Fingerprint Spectrum Detection Method and Fingerprint Spectrum of Angong Niuhuang Pills

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a fingerprint detection method for Angong Niuhuang Pills and a fingerprint thereof; a fingerprint detection method for Angong Niuhuang Pills, comprising the following steps: preparing a test solution: weighing the Angong Niuhuang Pills, crushing them, adding diatomaceous earth to mix them, and then performing microwave drying and freezing treatment, adding a methanol solution to perform ultrasonic extraction, and filtering to obtain a test solution; preparing a reference solution: respectively weighing musk, borneol, curcuma, gardenia, scutellaria, coptis root, and bezoar, adding a methanol solution to dissolve them, and obtaining a reference solution; determining the fingerprint of the Angong Niuhuang Pills by high performance liquid chromatography; the invention adopts high performance liquid chromatography and reasonably controls the chromatographic conditions, so that the fingerprint obtained by the invention can perform more comprehensive quality evaluation on the Angong Niuhuang Pills, and the detection method is simple and rapid, has high accuracy, good repeatability, good stability, and good durability.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a fingerprint spectrum detection method of Angong Niuhuang Pills and a fingerprint spectrum thereof. Background Art

[0002] The quality control research in the prior art mainly focuses on the content determination of 2-3 components in Angong Niuhuang Pills, which only focuses on a few components in Angong Niuhuang Pills and cannot fully reflect the overall quality of the preparation. Summary of the invention

[0003] In view of the deficiencies in the prior art, the object of the present invention is to provide a fingerprint detection method for Angong Niuhuang Pills and its fingerprint.

[0004] In the first aspect, the present application provides a fingerprint spectrum detection method of Angong Niuhuang Pills, which adopts the following technical scheme: A fingerprint spectrum detection method of Angong Niuhuang Pills, the steps are as follows:

[0005] (1) Preparation of test solution:

[0006] Take Angong Niuhuang Pills, weigh accurately, cut into pieces, add diatomaceous earth to disperse and mix, then perform microwave drying and freezing treatment; grind the frozen mixture evenly, put it in a stoppered conical flask, add methanol, weigh it, perform ultrasonic extraction, weigh it after cooling, make up the lost weight with methanol, shake well, filter through 0.22μm microporous filter membrane, and take the filtrate to obtain.

[0007] The microwave drying step is as follows: drying the mixture at a wavelength of 1 mm-1 m, a frequency of 500 MHz-1200 MHz, and a vacuum degree of -0.08 Mpa for 15-20 minutes.

[0008] The steps of freezing treatment are as follows: the microwave-dried mixture is frozen at -10°C to -20°C for 1-5 minutes.

[0009] (2) Preparation of reference solution:

[0010] Weigh artificial musk, borneol, curcuma, gardenia, scutellaria, coptis root, bezoar respectively, add methanol solution to dissolve, and obtain reference solution;

[0011] (3) Fingerprint of Angong Niuhuang Pills determined by HPLC:

[0012] The test solution and the reference solution were injected into the HPLC to determine the fingerprint.

[0013] The similarity between the obtained spectrum and the standard fingerprint spectrum calculated by software shall not be less than 90%.

[0014] By adopting the above technical scheme, the Angong Niuhuang Pills are subjected to microwave drying so that the material itself is heated, and the moisture in the material can strongly absorb microwaves, thereby evaporating part of the moisture in the Angong Niuhuang Pills, and the microwave-dried Angong Niuhuang Pills are frozen, and the dried material is quickly cooled and hardened, thereby reducing the viscosity of the Angong Niuhuang Pills, effectively preventing the material from having a certain influence on the product detection due to excessive viscosity, thereby ensuring the accuracy of the detection result.

[0015] Preferably, the chromatographic conditions in step (3) are as follows: chromatographic column: C18; mobile phase A: acetonitrile; mobile phase B: aqueous phosphoric acid solution with a volume concentration of 0.1%-0.5%; column temperature: 25-35°C; mobile phase flow rate: 0.5-1.2 mL / min; injection volume: 5-15 μl; detection wavelength: 200-260 nm.

[0016] Preferably, the chromatographic column is Agilent TC-C18 (250 mm×4.6 mm, 5 μm).

[0017] Preferably, the volume concentration of the mobile phase B phosphoric acid aqueous solution is 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%; further, the volume concentration of the mobile phase B phosphoric acid aqueous solution is 0.1%.

[0018] Preferably, the column temperature is 25°C, 28°C, 30°C, 32°C or 35°C; further, the column temperature is 30°C.

[0019] Preferably, the detection wavelength is 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm or 260 nm; further, the detection wavelength is 230 nm.

[0020] Preferably, the mobile phase flow rate is 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min or 1.2 mL / min; further, the mobile phase flow rate is 0.8 mL / min.

[0021] Preferably, the gradient elution conditions in step (3) are as follows: 0-2 min, 10% A; 2-25 min, 10-23% A; 25-35 min, 23% A; 35-55 min, 23-35% A; 55-60 min, 35-45% A; 60-70 min, 45-55% A; 70-80 min, 55-90% A; 80-85 min, 90-95% A; 85-105 min, 95% A.

[0022] Preferably, in step (3), the amount of the test solution added is 1-10 μl, and the amount of the reference solution added is 1-10 μl; further, the amount of the test solution added is 10 μl, and the amount of the reference solution added is 10 μl.

[0023] Preferably, in step (1), the amount of Angong Niuhuang Pills added is 0.3-0.9 g; the amount of diatomaceous earth added is 0.5-3.5 g; and the amount of methanol added is 15-30 mL.

[0024] Preferably, in step (1), the time of ultrasonic extraction is 25-35 min; the power of ultrasonic extraction is 200-300 w, and the frequency of ultrasonic extraction is 40-50 kHz; further, the time of ultrasonic extraction is 30 min; the power of ultrasonic extraction is 250 w, and the frequency of ultrasonic extraction is 45 kHz.

[0025] Preferably, the volume concentration of the methanol solution in step (1) is 50-80%; further, the volume concentration of the methanol solution is 70%.

[0026] By adopting the above technical scheme, the present application adopts high performance liquid chromatography and reasonably controls the chromatographic conditions, using acetonitrile as mobile phase A and a 0.1% volume concentration of phosphoric acid aqueous solution as mobile phase B for gradient elution; the detection method of the present application has good precision, repeatability, solution stability and durability.

[0027] In the second aspect, the present application provides a fingerprint spectrum of Angong Niuhuang Pills, using the following technical solution:

[0028] A fingerprint spectrum of Angong Niuhuang Pills, the fingerprint spectrum has 16 characteristic fingerprint peaks, and the standard fingerprint spectrum of Angong Niuhuang Pills has the following characteristic peaks: Peak 1: relative retention time 6.349, relative peak area 63906;

[0029] Peak 2: relative retention time 13.613, relative peak area 141891;

[0030] Peak 3: relative retention time 17.116, relative peak area 698190;

[0031] Peak 4: relative retention time 19.483, relative peak area 254609;

[0032] Peak 5: relative retention time 21.52, relative peak area 66065;

[0033] Peak 6: relative retention time 26.112, relative peak area 106583;

[0034] Peak 7: relative retention time 35.063, relative peak area 242052;

[0035] Peak 8: relative retention time 35.717, relative peak area 339870;

[0036] Peak 9: relative retention time 36.384, relative peak area 976049;

[0037] Peak 10: relative retention time 45.589, relative peak area 735320;

[0038] Peak 11: relative retention time 46.397, relative peak area 3525985;

[0039] Peak 12: relative retention time 48.444, relative peak area 107409;

[0040] Peak 13: relative retention time 49.478, relative peak area 70256;

[0041] Peak 14: relative retention time 51.084, relative peak area 202591;

[0042] Peak 15: relative retention time 53.703, relative peak area 379210;

[0043] Peak 16: relative retention time 69.119, relative peak area 156056;

[0044] By adopting the above technical scheme, the present application adopts high performance liquid chromatography and reasonably controls the chromatographic conditions, using acetonitrile as mobile phase A and a 0.1% volume concentration of phosphoric acid aqueous solution as mobile phase B for gradient elution, and a total of 55 common peaks are calibrated, and 16 chromatographic peaks are identified; the fingerprint spectrum obtained by the present application can conduct a more comprehensive quality evaluation of Angong Niuhuang Pills, and the time is short, which can improve the efficiency of quality detection. At the same time, the method is simple, accurate, repeatable, stable and durable, which can provide a scientific basis for the quality evaluation and control of Angong Niuhuang Pills.

[0045] In summary, the present application includes at least one of the following beneficial technical effects:

[0046] The present application adopts high performance liquid chromatography and reasonably controls the chromatographic conditions so that the fingerprint spectrum obtained in the present application can conduct a more comprehensive quality evaluation of Angong Niuhuang Pills. At the same time, the detection method of the present application is simple, rapid, highly accurate, repeatable, and durable, and can provide a scientific basis for the quality evaluation and control of Angong Niuhuang Pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the sample spectrum of ten batches of Angong Niuhuang Pills;

[0048] Figure 2This is the fingerprint map of Angong Niuhuang Pills. DETAILED DESCRIPTION

[0049] The raw materials involved in this application are all commercially available products, and the application is further described in detail below in conjunction with the embodiments and comparative examples.

[0050] Example:

[0051] 1. Experimental instruments and reagents

[0052] 1. Experimental instruments

[0053] LC-2030 high performance liquid chromatograph (SHIMADZU, Japan);

[0054] KQ-300DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0055] PRACTUM 124-1CN electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.);

[0056] Agilent TC-C18 chromatographic column (250 mm×4.6 mm, 5 μm).

[0057] 2. Experimental reagents: methanol, acetonitrile (chromatographic grade): Enko Chemical ACS, USA;

[0058] Methanol and phosphoric acid (analytical grade): Nanjing Chemical Reagent Co., Ltd.

[0059] Purified water: Hangzhou Wahaha Group Co., Ltd.

[0060] Angong Niuhuang Wan: Nanjing Tongrentang Pharmaceutical Co., Ltd. (Batch No.: 1805012).

[0061] 3. Reference substances and reference medicinal materials

[0062] Artificial musk 210703300 (Nanjing Tongrentang Pharmaceutical Co., Ltd.); borneol reference substance 110743-201706 (China Food and Drug Inspection Institute); reference medicinal materials: Curcuma 120949-201608, Gardenia 120986-201610, Scutellaria baicalensis 120955-201810, Coptis chinensis 120913-201611, Bezoar 121328-201202 (China Food and Drug Inspection Institute).

[0063] 4. Investigation of the properties of medicinal materials

[0064] Prepare a single-flavor reference medicinal material solution according to the medicinal material ratio in the prescription, and inject and measure according to the determined chromatographic conditions.

[0065] Curcuma has common peaks at 84.525min and 86.996min.

[0066] Bezoar has common peaks at 74.928min, 76.177min, 86.996min and 40.960min.

[0067] Musk has common peaks at 17.231min, 57.635min, and 86.996min.

[0068] Coptis chinensis has common peaks at 4.873min, 6.295min, 8.378min, 8.906min, 18.338min, 21.966min, 23.064min, 25.458min, 25.921min, 28.156min, 34.236min, 36.323min, 40.216min, 40.96min, 41.616min, 47.594min, 52.116min, 52.978min, and 86.996min.

[0069] Scutellaria baicalensis has common peaks at 25.458min, 30.886min, 32.341min, 32.683min, 33.249min, 34.236min, 34.493min, 35.023min, 37.182min, 38.032min, 43.752min, 46.297min, 50.078min, 54.659min, 57.635min, 59.223min, 60.816min, 61.962min, 62.493min, 66.316min, 73.198min, 74.477min, 81.914min, 82.647min, 84.023min and 86.996min.

[0070] Gardenia has common peaks at 9.181min, 16.195min, 20.265min, 31.759min, 34.493min, 36.323min, 39.790min, 86.996min, and 104.264min.

[0071] 2. Fingerprint Spectrum Detection Method of Angong Niuhuang Pills

[0072] 1. Preparation of test solution: Take about 0.5g of Angong Niuhuang Pills, weigh accurately, cut into pieces, add 1g of diatom to disperse; then microwave dry and freeze; grind the frozen mixture evenly, put it in a stoppered conical flask, add 25mL of methanol with a volume concentration of 75%, weigh it, and extract it ultrasonically (power 250w, frequency 45kHz) for 30min, weigh it after cooling, make up the lost weight with methanol, shake it evenly, filter it with 0.22μm microporous filter membrane, and take the filtrate.

[0073] The microwave drying step is as follows: the mixture is subjected to microwave drying for 18 minutes at a wavelength of 500 mm, a frequency of 900 MHz, and a vacuum degree of -0.08 Mpa.

[0074] The steps of freezing treatment are as follows: the microwave-dried mixture is frozen at -15°C for 3 min.

[0075] 2. Preparation of reference solution: Weigh 0.5 g each of artificial musk, borneol, curcuma, gardenia, scutellaria, coptis root and bezoar, add 25 mL of methanol with a volume concentration of 75% to obtain a reference solution;

[0076] 3. Determination of fingerprint of Angong Niuhuang Wan by high performance liquid chromatography: Accurately pipette 10μl of test solution and 10μl of reference solution, inject into high performance liquid chromatography instrument, and determine to obtain fingerprint, see Figure 2 .

[0077] The similarity between the obtained spectrum and the standard fingerprint spectrum calculated by software shall not be less than 90%.

[0078] The chromatographic conditions were as follows: chromatographic column: C18; mobile phase A: acetonitrile; mobile phase B: 0.1% by volume phosphoric acid aqueous solution; column temperature: 30°C; mobile phase flow rate: 0.8 mL / min; injection volume: 10 μl; detection wavelength: 230 nm.

[0079] The chromatographic column was Agilent TC-C18.

[0080] The gradient elution conditions were as follows: 0-2 min, 10% A; 2-25 min, 10-23% A; 25-35 min, 23% A; 35-55 min, 23-35% A; 55-60 min, 35-45% A; 60-70 min, 45-55% A; 70-80 min, 55-90% A; 80-85 min, 90-95% A; 85-105 min, 95% A.

[0081] 3. Methodological Investigation

[0082] 1. Precision test:

[0083] Accurately pipette the test solution of Angong Niuhuang Pills from the same batch and inject it 6 times continuously. Calculate the RSD value based on the retention time and peak area of ​​each characteristic peak.

[0084] The results showed that the retention time RSD of each characteristic peak was less than 0.73%, and the peak area RSD was less than 1.69%, indicating good precision (see Table 1).

[0085] Table 1 Precision test results

[0086]

[0087]

[0088] 2. Repeatability test

[0089] Take 6 portions of Angong Niuhuang Pills from the same batch, prepare the test solution according to the method, inject and measure them separately, and calculate the RSD value according to the retention time and peak area of ​​each characteristic peak.

[0090] The results showed that the retention time RSD of each characteristic peak was less than 0.20%, and the peak area RSD was less than 2.69%, indicating that the method had good repeatability (see Table 2).

[0091] Table 2 Repeatability test results

[0092]

[0093]

[0094] 3. Stability test

[0095] Accurately pipette the test solution of Angong Niuhuang Pills from the same batch, and measure it at 0, 3, 6, 9, 12, and 24 h, respectively. Calculate the RSD value based on the retention time and peak area of ​​each characteristic peak.

[0096] The results showed that the retention time RSD of each characteristic peak was less than 0.95%, and the peak area RSD was less than 4.62%, indicating that the test results of the test solution were stable within 24 hours, as shown in Table 3.

[0097] Table 2 Stability test results

[0098]

[0099]

[0100] 4. Durability test

[0101] The same batch of Angong Niuhuang Wan test solution was accurately drawn and injected into different HPLCs (Agilent LC1100 HPLC, Waters e2695 HPLC) according to the determined chromatographic conditions. The results showed that the similarity between the two spectra was greater than 90% under the experimental conditions. Therefore, this method is applicable to different HPLCs.

[0102] 5. Determination of ten batches of samples

[0103] Ten batches of Angong Niuhuang Wan samples were taken, and the test solution was prepared according to the method. The samples were injected in sequence and the spectra were recorded. The results were analyzed and processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" to generate fingerprint spectra. Figure 1 .

[0104] The present application adopts high performance liquid chromatography and reasonably controls the chromatographic conditions, uses acetonitrile as mobile phase A and a 0.1% volume concentration of phosphoric acid aqueous solution as mobile phase B for gradient elution, calibrates a total of 55 common peaks, and identifies 16 chromatographic peaks therein; the fingerprint spectrum obtained in the present application can conduct a more comprehensive quality evaluation of Angong Niuhuang Pills in a short time, and can improve the efficiency of quality detection. At the same time, the method is simple, accurate, repeatable, stable and durable, and can provide a scientific basis for the quality evaluation and control of Angong Niuhuang Pills.

[0105] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A fingerprint spectrum of Angong Niuhuang Pills obtained by using the fingerprint spectrum detection method of Angong Niuhuang Pills, characterized in that: The steps of the detection method are as follows: (1) Preparation of test solution; Weigh Angong Niuhuang Pills, crush them, add diatomaceous earth to mix, perform microwave drying and freezing treatment, add methanol solution to perform ultrasonic extraction, and filter to obtain a test solution; (2) Preparation of reference solution; (3) Determination of fingerprint of Angong Niuhuang Wan by HPLC; The test solution and the reference solution were injected into the HPLC to determine the fingerprints; The steps of microwave drying are as follows: drying the mixture at a wavelength of 1 mm-1 m, a frequency of 500 MHz-1200 MHz, and a vacuum degree of -0.08 Mpa for 15-20 min; The chromatographic conditions in step (3) are as follows: chromatographic column: C18; mobile phase A: acetonitrile; mobile phase B: aqueous phosphoric acid solution with a volume concentration of 0.1%-0.5%; Column temperature: 25-35°C; mobile phase flow rate: 0.5-1.2 mL / min; injection volume: 5-15 μl; detection wavelength: 200-260 nm; The gradient elution conditions in step (3) are as follows: 0-2 min, 10% A; 2-25 min, 10-23% A; 25-35 min, 23% A; 35-55 min, 23-35% A; 55-60 min, 35-45% A; 60-70 min, 45-55% A; 70-80 min, 55-90% A; 80-85 min, 90-95% A; 85-105 min, 95% A; In step (1), the amount of Angong Niuhuang Pill added is 0.3-0.9 g; the amount of diatomaceous earth added is 0.5-3.5 g; the amount of methanol solution added is 15-30 mL; and the volume concentration of the methanol solution is 50-80%; There are 16 common peaks in the fingerprint spectrum of Angong Niuhuang Pills, and the retention times of the common peaks are: Peak 1: relative retention time 6.349; Peak 2: relative retention time 13.613; Peak 3: relative retention time 17.116; Peak 4: relative retention time 19.483; Peak 5: relative retention time 21.52; Peak 6: relative retention time 26.112; Peak 7: relative retention time 35.063; Peak 8: relative retention time 35.717; Peak 9: relative retention time 36.384; Peak 10: relative retention time 45.589; Peak 11: relative retention time 46.397; Peak 12: relative retention time 48.444; Peak 13: relative retention time 49.478; Peak 14: relative retention time 51.084; Peak 15: relative retention time 53.703; Peak 16: relative retention time 69.

119.

2. The fingerprint of Angong Niuhuang Pill according to claim 1, characterized in that: The steps of freezing treatment are as follows: freeze the mixture at -10°C~-20°C for 1-5 minutes.

3. The fingerprint of Angong Niuhuang Pill according to claim 1, characterized in that: In step (3), the amount of the test solution added is 1-10 μl, and the amount of the reference solution added is 1-10 μl.

4. The fingerprint of Angong Niuhuang Pill according to claim 1, characterized in that: The ultrasonic extraction time in step (1) is 25-35 minutes; the ultrasonic extraction power is 200-300w, and the ultrasonic extraction frequency is 40-50kHz.

5. The fingerprint of Angong Niuhuang Pill according to claim 1, characterized in that: The preparation steps of the reference solution in step (2) are as follows: weigh musk, borneol, curcuma, gardenia, scutellaria, coptis, and bezoar respectively, add methanol solution to dissolve, and obtain a reference solution.

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