Quality control method for CDD-2101 particles

Abstract

The present invention provides a quality control method for CDD-2101 particles. The method comprises performing qualitative identification on immature orange fruit stir-fried with bran in CDD-2101 particles by means of thin-layer chromatography. An identification method comprises: spotting a test solution and a reference crude drug solution on the same silica gel thin-layer plate, putting the silica gel thin-layer plate into a developing agent for developing, and observing the distribution of fluorescent spots under ultraviolet light, wherein the developing agent is prepared by compounding cyclohexane, trichloromethane, ethyl acetate, and formic acid according to the volume ratio of 3:2:8:0.2. The method of the present invention has high sensitivity and good reproducibility, and a scientific basis is provided for accurately evaluating and effectively controlling the quality of the CDD-2101 particles from multiple aspects and multiple angles.

Description

Quality control method of CDD-2101 granules TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine ingredient analysis, and particularly relates to a quality control method of CDD-2101 granules. BACKGROUND

[0002] The CDD-2101 granules are derived from the ancient prescription of Mazi Ren Pill in Treatise on Febrile Diseases and Miscellaneous Diseases, and are composed of hemp seeds, rhubarb and the like, and have the effect of "purging without being too harsh and not damaging normal qi". Hemp seeds are used as the monarch drug, which is a drug for moistening the intestines and promoting defecation. Apricot kernels, Chinese peony and hemp seeds all have the effect of moistening the intestines. In addition to moistening the intestines, Chinese peony can also tonify yin and blood. Apricot kernels can lower lung qi and promote the flow of qi in the bowels, and are combined with roasted hovenia fruit and magnolia officinalis, which can lower lung qi and promote the flow of qi in the bowels, and reflect the lung and large intestine being in a paired relationship. The auxiliary drugs of rhubarb, roasted hovenia fruit and magnolia officinalis are combined to moisten the bowels and also have the effect of clearing away dry heat in the stomach and intestines. The whole prescription is combined to moisten the bowels and mainly moisten the intestines.

[0003] The CDD-2101 granules adopt a modern preparation process, which not only retains the characteristics of effective components of traditional decoctions, but also avoids the problems that traditional decoctions are difficult to be added or subtracted according to the condition and are inconvenient to carry. The preparation process method is as follows: according to the prescription, hemp seeds, rhubarb, bitter apricot kernels, magnolia officinalis, Chinese peony and roasted hovenia fruit are taken, and after being soaked in water for 2 hours, they are decocted for 2.5 hours. After the decocting liquid is collected by filtration, vacuum reduction and pressure concentration are performed to obtain a concentrated liquid with a relative density of 1.10-1.20 (75±2 DEG C). The concentrated liquid is subjected to spray drying to obtain dry paste powder. Magnesium stearate and dextrin are added to the dry paste powder and uniformly mixed, and dry granulation is performed to obtain the CDD-2101 granules. The compound prescription granules not only inherit the advantages of single prescription granules of traditional Chinese medicine, but also consider the interaction of decoction in the decoction process, which is in line with the traditional Chinese medicine theory and has great value.

[0004] SUMMARY

[0005] In order to overcome the shortcomings of the existing quality standard supervision, the purpose of the present application is to provide a quality control method of CDD-2101 granules, which provides a scientific basis for accurately evaluating and effectively controlling the quality of CDD-2101 granules.

[0006] In order to achieve the above-mentioned purpose, the present application provides a quality control method of CDD-2101 granules, wherein the raw materials of the CDD-2101 granules include the following medicinal materials: hemp seeds, rhubarb, magnolia officinalis, Chinese peony and roasted hovenia fruit. The quality control method includes qualitative identification of roasted hovenia fruit in the CDD-2101 granules by using thin layer chromatography, and the identification method includes the following steps:

[0007] The test sample solution and the control medicinal material solution are spotted on the same silica gel thin layer plate, developed in the bran-fried Citrus aurantium L. identification-developing agent, and then the distribution of fluorescent spots is observed under ultraviolet light; wherein the bran-fried Citrus aurantium L. identification-developing agent is composed of cyclohexane, chloroform, ethyl acetate and formic acid in a volume ratio of 3:2:8:0.2.

[0008] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification of bran-fried Citrus aurantium L., the spotting amount of the control medicinal material solution and the test sample solution is respectively 8-20 μl, more preferably 8 μl.

[0009] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification of bran-fried Citrus aurantium L., the solvent of the test sample solution and the control medicinal material solution is methanol.

[0010] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification of bran-fried Citrus aurantium L., the developing condition comprises: humidity of 32-88%, temperature of 3-40 ℃, and saturation time ≤30 min; the saturation time is more preferably 15-30 min.

[0011] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification of bran-fried Citrus aurantium L., when the test sample solution and the control medicinal material solution are spotted, a negative sample solution of bran-fried Citrus aurantium L. is also spotted.

[0012] According to the specific embodiment of the present application, preferably, the quality control method further comprises adopting the thin layer chromatography to perform qualitative identification on any one of Cannabis sativa L., Rhubarb, Magnolia officinalis, and Radix Paeoniae Alba in the CDD-2101 granules; the thin layer chromatography qualitative identification method comprises the following steps:

[0013] Preparation of the test sample solution and the control medicinal material solution;

[0014] The test sample solution and the control medicinal material solution are spotted on the same silica gel thin layer plate, developed in the bran-fried Citrus aurantium L. identification-developing agent, and then the distribution of fluorescent spots is observed under ultraviolet light; wherein the bran-fried Citrus aurantium L. identification-developing agent is composed of cyclohexane, chloroform, ethyl acetate and formic acid in a volume ratio of 3:2:8:0.2.

[0015] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification of bran-fried Citrus aurantium L., the solvent of the test sample solution and the control medicinal material solution is methanol.

[0016] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification of bran-fried Citrus aurantium L., the developing condition comprises: humidity of 32-88%, temperature of 3-40 ℃, and saturation time ≤30 min; the saturation time is more preferably 15-30 min.

[0017] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification of bran-fried Citrus aurantium L., the developing condition comprises: humidity of 32-88%, temperature of 3-40 ℃, and saturation time ≤30 min; the saturation time is more preferably 15-30 min.

[0018] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of Rhizoma et Radix Rhei, the sample volume of the control medicinal material solution and the sample solution is 8-25 μl respectively.

[0019] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of Magnoliae officinalis Cortex, the developing agent is composed of cyclohexane, ethyl acetate and formic acid with a volume ratio of 12:3:0.1.

[0020] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of Magnoliae officinalis Cortex, the sample volume of the control medicinal material solution and the sample solution is 15-30 μl respectively.

[0021] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of Radix Paeoniae Alba, the developing agent is composed of trichloromethane, ethyl acetate, methanol and 25-28 wt% ammonia solution with a volume ratio of 8:1:4:1.

[0022] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of Radix Paeoniae Alba, the sample volume of the control medicinal material solution and the sample solution is 8-18 μl respectively.

[0023] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of any one of the medicinal materials, the developing condition comprises: humidity of 32-88%, temperature of 3-40℃, and saturation time of ≤30 min.

[0024] According to the specific embodiment of the present application, preferably, the solvent of the sample solution and the control medicinal material solution of any one of the medicinal materials is methanol.

[0025] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of any one of Fructus Cannabis, Magnoliae officinalis Cortex and Radix Paeoniae Alba, the spot developing mode is: after the developed silica gel thin layer plate is dried, vanillin sulfuric acid solution is sprayed, and heating is performed until the spots are clear.

[0026] According to the specific embodiment of the present application, preferably, in the spot developing mode, the mass concentration of the vanillin sulfuric acid solution is 5%, and the heating temperature is 105℃.

[0027] According to the specific embodiment of the present application, preferably, in the thin layer chromatography identification method of Rhizoma et Radix Rhei, the spot developing mode is: after the silica gel thin layer plate is dried, it is placed under ultraviolet light for observation.

[0028] According to the specific embodiment of the present application, preferably, the thin layer chromatography identification method of the six medicinal materials of Fructus Cannabis, Rhizoma et Radix Rhei, Magnoliae officinalis Cortex, Radix Paeoniae Alba and Fructus Aurantii Immaturus Stir-baked with Wheat Bran is as follows:

[0029] (1) Take CDD-2101 granules, grind them, take 2g, add water 30ml, ultrasonic treatment for 30 minutes (power 500W, frequency 40kHz), centrifugal (4000r / min) for 10 minutes, take the supernatant, add water saturated n-butanol, shake extraction for 2 times, 30ml each time, combine the n-butanol extract, evaporate to dryness, add methanol 2ml to dissolve, as the test solution. Take 0.1g of cannabis seed control drug, add methanol 2ml, ultrasonic treatment for 10 minutes (power 500W, frequency 40kHz), take the supernatant as the cannabis seed control drug solution. Take 1-5μl of the cannabis seed control drug solution and 8-20μl of the test solution, respectively, and point them on the same silica gel G thin layer plate, with cyclohexane-ethyl acetate-formic acid (volume ratio 12:3:0.1) as the developing agent, humidity 32-88% and temperature 3-40℃, saturation time 0-30min, use Qingdao, Merck or Yantai plate to develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat at 105℃ until the spots develop clear color. In the test chromatogram, the same color spots appear at the corresponding positions of the control drug chromatogram.

[0030] (2) Take CDD-2101 granules, grind them, take 2g, add methanol 20ml, ultrasonic treatment for 30 minutes (power 500W, frequency 40kHz), filter, evaporate the filtrate to dryness, add methanol 2ml to dissolve, as the test solution. Take 0.3g of rhubarb control drug, add water 50ml, heat reflux for 60 minutes, centrifugal (4000r / min) for 10 minutes, take the supernatant, evaporate to dryness, add methanol 20ml, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add methanol 2ml to dissolve, as the control drug solution. Take 8-25μl of the rhubarb control drug solution and the test solution, respectively, and point them on the same silica gel G thin layer plate, with cyclohexane-ethyl acetate-formic acid (volume ratio 12:3:0.1) as the developing agent, humidity 32-88% and temperature 4-40℃, saturation time 0-30min, use Qingdao, Merck or Yantai plate to develop, take out, dry, and examine under ultraviolet light (365nm). In the test chromatogram, the same color fluorescent spots appear at the corresponding positions of the rhubarb control drug chromatogram.

[0031] (3) Qualitative identification of Magnoliae Officinalis Cortex by thin layer chromatography: Take CDD-2101 granules, grind them, take 2 g, add 20 ml of methanol, ultrasonic treatment for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate, add 2 ml of methanol to the residue to dissolve it, and use it as the test solution. Take another 0.5 g of Magnoliae Officinalis Cortex control drug, add 50 ml of water, heat reflux for 60 minutes, centrifuge (4000 r / min) for 10 minutes, take the supernatant, evaporate it, add 20 ml of methanol to the residue, ultrasonic treatment for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate, add 2 ml of methanol to the residue to dissolve it, and use it as the control drug solution. Take the magnolol and honokiol control substances, add methanol to make a mixture solution containing 0.5 mg of each per 1 ml, and use it as the control substance solution. Take 15-30 μl of each of the above three solutions, spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid (volume ratio 12:3:0.1) as the developing agent, the humidity is 32-88%, the temperature is 3-40°C, the saturation time is 0-30 min, use Qingdao, Merck or Yantai plate to develop, take them out, air dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots develop clearly. The test chromatogram shows the same color spots at the corresponding positions of the magnolol and honokiol control substances and the Magnoliae Officinalis Cortex control drug.

[0032] (4) Qualitative identification of Paeoniae Albus Radix by thin layer chromatography: Take CDD-2101 granules, grind them, take 2 g, add 20 ml of methanol, ultrasonic treatment for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate, add 2 ml of methanol to the residue to dissolve it, and use it as the test solution. Take another 0.25 g of Paeoniae Albus Radix control drug, add 50 ml of water, heat reflux for 60 minutes, centrifuge (4000 r / min) for 10 minutes, take the supernatant, evaporate it, add 20 ml of methanol to the residue, ultrasonic treatment for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate, add 2 ml of methanol to the residue to dissolve it, and use it as the control drug solution. Take 8-18 μl of the test solution and the above control drug solution, respectively, spot them on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-25-28 wt% concentrated ammonia solution (volume ratio 8:1:4:1) as the developing agent, the humidity is 32-88%, the temperature is 4-40°C, the saturation time is 0-30 min, use Qingdao, Merck or Yantai plate to develop, take them out, air dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots develop clearly. The test chromatogram shows the same color spots at the corresponding positions of the Paeoniae Albus Radix control drug.

[0033] (5) Qualitative identification of bran-fried bitter orange fruit by thin layer chromatography: take CDD-2101 granules, grind finely, take 2g, add methanol 20ml, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate, add methanol 2ml to dissolve, as test sample solution. Take another bran-fried bitter orange fruit negative sample, grind finely, take 2g, add methanol 20ml, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate, add methanol 2ml to dissolve, as negative sample solution. Take bran-fried bitter orange fruit control drug 0.3g, add water 50ml, heat reflux for 60 minutes, centrifuge (4000r / min) for 10 minutes, take the supernatant, evaporate, add methanol 20ml, ultrasonic treatment for 30 minutes (power 500W, frequency 40kHz), filter, evaporate the filtrate, add methanol 2ml to dissolve, as control drug solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four general 0502) experiment, take 8-20ul of bran-fried bitter orange fruit control drug solution and test sample solution, respectively, and point on the same silica gel G thin layer plate. With cyclohexane-trichloromethane-ethyl acetate-formic acid (3:2:8:0.2) as developing agent, the humidity is 32-88% and the temperature is 3-40℃, the saturation time is 0-30min, using Qingdao, Merck or Yantai plate development, take out, dry, under the ultraviolet lamp 365nm, observe. The test sample chromatogram shows the same color fluorescent main spot at the corresponding position of the control drug.

[0034] According to the specific embodiments of the present application, preferably, the quality control method further comprises: using liquid chromatography-tandem mass spectrometry to quantitatively determine at least one of the following index components in CDD-2101 granules: cannabamide B, emodin, rhein, chrysophanol, aloe-emodin, amygdalin, magnolol, honokiol, paeoniflorin, synephrine and hesperidin;

[0035] According to the specific embodiments of the present application, preferably, high performance liquid chromatography-tandem mass spectrometry is used to quantitatively determine at least one of the following index components in CDD-2101 granules: cannabamide B, emodin, amygdalin, magnolol, paeoniflorin, hesperidin, more preferably cannabamide B.

[0036] According to the specific embodiments of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of cannabamide B, the chromatographic conditions include:

[0037] The chromatographic column is ZORBAX Eclipse Plus C18 column (2.1mmx100mm, 1.8um, Agilent), and the column theoretical plate number is ≥1000; the column temperature is 40℃;

[0038] The mobile phase includes phase A and phase B, the phase A is acetonitrile, and the phase B is 0.1% formic acid aqueous solution, and the flow rate of the mobile phase is 0.4ml / min;

[0039] The elution mode is gradient elution, and the elution procedure is as follows:

[0040] The mass spectrometry conditions include:

[0041] A triple quadrupole mass spectrometry detector;

[0042] Electrospray ionization in negative mode;

[0043] Detection mode: multiple reaction monitoring;

[0044] Reaction monitoring ion pair: m / z 595.0→269.0 and m / z 595.0→432.0.

[0045] In the liquid chromatography-tandem mass spectrometry detection of the cannabinoidamide B of the application, the cannabinoidamide B molecular ion (m / z 595.0) is bombarded by energy, and the chemical bond site is broken to generate multiple ion fragments, among which the m / z 269.0 and m / z 432.0 fragments have the highest abundance.

[0046] According to the specific embodiment of the application, preferably, in the liquid chromatography-tandem mass spectrometry detection of chrysophanol, the chromatography conditions include:

[0047] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm×100 mm, 1.8 μm, Agilent), and the column theoretical plate number is ≥1000; the column temperature is 40°C;

[0048] The mobile phase includes phase A and phase B, the phase A is a 0.1% formic acid aqueous solution, the phase B is a 0.1% formic acid acetonitrile solution, and the flow rate is 0.4 ml / min;

[0049] The elution mode is gradient elution, and the elution procedure is as follows:

[0050] The mass spectrometry conditions include:

[0051] A triple quadrupole mass spectrometry detector;

[0052] Electrospray ionization in negative mode;

[0053] Detection mode: multiple reaction monitoring;

[0054] Reaction monitoring ion pair: m / z 253.0→225.0 and m / z 253.0→195.0.

[0055] According to the specific embodiment of the application, preferably, in the liquid chromatography-tandem mass spectrometry detection of chrysophanic acid, the chromatography conditions include:

[0056] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the column is ≥1000; the column temperature is 40°C;

[0057] The mobile phase comprises phase A and phase B, the phase A is 0.1% formic acid aqueous solution in volume fraction, the phase B is 0.1% formic acid acetonitrile solution in volume fraction, and the flow rate is 0.4 ml / min;

[0058] The elution mode is gradient elution, and the elution procedure is as follows:

[0059] The mass spectrometry condition comprises:

[0060] A triple quadrupole mass spectrometry detector;

[0061] Electrospray ionization in negative mode;

[0062] The detection mode is multiple reaction monitoring;

[0063] The reaction monitoring ion pair is m / z 283.0→239.0 and m / z 283.0→183.1.

[0064] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of emodin, the chromatographic condition comprises:

[0065] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the column is ≥1000; the column temperature is 40°C;

[0066] The mobile phase comprises phase A and phase B, the phase A is 0.1% formic acid aqueous solution in volume fraction, the phase B is 0.1% formic acid acetonitrile solution in volume fraction, and the flow rate is 0.4 ml / min;

[0067] The elution mode is gradient elution, and the elution procedure is as follows:

[0068] The mass spectrometry condition comprises:

[0069] A triple quadrupole mass spectrometry detector;

[0070] Electrospray ionization in negative mode;

[0071] The detection mode is multiple reaction monitoring;

[0072] The reaction monitoring ion pair is m / z 269.0→225.0 and m / z 269.0→241.0.

[0073] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of aloe-emodin, the chromatographic conditions comprise:

[0074] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the chromatographic column is ≥1000; the column temperature is 40℃;

[0075] The mobile phase comprises phase A and phase B, the phase A is a 0.1% formic acid aqueous solution, the phase B is a 0.1% formic acid acetonitrile solution, and the flow rate is 0.4 ml / min;

[0076] The elution mode is gradient elution, and the elution program is as follows:

[0077] The mass spectrometry conditions comprise:

[0078] A triple quadrupole mass spectrometry detector;

[0079] An electrospray ionization negative mode;

[0080] The detection mode is multiple reaction monitoring;

[0081] The reaction monitoring ion pair is m / z 269.0→240.0 and m / z 269.0→183.0.

[0082] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of amygdalin, the chromatographic conditions comprise:

[0083] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the chromatographic column is ≥1000; the column temperature is 40℃;

[0084] The mobile phase comprises phase A and phase B, the phase A is a 0.1% formic acid aqueous solution, the phase B is a 0.1% formic acid acetonitrile solution, and the flow rate is 0.4 ml / min;

[0085] The elution mode is gradient elution, and the elution program is as follows:

[0086] The mass spectrometry conditions comprise:

[0087] A triple quadrupole mass spectrometry detector;

[0088] An electrospray ionization negative mode;

[0089] The detection mode is multiple reaction monitoring;

[0090] Reaction monitoring ion pairs: m / z 456.0→ 323.0 and m / z 456.0→ 119.0.

[0091] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of honokiol, the chromatographic conditions comprise:

[0092] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the chromatographic column is ≥1000; and the column temperature is 40°C.

[0093] The mobile phase comprises phase A and phase B, the phase A is 0.1% formic acid aqueous solution by volume fraction, the phase B is 0.1% formic acid acetonitrile solution by volume fraction, and the flow rate is 0.4 ml / min.

[0094] The elution mode is gradient elution, and the elution program is as follows:

[0095] The mass spectrometry conditions comprise:

[0096] A triple quadrupole mass spectrometry detector.

[0097] Electrospray ionization in negative mode.

[0098] The detection mode is multiple reaction monitoring.

[0099] Reaction monitoring ion pairs: m / z 265.0→ 247.1 and m / z 265.0→ 245.0.

[0100] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of honokiol, the chromatographic conditions comprise:

[0101] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the chromatographic column is ≥1000; and the column temperature is 40°C.

[0102] The mobile phase comprises phase A and phase B, the phase A is 0.1% formic acid aqueous solution by volume fraction, the phase B is 0.1% formic acid acetonitrile solution by volume fraction, and the flow rate is 0.4 ml / min.

[0103] The elution mode is gradient elution, and the elution program is as follows:

[0104] The mass spectrometry conditions comprise:

[0105] A triple quadrupole mass spectrometry detector.

[0106] Electrospray ionization in negative mode.

[0107] Detection mode: multiple reaction monitoring

[0108] Reaction monitoring ion pairs: m / z 265.0→223.0 and m / z 265.0→249.0.

[0109] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of paeoniflorin, the chromatographic conditions comprise:

[0110] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the chromatographic column is ≥1000; the column temperature is 40℃.

[0111] The mobile phase comprises phase A and phase B, the phase A is a 0.1% formic acid aqueous solution, the phase B is a 0.1% formic acid acetonitrile solution, and the flow rate is 0.4 ml / min.

[0112] The elution mode is gradient elution, and the elution program is as follows:

[0113] The mass spectrometry conditions comprise:

[0114] A triple quadrupole mass spectrometry detector.

[0115] Electrospray ionization negative mode.

[0116] Detection mode: multiple reaction monitoring

[0117] Reaction monitoring ion pairs: m / z 525.0→121.0 and m / z 525.0→327.0.

[0118] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of synephrine, the chromatographic conditions comprise:

[0119] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the theoretical plate number of the chromatographic column is ≥1000; the column temperature is 40℃.

[0120] The mobile phase comprises phase A and phase B, the phase A is a 0.1% formic acid aqueous solution, the phase B is a 0.1% formic acid acetonitrile solution, and the flow rate is 0.4 ml / min.

[0121] The elution mode is gradient elution, and the elution program is as follows:

[0122] The mass spectrometry conditions comprise:

[0123] A triple quadrupole mass spectrometry detector.

[0124] Electrospray ionization negative mode

[0125] Detection mode: multiple reaction monitoring

[0126] Reaction monitoring ion pairs: m / z 168.0→ 150.0 and m / z 168.0→ 91.0.

[0127] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection of hesperidin, the chromatographic conditions comprise:

[0128] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent), and the column theoretical plate number is ≥1000; the column temperature is 40℃;

[0129] The mobile phase comprises phase A and phase B, the phase A is 0.1% formic acid aqueous solution by volume fraction, the phase B is 0.1% formic acid acetonitrile solution by volume fraction, and the flow rate is 0.4 ml / min;

[0130] The elution mode is gradient elution, and the elution program is as follows:

[0131] The mass spectrometry conditions comprise:

[0132] Triple quadrupole mass spectrometry detector

[0133] Electrospray ionization negative mode

[0134] Detection mode: multiple reaction monitoring

[0135] Reaction monitoring ion pairs: m / z 609.2→ 301.0 and m / z 609.2→ 286.0.

[0136] According to the specific embodiment of the present application, preferably, in the liquid chromatography-tandem mass spectrometry detection, the test sample solution and the standard solution are respectively prepared by using methanol as an extractant and / or a solvent.

[0137] Preferably, the content of the target substance is calculated by using an external standard method.

[0138] According to the specific embodiment of the present application, preferably, the specific steps of the liquid chromatography-tandem mass spectrometry of the 11 index components are as follows:

[0139] (1) Determination of the content of cannabamide B:

[0140] Chromatographic conditions and system suitability test:

[0141] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0142] The gradient elution was performed with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B according to the following procedure:

[0143] The column temperature was maintained at 40 °C.

[0144] The triple quadrupole mass spectrometry detector and electro-spray ionization negative mode (ESI - ) were used, and the multiple reaction monitoring (MRM) was used with ion pairs m / z 595.0→269.0 and m / z 595.0→432.0.

[0145] The theoretical plate number of the chromatographic column was not less than 1000, calculated with the peak of cannabimovone B.

[0146] Preparation of the reference solution:

[0147] An appropriate amount of cannabimovone B standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 1.0 mg of cannabimovone B per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.11 μg, 0.17 μg, 0.33 μg, 0.50 μg, and 1.0 μg of cannabimovone B per 1 ml, respectively.

[0148] Preparation of the test solution:

[0149] An appropriate amount of CDD-2101 granules was finely ground, about 0.1 g was accurately weighed and transferred to a conical flask with a stopper. 10 ml of 70% methanol was accurately added, and ultrasonic extraction (power 500 W, frequency 53 kHz) was performed for 30 minutes. The cooled and filtered. The above extraction was repeated once. The filtrate was mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate was used as the test solution.

[0150] Determination method:

[0151] 1 μl of the reference solution and the test solution was accurately pipetted, respectively, and injected into the liquid chromatograph for determination, and the content of cannabimovone B was obtained.

[0152] (2) Determination of the content of chrysophanol:

[0153] Chromatographic conditions and system suitability test:

[0154] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile solution was used as mobile phase B, and gradient elution was carried out according to the following program:

[0155] The column temperature was kept at 40℃.

[0156] A triple quadrupole mass spectrometer detector and an electrospray ionization negative mode (ESI - ) were used, and multiple reaction monitoring (MRM) ion pairs m / z 253.0→225.0 and m / z 253.0→195.0 were used.

[0157] The theoretical plate number of the chromatographic column was not less than 1000, calculated by the peak of chrysophanol.

[0158] Preparation of the reference solution:

[0159] An appropriate amount of chrysophanol standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 0.060 mg of chrysophanol per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.033, 0.17, 0.33, 0.50, and 1.0 μg of chrysophanol per 1 ml, respectively.

[0160] Preparation of the test solution:

[0161] An appropriate amount of CDD-2101 granules was finely ground, about 0.1 g was accurately weighed and transferred to a conical flask with a stopper. 10 ml of 70% methanol was accurately added, and ultrasonic extraction (power 500 W, frequency 53 kHz) was performed for 30 minutes. The cooled and filtered. The above extraction was repeated once. The filtrate was mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate was used as the test solution.

[0162] Determination method:

[0163] 1 μl of the reference solution and the test solution was accurately pipetted into the liquid chromatograph, and the determination was carried out, i.e. the determination was completed.

[0164] (3) Determination of the content of rhein:

[0165] Chromatographic conditions and system suitability test:

[0166] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0167] 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile solution was used as mobile phase B, and gradient elution was carried out according to the following program:

[0168] The column temperature was maintained at 40℃.

[0169] A triple quadrupole mass spectrometer detector and electrospray ionization negative mode (ESI - ), using multiple reaction monitoring (MRM) ion pairs m / z 283.0→239.0 and m / z 283.0→183.1.

[0170] The column theoretical plate number was not less than 1000, calculated by rheumic acid peak.

[0171] The preparation of the reference solution: accurately weighed a certain amount of rheumic acid standard, added methanol to prepare a standard stock solution containing 0.060 mg of rheumic acid per 1 ml. Accurately measured a certain amount of standard stock solution, added methanol to prepare a series of standard solutions containing 0.033 μg, 0.17 μg, 0.33 μg, 0.50 μg, 1.0 μg of rheumic acid per 1 ml, respectively;

[0172] Preparation of test solution:

[0173] Take CDD-2101 granules, grind them finely, take about 0.1 g, and accurately weigh. Accurately add 25 ml of methanol-hydrochloric acid (10:1) mixed solution and weigh. Reflux at 80℃ water bath for 60 minutes, take out and cool and weigh. Make up the weight loss with methanol, shake well and filter. Accurately measure 3.0 ml of continuous filtrate and transfer it to a 5 ml volumetric flask. Add 1.0 ml of 4% NaOH solution, dilute to the flask mark with methanol, mix well, filter, and the continuous filtrate is used as the test solution.

[0174] Determination method:

[0175] Accurately pipette 1 μl of the reference solution and 1 μl of the test solution into the liquid chromatograph, respectively, and determine, i.e. get.

[0176] (4) Determination of emodin content:

[0177] Chromatographic conditions and system suitability test:

[0178] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0179] 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, flow rate of 0.4 ml / min, gradient elution according to the following program:

[0180] The column temperature was maintained at 40℃.

[0181] A triple quadrupole mass spectrometer detector and electrospray ionization negative mode (ESI - ), using multiple reaction monitoring (MRM) ion pairs m / z 269.0→225.0 and m / z 269.0→241.0.

[0182] The column theoretical plate number is not less than 1000, calculated by the emodin peak.

[0183] Preparation of the control solution:

[0184] Accurately weigh a certain amount of emodin standard, add methanol to prepare a standard stock solution containing 0.060 mg of emodin per 1 ml. Accurately weigh a certain amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 0.033, 0.17, 0.33, 0.50, and 1.0 μg of emodin per 1 ml, respectively.

[0185] Preparation of the test solution:

[0186] Take a certain amount of CDD-2101 granules, grind them, take about 0.1 g, accurately weigh and transfer them to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, remove and cool, and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution.

[0187] Determination method: accurately pipette 1 μl of the control solution and the test solution, respectively, inject into the liquid chromatograph, and determine, i.e. get.

[0188] (5) Determination of aloe emodin content:

[0189] Chromatographic conditions and system suitability test:

[0190] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0191] 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, flow rate of 0.4 ml / min, gradient elution according to the following program:

[0192] Keep the column temperature at 40℃.

[0193] A triple quadrupole mass spectrometer detector and electrospray ionization negative mode (ESI - ), using multiple reaction monitoring (MRM) ion pairs m / z 269.0→240.0 and m / z 269.0→183.0.

[0194] The theoretical plate number of the chromatographic column is not less than 1000, calculated based on the peak of aloe-emodin.

[0195] Preparation of the control solution:

[0196] An appropriate amount of aloe-emodin standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 0.11 mg of aloe-emodin per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.035 μg, 0.18 μg, 0.35 μg, 0.50 μg, 1.1 μg, 1.6 μg, and 3.2 μg of aloe-emodin per 1 ml, respectively.

[0197] Preparation of the test solution: An appropriate amount of CDD-2101 granules was finely ground, and about 0.1 g was accurately weighed. 25 ml of a mixed solution of methanol-hydrochloric acid (10:1 by volume) was added and weighed. It was refluxed in a water bath at 80°C for 60 minutes, then cooled and weighed. The lost weight was made up with methanol, and after shaking well, it was filtered. 3.0 ml of the continuous filtrate was accurately measured and transferred to a 5 ml volumetric flask. 1.0 ml of 4% NaOH solution was added, diluted with methanol to the flask mark, mixed well, filtered, and the continuous filtrate was used as the test solution.

[0198] Determination method:

[0199] 1 μl of the control solution and the test solution was accurately pipetted, respectively, and injected into the liquid chromatograph for determination.

[0200] (6) Determination of amygdalin content:

[0201] Chromatographic conditions and system suitability test:

[0202] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0203] 0.1% formic acid in water was used as mobile phase A, and 0.1% formic acid in acetonitrile was used as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution was performed according to the following program:

[0204] The column temperature was maintained at 40°C.

[0205] A triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ) were used, and multiple reaction monitoring (MRM) ion pairs m / z 456.0→323.0 and m / z 456.0→119.0 were used.

[0206] The theoretical plate number of the chromatographic column is not less than 1000, calculated based on the peak of amygdalin.

[0207] Preparation of the control solution:

[0208] Accurately weigh a certain amount of amygdalin standard substance, add methanol to prepare a standard stock solution containing 1.0 mg of amygdalin per 1 ml. Accurately weigh a certain amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 0.89, 2.2, 3.3, 6.6, 10, and 20 μg of amygdalin per 1 ml, respectively.

[0209] Preparation of the test solution:

[0210] Take a certain amount of CDD-2101 granules, finely grind, accurately weigh about 0.1 g, and transfer it to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool, and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution.

[0211] Determination method:

[0212] Accurately pipette 1 μl of the control solution and the test solution, respectively, into the liquid chromatograph, and determine, i.e. obtain.

[0213] (7) Determination of Magnolol Content:

[0214] Chromatographic conditions and system suitability test:

[0215] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0216] 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, flow rate of 0.4 ml / min, gradient elution according to the following program:

[0217] Keep the column temperature at 40℃.

[0218] Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ), use multiple reaction monitoring (MRM) ion pairs m / z 265.0→247.1 and m / z 265.0→245.0.

[0219] The theoretical plate number of the chromatographic column is not less than 1000, calculated based on the magnolol peak.

[0220] Preparation of the control solution:

[0221] Accurately weigh a certain amount of Magnolol standard substance, add methanol to prepare a standard stock solution containing 0.30 mg of Magnolol per 1 ml. Accurately weigh a certain amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 6.0, 15.0, 30.0, 60.0, 90.0 μg of Magnolol per 1 ml, respectively;

[0222] Preparation of test solution:

[0223] Take a certain amount of CDD-2101 granules, finely grind, take about 0.1 g, accurately weigh and transfer into a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution.

[0224] Determination method:

[0225] Accurately pipette 1 μl of the control solution and the test solution, respectively, into the liquid chromatograph, and determine, to obtain.

[0226] (8) Determination of Magnolol content:

[0227] Chromatographic conditions and system suitability test:

[0228] The chromatographic column is ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0229] 0.1% formic acid aqueous solution is used as mobile phase A, and 0.1% formic acid acetonitrile solution is used as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution is carried out according to the following program:

[0230] Keep the column temperature at 40℃.

[0231] Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ), use multiple reaction monitoring (MRM) ion pairs m / z 265.0→223.0 and m / z 265.0→249.0.

[0232] The theoretical plate number of the chromatographic column is not less than 1000, calculated with Magnolol peak.

[0233] Preparation of control solution:

[0234] Accurately weigh a suitable quantity of honokiol standard substance into a 50-ml volumetric flask, dissolve in methanol to make a standard stock solution containing 0.30 mg of honokiol per 1 ml. Accurately weigh a suitable quantity of the standard stock solution into a 50-ml volumetric flask, dissolve in methanol to make a series of standard solutions containing 6.0, 15.0, 30.0, 60.0, 90.0 μg of honokiol per 1 ml, respectively.

[0235] Preparation of test solution:

[0236] Take CDD-2101 granules, grind them, take about 0.1 g, accurately weigh and transfer them into a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool, and filter. Repeat the above extraction once. Mix the filtrate in a 25-ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and use the continuous filtrate as the test solution.

[0237] Determination method:

[0238] Accurately pipette 1 μl of the control solution and the test solution, respectively, into the liquid chromatograph, and determine, to obtain.

[0239] (9) Determination of paeoniflorin content:

[0240] Chromatographic conditions and system suitability test:

[0241] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0242] 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution according to the following program:

[0243] Keep the column temperature at 40℃.

[0244] Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ), use multiple reaction monitoring (MRM) ion pairs m / z 525.0→121.0 and m / z 525.0→327.0.

[0245] The theoretical plate number of the chromatographic column is not less than 1000, calculated with the paeoniflorin peak.

[0246] Preparation of control solution:

[0247] Accurately weigh the appropriate amount of paeoniflorin standard substance, add methanol to prepare a standard stock solution containing 0.060 mg of paeoniflorin per 1 ml. Accurately weigh the appropriate amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 0.033, 0.17, 0.33, 0.50, 1.0 μg of paeoniflorin per 1 ml, respectively.

[0248] Preparation of test solution:

[0249] Take an appropriate amount of CDD-2101 granules, finely grind, take about 0.1 g, accurately weigh and transfer it to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution.

[0250] Determination method:

[0251] Accurately pipette 1 μl of the control solution and the test solution, respectively, into the liquid chromatograph, and determine, i.e. obtain.

[0252] (10) Determination of synephrine content:

[0253] Chromatographic conditions and system suitability test:

[0254] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0255] 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, with a flow rate of 0.4 ml / min, gradient elution according to the following program:

[0256] Keep the column temperature at 40℃.

[0257] Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ), use multiple reaction monitoring (MRM) ion pairs m / z 168.0→150.0 and m / z 168.0→91.0.

[0258] The minimum resolution of the peaks of synephrine and its coexisting components is not less than 1.5, calculated based on the synephrine peak.

[0259] Preparation of control solution:

[0260] Accurately weigh a suitable quantity of synephrine standard into a 10-ml volumetric flask, dilute to the mark with methanol, and mix to obtain a stock solution containing 1.0 mg of synephrine per 1 ml. Accurately pipette a suitable quantity of the stock solution into 10-ml volumetric flasks, dilute to the mark with methanol, and mix to obtain a series of standard solutions containing 0.017, 0.033, 0.067, 0.17, and 0.25 μg of synephrine per 1 ml, respectively.

[0261] Preparation of test solution:

[0262] Accurately weigh about 0.1 g of CDD-2101 granules into a conical flask with a stopper, add 10 ml of 70% methanol, and ultrasonically extract (power 500 W, frequency 53 kHz) for 30 min. Repeat the extraction once. Mix the filtrate in a 25-ml volumetric flask, dilute to the mark with methanol, mix well, filter, and use the continuous filtrate as the test solution.

[0263] Determination method:

[0264] Accurately pipette 1 μl of the control solution and 1 μl of the test solution into the liquid chromatograph, and determine, to obtain.

[0265] (11) Determination of hesperidin content:

[0266] Chromatographic conditions and system suitability test:

[0267] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent).

[0268] Use 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile solution as mobile phase B, with a flow rate of 0.4 ml / min, and perform gradient elution according to the following program:

[0269] Maintain the column temperature at 40°C.

[0270] Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ), and use multiple reaction monitoring (MRM) ion pairs m / z 609.2→301.0 and m / z 609.2→286.0.

[0271] The theoretical plate number of the chromatographic column is not less than 1000, calculated from the hesperidin peak.

[0272] Preparation of control solution:

[0273] Accurately weigh a certain amount of hesperidin standard substance, add methanol to prepare a standard stock solution containing 1.0 mg of hesperidin per 1 ml. Accurately weigh a certain amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 0.39 μg, 0.98 μg, 1.5 μg, 2.9 μg, 4.4 μg, and 8.8 μg of hesperidin per 1 ml, respectively.

[0274] Preparation of test sample solution:

[0275] A certain amount of CDD-2101 granules was finely ground, about 0.1 g was accurately weighed and transferred into a conical flask with a stopper. 10 ml of 70% methanol was accurately added, and ultrasonic extraction (power 500 W, frequency 53 kHz) was performed for 30 minutes. The flask was removed, cooled, and filtered. The above extraction was repeated once. The filtrate was mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate was used as the test solution.

[0276] Determination method:

[0277] 1 μl of the control sample solution and the test sample solution was accurately pipetted into the liquid chromatograph, and determination was performed, thereby obtaining the result.

[0278] According to the specific embodiment of the present application, preferably, the quality control method further comprises: obtaining a fingerprint of the CDD-2101 granules by sampling liquid chromatography-tandem mass spectrometry for consistency evaluation, and the detected components include one or more of synephrine, amygdalin, paeonol glycoside, paeoniflorin, naringenin, hesperidin, cannabamide B, aloe-emodin, rhein, emodin, honokiol, magnolol, and chrysophanol.

[0279] According to the specific embodiment of the present application, preferably, in the fingerprint detection, the chromatographic conditions include:

[0280] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); the column temperature is 40°C;

[0281] The mobile phase includes phase A and phase B, the phase A is 0.1% formic acid aqueous solution by volume, the phase B is 0.1% formic acid acetonitrile solution by volume, and the flow rate is 0.4 ml / min;

[0282] The elution mode is gradient elution, and the elution program is as follows:

[0283] The mass spectrometry conditions include:

[0284] Triple quadrupole mass spectrometry detector;

[0285] Electrospray ionization negative mode;

[0286] Detection mode: multiple reaction monitoring

[0287] Reaction monitoring ion pairs:

[0288] According to the specific embodiments of the present application, preferably, in the fingerprint detection, the test sample solution and the standard solution are respectively prepared with methanol as the extractant and / or solvent.

[0289] According to the specific embodiments of the present application, preferably, the specific steps of the fingerprint detection are as follows:

[0290] Chromatographic conditions and system suitability test:

[0291] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0292] 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, the flow rate is 0.4 ml / min, and gradient elution is carried out according to the following program:

[0293] The column temperature is maintained at 40°C.

[0294] A triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ) and multiple reaction monitoring (MRM) ion pairs are used, and the ion pairs are as follows:

[0295] Preparation of the reference solution:

[0296] An appropriate amount of standard (syringidol, amygdalin, paeonol glycoside, paeonol, naringenin, hesperidin, cannabamide B, aloe emodin, rhein, emodin, honokiol, magnolol and chrysophanol) is accurately weighed, methanol is added, and a mixed standard solution is prepared, containing 5 μg of each standard per 1 ml;

[0297] Preparation of the test sample solution:

[0298] An appropriate amount of CDD-2101 granules is finely ground, about 0.1 g is accurately weighed and transferred to a conical flask with a stopper. 10 ml of 70% methanol is accurately added, ultrasonic extraction (power 500 W, frequency 53 kHz) is carried out for 30 minutes, and then the flask is removed, cooled and filtered. The above extraction is repeated once. The filtrate is mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate is used as the test solution.

[0299] Determination method:

[0300] Accurately pipet 1ul of the control solution and the sample solution respectively into the liquid chromatograph, and then determine, so as to obtain.

[0301] The technical solution provided by the present application has the following beneficial effects:

[0302] The quality control method of the CDD-2101 granules has high sensitivity and good reproducibility, and provides scientific basis for accurately evaluating and effectively controlling the quality of the CDD-2101 granules from multiple aspects and multiple angles, supplements the defects of the existing standards, and ensures the safety and effectiveness of medication.

[0303] Regarding the optimization of the thin layer chromatography identification method of the torrefied Semen Citri Reticulatae in the CDD-2101 granules, different developing systems are designed and optimized, and through comparison, the separation effect of the cyclohexane-trichloromethane-ethyl acetate-formic acid (3:2:8:0.2) developing system is better than that of the developing system adopted in the pharmacopoeia.

[0304] Regarding the content determination, the pharmacopoeia only studies emodin and chrysophanol in the Rheum palmatum in the CDD-2101 granules (Maren Pill), which is relatively one-sided. The effective components of the other five medicinal materials are also controlled in the present application, especially the content control of cannabidiolamide B in the Fructus Cannabis. The content control of cannabidiolamide B has not been reported in the pharmacopoeia and the literature, so the method of the present application is more accurate and comprehensive, and provides an effective means for the quality control of the product.

[0305] In addition, the fingerprint spectrum of the CDD-2101 granules (Maren Pill) has not been reported in the literature. Through the establishment of the fingerprint spectrum, the complex chemical fingerprint qualitative and quantitative information in the CDD-2101 granules can be obtained to the maximum extent only by one sample preparation and analysis, and through detection and analysis, a plurality of effective components such as synephrine, amygdalin, paeonol glycoside, paeoniflorin, naringenin, hesperidin, cannabidiolamide B, aloe-emodin, rhein, emodin, honokiol, magnolol and chrysophanol are identified in the medicine of the present application. The sensitivity and accuracy of the detection method are greatly enhanced, so as to ensure the comprehensiveness of the quality evaluation of the medicine of the present application, and good precision and stability, and repeatability are obtained, which provides a new method for improving the quality control. BRIEF DESCRIPTION OF DRAWINGS

[0306] Fig. 1 is a thin layer identification chromatogram of Fructus Cannabis in Example 1, wherein 1 is the Fructus Cannabis control drug material-2ul (sample amount), and 2 is the sample-12ul (sample amount);

[0307] Fig. 2 is a thin layer identification chromatogram of Rheum palmatum in Example 2, wherein 1 is the Rheum palmatum control drug material-15ul, and 2 is the sample-5ul;

[0308] Figure 3 is a TLC chromatogram of Magnoliae officinalis Cortex in Example 3, wherein 1 is a reference substance-15 μl, 2 is a test sample-15 μl, and 3 is a control medicinal material-15 μl;

[0309] Figure 4 is a TLC chromatogram of Radix Paeoniae Alba in Example 4, wherein 1 is a Radix Paeoniae Alba control medicinal material-8 μl, and 2 is a test sample-8 μl;

[0310] Figure 5 is a TLC chromatogram of Fructus Aurantii Immaturus Stir-baked with Wheat Bran in Example 5, wherein 1 is a negative sample of Fructus Aurantii Immaturus Stir-baked with Wheat Bran-15 μl, 2 and 3 are Fructus Aurantii Immaturus Stir-baked with Wheat Bran control medicinal materials (15 μl and 20 μl, respectively), and 4 and 5 are Fructus Aurantii Immaturus Stir-baked with Wheat Bran test samples (15 μl and 20 μl, respectively);

[0311] Figure 6 is a TLC chromatogram for investigating sample application amount of Fructus Aurantii Immaturus Stir-baked with Wheat Bran in Example 6, wherein 1-5 are Fructus Aurantii Immaturus Stir-baked with Wheat Bran control medicinal materials (8 μl, 10 μl, 12 μl, 15 μl, and 20 μl, respectively), and 6-10 are test samples (8 μl, 10 μl, 12 μl, 15 μl, and 20 μl, respectively);

[0312] Figure 7 is a TLC chromatogram for investigating saturation time of Fructus Aurantii Immaturus Stir-baked with Wheat Bran in Example 6, wherein the saturation times of the three chromatograms from left to right are 0 (unsaturated), 15 min, and 30 min, respectively, 1 is a Fructus Aurantii Immaturus Stir-baked with Wheat Bran control medicinal material-8 μl, and 2 is a test sample-8 μl;

[0313] Figure 8 is a TLC chromatogram for investigating specificity of Fructus Aurantii Immaturus Stir-baked with Wheat Bran in Example 6, wherein 1 is a negative sample of Fructus Aurantii Immaturus Stir-baked with Wheat Bran-8 μl, 2 is a Fructus Aurantii Immaturus Stir-baked with Wheat Bran control medicinal material-8 μl, and 3 is a test sample-8 μl;

[0314] Figure 9 is a TLC chromatogram for investigating relative humidity of Fructus Aurantii Immaturus Stir-baked with Wheat Bran in Example 6, wherein the humidities (RH) of the three chromatograms from left to right are 32%, 68%, and 88%, respectively, 1 is a Fructus Aurantii Immaturus Stir-baked with Wheat Bran control medicinal material-8 μl, and 2 is a test sample-8 μl;

[0315] Figure 10 is a TLC chromatogram for investigating different temperatures of Fructus Aurantii Immaturus Stir-baked with Wheat Bran in Example 6, wherein the temperatures of the three chromatograms from left to right are 3°C, 23.3°C, and 40°C, respectively, 1 is a Fructus Aurantii Immaturus Stir-baked with Wheat Bran control medicinal material-8 μl, and 2 is a test sample-8 μl;

[0316] Figure 11 is a TLC chromatogram for investigating different brands of thin-layer plates of Fructus Aurantii Immaturus Stir-baked with Wheat Bran in Example 6, wherein the brands of the three chromatograms from left to right are Merck, Yantai, and Qingdao, respectively, 1 is a Fructus Aurantii Immaturus Stir-baked with Wheat Bran control medicinal material-8 μl, and 2 is a test sample-8 μl;

[0317] Figure 12 is a standard curve of Cannabinoidamide B in Example 8;

[0318] Figure 13 is a CDD-2101 particle fingerprint map in Example 9, wherein peaks 1-13 are octopine, amygdalin, paeonol glycoside, paeoniflorin, naringenin, hesperidin, cannabimide B, aloe-emodin, rhein, emodin, honokiol, magnolol and chrysophanol, respectively;

[0319] Figure 14 is a differential thin layer chromatogram of torrefied bitter orange fruit in Comparative Example 1 (developing agent 1), wherein 1 is a negative sample of torrefied bitter orange fruit without bran-15 μl, 2 is a torrefied bitter orange fruit control medicinal material-15 μl, 3 is a torrefied bitter orange fruit control medicinal material-20 μl, 4 is a test sample-15 μl, and 5 is a test sample-20 μl;

[0320] Figure 15 is a differential thin layer chromatogram of torrefied bitter orange fruit in Comparative Example 2 (developing agent 2), wherein 1 is a negative sample of torrefied bitter orange fruit without bran-10 μl, 2 is a torrefied bitter orange fruit control medicinal material-8 μl, 3 is a test sample-8 μl, and 4 is a test sample-10 μl;

[0321] Figure 16 is a differential thin layer chromatogram of torrefied bitter orange fruit in Comparative Example 3 (developing agent 3), wherein 1 is a negative sample of torrefied bitter orange fruit without bran-10 μl, 2 is a torrefied bitter orange fruit control medicinal material-8 μl, 3 is a test sample-8 μl, and 4 is a test sample-10 μl;

[0322] Figure 17 is a differential thin layer chromatogram of torrefied bitter orange fruit in Example 10 (developing agent 4), wherein 1 is a negative sample of torrefied bitter orange fruit without bran-5 μl, 2 is a torrefied bitter orange fruit control medicinal material-8 μl, and 3 is a test sample-8 μl. DETAILED DESCRIPTION

[0323] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the implementation of the present application.

[0324] Instruments and reagents

[0325] 1. Instruments

[0326] Linomat-5 (CAMAG, Switzerland); CAMAG TLC Visualizer (CAMAG, Switzerland); ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.); pre-coated silica gel G plates for TLC (Qingdao Haian Chemical Factory Branch, Yantai Chemical Industry Research Institute, Merck, Germany); ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometer (Agilent 1290 Infinity II-Agilent 6470 LC / TQ, Agilent Technologies, USA); electronic analytical balance (ml204 / 02, Mettler-Toledo); electronic analytical balance (XPR205, Mettler-Toledo); electronic analytical balance (E600-2, Changshu Shuangjie Testing Instrument Factory).

[0327] 2. Materials and reagents

[0328] Hemp seeds reference drug material (batch number: 121097-201104, China Institute for Food and Drug Control); Rhubarb (medicinal rhubarb) reference drug material (batch number: 120984-201202, China Institute for Food and Drug Control); Magnolia officinalis reference drug material (batch number: 110729-201513, China Institute for Food and Drug Control); White peony root reference drug material (batch number: 120905-201109, China Institute for Food and Drug Control); Fried Semen Aurantii Immaturi reference drug material (batch number: 120936-201005, China Institute for Food and Drug Control); Cynarin (batch number: 1102720, Dr. Ehrenstorfer); Amygdalin (batch number: YY90093, Shanghai Yuye Co., Ltd.); Paeonoside (batch number: O31GB166251, Shanghai Yuye Co., Ltd.); Paeoniflorin (batch number: 110736-201136, China Institute for Food and Drug Control); Naringenin (batch number: YY90146, Shanghai Yuye Co., Ltd.); Hesperidin (batch number: CFS202102, ChemFaces); Cannabinoid B Cannabinamide B (batch number: CFN95268, ChemFaces); Aloe-emodin (batch number: 20120412, Shanghai Yuye Co., Ltd.); Rhein (batch number: 110757-201607, China Institute for Food and Drug Control); Emodin (batch number: 110756-201913, China Institute for Food and Drug Control); Magnolol reference substance (batch number: 110729-202015, China Institute for Food and Drug Control); Honokiol reference substance (batch number: YY90181, Shanghai Yuye Co., Ltd.); Chrysophanol (batch number: 110796-201319, China Institute for Food and Drug Control); CDD-2101 granules (batch number: A210088810K, Peili (Nanning) Pharmaceutical Co., Ltd.) for the preparation of test sample solutions in Examples 1-6; CDD-2101 granules (batch number: A210173410, Peili (Nanning) Pharmaceutical Co., Ltd.) for the preparation of test sample solutions in Examples 7-9; Hemp seeds drug material negative sample, Rhubarb drug material negative sample, Magnolia officinalis drug material negative sample, White peony root drug material negative sample, Fried Semen Aurantii Immaturi drug material negative sample were all prepared by the Pharmaceutical Research Institute of Peili (Nanning) Pharmaceutical Co., Ltd.

[0329] Acetonitrile (OMINI) was chromatographically pure, phosphoric acid (Chongqing Chuandong Chemical Co., Ltd.) and methanol (Guangdong Guanghua Science and Technology Co., Ltd.) were both analytically pure, and water was ultrapure water.

[0330] The present application will be described in detail below in conjunction with specific embodiments and drawings.

[0331] Example 1

[0332] In this embodiment, thin layer chromatography is used for qualitative identification of Cannabis sativa L. in CDD-2101 granules, as follows:

[0333] Take CDD-2101 granules, grind them, take 2g, add water 30ml, ultrasonic treatment for 30 minutes (power 500W, frequency 40kHz), centrifuge (4000r / min) for 10 minutes, take the supernatant, add water to saturation, shake with n-butanol for 2 times, each time 30ml, combine the n-butanol extract, evaporate to dryness, add methanol 2ml to dissolve, as the test solution. Take Cannabis sativa L. control drug 0.1g, add methanol 2ml, ultrasonic treatment for 10 minutes (power 500W, frequency 40kHz), take the supernatant as the Cannabis sativa L. control drug solution. According to the thin layer chromatography experiment (Chinese Pharmacopoeia 2020 edition four general rules 0502), take 2ul of the above Cannabis sativa L. control drug solution and 12ul of the test solution, respectively, and point them on the same silica gel G thin layer plate. Use cyclohexane-ethyl acetate-formic acid (volume ratio 12:3:0.1) as the developing agent, the humidity is 68% and the temperature is 23.3℃, the saturation time is 15min, use Yantai plate to develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat at 105℃ until the spots develop clearly. As shown in Figure 1, the same colored spots appear in the test chromatogram at the corresponding positions of the control drug chromatogram;

[0334] Example 2

[0335] In this embodiment, thin layer chromatography is used for qualitative identification of Rheum officinale Baill. in CDD-2101 granules, as follows:

[0336] Take CDD-2101 granules, grind them, take 2g, add methanol 20ml, ultrasonic treatment for 30 minutes (power 500W, frequency 40kHz), filter, evaporate the filtrate to dryness, add methanol 2ml to dissolve, as the test solution. Take Rheum officinale Baill. control drug 0.3g, add water 50ml, heat reflux for 60 minutes, centrifuge (4000r / min) for 10 minutes, take the supernatant, evaporate to dryness, add methanol 20ml to the residue, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add methanol 2ml to dissolve, as the control drug solution. According to the thin layer chromatography experiment (Chinese Pharmacopoeia 2020 edition four general rules 0502), take 15ul of the Rheum officinale Baill. control drug solution and the test solution, respectively, and point them on the same silica gel G thin layer plate. Use cyclohexane-ethyl acetate-formic acid (volume ratio 12:3:0.1) as the developing agent, the humidity is 68% and the temperature is 23.3℃, the saturation time is 30min, use Yantai plate to develop, take out, dry, and observe under ultraviolet light (365nm). As shown in Figure 2, the same colored fluorescent spots appear in the test chromatogram at the corresponding positions of the Rheum officinale Baill. control drug chromatogram;

[0337] Example 3

[0338] In this embodiment, qualitative identification of Magnoliae officinalis Cortex in CDD-2101 granules is carried out by thin layer chromatography, as follows:

[0339] Take CDD-2101 granules, grind them, take 2 g, add 20 ml of methanol, ultrasonic treat for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate, add 2 ml of methanol to the residue to dissolve it, and use it as the test solution. Take another 0.5 g of Magnoliae officinalis Cortex control drug, add 50 ml of water, heat and reflux for 60 minutes, centrifuge (4000 r / min) for 10 minutes, take the supernatant, evaporate it, add 20 ml of methanol to the residue, ultrasonic treat for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate, add 2 ml of methanol to the residue to dissolve it, and use it as the control drug solution. Take magnolol and honokiol reference substances, add methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml, and use it as the reference substance solution. According to the thin layer chromatography method (2020 edition of Chinese Pharmacopoeia, General Test 0502), take 15 μl of each of the above three solutions, spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid (volume ratio 12:3:0.1) as the developing agent, the humidity is 68%, the temperature is 23.3℃, the saturation time is 15 min, use Yantai plate to develop, take it out, dry it, spray with 5% vanillin sulfuric acid solution, heat it at 105℃ until the spots develop clearly. As shown in Figure 3, in the test sample chromatogram, at the positions corresponding to the magnolol and honokiol reference substances and the Magnoliae officinalis Cortex control drug chromatogram, the same colored spots appear;

[0340] Example 4

[0341] In this embodiment, qualitative identification of Radix Paeoniae Alba in CDD-2101 granules is carried out by thin layer chromatography, as follows:

[0342] Take CDD-2101 granules, grind finely, take 2 g, add methanol 20 ml, ultrasonic treatment for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate to dryness, add methanol 2 ml to dissolve as the test solution. Another 0.25 g of white peony root control drug was taken, added with water 50 ml, heated to reflux for 60 minutes, centrifuged (4000 r / min) for 10 minutes, took the supernatant, evaporated to dryness, the residue was added with methanol 20 ml, ultrasonic treatment for 30 minutes (power 500 W, frequency 40 kHz), filtered, the filtrate was evaporated to dryness, the residue was added with methanol 2 ml to dissolve as the control drug solution. According to the thin layer chromatography method (2020 edition of Chinese Pharmacopoeia four general 0502) experiment, 8 μl of the test solution and the above control drug solution were taken and spotted on the same silica gel G thin layer plate, with chloroform-ethyl acetate-methanol-25-28% concentrated ammonia solution (volume ratio 8:1:4:1) as the developing agent, the humidity was 68% and the temperature was 23.3℃, the saturation time was 15 min, Yantai plate was used for development, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, heated at 105℃ until the spots were clear. As shown in Figure 4, the test chromatogram showed the same color spots at the corresponding positions of the white peony root control drug chromatogram;

[0343] Example 5

[0344] In this example, the pustulated bitter orange fruit in CDD-2101 granules was qualitatively identified by thin layer chromatography, as follows:

[0345] Take CDD-2101 granules, grind finely, take 2g, add methanol 20ml, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add methanol 2ml to dissolve as the test solution. Another negative sample of fried immature bitter orange without bran is taken, ground finely, 2g is taken, 20ml of methanol is added, ultrasonic treatment for 30 minutes, filtered, the filtrate is evaporated to dryness, the residue is dissolved with 2ml of methanol as the negative sample solution. Take 0.3g of fried immature bitter orange control drug, add water 50ml, heat reflux for 60 minutes, centrifuge (4000r / min) for 10 minutes, take the supernatant, evaporate to dryness, add methanol 20ml, ultrasonic treatment for 30 minutes (power 500W, frequency 40kHz), filter, evaporate the filtrate to dryness, add methanol 2ml to dissolve as the control drug solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four general rules 0502) experiment, 8μl of fried immature bitter orange control drug solution and test solution are taken and spotted on the same silica gel G thin layer plate. With cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) as the developing agent, the humidity is 68% and the temperature is 24.8℃, the saturation time is 15min, Yantai plate is used for development, take out, dry, and observe under ultraviolet lamp 365nm. As shown in Figure 5, the test sample chromatogram shows the same color fluorescent main spot at the corresponding position of the fried immature bitter orange control drug.

[0346] Example 6

[0347] This example investigates the methodology of the thin layer identification method of fried immature bitter orange in Example 5, as follows:

[0348] (1) Spotting amount investigation

[0349] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four general rules 0502) experiment, 8-20μl of test sample solution and control drug solution are taken and spotted on the same silica gel G thin layer plate, with cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) as the developing agent, develop, take out, dry, and observe under ultraviolet lamp (365nm). The test sample chromatogram shows the same color fluorescent main spot at the corresponding position of the control drug chromatogram. The results are shown in Figure 6. As can be seen from the results, the fried immature bitter orange control drug and the test sample have clear spots after development when the spotting amount is 8μl, without tailing phenomenon, so the spotting amount of fried immature bitter orange control drug solution and test sample solution is determined to be 8μl.

[0350] (2) Saturation time investigation

[0351] According to the thin layer chromatography method (ChP 2020 Vol. 4 General Rule 0502), 8 μl of the test sample solution and 8 μl of the negative sample solution and 8 μl of the control medicinal material solution were spotted on the same silica gel G thin layer plate, cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) was used as the developing agent, the thin layer plate was saturated for 15 minutes, 30 minutes and not saturated respectively, developed, taken out, air dried, and observed under ultraviolet light (365 nm). The results are shown in Figure 7. It can be seen from the results that under different saturation times, saturation for more than 15 minutes can obtain better development effect, and the spots are clear and well separated, therefore saturation for 15 minutes is adopted.

[0352] (3) Specificity test

[0353] A certain amount of negative sample of Qian Gu Chao Zhi Shi was finely ground, 2 g of which was taken and added with 20 ml of methanol, ultrasonically treated for 30 minutes (power 500 W, frequency 40 kHz), filtered, and the filtrate was evaporated to dryness. The residue was dissolved with 2 ml of methanol to serve as the negative sample solution.

[0354] According to the thin layer chromatography method (ChP 2020 Vol. 4 General Rule 0502), 8 μl of the test sample solution and 8 μl of the negative sample solution and 8 μl of the control medicinal material solution were spotted on the same silica gel G thin layer plate, cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) was used as the developing agent, developed, taken out, air dried, and observed under ultraviolet light (365 nm). The results are shown in Figure 8. It can be seen from the results that in the test sample chromatogram, a fluorescent main spot of the same color as that of the control medicinal material of Fen Chao Zhi Shi appeared at the corresponding position, the spot was clear, and the negative sample did not interfere, indicating that the method has good specificity.

[0355] (4) Durability-Relative Humidity Investigation

[0356] According to the thin layer chromatography method (ChP 2020 Vol. 4 General Rule 0502), 8 μl of the test sample solution and 8 μl of the control medicinal material solution were spotted on the same silica gel G thin layer plate, cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) was used as the developing agent, developed at relative humidity (RH) of 32%, 68% and 88% respectively, taken out, air dried, and observed under ultraviolet light (365 nm). The results are shown in Figure 9. It can be seen from the results that under different relative humidity, in the test sample chromatogram, a fluorescent main spot of the same color as that of the control medicinal material of Fen Chao Zhi Shi appeared at the corresponding position, the spot was clear, indicating that the method has good durability under different relative humidity.

[0357] (5) Durability-Temperature Investigation

[0358] The test solution and the control drug solution were each taken 8 μl and spotted on the same silica gel G thin layer plate. Cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) was used as the developing agent, and the plate was developed at 3°C, room temperature (24.8°C) and high temperature (40°C) respectively. After taking out, air-drying, and placing under ultraviolet light (365 nm) for inspection, the results were as shown in FIG. 10. It can be seen from the results that, under different temperature development, the test sample chromatogram showed the same color fluorescent main spot at the position corresponding to the control drug chromatogram of Fried Fritillariae Thunbergii Bulbus, and the spot was clear, indicating that the method has good robustness under different temperature development.

[0359] (6) Robustness - Investigation of Different Brands of Thin Layer Plates

[0360] The test solution and the control drug solution were each taken 8 μl and spotted on the same silica gel G thin layer plate (brands were Qingdao Haoyang, Yantai Chemical, and Germany Merck, respectively). Cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) was used as the developing agent, and the plate was developed, taken out, air-dried, and placed under ultraviolet light (365 nm) for inspection. The results were as shown in FIG. 11. It can be seen from the results that, under development of different brands of thin layer plates, the test sample chromatogram showed the same color fluorescent main spot at the position corresponding to the control drug chromatogram of Fried Fritillariae Thunbergii Bulbus, and the separation was good on Merck and Yantai plates, but poor on Qingdao plate. Therefore, the silica gel G thin layer plates of Merck and Yantai were preferentially selected for development.

[0361] Example 7

[0362] In this example, liquid chromatography-tandem mass spectrometry was used to detect the content of Cannabicyclol in CDD-2101 granules, as follows:

[0363] Chromatographic conditions and system suitability test:

[0364] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0365] Acetonitrile was used as mobile phase A, and 0.1% (volume fraction) formic acid aqueous solution was used as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution was performed according to the following program:

[0366] The column temperature was maintained at 40°C.

[0367] A triple quadrupole mass spectrometer detector and electrospray ionization negative mode (ESI -), using multiple reaction monitoring (MRM) ion pairs m / z 595.0→269.0 and m / z 595.0→432.0.

[0368] The column shall have not less than 1000 theoretical plates, calculated as the peak for Cannabinoidamide B.

[0369] Preparation of the reference solution:

[0370] Accurately weigh a suitable amount of Cannabinoidamide B standard, add methanol to prepare a stock solution containing 1.0 mg of Cannabinoidamide B per 1 ml. Accurately weigh a suitable amount of the stock solution, add methanol to prepare a series of standard solutions containing 0.11 μg, 0.17 μg, 0.33 μg, 0.50 μg, 1.0 μg of Cannabinoidamide B per 1 ml, respectively.

[0371] Preparation of the test solution:

[0372] Take a suitable amount of CDD-2101 granules, grind them, take about 0.1 g, accurately weigh and transfer it to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution;

[0373] Determination method:

[0374] Accurately pipette 1 μl of the reference solution and 1 μl of the test solution, respectively, into the liquid chromatograph for determination, and the content of Cannabinoidamide B is obtained.

[0375] Example 8

[0376] This example investigates the method for detecting the content of Cannabinoidamide B in the CDD-2101 granules of Example 7, as follows:

[0377] (1) Investigation of the linear range

[0378] Accurately weigh a suitable amount of Cannabinoidamide B standard, add methanol to prepare a stock solution containing 1.0 mg of Cannabinoidamide B per 1 ml. Accurately weigh a suitable amount of the stock solution, add methanol to prepare a series of standard solutions S1-S5 containing 0.12 μg, 0.37 μg, 1.1 μg, 3.3 μg, 10.0 μg of Cannabinoidamide B per 1 ml, respectively.

[0379] Accurately pipette 2 μl of S1-S5 reference solutions, respectively, into the high performance liquid chromatograph for determination. With the peak area of Cannabinoidamide B measured by the reference solution as the ordinate and the corresponding concentration as the abscissa, a standard curve is drawn. The regression equation of the Cannabinoidamide B reference is: y=543136x-32033 (R2=0.9999). The linear range of Cannabinoidamide B is 0.12-10.0 μg / ml.2 = 1).

[0380] The results show that the injection amount of cannabinamide B is in the range of 0.12-10 μg / ml, and the injection amount and the peak area have a good linear relationship, indicating that the external standard method can be used for measurement and calculation. The above test results are shown in Table 1 below, and the standard curve is shown in Figure 12.

[0381] Table 1 Linear range test results

[0382] (2) Precision test

[0383] Precisely take 2 μl of the above line 1 reference solution, repeat the sampling for 6 times, and determine the peak area. The results are shown in Table 2 below.

[0384] Table 2 Precision test results

[0385] The results show that the RSD values of the peak area of cannabinamide B are all less than 3.0%, indicating that the instrument precision is good.

[0386] (3) Stability test

[0387] Take the same test sample solution, sample at 0 h, 2.5 h, 5 h, 9 h, 13 h, and 24 h, respectively, and determine according to the method. The results are shown in Table 3 below.

[0388] Table 3 Stability test results

[0389] The results show that the RSD values of the peak area of cannabinamide B are all less than 3.0%, indicating that the test sample solution is stable within 24 hours.

[0390] (4) Reproducibility test

[0391] Take the same batch of sample, grind it finely, take 6 portions, each 0.1 g, accurately weigh, prepare the test sample solution according to the "preparation of test sample solution", accurately take 2 μl of the test sample solution for sampling, determine the peak area, and calculate the content according to the standard curve of cannabinamide B in the "linear range test". The results are shown in Table 4 below.

[0392] Table 4 Reproducibility test results

[0393] The results show that the RSD values of the content of cannabinamide B are all less than 4.0%, indicating that the method has good reproducibility.

[0394] (5) Recovery test

[0395] Take 6 dry and clean conical flasks, respectively, accurately add 0.1 ml of the concentration of 0.33 mg / ml cannabinamide B reference substance solution, place it on the water bath to evaporate the solvent, and then respectively add a known amount of sample (cannabinamide B content is 0.20 mg / g), about 0.1 g each, accurately weigh and place in a stoppered conical flask, according to the method of "preparation of test solution", from "accurately add 10 ml of 70% methanol", prepare the test solution, sample determination peak area, according to the "linear range of the investigation" of cannabinamide B standard curve to calculate the recovery rate, the results are shown in the following table 5.

[0396] Table 5 cannabinamide B recovery rate test results

[0397] The results show that the average recovery rate of cannabinamide B is between 85-110%, and the RSD value is 4.5%, which meets the requirements of the pharmacopoeia, indicating that the recovery rate of this method is good.

[0398] Example 9

[0399] This example uses fingerprint to evaluate the quality of CDD-2101 granules, as follows:

[0400] Chromatographic conditions and system suitability test:

[0401] The chromatographic column is ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent);

[0402] 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, flow rate of 0.4 ml / min, gradient elution according to the following program:

[0403] Keep the column temperature at 40℃.

[0404] Use triple quadrupole mass spectrometry detector and electrospray ionization negative mode (ESI - ) and multiple reaction monitoring (MRM) ion pairs, ion pairs as follows:

[0405] Preparation of reference solution:

[0406] Accurately weigh a certain amount of standard (syringon, amygdalin, peonin, paeoniflorin, naringenin, hesperidin, cannabinamide B, aloe emodin, rhein, emodin, honokiol, magnolol and chrysophanol), add methanol to prepare a mixed standard solution, containing 5 μg of each standard per 1 ml.

[0407] Preparation of test solution:

[0408] Take CDD-2101 granules, grind finely, take about 0.1 g, accurately weigh and transfer to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute to the flask mark with methanol, mix well, filter, and the continuous filtrate is used as the test solution.

[0409] Determination method:

[0410] Accurately pipette 1 μl of the reference solution and the test solution respectively into the liquid chromatograph, and determine, i.e. the chromatogram obtained with the test solution corresponds to the chromatogram obtained with the reference solution in peak retention time. According to the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (China Pharmacopoeia Committee, version number 2012.130723), the similarity of the peak area in the fingerprint of different batches of test solution is calculated. The results are shown in Figure 13.

[0411] Example 10

[0412] This comparative example provides a qualitative identification of Fried Semen Aurantii in CDD-2101 granules by thin layer chromatography, as follows:

[0413] Take CDD-2101 granules, grind finely, take 2 g, add 20 ml of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add 2 ml of methanol to dissolve the residue as the test solution. Take another negative sample of Fried Semen Aurantii without bran, grind finely, take 2 g, add 20 ml of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add 2 ml of methanol to dissolve the residue as the negative sample solution. Take another 0.3 g of Fried Semen Aurantii control drug, add 50 ml of water, heat reflux for 60 minutes, centrifuge (4000 r / min) for 10 minutes, take the supernatant, evaporate to dryness, add 20 ml of methanol to the residue, ultrasonic treatment for 30 minutes (power 500 W, frequency 40 kHz), filter, evaporate the filtrate to dryness, add 2 ml of methanol to dissolve the residue as the control drug solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four general 0502), take an appropriate amount of test solution and control drug solution, respectively, and point them on the same silica gel G thin layer plate. Take cyclohexane-trichloromethane-ethyl acetate-formic acid (volume ratio 3:2:8:0.2) as the developing agent (recorded as developing agent 4), develop, take out, dry, and observe under ultraviolet light 365 nm.

[0414] Comparative Example 1

[0415] The comparative example provides qualitative identification of Fructus Aurantii Immaturus in CDD-2101 granules by thin layer chromatography. The specific method is the same as that of Example 5, except that the developing agent (denoted as developing agent 2) is toluene-ethyl acetate-formic acid (volume ratio 7:2.5:0.5).

[0416] Comparative Example 2

[0417] The comparative example provides qualitative identification of Fructus Aurantii Immaturus in CDD-2101 granules by thin layer chromatography. The specific method is the same as that of Example 5, except that the developing agent (denoted as developing agent 2) is toluene-ethyl acetate-formic acid (volume ratio 7:2.5:0.5).

[0418] Comparative Example 3

[0419] The comparative example provides qualitative identification of Fructus Aurantii Immaturus in CDD-2101 granules by thin layer chromatography. The specific method is the same as that of Example 5, except that the developing agent (denoted as developing agent 3) is cyclohexane-acetone (volume ratio 10:3).

[0420] The thin layer chromatography developing results of the above Comparative Examples 1-3 and Example 10 are shown in Figures 14-17, respectively.

[0421] It can be seen from the results that under the conditions of developing agent 1 (Comparative Example 1), the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions of the control medicinal material chromatogram, but the spot separation effect is poor; under the conditions of developing agent 2 (Comparative Example 2), the spots in the control medicinal material chromatogram are clustered in the lower part and cannot be well separated; under the conditions of developing agent 3 (Comparative Example 3), the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions of the control medicinal material chromatogram, but there are fewer corresponding spots, and some spots are negative and interfere; under the conditions of developing agent 4 (Example 10), the test sample chromatogram shows fluorescent main spots of the same color at the corresponding positions of the control medicinal material, and the spots are clear and there is no interference in the negative, and the separation effect is the best among the above four developing systems. Therefore, this developing agent is selected for development. Among the above four developing systems, cyclohexane-ethyl acetate-formic acid (5.5:4.5:0.1) is the developing system adopted by the Pharmacopoeia.

[0422] Example 11 Determination of Chrysophanol Content

[0423] In this example, liquid chromatography-tandem mass spectrometry is used to detect the content of chrysophanol in CDD-2101 granules, as follows:

[0424] Chromatographic conditions and system suitability test:

[0425] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% (by volume, the same below) formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile solution was used as mobile phase B, the flow rate was 0.4 ml / min, and gradient elution was carried out according to the following program:

[0426] The column temperature was kept at 40℃. A triple quadrupole mass spectrometry detector and an electro-spray ionization negative mode (ESI - ) were used, and the multiple reaction monitoring (MRM) ion pairs m / z 253.0→225.0 and m / z 253.0→195.0 were used. The chromatographic column theoretical plate number was not less than 1000, calculated by the peak of chrysophanol.

[0427] Preparation of the control solution:

[0428] An appropriate amount of chrysophanol standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 0.060 mg of chrysophanol per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.033 μg, 0.17 μg, 0.33 μg, 0.50 μg, and 1.0 μg of chrysophanol per 1 ml, respectively.

[0429] Preparation of the test solution:

[0430] An appropriate amount of CDD-2101 granules was finely ground, about 0.1 g was accurately weighed and transferred into a conical flask with a stopper. 10 ml of 70% methanol was accurately added, and ultrasonic extraction (power 500 W, frequency 53 kHz) was performed for 30 minutes. The flask was removed, cooled and filtered. The above extraction was repeated once. The filtrate was mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate was used as the test solution.

[0431] Determination method:

[0432] 1 μl of the control solution and the test solution was accurately pipetted, respectively, and injected into the liquid chromatograph, and determination was performed, and then the content of chrysophanol was obtained.

[0433] Example 12 Determination of the content of rhein

[0434] In this example, the content of rhein in CDD-2101 granules was detected by liquid chromatography-tandem mass spectrometry, as follows:

[0435] Chromatographic conditions and system suitability test:

[0436] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile solution was used as mobile phase B, the flow rate was 0.4 ml / min, and gradient elution was carried out according to the following program:

[0437] The column temperature was kept at 40℃. A triple quadrupole mass spectrometry detector and an electro-spray ionization negative mode (ESI - ) were used, and the multiple reaction monitoring (MRM) ion pairs m / z 283.0→239.0 and m / z 283.0→183.1 were used. The chromatographic column theoretical plate number was not less than 1000, calculated by the rhubarb acid peak.

[0438] Preparation of the control solution:

[0439] An appropriate amount of rhubarb acid standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 0.060 mg of rhubarb acid per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.033 μg, 0.17 μg, 0.33 μg, 0.50 μg, and 1.0 μg of rhubarb acid per 1 ml, respectively.

[0440] Preparation of the test solution:

[0441] An appropriate amount of CDD-2101 granules was finely ground, and about 0.1 g was accurately weighed. 25 ml of a methanol-hydrochloric acid (volume ratio 10:1) mixed solution was accurately added and weighed. It was refluxed in a 80℃ water bath for 60 minutes, then cooled and weighed. The lost weight was made up with methanol, shaken well and filtered. 3.0 ml of the continuous filtrate was accurately measured and transferred to a 5 ml volumetric flask. 1.0 ml of 4% NaOH solution was added, diluted with methanol to the flask mark, mixed well, filtered, and the continuous filtrate was used as the test solution.

[0442] The control solution and the test solution were each precisely pipetted 1 μl, injected into the liquid chromatograph, and determined, and the determination was completed.

[0443] Example 13 Determination of emodin content

[0444] In this example, the emodin content in CDD-2101 granules was detected by liquid chromatography-tandem mass spectrometry, as follows:

[0445] Chromatographic conditions and system suitability test:

[0446] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile solution was used as mobile phase B, the flow rate was 0.4 ml / min, and gradient elution was carried out according to the following program:

[0447] The column temperature was kept at 40℃. A triple quadrupole mass spectrometer detector and an electrospray ionization negative mode (ESI - ) were used, and the multiple reaction monitoring (MRM) ion pairs m / z 269.0→225.0 and m / z 269.0→241.0 were used. The theoretical plate number of the chromatographic column was not less than 1000, calculated by the emodin peak.

[0448] Preparation of the control solution:

[0449] An appropriate amount of emodin standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 0.060 mg of emodin per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.033, 0.17, 0.33, 0.50, and 1.0 μg of emodin per 1 ml, respectively.

[0450] Preparation of the test solution:

[0451] An appropriate amount of CDD-2101 granules was finely ground, about 0.1 g was accurately weighed and transferred to a conical flask with a stopper. 10 ml of 70% methanol was accurately added, and ultrasonic extraction (power 500 W, frequency 53 kHz) was performed for 30 minutes. The cooled and filtered. The above extraction was repeated once. The filtrate was mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate was used as the test solution.

[0452] Determination method:

[0453] 1 μl of the control solution and the test solution was accurately pipetted respectively, injected into the liquid chromatograph, and determined, and the content of emodin was obtained.

[0454] Example 14 Determination of the content of aloe emodin

[0455] In this example, the content of aloe emodin in CDD-2101 granules was detected by liquid chromatography-tandem mass spectrometry, as follows:

[0456] Chromatographic conditions and system suitability test:

[0457] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile solution was used as mobile phase B, the flow rate was 0.4 ml / min, and gradient elution was carried out according to the following program:

[0458] The column temperature was kept at 40℃. A triple quadrupole mass spectrometry detector and an electro-spray ionization negative mode (ESI - ) were used, and the multiple reaction monitoring (MRM) ion pairs m / z 269.0→240.0 and m / z 269.0→183.0 were used. The chromatographic column theoretical plate number was not less than 1000, calculated by aloe emodin peak.

[0459] Preparation of the control solution:

[0460] An appropriate amount of aloe emodin standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 0.11 mg of aloe emodin per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.035 μg, 0.18 μg, 0.35 μg, 0.50 μg, 1.1 μg, 1.6 μg, and 3.2 μg of aloe emodin per 1 ml, respectively.

[0461] Preparation of the test solution:

[0462] An appropriate amount of CDD-2101 granules was finely ground, and about 0.1 g was accurately weighed. 25 ml of a mixed solution of methanol-hydrochloric acid (volume ratio 10:1) was accurately added and weighed. It was refluxed in a 80℃ water bath for 60 minutes, then cooled and weighed. The lost weight was made up with methanol, shaken well, and filtered. 3.0 ml of the continuous filtrate was accurately measured and transferred to a 5 ml volumetric flask. 1.0 ml of 4% NaOH solution was added, diluted with methanol to the flask mark, mixed well, filtered, and the continuous filtrate was used as the test solution.

[0463] Determination method:

[0464] 1 μl of the control solution and the test solution was accurately pipetted respectively, injected into the liquid chromatograph, and determined, and the results were obtained.

[0465] Example 15 Determination of amygdalin content

[0466] In this example, the liquid chromatography-tandem mass spectrometry method was used to detect the amygdalin content in CDD-2101 granules, as follows:

[0467] Chromatographic conditions and system suitability test:

[0468] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution was used as mobile phase A, 0.1% formic acid acetonitrile solution was used as mobile phase B, the flow rate was 0.4 ml / min, and gradient elution was carried out according to the following procedure:

[0469] The column temperature was kept at 40°C. A triple quadrupole mass spectrometer detector and an electrospray ionization negative mode (ESI - ) were used, and the multiple reaction monitoring (MRM) ion pairs m / z 456.0→323.0 and m / z 456.0→119.0 were used. The chromatographic column had a theoretical plate number of not less than 1000, calculated by the amygdalin peak.

[0470] Preparation of the control solution:

[0471] An appropriate amount of amygdalin standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 1.0 mg of amygdalin per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 0.89 μg, 2.2 μg, 3.3 μg, 6.6 μg, 10 μg, and 20 μg of amygdalin per 1 ml, respectively.

[0472] The test solution was prepared by taking an appropriate amount of CDD-2101 granules, grinding them finely, accurately weighing about 0.1 g, and transferring them to a conical flask with a stopper. 10 ml of 70% methanol was accurately added, and ultrasonic extraction (power 500 W, frequency 53 kHz) was performed for 30 minutes. The cooled and filtered solution was repeated once. The filtrate was mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate was used as the test solution.

[0473] Determination method:

[0474] 1 μl of the control solution and the test solution was accurately pipetted into the liquid chromatograph, and determination was performed, and the results were obtained.

[0475] Example 16 Determination of Magnolol Content

[0476] In this example, liquid chromatography-tandem mass spectrometry was used to detect the content of magnolol in CDD-2101 granules, as follows:

[0477] Chromatographic conditions and system suitability test:

[0478] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution was used as mobile phase A, 0.1% formic acid acetonitrile solution was used as mobile phase B, the flow rate was 0.4 ml / min, and gradient elution was carried out according to the following procedure:

[0479] The column temperature was maintained at 40℃. A triple quadrupole mass spectrometry detector and electrospray ionization negative mode (ESI - ) were used with multiple reaction monitoring (MRM) ion pairs m / z 265.0→247.1 and m / z 265.0→245.0. The column theoretical plate number was not less than 1000, calculated with magnolol peak.

[0480] Preparation of the control solution:

[0481] An appropriate amount of magnolol standard was accurately weighed, and methanol was added to prepare a standard stock solution containing 0.30 mg of magnolol per 1 ml. An appropriate amount of the standard stock solution was accurately measured, and methanol was added to prepare a series of standard solutions containing 6.0 μg, 15.0 μg, 30.0 μg, 60.0 μg, and 90.0 μg of magnolol per 1 ml, respectively.

[0482] Preparation of the test solution:

[0483] An appropriate amount of CDD-2101 granules was finely ground, about 0.1 g was accurately weighed and transferred to a conical flask with a stopper. 10 ml of 70% methanol was accurately added, and ultrasonic extraction (power 500 W, frequency 53 kHz) was performed for 30 minutes. The flask was removed, cooled, and filtered. The above extraction was repeated once. The filtrate was mixed in a 25 ml volumetric flask, diluted with methanol to the flask mark, mixed uniformly, filtered, and the continuous filtrate was used as the test solution.

[0484] Determination method:

[0485] 1 μl of the control solution and the test solution was accurately pipetted into the liquid chromatograph, respectively, and determined, and the content of magnolol was obtained.

[0486] Determination of the content of magnolol in Example 17

[0487] In this example, the content of magnolol in CDD-2101 granules was detected by liquid chromatography-tandem mass spectrometry, as follows:

[0488] Chromatographic conditions and system suitability test:

[0489] The chromatographic column was ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile solution was used as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution was performed according to the following program:

[0490] The column temperature was maintained at 40℃. A triple quadrupole mass spectrometry detector and electrospray ionization negative mode (ESI -), using multiple reaction monitoring (MRM) ion pairs m / z 265.0→223.0 and m / z 265.0→249.0. The column efficiency was not less than 1000 plates with honokiol peak.

[0491] Preparation of the control solution:

[0492] Accurately weigh a certain amount of honokiol standard, add methanol to prepare a standard stock solution containing 0.30 mg honokiol per 1 ml. Accurately weigh a certain amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 6.0 μg, 15.0 μg, 30.0 μg, 60.0 μg, and 90.0 μg honokiol per 1 ml, respectively.

[0493] Preparation of the test solution:

[0494] Take a certain amount of CDD-2101 granules, finely grind, accurately weigh about 0.1 g, and transfer it to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool, and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution.

[0495] Determination method:

[0496] Accurately pipette 1 μl of the control solution and the test solution, respectively, into the liquid chromatograph, and determine, i.e. obtain.

[0497] Example 18 Determination of the content of paeoniflorin

[0498] In this example, the content of paeoniflorin in CDD-2101 granules is detected by liquid chromatography-tandem mass spectrometry, as follows:

[0499] Chromatographic conditions and system suitability test:

[0500] The chromatographic column is ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution is used as mobile phase A, and 0.1% formic acid acetonitrile solution is used as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution is performed according to the following program:

[0501] Keep the column temperature at 40℃. Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI -), using multiple reaction monitoring (MRM) ion pairs m / z 525.0→121.0 and m / z 525.0→327.0. The column efficiency was not less than 1000 theoretical plates, calculated with the peak of paeoniflorin.

[0502] Preparation of the control solution:

[0503] Accurately weigh a certain amount of paeoniflorin standard, add methanol to prepare a standard stock solution containing 0.060 mg of paeoniflorin per 1 ml. Accurately weigh a certain amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 0.033 μg, 0.17 μg, 0.33 μg, 0.50 μg, and 1.0 μg of paeoniflorin per 1 ml, respectively.

[0504] Preparation of the test solution:

[0505] Take a certain amount of CDD-2101 granules, finely grind, accurately weigh about 0.1 g, and transfer it to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution.

[0506] Determination method:

[0507] Accurately pipette 1 μl of the control solution and the test solution, respectively, into the liquid chromatograph, and determine, i.e. obtain.

[0508] Determination of the content of synephrine

[0509] This example uses liquid chromatography-tandem mass spectrometry to detect the content of synephrine in CDD-2101 granules, as follows:

[0510] Chromatographic conditions and system suitability test:

[0511] The chromatographic column is a ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution is used as mobile phase A, and 0.1% formic acid acetonitrile solution is used as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution is performed according to the following program:

[0512] Keep the column temperature at 40℃. Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ), using multiple reaction monitoring (MRM) ion pairs m / z 525.0→121.0 and m / z 525.0→327.0. The column efficiency was not less than 1000 theoretical plates, calculated with the peak of paeoniflorin. - ), using multiple reaction monitoring (MRM) ion pairs m / z 525.0→121.0 and m / z 525.0→327.0. The column efficiency was not less than 1000 theoretical plates, calculated with the peak of paeoniflorin.

[0513] Preparation of the standard solution:

[0514] Accurately weigh a certain amount of synephrine standard, add methanol to prepare a standard stock solution containing 1.0 mg of synephrine per 1 ml. Accurately weigh a certain amount of the standard stock solution, add methanol to prepare a series of standard solutions containing 0.017 μg, 0.033 μg, 0.067 μg, 0.17 μg, and 0.25 μg of synephrine per 1 ml, respectively.

[0515] Preparation of the sample solution:

[0516] Take a certain amount of CDD-2101 granules, finely grind, accurately weigh about 0.1 g, and transfer it to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool, and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute with methanol to the flask mark, mix well, filter, and the continuous filtrate is used as the test solution.

[0517] Determination method:

[0518] Accurately pipette 1 μl of the standard solution and the sample solution, respectively, into the liquid chromatograph, and determine, i.e. obtain.

[0519] Example 20 Determination of the content of hesperidin

[0520] In this example, the content of hesperidin in CDD-2101 granules is detected by liquid chromatography-tandem mass spectrometry, as follows:

[0521] Chromatographic conditions and system suitability test:

[0522] The chromatographic column is ZORBAX Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 μm, Agilent); 0.1% formic acid aqueous solution is used as mobile phase A, and 0.1% formic acid acetonitrile solution is used as mobile phase B, with a flow rate of 0.4 ml / min, and gradient elution is performed according to the following program:

[0523] Keep the column temperature at 40℃. Use a triple quadrupole mass spectrometer detector and an electro-spray ionization negative mode (ESI - ), use multiple reaction monitoring (MRM) ion pairs m / z 609.2→301.0 and m / z 609.2→286.0. The theoretical plate number of the chromatographic column is not less than 1000, calculated by the hesperidin peak.

[0524] Preparation of the standard solution:

[0525] Accurately weigh an appropriate amount of hesperidin standard, add methanol to prepare a stock solution containing 1.0 mg of hesperidin per 1 ml. Accurately weigh an appropriate amount of the stock solution, add methanol to prepare a series of standard solutions containing 0.39 μg, 0.98 μg, 1.5 μg, 2.9 μg, 4.4 μg, 8.8 μg of hesperidin per 1 ml, respectively.

[0526] Preparation of test sample solution:

[0527] Take an appropriate amount of CDD-2101 granules, finely grind, take about 0.1 g, accurately weigh and transfer to a conical flask with a stopper. Accurately add 10 ml of 70% methanol, ultrasonic (power 500 W, frequency 53 kHz) for 30 minutes, take out and cool and filter. Repeat the above extraction once. Mix the filtrate in a 25 ml volumetric flask, dilute to the flask mark with methanol, mix well, filter, and the continuous filtrate is used as the test solution.

[0528] Determination method:

[0529] Accurately pipette 1 μl of the control solution and the test sample solution, respectively, into the liquid chromatograph for determination, and the result is obtained.

Claims

1. A method for quality control of CDD-2101 particles, wherein: The raw materials of the CDD-2101 granules include the following medicinal materials: hemp seed, rhubarb, magnolia bark, white peony root, and stir-fried immature bitter orange; the quality control method includes using thin layer chromatography to qualitatively identify the stir-fried immature bitter orange in the CDD-2101 granules, and the identification method includes the following steps: The test sample solution and the control medicinal material solution are spotted on the same silica gel thin layer plate, placed in a bran-fried Citrus aurantium identification-developing agent for development, and then the distribution of fluorescent spots is observed under ultraviolet light; wherein the bran-fried Citrus aurantium identification-developing agent is composed of cyclohexane, chloroform, ethyl acetate, and formic acid in a volume ratio of 3:2:8:0.

2.

2. The quality control method of CDD-2101 particles according to claim 1, wherein: In the thin layer chromatography identification of fried aurantium immaturum with bran, the sample volume of the control medicinal material solution and the test sample solution is 8-20μl respectively.

3. The quality control method of CDD-2101 particles according to claim 1, wherein: In the thin layer chromatography identification of fried aurantium immaturum with bran, the development conditions include: humidity of 32-88%, temperature of 3-40℃, and saturation time ≤30min.

4. The method for quality control of CDD-2101 particles according to claim 1, wherein: The quality control method further comprises using thin layer chromatography to qualitatively identify any one of the medicinal materials selected from hemp seed, rhubarb, magnolia bark, and white peony root in the CDD-2101 granules; the thin layer chromatography qualitative identification method comprises the following steps: Prepare test sample solution and control medicinal material solution; Spot the test solution and the control medicinal material solution on the same silica gel thin layer plate, place it in a developing agent for development, and then observe the distribution of the spots on the silica gel thin layer plate after color development.

5. The method for quality control of CDD-2101 particles according to claim 4, wherein: In the thin layer chromatography identification method of hemp seed, the developing solvent is composed of cyclohexane, ethyl acetate and formic acid in a volume ratio of 12:3:0.

1.

6. The method for quality control of CDD-2101 particles according to claim 4, wherein: In the thin layer chromatography identification method of rhubarb, the developing solvent is composed of cyclohexane, ethyl acetate and formic acid in a volume ratio of 12:3:0.

1.

7. The method for quality control of CDD-2101 particles according to claim 4, wherein: In the thin layer chromatography identification method of Magnolia officinalis, the developing solvent is composed of cyclohexane, ethyl acetate and formic acid in a volume ratio of 12:3:0.

1.

8. The method for quality control of CDD-2101 particles according to claim 4, wherein: In the thin-layer chromatography identification method for white peony root, a developing solvent is prepared by mixing chloroform, ethyl acetate, methanol and 25-28 wt% concentrated ammonia solution in a volume ratio of 8:1:4:

1.

9. The method for controlling the quality of CDD-2101 particles according to claim 4, wherein: In the thin-layer chromatography identification method for any medicinal material, the development conditions include: humidity of 32-88%, temperature of 3-40° C., and saturation time of ≤30 min.

10. The method for quality control of CDD-2101 particles according to claim 4, wherein: The spot color development method for thin layer chromatography identification of any one of the medicinal materials of hemp seed, magnolia bark, and white peony root is as follows: the developed silica gel thin layer plate is dried, sprayed with vanillin sulfuric acid solution, and heated until the spots are clear.

11. The method for quality control of CDD-2101 particles according to claim 1, wherein: The quality control method also includes: using liquid chromatography-tandem mass spectrometry to quantitatively determine at least one of the following indicator components in CDD-2101 granules: cannabinamide B, emodin, rhein, chrysophanol, aloe-emodin, amygdalin, magnolol, honokiol, paeoniflorin, synephrine and hesperidin.

12. The method for controlling the quality of CDD-2101 particles according to claim 11, wherein: High performance liquid chromatography-tandem mass spectrometry was used to quantitatively determine at least one of the following index components in CDD-2101 granules: cannabinamide B, emodin, amygdalin, magnolol, paeoniflorin, and hesperidin.

13. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of cannabinamide B, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is acetonitrile, phase B is a 0.1% volume fraction of formic acid aqueous solution, and the flow rate of the mobile phase is 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 595.0→269.0 and m / z 595.0→432.

0.

14. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of chrysophanol, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 253.0→225.0 and m / z 253.0→195.

0.

15. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of rhein, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 283.0→239.0 and m / z 283.0→183.

1.

16. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of emodin, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 269.0→225.0 and m / z 269.0→241.

0.

17. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of aloe-emodin, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 269.0→240.0 and m / z 269.0→183.

0.

18. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of amygdalin, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 456.0→323.0 and m / z 456.0→119.

0.

19. The method for controlling the quality of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of magnolol, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 265.0→247.1 and m / z 265.0→245.

0.

20. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of honokiol, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 265.0→223.0 and m / z 265.0→249.

0.

21. The method for controlling the quality of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of paeoniflorin, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 525.0→121.0 and m / z 525.0→327.

0.

22. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of synephrine, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 168.0→150.0 and m / z 168.0→91.

0.

23. The method for quality control of CDD-2101 particles according to claim 11, wherein: In the liquid chromatography-tandem mass spectrometry detection of hesperidin, the chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) with a theoretical plate number ≥ 1000; the column temperature was 40°C; The mobile phase includes phase A and phase B, phase A is a 0.1% by volume formic acid aqueous solution, and phase B is a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction monitoring ion transitions: m / z 609.2→301.0 and m / z 609.2→286.

0.

24. The method for quality control of CDD-2101 particles according to claim 1, wherein: The quality control method also includes: obtaining a fingerprint of the CDD-2101 particles by sampling liquid chromatography-tandem mass spectrometry for consistency evaluation, and the detected components include: synephrine, amygdalin, paeoniflorin, paeoniflorin, naringenin, hesperidin, cannabinamide B, aloe-emodin, rhein, emodin, honokiol, magnolol, and chrysophanol. One or more of the following:

25. The method for quality control of CDD-2101 particles according to claim 24, wherein: In fingerprint detection, chromatographic conditions include: The chromatographic column was a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent); the column temperature was 40 °C; The mobile phases included phase A and phase B, phase A was a 0.1% by volume formic acid aqueous solution, and phase B was a 0.1% by volume formic acid acetonitrile solution, with a flow rate of 0.4 ml / min; The elution method is gradient elution, and the elution procedure is as follows: Mass spectrometry conditions included: triple quadrupole mass spectrometer; electrospray ionization negative mode; Detection method: Multiple reaction monitoring; Reaction Monitoring Ion Pairs:

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