Identification and application of periostracum cicadae medicinal materials, pieces, standard decoction, formula granules and Chinese patent drugs

Abstract

The application discloses a kind of cicada slough medicinal materials, decoction piece, standard decoction, formula granule and Chinese patent medicine identification method, it is related to traditional Chinese medicine identification technical field.Detailedly, the identification method includes: with m / z double charge 493.77→572.35, 493.77→798.45 and m / z three electric charges 918.75→798.45, 918.75→885.48, 988.51→1380.73 and 988.51→696.43 as detection ion pair detection;If there is the chromatographic peak corresponding to cicada slough control medicinal materials in ion flow chromatogram, and signal-to-noise ratio is greater than 3:1, then the sample to be detected contains cicada slough medicinal materials, cicada slough decoction piece, cicada slough standard decoction and / or cicada slough formula granule.The present application can effectively identify cicada slough medicinal materials and various preparations thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine identification, and particularly relates to a method for identifying Cicada Slough medicinal materials, decoction pieces, standard decoction, formula granules and Chinese patent medicines and application thereof. BACKGROUND

[0002] The Chinese Pharmacopoeia 2020 edition stipulates that the base of Cicada Slough is Cryptotympana pustulata Fabricius of Cicadidae. Cicada Slough is named Cicada, which is first recorded in Shennong's Herbal Classic and listed as medium grade. It has the effects of dispersing wind-heat, benefiting the throat, promoting rash, clearing the eyes, relieving convulsions and tetanus. It is often used in clinical practice for treating wind-heat cold, sore throat, hoarseness, measles, wind, itching, red eyes, and convulsions. Modern pharmacological studies have shown that Cicada Slough has anticonvulsant, sedative and antipyretic activities.

[0003] Cicada Slough is an animal-based traditional Chinese medicine with a large amount of use. There are many counterfeit or adulterated products on the market. Common counterfeits include mountain cicadas, southern cicadas, Chinese cicadas, and chirping cicadas. They are similar in appearance and shape, and it is difficult to accurately identify them using traditional identification methods, especially after being extracted with water to form standard decoction or other dosage forms. It is more difficult to effectively identify them, especially for similar species. In order to ensure the quality and efficacy of Cicada Slough, a method is needed to accurately and quickly identify the genuine and counterfeit products of Cicada Slough. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for identifying Cicada Slough medicinal materials, decoction pieces, standard decoction and formula granules, which can effectively identify Cicada Slough and its counterfeit products.

[0005] The present application also solves the technical problem of providing a method for identifying Cicada Slough Chinese patent medicines.

[0006] The present application also solves the technical problem of providing a method for identifying Cicada Slough Chinese patent medicines.

[0007] In order to solve the above technical problems, the present application provides a method for identifying Cicada Slough medicinal materials, decoction pieces, standard decoction and formula granules, which comprises:

[0008] The sample to be detected is extracted with an extraction solution and then trypsin is added for enzymolysis to obtain a first test solution;

[0009] The Cicada Slough control medicinal material is extracted with an extraction solution and then trypsin is added for enzymolysis to obtain a control medicinal material solution;

[0010] The first test solution and the control medicinal material solution are injected into a liquid chromatograph-mass spectrometer for detection to obtain an ion flow chromatogram;

[0011] The mass spectrometer adopts positive ion mode for multiple reaction monitoring, and m / z double charge 493.77→572.35, 493.77→798.45, m / z triple charge 918.75→798.45, 918.75→885.48, 988.51→1380.73 and 988.51→696.43 are selected as detection ion pairs for detection.

[0012] If there is a chromatographic peak corresponding to the cicada slough control medicinal material in the ion chromatogram, and the signal-to-noise ratio is greater than 3:1, then the sample to be detected contains cicada slough medicinal material, cicada slough decoction pieces, cicada slough standard decoction and / or cicada slough formula granules.

[0013] Correspondingly, the application also discloses a method for identifying cicada slough Chinese patent medicine, which comprises the following steps:

[0014] The sample to be detected is extracted by the extraction solution, and then trypsin is added for enzymolysis to obtain a second sample solution;

[0015] The cicada slough control medicinal material is extracted by the extraction solution, and then trypsin is added for enzymolysis to obtain a control medicinal material solution;

[0016] The second sample solution and the control medicinal material solution are injected into a liquid chromatograph-mass spectrometer for detection to obtain an ion chromatogram;

[0017] The mass spectrometer adopts positive ion mode for multiple reaction monitoring, and m / z double charge 493.77→572.35 and 493.77→798.45 are selected as detection ion pairs for detection.

[0018] If there is a chromatographic peak corresponding to the cicada slough control medicinal material in the ion chromatogram, and the signal-to-noise ratio is greater than 3:1, then the sample to be detected contains cicada slough medicinal material, cicada slough standard decoction and / or cicada slough formula granules.

[0019] As an improvement of the above technical scheme, the detection conditions of the liquid chromatograph comprise: gradient elution is carried out by taking acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B; wherein the volume concentration of the formic acid aqueous solution is 0.05-0.3%.

[0020] The gradient elution is carried out according to the following procedure:

[0021] 0-6 min, mobile phase A from 10% to 30%, and mobile phase B from 90% to 70%;

[0022] 6-8 min, mobile phase A from 30% to 90%, and mobile phase B from 70% to 10%;

[0023] 8-10 min, mobile phase A is 90%, and mobile phase B is 10%;

[0024] 10-11 min, mobile phase A from 90% to 10%, mobile phase B from 10% to 90%;

[0025] 11-16 min, mobile phase A is 10%, mobile phase B is 90%.

[0026] As an improvement of the above technical solution, the volume concentration of the formic acid aqueous solution is 0.1%.

[0027] As an improvement of the above technical solution, the detection conditions of the liquid chromatography include: the stationary phase is octadecylsilane bonded silica gel, the particle size is 1.6-1.9 μm; the column length of the chromatographic column is 50-150 mm, the column diameter is 2.1-4.6 mm, the column temperature is 28-32℃, the flow rate is 0.1-0.5 mL / min, and the injection volume is 1-3 μL.

[0028] As an improvement of the above technical solution, the detection conditions of the liquid chromatography include: the stationary phase is octadecylsilane bonded silica gel, the particle size is 1.6-1.9 μm, the column length of the chromatographic column is 100 mm, the column diameter is 2.1 mm, the column temperature is 30℃, the flow rate is 0.3 mL / min, and the injection volume is 2 μL.

[0029] As an improvement of the above technical solution, the detection conditions of the mass spectrometer include: the flow rate of atomizing gas is 1-5 L / min, the flow rate of heater is 8-12 L / min, the interface temperature is 280-320℃, the desolvation temperature is 520-530℃, the DL temperature is 240-260℃, the heating block temperature is 380-420℃, and the flow rate of drying gas is 8-12 L / min.

[0030] As an improvement of the above technical solution, the detection conditions of the mass spectrometer include: the flow rate of atomizing gas is 3 L / min, the flow rate of heating gas is 10 L / min, the interface temperature is 300℃, the desolvation temperature is 526℃, the DL temperature is 250℃, the heating block temperature is 400℃, and the flow rate of drying gas is 10 L / min.

[0031] As an improvement of the above technical solution, the detection conditions of the mass spectrometer include: the collision energy of the ion pair 493.77→572.35 is -27 V, the Q1 deviation is -20, and the Q3 deviation is -40;

[0032] The collision energy of the ion pair m / z 493.77→798.45 is -17 V, the Q1 deviation is -19, and the Q3 deviation is -40;

[0033] The collision energy of the ion pair m / z 918.75→798.45 is -40 V, the Q1 deviation is -28, and the Q3 deviation is -40;

[0034] The collision energy of the detected ion pair m / z 988.51→1380.73 is -25V, the Q1 deviation is -24, and the Q3 deviation is -48;

[0035] The collision energy of the detected ion pair m / z 988.51→1380.73 is -25V, the Q1 deviation is -24, and the Q3 deviation is -48;

[0036] The collision energy of the detected ion pair m / z 988.51→1380.73 is -25V, the Q1 deviation is -24, and the Q3 deviation is -48.

[0037] As an improvement of the above technical solution, the preparation method of the control medicinal material solution is:

[0038] Take 0.2-1g of Periostracum Cicadae control medicinal material, add 0.5-3vol% ammonium bicarbonate solution 20-30mL, heat reflux for 10-30 minutes, filter with 0.22μm microporous filter membrane, take 0.8-1.5mL of the filtrate, add 300-600μL of trypsin solution, shake well, and enzymolysis at 30-40℃ for 10-24 hours, and it is obtained.

[0039] The preparation method of the trypsin solution is: take trypsin, add 1vol% ammonium bicarbonate solution to prepare a solution containing 1mg per 1mL, and it is obtained.

[0040] As an improvement of the above technical solution, the preparation method of the control medicinal material solution is:

[0041] Take 0.5g of Periostracum Cicadae control medicinal material, add 1vol% ammonium bicarbonate solution 25mL, heat reflux for 15 minutes, filter with 0.22μm microporous filter membrane, take 1mL of the filtrate, add 400μL of trypsin solution, shake well, and enzymolysis at 37℃ for 12 hours, and it is obtained.

[0042] The preparation method of the trypsin solution is: take trypsin, add 1vol% ammonium bicarbonate solution to prepare a solution containing 1mg per 1mL, and it is obtained.

[0043] As an improvement of the above technical solution, the preparation method of the control medicinal material solution is:

[0044] Take 0.1-1g of Periostracum Cicadae control medicinal material, add 0.5-3vol% ammonium bicarbonate solution 20-30mL, heat reflux for 10-30 minutes, filter with 0.22μm microporous filter membrane, take 0.8-1.5mL of the filtrate, add 300-600μL of trypsin solution, shake well, and enzymolysis at 30-40℃ for 10-24 hours, and it is obtained.

[0045] The preparation method of the trypsin solution is as follows: taking trypsin, adding 1vol% ammonium bicarbonate solution to prepare a solution containing 1mg per 1mL.

[0046] The sample to be detected is medicinal materials or decoction pieces.

[0047] As an improvement of the above technical solution, the preparation method of the first test solution is as follows:

[0048] Taking 0.5g of the sample to be detected, adding 25mL of 1vol% ammonium bicarbonate solution, heating and refluxing for 15 minutes, filtering through a 0.22mu m microporous filter, taking 1mL of the filtrate, adding 400mu L of the trypsin solution, shaking uniformly, and enzymolysis at 37 DEG C for 12 hours, the first test solution is obtained.

[0049] The preparation method of the trypsin solution is as follows: taking trypsin, adding 1vol% ammonium bicarbonate solution to prepare a solution containing 1mg per 1mL.

[0050] The sample to be detected is medicinal materials or decoction pieces.

[0051] As an improvement of the above technical solution, the preparation method of the first test solution is as follows:

[0052] Taking 0.5g of the sample to be detected, adding 25mL of 1vol% ammonium bicarbonate solution, heating and refluxing for 15 minutes, filtering through a 0.22mu m microporous filter, taking 1mL of the filtrate, adding 400mu L of the trypsin solution, shaking uniformly, and enzymolysis at 37 DEG C for 12 hours, the first test solution is obtained.

[0053] The preparation method of the trypsin solution is as follows: taking trypsin, adding 1vol% ammonium bicarbonate solution to prepare a solution containing 1mg per 1mL.

[0054] The sample to be detected is standard decoction or formula granules.

[0055] As an improvement of the above technical solution, the preparation method of the first test solution is as follows:

[0056] Taking 0.5g of the sample to be detected, adding 25mL of 1vol% ammonium bicarbonate solution, heating and refluxing for 15 minutes, filtering through a 0.22mu m microporous filter, taking 1mL of the filtrate, adding 400mu L of the trypsin solution, shaking uniformly, and enzymolysis at 37 DEG C for 12 hours, the first test solution is obtained.

[0057] The preparation method of the trypsin solution is as follows: taking trypsin, adding 1vol% ammonium bicarbonate solution to prepare a solution containing 1mg per 1mL.

[0058] The sample to be detected is standard decoction.

[0059] As the improvement of the above technical scheme, the preparation method of the first test solution is:

[0060] 0.1g of the sample to be detected is taken, 50mL of 1vol% ammonium bicarbonate solution is added, ultrasonic treatment is performed for 30 minutes, filtration is performed through a 0.22mu m microporous filter, 1mL of the filtrate is taken, 100mu L of trypsin solution is added, shaking is performed, and 12 hours of constant temperature enzymolysis is performed at 37 DEG C, and the solution is obtained;

[0061] The preparation method of the trypsin solution is as follows: trypsin is taken, 1vol% ammonium bicarbonate solution is added, and a solution containing 1mg per 1mL is prepared, and the solution is obtained.

[0062] The sample to be detected is a formula granule.

[0063] As the improvement of the above technical scheme, the preparation method of the second test solution is:

[0064] 0.01-1g of the sample to be detected is taken, 20-40mL of 0.5-3vol% ammonium bicarbonate solution is added, ultrasonic treatment is performed for 20-40 minutes, filtration is performed through a 0.22mu m microporous filter, 0.8-1.5mL of the filtrate is taken, 50-300mu L of trypsin solution is added, shaking is performed, 10-24 hours of constant temperature enzymolysis is performed at 30-40 DEG C, and the solution is obtained.

[0065] The preparation method of the trypsin solution is as follows: trypsin is taken, 1vol% ammonium bicarbonate solution is added, and a solution containing 1mg per 1mL is prepared, and the solution is obtained.

[0066] The sample to be detected is a Chinese patent medicine.

[0067] Correspondingly, the application also discloses application of the identification method in identification of cicadas and counterfeits, wherein the cicadas are cicada medicinal materials, cicada decoction pieces, cicada standard decoction and / or cicada formula granules.

[0068] As the improvement of the above technical scheme, the counterfeits are selected from one or more of the following: liang cicadas, tettigoniids, calling cicadas, sinomaenidae, and phasmatodea. It should be noted that the counterfeits identifiable by the application are not limited to the above-mentioned species, for example, the application can also identify the following counterfeits: mountain cicadas, southern acridids, mongolian cold cicadas, and south fine cicadas, but are not limited thereto.

[0069] The counterfeits are prepared into medicinal materials, decoction pieces, standard decoction or formula granules.

[0070] The application has the following beneficial effects:

[0071] The identification method of the tussock caterpillar, the standard decoction and the formula granules of the application can effectively distinguish common counterfeits of the tussock caterpillar, such as the common cicada, the mole cricket, the singing cicada, the Sinian horse cicada and the weevil cicada, and provides a good guide for the quality control of the tussock caterpillar medicinal material. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 is the mass spectrum identification diagram (m / z 493.77) of the control medicinal material, G1-G7 samples in Example 2, wherein the left side is m / z 493.77→572.35; and the right side is m / z 493.77→798.45;

[0073] Figure 2 is the mass spectrum identification diagram (m / z 493.77) of G8-G15 samples in Example 2, wherein the left side is m / z 493.77→572.35; and the right side is m / z 493.77→798.45;

[0074] Figure 3 is the mass spectrum identification diagram (m / z 493.77) of G16-G17, GT1-GT6 samples in Example 2, wherein the left side is m / z 493.77→572.35; and the right side is m / z 493.77→798.45;

[0075] Figure 4 is the mass spectrum identification diagram (m / z 493.77) of GT7-GT14 samples in Example 2, wherein the left side is m / z 493.77→572.35; and the right side is m / z 493.77→798.45;

[0076] Figure 5 is the mass spectrum identification diagram (m / z 493.77) of GT15-GT17, CG1-CG3 samples in Example 2, wherein the left side is m / z 493.77→572.35; and the right side is m / z 493.77→798.45;

[0077] Figure 6 is the mass spectrum identification diagram (m / z 918.75) of the control medicinal material, G1-G7 samples in Example 2, wherein the left side is m / z 918.75→798.45; and the right side is m / z 918.75→885.48;

[0078] Figure 7 is the mass spectrum identification diagram (m / z 918.75) of G8-G15 samples in Example 2, wherein the left side is m / z 918.75→798.45; and the right side is m / z 918.75→885.48;

[0079] Figure 8is the mass spectrum identification chart (m / z 918.75) of G16-G17, GT1-GT6 samples in Example 2, wherein the left side is m / z 918.75→798.45; the right side is m / z 918.75→885.48;

[0080] Figure 9 is the mass spectrum identification chart (m / z 918.75) of GT7-GT14 samples in Example 2, wherein the left side is m / z 918.75→798.45; the right side is m / z 918.75→885.48;

[0081] Figure 10 is the mass spectrum identification chart (m / z 918.75) of GT15-GT17, CG1-CG3 samples in Example 2, wherein the left side is m / z 918.75→798.45; the right side is m / z 918.75→885.48;

[0082] Figure 11 is the mass spectrum identification chart (m / z 988.51) of control medicinal materials, G1-G7 samples in Example 2, wherein the left side is m / z 988.51→1380.73; the right side is m / z 988.51→696.43;

[0083] Figure 12 is the mass spectrum identification chart (m / z 988.51) of G8-G15 samples in Example 2, wherein the left side is m / z 988.51→1380.73; the right side is m / z 988.51→696.43;

[0084] Figure 13 is the mass spectrum identification chart (m / z 988.51) of G16-G17, GT1-GT6 samples in Example 2, wherein the left side is m / z 988.51→1380.73; the right side is m / z 988.51→696.43;

[0085] Figure 14 is the mass spectrum identification chart (m / z 988.51) of GT7-GT14 samples in Example 2, wherein the left side is m / z 988.51→1380.73; the right side is m / z 988.51→696.43;

[0086] Figure 15 is the mass spectrum identification chart (m / z 988.51) of GT15-GT17, CG1-CG3 samples in Example 2, wherein the left side is m / z 988.51→1380.73; the right side is m / z 988.51→696.43;

[0087] Figure 16is the mass spectrum identification diagram (m / z 918.75) of the control medicinal materials, cicada, katydid, ching-ching cicada, sinian horse cicada, and cicada in Example 2, wherein the left side is m / z 918.75→798.45; and the right side is m / z 918.75→885.48;

[0088] Figure 17 is the mass spectrum identification diagram (m / z 918.75) of the control medicinal materials, cicada, katydid, ching-ching cicada, sinian horse cicada, and cicada in Example 2, wherein the left side is m / z 918.75→798.45; and the right side is m / z 918.75→885.48;

[0089] Figure 18 is the mass spectrum identification diagram (m / z 918.75) of the control medicinal materials, cicada, katydid, ching-ching cicada, sinian horse cicada, and cicada in Example 2, wherein the left side is m / z 918.75→798.45; and the right side is m / z 918.75→885.48;

[0090] Figure 19 is the mass spectrum identification diagram (m / z 918.75) of the control medicinal materials, cicada, katydid, ching-ching cicada, sinian horse cicada, and cicada in Example 2, wherein the left side is m / z 918.75→798.45; and the right side is m / z 918.75→885.48. DETAILED DESCRIPTION

[0091] In order to make the object, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with the aid of drawings and specific embodiments.

[0092] Establishment of the liquid chromatography-mass spectrometry identification method for cicada slough medicinal materials, decoction pieces, standard decoction and formula granules

[0093] I. Preparation of test sample solution and control medicinal material solution

[0094] Cicada slough medicinal materials / decoction pieces, counterfeit products: take the powder (pass through No. 3 sieve) of the product, take about 0.5 g, add 1% ammonium bicarbonate solution 25 mL, heat reflux for 15 minutes, filter with a microporous filter membrane, take 1 mL of the filtrate, put it into a sample bottle, add trypsin solution 400 μL (take sequence analysis trypsin, add 1% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL, prepare immediately before use), shake well, and incubate at 37°C for 12 hours, and then obtain.

[0095] Cicada slough standard decoction: Take the product in proper amount, grind finely, take about 0.1 g, add 1% ammonium bicarbonate solution 25 mL, ultrasonic treatment for 15 minutes, cool, shake well, filter with microporous filter membrane, take 1 mL of the filtrate, put into a sample bottle, add trypsin solution 200 μL (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL, prepare immediately before use), shake well, enzymolysis at 37°C for 12 hours.

[0096] Cicada slough formula granules: Take the product in proper amount, grind finely, take about 0.1 g, add 1% ammonium bicarbonate solution 50 mL, ultrasonic treatment for 30 minutes, filter with microporous filter membrane, take 1 mL of the filtrate, put into a sample bottle, add trypsin solution 100 μL (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL, prepare immediately before use), shake well, enzymolysis at 37°C for 12 hours.

[0097] Cicada slough formula granules: Take the product in proper amount, grind finely, take about 0.1 g, add 1% ammonium bicarbonate solution 50 mL, ultrasonic treatment for 30 minutes, filter with microporous filter membrane, take 1 mL of the filtrate, put into a sample bottle, add trypsin solution 100 μL (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL, prepare immediately before use), shake well, enzymolysis at 37°C for 12 hours.

[0098] II. Preparation of control medicinal material solution

[0099] Take cicada slough control medicinal material about 0.5 g, add 1% ammonium bicarbonate solution 25 mL, heat reflux for 15 minutes, filter with microporous filter membrane, take 1 mL of the filtrate, put into a sample bottle, add trypsin solution 400 μL (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL, prepare immediately before use), shake well, enzymolysis at 37°C for 12 hours.

[0100] III. Chromatography and mass spectrometry conditions

[0101] Chromatography conditions: octadecylsilane bonded silica gel as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 to 1.9 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to the provisions in Table 1; flow rate is 0.3 mL per minute; column temperature is 30°C; injection volume is 2 μL.

[0102] Table 1 Gradient elution table

[0103]

[0104] Mass spectrometry conditions: mass spectrometry detector, electrospray positive ion mode (ESI+); atomizing gas flow 3 L / min; heating gas flow 10 L / min; interface temperature 300°C; desolvation temperature 526°C; DL temperature 250°C; heating block temperature 400°C; dry gas flow 10 L / min; collision energy and Q1, Q3 deviation as shown in Table 2. Multiple reaction monitoring (MRM) was performed, and the mass-to-charge ratio (m / z) 493.77 (double charge)→572.35, m / z 493.77 (double charge)→798.45, m / z 918.75 (triple charge)→798.45, m / z 918.75 (triple charge)→885.48, m / z 988.51 (triple charge)→1380.73 and m / z 988.51 (triple charge)→696.43 were selected as the detection ion pairs. The cicada slough control drug material solution was taken, 2 μL was injected, and the signal-to-noise ratio of the MRM chromatographic peak determined by the above detection ion pairs should all be greater than 3:1.

[0105] Table 2 Collision energy and Q1, Q3 deviation of detection ion pairs

[0106]

[0107] Four, determination method

[0108] Respectively, 2 μL of the test sample solution and the control drug material solution was precisely taken and injected into the high performance liquid chromatography-mass spectrometry instrument for determination.

[0109] Example 2 Determination and identification of cicada slough drug material, standard decoction, formula granules and counterfeit

[0110] The samples in this example were a total of 42 batches, including 1 batch of cicada slough control drug material, 17 batches of cicada slough drug material (batch numbers: G1-G17), 17 batches of standard decoction (batch numbers: GT1-GT17), 3 batches of cicada slough formula granules (batch numbers: CG1-CG3), and counterfeit products including liao cicada, tree cricket, singing cicada, Sinian horse cicada and stink cicada.

[0111] The samples were identified by the identification method established in Example 1. The results are shown in Table 3 and Figures 1-18 .

[0112] Table 3 Mass spectrometry determination results of cicada slough drug material, standard decoction, formula granule samples and counterfeit products

[0113]

[0114]

[0115] From Table 3, Figures 1-18As can be seen from the above table, the ion chromatogram of the test sample of Cicada Slough medicinal material, Cicada Slough standard decoction and Cicada Slough dispensing granules shows the chromatographic peaks with the same retention time as the chromatographic peaks of the Cicada Slough control medicinal material solution, while the ion chromatogram of the test sample of the counterfeit (Liaochan, Oedipoda spp., Chionaspis spp., Sinochelidonium spp., and Tangchun) does not show any detectable chromatographic peaks.

[0116] Example 3: Establishment of a liquid chromatography-mass spectrometry identification method for Cicada Slough Chinese patent medicines

[0117] I. Preparation of test sample solution and control medicinal material solution

[0118] Cicada Slough-containing Chinese patent medicines and counterfeit-containing Chinese patent medicines: Take the product by an appropriate amount, add 25 mL of 1% ammonium bicarbonate solution, and ultrasonically treat for 30 minutes. After cooling, shake well, filter through a microporous filter, take 1 mL of the filtrate, and place it in a sample bottle. Add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL, and prepare it immediately before use), shake well, and incubate at 37°C for 12 hours to obtain the test sample solution.

[0119] II. Preparation of control medicinal material solution

[0120] Take about 0.5 g of Cicada Slough control medicinal material, add 25 mL of 1% ammonium bicarbonate solution, heat and reflux for 15 minutes, filter through a microporous filter, take 1 mL of the filtrate, and place it in a sample bottle. Add 400 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL, and prepare it immediately before use), shake well, and incubate at 37°C for 12 hours to obtain the test sample solution.

[0121] III. Chromatographic and mass spectrometric conditions

[0122] Chromatographic conditions: octadecylsilane-bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 to 1.9 μm); acetonitrile as the mobile phase A, and 0.1% formic acid solution as the mobile phase B, gradient elution according to the provisions in Table 4; flow rate 0.3 mL per minute; column temperature 30°C; injection volume 2 μL.

[0123] Table 4: Gradient elution table

[0124]

[0125] Mass spectrometry conditions: mass spectrometry detector, electrospray positive ion mode (ESI+); atomization gas flow 3 L / min; heating gas flow 10 L / min; interface temperature 300°C; desolvation temperature 526°C; DL temperature 250°C; heating block temperature 400°C; dry gas flow 10 L / min; collision energy and Q1, Q3 deviation as shown in Table 5. Multiple reaction monitoring (MRM) was performed, and the mass-to-charge ratio (m / z) 493.77 (double charge) → 572.35, m / z 493.77 (double charge) → 798.45 were selected as the detection ion pairs. The cicada slough control drug material solution was injected 2 μL, and the signal-to-noise ratio of the MRM chromatographic peak determined by the above detection ion pairs should be greater than 3:1.

[0126] Table 5 Collision energy and Q1, Q3 deviation of detection ion pairs

[0127]

[0128] Four, determination method

[0129] Respectively, 2 μL of the test sample solution and the control drug material solution was precisely taken and injected into the high performance liquid chromatography-mass spectrometry instrument for determination.

[0130] Example 4 Determination of cicada slough Chinese patent medicines

[0131] The samples studied in this example are: Jinyaliyan pills, Ganjie Bingmei tablets, Jinchenzhiyang granules, Mingmushangqing tablets, and Xiaoerqizhen pills.

[0132] The samples were identified by the identification method established in Example 3. The results are shown in Table 6 and Figure 19 .

[0133] Table 6 Mass spectrometry determination results of cicada slough drug materials, standard decoction, formula granules samples and counterfeit products

[0134]

[0135]

[0136] From Table 6, Figure 19 it can be seen that the Chinese patent medicines containing cicada slough all show chromatographic peaks consistent with the chromatographic retention time of the cicada slough control drug material solution.

[0137] The above is the preferred embodiment of the application. It should be noted that for those skilled in the art, without departing from the principles of the application, a number of improvements and refinements can be made, which are also considered within the scope of protection of the application.

Claims

1. A method for identifying cicada molting medicinal materials, processed slices, standard decoctions, and formulated granules, characterized in that, include: The sample to be tested was extracted with an extraction solution and then digested with trypsin to obtain the first test solution. The cicada molting control material was extracted with an extraction solution and then hydrolyzed with trypsin to obtain the control material solution; The first test solution and the reference medicinal material solution were injected into a liquid chromatograph-mass spectrometer for detection, and ion chromatograms were obtained. The mass spectrometer used positive ion mode for multiple reaction monitoring, selecting m / z double charges 493.77→572.35, 493.77→798.45 and m / z triple charges 918.75→798.45, 918.75→885.48, 988.51→1380.73 and 988.51→696.43 as detection ion pairs for detection. If a chromatographic peak corresponding to the cicada molting reference material is present in the ion chromatogram and the signal-to-noise ratio is greater than 3:1, then the sample to be tested contains cicada molting medicinal material, cicada molting slices, cicada molting standard decoction and / or cicada molting formula granules. The detection conditions for liquid chromatography included: an octadecylsilane-bonded silica gel stationary phase with a particle size of 1.6–1.9 μm; a column length of 100 mm and a column diameter of 2.1 mm; gradient elution using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B; wherein the volume concentration of the formic acid aqueous solution was 0.05–0.3%. The gradient elution is performed according to the following procedure: From 0 to 6 minutes, mobile phase A decreased from 10% to 30%, and mobile phase B decreased from 90% to 70%. Over 6-8 minutes, mobile phase A decreased from 30% to 90%, and mobile phase B decreased from 70% to 10%. 8-10 min, mobile phase A is 90%, mobile phase B is 10%; Over 10-11 minutes, mobile phase A decreased from 90% to 10%, and mobile phase B decreased from 10% to 90%. 11-16 min, mobile phase A is 10%, mobile phase B is 90%; The detection conditions of the mass spectrometer include: nebulizing gas flow rate of 1~5L / min, heater flow rate of 8~12L / min, interface temperature of 280~320℃, desolventizing temperature of 520~530℃, DL temperature of 240~260℃, heating block temperature of 380~420℃, and drying gas flow rate of 8~12L / min. Wherein, when the sample to be tested is a medicinal material or decoction piece, the preparation method of the first test solution is as follows: Take 0.1-1g of the sample to be tested, add 20-30mL of 0.5-3vol% ammonium bicarbonate solution, heat under reflux for 10-30 minutes, filter through a 0.22μm microporous membrane, take 0.8-1.5mL of the filtrate, add 300-600μL of trypsin solution, shake well, and enzymatically hydrolyze at 30-40℃ for 10-24 hours to obtain the sample. When the test sample is a standard decoction or formula granules, take 0.05~0.5g of the sample to be tested, add 20~60mL of 0.5~3vol% ammonium bicarbonate solution, sonicate for 10~40 minutes, filter through a 0.22μm microporous membrane, take 0.8~1.5mL of the filtrate, add 50~400μL of trypsin solution, shake well, and enzymatically hydrolyze at 30~40℃ for 10~24 hours to obtain the final product. The trypsin solution is prepared by taking trypsin and adding 1 vol% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL.

2. The identification method as described in claim 1, characterized in that, The volume concentration of the formic acid aqueous solution is 0.1%.

3. The identification method as described in claim 1, characterized in that, The detection conditions for liquid chromatography include: column temperature of 28~32℃, flow rate of 0.1~0.5mL / min, and injection volume of 1~3μL.

4. The identification method as described in claim 1, characterized in that, The detection conditions for liquid chromatography include: column temperature of 30℃, flow rate of 0.3 mL / min, and injection volume of 2 μL.

5. The identification method as described in claim 1, characterized in that, The detection conditions of the mass spectrometer include: nebulizing gas flow rate of 3 L / min, heating gas flow rate of 10 L / min, interface temperature of 300℃, desolvation temperature of 526℃, DL temperature of 250℃, heating block temperature of 400℃, and drying gas flow rate of 10 L / min.

6. The identification method as described in claim 1, characterized in that, The detection conditions of the mass spectrometer include: the collision energy of the detected ion pair m / z 493.77→572.35 is -27V, the Q1 deviation is -20, and the Q3 deviation is -40. The collision energy of the ion pair from m / z 493.77 to 798.45 was -17 V, the Q1 deviation was -19, and the Q3 deviation was -40. The collision energy of the ion pair from m / z 918.75 to 798.45 was -40 V, the Q1 deviation was -28 V, and the Q3 deviation was -40 V. The collision energy of the ion pair from m / z 918.75 to 885.48 was -35V, the Q1 deviation was -28, and the Q3 deviation was -26. The collision energy of the ion pair from m / z 988.51 to 1380.73 was -25 V, the Q1 deviation was -24 V, and the Q3 deviation was -48 V. The collision energy of the ion pair from m / z 988.51 to 696.43 was -54 V, the Q1 deviation was -24 V, and the Q3 deviation was -20 V.

7. The identification method as described in claim 1, characterized in that, The method for preparing the reference medicinal material solution is as follows: Take 0.2-1g of cicada molting reference material, add 20-30mL of 0.5-3vol% ammonium bicarbonate solution, heat under reflux for 10-30 minutes, filter through a 0.22μm microporous membrane, take 0.8-1.5mL of the filtrate, add 300-600μL of trypsin solution, shake well, and enzymatically hydrolyze at 30-40℃ for 10-24 hours to obtain the final product. The trypsin solution is prepared by taking trypsin and adding 1 vol% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL.

8. The identification method as described in claim 1, characterized in that, The method for preparing the reference medicinal material solution is as follows: Take 0.5g of cicada molting reference material, add 25mL of 1vol% ammonium bicarbonate solution, heat under reflux for 15 minutes, filter through a 0.22μm microporous membrane, take 1mL of the filtrate, add 400μL of trypsin solution, shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the final product. The trypsin solution is prepared by taking trypsin and adding 1 vol% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL.

9. The identification method as described in claim 1, characterized in that, The preparation method of the first test solution is as follows: Take 0.5g of the sample to be tested, add 25mL of 1vol% ammonium bicarbonate solution, heat under reflux for 15 minutes, filter through a 0.22μm microporous membrane, take 1mL of the filtrate, add 400μL of trypsin solution, shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the sample. The method for preparing the trypsin solution is as follows: take trypsin and add 1 vol% ammonium bicarbonate solution to prepare a solution containing 1 mg per 1 mL. The sample to be tested is a medicinal material or a decoction piece.

10. The identification method as described in claim 1, characterized in that, The preparation method of the first test solution is as follows: Take 0.1g of the sample to be tested, add 25mL of 1vol% ammonium bicarbonate solution, sonicate for 15 minutes, filter through a 0.22μm microporous membrane, take 1mL of the filtrate, add 200μL of trypsin solution, shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the sample. The sample to be tested was a standard decoction.

11. The identification method as described in claim 1, characterized in that, The preparation method of the first test solution is as follows: Take 0.1g of the sample to be tested, add 50mL of 1vol% ammonium bicarbonate solution, sonicate for 30 minutes, filter through a 0.22μm microporous membrane, take 1mL of the filtrate, add 100μL of trypsin solution, shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the sample. The sample to be tested is a formulation granule.

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