Method for determining content of aconitum alkaloid in traditional Chinese medicine composition containing cassia twig and application of aconitum alkaloid

Abstract

The invention discloses a method for determining the content of aconitum alkaloid in a traditional Chinese medicine composition containing cassia twigs, and the method comprises the following steps: taking a traditional Chinese medicine composition test solution and a reference substance solution for detection, and obtaining the content information of the reference substance of the traditional Chinese medicine composition according to the detection result; wherein the traditional Chinese medicine composition comprises cassia twig, radix paeoniae alba, liquorice, ephedra, ginger, bighead atractylodes rhizome, rhizoma anemarrhenae, radix saposhnikoviae and black prepared common monkshood daughter root, and the reference substances are benzoyl mesaconitine, benzoyl aconitine, benzoyl hypaconitine, mesaconitine, hypaconitine and aconitine. According to the method, content monitoring research on monoester alkaloids of black prepared tablets in a reference sample of the traditional Chinese medicine composition containing cassia twigs is carried out, meanwhile, limited control research on diester alkaloids is carried out, and the method has important quality control significance on research on the reference sample of the traditional Chinese medicine composition containing cassia twigs from multiple dimensions such as safety and effectiveness.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for determining the content of aconitum alkaloids in a traditional Chinese medicine composition containing cassia twig and an application thereof. Background Art

[0002] The main active and toxic components in Heishun Tablets are aconitine compounds, which can be divided into diester aconitine (aconitine, mesaconitine, mesaconitine), monoester aconitine (benzoylaconitine, benzoylmeaconitine, benzoylmeaconitine, etc.) and alcoholamine alkaloids (aconitine, mesaconitine, mesaconitine, etc.) according to their chemical structures. Since there are few literature reports on alcoholamine aconitine alkaloids, they have no absorption under ultraviolet light and cannot be measured by high performance liquid chromatography ultraviolet detectors, it is not planned to study alcoholamine alkaloids.

[0003] Monoester aconitine is less toxic, only 1 / 50-1 / 500 of aconitine, and has good biological activity, making it the main pharmacological substance basis in clinical applications; alcoholamine alkaloids are even less toxic, only 1 / 2000-1 / 4000 of aconitine, but their efficacy is also weaker.

[0004] Diester aconitine is highly toxic, and the toxic dose is close to the therapeutic dose. Aconitine has extremely strong cardiotoxicity and neurotoxicity, and is the main toxic component of aconite drugs. Oral administration of 0.2 mg can cause poisoning in humans, and 2-5 mg can cause death. Aconitine is easily absorbed from the digestive tract, and its low protein binding rate causes poisoning very quickly. Symptoms of poisoning can appear within minutes after accidental ingestion or overdose, and death often occurs within 3-4 hours after poisoning. Autopsies are mostly suffocation deaths without special pathological changes. Related studies have shown that diester alkaloids first affect the central nervous system, and then cause damage to heart function in many ways. When the dosage is 0.030-0.038 mg / kg, it will induce arrhythmia symptoms. When the dosage is greater than 0.100 mg / kg, it will affect the patient's quality of life. Its therapeutic window is narrow and poisoning is prone to occur.

[0005] There is no report in the prior art on the method for determining the content of aconitum alkaloids in the traditional Chinese medicine composition containing cassia twig and its application. Summary of the invention

[0006] Based on this, the present invention provides a method for determining the content of aconite alkaloids in a traditional Chinese medicine composition containing cassia twig, and the determination method comprises the following steps:

[0007] Take the test solution and the reference solution of the Chinese medicine composition for testing.

[0008] The chromatographic conditions of the detection are as follows: a chromatographic column with octadecylsilane bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is: 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; flow rate is 0.5-1.5 ml / min, column temperature is 40-42° C., detection wavelength is 180-360 nm, and injection volume is 10-30 μl;

[0009] According to the test results, obtaining the content information of the reference substance of the Chinese medicine composition;

[0010] Among them, the Chinese medicine composition includes cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes, rhizome of Anemarrhena, saposhnikovia and heishun tablets, and the reference substances are benzoylmesaconitine, benzoylmesaconitine, benzoylhypaconitine, aconitine, hypaconitine and aconitine.

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

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

[0013] Furthermore, the linear equation of the benzoylaconitine is y=33.75x+0.8969, R 2 =1.0000.

[0014] Furthermore, the linear equation of the benzoylhypaconitine is y=35.495x-7.1817, R 2 =0.9997.

[0015] Furthermore, the linear equation of the new aconitine is y=29.6145x-14.7747, R 2 =0.9990.

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

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

[0018] Furthermore, the preparation method of the test solution of the Chinese medicine composition includes: taking an appropriate amount of a decoction of the Chinese medicine composition containing cassia twigs and placing it in a container, adding a first solvent, weighing the weight, ultrasonically treating for a period of time, cooling, weighing the weight again, using the first solvent to make up for the lost weight, shaking, centrifuging, filtering, taking an appropriate amount of subsequent filtrate, eluting the subsequent filtrate after adsorption using a solid phase extraction column, collecting the eluate, recovering under reduced pressure, drying to obtain a residue, adding a second solvent to the residue to dissolve and dilute it, shaking, filtering, and obtaining the test solution of the Chinese medicine composition.

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

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

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

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

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

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

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

[0026] Furthermore, the ultrasonic treatment time is 20 to 60 minutes, for example, about 40 minutes.

[0027] Furthermore, the centrifugal speed is 4000-6000 rpm / min, for example, about 5000 rpm / min.

[0028] Furthermore, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0029] Furthermore, the volume ratio of the subsequent filtrate to the decoction of the traditional Chinese medicine composition is 0.5-1, for example, about 0.875.

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

[0031] Furthermore, the temperature of the reduced pressure recovery is not higher than 40°C.

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

[0033] Furthermore, 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] Furthermore, the preparation method of the solid phase extraction column comprises: using a mixed cation exchange reverse phase adsorbent as a filler, and using acetonitrile and water in sequence for elution to obtain the solid phase extraction column.

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

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

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

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

[0040] Furthermore, in the preparation method of the test solution of the traditional Chinese medicine composition, the elution method includes: using water, ammonia solution (5→100) for the first time, water, methanol, and acetonitrile for the second time for elution in sequence, leaving the liquid for a period of time after it has flowed out, eluting with a third solvent, and collecting the eluate.

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

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

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

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

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

[0046] Furthermore, the placement time is 4 to 6 minutes, for example, about 5 minutes.

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

[0048] Furthermore, 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] Furthermore, the volume of the third solvent is 10-30 ml, for example, about 20 ml.

[0050] Furthermore, the preparation method of the reference solution includes: weighing appropriate amounts of aconitine benzoylnesaconitine reference substance, aconitine benzoylnesaconitine reference substance, aconitine diester-type alkaloid reference substance and aconitine diester-type alkaloid reference extract, adding a second solvent to prepare the reference solution in which the concentrations of aconitine benzoylnesaconitine, benzoylnesaconitine, benzoylnesaconitine, aconitine, aconitine and aconitine are 1 to 50 μg / ml, respectively.

[0051] Furthermore, the aconitine diester-type alkaloid control extract contains mesaconitine, hypoaconitine and aconitine.

[0052] Furthermore, the concentration of benzoylaconitum in the reference solution is about 20 μg / ml.

[0053] Furthermore, the concentration of benzoylaconitine in the reference solution is about 3 μg / ml.

[0054] Furthermore, the concentration of benzoylhypoconitine in the reference solution is about 3 μg / ml.

[0055] Furthermore, the concentration of neoaconitine in the reference solution is about 5 μg / ml.

[0056] Furthermore, the concentration of hypoaconitine in the reference solution is about 5 μg / ml.

[0057] Furthermore, the concentration of aconitine in the reference solution is about 5 μg / ml.

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

[0059] Furthermore, the concentration of the 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 the acetonitrile to the ammonium acetate is acetonitrile:ammonium acetate=about 20:about 80.

[0061] Furthermore, the preparation method of the decoction of the traditional Chinese medicine composition containing cinnamon twig comprises: weighing appropriate amounts of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes, anemarrhena, siler and heishunpian, placing them in a container, adding water for soaking, decocting over high heat until boiling, decocting 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.

[0062] Furthermore, the container is a fully automatic ceramic medicine decoction pot.

[0063] Furthermore, the volume of the water is 1000-2000 ml, for example, about 1400 ml.

[0064] Furthermore, the ratio of the sum of the masses of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, siler and heishunpian to the mass / volume (g / ml) of water is 0.1-0.5, for example, about 0.31.

[0065] Furthermore, the soaking time is 30 to 60 minutes, for example, about 45 minutes.

[0066] Furthermore, the slow-cooking time is 30 to 90 minutes, for example, about 60 minutes.

[0067] Furthermore, the filtration is performed through a 200-mesh nylon filter cloth.

[0068] Furthermore, the mass ratios of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the anemarrhena and the liquorice are (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3).

[0069] Furthermore, the mass ratios of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the anemarrhena and the liquorice are (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] Furthermore, the mass ratio of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the anemarrhena and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2.

[0071] Furthermore, the mass range of the cassia twig is 40 to 70 g, for example, about 55.20 g.

[0072] Furthermore, the mass range of the black tablet is 20-40 g, for example, about 27.60 g.

[0073] Furthermore, the weight of the ginger is in the range of 50 to 90 g, for example, about 69.00 g.

[0074] Furthermore, the mass of the windshield is in the range of 40 to 70 g, for example, about 55.20 g.

[0075] Furthermore, the mass range of the ephedra is 20-40 g, for example, about 27.60 g.

[0076] Furthermore, the mass range of the Atractylodes macrocephala is 50 to 90 g, for example, about 69.00 g.

[0077] Furthermore, the mass range of the white peony root is 30 to 50 g, for example, about 41.40 g.

[0078] Furthermore, the mass range of the Anemarrhena Rhizoma is 40-70 g, for example, about 55.20 g.

[0079] Furthermore, the mass of the licorice is in the range of 20 to 40 g, for example, about 27.60 g.

[0080] Further, the flow rate is 0.6-1.0 ml / min, such as 0.75-0.85 ml / min, such as about 0.8 ml / min.

[0081] Furthermore, the detection wavelength is 200-300 nm, for example, about 235 nm.

[0082] Furthermore, the injection volume is 15-25 μl, for example, about 20 μl.

[0083] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the benzoyl aconitine is not less than 3000.

[0084] Furthermore, the chromatographic column is a Waters T3 chromatographic column, an Agilent InfintyLab Poroshell120Aq-C18 chromatographic column, or a YMC C18 chromatographic column.

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

[0086] Furthermore, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

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

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

[0089] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0090] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.

[0091] Furthermore, the buffered saline solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0092] Further, the buffered saline solution is an ammonium acetate solution and about 0.5 ml of glacial acetic acid is added per 1000 ml of the ammonium acetate solution.

[0093] Furthermore, 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 buffered saline solution is not greater than 7.0.

[0095] Furthermore, the detection limit of benzoyl aconitine is about 48.2 ng / ml.

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

[0097] Furthermore, the detection limit of benzoylhypoconitine is about 108.2 ng / ml.

[0098] Furthermore, the detection limit of the neoaconitine is about 139.7 ng / ml.

[0099] Furthermore, the detection limit of aconitine is about 131.2 ng / ml.

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

[0101] Furthermore, the limit of quantification of benzoyl aconitine is about 0.1927 μg / ml.

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

[0103] Furthermore, the limit of quantification of benzoylhypoconitine is about 0.2705 μg / ml.

[0104] Furthermore, the limit of quantification of the neoaconitine is about 0.2234 μg / ml.

[0105] Furthermore, the limit of quantification of the aconitine was about 0.2099 μg / ml.

[0106] Furthermore, the limit of quantification of aconitine was about 0.2221 μg / ml.

[0107] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method in quality detection and / or quality evaluation and / or quality control of aconitum alkaloids in a traditional Chinese medicine composition comprising cinnamon twig.

[0108] Beneficial effects of the present invention:

[0109] The present invention conducts content monitoring research on monoester alkaloids of Heishunpian medicinal flavor in a benchmark sample of a traditional Chinese medicine composition containing cassia twig, and simultaneously conducts limit control research on diester alkaloids. The research on the benchmark sample of the traditional Chinese medicine composition containing cassia twig has important quality control significance from multiple dimensions such as safety and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0111] Figure 1 This is the HPLC chromatogram under "2.2, initial conditions". Among them, A is the monoester alkaloid reference solution, B is the aconitum diester alkaloid reference extract solution; 1: benzoyl aconitine, 2: benzoyl aconitine and benzoyl hypoaconitine, 3: metaaconitine, 4: hypoaconitine, 5: aconitine.

[0112] Figure 2 This is the HPLC chromatogram under "2.3, mobile phase gradient adjustment". Among them, A is the mixed reference solution of aconitum alkaloids, B is the test solution; 1: benzoyl meaconitine, 2: benzoyl aconitine, 3: benzoyl mesaconitine, 4: meaconitine, 5: mesaconitine, 6: aconitine.

[0113] Figure 3 This is the HPLC chromatogram under "2.4, Optimization of the preparation method of the test solution". Among them, A is the mixed reference solution of aconitum alkaloids, B is the test solution; 1: benzoyl meaconitine, 2: benzoyl aconitine, 3: benzoyl mesaconitine, 4: meaconitine, 5: mesaconitine, 6: aconitine.

[0114] Figure 4This is the HPLC chromatogram under "2.5, adjust the proportion of organic phase". Among them, A is the mixed reference solution of aconitum alkaloids, B is the test solution; 1: benzoyl meaconitine, 2: benzoyl aconitine, 3: benzoyl mesaconitine, 4: meaconitine, 5: mesaconitine, 6: aconitine.

[0115] Figure 5 This is the HPLC chromatogram under "2.6, further optimization of chromatographic conditions". Among them, A is the mixed reference solution of aconitum alkaloids, B is the test solution; 1: benzoyl meaconitine, 2: benzoyl aconitine, 3: benzoyl mesaconitine, 4: meaconitine, 5: mesaconitine, 6: aconitine.

[0116] Figure 6 This is the HPLC chromatogram of the spiked test solution under "2.9, chromatographic conditions optimized again". Among them, 1: benzoyl aconitine, 2: benzoyl aconitine, 3: benzoyl mesaconitine, 4: mesaconitine, 5: mesaconitine, 6: aconitine. DETAILED DESCRIPTION

[0117] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0118] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0119] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention 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 invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0122] As described in the background technology section, there is no report on the method for determining the content of aconitum alkaloids in a traditional Chinese medicine composition containing cinnamon twigs and its application in the prior art. In order to solve the above problems, the present invention provides a method for determining the content of aconitum alkaloids in a traditional Chinese medicine composition containing cinnamon twigs, and the determination method comprises the following steps:

[0123] Take the test solution and the reference solution of the Chinese medicine composition for testing.

[0124] The chromatographic conditions of the detection are: a chromatographic column with octadecylsilane bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is acid aqueous solution, alkali aqueous solution and / or buffered saline solution, and the gradient elution program is: 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° C., the detection wavelength is 180-360 nm, and the injection volume is 10-30 μl.

[0125] In the present invention, when time, concentration, ratio, volume, power, frequency, rotation speed, mass, wavelength, flow rate, injection amount, temperature, or other values ​​or parameters are expressed as ranges, preferred ranges, or ranges limited by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "10-30" is disclosed, the described range should be interpreted as including the range "10-30", "10-25", "10-20", "10-15", "15-30", "15-25", "15-20", "20-30", "20-25", "25-30", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0126] According to the test results, obtaining the content information of the reference substance of the Chinese medicine composition;

[0127] Among them, the Chinese medicine composition includes cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes, rhizome of Anemarrhena, saposhnikovia and heishun tablets, and the reference substances are benzoylmesaconitine, benzoylmesaconitine, benzoylhypaconitine, aconitine, hypaconitine and aconitine.

[0128] In a preferred embodiment, the information is the content of the reference substance in the traditional Chinese medicine composition calculated according to the external standard method based on the corresponding peak areas in the recorded 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 aconitine is y=34.492x-9.3909, R 2 =0.9999.

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

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

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

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

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

[0135] In a preferred embodiment, the preparation method of the test solution of the Chinese medicine composition comprises: taking an appropriate amount of a decoction of the Chinese medicine composition containing cassia twigs and placing it in a container, adding a first solvent, weighing the weight, ultrasonically treating for a period of time, cooling, weighing the weight again, using the first solvent to make up for the lost weight, shaking, centrifuging, filtering, taking an appropriate amount of subsequent filtrate, eluting the subsequent filtrate after adsorption using a solid phase extraction column, collecting the eluate, recovering under reduced pressure, drying to obtain a residue, adding a second solvent to the residue to dissolve and dilute it, shaking, filtering, and obtaining the test solution of the 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, for example, about 0.5 mol / L.

[0139] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0140] In a preferred embodiment, the volume ratio between the decoction of the traditional Chinese medicine composition and the first solvent is 2-6, for example, about 4.

[0141] In the present invention, "about" refers to a value within a range of ±5% of a specific 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, for example, about 80 ml.

[0143] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 300-500W, such as about 400W.

[0145] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 to 50 kHz, such as about 40 kHz.

[0147] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

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

[0149] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

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

[0151] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5000" includes ±5% of 5000, or from 4750 to 5250.

[0152] In a preferred embodiment, the centrifugation time is 5 to 15 min, such as about 10 min.

[0153] In the present 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.

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

[0155] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 subsequent filtrate is 60-80 ml, for example, about 70 ml.

[0157] In the present invention, "about" refers to a value within a range of ±5% of a specific 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°C.

[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" refers to a value within the range of ±5% of a specific 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 the acetonitrile to the ammonium acetate is acetonitrile:ammonium acetate=about 20:about 80.

[0163] In the present 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; "about 80" includes ±5% of 80, or from 76 to 84.

[0164] In a preferred embodiment, the preparation method of the solid phase extraction column comprises: using a mixed cation exchange reverse phase adsorbent as a filler, and eluting with acetonitrile and water in sequence to obtain the solid phase extraction column.

[0165] In a preferred embodiment, the mass of the mixed cation exchange reverse phase adsorbent is 400-600 mg, for example, about 500 mg.

[0166] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0167] In a preferred embodiment, the capacity of the solid phase extraction column is 4-8 ml, for example 6 ml.

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

[0169] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

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

[0171] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0172] In a preferred embodiment, in the method for preparing the test solution of the traditional Chinese medicine composition, the elution method comprises: eluting in sequence using water, ammonia solution (5→100) for the first time, water, methanol, and acetonitrile for the second time, leaving the liquid for a period of time after it has flowed out, eluting with a third solvent, and collecting the eluate.

[0173] Here, "ammonia solution (5→100)" means "5% ammonia solution", and its preparation method is to dilute 5 ml of ammonia water to 100 ml with water.

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

[0175] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0176] In a preferred embodiment, the volume of the ammonia solution is 5-15 ml, for example, about 10 ml.

[0177] In the present 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 5-15 ml, for example, about 10 ml.

[0179] In the present 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 the methanol is 5-15 ml, such as about 10 ml.

[0181] In the present 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 acetonitrile is 5-15 ml, such as about 10 ml.

[0183] In the present 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 standing time is 4 to 6 minutes, such as about 5 minutes.

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

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

[0187] In a preferred embodiment, 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.

[0188] In the present invention, "about" refers to a value within the 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 10-30 ml, for example, about 20 ml.

[0190] In the present 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 preparation method of the reference solution comprises: weighing appropriate amounts of aconitine benzoylnesaconitine reference substance, aconitine benzoylnesaconitine reference substance, aconitine diester-type alkaloid reference substance and aconitine diester-type alkaloid reference extract, adding a second solvent to prepare the reference solution in which the concentrations of aconitine benzoylnesaconitine, aconitine benzoylnesaconitine, aconitine, aconitine and aconitine are 1 to 50 μg / ml, respectively.

[0192] In a preferred embodiment, the aconitine diester-type alkaloid control extract comprises metaaconitine, hypoaconitine and aconitine.

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

[0194] In the present 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.

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

[0196] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

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

[0198] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

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

[0200] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

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

[0202] In the present invention, "about" refers to a value within a range of ±5% of a particular 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 reference solution is about 5 μg / ml.

[0204] In the present invention, "about" refers to a value within a range of ±5% of a particular 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" refers to a value within the range of ±5% of a specific 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 the acetonitrile to the ammonium acetate is acetonitrile:ammonium acetate=about 20:about 80.

[0209] In the present 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; "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 cinnamon twig comprises: weighing appropriate amounts of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes, anemarrhena, siler and heishunpian, placing them in a container, adding water for soaking, decocting over high heat until boiling, decocting 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.

[0211] In a preferred embodiment, the container is a fully automatic ceramic medicine decoction pot.

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

[0213] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 masses of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, siler and heishunpian to the mass / volume (g / ml) of water is 0.1-0.5, for example, about 0.31.

[0215] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.31" includes ±5% of 0.31, or from 0.2945 to 0.3255.

[0216] In a preferred embodiment, the soaking time is 30 to 60 minutes, such as about 45 minutes.

[0217] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 45" includes ±5% of 45, or from 42.75 to 47.25.

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

[0219] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

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

[0221] In a preferred embodiment, the mass ratio of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the anemarrhena and the liquorice 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 cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the anemarrhena and the liquorice 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 of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the anemarrhena and the liquorice 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" refers to a value within a range of ±5% of a specific 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 mass range of the cassia twig is 40-70 g, for example, about 55.20 g.

[0226] In the present 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.

[0227] In a preferred embodiment, the mass of the black tablets ranges from 20 to 40 g, such as about 27.60 g.

[0228] In the present 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.

[0229] In a preferred embodiment, the weight of the ginger is in the range of 50 to 90 g, for example, about 69.00 g.

[0230] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 69.00" includes ±5% of 69.00, or from 65.55 to 72.45.

[0231] In a preferred embodiment, the windshield has a mass range of 40 to 70 g, such as about 55.20 g.

[0232] In the present 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.

[0233] In a preferred embodiment, the mass of the ephedra is in the range of 20 to 40 g, for example, about 27.60 g.

[0234] In the present 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.

[0235] In a preferred embodiment, the mass range of the Atractylodes macrocephala is 50-90 g, for example, about 69.00 g.

[0236] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 69.00" includes ±5% of 69.00, or from 65.55 to 72.45.

[0237] In a preferred embodiment, the mass range of the white peony root is 30-50 g, for example, about 41.40 g.

[0238] In the present 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 range of the Anemarrhena Rhizoma is 40-70 g, for example, about 55.20 g.

[0240] In the present 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 mass of the liquorice is in the range of 20 to 40 g, such as about 27.60 g.

[0242] In the present 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 0.6-1.0 ml / min, such as 0.75-0.85 ml / min, such as about 0.8 ml / min.

[0244] In the present 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 the present 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 15-25 μl, such as about 20 μl.

[0248] In the present 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 benzoylaconitum is not less than 3000.

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

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

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

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

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

[0255] In the present invention, "about" refers to a value within the range of ±5% of a specific 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 acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0257] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.

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

[0259] In a preferred embodiment, the buffered saline 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 invention, "about" refers to a value within a range of ±5% of a specific 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" refers to a value within the range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

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

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

[0265] In the present invention, "about" refers to a value within a range of ±5% of a specific 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" refers to a value within a range of ±5% of a specific 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 benzoylhypoconitine is about 108.2 ng / ml.

[0269] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 the neoaconitine is about 139.7 ng / ml.

[0271] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 hypoaconitine is about 131.2 ng / ml.

[0273] In the present invention, "about" refers to a value within a range of ±5% of a specific 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" refers to a value within a range of ±5% of a specific 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 quantification of benzoylaconitum is about 0.1927 μg / ml.

[0277] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 quantification of benzoylaconitine is about 0.1955 μg / ml.

[0279] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 quantification of benzoylhypoconitine is about 0.2705 μg / ml.

[0281] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 quantification of the neoaconitine is about 0.2234 μg / ml.

[0283] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 quantification of hypoaconitine is about 0.2099 μg / ml.

[0285] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 quantification of aconitine is about 0.2221 μg / ml.

[0287] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 invention, there is provided a use of the above-mentioned determination method in quality detection and / or quality evaluation and / or quality control of aconitum alkaloids in a traditional Chinese medicine composition comprising cinnamon twig.

[0289] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0290] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

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

[0292] Example

[0293] 1. Instruments and test drugs

[0294] 1.1 Instruments

[0295] Electronic balance (specification: 1 / 10,000, model: BCE124I-1CCN / BSA124, number: 20501-012 / 010, manufacturer: Sartorius Scientific Instrument Co., Ltd.); Electronic balance (specification: 1 / 100,000, model: MS105DU, number: 20501-006, manufacturer: Mettler Toledo Instrument Co., Ltd.); Electronic balance (specification: 1 / 100,000, model: JY2002, number: 20502-017, manufacturer: Shanghai Puchun Measuring Instrument Co., Ltd.); Decoction pot (model: 2.5L, number: 20602-069 / 072, manufacturer: Kangyashun Electric Co., Ltd.); liquid heater (model: 30B2, number: 20304-081 / 080, manufacturer: Kangyashun Electric Co., Ltd.); decoction pot (model: 3.0L, number: 20602-016 / 018 / 019, manufacturer: Chaozhou Chaoan District Kangyashun Electric Co., Ltd.); temperature control heating plate (model: KDMB, number: 20302-036 / 034, manufacturer: Shandong Juancheng Hualu Electric Heating Instrument Co., Ltd.); high performance liquid chromatograph (model: Aglient 1100-DAD, No.: 20101-007 / 008, Manufacturer: Agilent Technologies, Inc.); High Performance Liquid Chromatography (Model: Aglient 1290-DAD, No.: 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 Instrument Co., Ltd.); chromatographic column (model: Waters T 3 , 4.6mm×150mm, 2.7μm, SN01623401818432, manufacturer: Changsha Jiedao Scientific Instrument Co., Ltd.); chromatographic 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, 4.6mm×150mm, 2.7μm, SNMC005032, manufacturer: Changsha Kemei Analytical Instrument Co., Ltd.); CNC ultrasonic cleaner (model: KQ-500DB, number: 21101-035, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.); centrifuge (model: Yingtai-TD4N, number: 21101-003, manufacturer: Changsha Yingtai Instrument Co., Ltd.); rotary evaporator (model: R-1001VN, number: 20603-001, manufacturer: Zhengzhou Great Wall Science and Technology Co., Ltd.); 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 Experiment Technology Co., Ltd.); solid phase extraction column (model: mixed strong cation exchange solid phase extraction column (150 mg, 6 ml), S / N: I0860042, manufacturer: Shanghai Anpu Experiment Technology Co., Ltd.).

[0296] 1.2. Drug testing

[0297] 1.2.1 Reagents

[0298] Water (Batch No.: 20231126, 20240510, 20240713, Manufacturer: Hunan Xiangwa Beverage Technology Co., Ltd., Grade: Purified Water); Acetonitrile (Batch No.: WXBF2252V, WXBF1923V, WXBF2846V, Manufacturer: SIGMA, Grade: Chromatographic Grade, Batch No.: 013424040417, Manufacturer: Shanghai Xingke High Purity Solvent Co., Ltd., Grade: Chromatographic Grade); Methanol (Batch No.: WX BF2172V, manufacturer: SIGMA, grade: chromatography grade, grade: chromatography grade, batch number: 0234240411D, manufacturer: Shanghai Xingke High Purity Solvent Co., Ltd., grade: chromatography grade); ammonia (batch number: 20230830, 20231103, 20231101, manufacturer: Sinopharm Chemical Reagent Co., Ltd., grade: analytical grade); ammonium acetate (batch number: 20220622, 20231017, manufacturer: Sinopharm Chemical Reagent Co., Ltd. Sinopharm Chemical Reagent Co., Ltd., grade: analytical grade); hydrochloric acid (batch number: 20230313, 20230905, manufacturer: Sinopharm Chemical Reagent Co., Ltd., grade: analytical grade); tetrahydrofuran (batch number: 23085314, manufacturer: Anhui Tiandi High Purity Solvent Co., Ltd., grade: chromatographic grade); glacial acetic acid (batch number: 20220929, manufacturer: Sinopharm Chemical Reagent Co., Ltd., grade: analytical grade); isopropanol (batch number: 23085307, manufacturer: Anhui Tiandi High Purity Solvent Co., Ltd., grade: chromatographic grade); dichloromethane (batch number: 20230511, manufacturer: Sinopharm Chemical Reagent Co., Ltd., grade: analytical grade); ether (batch number: 20230530, manufacturer: Sinopharm Chemical Reagent Co., Ltd., grade: analytical grade); ammonium dihydrogen phosphate (batch number: C15317262, manufacturer: Shanghai MacLean Biochemical Technology Co., Ltd., grade: chromatographic grade).

[0299] 1.2.2. Reference substances and reference extracts

[0300] Benzoylneaconitine (batch number: 111795-202106, content is 96.3%, source: China Food and Drug Inspection Institute); Benzoylneaconitine (batch number: 111794-202307, content is 98.0%, source: China Food and Drug Inspection Institute); Benzoylhyaconitine (batch number: 111796-202207, content is 96.4%, source: China Food and Drug Inspection Institute); Aconitum diester type alkaloid control extract (batch number: 112029-2023 02, the content is calculated as 32.9% neoaconitine, 30.9% hypoaconitine, and 32.7% aconitine, source: China Food and Drug Inspection Institutes); Aconitum diester type alkaloid control extract (batch number: 112029-201601, the content is calculated as 31.7% neoaconitine, 30.0% hypoaconitine, and 31.8% aconitine, source: China Food and Drug Inspection Institutes); Benzoylaconitine (batch number: 111794-202006, the content is calculated as 99.5%, source: China Food and Drug Inspection Institutes).

[0301] 1.2.3. Medicinal pieces information

[0302] Cinnamon twig (batch number: ZA00923070504, origin: Rong County, Yulin City, Guangxi Zhuang Autonomous Region, source: homemade); White peony root (batch number: ZA00123070503, origin: Qiaocheng District, Bozhou City, Anhui Province, source: homemade); Atractylodes macrocephala (batch number: ZA04923070707, origin: She County, Huangshan City, Anhui Province, source: homemade); Atractylodes macrocephala (batch number: ZA04923080104, origin: Zhejiang Xinchang County, Shaoxing City, Hebei Province, Source: Homemade); Anemarrhena (Batch No.: ZA08023070701, Origin: Boye County, Baoding City, Hebei Province, Source: Homemade); Anemarrhena (Batch No.: ZA08023070704, Origin: Anguo City, Baoding City, Hebei Province, Source: Homemade); Heishun Tablets (Batch No.: ZA1732307001, Origin: Jiangyou City, Mianyang City, Sichuan Province, Source: Homemade); Licorice (Batch No.: ZA004 23071001, origin: Guazhou County, Jiuquan City, Gansu Province, source: homemade); Licorice (Batch No.: ZA00423081513, origin: Hangjin Banner, Ordos City, Inner Mongolia Autonomous Region, source: homemade); Saposhnikovia divaricata (Batch No.: ZA03923070703, origin: Tailai County, Qiqihar City, Heilongjiang Province, source: homemade); Saposhnikovia divaricata (Batch No.: ZA03923111301, origin: Inner Mongolia Autonomous Region Tuquan County, Xing'an League, source: homemade); Ephedra (batch number: 01-21030201, origin: Alukeerqin Banner, Chifeng City, Inner Mongolia Autonomous Region, source: Anhui Runfurong Pharmaceutical Co., Ltd.); Ginger (batch number: ZA00523102601, origin: Jianwei County, Leshan City, Sichuan Province, source: homemade); Ginger (batch number: ZA00523091101, origin: Jianwei County, Leshan City, Sichuan Province, source: homemade).

[0303] 1.2.4 Preparation of reference samples

[0304] Benchmark sample: weigh 55.20g of cinnamon twig, 41.40g of white peony root, 27.60g of liquorice, 27.60g of ephedra, 69.00g of ginger, 69.00g of atractylodes, 55.20g of rhizoma anemarrhenae, 55.20g of saposhnikovia divaricata and 27.60g of Heishunpian, put them in a 3.0L fully automatic ceramic decoction pot, add 1400ml of water, soak for 45 minutes, heat to boiling over high heat, keep boiling for 60 minutes over low heat, filter with 200 mesh nylon filter cloth while hot, collect the filtrate, and obtain. Negative control decoction lacking Heishunpian: The prescription lacks the taste of Heishunpian, and is prepared in the same way as the preparation method of the benchmark sample. Heishunpian single decoction: weigh 27.60g of Heishunpian, and prepare it in the same way as the preparation method of the benchmark sample.

[0305] 1.2.5. Decoction Information

[0306] A Chinese medicine composition containing cinnamon twig compound decoction (batch number: GZSYZMKLST-2023120601-L, batch number: GZSYZMKLST-2024011003-L, GZSYZMKLST-2024022604-L, GZSYZMKLST-2024030301-L, GZSYZMKLST-2024030302-L); Heishun tablets Taste decoction (batch number: GZSYZMKLST-2024022601-L, GZSYZMKLST-2024070504-L, GZSYZMKLST-2024052101-L); negative control decoction of Quheishun tablets (batch number: GZSYZMKLST-2024022605-L, GZSYZMKLST-2024052102-L, GZSY ZMKLST-2024052401-L, GZSYZMKLST-2024070505-L). Benchmark sample (batch number: GZSYZMKLST-2024052103-L, GZSYZMKLST-2024052104-L, GZSY ZMKLST-2024052105-L, GZSYZMKLST-2024052106-L, GZSYZMKLST-2024052107-L, GZSYZMKLST-2024 052108-L, GZSYZMKLST-2024060301-L, GZSYZMKLST-2024061503-L, GZSYZMKLST-2024061504-L, GZS YZMKLST-2024061505-L, GZSYZMKLST-2024061506-L, GZSYZMKLST-2024061507-L, GZSYZMKLST-202 4061508-L, GZSYZMKLST-2024070501-L, GZSYZMKLST-2024070502-L, GZSYZMKLST-2024070503-L, GZ SYZMKLST-2024071401-L, GZSYZMKLST-2024071402-L).

[0307] 2. Preliminary establishment of content method

[0308] 2.1. Selection of detection wavelength

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

[0310] 2.2 Initial conditions

[0311] Chromatographic conditions: Octadecylsilane bonded silica gel was used as filler (HALO AQ-C 18, 4.6mm×150mm, 2.7μm); acetonitrile-tetrahydrofuran (25:15) was used as mobile phase A, 0.1mol / L ammonium acetate solution (0.5ml of glacial acetic acid was added for every 1000ml) was used as mobile phase B, gradient elution was performed according to the provisions in Table 1, the flow rate was 0.6ml per minute, and the detection wavelength was 235nm.

[0312] Table 1 Gradient elution conditions 1

[0313] Time (minutes) 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: Take appropriate amount of benzoyl-neoaconitine reference substance, benzoyl-aconitine reference substance, benzoyl-hypoaconitine reference substance, and aconitine diester type alkaloid reference extract, accurately weigh them, and add isopropanol-dichloromethane (1:1) mixed solution to prepare appropriate concentration stock solutions; accurately measure appropriate amount of benzoyl-neoaconitine reference substance stock solution, benzoyl-aconitine reference substance stock solution, and benzoyl-hypoaconitine reference substance stock solution, respectively, place them in a volumetric bottle of appropriate specifications, and add isopropanol-dichloromethane (1:1) mixed solution to prepare a solution containing 10 μg of each per 1 ml, as a monoester type alkaloid reference solution. Accurately measure appropriate amount of aconitine diester type alkaloid reference extract stock solution, place it in a volumetric bottle of appropriate specifications, and add isopropanol-dichloromethane (1:1) mixed solution to prepare a solution containing 10 μg of each of neoaconitine, hypoaconitine, and aconitine per 1 ml, and obtain.

[0315] Preparation of test solution: Accurately measure 100 ml of this product (the compound decoction of Chinese medicine composition containing cinnamon twig in "1.2.5, decoction information", batch number: GZSYZMKLST-2023120601-L), put it in a separatory funnel, add ether and 0.1 mol / L hydrochloric acid solution, shake and extract twice, each time with 140 ml of ether and 20 ml of 0.1 mol / L hydrochloric acid solution, discard the ether solution, and place it in the following After adding ammonia test solution and ether to the acid solution, shake and extract 4 times, each time with 8 ml of ammonia test solution and 140 ml of ether, combine the ether solutions, recover the solvent under reduced pressure below 40°C to dryness, add a small amount of acetonitrile-0.02 mol / L diammonium phosphate solution (1:1) to the residue to dissolve it, transfer it to a 10 ml volumetric flask, dilute to the scale with acetonitrile-0.02 mol / L diammonium phosphate solution (1:1), shake well, filter, and take the filtrate to obtain the product.

[0316] Determination method: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0317] According to the preliminary proposed content determination method, the results are shown in Table 2 and Figure 1 shown.

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

[0319]

[0320] The results showed that under the chromatographic conditions, the monoester alkaloid mixed reference solution only showed two chromatographic peaks, which was inconsistent with the situation that the mixed reference solution contained three components. It was speculated that under this condition, the chromatographic peaks of benzoylaconitine and benzoylhyaconitine co-eluted. Since the separation of the chromatographic peaks of the reference substance could not meet the requirements of content determination, the test sample was not analyzed, and the next step was to adjust the mobile phase gradient.

[0321] 2.3. Mobile phase gradient adjustment

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

[0323] Table 3 Gradient elution conditions 2

[0324]

[0325]

[0326] Preparation of reference solution: Take appropriate amount of benzoylmeaconitine reference substance, benzoylaconitine reference substance, benzoylhysaconitine reference substance, and aconitine diester type alkaloid reference extract, accurately weigh, add acetonitrile-0.02 mol / L diammonium phosphate solution (1:1) to make a mixed solution containing 50 μg of benzoylmeaconitine, 10 μg of benzoylaconitine, 10 μg of benzoylhysaconitine, 3 μg of meaconitine, 3 μg of hypoaconitine, and 3 μg of aconitine per 1 ml, as the mixed reference solution of aconitine alkaloids.

[0327] Table 4 Chromatographic peak parameters of aconitum alkaloids mixed control and test products

[0328]

[0329] The results showed that under this gradient, six chromatographic peaks appeared in the mixed control solution of aconite alkaloids, and the separation was significantly improved. However, the chromatographic peak separation of both monoester alkaloids and diester alkaloids in the test sample was poor, and baseline separation was not achieved, which could not meet the requirements of content determination analysis.

[0330] After analyzing the preparation method of the test solution, it is speculated that the properties of the above impurity components may be similar to those of aconite alkaloids. It is difficult to make the above 6 components meet the requirements of content determination only by optimizing the chromatographic conditions. Therefore, it is considered to optimize the preparation method of the test solution in order to improve the separation by purifying the test sample.

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

[0332] Preparation method of test solution: accurately measure 80ml of the product, place it in a 100ml volumetric flask, add 20ml of 0.5mol / L hydrochloric acid solution to the scale, weigh it, ultrasonically treat it (power 400W, frequency 40kHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with 0.5mol / L hydrochloric acid solution, shake it well, transfer it to a centrifuge tube, centrifuge it (speed is 5000 rpm) for 10 minutes, filter it, accurately measure 10ml of the filtrate and add it to a solid phase extraction column (with mixed cation exchange reverse phase adsorbent as filler, 15 0mg, volume is 6ml, pre-eluted with 6ml of acetonitrile and water), eluted with 3ml of water, ammonia solution (5→100), water, methanol, 5ml of acetonitrile, after the eluent has flowed out, let it stand for 5 minutes, and then elute with 10ml of a mixed solution of acetonitrile-concentrated ammonia test solution (90:10), collect the eluent, recover the solvent under reduced pressure below 40°C to dryness, accurately add a mixed solution of acetonitrile-0.1% phosphoric acid solution (20:80) to dissolve the residue, put it in a 5ml volumetric flask, and dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0333] At the same time, the analysis was performed under the chromatographic conditions of Table 3 above, and the results are shown in Tables 5 and Figure 3 shown.

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

[0335]

[0336] The results showed that for monoester alkaloids, compared with the control, only the chromatographic peak of benzoylmesaconitine was present in the monoester alkaloid components in the test sample, and no chromatographic peaks were detected at the corresponding retention times of benzoylmesaconitine and benzoylmesaconitine, indicating 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 chromatographic peak; the diester alkaloids were analyzed, and combined with experience and component content analysis, it was inferred that there was an obvious interference peak at the corresponding retention time of the control with meaconitine, so it was considered to increase the sample loading amount and adjust the mobile phase ratio at the same time in order to obtain better chromatographic behavior.

[0337] 2.5. Adjust the proportion of organic phase

[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 chromatographic conditions after adjustment are shown in Table 6, and the results are shown in Tables 7 and Figure 4 As shown, at the same time, 30 ml of the filtrate of the test solution was added to the solid phase extraction column for corresponding treatment to prepare the test solution.

[0339] Octadecylsilane bonded silica as filler (HALO AQ-C 18 , 4.6mm×150mm, 2.7μm); acetonitrile-tetrahydrofuran (9:1) was used as mobile phase A, 0.1mol / L ammonium acetate solution (0.5ml of glacial acetic acid was added for every 1000ml) was used as mobile phase B, gradient elution was performed according to the provisions in Table 6, the column temperature was 30°C, the flow rate was 0.6ml per minute, and the detection wavelength was 235nm.

[0340] Table 6 Gradient elution conditions 3

[0341] Time (minutes) 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 Chromatographic peak parameters of aconite alkaloids mixed control and test monoester alkaloids

[0343]

[0344]

[0345] The results showed that benzoylmeaconitine, benzoylaconitine and benzoylhyaconitine were all detected in the test sample, and no chromatographic peak appeared at the corresponding retention time of the diester alkaloid control, but the separation of benzoylaconitine was not good, so the chromatographic conditions needed to be further optimized.

[0346] 2.6. Further optimization of chromatographic conditions

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

[0348] Table 8 Gradient elution conditions 4

[0349] Time (minutes) 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 Chromatographic peak parameters of monoester alkaloids of the test samples

[0351]

[0352] The results showed that under the chromatographic conditions, the three components of monoester alkaloids in the test sample were well separated, and there was no interference at the corresponding retention time of the diester alkaloid control, which met the requirements for separation of content determination.

[0353] The chromatographic conditions were tentatively determined and used for the subsequent optimization of the test solution preparation method.

[0354] 2.7. Optimization of the preparation method of the test solution

[0355] The chromatographic peak areas of benzoyl aconitine and benzoyl mesaconitine in the test sample obtained by the above test sample preparation method are small, and the sensitivity of the instrument is required to be 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 to improve the response of the chromatographic peak. Therefore, it is necessary to investigate the specifications of the solid phase extraction column, the sample volume of different subsequent filtrates, and the corresponding different eluent volumes, so as to meet the requirements of content determination and ensure that the detection of the method is accurate and reproducible.

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

[0357] The adsorption capacity of aconite alkaloids by solid phase extraction columns with loading amounts of 150 mg and 500 mg was investigated. The results showed that the total peak area of ​​monoester alkaloids of the test sample obtained by using a solid phase extraction column with a loading amount of 500 mg was greater than that of the test sample obtained by using a solid phase extraction column with a loading amount of 150 mg, indicating that when a solid phase extraction column with a loading amount of 150 mg was used and the filtrate loading volume was 30 ml, it had exceeded its adsorption capacity range and might cause overloading, so a solid phase extraction column with a loading amount of 500 mg was used for subsequent research.

[0358] 2.7.2. Investigation of different reconstitution solvents

[0359] Because the 0.1% phosphoric acid solution in the original reconstitution solvent is different from the 0.1 mol / L ammonium acetate solution used in the mobile phase aqueous 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 reconstitution solvent on the chromatographic peaks in the test sample were investigated.

[0360] Combined with "2.6 Further optimization of chromatographic conditions", the retention time of aconitine in the chromatogram of the mixed reference solution of aconitine alkaloids is between 36 and 38 minutes. It can be seen from the results that the impurity peak shape of the test sample obtained using the mixed solution of acetonitrile-0.1 mol / L ammonium acetate solution (20:80) in this time period is better than the impurity peak of the mixed solution of acetonitrile-0.1% phosphoric acid solution (20:80), which is beneficial to the separation of impurity peaks and aconitine in the later spiked samples. Therefore, the mixed solution of acetonitrile-0.1 mol / L ammonium acetate solution (20:80) is selected as the re-dissolution solvent for the test sample.

[0361] 2.7.3. Investigation of different loading volumes

[0362] Combined with the results of "2.7.2", when the sample volume of the subsequent filtrate is 30ml, basically no benzoylhypaconitine is 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 showed that with the increase of sample volume, the peak areas of chromatographic peaks of benzoylmeaconitine, benzoylmeaconitine and benzoylmeaconitine gradually increased; when the sample volume was 70 ml, the peak area of ​​benzoylmeaconitine was 898.034, the peak area of ​​benzoylmeaconitine was 101.606, and the peak area of ​​benzoylmeaconitine was 69.751, and the response was good. At the same time, combined with the optional specifications of the solid phase extraction column and the content of the Heishun tablets themselves, the sample volume of the subsequent filtrate was selected as 70 ml.

[0364] 2.7.4. Investigation of different eluent volumes

[0365] The amount of acetonitrile-concentrated ammonia test solution (90:10) mixed solution for elution was determined by using 10 ml of the mixed solution of acetonitrile-concentrated ammonia test solution (90:10) in batches and monitoring the elution status of each time in a timely manner.

[0366] The results showed that after the solid phase extraction column was eluted for the second time with a mixed solution of acetonitrile-concentrated ammonia test solution (90:10), part of the test sample still eluted out of the column, and no monoester alkaloid chromatographic peak was detected in the test sample after the third elution, indicating that the aconitum alkaloid components of the solid phase extraction column could be completely eluted in the second time. Therefore, the elution amount of the mixed solution of acetonitrile-concentrated ammonia test solution (90:10) was determined to be 20 ml.

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

[0368] 2.8. Tentative Detection Method

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

[0370] Table 10 Gradient elution conditions 5

[0371] Time (minutes) 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: accurately measure 80 ml of the product, place it in a 100 ml volumetric flask, add 20 ml of 0.5 mol / L hydrochloric acid solution to the mark, weigh it, ultrasonically treat it (power 400 W, frequency 40 kHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with 0.5 mol / L hydrochloric acid solution, shake it well, transfer it to a centrifuge tube, centrifuge it (speed is 5000 rpm) for 10 minutes, filter it, accurately measure 70 ml of the filtrate, add it to a solid phase extraction column (with mixed cation exchange reverse phase adsorbent as filler, 500 mg, capacity is 6ml, pre-eluted with acetonitrile and water 12ml each), eluted with water 6ml, ammonia solution (5→100), water, methanol, acetonitrile 10ml each, after the eluent has flowed out, let it stand for 5 minutes, and then elute with acetonitrile-concentrated ammonia test solution (90:10) mixed solution 20ml, collect the eluent, recover the solvent to dryness under reduced pressure below 40℃, add acetonitrile-0.1mol / L ammonium acetate (20:80) mixed solution to dissolve the residue, put it in a 5ml volumetric flask, and dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0373] Preparation of spiked test solution: accurately measure 80 ml of this product, accurately add 30 μg of aconitum diester type alkaloid control extract, place in a 100 ml volumetric flask, add 20 ml of 0.5 mol / L hydrochloric acid solution to the scale, weigh the weight, ultrasonically treat (power 400 W, frequency 40 kHz) for 40 minutes, cool, weigh again, make up the lost weight with 0.5 mol / L hydrochloric acid solution, shake well, centrifuge (speed is 5000 rpm) for 10 minutes, filter, accurately measure 70 ml of the filtrate, add to the solid phase extraction column (mixed cation exchange reverse phase adsorption The agent is a filler, 500 mg, and the capacity is 6 ml. It is pre-eluted with 12 ml of acetonitrile and water respectively), and then eluted with 6 ml of water, ammonia solution (5→100), water, methanol, and acetonitrile respectively. After the eluent has flowed out, let it stand for 5 minutes, and then elute with 20 ml of a mixed solution of acetonitrile-concentrated ammonia solution (90:10), collect the eluent, and recover the solvent to dryness under reduced pressure below 40°C. The residue is added with a mixed solution of acetonitrile-0.1 mol / L ammonium acetate (20:80) to dissolve, placed in a 5 ml volumetric flask, and diluted to the scale, shaken well, filtered, and the filtrate is obtained.

[0374] Determination method: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0375] 2.9. Chromatographic conditions were optimized again

[0376] When using "2.8, tentative detection method" for methodological validation, it was found that non-target substances in the spiked test solution (an appropriate amount of aconitum diester-type alkaloid control extract was added during the preparation of the test solution) were mutually converted, and the converted substances co-eluted with the target substances in the chromatogram, making it impossible to accurately perform quantitative analysis of the target substances.

[0377] After analyzing the above phenomenon, it was determined that it was not related to reagents or equipment.

[0378] Secondly, in order to improve the separation of target substances and non-target substances, the preparation method of the test solution was re-examined and optimized, but to no avail; therefore, the chromatographic conditions were readjusted and optimized. The optimized chromatographic conditions and results are shown in Tables 11 to 13 and Figure 6 As shown:

[0379] Octadecylsilane bonded silica was used as filler (Agilent InfintyLab Poroshell 120Aq-C 18 , 4.6mm×150mm, 2.7μm); acetonitrile-tetrahydrofuran (98:2) as mobile phase A, 0.1mol / L ammonium acetate solution (0.5ml of glacial acetic acid per 1000ml) as mobile phase B, gradient elution according to the provisions in Table 11; flow rate 0.8ml per minute; column temperature 40℃; detection wavelength 235nm. The theoretical plate number calculated based on the benzoylmonaconitine peak should not be less than 3000.

[0380] Table 11 Gradient elution conditions 6

[0381] Time (minutes) 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 index components in spiked test samples

[0383]

[0384] Table 13 System suitability parameters for chromatographic peaks of index components in spiked test samples

[0385]

[0386]

[0387] The results showed that under the chromatographic conditions, the separation of monoester alkaloids and diester alkaloids in the spiked test solution was good, meeting the requirements of the separation degree of content determination.

[0388] 3. Methodological validation

[0389] According to the above-mentioned tentative detection method, a systematic methodological validation was carried out on the monoester alkaloid content determination and diester alkaloid limit inspection methods of the benchmark samples of traditional Chinese medicine compositions containing cinnamon twigs in accordance with the 2020 edition of Part IV 9101 Analytical Method Validation Guidelines.

[0390] 3.1 Chromatographic conditions and solution preparation

[0391] Since aconitine and metaconitine were not detected in the test sample during development, in order to determine the feasibility of the limit test method for diester-type alkaloids in the test sample solution of the traditional Chinese medicine composition containing cassia twig, the spiked test sample solution will be prepared using the spiking method (since the China Food Inspection and Quarantine Institutes no longer provides aconitine reference substance, the aconitum diester-type alkaloid reference extract will be used for spiking), and a systematic methodological study on the limit test of diester-type alkaloids will be conducted using spiked test samples.

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

[0393] Preparation of reference solution: Take appropriate amount of benzoylneaconitine reference substance, benzoylaconitine reference substance, benzoylhypaconitine reference substance, and aconitine diester type alkaloid reference extract, accurately weigh them, add acetonitrile-0.1mol / L ammonium acetate (20:80) mixed solution to prepare appropriate concentration stock solution, accurately weigh benzoylneaconitine reference substance stock solution, benzoylaconitine reference substance stock solution, benzoylhypaconitine reference substance stock solution, and acetonitrile-0.1mol / L ammonium acetate mixed solution to prepare appropriate concentration stock solution. Place an appropriate amount of aconitine reference stock solution and aconitine diester type alkaloid reference extract stock solution in a volumetric flask of appropriate specifications, add a mixed solution of acetonitrile-0.1 mol / L ammonium acetate (20:80), dilute to the scale, shake well, and obtain (each 1 ml contains 20 μg of benzoylmeaconitine, 3 μg of benzoylaconitine, 3 μg of benzoylhypaconitine, 5 μg of meaconitine, 5 μg of hypoaconitine, and 5 μg of aconitine).

[0394] Preparation of test solution: accurately measure 80 ml of the product, place it in a 100 ml volumetric flask, add 20 ml of 0.5 mol / L hydrochloric acid solution to the scale, weigh the weight, ultrasonic (power 400 W, frequency 40 kHz) for 40 minutes, let cool, weigh again, make up the lost weight with 0.5 mol / L hydrochloric acid solution, shake well, transfer to a centrifuge tube, centrifuge (speed 5000 rpm) for 10 minutes, filter, accurately measure 70 ml of the filtrate, add it to a solid phase extraction column (with mixed cation exchange reverse phase adsorbent as filler, 500 m g, capacity is 6ml, pre-eluted with acetonitrile and water 12ml each), eluted with water 6ml, ammonia solution (5→100), water, methanol, acetonitrile 10ml each, after the eluent has flowed out, let it stand for 5 minutes, and then elute with 20ml of a mixed solution of acetonitrile-concentrated ammonia solution (90:10), collect the eluent, recover the solvent to dryness under reduced pressure below 40°C, add a mixed solution of acetonitrile-0.1mol / L ammonium acetate (20:80) to dissolve the residue, put it in a 5ml volumetric flask, and dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0395] Preparation of spiked test solution: accurately measure 80 ml of this product, accurately add 30 μg of aconitum diester type alkaloid control extract, place in a 100 ml volumetric flask, add 20 ml of 0.5 mol / L hydrochloric acid solution to the scale, weigh the weight, ultrasonically treat (power 400 W, frequency 40 kHz) for 40 minutes, cool, weigh again, make up the lost weight with 0.5 mol / L hydrochloric acid solution, shake well, centrifuge (speed is 5000 rpm) for 10 minutes, filter, accurately measure 70 ml of the filtrate, add to the solid phase extraction column (mixed cation exchange reverse phase adsorption The agent is a filler, 500 mg, and the capacity is 6 ml. It is pre-eluted with 12 ml of acetonitrile and water respectively), and then eluted with 6 ml of water, ammonia solution (5→100), water, methanol, and acetonitrile respectively. After the eluent has flowed out, let it stand for 5 minutes, and then elute with 20 ml of a mixed solution of acetonitrile-concentrated ammonia solution (90:10), collect the eluent, and recover the solvent to dryness under reduced pressure below 40°C. The residue is added with a mixed solution of acetonitrile-0.1 mol / L ammonium acetate (20:80) to dissolve, placed in a 5 ml volumetric flask, and diluted to the scale, shaken well, filtered, and the filtrate is obtained.

[0396] Determination method: Accurately pipette 20 μl of reference solution, test solution and spiked test solution respectively, inject into liquid chromatograph, and determine.

[0397] 3.2. Exclusivity

[0398] Negative control solution of Quheishun tablets: Take the negative control decoction of Quheishun tablets and prepare it in the same way as 3.1 “Preparation of test solution”.

[0399] Heishunpian single-flavor test sample solution: Take Heishunpian single-flavor decoction and prepare it in the same way as 3.1 “Preparation of test sample solution”.

[0400] Blank solution: Take purified water and prepare the same as 3.1 “Preparation of test solution”.

[0401] Take the test sample solution, the spiked test sample solution, the negative control solution lacking Heishunpian, the test sample solution of Heishunpian alone, the reference solution, the blank solution and the blank solvent (acetonitrile-0.1mol / L ammonium acetate (20:80) mixed solution), and perform HPLC analysis according to the tentative chromatographic conditions, record the chromatogram and the ultraviolet absorption spectra of the reference and test samples, and examine whether the test sample solution lacking the negative control decoction of Heishunpian shows a negative reaction. If diester-type alkaloids are not detected in the test sample, determine the position of the diester-type alkaloid chromatographic peak by spiked test sample.

[0402] The results showed that there was no interference in the blank solvent, blank solution and negative control solution lacking Heishun tablets; the monoester alkaloids and diester alkaloids in the test solution came from the taste of Heishun tablets in the benchmark sample of the traditional Chinese medicine composition containing cassia twig; the separation of each chromatographic peak of the monoester alkaloids and diester alkaloids in the spiked test sample was greater than 1.5, which met the requirements of high performance liquid chromatography content determination; the ultraviolet absorption wavelengths of each component in the reference solution and the test solution were relatively consistent; in summary, it was shown that the method had good specificity.

[0403] 3.3 Precision

[0404] 3.3.1. Instrument precision test

[0405] Take the reference solution and repeat the injection 6 times according to the proposed chromatographic conditions for content determination, determine the chromatographic peak areas of monoester alkaloids and diester alkaloids and calculate the RSD (RSD≤2.0%).

[0406] The results showed that the reference solution was injected six times under the chromatographic conditions, and the RSD values ​​of the peak areas of the monoester alkaloids and diester alkaloids were less than 2%, indicating that the chromatographic system met the accuracy level that the analytical method could achieve.

[0407] 3.3.2 Repeatability test

[0408] Prepare 6 spiked test solutions, inject them according to the proposed chromatographic conditions, and determine the RSD of the content of monoester alkaloids and diester alkaloids in each test solution (RSD of the total amount of monoester alkaloids ≤ 8.0%, RSD of the total amount of diester alkaloids ≤ 15.0%).

[0409] The results showed that among the six samples prepared, the RSD value of the total amount of monoester alkaloids was 3.1%, less than 8.0%; the RSD value of the total amount of diester alkaloids was 4.2%, less than 15.0%, which met the requirements and showed that the method had good repeatability.

[0410] 3.3.3 Intermediate precision

[0411] The same batch of GZSYZMKL reference samples were taken and independently operated by different experimenters on different dates according to the preparation method of spiked test sample solution. Experimenter A used Agilent 1290-DAD HPLC and Experimenter B used Agilent 1100-DAD HPLC to determine the total content of monoester alkaloids and diester alkaloids and calculate the RSD of 12 samples. (RSD of total monoester alkaloids ≤ 8.0%, RSD of total diester alkaloids ≤ 15.0%).

[0412] The results showed that among the 12 prepared samples, the RSD value of the total amount of monoester alkaloids was 2.6%, less than 8.0%; the RSD value of the total amount of diester alkaloids was 10.6%, less than 15.0%, which met the specified requirements, indicating that the intermediate precision of this method was good.

[0413] 3.4 Linear

[0414] Take appropriate amount of benzoyl-neoaconitine reference substance, benzoyl-aconitine reference substance, benzoyl-hypoaconitine reference substance, and aconitine diester alkaloid extract, accurately weigh, add acetonitrile-0.1mol / L ammonium acetate (20:80) mixed solution to prepare mixed control solutions of different concentrations containing benzoyl-neoaconitine, benzoyl-hypoaconitine, benzoyl-aconitine, aconitine, aconitine, and aconitine (if the concentration does not reach the quantitative limit of the detection component, it will be further diluted). Accurately draw 20μl of mixed control solutions of different concentrations, inject into liquid chromatograph, measure peak area, and use the concentration of benzoyl-neoaconitine, benzoyl-aconitine, benzoyl-hypoaconitine, aconitine, aconitine, and aconitine as the horizontal coordinate and the peak area as the vertical coordinate to draw standard curves, and list regression equations, correlation coefficients r (r≥0.999), residual sum of squares, and linear graphs.

[0415] The results showed that the linear regression equation of benzoyl aconitine was y=34.492x-9.3909, the correlation coefficient r was 0.9999, and the residual sum of squares RSS was 1761.372, indicating that benzoyl aconitine had a good linear relationship in the range of 0.1927μg / ml to 96.3385μg / ml; the linear regression equation of benzoyl aconitine was y=33.75x+0.8969, and the correlation coefficient r was 1.0000. The residual sum of squares RSS is 12.096, indicating that benzoylmeaconitine has a good linear relationship in the range of 0.1955μg / ml to 16.7580μg / ml; the linear regression equation of benzoylmeaconitine 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 benzoylmeaconitine has a good linear relationship in the range of 0.2705μg / ml to 16.2299μg / ml. The linear relationship is good in the range of ml; the linear regression equation of neoaconitine 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 the linear relationship of neoaconitine is good in the range of 0.2234μg / ml to 27.9321μg / ml; the linear regression equation of mesaconitine is y=32.032x-5.855, the correlation coefficient r is 0.9998, and the residual sum of squares RSS is 1075.005. The sum of squares RSS is 185.032, indicating that aconitine has a good linear relationship in the range of 0.2099μg / ml to 27.7623μg / 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 0.2221μg / ml to 27.7623μg / ml.

[0416] 3.5 Detection Limit

[0417] The detection limit is determined in accordance with the guidance for validation of analytical methods for pharmaceutical quality standards in the four general chapters of the 2020 edition of the "Chinese Pharmacopoeia". The signal measured by the known concentration reference (diluted by the lowest concentration linear reference or the quantitative limit reference) is compared with the signal measured by the blank sample. The signal-to-noise ratio is generally 3:1, thereby determining the lowest concentration of the substance being measured.

[0418] The results showed that the lowest concentrations of benzoylmesaconitine, benzoylmesaconitine and benzoylhypaconitine that could be reliably detected were 48.2 ng / ml, 111.7 ng / ml and 108.2 ng / ml respectively; the lowest concentrations of metaconitine, hypoaconitine and aconitine were 139.7 ng / m, 131.2 ng / ml and 138.8 ng / ml respectively.

[0419] 3.6 Limit of Quantitation

[0420] The detection limit is determined in accordance with the guidance for validation of analytical methods for pharmaceutical quality standards in the fourth general chapter of the 2020 edition of the Chinese Pharmacopoeia. The signal measured by the known concentration reference (diluted by the lowest concentration linear reference) is compared with the signal measured by the blank sample. The signal-to-noise ratio is generally 10:1, thereby determining the lowest concentration of the substance to be measured that can be reliably quantified.

[0421] The results showed that the lowest concentrations of benzoylmesaconitine, benzoylmesaconitine and benzoylhypaconitine that could be reliably quantified were 0.1927μg / ml, 0.1955μg / ml and 0.2705μg / ml, respectively; the lowest concentrations of metaconitine, hypoaconitine and aconitine were 0.2234μg / ml, 0.2099μg / ml and 0.2221μg / ml, respectively.

[0422] 3.7 Accuracy test

[0423] That is, the sample recovery test, 9 portions are taken from the same batch of GZSYZMKL reference samples, 40ml of each portion is accurately measured and placed in 100ml, 20ml of pure water is added, and three different concentrations of high, medium and low are added (the amount of reference substance added needs to be determined according to the results of the content of each component of the test solution. If the diester type alkaloids are not detected in the test solution, the amount of diester type alkaloids added is determined according to the reference literature "diester type aconitine is highly toxic, and the toxic dose is close to the therapeutic dose. Among them, aconitine has extremely strong cardiotoxicity and neurotoxicity. It is the main toxic component of aconitum drugs. Human Oral administration of 0.2 mg can cause poisoning". After conversion, it can be concluded that the content of aconitine in a sample should not exceed 32 μg. Combined with the production ratio of aconitine in the diester type control extract of aconitum, the addition amount of the diester type control extract of aconitum was designed to be 90 μg, 60 μg, and 30 μg from high concentration to low concentration, respectively. Each reference substance was accurately added at a ratio of 1:1.5, 1:1, and 1:0.5, and diluted to volume with 0.5 mol / l hydrochloric acid. The solution was prepared according to the proposed method for preparing the test solution. Three test solutions were prepared for each concentration for determination. At the same time, two accompanying test solutions were prepared for content determination. The content of the accompanying test sample solution was used to calculate the sample recovery rate of the total amount of monoester alkaloids and diester alkaloids (total amount of monoester alkaloids 75%~120%, total amount of diester alkaloids 70%~125%) and RSD (total amount of monoester alkaloids RSD≤8.0%, total amount of diester alkaloids RSD≤15.0%).

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

[0425] 3.8 Stability Study

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

[0427] The results showed that within 72 hours, the RSDs of the peak areas of chromatographic peaks of benzoylmesaconitine, benzoylmesaconitine, benzoylhypaconitine, mesaconitine, hypaconitine and aconitine were all less than 2.0%. Although the RSD of the peak area of ​​benzoylmesaconitine was greater than 2.0%, the peak area change did not show a certain regularity due to its small peak area. It is speculated that the reason for the change should be unrelated to the change of the substance itself, so the RSD value requirement can be appropriately relaxed; this shows that these components in the test sample are basically stable within 72 hours.

[0428] 3.9 Durability Test

[0429] 3.9.1. Investigation of different flow rates

[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, and calculate the content and RSD of the total amount of monoester alkaloids and diester alkaloids (RSD of the total amount of monoester alkaloids ≤8.0%, RSD of the total amount of diester alkaloids ≤15.0%).

[0431] The results showed that under the flow rate of 0.75ml / min~0.85ml / min, the RSD of the total content of monoester alkaloids in the test solution was 0.5%, not more than 8.0%; the RSD of the total content of diester alkaloids was 1.5%, not more than 15.0%, which met the requirements of 9101 of the General Rules of Part IV of the 2020 edition of the "Chinese Pharmacopoeia", indicating that the method has good durability (different flow rates).

[0432] 3.9.2. Investigation of different column temperatures

[0433] During the early method development process, there was a certain interference when the column temperature was between 30℃ and 35℃, so the column temperature range was narrowed. The same batch of spiked test solution was injected at different column temperatures of 38℃, 40℃ and 42℃, and the content and RSD of the total amount of monoester alkaloids and 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 conditions of column temperature of 38℃~42℃, the RSD of the total content of monoester alkaloids in the spiked test solution was 0.6%, not more than 8.0%; the RSD of the total content of diester alkaloids was 0.2%, not more than 15.0%, which numerically met the requirements of 9101 of the General Rules of Part IV of the 2020 edition of the "Chinese Pharmacopoeia"; but when the column temperature was 38℃, the separation degree of the chromatographic peak of benzoylaconitine and impurity peak 2 in the spiked test sample was 0.62, which was significantly less than 1.5, and did not meet the requirements for the determination of benzoylaconitine content under this condition; in summary, this method has good durability under the conditions of column temperature of 40℃~42℃ (different column temperatures).

[0435] 3.9.3. Chromatographic columns of different brands

[0436] Take the same batch of spiked test solution and examine different brands of chromatographic columns according to the determined chromatographic conditions: Column 1 (Waters T 3 , 4.6 mm × 150 mm, 2.7 μm, SN0162340181432), chromatographic column 2 (Agilent InfintyLabPoroshell 120Aq-C 18 , 4.6mm×150mm, 2.7μm, SNUSKTM01334), chromatographic column 3 (YMC C 18 , 4.6mm×150mm, 2.7μm, SNMC005032), calculate the content and RSD of the total amount of monoester alkaloids and the total amount of diester alkaloids (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 conditions of different brands of chromatographic columns, the RSD of the total content of monoester alkaloids in the test solution was 5.6%, less than 8.0%, and the RSD of the total content of diester alkaloids was 6.8%, less than 15.0%, which met the requirements of 9101 of the General Rules of Part IV of the 2020 edition of the "Chinese Pharmacopoeia", indicating that this method has good durability (different brands of chromatographic columns).

[0438] 4. Method determination

[0439] The above methodological research results show that this method meets the requirements of the 9101 analytical method validation guidelines of Part IV of the 2020 edition of the Chinese Pharmacopoeia, and can be used to accurately determine the contents of benzoylmesaconitine, benzoylmesaconitine, benzoylhypaconitine, mesaconitine, hypaconitine and aconitine in the reference sample of the traditional Chinese medicine composition containing cinnamon twig. Therefore, the method for determining the content of monoester alkaloids and the limit inspection method for diester alkaloids in the reference sample of the traditional Chinese medicine composition containing cinnamon twig is determined as follows:

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

[0441] Preparation of reference solution: Take appropriate amounts of benzoylmeaconitine reference substance, benzoylaconitine reference substance, benzoylhysaconitine reference substance, and aconitine diester-type alkaloid reference extract, accurately weigh them, and add a mixed solution of acetonitrile-0.1 mol / L ammonium acetate (20:80) containing 20 μg of benzoylmeaconitine, 3 μg of benzoylaconitine, 3 μg of benzoylhysaconitine, 5 μg of meaconitine, 5 μg of hypoaconitine, and 5 μg of aconitine per 1 ml.

[0442] Preparation of test solution: Accurately measure 80 ml of the 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 it, ultrasonically treat it (power 400 W, frequency 40 kHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with 0.5 mol / L hydrochloric acid solution, shake it well, transfer it to a centrifuge tube, centrifuge it (speed is 5000 rpm) for 10 minutes, filter it, accurately measure 70 ml of the filtrate, add it to a solid phase extraction column (with mixed cation exchange reverse phase adsorbent as filler, 500 m g, capacity is 6ml, pre-eluted with acetonitrile and water 12ml each), eluted with water 6ml, ammonia solution (5→100), water, methanol, acetonitrile 10ml each, after the eluent has flowed out, let it stand for 5 minutes, and then elute with acetonitrile-concentrated ammonia test solution (90:10) mixed solution 20ml, collect the eluent, recover the solvent to dryness under reduced pressure below 40℃, add acetonitrile-0.1mol / L ammonium acetate (20:80) mixed solution to dissolve the residue, put it in a 5ml volumetric flask, and dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0443] Determination method: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0444] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for determining the content of aconite alkaloids in a traditional Chinese medicine composition containing cassia twig, characterized in that: The determination method comprises the following steps: Take the test solution and the reference solution of the Chinese medicine composition for testing. The chromatographic conditions of the detection are: a chromatographic column with octadecylsilane bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is acid aqueous solution, alkali aqueous solution and / or buffered saline solution, and the gradient elution program is: 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; flow rate is 0.5-1.5 ml / min, column temperature is 40-42° C., detection wavelength is 180-360 nm, and injection volume is 10-30 μl; According to the test results, obtaining the content information of the reference substance of the Chinese medicine composition; Among them, the traditional Chinese medicine composition includes cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes, rhizome of Anemarrhena, saposhnikovia and heishun tablets, and the reference substances are benzoylmesaconitine, benzoylmesaconitine, benzoylhypaconitine, aconitine, hypaconitine and aconitine.

2. The measuring method according to claim 1, characterized in that The information is the content of the reference substance in the Chinese medicine composition calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Chinese medicine composition and the chromatogram of the reference substance solution; Preferably, the linear equation of the benzoyl aconitine is y=34.492x-9.3909, R 2 =0.9999; Preferably, the linear equation of the benzoylaconitine is y=33.75x+0.8969, R 2 =1.0000; Preferably, the linear equation of the benzoylhypaconitine is y=35.495x-7.1817, R 2 =0.9997; Preferably, the linear equation of the neoaconitine is y=29.6145x-14.7747, R 2 =0.9990; Preferably, the linear equation of the hypaconitine is y=32.032x-5.855, R 2 =0.9998; Preferably, the linear equation of aconitine is y=29.8871x-15.1308, R 2 =0.9990.

3. The measuring method according to claim 1, characterized in that The preparation method of the Chinese medicine composition test solution comprises: taking an appropriate amount of a decoction of the Chinese medicine composition containing cassia twigs and placing it in a container, adding a first solvent, weighing the weight, ultrasonically treating for a period of time, cooling, weighing the weight again, using the first solvent to make up for the lost weight, shaking, centrifuging, filtering, taking an appropriate amount of subsequent filtrate, eluting the subsequent filtrate after adsorption using a solid phase extraction column, collecting the eluate, recovering under reduced pressure, drying to obtain a residue, adding a second solvent to the residue to dissolve and dilute it, shaking, filtering, and obtaining the Chinese medicine composition test solution; Preferably, the container is a volumetric flask; Preferably, the first solvent is hydrochloric acid; More preferably, the concentration of the hydrochloric acid is 0.4 to 0.6 mol / L, for example, about 0.5 mol / L; Preferably, the volume ratio between the decoction of the traditional Chinese medicine composition and the first solvent is 2 to 6, for example, about 4; Preferably, the volume of the decoction of the Chinese medicine composition is 60-100 ml, for example, about 80 ml; Preferably, the power of the ultrasonic treatment is 300-500W, for example, about 400W; Preferably, the frequency of the ultrasonic treatment is 30 to 50 kHz, for example, about 40 kHz; Preferably, the ultrasonic treatment time is 20 to 60 minutes, for example, about 40 minutes; Preferably, the centrifugal speed is 4000-6000 rpm / min, for example, about 5000 rpm / min; Preferably, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes; More preferably, the volume ratio between the subsequent filtrate and the decoction of the traditional Chinese medicine composition is 0.5 to 1, for example, about 0.875; More preferably, the volume of the subsequent filtrate is 60 to 80 ml, for example, about 70 ml; More preferably, the temperature of the reduced pressure recovery is not higher than 40°C; Still more preferably, the second solvent is a mixed solution of acetonitrile and ammonium acetate; Particularly preferably, the concentration of the ammonium acetate is 0.05 to 0.2 mol / L, for example, about 0.1 mol / L; Particularly preferably, in the second solvent, the volume ratio of the acetonitrile to the ammonium acetate is acetonitrile:ammonium acetate=about 20:about 80.

4. The measuring method according to claim 3, characterized in that The preparation method of the solid phase extraction column comprises: using a mixed cation exchange reverse phase adsorbent as a filler, and sequentially using acetonitrile and water for elution, to obtain; Preferably, the mass of the mixed cation exchange reverse phase adsorbent is 400-600 mg, for example, about 500 mg; Preferably, the capacity of the solid phase extraction column is 4 to 8 ml, for example 6 ml; More preferably, the volume of acetonitrile is 10 to 15 ml, for example about 12 ml; More preferably, the volume of the water is 10-15 ml, for example about 12 ml; Preferably, in the method for preparing the test solution of the traditional Chinese medicine composition, the elution method comprises: using water, ammonia solution (5→100) for the first time, water, methanol, and acetonitrile for the second time for elution in sequence, leaving the solution for a period of time after the liquid has drained away, eluting with a third solvent, and collecting the eluate; More preferably, the volume of the first water is 4-8 ml, for example, about 6 ml; Still more preferably, the volume of the ammonia solution is 5 to 15 ml, for example about 10 ml; More preferably, the volume of the second water is 5 to 15 ml, for example, about 10 ml; Still more preferably, the volume of the methanol is 5 to 15 ml, for example about 10 ml; Still more preferably, the volume of acetonitrile is 5 to 15 ml, for example about 10 ml; Particularly preferably, the placement time is 4 to 6 minutes, for example, about 5 minutes; Particularly preferably, the third solvent is a mixed solution of acetonitrile and aqueous ammonia; Particularly preferably, 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; Particularly preferably, the volume of the third solvent is 10-30 ml, for example, about 20 ml.

5. The measuring method according to claim 1, characterized in that The preparation method of the reference substance solution comprises: weighing appropriate amounts of benzoylmeaconitine reference substance, benzoylmeaconitine reference substance, benzoylhypaconitine reference substance and aconitine diester type alkaloid reference extract, adding a second solvent to prepare the reference substance solution with the concentrations of benzoylmeaconitine, benzoylmeaconitine, benzoylhypaconitine, meaconitine, hypaconitine and aconitine being 1 to 50 μg / ml respectively; Preferably, the aconitine diester-type alkaloid control extract comprises mesaconitine, mesaconitine and aconitine; Preferably, the concentration of benzoylaconitum in the reference solution is about 20 μg / ml; Preferably, the concentration of benzoylaconitine in the reference solution is about 3 μg / ml; Preferably, the concentration of benzoylhypoconitine in the reference solution is about 3 μg / ml; Preferably, the concentration of neoaconitine in the reference solution is about 5 μg / ml; Preferably, the concentration of hypaconitine in the reference solution is about 5 μg / ml; Preferably, the concentration of aconitine in the reference solution is about 5 μg / ml; More preferably, the second solvent is a mixed solution of acetonitrile and ammonium acetate; Also preferably, the concentration of the ammonium acetate is 0.05 to 0.2 mol / L, for example, about 0.1 mol / L; Also preferably, in the second solvent, the volume ratio of the acetonitrile to the ammonium acetate is acetonitrile:ammonium acetate=about 20:about 80.

6. The measuring method according to claim 3, characterized in that The preparation method of the decoction of the traditional Chinese medicine composition containing cinnamon twigs comprises: weighing appropriate amounts of cinnamon twigs, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishunpian, placing the decoction in a container, adding water for soaking, boiling over high heat, boiling over low heat for a period of time, filtering, collecting the filtrate, and obtaining the decoction of the traditional Chinese medicine composition containing cinnamon twigs; Preferably, the container is a fully automatic ceramic medicine decoction pot; Preferably, the volume of the water is 1000-2000 ml, for example about 1400 ml; Preferably, the ratio of the sum of the mass of the cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and Heishun tablets to the mass / volume (g / ml) of the water is 0.1 to 0.5, for example, about 0.31; Preferably, the soaking time is 30 to 60 minutes, for example, about 45 minutes; Preferably, the slow-cooking time is 30 to 90 minutes, for example, about 60 minutes; Preferably, the filtration is 200 mesh nylon filter cloth filtration; More preferably, the mass ratio of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3); Preferably, the mass ratio of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice 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); More preferably, the mass ratio of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2; Particularly preferably, the mass range of the cassia twig is 40 to 70 g, for example, about 55.20 g; Particularly preferably, the mass range of the black shun tablets is 20 to 40 g, for example, about 27.60 g; Particularly preferably, the weight of the ginger is in the range of 50 to 90 g, for example, about 69.00 g; Particularly preferably, the mass of the windshield is in the range of 40 to 70 g, for example about 55.20 g; Particularly preferably, the mass range of the ephedra is 20 to 40 g, for example, about 27.60 g; Particularly preferably, the mass range of the Atractylodes macrocephala is 50 to 90 g, for example, about 69.00 g; Particularly preferably, the mass range of the white peony root is 30 to 50 g, for example, about 41.40 g; Particularly preferably, the mass range of the Anemarrhena Rhizoma is 40 to 70 g, for example, about 55.20 g; Particularly preferably, the mass of the liquorice is in the range of 20 to 40 g, such as about 27.60 g.

7. The measuring method according to claim 1, characterized in that The flow rate is 0.6 to 1.0 ml / min, such as 0.75 to 0.85 ml / min, such as about 0.8 ml / min; Preferably, the detection wavelength is 200-300 nm, for example, about 235 nm; Preferably, the injection volume is 15 to 25 μl, for example, about 20 μl; More preferably, the theoretical plate number of the chromatographic peak corresponding to the benzoyl aconitine is not less than 3000; Preferably, the chromatographic column is a Waters T3 chromatographic column, an Agilent InfintyLab Poroshell 120Aq-C18 chromatographic column, or a YMC C18 chromatographic column; Still more preferably, the chromatographic column is an Agilent InfintyLab Poroshell 120Aq-C18 chromatographic column; Even more preferably, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

8. The measuring method according to claim 1, characterized in that Mobile phase A is a mixed solution of acetonitrile and tetrahydrofuran; Preferably, in the mobile phase A, the volume ratio of the acetonitrile to the tetrahydrofuran is acetonitrile:tetrahydrofuran=about 98:about 2; Preferably, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations; More preferably, the buffered saline solution is an aqueous acetate solution and / or an aqueous acetate solution; Also preferably, the buffered saline solution is an ammonium acetate solution and about 0.5 ml of glacial acetic acid is added per 1000 ml of ammonium acetate solution; Still more preferably, the concentration of the ammonium acetate solution is 0.05 to 0.2 mol / L, for example, about 0.1 mol / L; Even more preferably, the pH value of the buffered saline solution is no greater than 7.

0.

9. The measuring method according to claim 1, characterized in that The detection limit of the benzoyl aconitine is about 48.2 ng / ml; Preferably, the detection limit of benzoylaconitine is about 111.7 ng / ml; Preferably, the detection limit of benzoylhypaconitine is about 108.2 ng / ml; Preferably, the detection limit of the neoaconitine is about 139.7 ng / ml; Preferably, the detection limit of the hypaconitine is about 131.2 ng / ml; Preferably, the detection limit of aconitine is about 138.8 ng / ml; More preferably, the limit of quantification of benzoyl aconitine is about 0.1927 μg / ml; More preferably, the limit of quantification of benzoylaconitine is about 0.1955 μg / ml; More preferably, the limit of quantification of benzoylhypaconitine is about 0.2705 μg / ml; More preferably, the limit of quantification of the neoaconitine is about 0.2234 μg / ml; More preferably, the limit of quantification of the hypoaconitine is about 0.2099 μg / ml; More preferably, the limit of quantification of aconitine is about 0.2221 μg / ml.

10. Use of the determination method according to any one of claims 1 to 9 in the quality detection and / or quality evaluation and / or quality control of aconitum alkaloids in a traditional Chinese medicine composition comprising cinnamon twig.

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