Fingerprint Spectrum Detection Method and Fingerprint Spectrum of Niuhuang Qingxin Pill

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a fingerprint detection method for Niuhuang Qingxin Pills and a fingerprint thereof; a fingerprint detection method for Niuhuang Qingxin Pills, comprising the following steps: preparation of a test solution: weighing Niuhuang Qingxin Pills, crushing them, adding diatomaceous earth to mix them, and obtaining a mixture; adding the mixture to a methanol solution, performing ultrasonic extraction, filtering, and obtaining a test solution; preparation of a reference solution; and determination of the fingerprint of Niuhuang Qingxin Pills by high performance liquid chromatography: injecting the test solution and the reference solution into a high performance liquid chromatograph respectively to determine the fingerprint; the application adopts high performance liquid chromatography and reasonably controls the chromatographic conditions, and the fingerprint obtained can perform more comprehensive quality evaluation on Niuhuang Qingxin Pills, thereby providing a scientific basis for quality evaluation and control of Niuhuang Qingxin Pills.

Description

Technical Field

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

[0002] Niuhuang Qingxin Pills are a traditional Chinese medicine preparation composed of six medicinal ingredients: bezoar, cinnabar, coptis, gardenia, curcuma, and scutellaria. It has the effects of clearing away heat and detoxifying, opening the orifices and tranquilizing the mind. It is mainly used to treat internal invasion of warm evil, heat entering the pericardium, phlegm congestion, restlessness and coma, delirium and convulsions. It is clinically used to clear away heat and detoxify, calm the nerves and tranquilize the mind.

[0003] Niuhuang Qingxin Pills are included in the 2020 edition of the "Chinese Pharmacopoeia", but its current standard only stipulates the determination method of berberine hydrochloride content in cinnabar and coptis chinensis. The active ingredients such as baicalin and gardenia jasminoides can only be qualitatively identified by TLC. At present, there are no active ingredients contained in bezoar, scutellaria, and gardenia, and the literature only involves berberine hydrochloride, baicalin, and gardenia jasminoides with higher content.

[0004] Therefore, the quality control research in the prior art only measures the content of 2 to 3 components in Niuhuang Qingxin Pills, which cannot fully reflect the overall quality of Niuhuang Qingxin Pills preparations. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a fingerprint detection method for Niuhuang Qingxin Pills and its fingerprint.

[0006] In the first aspect, the present application provides a fingerprint spectrum detection method of Niuhuang Qingxin Pills, which adopts the following technical scheme: A fingerprint spectrum detection method of Niuhuang Qingxin Pills, the steps are as follows:

[0007] (1) Preparation of test solution

[0008] S1, weighing Niuhuang Qingxin Pills, crushing them, adding diatomaceous earth to mix them, and obtaining a mixture;

[0009] S2. Add methanol solution to the mixture, perform ultrasonic extraction, and filter to obtain a test solution;

[0010] (2) Preparation of reference solution

[0011] Weigh borneol, antelope horn, musk, pollen, cinnabar, bezoar, realgar, buffalo horn, yam, dried ginger, jujube, chuanxiong, ginseng, ophiopogon, cinnamon, bupleurum, saposhnikovia, scutellaria, donkey-hide gelatin, atractylodes, liushenqu, atractylodes, bitter almond, licorice, angelica, soybean yellow roll, white peony root, tuckahoe, platycodon, add methanol solution to dissolve, and obtain a reference solution;

[0012] (3) Determination of the fingerprint of Niuhuang Qingxin Pills by HPLC

[0013] The test solution and the reference solution were injected into the HPLC to determine the fingerprint.

[0014] Preferably, the mixture prepared in step S1 is post-treated as follows: the mixture is frozen at -5 to -15°C for 10-15 minutes, and then the mixture is dried at a vacuum pressure of 1-20 Pa and a temperature of 35-55°C for 40-60 minutes.

[0015] By adopting the above technical scheme, the Niuhuang Qingxin Pills are cooled, and the material becomes hard after rapid cooling. The cooled material is then vacuum dried to remove part of the moisture in the material, thereby reducing the viscosity of the Niuhuang Qingxin Pills, effectively preventing the material from having a certain impact on the product detection due to excessive viscosity, thereby ensuring the accuracy of the test results.

[0016] 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 0.01%-0.05%; column temperature: 30-40°C; mobile phase flow rate: 0.5-1.5mL / min; injection volume: 5-15μl; detection wavelength: 200-360nm.

[0017] Preferably, the chromatographic column is Agilent TC-C18.

[0018] Preferably, the volume concentration of the phosphoric acid aqueous solution in the mobile phase B is 0.01%, 0.02%, 0.03%, 0.04% or 0.05%; further, the volume concentration of the phosphoric acid aqueous solution is 0.03%.

[0019] Preferably, the column temperature is 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C; further, the column temperature is 35°C.

[0020] Preferably, the mobile phase flow rate is 0.5 mL / min, 0.8 mL / min, 1 mL / min, 1.2 mL / min or 1.5 mL / min; further, the mobile phase flow rate is 1 mL / min.

[0021] Preferably, the detection wavelength is 200 nm, 210 nm, 250 nm or 360 nm; further, the detection wavelength is 250 nm.

[0022] Preferably, the gradient elution conditions in step (3) are as follows: 0-30 min, 12% A; 30-43 min, 12-17% A; 43-63 min, 17% A; 63-85 min, 17-25% A; 85-95 min, 25% A; 95-110 min, 25-31% A; 110-165 min, 31-45% A; 165-210 min, 45-70% A.

[0023] 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.

[0024] Preferably, the mass ratio of Niuhuang Qingxin Pills to diatomaceous earth in step S1 is 1:(1-2.5); the amount of the mixture added in step S2 is 3-12 g; and the amount of the methanol solution added is 15-30 mL.

[0025] Preferably, the volume concentration of the methanol solution in step S2 is 50-80%; further, the volume concentration of the methanol solution is 75%.

[0026] Preferably, the ultrasonic extraction time in step S2 is 25-40 min.

[0027] 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 0.03% volume concentration of 0.03% phosphoric acid aqueous solution as mobile phase B for gradient elution. A total of 34 common peaks are calibrated, and 7 chromatographic peaks are identified. The detection method of the present application has good precision, repeatability, solution stability and durability.

[0028] In the second aspect, the present application provides a fingerprint of Niuhuang Qingxin Pills, using the following technical solution:

[0029] A fingerprint spectrum of Niuhuang Qingxin Pills, wherein there are 7 common peaks in the fingerprint spectrum of Niuhuang Qingxin Pills, and the retention times of the common peaks are:

[0030] Peak 1: relative retention time 22.121;

[0031] Peak 2: relative retention time 43.267;

[0032] Peak 3: relative retention time 57.186;

[0033] Peak 4: relative retention time 79.595;

[0034] Peak 5: relative retention time 91.140;

[0035] Peak 6: relative retention time 96.082;

[0036] Peak 7: relative retention time 132.415.

[0037] 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 phosphoric acid aqueous solution with a volume concentration of 0.03% as mobile phase B for gradient elution, and a total of 34 common peaks are calibrated, and 7 chromatographic peaks are identified; the fingerprint spectrum obtained by the present application can perform a more comprehensive quality evaluation of Niuhuang Qingxin Pills, and the time is short, which can improve the quality detection efficiency. 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 Niuhuang Qingxin Pills.

[0038] This application simultaneously determines the contents of gardenia glycoside, baicalin, epiberberine, coptisine hydrochloride, palmatine hydrochloride, berberine hydrochloride and bilirubin in the preparation. Compared with the pharmacopoeia method, this method is simpler and easier to operate, and can more comprehensively evaluate the quality of Niuhuang Qingxin Pills.

[0039] In summary, the present application includes at least one of the following beneficial technical effects:

[0040] This application adopts high performance liquid chromatography and reasonably controls the chromatographic conditions. The fingerprint spectrum obtained can conduct a more comprehensive quality evaluation of Niuhuang Qingxin Pills. 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 Niuhuang Qingxin Pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the sample spectrum of ten batches of Niuhuang Qingxin Pills;

[0042] Figure 2 It is the fingerprint of Niuhuang Qingxin Pills. DETAILED DESCRIPTION

[0043] 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.

[0044] Example:

[0045] 1. Experimental instruments and reagents

[0046] 1. Experimental instruments

[0047] Waters e2695 high performance liquid chromatograph;

[0048] JIEMENG brand ultrasonic cleaner JP-080S;

[0049] Agilent5 TC-C18 column (4.6×250 mm, 4.6 μm).

[0050] 2. Experimental reagent acetonitrile: ACS, USA;

[0051] Phosphoric acid: Sinopharm Chemical Reagent Co., Ltd.;

[0052] Methanol: Nanjing Chemical Reagent Co., Ltd.;

[0053] Water: laboratory purified water;

[0054] Niuhuang Qingxin Pills: Nanjing Tongrentang Pharmaceutical Co., Ltd. (180608).

[0055] 3. Reference substances and reference medicinal materials

[0056] Angelica (120927-201617), Cinnamon (121363-201703), Dried Ginger (120942-201510), Bitter Almond (121554-201204), Bupleurum (120992-201509), Scutellaria (120955-201810),

[0057] Bletilla striata (121498-201804), Ligusticum chuanxiong (120918-201813), Platycodon grandiflorum (121028-20112),

[0058] Licorice (120904-202021), Pollen Typhae (121225-201804), White Peony Root (120905-201610),

[0059] Saposhnikovia divaricata (120947-201810), Ginseng (120917-201712), Atractylodes macrocephala (120925-201812),

[0060] Bezoar (121328-201202), Ophiopogon japonicus (121013-201711), Borneol (110743-20106),

[0061] Antelope horn (121064-201004), Chinese yam (121137-201807), donkey-hide gelatin (121271-201904), Poria cocos (121117-201910), jujube (121040-201809), buffalo horn concentrated powder (121627-202002): China Food and Drug Administration;

[0062] Soybean yellow roll (201100300), Liushenqu (210111801), artificial musk (210703300), cinnabar (210402000), realgar (201107600): Nanjing Tongrentang Co., Ltd. medicinal materials;

[0063] 2. Fingerprint Detection Method of Niuhuang Qingxin Pills

[0064] 1. Preparation of test solution:

[0065] S1. Take 3 g of Niuhuang Qingxin Pills, accurately weigh, chop and mix, add 4.5 g of diatomaceous earth, mix and grind to obtain a mixture; S1.1. Post-treat the mixture prepared in step S1, the steps are as follows: freeze the mixture at -10 ° C for 12 minutes, and then dry the mixture at a vacuum pressure of 10 Pa and a temperature of 45 ° C for 50 minutes;

[0066] S2. Take 6 g of the mixture prepared in step S1.1, weigh it accurately, put it in a stoppered brown conical flask, add 25 mL of 75% methanol by volume, extract by ultrasonic for 30 min, filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0067] 2. Preparation of reference solution: Weigh 0.5 g each of borneol, antelope horn, musk, pollen, cinnabar, bezoar, realgar, buffalo horn, yam, dried ginger, jujube, chuanxiong, ginseng, ophiopogon, cinnamon, bupleurum, saposhnikovia, scutellaria, donkey-hide gelatin, atractylodes, liushenqu, atractylodes, bitter almond, licorice, angelica, soybean yellow roll, white peony root, poria, and platycodon, add 25 mL of methanol with a volume concentration of 75% to obtain a reference solution;

[0068] 3. Determination of the fingerprint of Niuhuang Qingxin Pills by HPLC: 10 μl of the test solution and 10 μl of the reference solution were injected into the HPLC instrument respectively to determine the fingerprint. Figure 2 The similarity between the obtained fingerprint spectrum and the standard fingerprint spectrum calculated by software shall not be less than 90%.

[0069] The chromatographic conditions were as follows: chromatographic column: C18; mobile phase A: acetonitrile; mobile phase B: 0.03% phosphoric acid aqueous solution; column temperature: 35°C; mobile phase flow rate: 1 mL / min; injection volume: 10 μl; detection wavelength: 250 nm.

[0070] The chromatographic column was Agilent TC-C18.

[0071] The gradient elution conditions were as follows: 0-30 min, 12% A; 30-43 min, 12-17% A; 43-63 min, 17% A; 63-85 min, 17-25% A; 85-95 min, 25% A; 95-110 min, 25-31% A; 110-165 min, 31-45% A; 165-210 min, 45-70% A.

[0072] 3. Methodological Investigation

[0073] 1. Stability test

[0074] Accurately pipette 10 μL of the test solution from the same batch, and measure at 0 h, 4 h, 8 h, 16 h, 20 h, and 24 h, respectively. Calculate the RSD value based on the retention time and peak area of ​​each characteristic peak.

[0075] The results showed that the retention time RSD of each characteristic peak was less than 2%, and the RSD value of the selected common peak area within 24 hours was less than 5%, indicating that under this condition, the test results of the test solution were stable within 24 hours, as shown in Table 1.

[0076] Table 1 Stability test results

[0077]

[0078] 2. Precision test

[0079] Accurately pipette 10 μl of the test solution 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.

[0080] The results showed that the retention time RSD of each characteristic peak was less than 1%, the peak area RSD was less than 3%, and the precision was good (see Table 2).

[0081] Table 2 Precision test results

[0082]

[0083] 3. Repeatability test: Take 6 samples of the same batch number, operate according to the test solution preparation method, make test solutions, inject samples for measurement, and 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 1%, and the peak area RSD was less than 5%, indicating that the method had good repeatability (see Table 3).

[0085] Table 3 Repeatability test results

[0086]

[0087] 4. Durability

[0088] Take the same batch of samples and operate according to the method under the item of test solution preparation to prepare the test solution. Under the same chromatographic conditions, measure them on Waters e2695 HPLC and Agilent 1100 HPLC, and compare the similarity between the two spectra.

[0089] 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 liquid chromatographs.

[0090] 5. Determination of ten batches of samples

[0091] Ten different batches of Niuhuang Qingxin Pills samples were obtained within the validity period and were prepared according to the test solution preparation method. The test solutions were determined according to the method, the common peak areas were calculated and the fingerprints were generated. Figure 1 .

[0092] The present application adopts high performance liquid chromatography and reasonably controls the chromatographic conditions, uses acetonitrile as mobile phase A and a 0.03% volume concentration of phosphoric acid aqueous solution as mobile phase B for gradient elution, calibrates a total of 34 common peaks, and identifies 7 chromatographic peaks therein; the fingerprint spectrum obtained in the present application can conduct a more comprehensive quality evaluation of Niuhuang Qingxin Pills in a short time, and can improve the efficiency of quality detection. At the same time, the method is simple, accurate, repeatable and durable, and can provide a scientific basis for the quality evaluation and control of Niuhuang Qingxin Pills.

[0093] 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 detection method for Niuhuang Qingxin Pills, characterized in that: Here are the steps: (1) Preparation of test solution; S1, weighing Niuhuang Qingxin Pills, crushing them, adding diatomaceous earth to mix them, and obtaining a mixture; S2. Add methanol solution to the mixture, perform ultrasonic extraction, and filter to obtain a test solution; (2) Preparation of reference solution; (3) Determination of the fingerprint of Niuhuang Qingxin Pills by HPLC; The test solution and the reference solution were injected into the HPLC to determine the fingerprints; 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 0.01%-0.05%; Column temperature: 30-40℃; mobile phase flow rate: 0.5-1.5mL / min; injection volume: 5-15μl; detection wavelength: 200-360nm; The gradient elution conditions in step (3) are as follows: 0-30 min, 12% A; 30-43 min, 12-17% A; 43-63 min, 17% A; 63-85 min, 17-25% A; 85-95 min, 25% A; 95-110 min, 25-31% A; 110-165 min, 31-45% A; 165-210 min, 45-70% A; The mass ratio of Niuhuang Qingxin Pills to diatomaceous earth in step S1 is 1:(1-2.5); the amount of the mixture added in step S2 is 3-12 g; the amount of the methanol solution added is 15-30 mL; The volume concentration of the methanol solution in step S2 is 50-80%.

2. The fingerprint detection method of Niuhuang Qingxin Pills according to claim 1, characterized in that: The mixture prepared in step S1 is post-treated as follows: the mixture is frozen at -5 to -15°C for 10-15 minutes, and then the mixture is dried at a vacuum pressure of 1-20 Pa and a temperature of 35-55°C for 40-60 minutes.

3. The fingerprint detection method of Niuhuang Qingxin Pills 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 detection method of Niuhuang Qingxin Pills according to claim 1, characterized in that: The time of ultrasonic extraction in step S2 is 25-40 minutes.

5. The fingerprint detection method of Niuhuang Qingxin Pills according to claim 1, characterized in that: The preparation steps of the reference solution in step (2) are as follows: weigh borneol, antelope horn, musk, pollen, cinnabar, bezoar, realgar, buffalo horn, yam, dried ginger, jujube, chuanxiong, ginseng, ophiopogon, cinnamon, bupleurum, saposhnikovia, scutellaria, donkey-hide gelatin, atractylodes, six-spirit song, atractylodes, bitter almond, licorice, angelica, soybean yellow roll, white peony root, poria, platycodon, add methanol solution to dissolve, and obtain a reference solution.

6. A fingerprint detection method for Niuhuang Qingxin Pills according to claim 1, characterized in that: There are 7 common peaks in the fingerprint of Niuhuang Qingxin Pills, and the retention times of the common peaks are: Peak 1: relative retention time 22.121; Peak 2: relative retention time 43.267; Peak 3: relative retention time 57.186; Peak 4: relative retention time 79.595; Peak 5: relative retention time 91.140; Peak 6: relative retention time 96.082; Peak 7: relative retention time 132.415.

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