzoylhypaconitine control, and aconitum-biesters alkaloid control extract, and adding a second solvent to prepare the control solution with benzoylmesaconitine, benzoylaconitine, benzoylhypaconitine, mesaconitine, hypaconitine, and aconitine concentrations of 1-50 μg / ml, respectively.

[0192] In a preferred embodiment, the aconitum-biesters alkaloid control extract comprises mesaconitine, hypaconitine, and aconitine.

[0193] In a preferred embodiment, the concentration of benzoylmesaconitine in the control solution is about 20 μg / ml.

[0194] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0195] In a preferred embodiment, the concentration of benzoylaconitine in the control solution is about 3 μg / ml.

[0196] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0197] In a preferred embodiment, the concentration of benzoylhypaconitine in the control solution is about 3 μg / ml.

[0198] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0199] In a preferred embodiment, the concentration of mesaconitine in the control solution is about 5 μg / ml.

[0200] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0201] In a preferred embodiment, the concentration of hypaconitine in the control solution is about 5 μg / ml.

[0202] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0203] In a preferred embodiment, the concentration of aconitine in the control solution is about 5 μg / ml.

[0204] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0205] In a preferred embodiment, the second solvent is a mixed solution of acetonitrile and ammonium acetate.

[0206] In a preferred embodiment, the concentration of ammonium acetate is 0.05-0.2 mol / L, for example about 0.1 mol / L.

[0207] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0208] In a preferred embodiment, in the second solvent, the volume ratio of acetonitrile to ammonium acetate is acetonitrile:ammonium acetate = about 20:about 80.

[0209] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 20" includes ±5% of 20, or from 19 to 21; "about 80" includes ±5% of 80, or from 76 to 84.

[0210] In a preferred embodiment, the preparation method of the decoction of the traditional Chinese medicine composition containing cassia twig comprises: weighing appropriate amounts of cassia twig, white peony root, licorice, ephedra, ginger, atractylodes, anemarrhena, siler and black ginseng, placing them in a container, adding water for soaking, boiling under strong fire, simmering for a period of time under weak fire, filtering, collecting the filtrate, and obtaining the decoction of the traditional Chinese medicine composition containing cassia twig.

[0211] In a preferred embodiment, the container is a full-automatic ceramic medicine decocting pot.

[0212] In a preferred embodiment, the volume of water is 1000-2000 ml, for example about 1400 ml.

[0213] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 1400" includes ±5% of 1400, or from 1330 to 1470.

[0214] In a preferred embodiment, the ratio of the sum of the mass of cassia twig, white peony root, licorice, ephedra, ginger, atractylodes, anemarrhena, siler and black ginseng to the mass / volume (g / ml) of water is 0.1-0.5, for example about 0.31.

[0215] In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 0.31" includes ±5% of 0.31, or from 0.2945 to 0.3255.

[0216] In a preferred embodiment, the time of the steeping is 30-60 min, such as about 45 min.

[0217] In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 45" includes ±5% of 45, or from 42.75 to 47.25.

[0218] In a preferred embodiment, the time of the slow-cooking is 30-90 min, such as about 60 min.

[0219] In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0220] In a preferred embodiment, the filtration is a 200 mesh nylon cloth filtration.

[0221] In a preferred embodiment, the mass ratio between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Radix Paeoniae Alba, the Anemarrhena Asphodeloides, and the Glycyrrhiza is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3).

[0222] In a preferred embodiment, the mass ratio between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Radix Paeoniae Alba, the Anemarrhena Asphodeloides, and the Glycyrrhiza is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5).

[0223] In a preferred embodiment, the mass ratio between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Radix Paeoniae Alba, the Anemarrhena Asphodeloides, and the Glycyrrhiza is about 4:about 2:about 5:about 4:about 2:about 5:about 3:about 4:about 2.

[0224] In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0225] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g.

[0226] In the present application, "about" means a value in the range of ±5% of a particular value. For example, "about 41.40" includes ±5% of 41.40, or from 39.54 to 43.26.

[0227] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g.

[0228] In the present application, "about" means a value in the range of ±5% of a particular value. For example, "about 41.40" includes ±5% of 41.40, or from 39.54 to 43.26.

[0229] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g.

[0230] In the present application, "about" means a value in the range of ±5% of a particular value. For example, "about 41.40" includes ±5% of 41.40, or from 39.54 to 43.26.

[0231] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g.

[0232] In the present application, "about" means a value in the range of ±5% of a particular value. For example, "about 41.40" includes ±5% of 41.40, or from 39.54 to 43.26.

[0233] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g.

[0234] In the present application, "about" means a value in the range of ±5% of a particular value. For example, "about 41.40" includes ±5% of 41.40, or from 39.54 to 43.26.

[0235] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g.

[0236] In the present application, "about" means a value in the range of ±5% of a particular value. For example, "about 41.40" includes ±5% of 41.40, or from 39.54 to 43.26.

[0237] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g.

[0238] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0239] In a preferred embodiment, the mass of the Anemarrhena asphodeloides ranges from 40 to 70 g, for example, about 55.20 g.

[0240] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55.20" includes ±5% of 55.20, or from 52.44 to 57.96.

[0241] In a preferred embodiment, the licorice has a mass range of 20 to 40 g, for example, about 27.60 g.

[0242] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0243] In a preferred embodiment, the flow rate is 0.6 to 1.0 ml / min, for example 0.75 to 0.85 ml / min, for example about 0.8 ml / min.

[0244] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.8" includes ±5% of 0.8, or from 0.76 to 0.84.

[0245] In a preferred embodiment, the detection wavelength is 200–300 nm, for example, about 235 nm.

[0246] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 235" includes ±5% of 235, or from 223.25 to 246.75.

[0247] In a preferred embodiment, the injection volume is 15 to 25 μl, for example, about 20 μl.

[0248] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0249] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the benzoyl neoaconitine is not less than 3000.

[0250] In a preferred embodiment, the chromatographic column is a Waters T3 column, an Agilent InfintyLabPoroshell 120Aq-C18 column, or a YMC C18 column.

[0251] In a preferred embodiment, the chromatographic column is an Agilent InfintyLab Poroshell 120 Aq-C18 chromatographic column.

[0252] In a preferred embodiment, the chromatographic column has the following specifications: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

[0253] In a preferred embodiment, the mobile phase A is a mixture of acetonitrile and tetrahydrofuran.

[0254] In a preferred embodiment, in the mobile phase A, the volume ratio of acetonitrile to tetrahydrofuran is acetonitrile: tetrahydrofuran = about 98: about 2.

[0255] In the present application, "about" means a value within ±5% of a specified value. For example, "about 98" includes ±5% of 98, or from 93.1 to 102.9; "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0256] In a preferred embodiment, the aqueous acid solution, aqueous base solution and / or aqueous buffer salt solution is selected from one or more of weak acids and their salts, weak bases and their salts at different concentrations.

[0257] In a preferred embodiment, the aqueous acid solution, aqueous base solution and / or aqueous buffer salt solution is selected from formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid at different concentrations.

[0258] In a preferred embodiment, the aqueous buffer salt solution is an aqueous acetate solution and / or an aqueous acetate solution.

[0259] In a preferred embodiment, the aqueous buffer salt solution is an ammonium acetate solution and about 0.5 ml of glacial acetic acid is added per 1000 ml of ammonium acetate solution.

[0260] In the present application, "about" means a value within ±5% of a specified value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0261] In a preferred embodiment, the concentration of the ammonium acetate solution is 0.05-0.2 mol / L, for example about 0.1 mol / L.

[0262] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0263] In a preferred embodiment, the buffered saline solution has a pH of no more than 7.0.

[0264] In a preferred embodiment, the detection limit of benzoylmesaconitine is about 48.2 ng / ml.

[0265] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 48.2" includes ±5% of 48.2, or from 45.79 to 50.61.

[0266] In a preferred embodiment, the detection limit of benzoylaconitine is about 111.7 ng / ml.

[0267] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 111.7" includes ±5% of 111.7, or from 106.115 to 117.285.

[0268] In a preferred embodiment, the detection limit of benzoylhypaconitine is about 108.2 ng / ml.

[0269] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 108.2" includes ±5% of 108.2, or from 102.79 to 113.61.

[0270] In a preferred embodiment, the detection limit of mesaconitine is about 139.7 ng / ml.

[0271] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 139.7" includes ±5% of 139.7, or from 132.715 to 146.685.

[0272] In a preferred embodiment, the detection limit of hypaconitine is about 131.2 ng / ml.

[0273] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 131.2" includes ±5% of 131.2, or from 124.64 to 137.76.

[0274] In a preferred embodiment, the detection limit of aconitine is about 138.8 ng / ml.

[0275] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 138.8" includes ±5% of 138.8, or from 131.86 to 145.74.

[0276] In a preferred embodiment, the limit of quantitation for benzoylmesaconitine is about 0.1927 μg / ml.

[0277] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.1927" includes ±5% of 0.1927, or from 0.183065 to 0.202335.

[0278] In a preferred embodiment, the limit of quantitation for benzoylaconitine is about 0.1955 μg / ml.

[0279] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.1955" includes ±5% of 0.1955, or from 0.185725 to 0.205275.

[0280] In a preferred embodiment, the limit of quantitation for benzoylhypaconitine is about 0.2705 μg / ml.

[0281] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.2705" includes ±5% of 0.2705, or from 0.256975 to 0.284025.

[0282] In a preferred embodiment, the limit of quantitation for mesaconitine is about 0.2234 μg / ml.

[0283] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.2234" includes ±5% of 0.2234, or from 0.21223 to 0.23457.

[0284] In a preferred embodiment, the limit of quantitation for hypaconitine is about 0.2099 μg / ml.

[0285] In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.2099" includes ±5% of 0.2099, or from 0.199405 to 0.220395.

[0286] In a preferred embodiment, the limit of quantitation for aconitine is about 0.2221 μg / ml.

[0287] In the present application, "about" means a value within ±5% of a specified value. For example, "about 0.2221" includes ±5% of 0.2221, or from 0.210995 to 0.233205.

[0288] According to another aspect of the present application, there is provided a use of the above-mentioned determination method in the quality detection and / or quality evaluation and / or quality control of aconitum alkaloids in a traditional Chinese medicine composition comprising Ramulus Cinnamomi.

[0289] The present application will be further described with reference to the following examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions or the conditions suggested by the manufacturers.

[0290] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are intended to be illustrative only and are not intended to be limiting.

[0291] The above-mentioned features mentioned in the present application, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in the present patent specification can be used with any combination, and each feature disclosed in the present specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0292] Examples

[0293] 1. Instruments and reagents

[0294] 1.1. Instruments

[0295] Electronic balance (specification: 0.01%, model: BCE124I-1CCN / BSA124, serial number: 20501-012 / 010, manufacturer: Sartorius Instruments Co., Ltd.); Electronic balance (specification: 0.01%, model: MS105DU, serial number: 20501-006, manufacturer: Mettler Toledo Instruments Co., Ltd.); Electronic balance (specification: 0.1%, model: JY2002, serial number: 20502-017, manufacturer: Shanghai Puchun Metrology Instruments Co., Ltd.); Decoction pot (model: 2.5L, serial number: 20602-069 / 072 (Manufacturer: Kangyashun Electric Appliance Co., Ltd.); Liquid heater (Model: 30B2, Serial Number: 20304-081 / 080, Manufacturer: Kangyashun Electric Appliance Co., Ltd.); Decoction pot (Model: 3.0L, Serial Number: 20602-016 / 018 / 019, Manufacturer: Chaozhou City Chaoan District Kangyashun Electric Appliance Co., Ltd.); Temperature-adjustable heating plate (Model: KDMB, Serial Number: 20302-036 / 034, Manufacturer: Shandong Juancheng Hualu Electric Heating Instrument Co., Ltd.); High-performance liquid chromatograph (Model: Aglient) 1100-DAD, serial number: 20101-007 / 008, manufacturer: Agilent Technologies, Inc.; High-performance liquid chromatograph (model: Agilent 1290-DAD, serial number: 20101-014 / 049, manufacturer: Agilent Technologies, Inc.); Chromatographic column (model: HALO 90A AQ-C). 18 4.6mm × 150mm, 2.7μm, S / N: USAQT001536, USAQT001619, Manufacturer: Changsha Kemei Analytical Instruments Co., Ltd.; Column (Model: Waters) T3, 4.6mm×150mm, 2.7μm, SN01623401818432, Manufacturer: Changsha Jiedao Scientific Instruments Co., Ltd.; Column (Model: Agilent InfintyLab Poroshell 120Aq-C) 18 4.6mm×150mm, 2.7μm, SNUSKTM01043, manufacturer: Beijing Bafang Century Technology Co., Ltd.; chromatographic column (model: YMC MeteoricCore C). 18, 4.6 mm x 150 mm, 2.7 μm, S.N. MC005032, manufacturer: Changsha Kromasil Analytical Instrument Co., Ltd.); a numerical control ultrasonic cleaner (model: KQ-500DB, number: 21101-035, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.); a centrifuge (model: Yingtai-TD4N, number: 21101-003, manufacturer: Changsha Yingtai Instrument Co., Ltd.); a rotary evaporator (model: R-1001VN, number: 20603-001, manufacturer: Zhengzhou Greatwall Scientifics Co., Ltd.); a solid phase extraction column (model: mixed strong cation exchange solid phase extraction column (500 mg, 6 ml), S / N: N1750042, N1750031, R5540032, R5540043, T4480029, manufacturer: Shanghai Anpu Experimental Technology Co., Ltd.); a solid phase extraction column (model: mixed strong cation exchange solid phase extraction column (150 mg, 6 ml), S / N: I0860042, manufacturer: Shanghai Anpu Experimental Technology Co., Ltd.).

[0296] 1.2, reagents

[0297] 1.2.1, reagents

[0298] Water (batch number: 20231126, 20240510, 20240713, manufacturer: Hunan Xiangwa Beverage Science and Technology Limited Company, grade: purified water); acetonitrile (batch number: WXBF2252V, WXBF1923V, WXBF2846V, manufacturer: SIGMA, grade: chromatographic grade, batch number: 013424040417, manufacturer: Shanghai Skyrun High-Purity Solvent Co., Ltd., grade: chromatographic grade); methanol (batch number: WXBF2172V, manufacturer: SIGMA, grade: chromatographic grade, grade: chromatographic grade, batch number: 0234240411D, manufacturer: Shanghai Skyrun High-Purity Solvent Co., Ltd., grade: chromatographic grade); ammonia water (batch number: 20230830, 20231103, 20231101, manufacturer: National Pharmaceutical Group Chemical Reagent Co., Ltd., grade: analytical pure); ammonium acetate (batch number: 20220622, 20231017, manufacturer: National Pharmaceutical Group Chemical Reagent Co., Ltd., grade: analytical pure); hydrochloric acid (batch number: 20230313, 20230905, manufacturer: National Pharmaceutical Group Chemical Reagent Co., Ltd., grade: analytical pure); tetrahydrofuran (batch number: 23085314, manufacturer: Anhui Tiandi High-Purity Solvent Co., Ltd., grade: chromatographic grade); glacial acetic acid (batch number: 20220929, manufacturer: National Pharmaceutical Group Chemical Reagent Co., Ltd., grade: analytical pure); isopropyl alcohol (batch number: 23085307, manufacturer: Anhui Tiandi High-Purity Solvent Co., Ltd., grade: chromatographic grade); dichloromethane (batch number: 20230511, manufacturer: National Pharmaceutical Group Chemical Reagent Co., Ltd., grade: analytical pure); diethyl ether (batch number: 20230530, manufacturer: National Pharmaceutical Group Chemical Reagent Co., Ltd., grade: analytical pure); ammonium dihydrogen phosphate (batch number: C15317262, manufacturer: Shanghai Maikelin Biochemical Technology Co., Ltd., grade: chromatographic grade).

[0299] 1.2.2, Control and control extract

[0300] benzoylmesaconitine (batch number: 111795-202106, content 96.3%, source: China Institute for Food and Drug Control); benzoylaconine (batch number: 111794-202307, content 98.0%, source: China Institute for Food and Drug Control); benzoylhypaconitine (batch number: 111796-202207, content 96.4%, source: China Institute for Food and Drug Control); aconitum biflavoide alkaloid reference extract (batch number: 112029-202302, content 32.9% of mesaconitine, 30.9% of hypaconitine, 32.7% of aconitine, source: China Institute for Food and Drug Control); aconitum biflavoide alkaloid reference extract (batch number: 112029-201601, content 31.7% of mesaconitine, 30.0% of hypaconitine, 31.8% of aconitine, source: China Institute for Food and Drug Control); benzoylaconine (batch number: 111794-202006, content 99.5%, source: China Institute for Food and Drug Control).

[0301] 1.2.3, information of medicinal materials

[0302] Ramulus cinnamomi (batch number: ZA00923070504, place of origin: Rongxian County, Yulin City, Guangxi Zhuang Autonomous Region, source: self-made); Radix paeoniae alba (batch number: ZA00123070503, place of origin: Qiaocheng District, Bozhou City, Anhui Province, source: self-made); Rhizoma atractylodis macrocephalae (batch number: ZA04923070707, place of origin: Shexian County, Huangshan City, Anhui Province, source: self-made); Rhizoma atractylodis macrocephalae (batch number: ZA04923080104, place of origin: Xinchang County, Shaoxing City, Zhejiang Province, source: self-made); Rhizoma anemarrhenae (batch number: ZA08023070701, place of origin: Boye County, Baoding City, Hebei Province, source: self-made); Rhizoma anemarrhenae (batch number: ZA08023070704, place of origin: Anguo City, Baoding City, Hebei Province, source: self-made); Heishunpian (batch number: ZA1732307001, place of origin: Jiangyou City, Mianyang City, Sichuan Province, source: self-made); Radix glycyrrhizae (batch number: ZA00423071001, place of origin: Guazhou County, Jiuquan City, Gansu Province, source: self-made); Radix glycyrrhizae (batch number: ZA00423081513, place of origin: Hangjin Banner, Ordos City, Inner Mongolia Autonomous Region, source: self-made); Saposhnikovia divaricata (Turcz.) Schischk. (batch number: ZA03923070703, place of origin: Tailai County, Qiqihar City, Heilongjiang Province, source: self-made); Saposhnikovia divaricata (Turcz.) Schischk. (batch number: ZA03923111301, place of origin: Tuquan County, Xing'anmeng, Inner Mongolia Autonomous Region, source: self-made); Ephedra sinica Stapf (batch number: 01-21030201, place of origin: Arhorqin Banner, Chifeng City, Inner Mongolia Autonomous Region, source: Anhui Runfuryong Pharmaceutical Co., Ltd.); Zingiber officinale Roscoe (batch number: ZA00523102601, place of origin: Jiangxi County, Leshan City, Sichuan Province, source: self-made); Zingiber officinale Roscoe (batch number: ZA00523091101, place of origin: Jiangxi County, Leshan City, Sichuan Province, source: self-made).

[0303] 1.2.4. Preparation of the reference sample

[0304] Reference sample: 55.20 g of Ramulus cinnamomi, 41.40 g of Radix paeoniae alba, 27.60 g of Radix glycyrrhizae, 27.60 g of Ephedra sinica Stapf, 69.00 g of Zingiber officinale Roscoe, 69.00 g of Rhizoma atractylodis macrocephalae, 55.20 g of Rhizoma anemarrhenae, 55.20 g of Saposhnikovia divaricata (Turcz.) Schischk. and 27.60 g of Heishunpian were weighed into a 3.0 L automatic ceramic decocting kettle, 1400 ml of water was added, soaked for 45 minutes, heated to boiling with strong fire, and kept boiling with weak fire for 60 minutes. The filtrate was collected by hot filtration with 200 mesh nylon filter cloth, and the reference sample was obtained. The negative control decoction without Heishunpian: the Heishunpian was omitted from the prescription, and the decoction was prepared according to the preparation method of the reference sample. The single Heishunpian decoction: 27.60 g of Heishunpian was weighed and prepared according to the preparation method of the reference sample.

[0305] 1.2.5. Information of decoction

[0306] Traditional Chinese medicine composition compound decoction containing Ramulus cinnamomi (batch number: GZSYZMKLST-2023120601-L, batch number: GZSYZMKLST-2024011003-L, GZSYZMKLST-2024022604-L, GZSYZMKLST-2024030301-L, GZSYZMKLST-2024030302-L); He Shun tablet single decoction (batch number: GZSYZMKLST-2024022601-L, GZSYZMKLST-2024070504-L, GZSYZMKLST-2024052101-L); He Shun tablet negative control decoction (batch number: GZSYZMKLST-2024022605-L, GZSYZMKLST-2024052102-L, GZSYZMKLST-2024052401-L, GZSYZMKLST-2024070505-L). Reference sample (batch number: GZSYZMKLST-2024052103-L, GZSYZMKLST-2024052104-L, GZSYZMKLST-2024052105-L, GZSYZMKLST-2024052106-L, GZSYZMKLST-2024052107-L, GZSYZMKLST-2024052108-L, GZSYZMKLST-2024060301-L, GZSYZMKLST-2024061503-L, GZSYZMKLST-2024061504-L, GZSYZMKLST-2024061505-L, GZSYZMKLST-2024061506-L, GZSYZMKLST-2024061507-L, GZSYZMKLST-2024061508-L, GZSYZMKLST-2024070501-L, GZSYZMKLST-2024070502-L, GZSYZMKLST-2024070503-L, GZSYZMKLST-2024071401-L, GZSYZMKLST-2024071402-L).

[0307] 2. Preliminary establishment of content method

[0308] 2.1. Selection of detection wavelength

[0309] The present application selects the detection wavelength as 235 nm.

[0310] 2.2. Initial conditions

[0311] Chromatographic conditions: octadecylsilane bonded silica gel as filler (HALO AQ-C 18Column:Chromolith Speed-Rod RP-18e (4.6 mm x 150 mm, 2.7 μm); mobile phase A: acetonitrile-tetrahydrofuran (25:15), mobile phase B: 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml), gradient elution as specified in Table 1, flow rate: 0.6 ml / min, detection wavelength: 235 nm.

[0312] Table 1 Gradient elution conditions 1

[0313] Time (min) Mobile phase A (%) Mobile phase B (%) 0～48 15→26 85→74 48～49 26→35 74→65 49～58 35 65 58～65 35→15 65→85

[0314] Preparation of reference solution: an appropriate amount of reference substance of benzoyl new aconine, benzoyl aconine, benzoyl hypaconine, and aconitum bis-ester alkaloid reference extract were precisely weighed and measured, and dissolved in isopropyl alcohol-dichloromethane (1:1) to prepare a stock solution of appropriate concentration; an appropriate amount of the stock solution of benzoyl new aconine, benzoyl aconine, and benzoyl hypaconine was precisely measured and placed in a suitable size volumetric flask, and isopropyl alcohol-dichloromethane (1:1) was added to prepare a solution containing 10 μg of each per 1 ml, which was used as the reference solution of mono-ester alkaloids. An appropriate amount of the stock solution of aconitum bis-ester alkaloid reference extract was precisely measured and placed in a suitable size volumetric flask, and isopropyl alcohol-dichloromethane (1:1) was added to prepare a solution containing 10 μg of new aconine, hypaconine, and aconine per 1 ml, which was obtained.

[0315] Preparation of test solution: 100 ml of the product ("1.2.5, Decoction Information" containing the traditional Chinese medicine composition compound decoction containing Ramulus Cinnamomi, batch number: GZSYZMKLST-2023120601-L) was precisely measured and placed in a separatory funnel, and then extracted twice by shaking with 140 ml of diethyl ether and 20 ml of 0.1 mol / L hydrochloric acid solution, and the diethyl ether liquid was discarded. Then, 8 ml of ammonia test solution and 140 ml of diethyl ether were added to the lower acid solution, and extracted by shaking four times, and the diethyl ether liquid was combined. The solvent was recovered to dryness under reduced pressure below 40°C, and then a small amount of acetonitrile-0.02 mol / L ammonium dihydrogen phosphate solution (1:1) was added to dissolve the residue, which was transferred to a 10 ml volumetric flask and diluted to the mark with acetonitrile-0.02 mol / L ammonium dihydrogen phosphate solution (1:1), shaken well, filtered, and the filtrate was collected, which was obtained.

[0316] Determination method: 20 μl of the reference solution and the test solution was precisely taken and injected into the liquid chromatograph, and then determined, which was obtained.

[0317] The results are shown in Table 2 and Figure 1 Table 2.

[0318] Table 2. Chromatographic data of monoester and diester alkaloid controls under initial conditions.

[0319]

[0320] The results showed that under these chromatographic conditions, the mixed control solution of monoester-type alkaloids only exhibited two chromatographic peaks, which is inconsistent with the presence of three components in the mixed control solution. It is speculated that under these conditions, the chromatographic peaks of benzoyl aconitine and benzoyl hypoconitine co-elute. Since the separation of the chromatographic peaks of the reference standard could not meet the requirements for content determination, the test sample was not analyzed. The next step is to adjust the mobile phase gradient.

[0321] 2.3 Adjustment of mobile phase gradient

[0322] The adjusted mobile phase gradient is shown in Table 3, and the results are shown in Table 4. Figure 2 As shown.

[0323] Table 3 Gradient elution conditions 2

[0324]

[0325]

[0326] Preparation of reference solutions: Take appropriate amounts of benzoyl neoaconitine reference standard, benzoyl aconitine reference standard, benzoyl hypoaconitine reference standard, and aconitine diester type alkaloid reference extract, accurately weigh them, and add acetonitrile-0.02mol / L ammonium dihydrogen phosphate solution (1:1) to prepare a solution containing 50μg of benzoyl neoaconitine, 10μg of benzoyl aconitine, 10μg of benzoyl hypoaconitine, 3μg of neoaconitine, 3μg of hypoaconitine, and 3μg of aconitine per ml, as a mixed reference solution of aconitine alkaloids.

[0327] Table 4. Chromatographic peak parameters of aconite alkaloid mixed control and test sample

[0328]

[0329] The results showed that under this gradient, the mixed control solution of aconitine alkaloids exhibited 6 chromatographic peaks, indicating a significant improvement in separation. However, the separation of chromatographic peaks in the test sample, whether monoester or diester alkaloids, was poor, failing to reach baseline separation and thus not meeting the requirements for content determination analysis.

[0330] After analyzing the preparation method of the test solution, it was speculated that the properties of the above-mentioned impurity components may be similar to those of aconite alkaloids. It would be difficult to make the above six components meet the content determination requirements by optimizing the chromatographic conditions alone. Therefore, it was considered to optimize the preparation method of the test solution in order to improve the separation through test sample purification.

[0331] 2.4 Optimization of the preparation method of the test solution

[0332] Preparation of the test solution: Accurately measure 80 ml of this product and place it in a 100 ml volumetric flask. Add 20 ml of 0.5 mol / L hydrochloric acid solution to the mark, weigh the solution, sonicate (400 W, 40 kHz) for 40 minutes, cool, weigh again, replenish the lost weight with 0.5 mol / L hydrochloric acid solution, shake well, transfer to a centrifuge tube, centrifuge (5000 rpm) for 10 minutes, filter, accurately measure 10 ml of the filtrate and add it to a solid-phase extraction column (using a mixed cation exchange reversed-phase adsorbent as the packing material, 15...). 0 mg, volume 6 ml, pre-eluted with 6 ml each of acetonitrile and water, then eluted sequentially with 3 ml of water, ammonia solution (5→100), water, methanol, and 5 ml each of acetonitrile. After the eluent has drained, let stand for 5 minutes, then elute with 10 ml of a mixed solution of acetonitrile-concentrated ammonia (90:10). Collect the eluent, and recover the solvent under reduced pressure below 40°C until dry. Accurately dissolve the residue in a mixed solution of acetonitrile-0.1% phosphoric acid (20:80), place in a 5 ml volumetric flask, dilute to the mark, shake well, filter, and collect the filtrate.

[0333] Simultaneously, the analysis was performed under the chromatographic conditions described in Table 3 above, and the results are shown in Table 5 and... Figure 3 As shown.

[0334] Table 5. Chromatographic peak parameters of monoester alkaloids in mixed control and test samples of aconitine alkaloids.

[0335]

[0336] The results showed that, for monoester-type alkaloids, compared with the control, only benzoylneopioid peaks were observed in the test sample, while no peaks were detected at the corresponding retention times of benzoylneopioid and benzoylhypoaconitine. This indicated that the sample loading was too small and the concentration of the corresponding components in the test sample was too low, resulting in the failure to detect the target peaks. For diester-type alkaloids, combined with experience and component content analysis, it was inferred that there was a significant interfering peak at the corresponding retention time of the neoaconitine control. Therefore, it was considered to simultaneously increase the sample loading and adjust the mobile phase ratio to obtain better chromatographic behavior.

[0337] 2.5 Adjusting the organic phase ratio

[0338] The ratio of acetonitrile-tetrahydrofuran (25:15) was adjusted to acetonitrile-tetrahydrofuran (9:1) for trial, and the elution gradient was adjusted accordingly. The adjusted chromatographic conditions are shown in Table 6, and the results are shown in Table 7. Figure 4 As shown, 30 ml of the filtrate of the test sample solution was added to a solid-phase extraction column for appropriate processing to prepare the test sample solution.

[0339] Octadecylsilane bonded silica gel as the packing agent (HALO AQ-C 18 Agilent ZORBAX SB-C18 column, 4.6 mm x 150 mm, 2.7 μm; acetonitrile-tetrahydrofuran (9:1) as mobile phase A, 0.1 mol / L ammonium acetate solution (add 0.5 ml of glacial acetic acid to 1000 ml) as mobile phase B, gradient elution as specified in Table 6, column temperature 30 °C, flow rate 0.6 ml per minute, detection wavelength 235 nm.

[0340] Table 6 Gradient elution conditions 3

[0341] Time (min) Mobile phase A (%) Mobile phase B (%) 0～10 15→24 85→76 10～28 24 76 28～32 24→27 76→73 32～55 27 73 55～58 27→60 73→40 58～60 60→15 40→85 60～90 15 85

[0342] Table 7 Aconite alkaloid mixture control and test sample monoester type alkaloid chromatographic peak parameters

[0343]

[0344]

[0345] The results show that benzoyl neoaconitine, benzoyl aconitine and benzoyl hypaconitine are detected in the test sample, and no chromatographic peak appears at the corresponding retention time of diester type alkaloid control, but the separation of benzoyl aconitine is not good, so the chromatographic conditions need to be further optimized.

[0346] 2.6, Further optimization of chromatographic conditions

[0347] The adjusted mobile phase gradient is shown in Table 8, and since increasing the column temperature can improve separation, the column temperature is also adjusted to 40 °C, and the results are shown in Table 9 and Figure 5 .

[0348] Table 8 Gradient elution conditions 4

[0349] Time (min) Mobile phase A (%) Mobile phase B (%) 0～15 15→24 85→76 15～28 24 76 28～30 24→26 76→74 30～50 26→28 74→72 50～52 28→15 72→85

[0350] Table 9 Test sample monoester type alkaloid chromatographic peak parameters

[0351]

[0352] The results show that under this chromatographic condition, the separation of the three components of monoester type alkaloids in the test sample is good, and there is no interference at the corresponding retention time of diester type alkaloid control, meeting the requirements of separation degree for content determination.

[0353] This chromatographic condition is tentatively determined, and is used for further optimization of the preparation method of test sample solution.

[0354] 2.7, Further optimization of the preparation method of test sample solution

[0355] The peak area of benzoyl protoaconitine and benzoyl hypaconitine in the test sample obtained by the above test sample preparation method is small, and the sensitivity of the instrument is high, which is not suitable for quantitative analysis. Therefore, it is necessary to continue to increase the volume of the filtrate added to the solid phase extraction column, that is, the sample volume, so as to increase the concentration of the target component in the obtained test sample, and to improve the response of the chromatographic peak. Therefore, the specifications of the solid phase extraction column, different sample volumes of the subsequent filtrate and different volumes of the eluent are investigated to meet the requirements of content determination and ensure the detection accuracy and reproducibility of the method.

[0356] 2.7.1 Investigation of solid phase extraction columns with different loading amounts

[0357] The solid phase extraction columns with loading amounts of 150 mg and 500 mg were investigated for their adsorption capacity for aconitum alkaloids. The results showed that the total peak area of the monoester type alkaloids in the test sample obtained using the solid phase extraction column with a loading amount of 500 mg was greater than that obtained using the solid phase extraction column with a loading amount of 150 mg, indicating that when the solid phase extraction column with a loading amount of 150 mg was used and the sample volume of the filtrate was 30 ml, it had exceeded its adsorption capacity and might have caused overload. Therefore, the solid phase extraction column with a loading amount of 500 mg was used for subsequent research.

[0358] 2.7.2 Investigation of different redissolution solvents

[0359] Since the 0.1% phosphoric acid solution in the original redissolution solvent is different from the 0.1 mol / L ammonium acetate solution used in the mobile phase water phase, the effects of using a mixed solution of acetonitrile-0.1% phosphoric acid solution (20:80) and a mixed solution of acetonitrile-0.1 mol / L ammonium acetate solution (20:80) as the redissolution solvent on each chromatographic peak in the test sample were investigated.

[0360] Based on the retention time of hypaconitine in the chromatogram of the aconitum alkaloid mixture control solution in "2.6 Further optimization of chromatographic conditions", the results showed that the peak shape of the test sample obtained using the mixed solution of acetonitrile-0.1 mol / L ammonium acetate solution (20:80) was better than that of the mixed solution of acetonitrile-0.1% phosphoric acid solution (20:80) at this time period, which was beneficial to the separation of the impurity peak and hypaconitine in the later spiked sample. Therefore, the mixed solution of acetonitrile-0.1 mol / L ammonium acetate solution (20:80) was selected as the redissolution solvent for the test sample.

[0361] 2.7.3 Investigation of different sample volumes

[0362] Based on the results of "2.7.2", when the sample volume of the subsequent filtrate was 30 ml, benzoyl hypaconitine was not detected in the test sample. Therefore, it is necessary to increase the sample volume of the subsequent filtrate for investigation and determine its sample volume.

[0363] The results show that with the increase of sample volume, the peak areas of benzoyl new protoaconitine, benzoyl protoaconitine and benzoyl hypaconitine gradually increase; when the sample volume is 70 ml, the peak area of benzoyl new protoaconitine is 898.034, the peak area of benzoyl protoaconitine is 101.606, and the peak area of benzoyl hypaconitine is 69.751, and the response is good. At the same time, combined with the optional specifications of the solid phase extraction column and the content of the black aconite medicinal material itself, the sample volume of the filtrate is selected as 70 ml.

[0364] 2.7.4, Investigation of different eluent volumes

[0365] By using the mixed solution of acetonitrile-conc. ammonia test solution (90:10) for elution in batches, each time 10 ml, and timely monitoring the elution of each time, the use amount of the mixed solution of acetonitrile-conc. ammonia test solution (90:10) for elution is determined.

[0366] The results show that after the solid phase extraction column is eluted for the second time by the mixed solution of acetonitrile-conc. ammonia test solution (90:10), part of benzoyl new protoaconitine in the test sample is still eluted out of the column, and after the third elution, no chromatographic peak of monoester type alkaloid is detected in the test sample, indicating that the aconite alkaloid components can be completely eluted in the second time of using the solid phase extraction column. Therefore, the use amount of the mixed solution of acetonitrile-conc. ammonia test solution (90:10) for elution is determined as 20 ml.

[0367] Combined with the above elution amount investigation results of the mixed solution of acetonitrile-conc. ammonia test solution (90:10), in order to ensure that the target components can be completely eluted under the above sample amount and at the same time meet the requirements of impurity removal, the use amounts of water, ammonia solution (5→100), methanol and acetonitrile in the elution process are all increased by 1 times. That is, before use, the solid phase extraction column is pre-eluted with acetonitrile and water each for 12 ml, and after the filtrate is loaded, it is eluted with water 6 ml, ammonia solution (5→100), water, methanol and acetonitrile each for 10 ml, and after standing, it is eluted with the mixed solution of acetonitrile-conc. ammonia test solution (90:10) for 20 ml.

[0368] 2.8, Tentative detection method

[0369] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as filler (HALO AQ-C 18 , 4.6 mm x 150 mm, 2.7 μm); acetonitrile-tetrahydrofuran (9:1) as mobile phase A, 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) as mobile phase B, gradient elution according to the provisions in Table 10; flow rate is 0.6 ml per minute; column temperature is 40℃; detection wavelength is 235 nm. The theoretical plate number calculated according to the peak of benzoyl new protoaconitine should not be less than 3000.

[0370] Table 10 Gradient elution condition 5

[0371] Time (min) Mobile phase A (%) Mobile phase B (%) 0～15 15→24 85→76 15～28 24 76 28～30 24→26 76→74 30～50 26→28 74→72

[0372] Preparation of test solution: Take 80 ml of the sample, add 20 ml of 0.5 mol / L hydrochloric acid solution to the 100 ml flask, weigh, ultrasonic treatment (power 400 W, frequency 40 kHz) for 40 minutes, cool, re-weigh, add 0.5 mol / L hydrochloric acid solution to make up the weight loss, shake, centrifuge (5000 rpm) for 10 minutes, filter, take 70 ml of the filtrate, add to the solid phase extraction column (mixed cation exchange reverse phase adsorbent as filler, 500 mg, 6 ml in volume, previously eluted with acetonitrile and water 12 ml each), elute with water 6 ml, ammonia solution (5→100), water, methanol, acetonitrile 10 ml each, after the eluent flows out, place for 5 minutes, then elute with acetonitrile-concentrated ammonia solution (90:10) mixed solution 20 ml, collect the eluent, recover the solvent to dryness under reduced pressure below 40℃, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution to dissolve the residue, place in a 5 ml flask, dilute to the mark, shake, filter, take the filtrate, and you get it.

[0373] Preparation of test solution: Take 80 ml of the sample, add 20 ml of 0.5 mol / L hydrochloric acid solution to the 100 ml flask, weigh, ultrasonic treatment (power 400 W, frequency 40 kHz) for 40 minutes, cool, re-weigh, add 0.5 mol / L hydrochloric acid solution to make up the weight loss, shake, centrifuge (5000 rpm) for 10 minutes, filter, take 70 ml of the filtrate, add to the solid phase extraction column (mixed cation exchange reverse phase adsorbent as filler, 500 mg, 6 ml in volume, previously eluted with acetonitrile and water 12 ml each), elute with water 6 ml, ammonia solution (5→100), water, methanol, acetonitrile 10 ml each, after the eluent flows out, place for 5 minutes, then elute with acetonitrile-concentrated ammonia solution (90:10) mixed solution 20 ml, collect the eluent, recover the solvent to dryness under reduced pressure below 40℃, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution to dissolve the residue, place in a 5 ml flask, dilute to the mark, shake, filter, take the filtrate, and you get it.

[0374] Determination: Take 20 μl of the control solution and test solution respectively, inject into the liquid chromatograph, and determine.

[0375] 2.9, Optimization of chromatographic conditions again

[0376] When the methodology was validated using “2.8, Provisional Detection Method”, it was found that non-target substances in the spiked test solution (with an appropriate amount of aconitine diester alkaloid reference extract added during the preparation of the test solution) interconverted, and the converted substances and target substances co-eluted in the chromatogram, making it impossible to accurately quantify the target substances.

[0377] Analysis of the above phenomenon revealed that it was unrelated to the reagents or equipment.

[0378] Secondly, in order to improve the separation of target and non-target substances, attempts were made to re-examine and optimize the preparation method of the test sample solution, but without success. Therefore, the chromatographic conditions were readjusted and optimized, and the optimized chromatographic conditions and results are shown in Tables 11-13. Figure 6 As shown:

[0379] Using octadecylsilane-bonded silica gel as a filler (Agilent InfintyLab Poroshell 120Aq-C) 18 The column was 4.6 mm × 150 mm, 2.7 μm in diameter. Acetonitrile-tetrahydrofuran (98:2) was used as mobile phase A, and 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) was used as mobile phase B. Gradient elution was performed according to the specifications in Table 11. The flow rate was 0.8 ml / min; the column temperature was 40 °C; and the detection wavelength was 235 nm. The theoretical plate number, calculated based on the benzoyl neoaconitine peak, should be no less than 3000.

[0380] Table 11 Gradient elution conditions 6

[0381] Time (min) Mobile phase A (%) Mobile phase B (%) 0～25 15→20 85→80 25～40 20→21 80→79 40～42 21→26 79→74 42～60 26→37 74→63

[0382] Table 12 System Suitability Parameters for Chromatographic Peaks of Indicator Components in Spiked Test Samples

[0383]

[0384] Table 13 System Suitability Parameters for Chromatographic Peaks of Indicator Components in Spiked Test Samples

[0385]

[0386]

[0387] The results showed that under these chromatographic conditions, the monoester and diester alkaloids in the spiked test solution were well separated, meeting the resolution requirements for content determination.

[0388] 3. Methodological Validation

[0389] According to the above tentative detection method, the content of single ester alkaloids in the reference sample of traditional Chinese medicine composition containing cassia twig and the limit test method of double ester alkaloids were systematically methodologically verified according to the analysis method verification guiding principles of Chinese Pharmacopoeia 2020 edition volume 4 9101.

[0390] 3.1, Chromatographic conditions and solution preparation

[0391] Since aconitine and new aconitine were not detected in the test sample during development, in order to determine the feasibility of the double ester alkaloid limit test method for the test sample solution of traditional Chinese medicine composition containing cassia twig, a standard addition method was used to prepare a standard addition test sample solution (since the aconitine reference substance was no longer provided by the China Institute for Food and Drug Control, aconitine double ester alkaloid reference extract was used for standard addition), and a systematic methodological study was conducted on the double ester alkaloid limit test of the standard addition test sample.

[0392] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as filler (Agilent InfintyLab Poroshell 120Aq-C 18 , 4.6 mm x 150 mm, 2.7 μm); acetonitrile-tetrahydrofuran (98:2) as mobile phase A, 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) as mobile phase B, gradient elution according to the provisions in Table 11; flow rate of 0.8 ml per minute; column temperature of 40℃; detection wavelength of 235 nm. The theoretical plate number should not be less than 3000 calculated by the benzoyl new aconine peak.

[0393] Preparation of reference substance solution: take benzoyl new aconine reference substance, benzoyl aconine reference substance, benzoyl hypaconine reference substance, aconitine double ester alkaloid reference extract, accurately weigh, respectively, prepare appropriate concentration stock solution with acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution, accurately take benzoyl new aconine reference substance stock solution, benzoyl aconine reference substance stock solution, benzoyl hypaconine reference substance stock solution, aconitine double ester alkaloid reference extract stock solution, respectively, in a suitable size volumetric flask, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution, dilute to the mark, shake well, and obtain (each 1 ml contains benzoyl new aconine 20 μg, benzoyl aconine 3 μg, benzoyl hypaconine 3 μg, new aconitine 5 μg, hypaconitine 5 μg, aconitine 5 μg).

[0394] Preparation of test solution: precisely take 80 ml of the product, add 20 ml of 0.5 mol / L hydrochloric acid solution to the mark in a 100 ml volumetric flask, weigh, ultrasonic (power 400 W, frequency 40 kHz) for 40 minutes, cool, re-weigh, add 0.5 mol / L hydrochloric acid solution to make up the weight loss, shake, transfer to a centrifuge tube, centrifuge (speed 5000 rpm per minute) for 10 minutes, filter, precisely take 70 ml of the filtrate, add to a solid phase extraction column (with mixed cation exchange reversed phase adsorbent as filler, 500 mg, capacity 6 ml, previously eluted with acetonitrile, water 12 ml each), eluted with water 6 ml, ammonia solution (5→100), water, methanol, acetonitrile 10 ml each, after the eluent flows out, place for 5 minutes, then eluted with acetonitrile-concentrated ammonia solution (90:10) mixed solution 20 ml, collect the eluent, recover the solvent to dryness under reduced pressure below 40℃, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution to dissolve the residue, place in a 5 ml volumetric flask, and dilute to the mark, shake, filter, take the filtrate, and obtain.

[0395] Preparation of spiked test solution: precisely take 80 ml of the product, precisely add 30 μg of aconitum biflavoide extract, place in a 100 ml volumetric flask, add 20 ml of 0.5 mol / L hydrochloric acid solution to the mark, weigh, ultrasonic treatment (power 400 W, frequency 40 kHz) for 40 minutes, cool, re-weigh, add 0.5 mol / L hydrochloric acid solution to make up the weight loss, shake, centrifuge (speed 5000 rpm per minute) for 10 minutes, filter, precisely take 70 ml of the filtrate, add to a solid phase extraction column (with mixed cation exchange reversed phase adsorbent as filler, 500 mg, capacity 6 ml, previously eluted with acetonitrile, water 12 ml each), eluted with water 6 ml, ammonia solution (5→100), water, methanol, acetonitrile 10 ml each, after the eluent flows out, place for 5 minutes, then eluted with acetonitrile-concentrated ammonia solution (90:10) mixed solution 20 ml, collect the eluent, recover the solvent to dryness under reduced pressure below 40℃, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution to dissolve the residue, place in a 5 ml volumetric flask, and dilute to the mark, shake, filter, take the filtrate, and obtain.

[0396] Determination method: precisely take 20 μl of the control solution, test solution and spiked test solution respectively, inject into the liquid chromatograph, determine, and obtain.

[0397] 3.2, specificity

[0398] Negative control solution of Heishunpian: take the negative control decoction of Heishunpian, and obtain according to 3.1 "preparation of test solution".

[0399] He Shunpian single test sample solution: take He Shunpian single decoction, same as 3.1 "preparation of test sample solution", namely.

[0400] Blank solution: take purified water, same as 3.1 "preparation of test sample solution", namely.

[0401] Take the test sample solution, the standard addition test sample solution, the He Shunpian negative control solution, the He Shunpian single test sample solution, the control sample solution, the blank solution, and the blank solvent (acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution), perform HPLC analysis under the tentative chromatographic conditions, record the chromatogram and the ultraviolet absorption spectrum of the control sample and the test sample, and investigate whether the He Shunpian negative control decoction test sample solution is negative. If the diester type alkaloids are not detected in the test sample, determine the diester type alkaloid chromatographic peak position by adding the standard addition test sample.

[0402] The results show that the blank solvent, the blank solution, and the He Shunpian negative control solution have no interference; the monoester type alkaloids and the diester type alkaloids in the test sample solution are derived from the He Shunpian medicinal ingredient in the Chinese medicine composition reference sample containing Guizhi; the separation degree of each chromatographic peak of the monoester type alkaloids and the diester type alkaloids in the standard addition test sample is greater than 1.5, meeting the requirements of high performance liquid chromatography content determination; the ultraviolet absorption wavelengths of each component in the control sample solution and the test sample solution are consistent; and in summary, the method has good specificity.

[0403] 3.3. Precision

[0404] 3.3.1. Instrument precision test

[0405] Take the control sample solution, repeat the sample injection 6 times under the proposed content determination chromatographic conditions, determine the chromatographic peak peak area of the monoester type alkaloids and the diester type alkaloids, and calculate the RSD (RSD≤2.0%).

[0406] The results show that the control sample solution is repeatedly injected 6 times under the chromatographic conditions, and the RSD value of the chromatographic peak peak area of the monoester type alkaloids and the diester type alkaloids is less than 2%, indicating that the chromatographic system meets the precision level that the analysis method can achieve.

[0407] 3.3.2. Reproducibility test

[0408] Prepare 6 standard addition test sample solutions, inject under the proposed chromatographic conditions, and determine the content RSD of the monoester type alkaloids and the diester type alkaloids in each test sample solution (the total amount of monoester type alkaloids RSD≤8.0%, the total amount of diester type alkaloids RSD≤15.0%).

[0409] The results show that in the 6 prepared test samples, the RSD value of the total amount of monoester alkaloids is 3.1%, less than 8.0%; the RSD value of the total amount of diester alkaloids is 4.2%, less than 15.0%; which meets the requirements, indicating that the method has good repeatability.

[0410] 3.3.3. Intermediate precision

[0411] Take the same batch of GZSYZMKL reference sample, on different dates, independently operated by different experimenters according to the preparation method of the spiked test sample solution, the first experimenter uses Agilent 1290-DAD high performance liquid chromatograph, the second experimenter uses Agilent 1100-DAD high performance liquid chromatograph, to determine the content of the total amount of monoester alkaloids and the total amount of diester alkaloids and calculate the RSD of 12 test samples. (The RSD of the total amount of monoester alkaloids is ≤8.0%, and the RSD of the total amount of diester alkaloids is ≤15.0%).

[0412] The results show that in the 12 prepared test samples, the RSD value of the total amount of monoester alkaloids is 2.6%, less than 8.0%; the RSD value of the total amount of diester alkaloids is 10.6%, less than 15.0%; which meets the requirements, indicating that the intermediate precision of the method is good.

[0413] 3.4. Linearity

[0414] Take benzoyl neoline reference substance, benzoylaconine reference substance, benzoylhypaconine reference substance, and aconitum diester alkaloid extract, accurately weigh, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution to prepare mixed control solution containing benzoyl neoline, benzoylhypaconine, benzoylaconine, neoline, hypaconine, and aconine at different concentrations (if the concentration does not reach the detection component quantitative limit standard, further dilution will be carried out). Precisely take 20 μl of mixed control solution of different concentrations, inject into the liquid chromatograph, measure the peak area, take the concentration of benzoyl neoline, benzoylaconine, benzoylhypaconine, neoline, hypaconine, and aconine as the horizontal coordinate, and the peak area as the vertical coordinate, draw the standard curve, and list the regression equation, correlation coefficient r (r≥0.999), residual sum of squares, and linear graph.

[0415] The results show that the linear regression equation of benzoyl neoprotoaconitine is y = 34.492x - 9.3909, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 1761.372, indicating that benzoyl neoprotoaconitine has a good linear relationship in the range of 0.1927 μg / ml to 96.3385 μg / ml; the linear regression equation of benzoyl protoaconitine is y = 33.75x + 0.8969, the correlation coefficient r is 1.0000, and the residual sum of squares RSS is 12.096, indicating that benzoyl protoaconitine has a good linear relationship in the range of 0.1955 μg / ml to 16.7580 μg / ml; the linear regression equation of benzoyl hypoprotoaconitine is y = 35.495x - 7.1817, the correlation coefficient r is 0.9997, and the residual sum of squares RSS is 148.971, indicating that benzoyl hypoprotoaconitine has a good linear relationship in the range of 0.2705 μg / ml to 16.2299 μg / ml; the linear regression equation of neoprotoaconitine is y = 29.6145x - 14.7747, the correlation coefficient r is 0.9990, and the residual sum of squares RSS is 1075.005, indicating that neoprotoaconitine has a good linear relationship in the range of 0.2234 μg / ml to 27.9321 μg / ml; the linear regression equation of hypoprotoaconitine is y = 32.032x - 5.855, the correlation coefficient r is 0.9998, and the residual sum of squares RSS is 185.032, indicating that hypoprotoaconitine has a good linear relationship in the range of 0.2099 μg / ml to 27.7623 μg / ml; the linear regression equation of aconitine is y = 29.8871x - 15.1308, the correlation coefficient r is 0.9990, and the residual sum of squares RSS is 1048.461, indicating that aconitine has a good linear relationship in the range of 0.2221 μg / ml to 27.7623 μg / ml.

[0416] 3.5, Detection limit

[0417] According to the detection limit in the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the fourth edition of "Chinese Pharmacopoeia" 2020, the signal measured by the known concentration reference substance (diluted from the lowest concentration linear reference substance or the limit of quantification reference substance) is compared with the signal measured by the blank sample, and the signal-to-noise ratio is generally 3:1, so as to determine the minimum concentration of the measured substance.

[0418] The results show that the minimum concentrations of benzoyl neoprotoaconitine, benzoyl protoaconitine and benzoyl hypoprotoaconitine that can be reliably detected are 48.2 ng / ml, 111.7 ng / ml and 108.2 ng / ml respectively; the minimum concentrations of neoprotoaconitine, hypoprotoaconitine and aconitine are 139.7 ng / ml, 131.2 ng / ml and 138.8 ng / ml respectively.

[0419] 3.6, Limit of quantification

[0420] The detection limit was determined according to the detection limit in the Guidelines for Validation of Analytical Methods for Drug Quality Standards in Chinese Pharmacopoeia 2020, the signal measured by the known concentration reference substance (diluted from the lowest concentration linear reference substance) was compared with the signal measured by the blank sample, and the signal-to-noise ratio was generally 10:1, so as to determine the lowest concentration of the measured substance that could be reliably quantified.

[0421] The results showed that the lowest concentrations of benzoyl neopine, benzoyl protopine and benzoyl hypopine that could be reliably quantified were 0.1927 μg / ml, 0.1955 μg / ml and 0.2705 μg / ml respectively; the lowest concentrations of neopine, hypopine and aconitine were 0.2234 μg / ml, 0.2099 μg / ml and 0.2221 μg / ml respectively.

[0422] 3.7. Accuracy investigation

[0423] That is, the sample recovery test, 9 portions were taken from the same batch of GZSYZMKL reference sample, each portion was precisely measured 40 ml and placed in 100 ml, 20 ml of pure water was added, high, medium and low three different concentrations (the amount of reference substance added needs to be determined according to the content results of each component in the test solution, if the double ester type alkaloids in the test solution are not detected, the amount of double ester type alkaloids is calculated according to the reference literature "double ester type aconitine is highly toxic, the toxic dose is close to the therapeutic dose, among which aconitine has extremely strong cardiotoxicity and neurotoxicity, which is the main toxic component of aconite drugs, and 0.2 mg of human oral administration can cause poisoning", after conversion, it can be concluded that the aconitine contained in a sample should not exceed 32 μg, combined with the yield ratio of aconite double ester type reference extract, the addition amount of aconite double ester type reference extract is designed from high concentration to low concentration as 90 μg, 60 μg, 30 μg respectively, each reference substance is precisely added in the proportion of 1:1.5, 1:1, 1:0.5, diluted to constant volume with 0.5 mol / l hydrochloric acid, and the test solution is prepared according to the proposed preparation method of test solution, 3 test solutions are prepared for each concentration for determination. At the same time, 2 test solutions were prepared for determination. The content of the test solution was calculated, the sample recovery rate of the total amount of single ester type alkaloids and the total amount of double ester type alkaloids (the total amount of single ester type alkaloids was 75% to 120%, the total amount of double ester type alkaloids was 70% to 125%) and RSD (the RSD of the total amount of single ester type alkaloids was less than or equal to 8.0%, the RSD of the total amount of double ester type alkaloids was less than or equal to 15.0%) were calculated.

[0424] The results showed that the total recovery rate (%) of monoester alkaloids in the test solution was between 91.8% and 110.4%, with an RSD of 6.3%; the total recovery rate of diester alkaloids was between 97.3% and 105.0%, with an RSD of 3.6%. Referring to the recovery limit requirements in the Validation Guidelines for Analytical Methods of Drug Quality Standards in Part IV of the 2020 Edition of the Chinese Pharmacopoeia 9101, the accuracy of this method can be judged to be good.

[0425] 3.8 Stability Test

[0426] Take the same spiked test solution and inject it at 0, 4, 8, 12, 24, 36, 48 and 72 hours according to the planned chromatographic conditions. Measure the peak area of ​​monoester and diester alkaloids and calculate the RSD (≤2.0%).

[0427] The results showed that within 72 hours, the peak area RSDs of benzoylneopioid, benzoylaconitine, benzoylhypoaconitine, neoaconitine, hypoaconitine, and aconitine were all less than 2.0%. Although the peak area RSD of benzoylhypoaconitine was greater than 2.0%, its peak area was relatively small, and the peak area change did not show a certain pattern. It is speculated that the change was not related to the change of the substance itself, so the RSD value requirement can be appropriately relaxed. This indicates that these components are basically stable in the test sample within 72 hours.

[0428] 3.9 Durability Test

[0429] 3.9.1 Investigation of different flow velocities

[0430] Take the same batch of spiked test solution and conduct experiments at flow rates of 0.75 ml, 0.80 ml and 0.85 ml per minute, respectively. Calculate the total content of monoester alkaloids, the total content of diester alkaloids and the RSD (total monoester alkaloids RSD≤8.0%, total diester alkaloids RSD≤15.0%).

[0431] The results showed that, under flow rates of 0.75 ml / min to 0.85 ml / min, the RSD of the total content of monoester alkaloids in the test solution was 0.5%, not exceeding 8.0%; and the RSD of the total content of diester alkaloids was 1.5%, not exceeding 15.0%, which met the requirements of General Chapter 9101, Part IV, Chinese Pharmacopoeia 2020 Edition; indicating that the method has good robustness (at different flow rates).

[0432] 3.9.2 Investigation at different column temperatures

[0433] During the early method development process, there was some interference when the column temperature was 30-35°C, so the column temperature range was narrowed. The same batch of spiked sample solution was injected at different column temperatures of 38°C, 40°C and 42°C, and the content and RSD of the total amount of monoester alkaloids and the total amount of diester alkaloids were calculated (RSD of the total amount of monoester alkaloids ≤8.0%, RSD of the total amount of diester alkaloids ≤15.0%).

[0434] The results showed that under the condition of column temperature 38-42°C, the RSD of the total amount of monoester alkaloids in the spiked sample solution was 0.6%, which was not greater than 8.0%; the RSD of the total amount of diester alkaloids was 0.2%, which was not greater than 15.0%, which met the requirements of General Test 9101 in Chinese Pharmacopoeia 2020 edition in numerical value; but the separation degree of benzoyl aconine and impurity 2 in the spiked sample was 0.62 at the column temperature of 38°C, which was significantly less than 1.5, and did not meet the requirements of benzoyl aconine content determination under this condition; in summary, the method had good durability (different column temperatures) under the condition of column temperature 40-42°C.

[0435] 3.9.3, Different brands of chromatographic columns

[0436] The same batch of spiked sample solution was taken, and different brands of chromatographic columns were investigated according to the determined chromatographic conditions: chromatographic column 1 (Waters T3, 4.6 mm x 150 mm, 2.7 μm, S.N. 0162340181432), chromatographic column 2 (Agilent InfintyLab Poroshell 120Aq-C 18 , 4.6 mm x 150 mm, 2.7 μm, S.N. USKTM01334), chromatographic column 3 (YMC C 18 , 4.6 mm x 150 mm, 2.7 μm, S.N. MC005032), and the content and RSD of the total amount of monoester alkaloids and the total amount of diester alkaloids were calculated (RSD of the total amount of monoester alkaloids ≤8.0%, RSD of the total amount of diester alkaloids ≤15.0%).

[0437] The results showed that under the condition of different brands of chromatographic columns, the RSD of the total amount of monoester alkaloids in the sample solution was 5.6%, which was less than 8.0%, and the RSD of the total amount of diester alkaloids was 6.8%, which was less than 15.0%, which met the requirements of General Test 9101 in Chinese Pharmacopoeia 2020 edition; it was proved that the method had good durability (different brands of chromatographic columns).

[0438] 4, Method determination

[0439] The results of the above methodological study show that the method meets the requirements of the Analysis Method Verification Guiding Principles in the Fourth Volume of the Chinese Pharmacopoeia 2020 edition, and can be used to accurately determine the contents of benzoyl neoconhydrine, benzoyl conhydrine, benzoyl epiconhydrine, neconhydrine, epiconhydrine and conhydrine in the reference samples of traditional Chinese medicine compositions containing Ramulus Cinnamomi. Therefore, the content determination method of single ester type alkaloids and the limit test method of double ester type alkaloids for the reference samples of traditional Chinese medicine compositions containing Ramulus Cinnamomi are as follows:

[0440] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; acetonitrile-tetrahydrofuran (98:2) as mobile phase A, 0.1 mol / L ammonium acetate solution (add 0.5 ml of glacial acetic acid to 1000 ml) as mobile phase B, gradient elution according to the provisions in Table 11; flow rate of 0.8 ml per minute; column temperature of 40°C; detection wavelength of 235 nm. The theoretical plate number should not be less than 3000 calculated by the benzoyl neoconhydrine peak.

[0441] Preparation of reference solution: take appropriate amounts of benzoyl neoconhydrine reference substance, benzoyl conhydrine reference substance, benzoyl epiconhydrine reference substance, and aconitine double ester type alkaloid reference extract, accurately weigh, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution, each 1 ml contains benzoyl neoconhydrine 20 μg, benzoyl conhydrine 3 μg, benzoyl epiconhydrine 3 μg, neconhydrine 5 μg, epiconhydrine 5 μg, and conhydrine 5 μg mixed solution, and you get it.

[0442] Preparation of test solution: accurately take 80 ml of the product into a 100 ml volumetric flask, add 20 ml of 0.5 mol / L hydrochloric acid solution to the mark, weigh, ultrasonic treat (power 400 W, frequency 40 kHz) for 40 minutes, cool, re-weigh, make up the weight loss with 0.5 mol / L hydrochloric acid solution, shake well, transfer to a centrifuge tube, centrifuge (speed 5000 rpm per minute) for 10 minutes, filter, accurately take 70 ml of the filtrate, add it to a solid phase extraction column (with mixed cation exchange reversed phase adsorbent as the filler, 500 mg, capacity 6 ml, pre-eluted with acetonitrile and water 12 ml each), elute with water 6 ml, ammonia solution (5→100), water, methanol, acetonitrile 10 ml each, after the eluent flows out, place for 5 minutes, then elute with acetonitrile-concentrated ammonia test solution (90:10) mixed solution 20 ml, collect the eluent, recover the solvent to dryness under reduced pressure below 40°C, add acetonitrile-0.1 mol / L ammonium acetate (20:80) mixed solution to dissolve the residue, place in a 5 ml volumetric flask, dilute to the mark, shake well, filter, take the filtrate, and you get it.

[0443] Determination method: accurately pipette 20 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and you get it.

[0444] The above has described the embodiments of the present application in detail, and the principles and implementation manners of the present application are described by applying specific examples. The above description of the embodiments is only used to help understand the method of the present application and its core idea. Meanwhile, the changes or deformations made by the person skilled in the art according to the idea of the present application, based on the specific implementation manners and application scope of the present application, all belong to the protection scope of the present application. In summary, the content of the present description should not be understood as a limitation of the present application.

Claims

1. A method for determining the content of aconitine alkaloids in a traditional Chinese medicine composition containing cinnamon twig, characterized in that, The determination method includes the following steps: Take the test solution and reference solution of the traditional Chinese medicine composition for testing. The chromatographic conditions for the detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was a mixed solution of acetonitrile and tetrahydrofuran; mobile phase B was an ammonium acetate solution with approximately 0.5 ml of glacial acetic acid added per 1000 ml of ammonium acetate solution; the gradient elution program was as follows: 0–25 min, 85% B → 80% B; 25–40 min, 80% B → 79% B; 40–42 min, 79% B → 74% B; 42–60 min, 74% B → 63% B; the flow rate was 0.5–1.5 ml / min; the column temperature was 40–42 °C; the detection wavelength was 180–360 nm; and the injection volume was 10–30 μl. The volume ratio of acetonitrile to tetrahydrofuran in the mixed solution was approximately 98:approximately 2. Based on the test results, the content information of the reference standard of the traditional Chinese medicine composition is obtained; The traditional Chinese medicine composition includes cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena rhizome, saposhnikovia root, and black aconite tablets; the reference standards are benzoyl neoaconitine, benzoyl aconitine, benzoyl hypoaconitine, neoaconitine, hypoaconitine, and aconitine. The preparation method of the test solution of the traditional Chinese medicine composition includes: taking an appropriate amount of the decoction of the traditional Chinese medicine composition containing cinnamon twig and placing it in a container, adding hydrochloric acid, weighing, sonicating for a period of time, cooling, weighing again, using hydrochloric acid to replenish the lost weight, shaking, centrifuging, filtering, taking an appropriate amount of the filtrate, using a solid phase extraction column to adsorb and elute the filtrate, collecting the eluent, recovering under reduced pressure, drying to obtain the residue, adding a mixed solution of acetonitrile and ammonium acetate to the residue to dissolve and dilute, shaking, filtering, and obtaining the test solution of the traditional Chinese medicine composition; The solid-phase extraction column is prepared by using a mixed cation exchange reversed-phase adsorbent as a packing material and eluting with acetonitrile and water in sequence. In the preparation method of the test solution of the traditional Chinese medicine composition, the elution method includes: eluting with water, ammonia solution (5→100), water, methanol, and acetonitrile in sequence; after the liquid has drained, the solution is left to stand for a period of time; eluting is then performed with a mixed solution of acetonitrile and ammonia; and the eluent is collected.

2. The determination method according to claim 1, characterized in that, The information is obtained by calculating the content of the reference standard in the traditional Chinese medicine composition according to the external standard method based on the corresponding peak areas in the chromatograms of the test solution and the reference solution of the recorded traditional Chinese medicine composition.

3. The determination method according to claim 1, characterized in that, The linear equation for benzoylneoprothiolane is y = 34.492x - 9.3909, R 2 =0.9999.

4. The determination method according to claim 1, characterized in that, The linear equation for benzoyl aconitine is y = 33.75x + 0.8969, R 2 =1.0000.

5. The determination method according to claim 1, characterized in that, The linear equation for benzoyl aconitine is y = 35.495x - 7.1817, R 2 =0.9997.

6. The determination method according to claim 1, characterized in that, The linear equation for the neoaconitine is y = 29.6145x - 14.7747, R0 2 =0.9990.

7. The determination method according to claim 1, characterized in that, The linear equation for the aconitine is y = 32.032x - 5.855, R0 2 =0.9998.

8. The determination method according to claim 1, characterized in that, The linear equation for aconitine is y = 29.8871x - 15.1308, R0 2 =0.9990.

9. The determination method according to claim 1, characterized in that, The container is a volumetric flask.

10. The determination method according to claim 1, characterized in that, The concentration of the hydrochloric acid is 0.4–0.6 mol / L.

11. The determination method according to claim 10, characterized in that, The concentration of the hydrochloric acid is approximately 0.5 mol / L.

12. The determination method according to claim 1, characterized in that, The volume ratio between the decoction of the traditional Chinese medicine composition and the hydrochloric acid is 2 to 6.

13. The determination method according to claim 12, characterized in that, The volume ratio between the decoction of the traditional Chinese medicine composition and the hydrochloric acid is approximately 4.

14. The determination method according to claim 1, characterized in that, The volume of the decoction of the traditional Chinese medicine composition is 60-100 ml.

15. The determination method according to claim 14, characterized in that, The volume of the decoction of the traditional Chinese medicine composition is approximately 80 ml.

16. The determination method according to claim 1, characterized in that, The power of the ultrasonic treatment is 300-500W.

17. The determination method according to claim 16, characterized in that, The power of the ultrasonic treatment is approximately 400W.

18. The determination method according to claim 1, characterized in that, The frequency of the ultrasonic treatment is 30–50 kHz.

19. The determination method according to claim 18, characterized in that, The frequency of the ultrasonic treatment is approximately 40 kHz.

20. The determination method according to claim 1, characterized in that, The ultrasonic treatment time is 20 to 60 minutes.

21. The determination method according to claim 20, characterized in that, The ultrasonic treatment lasted for approximately 40 minutes.

22. The determination method according to claim 1, characterized in that, The centrifuge speed is 4000-6000 rpm / min.

23. The determination method according to claim 22, characterized in that, The centrifuge speed is approximately 5000 rpm / min.

24. The determination method according to claim 1, characterized in that, The centrifugation time is 5 to 15 minutes.

25. The determination method according to claim 1, characterized in that, The centrifugation time is approximately 10 minutes.

26. The determination method according to claim 1, characterized in that, The volume ratio between the filtrate and the decoction of the traditional Chinese medicine composition is 0.5 to 1.

27. The determination method according to claim 26, characterized in that, The volume ratio between the filtrate and the decoction of the traditional Chinese medicine composition is approximately 0.

875.

28. The determination method according to claim 1, characterized in that, The volume of the filtrate is 60-80 ml.

29. The determination method according to claim 28, characterized in that, The volume of the subsequent filtrate is approximately 70 ml.

30. The determination method according to claim 1, characterized in that, The temperature of the decompression recovery is not higher than 40°C.

31. The determination method according to claim 1, characterized in that, In the mixed solution of acetonitrile and ammonium acetate, the concentration of ammonium acetate is 0.05–0.2 mol / L.

32. The determination method according to claim 31, characterized in that, The concentration of the ammonium acetate is approximately 0.1 mol / L.

33. The determination method according to claim 1, characterized in that, In the mixed solution of acetonitrile and ammonium acetate, the volume ratio of acetonitrile to ammonium acetate is approximately 20: approximately 80.

34. The determination method according to claim 1, characterized in that, The mass of the mixed cation exchange reversed-phase adsorbent is 400–600 mg.

35. The determination method according to claim 34, characterized in that, The mass of the mixed cation exchange reversed-phase adsorbent is approximately 500 mg.

36. The determination method according to claim 1, characterized in that, The solid-phase extraction column has a capacity of 4–8 ml.

37. The determination method according to claim 36, characterized in that, The solid-phase extraction column has a capacity of 6 ml.

38. The determination method according to claim 1, characterized in that, In the preparation method of the solid phase extraction column, the volume of the acetonitrile is 10-15 ml.

39. The determination method according to claim 38, characterized in that, The volume of the acetonitrile is approximately 12 ml.

40. The determination method according to claim 1, characterized in that, In the preparation method of the solid phase extraction column, the volume of water is 10-15 ml.

41. The determination method according to claim 40, characterized in that, The volume of the water is approximately 12 ml.

42. The determination method according to claim 1, characterized in that, The volume of the first water is 4-8 ml.

43. The determination method according to claim 42, characterized in that, The volume of the first batch of water was approximately 6 ml.

44. The determination method according to claim 1, characterized in that, The volume of the ammonia solution is 5–15 ml.

45. The determination method according to claim 44, characterized in that, The volume of the ammonia solution is approximately 10 ml.

46. ​​The determination method according to claim 1, characterized in that, The volume of the second water is 5-15 ml.

47. The determination method according to claim 46, characterized in that, The volume of the second batch of water was approximately 10 ml.

48. The determination method according to claim 1, characterized in that, The volume of methanol is 5-15 ml.

49. The determination method according to claim 48, characterized in that, The volume of methanol is approximately 10 ml.

50. The determination method according to claim 1, characterized in that, In the elution method, when acetonitrile is used for elution, the volume of acetonitrile is 5 to 15 ml.

51. The determination method according to claim 50, characterized in that, The volume of the acetonitrile is approximately 10 ml.

52. The determination method according to claim 1, characterized in that, The placement time is 4 to 6 minutes.

53. The determination method according to claim 52, characterized in that, The placement time is approximately 5 minutes.

54. The determination method according to claim 1, characterized in that, In the mixed solution of acetonitrile and ammonia, the volume ratio of acetonitrile to concentrated ammonia solution is approximately 90:

10.

55. The determination method according to claim 1, characterized in that, The volume of the mixed solution of acetonitrile and ammonia is 10-30 ml.

56. The determination method according to claim 55, characterized in that, The volume of the mixed solution of acetonitrile and ammonia is approximately 20 ml.

57. The determination method according to claim 1, characterized in that, The method for preparing the reference solution includes: weighing appropriate amounts of benzoyl neoaconitine reference standard, benzoyl aconitine reference standard, benzoyl hypoaconitine reference standard and aconitine diester type alkaloid reference extract, adding a second solvent to prepare the reference solution with concentrations of benzoyl neoaconitine, benzoyl aconitine, benzoyl hypoaconitine, neoaconitine, hypoaconitine and aconitine of 1-50 μg / ml.

58. The determination method according to claim 57, characterized in that, The aconitine diester-type alkaloid control extract contains neoaconitine, hypoaconitine, and aconitine.

59. The determination method according to claim 57, characterized in that, The concentration of benzoylneoprothiolane in the reference solution was approximately 20 μg / ml.

60. The determination method according to claim 57, characterized in that, The concentration of benzoyl aconitine in the reference solution was approximately 3 μg / ml.

61. The determination method according to claim 57, characterized in that, The concentration of benzoyl aconitine in the reference solution was approximately 3 μg / ml.

62. The determination method according to claim 57, characterized in that, The concentration of neoaconitine in the reference solution was approximately 5 μg / ml.

63. The determination method according to claim 57, characterized in that, The concentration of aconitine in the reference solution was approximately 5 μg / ml.

64. The determination method according to claim 57, characterized in that, The concentration of aconitine in the reference solution was approximately 5 μg / ml.

65. The determination method according to claim 57, characterized in that, The second solvent is a mixed solution of acetonitrile and ammonium acetate.

66. The determination method according to claim 65, characterized in that, The concentration of the ammonium acetate is 0.05–0.2 mol / L.

67. The determination method according to claim 66, characterized in that, The concentration of the ammonium acetate is approximately 0.1 mol / L.

68. The determination method according to claim 65, characterized in that, In the second solvent, the volume ratio of acetonitrile to ammonium acetate is approximately 20: approximately 80.

69. The determination method according to claim 1, characterized in that, The preparation method of the decoction of the traditional Chinese medicine composition containing cinnamon twig includes: weighing appropriate amounts of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena rhizome, saposhnikovia root, and black aconite root slices, placing them in a container, adding water to soak them, boiling over high heat, simmering over low heat for a period of time, filtering, collecting the filtrate, and obtaining the decoction of the traditional Chinese medicine composition containing cinnamon twig.

70. The determination method according to claim 69, characterized in that, The container is a fully automatic ceramic decoction pot.

71. The determination method according to claim 69, characterized in that, The volume of the water is 1000-2000 ml.

72. The determination method according to claim 71, characterized in that, The volume of the water is approximately 1400 ml.

73. The determination method according to claim 69, characterized in that, The ratio of the sum of the masses of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata, and aconite root slices to the mass / volume of water is 0.1 to 0.5, in g / ml.

74. The determination method according to claim 73, characterized in that, The ratio of the sum of the masses of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata, and aconite root slices to the mass / volume of the water is approximately 0.31, in g / ml.

75. The determination method according to claim 69, characterized in that, The soaking time is 30 to 60 minutes.

76. The determination method according to claim 75, characterized in that, The soaking time is approximately 45 minutes.

77. The determination method according to claim 69, characterized in that, The simmering time is 30 to 90 minutes.

78. The determination method according to claim 77, characterized in that, The simmering time is approximately 60 minutes.

79. The determination method according to claim 69, characterized in that, The filtration is performed using 200-mesh nylon filter cloth.

80. The determination method according to claim 69, characterized in that, The mass ratio of the cinnamon twig, the black aconite slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3).

81. The determination method according to claim 80, characterized in that, The mass ratio of the cinnamon twig, the black aconite slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5).

82. The determination method according to claim 81, characterized in that, The mass ratio of the cinnamon twig, the black aconite root slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root is approximately 4:2:5:4:2:5:3:4:

2.

83. The determination method according to claim 82, characterized in that, The weight range of the cinnamon twigs is 40–70g.

84. The determination method according to claim 83, characterized in that, The weight of the cinnamon twig is approximately 55.20g.

85. The determination method according to claim 82, characterized in that, The weight range of the black shiitake slices is 20-40g.

86. The determination method according to claim 85, characterized in that, The weight of the black shiitake slices is approximately 27.60g.

87. The determination method according to claim 82, characterized in that, The weight range of the ginger is 50-90g.

88. The determination method according to claim 87, characterized in that, The ginger weighs approximately 69.00g.

89. The determination method according to claim 82, characterized in that, The weight range of the windproof material is 40–70g.

90. The determination method according to claim 89, characterized in that, The windproof material weighs approximately 55.20g.

91. The determination method according to claim 82, characterized in that, The mass range of the ephedra is 20–40 g.

92. The determination method according to claim 91, characterized in that, The mass of the ephedra is approximately 27.60g.

93. The determination method according to claim 82, characterized in that, The weight range of Atractylodes macrocephala is 50-90g.

94. The determination method according to claim 93, characterized in that, The mass of the Atractylodes macrocephala is approximately 69.00g.

95. The determination method according to claim 82, characterized in that, The weight range of the white peony root is 30-50g.

96. The determination method according to claim 95, characterized in that, The weight of the white peony root is approximately 41.40g.

97. The determination method according to claim 82, characterized in that, The weight range of Anemarrhena asphodeloides is 40-70g.

98. The determination method according to claim 97, characterized in that, The mass of the Anemarrhena asphodeloides is approximately 55.20g.

99. The determination method according to claim 83, characterized in that, The weight range of the licorice is 20-40g.

100. The determination method according to claim 99, characterized in that, The weight of the licorice is approximately 27.60g.

101. The determination method according to claim 1, characterized in that, The flow rate is 0.6–1.0 ml / min.

102. The determination method according to claim 101, characterized in that, The flow rate is 0.75–0.85 ml / min.

103. The determination method according to claim 102, characterized in that, The flow rate is approximately 0.8 ml / min.

104. The determination method according to claim 1, characterized in that, The detection wavelength is 200–300 nm.

105. The determination method according to claim 104, characterized in that, The detection wavelength is approximately 235 nm.

106. The determination method according to claim 1, characterized in that, The injection volume is 15–25 μl.

107. The determination method according to claim 106, characterized in that, The injection volume is approximately 20 μl.

108. The determination method according to claim 1, characterized in that, The theoretical plate number of the chromatographic peak corresponding to the benzoyl neoaconitine is not less than 3000.

109. The determination method according to claim 1, characterized in that, The chromatographic column is a Waters T3 column, an Agilent InfintyLab Poroshell 120Aq-C18 column, or a YMC C18 column.

110. The determination method according to claim 1, characterized in that, The chromatographic column was an Agilent Infinty LabPoroshell 120Aq-C18 column.

111. The determination method according to claim 1, characterized in that, The specifications of the chromatographic column are as follows: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

112. The determination method according to claim 1, characterized in that, In the chromatographic conditions of the detection, the concentration of the ammonium acetate solution is 0.05–0.2 mol / L.

113. The determination method according to claim 112, characterized in that, The concentration of the ammonium acetate solution is approximately 0.1 mol / L.

114. The determination method according to claim 1, characterized in that, The detection limit for benzoylneoprothiolane is approximately 48.2 ng / ml.

115. The determination method according to claim 1, characterized in that, The detection limit for benzoyl aconitine is approximately 111.7 ng / ml.

116. The determination method according to claim 1, characterized in that, The detection limit for benzoyl aconitine is approximately 108.2 ng / ml.

117. The determination method according to claim 1, characterized in that, The detection limit for the neoaconitine was approximately 139.7 ng / ml.

118. The determination method according to claim 1, characterized in that, The detection limit for aconitine was approximately 131.2 ng / ml.

119. The determination method according to claim 1, characterized in that, The detection limit for aconitine was approximately 138.8 ng / ml.

120. The determination method according to claim 1, characterized in that, The limit of quantification for the benzoylneoprothiolane is approximately 0.1927 μg / ml.

121. The determination method according to claim 1, characterized in that, The limit of quantification for the benzoyl aconitine is approximately 0.1955 μg / ml.

122. The determination method according to claim 1, characterized in that, The limit of quantification for the benzoyl aconitine is approximately 0.2705 μg / ml.

123. The determination method according to claim 1, characterized in that, The limit of quantification for the neoaconitine is approximately 0.2234 μg / ml.

124. The determination method according to claim 1, characterized in that, The limit of quantification for the aconitine is approximately 0.2099 μg / ml.

125. The determination method according to claim 1, characterized in that, The limit of quantification for aconitine is approximately 0.2221 μg / ml.

126. Use of the determination method according to any one of claims 1 to 125 in the quality detection and / or quality evaluation and / or quality control of aconitine alkaloids in traditional Chinese medicine compositions containing cinnamon twig.

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