Identification method and application of zaocys dhumnade and product thereof

Abstract

The invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, in particular to an identification method and application of zaocys dhumnade and products thereof. The method for identifying the zaocys dhumnade and the zaocys dhumnade product comprises the following steps: preparing a reference medicinal material solution and a test solution; and determining the reference medicinal material solution and the test solution by using a high performance liquid chromatography-mass spectrometry method, and carrying out gradient elution by using acetonitrile as a mobile phase A and a formic acid aqueous solution as a mobile phase B. According to the present invention, the specificity identification method for the zaocys dhumnade and the zaocys dhumnade product is established through the high performance liquid chromatography-mass spectrometry method, such that the problem of difficult identification due to the lack of the appearance character of the zaocys dhumnade standard decoction, the zaocys dhumnade formula particle, the zaocys dhumnade-containing Chinese patent medicine and other products is solved; and a basis is provided for quality control and evaluation of the zaocys dhumnade and the zaocys dhumnade product, especially the zaocys dhumnade aqueous extract

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and particularly to a method for identifying Zaocys dhumnades and its products and applications thereof. Background Art

[0002] The Chinese Pharmacopoeia (2020 Edition) stipulates that the original plant of Zaocys dhumnades is the dried body of the animal Zaocys dhumnades (Cantor) of the family Colubridae. Zaocys dhumnades was first recorded in "Leigong's Treatise on Processing of Medicinal Materials", which states that "Zaocys dhumnades from Qizhou weighing only three to one tael is excellent, with the head and tail intact, eyes not closed as if alive, and there are reverse hairs on the head for two inches in a row, about half a fen long, with the head and tail facing each other". Zaocys dhumnades has the effects of dispelling wind, dredging collaterals, and stopping convulsions. Clinically, it is used for stubborn wind-damp arthralgia, numbness and contracture, deviation of the mouth and eyes due to stroke, hemiplegia, convulsions and spasms, tetanus, leprosy, scabies and tinea. Modern pharmacological research shows that Zaocys dhumnades has anti-inflammatory, analgesic and other effects.

[0003] As a kind of medicinal snake, due to its remarkable curative effect, limited resources and high price, Zaocys dhumnades is often used as medicine in the form of dried body or powder in the market, losing its original shape and being difficult to distinguish the true from the false with the naked eye. The phenomenon of counterfeiting by lawbreakers is endless, seriously affecting the effectiveness and safety of drugs. Therefore, it is difficult to rely on traditional identification methods, and the accuracy is low. Especially after being made into standard decoctions or other dosage forms by water extraction, it is even more difficult to conduct effective identification.

[0004] With the development of proteomics and molecular biology, breakthrough progress has been made in the specific identification research at the polypeptide and nucleic acid levels, and the identification technologies for various animal-based traditional Chinese medicines are becoming increasingly perfect. Many researchers have used the PCR method (polymerase chain reaction method) to identify Zaocys dhumnades medicinal materials. However, there are few studies on the specific identification and identification of characteristic ion pairs of water extracts of Zaocys dhumnades, such as standard decoctions, traditional Chinese medicine formula granules or other proprietary Chinese medicines containing Zaocys dhumnades.

[0005] Therefore, how to specifically identify Zaocys dhumnades and its products to meet the quality control needs is an urgent technical problem to be solved, and it is also an urgent technical problem to be overcome in the field of quality control of Zaocys dhumnades medicinal materials and their products. Summary of the Invention

[0006] Based on this, the present invention provides a method for identifying Zaocys dhumnades and its products, including the following steps:

[0007] Prepare a reference medicinal material solution and a test sample solution;

[0008] Determine the reference medicinal material solution and the test sample solution by high performance liquid chromatography-mass spectrometry (HPLC-MS), using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution.

[0009] In some of these embodiments, the gradient elution procedure is as follows:

[0010] From 0 to 5 minutes, the volume percentage of mobile phase A increases from 8% to 15%.

[0011] From 5 minutes to 8 minutes, the volume percentage of mobile phase A increases from 15% to 40%.

[0012] In some of these embodiments, in the aqueous formic acid solution, the volume percentage of formic acid is 0.05% - 0.15%.

[0013] In some of these embodiments, the conditions for high performance liquid chromatography include: a chromatographic column filled with octadecylsilane-bonded silica gel.

[0014] In some of these embodiments, the conditions for high performance liquid chromatography include: a flow rate of 0.25 mL / min - 0.35 mL / min.

[0015] In some of these embodiments, the conditions for high performance liquid chromatography include: a column temperature of 25°C - 35°C.

[0016] In some of these embodiments, the conditions for high performance liquid chromatography include: an injection volume of 1 μL - 3 μL.

[0017] In some of these embodiments, the mass spectrometry conditions include: using mass-to-charge ratios m / z 674.00→747.37, m / z 674.00→917.96, m / z 700.33→607.29, and m / z 700.33→869.44 as the detected ion pairs.

[0018] In some of these embodiments, the mass spectrometry conditions include: using the electrospray positive ion mode.

[0019] In some of these embodiments, the mass spectrometry conditions include: multiple reaction monitoring scanning.

[0020] In some of these embodiments, the mass spectrometry conditions include: a nebulizing gas flow rate of 2 L / min - 3 L / min.

[0021] In some of these embodiments, the mass spectrometry conditions include: a heating gas flow rate of 8 L / min - 10 L / min.

[0022] In some of these embodiments, the mass spectrometry conditions include: an interface temperature of 300°C - 400°C.

[0023] In some of these embodiments, the mass spectrometry conditions include: a desolvation temperature of 526°C.

[0024] In some of these embodiments, the mass spectrometry conditions include: a DL temperature of 150°C - 250°C.

[0025] In some of these embodiments, the mass spectrometry conditions include: the heating block temperature is 350°C to 450°C.

[0026] In some of these embodiments, the mass spectrometry conditions include: the drying gas flow rate is 8 L / min to 10 L / min.

[0027] In some of these embodiments, preparing the control medicinal material solution includes the following steps:

[0028] Mix the control medicinal material of Zaocys dhumnades with the first ammonium bicarbonate aqueous solution, perform the first extraction, and take the filtrate and mix it with the first trypsin solution for the first enzymatic hydrolysis.

[0029] In some of these embodiments, in the first ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 0.5% to 1.5%.

[0030] In some of these embodiments, the first extraction is performed by heating under reflux or ultrasonic extraction.

[0031] In some of these embodiments, the first trypsin solution is prepared by mixing 1 mg of trypsin with 1 mL of ammonium bicarbonate aqueous solution with a mass fraction of 0.5% to 1.5%.

[0032] In some of these embodiments, the temperature of the first enzymatic hydrolysis is 35°C to 40°C, and the time of the first enzymatic hydrolysis is 6 h to 14 h.

[0033] In some of these embodiments, preparing the test sample solution includes the following steps:

[0034] Mix the test sample with the second ammonium bicarbonate aqueous solution, perform the second extraction, and take the filtrate and mix it with the second trypsin solution for the second enzymatic hydrolysis.

[0035] In some of these embodiments, in the second ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 0.5% to 1.5%.

[0036] In some of these embodiments, the second extraction is performed by heating under reflux or ultrasonic extraction.

[0037] In some of these embodiments, the second trypsin solution is prepared by mixing 1 mg of trypsin with 1 mL of ammonium bicarbonate aqueous solution with a mass fraction of 0.5% to 1.5%.

[0038] In some of these embodiments, the temperature of the second enzymatic hydrolysis is 35°C to 40°C, and the time of the second enzymatic hydrolysis is 6 h to 14 h.

[0039] In some of these embodiments, the test samples include one or more of the medicinal materials of Zaocys dhumnades, the standard decoction of Zaocys dhumnades, the formula granules of Zaocys dhumnades, the cut pieces of Zaocys dhumnades, the cut pieces of Zaocys dhumnades processed with wine, the standard decoction of Zaocys dhumnades processed with wine, the formula granules of Zaocys dhumnades processed with wine, the Chinese patent medicines containing Zaocys dhumnades, and the counterfeits.

[0040] In some of these embodiments, the counterfeits include one or more of Elaphe carinata, Elaphe taeniura, Elaphe moellendorffi, Ptyas korros, Ptyas mucosus, Agkistrodon acutus, and Naja naja.

[0041] The present invention also provides the application of the above-mentioned identification method in the authenticity identification and detection of the medicinal materials, cut pieces, standard decoctions, and formula granules of Zaocys dhumnades.

[0042] The present invention also provides the application of the above-mentioned identification method in the authenticity identification and detection of the cut pieces, standard decoctions, and formula granules of Zaocys dhumnades processed with wine.

[0043] The present invention also provides the application of the above-mentioned identification method in the authenticity identification and detection of the Chinese patent medicines containing Zaocys dhumnades.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention has established a specific identification method for Zaocys dhumnades and its products by high performance liquid chromatography-mass spectrometry (HPLC-MS), solving the problem of difficult identification due to the lack of appearance characteristics of the standard decoction, formula granules, and Chinese patent medicines containing Zaocys dhumnades. The identification method of the present invention has strong specificity and provides a basis for the quality control and evaluation of Zaocys dhumnades and its products, especially the water extract products of Zaocys dhumnades. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the secondary mass spectrometry diagram of the characteristic ions of Zaocys dhumnades;

[0047] Figure 2 It is the mass spectrometry identification diagram (m / z 674.00) of the medicinal materials of Zaocys dhumnades (a), where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0048] Figure 3 It is the mass spectrometry identification diagram (m / z 674.00) of the medicinal materials of Zaocys dhumnades (b), where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0049] Figure 4 It is the mass spectrometry identification diagram (m / z 674.00) of the cut pieces of Zaocys dhumnades (a), where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0050] Figure 5 Mass spectrometry identification diagram of Zaocys dhumnades decoction pieces (m / z 674.00) (b). Among them, for the left diagram: m / z 674.00 → 747.37, and for the right diagram: m / z 674.00 → 917.96;

[0051] Figure 6 Mass spectrometry identification diagram of standard decoction of Zaocys dhumnades (m / z 674.00) (a). Among them, for the left diagram: m / z 674.00 → 747.37, and for the right diagram: m / z 674.00 → 917.96;

[0052] Figure 7 Mass spectrometry identification diagram of standard decoction of Zaocys dhumnades (m / z 674.00) (b). Among them, for the left diagram: m / z 674.00 → 747.37, and for the right diagram: m / z 674.00 → 917.96;

[0053] Figure 8 Mass spectrometry identification diagram of formula granules of Zaocys dhumnades (m / z 674.00). Among them, for the left diagram: m / z 674.00 → 747.37, and for the right diagram: m / z 674.00 → 917.96;

[0054] Figure 9 Mass spectrometry identification diagram of Zaocys dhumnades medicinal materials (m / z 700.33) (a). Among them, for the left diagram: m / z 700.33 → 607.29, and for the right diagram: m / z 700.33 → 869.44;

[0055] Figure 10 Mass spectrometry identification diagram of Zaocys dhumnades medicinal materials (m / z 700.33) (b). Among them, for the left diagram: m / z 700.33 → 607.29, and for the right diagram: m / z 700.33 → 869.44;

[0056] Figure 11 Mass spectrometry identification diagram of Zaocys dhumnades decoction pieces (m / z 700.33) (a). Among them, for the left diagram: m / z 700.33 → 607.29, and for the right diagram: m / z 700.33 → 869.44;

[0057] Figure 12 Mass spectrometry identification diagram of Zaocys dhumnades decoction pieces (m / z 700.33) (b). Among them, for the left diagram: m / z 700.33 → 607.29, and for the right diagram: m / z 700.33 → 869.44;

[0058] Figure 13 Mass spectrometry identification diagram of standard decoction of Zaocys dhumnades (m / z 700.33) (a). Among them, for the left diagram: m / z 700.33 → 607.29, and for the right diagram: m / z 700.33 → 869.44;

[0059] Figure 14 Mass spectrometry identification diagram (m / z 700.33) (b) of the standard decoction of Zaocys dhumnades, where the left figure: m / z 700.33 → 607.29, and the right figure: m / z 700.33 → 869.44;

[0060] Figure 15 Mass spectrometry identification diagram (m / z 700.33) of the formula granules of Zaocys dhumnades, where the left figure: m / z 700.33 → 607.29, and the right figure: m / z 700.33 → 869.44;

[0061] Figure 16 Mass spectrometry identification diagram (m / z 674.00) (a) of the processed Zaocys dhumnades slices, where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0062] Figure 17 Mass spectrometry identification diagram (m / z 674.00) (b) of the processed Zaocys dhumnades slices, where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0063] Figure 18 Mass spectrometry identification diagram (m / z 674.00) (a) of the standard decoction of the processed Zaocys dhumnades, where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0064] Figure 19 Mass spectrometry identification diagram (m / z 674.00) (b) of the standard decoction of the processed Zaocys dhumnades, where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0065] Figure 20 Mass spectrometry identification diagram (m / z 674.00) of the formula granules of the processed Zaocys dhumnades, where the left figure: m / z 674.00 → 747.37, and the right figure: m / z 674.00 → 917.96;

[0066] Figure 21 Mass spectrometry identification diagram (m / z 700.33) (a) of the processed Zaocys dhumnades slices, where the left figure: m / z 700.33 → 607.29, and the right figure: m / z 700.33 → 869.44;

[0067] Figure 22Mass spectrometry identification diagram of processed Zaocys dhumnades decoction pieces (m / z 700.33) (b). Among them, the left figure: m / z 700.33 → 607.29, the right figure: m / z 700.33 → 869.44;

[0068] Figure 23 Mass spectrometry identification diagram of standard decoction of processed Zaocys dhumnades (m / z 700.33) (a). Among them, the left figure: m / z 700.33 → 607.29, the right figure: m / z 700.33 → 869.44;

[0069] Figure 24 Mass spectrometry identification diagram of standard decoction of processed Zaocys dhumnades (m / z 700.33) (b). Among them, the left figure: m / z 700.33 → 607.29, the right figure: m / z 700.33 → 869.44;

[0070] Figure 25 Mass spectrometry identification diagram of formula granules of processed Zaocys dhumnades (m / z 700.33). Among them, the left figure: m / z 700.33 → 607.29, the right figure: m / z 700.33 → 869.44;

[0071] Figure 26 Mass spectrometry identification diagram of Zaocys dhumnades and other counterfeits (m / z 674.00); among them, the left figure: m / z 674.00 → 747.37; the right figure: m / z 674.00 → 917.96;

[0072] Figure 27 Mass spectrometry identification diagram of Zaocys dhumnades and other counterfeits (m / z 700.33). Among them, the left figure: m / z 700.33 → 607.29, the right figure: m / z 700.33 → 869.44;

[0073] Figure 28 Mass spectrometry identification diagram of Chinese patent medicine containing Zaocys dhumnades (m / z 674.00). Among them, the left figure: m / z 674.00 → 747.37, the right figure: m / z 674.00 → 917.96;

[0074] Figure 29 Mass spectrometry identification diagram of Chinese patent medicine containing Zaocys dhumnades (m / z 700.33). Among them, the left figure: m / z 700.33 → 607.29; the right figure: m / z 700.33 → 869.44. Detailed implementation methods

[0075] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0077] Medicinal snake traditional Chinese medicine is an important part of traditional Chinese medicine in China. As one of the medicinal snakes, Zaocys dhumnades has significant curative effects, limited resources, and high prices. In the market, it is often used as medicine in the form of dried bodies or powders, losing its original properties and being difficult to distinguish between genuine and fake with the naked eye. The phenomenon of counterfeiting by lawbreakers is endless, seriously affecting the effectiveness and safety of drugs. There are now many counterfeits or adulterated products on the market. Common counterfeits include Elaphe carinata, Elaphe taeniura, Elaphe moellendorffi, Ptyas korros, Ptyas mucosus, Agkistrodon acutus, Naja atra, etc. In terms of morphological identification, snake medicinal materials are mainly distinguished by the head, dorsal and ventral parts, and tail scales. However, some commercially available Zaocys dhumnades and their counterfeits have had their heads, skins, and scales removed, and these characteristics have disappeared significantly. It is difficult to rely on traditional identification methods, with low accuracy. Especially after being made into standard decoctions or other dosage forms through water extraction, it is even more difficult to conduct effective identification, especially for the detection of doping with morphologically similar species. To ensure the quality and curative effects of Zaocys dhumnades medicinal materials, there is an urgent need for a method that can accurately and quickly identify genuine Zaocys dhumnades and its counterfeits.

[0078] The present invention provides a method for specifically identifying Zaocys dhumnades and its counterfeits, which is mainly used for the rapid identification of Zaocys dhumnades in Zaocys dhumnades medicinal materials, decoction pieces, standard decoctions, their formula granules, and other Zaocys dhumnades products.

[0079] The technical solution of the present invention is as follows:

[0080] A method for identifying Zaocys dhumnades and its products, comprising the following steps:

[0081] Preparing a control medicinal material solution and a test sample solution;

[0082] Measuring the control medicinal material solution and the test sample solution by high performance liquid chromatography-mass spectrometry (HPLC-MS), using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution.

[0083] In some examples, the gradient elution program is:

[0084] From 0 to 5 min, the volume percentage of mobile phase A increases from 8% to 15%.

[0085] From 5 min to 8 min, the volume percentage of mobile phase A increases from 15% to 40%.

[0086] In some examples, in the aqueous formic acid solution, the volume percentage of formic acid is 0.05% - 0.15%, for example, it can be 0.05%, 0.08%, 0.1%, 0.12% or 0.15%. Preferably, the volume percentage of formic acid is 0.1%.

[0087] In some examples, the conditions of high - performance liquid chromatography include: a chromatographic column filled with octadecylsilyl - bonded silica gel.

[0088] Specifically, the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.6 μm - 1.9 μm.

[0089] In some examples, the conditions of high - performance liquid chromatography include: the flow rate is 0.25 mL / min - 0.35 mL / min, for example, it can be 0.25 mL / min, 0.26 mL / min, 0.27 mL / min, 0.28 mL / min, 0.29 mL / min, 0.3 mL / min, 0.31 mL / min, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min or 0.35 mL / min. Preferably, the flow rate is 0.3 mL / min.

[0090] In some examples, the conditions of high - performance liquid chromatography include: the column temperature is 25°C - 35°C. For example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C. Preferably, the column temperature is 30°C.

[0091] In some examples, the conditions of high - performance liquid chromatography include: the injection volume is 1 μL - 3 μL, for example, it can be 1 μL, 2 μL or 3 μL. Preferably, the injection volume is 2 μL.

[0092] In some examples, the mass spectrometry conditions include: using mass - to - charge ratios m / z 674.00→747.37, m / z 674.00→917.96, m / z 700.33→607.29 and m / z 700.33→869.44 as detection ion pairs.

[0093] In the ion current chromatogram of the test sample extracted with the ion pairs of mass-to-charge ratios m / z 674.00 (triple charge) → 747.37 and m / z 674.00 (triple charge) → 917.96, m / z 700.33 (double charge) → 607.29 and m / z 700.33 (double charge) → 869.44, chromatographic peaks consistent with the retention time of the chromatogram of the control medicinal material should simultaneously appear in the medicinal material, cut crude drug, standard decoction and its formula granules, and proprietary Chinese medicines containing Zaocys dhumnades; no chromatographic peak consistent with the retention time of the chromatogram of the control medicinal material should appear in the counterfeit.

[0094] The present invention uses characteristic ion pairs to specifically identify the components of Zaocys dhumnades in Zaocys dhumnades and its products, solving the problem of difficult identification of products such as the standard decoction of Zaocys dhumnades, formula granules and proprietary Chinese medicines containing Zaocys dhumnades due to the lack of appearance characteristics.

[0095] In some of these examples, the conditions for mass spectrometry detection include: using the electrospray positive ion mode.

[0096] In some of these examples, the conditions for mass spectrometry detection include: multiple reaction monitoring scanning.

[0097] In some of these examples, the conditions for mass spectrometry detection include: the nebulizing gas flow rate is 2 L / min to 3 L / min, for example, it can be 2 L / min, 2.5 L / min and 3 L / min. Preferably, the nebulizing gas flow rate is 3 L / min.

[0098] In some of these examples, the conditions for mass spectrometry detection include: the heating gas flow rate is 8 L / min to 10 L / min, for example, it can be 8 L / min, 9 L / min and 10 L / min. Preferably, the heating gas flow rate is 10 L / min.

[0099] In some of these examples, the conditions for mass spectrometry detection include: the interface temperature is 300 °C to 400 °C, for example, it can be 300 °C, 320 °C, 350 °C, 380 °C and 400 °C. Preferably, the interface temperature is 300 °C.

[0100] In some of these examples, the conditions for mass spectrometry detection include: the desolvation temperature is 526 °C.

[0101] In some of these examples, the conditions for mass spectrometry detection include: the DL temperature is 150 °C to 250 °C, for example, it can be 150 °C, 180 °C, 200 °C, 220 °C and 250 °C. Preferably, the DL temperature is 250 °C.

[0102] In some of these examples, the conditions for mass spectrometry detection include: the temperature of the heating block is 350°C to 450°C, for example, it can be 350°C, 360°C, 380°C, 400°C, 420°C, and 450°C. Preferably, the temperature of the heating block is 400°C.

[0103] In some of these examples, the conditions for mass spectrometry detection include: the flow rate of the drying gas is 8 L / min to 10 L / min, for example, it can be 8 L / min, 9 L / min, and 10 L / min. Preferably, the flow rate of the drying gas is 10 L / min.

[0104] In some of these examples, preparing the control crude drug solution includes the following steps:

[0105] Mix the Zaocys dhumnades control crude drug with the first ammonium bicarbonate aqueous solution, perform the first extraction, and take the filtrate and mix it with the first trypsin solution for the first enzymatic hydrolysis.

[0106] In some specific examples, in the first ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 0.5% to 1.5%, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%. Preferably, in the first ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 1%.

[0107] In some specific examples, in the step of preparing the control crude drug solution, the mass-to-volume ratio of the Zaocys dhumnades control crude drug to the first ammonium bicarbonate aqueous solution is (0.15 to 0.25) g:25 mL. Preferably, the mass-to-volume ratio is 0.2 g:25 mL.

[0108] In some specific examples, the way of the first extraction is heating under reflux extraction or ultrasonic extraction. Preferably, the way of the first extraction is heating under reflux.

[0109] In some specific examples, the heating reflux temperature is 90°C to 100°C, and the heating reflux time is 25 min to 35 min. Preferably, the heating reflux temperature is 100°C, and the heating reflux time is 30 min.

[0110] In some specific examples, the first trypsin solution is prepared by mixing 1 mg of trypsin with 1 mL of ammonium bicarbonate aqueous solution with a mass fraction of 0.5% to 1.5%.

[0111] More specifically, the trypsin is trypsin for sequence analysis.

[0112] In some specific examples, in the step of preparing the reference medicinal material solution, the volume ratio of the filtrate to the first trypsin solution is (4 - 6):1. Preferably, the volume ratio of the filtrate to the first trypsin solution is 5:1.

[0113] In some specific examples, the temperature of the first enzymatic hydrolysis is 35°C - 40°C, and the time of the first enzymatic hydrolysis is 6h - 14h. Preferably, the temperature of the first enzymatic hydrolysis is 37°C, and the time of the first enzymatic hydrolysis is 12h.

[0114] It can be understood that the first enzymatic hydrolysis of the present invention is carried out under constant temperature conditions.

[0115] In some of these examples, preparing the test sample solution includes the following steps:

[0116] Mix the test sample with the second ammonium bicarbonate aqueous solution for a second extraction, take the filtrate and mix it with the second trypsin solution for a second enzymatic hydrolysis.

[0117] In some of these examples, in the second ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 0.5% - 1.5%, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%. Preferably, in the second ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 1%.

[0118] In some specific examples, the second trypsin solution is prepared by mixing 1 mg of trypsin with 1 mL of ammonium bicarbonate aqueous solution with a mass fraction of 0.5% - 1.5%.

[0119] More specifically, the trypsin is trypsin for sequence analysis.

[0120] In some specific examples, in the step of preparing the test sample solution, the volume ratio of the filtrate to the second trypsin solution is (4 - 6):1. Preferably, the volume ratio of the filtrate to the second trypsin solution is 5:1.

[0121] In some specific examples, the temperature of the second enzymatic hydrolysis is 35°C - 40°C, and the enzymatic hydrolysis time is 6h - 14h. Preferably, the temperature of the second enzymatic hydrolysis is 37°C, and the time of the second enzymatic hydrolysis is 12h.

[0122] It can be understood that the second enzymatic hydrolysis of the present invention is carried out under constant temperature conditions.

[0123] In some specific examples, the test sample is one or more of Zaocys dhumnades medicinal materials, Zaocys dhumnades decoction pieces, Zaocys dhumnades decoction pieces processed with wine, or counterfeits. The mass-volume ratio of the test sample to the second ammonium bicarbonate solution is (0.1 - 0.3) g: 25 mL, preferably 0.2 g: 25 mL.

[0124] In some specific examples, the test sample is one or more of Zaocys dhumnades medicinal materials, Zaocys dhumnades decoction pieces, Zaocys dhumnades decoction pieces processed with wine, or counterfeits. The second extraction method is heating under reflux extraction or ultrasonic extraction. Preferably, heating under reflux extraction is adopted.

[0125] In some specific examples, the temperature of heating under reflux is 90 °C - 100 °C, and the time of heating under reflux is 0.5 h - 5 h. Preferably, the temperature of heating under reflux is 100 °C, and the time of heating under reflux is 2 h.

[0126] In some specific examples, the test sample is one or more of Zaocys dhumnades standard decoction or Zaocys dhumnades standard decoction processed with wine. The mass-volume ratio of the test sample to the second ammonium bicarbonate solution is (0.1 - 0.2) g: 50 mL, preferably 0.1 g: 50 mL.

[0127] In some specific examples, the test sample is one or more of Zaocys dhumnades standard decoction or Zaocys dhumnades standard decoction processed with wine. The second extraction method is heating under reflux extraction or ultrasonic extraction. Preferably, ultrasonic extraction is adopted.

[0128] In some specific examples, the power of ultrasonic extraction is 200 W - 400 W, the frequency is 35 kHz - 45 kHz, and the ultrasonic time is 15 min - 45 min. Preferably, the power of ultrasonic extraction is 300 W, the frequency is 40 kHz, and the ultrasonic time is 30 min.

[0129] In some specific examples, the test sample is one or more of Zaocys dhumnades formula granules or Zaocys dhumnades formula granules processed with wine. The mass-volume ratio of the test sample to the second ammonium bicarbonate solution is (0.1 - 0.2) g: 25 mL, preferably 0.1 g: 25 mL.

[0130] In some specific examples, the test sample is one or more of Zaocys dhumnades formula granules or Zaocys dhumnades formula granules processed with wine. The second extraction method is heating under reflux extraction or ultrasonic extraction. Preferably, ultrasonic extraction is adopted.

[0131] In some specific examples, the power of ultrasonic extraction is 200 W - 400 W, the frequency is 35 - 45 kHz, and the ultrasonic time is 15 min - 45 min. Preferably, the power of ultrasonic extraction is 300 W, the frequency is 40 kHz, and the ultrasonic time is 30 min.

[0132] In some specific examples, the test sample is a Chinese patent medicine containing Zaocys dhumnades, and the mass-volume ratio of the test sample to the second ammonium bicarbonate solution is (1-2) g: 20 ml, preferably 1 g: 20 ml.

[0133] In some specific examples, the test sample is a Chinese patent medicine containing Zaocys dhumnades, and the second extraction method is heating under reflux extraction or ultrasonic extraction. Preferably, heating under reflux extraction is adopted.

[0134] In some specific examples, the temperature of heating under reflux is 90 °C - 100 °C, and the time of heating under reflux is 0.5 h - 5 h. Preferably, the temperature of heating under reflux is 100 °C, and the time of heating under reflux is 1 h.

[0135] In some specific examples, before mixing the test sample with the second ammonium bicarbonate aqueous solution, the step of grinding the test sample finely is further included.

[0136] In the preparation method of the test sample solution of the present invention, selecting an ammonium bicarbonate aqueous solution with a mass fraction of 0.5% - 1.5% as the extraction solvent can, to a certain extent, solve the problem that the Zaocys dhumnades products extracted by water only retain the water-soluble substance information, resulting in incomplete extraction components. The small molecule polypeptide substances obtained after the test sample is decomposed by trypsin are relatively stable, which can avoid the problem that the structures of substances such as proteins, polypeptides, and DNA are damaged due to heating during the extraction process of Zaocys dhumnades products. It also avoids the interference in the extraction process of proteins, polypeptides, and DNA caused by the addition of excipients or the compatibility with other traditional Chinese medicines during the production process of Zaocys dhumnades products.

[0137] In some specific examples, the test sample includes one or more of Zaocys dhumnades medicinal materials, Zaocys dhumnades standard decoctions, Zaocys dhumnades formula granules, Zaocys dhumnades cut pieces, stir-fried Zaocys dhumnades cut pieces with wine, stir-fried Zaocys dhumnades standard decoctions with wine, stir-fried Zaocys dhumnades formula granules with wine, Chinese patent medicines containing Zaocys dhumnades, and counterfeits.

[0138] In some specific examples, the counterfeits include one or more of Elaphe carinata, Elaphe taeniura, Elaphe moellendorffi, Ptyas korros, Ptyas mucosus, Agkistrodon acutus, and Naja naja.

[0139] The present invention also provides the application of the above identification method in the authenticity identification and detection of Zaocys dhumnades medicinal materials, cut pieces, standard decoctions, and their formula granules.

[0140] The present invention also provides the application of the above identification method in the authenticity identification and detection of stir-fried Zaocys dhumnades cut pieces with wine, stir-fried Zaocys dhumnades standard decoctions with wine, and their formula granules.

[0141] The present invention also provides the application of the above identification method in the authenticity identification and detection of Chinese patent medicines containing Zaocys dhumnades.

[0142] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions in the following examples, the guidance given in the present invention is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or referring to the experimental methods known in the art.

[0143] In the following specific examples, for the measurement parameters of raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0144] The instruments and reagents used in the embodiments of the present invention are as follows:

[0145] 1 Instruments

[0146] Shimadzu LCMS-8045 triple quadrupole liquid chromatography-mass spectrometry instrument (Shimadzu Corporation); Waters Xevo TQ-S Cronos triple quadrupole liquid chromatography-mass spectrometry instrument (Waters Corporation); Waters BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm); Electrothermal constant temperature water bath (model HWS-28, Shanghai Yiheng Scientific Instrument Co., Ltd.); Numerical control ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.); Drug stability test chamber (SHH-250SD, Chongqing Yongsheng Experimental Instrument Factory); One ten-thousandth balance (ME204E, Mettler-Toledo Co., Ltd.); One millionth balance (Mettler-Toledo Co., Ltd., XP26); Ultra-pure water system (Milli-Q Direct, Merck KGaA).

[0147] 2 Reagents

[0148] Formic acid (H5330100, Shanghai Anpu Experimental Technology Co., Ltd.), acetonitrile (V23ACY0401VH, Anaqua), ammonium bicarbonate (C0850010, Shanghai Anpu Experimental Technology Co., Ltd.), trypsin (T1426, Sigma).

[0149] Example 1

[0150] 1. Characteristic ions

[0151] A set of characteristic ions for identifying the components of Zaocys dhumnades and its products. The secondary mass spectrometry diagrams of the characteristic ions are shown in Figure 1The monitored precursor ion mass-to-charge ratio and product ion mass-to-charge ratio corresponding to the characteristic ions are as follows: m / z 674.00 (triple charge) → 747.37 and m / z 674.00 (triple charge) → 917.96, m / z 700.33 (double charge) → 607.29 and m / z 700.33 (double charge) → 869.44.

[0152] 2. Research samples

[0153] There were a total of 93 batches of samples in this study, including 1 batch of the control medicinal material of Zaocys dhumnades, 15 batches each of Zaocys dhumnades medicinal materials (batch numbers: G1 - G15), Zaocys dhumnades decoction pieces (batch numbers: GP1 - GP15), standard decoctions (batch numbers: GT1 - GT15), 3 batches of Zaocys dhumnades formula granules (batch numbers: CG1 - CG3), 15 batches each of stir-fried Zaocys dhumnades with wine decoction pieces (batch numbers: GP16 - GP30), standard decoctions (batch numbers: GT16 - GT30), 3 batches of stir-fried Zaocys dhumnades with wine formula granules (batch numbers: CG4 - CG6). Chinese patent medicines containing Zaocys dhumnades include Tongbi Tablets, Shexiang Fengshi Capsules, Vitiligo Capsules, and Yixian Pills. The counterfeits include Elaphe carinata, Elaphe taeniura, Elaphe moellendorffi, Ptyas korros, Ptyas mucosus, Deinagkistrodon acutus, and Naja naja. As shown in Table 1:

[0154] Table 1. Sample information table

[0155]

[0156] 3. Preparation of control medicinal material solution and test solution

[0157] 3.1 Preparation of control medicinal material solution

[0158] Take about 0.2 g of the control medicinal material of Zaocys dhumnades, add 25 mL of 1% ammonium bicarbonate solution, heat under reflux for 30 minutes, filter through a microporous membrane, take 1 mL of the subsequent filtrate, place it in a sample injection vial, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, and enzymatically hydrolyze at 37°C for 12 hours to obtain the solution.

[0159] 3.2 Preparation of test solution

[0160] 3.2.1 Zaocys dhumnades medicinal materials, Zaocys dhumnades decoction pieces, stir-fried Zaocys dhumnades with wine decoction pieces, counterfeits (Elaphe carinata, Elaphe taeniura, Elaphe moellendorffi, Ptyas korros, Ptyas mucosus, Deinagkistrodon acutus, Naja naja)

[0161] Take the powder of this product (passed through No. 3 sieve), weigh about 0.2 g, add 25 mL of 1% ammonium bicarbonate solution, and heat under reflux for 2 hours. Filter through a microporous membrane filter, take 1 mL of the subsequent filtrate, place it in an injection vial, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, and enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample.

[0162] 3.2.2 Standard Decoction of Zaocys dhumnades and Wine-processed Standard Decoction of Zaocys dhumnades

[0163] Take an appropriate amount of this product, grind it finely, weigh about 0.1 g, add 50 mL of 1% ammonium bicarbonate solution, sonicate for 30 minutes, cool, and shake well. Filter through a microporous membrane filter, take 1 mL of the subsequent filtrate, place it in an injection vial, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, and enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample.

[0164] 3.2.3 Formula Granules of Zaocys dhumnades and Wine-processed Formula Granules of Zaocys dhumnades

[0165] Take an appropriate amount of this product, grind it finely, weigh about 0.1 g, add 25 mL of 1% ammonium bicarbonate solution, sonicate for 30 minutes, cool, and shake well. Filter through a microporous membrane filter, take 1 mL of the subsequent filtrate, place it in an injection vial, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, and enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample.

[0166] 3.2.4 Chinese Patent Medicines Containing Zaocys dhumnades

[0167] Take an appropriate amount of this product, grind it finely, weigh about 1 g, place it in a conical flask, add 20 mL of 1% ammonium bicarbonate solution, heat under reflux for 1 hour, cool, shake well, centrifuge (at a speed of 12,000 revolutions per minute) for 5 minutes, aspirate the supernatant, filter through a microporous membrane filter, take 1 mL of the subsequent filtrate, place it in an injection vial, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, and enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample.

[0168] 4. Chromatographic and Mass Spectrometric Conditions

[0169] 4.1 Chromatographic Conditions

[0170] Use a Waters BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm); use acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 2 below; the flow rate is 0.30 mL per minute; the column temperature is 30 °C; the injection volume is 2 μL.

[0171] Table 2 Gradient elution table

[0172]

[0173] 4.2 Mass spectrometry conditions

[0174] The mass spectrometer detector was used in electrospray positive ion mode (ESI+); the nebulizer gas flow rate was 3 L / min; the heating gas flow rate was 10 L / min; the interface temperature was 300 °C; the desolvation temperature was 526 °C; the DL temperature was 250 °C; the heating block temperature was 400 °C; the drying gas flow rate was 10 L / min; the collision energy and Q1 and Q3 deviations are shown in Table 3. Multiple reaction monitoring (MRM) was performed, and the mass-to-charge ratios of m / z 674.00 (triply charged) → 747.37 and m / z 674.00 (triply charged) → 917.96, m / z 700.33 (doubly charged) → 607.29 and m / z 700.33 (doubly charged) → 869.44 were selected as the detection ion pairs. Take the control medicinal material solution of the black-striped snake and inject 2 μl. The signal-to-noise ratio of the MRM chromatographic peak determined by the above detection ion pairs should be greater than 3:1. Pipette 2 μl of the test solution and inject it into the HPLC-MS / MS instrument for determination.

[0175] Table 3 Collision energy and Q1, Q3 deviation

[0176]

[0177] 5. Optimization of test sample preparation method:

[0178] 5.1 Investigation of the pre-treatment methods of japanese snake medicinal materials, japanese snake decoction pieces, japanese snake decoction pieces in wine, and counterfeit products (king snake, black-browed rat snake, elaphe snake, gray rat snake, smooth rat snake, acutus, cobra) (taking japanese snake medicinal materials as an example)

[0179] (1) Investigation of extraction methods

[0180] The two main extraction methods, reflux and ultrasound, were investigated. The effects of different extraction methods on the material spectrum identification of the garter snake were compared by calculating the "peak area / sample weight" of four pairs of characteristic ion pairs, and the best extraction method was selected.

[0181] Take an appropriate amount of Zaocys dhumnades (sieved through No. 3 sieve, batch number: G7), weigh about 0.2 g accurately, make 2 parallel groups with 2 portions in each group, add 25 mL of 1% ammonium bicarbonate solution to each portion respectively, weigh, subject one group to ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes for extraction, and subject the other group to heating under reflux for 30 minutes for extraction. After cooling, make up the lost weight with 1% ammonium bicarbonate, shake well, filter through a microporous membrane, transfer 1 mL into an injection vial, add 100 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, and enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample. The experimental results are shown in Table 4.

[0182] Table 4 Results of Mass Spectrometric Identification and Determination with Different Extraction Methods

[0183]

[0184] The results show that the "peak area / sample weight" of heating under reflux extraction is significantly higher than that of ultrasonic extraction, indicating that the reflux extraction method is more effective. Therefore, the reflux extraction method was selected.

[0185] (2) Investigation of extraction time

[0186] Mainly investigate different extraction times, and compare the effects of different extraction times on the mass spectrometric identification of Zaocys dhumnades by calculating the "peak area / sample weight" of 4 pairs of characteristic ion pairs to select the optimal extraction time.

[0187] Take an appropriate amount of Zaocys dhumnades (sieved through No. 3 sieve, batch number: G7), weigh about 0.2 g accurately, make 4 parallel groups with 2 portions in each group, add 25 ml of 1% ammonium bicarbonate solution to each portion respectively, weigh, reflux for 0.5 hour, 1 hour, 2 hours, and 4 hours respectively, after cooling, make up the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22 μm microporous membrane, transfer 1 ml into an injection vial, add 100 μl of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, and enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample. The experimental results are shown in Table 5.

[0188] Table 5 Results of Mass Spectrometric Identification and Determination with Different Extraction Times

[0189]

[0190] The results show that as the extraction time increases, the ratio of "peak area / sample weight" of 4 pairs of characteristic ion pairs shows an upward trend. When the extraction time reaches 2 hours, the ratio of "peak area / sample weight" basically no longer increases, indicating that the extraction is basically complete after 2 hours. Therefore, the extraction time was selected as 2 hours.

[0191] (3) Investigation of trypsin dosage

[0192] The effects of different trypsin dosages on the mass spectrometry identification of Zaocys dhumnades were mainly investigated. The "peak area / sample weight * dilution factor" of 4 pairs of characteristic ion pairs was calculated to compare the effects of different trypsin dosages on the mass spectrometry identification of Zaocys dhumnades, and the optimal trypsin dosage was selected to achieve the best enzymatic hydrolysis effect.

[0193] Appropriately weigh about 0.2 g of Zaocys dhumnades herbs (passed through No. 3 sieve, batch number: G7), in parallel for 4 groups, 2 portions in each group. Add 25 mL of 1% ammonium bicarbonate solution to each, weigh, reflux for 2 hours, cool, make up the lost weight with 1% ammonium bicarbonate solution, shake well, filter through a 0.22 μm microporous membrane filter, transfer 1 mL into an injection vial, and add 50 μL, 100 μL, 200 μL, 400 μL of trypsin solution respectively (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample. Inject for analysis, record the peak area, and calculate the "peak area / sample weight * dilution factor". The experimental results are shown in Table 6.

[0194] Table 6 Mass spectrometry identification determination results with different trypsin addition amounts

[0195]

[0196] The results showed that when the trypsin addition amounts were 200 μL and 400 μL, the "peak area / sample weight * dilution factor" values of the detected ions were relatively large. In order to ensure complete enzymatic hydrolysis and avoid inhibitory reactions caused by excessive enzyme, 200 μL was selected as the trypsin addition amount for this method.

[0197] According to the experimental results of the sample pretreatment investigation, the preparation method of the Zaocys dhumnades herb test sample can be determined as follows: take the powder of this product (passed through No. 3 sieve), weigh about 0.2 g, add 25 mL of 1% ammonium bicarbonate solution, heat under reflux for 2 hours. Filter through a microporous membrane, take 1 mL of the continued filtrate, place it in an injection vial, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the sample.

[0198] 5.2 Investigation on the pretreatment methods of the standard decoction of Zaocys dhumnades and the standard decoction of Zaocys dhumnades with wine (taking the standard decoction of Zaocys dhumnades as an example)

[0199] (1) Investigation on the extraction method

[0200] The reflux and ultrasonic extraction methods were mainly investigated. The "peak area / sample weight" of 4 pairs of characteristic ion pairs was calculated to compare the effects of different extraction methods on the mass spectrometry identification of the standard decoction of Zaocys dhumnades, and the optimal extraction method was selected.

[0201] Take an appropriate amount of the standard decoction of Zaocys dhumnades (batch number: GT2), grind it finely, take about 0.1 g, weigh it precisely, make 2 parallel groups with 2 portions in each group, add 50 ml of 1% ammonium bicarbonate solution to each portion respectively, weigh, subject one group to ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes for extraction, subject the other group to heating under reflux for 30 minutes for extraction, let it cool, make up the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22 μm microporous membrane, transfer 1 mL to an injection vial, add 100 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, enzymatically hydrolyze at 37 °C for 12 hours to obtain the sample. The experimental results are shown in Table 7.

[0202] Table 7 Results of mass spectrometry identification and determination with different extraction methods

[0203]

[0204] The results show that the difference in the ratio of "peak area / sample weight" between ultrasonic extraction and reflux extraction is not obvious. For the sake of simple operation, ultrasonic extraction method is selected.

[0205] (2) Investigation of extraction time

[0206] Mainly investigate three extraction times of 15 minutes, 30 minutes, and 45 minutes. By calculating the ratio of "peak area / sample weight" of 4 pairs of characteristic ion pairs, compare the influence of different extraction times on the mass spectrometry identification of the standard decoction of Zaocys dhumnades, and select the optimal extraction time.

[0207] Take an appropriate amount of the standard decoction of Zaocys dhumnades (batch number: GT2), grind it finely, take about 0.1 g, weigh it precisely, make 3 parallel groups with 2 portions in each group, add 50 ml of 1% ammonium bicarbonate solution to each portion respectively, weigh, subject them to ultrasonic treatment (power 300 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, let it cool, make up the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22 μm microporous membrane, transfer 1 ml to an injection vial, add 100 μl of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, enzymatically hydrolyze at 37 °C for 12 hours to obtain the sample. The experimental results are shown in Table 8.

[0208] Table 8 Results of mass spectrometry identification and determination with different extraction times

[0209]

[0210] The results show that the difference in the ratio of "peak area / sample weight" of the monitored ions at different extraction times is not obvious. To ensure complete extraction, the ultrasonic time is selected as 30 minutes.

[0211] (3) Investigation of trypsin dosage

[0212] Mainly investigate the relationship between the dosage of trypsin and the sampling amount of the standard decoction of Zaocys dhumnades, and investigate the dosage of trypsin that can achieve the best enzymatic hydrolysis effect under a certain sampling amount of the standard decoction of Zaocys dhumnades. Design to add 50 μl, 100 μl, 200 μl, 400 μl, and 600 μl of trypsin respectively to carry out enzymatic hydrolysis reaction under the sampling amount of 0.1 g of the standard decoction of Zaocys dhumnades. Finally, compare by calculating the "peak area / sampling amount * dilution factor" of 4 pairs of detected ion pairs, and select the best dosage of trypsin.

[0213] Take an appropriate amount of the standard decoction of Zaocys dhumnades (batch number: GT2), grind it finely, take about 0.1 g, weigh it accurately, place it in a conical flask, add 50 ml of 1% ammonium bicarbonate solution, weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes, let it cool, make up the lost weight with 1% ammonium bicarbonate, shake well, filter it through a 0.22 μm microporous filter membrane, transfer 1 ml to an injection vial, make 5 parallel groups, 2 portions in each group, and add 50 μl, 100 μl, 200 μl, 400 μl, and 600 μl of trypsin solution respectively (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, and enzymatically hydrolyze at 37 °C for 12 hours to obtain the sample. Inject for analysis, record the peak area, and calculate the "peak area / sampling amount * dilution factor". The experimental results are shown in Table 9.

[0214] Table 9 Mass spectrometry identification and determination results with different dosages of trypsin added

[0215]

[0216] The results show that adding different dosages of trypsin has little effect on the ratio of "peak area / sampling amount * dilution factor" of the detected ions. In order to ensure complete enzymatic hydrolysis and avoid inhibitory reactions caused by excessive enzymes, 200 μL is selected as the added amount of trypsin for the method.

[0217] According to the experimental results of the sample pretreatment investigation, the preparation method of the test sample of the standard decoction of Zaocys dhumnades can be determined as follows: Take an appropriate amount of this product, grind it finely, take about 0.1 g, add 50 ml of 1% ammonium bicarbonate solution, ultrasonically treat it for 30 minutes, let it cool, and shake well. Filter it through a microporous filter membrane, take 1 ml of the subsequent filtrate, place it in an injection vial, add 200 μl of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, and enzymatically hydrolyze at 37 °C for 12 hours to obtain the sample.

[0218] 5.3 Investigation on the pretreatment methods of the formula granules of Zaocys dhumnades and the formula granules of Zaocys dhumnades processed with wine (taking the formula granules of Zaocys dhumnades as an example)

[0219] (1) Investigation on the extraction method

[0220] Two extraction methods, reflux and ultrasonic extraction, were mainly investigated. By calculating the "peak area / sample weight" of 4 pairs of characteristic ion pairs, the effects of different extraction methods on the mass spectrometry identification of Zaocys dhumnades granules were compared to select the optimal extraction method.

[0221] Appropriately weigh an amount of Zaocys dhumnades formula granules (batch number: CG2), grind them finely, take about 0.1 g, accurately weigh, place in a conical flask, with 2 parallel groups, 2 portions in each group. Add 25 ml of 1% ammonium bicarbonate solution to each, weigh, subject one group to ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes for extraction, and the other group to reflux for 30 minutes for extraction. Take out, let cool, make up the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22 μm microporous membrane. Transfer 1 ml of the continued filtrate to a micro-sampling vial, add 100 μl of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, and enzymatically hydrolyze at 37 °C for 12 hours to obtain the sample. The experimental results are shown in Table 10.

[0222] Table 10 Determination results of mass spectrometry identification with different extraction methods

[0223]

[0224] The results showed that the "peak area / sample weight" of ultrasonic extraction was slightly higher than that of reflux extraction, indicating that the ultrasonic extraction method was more effective. Therefore, ultrasonic extraction was selected as the extraction method.

[0225] (2)Investigation of extraction time

[0226] In this experiment, the effects of different extraction times on the mass spectrometry identification of Zaocys dhumnades formula granules were investigated. Three extraction times, 15 minutes, 30 minutes, and 45 minutes, were mainly investigated. By calculating the "peak area / sample weight" of 4 pairs of characteristic ion pairs, the effects of different extraction times on the mass spectrometry identification of Zaocys dhumnades formula granules were compared to select the optimal extraction time.

[0227] Appropriately weigh an amount of Zaocys dhumnades formula granules (batch number: CG2), grind them finely, take about 0.1 g, accurately weigh, place in a conical flask, with 3 parallel groups, 2 portions in each group. Add 25 ml of 1% ammonium bicarbonate solution to each, weigh, subject them to ultrasonic treatment (power 300 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Take out, let cool, make up the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22 μm microporous membrane. Transfer 1 ml of the continued filtrate to a micro-sampling vial, add 100 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, prepared before use), shake well, and enzymatically hydrolyze at 37 °C for 12 hours to obtain the sample. The experimental results are shown in Table 11.

[0228] Table 11 Determination results of mass spectrometry identification with different extraction times

[0229]

[0230] The results showed that there was no obvious difference in the ratio of "peak area / sample weight" of the monitored ions at different extraction times. Extraction was basically complete in 15 minutes. To ensure the durability of the method, the ultrasonic extraction time was selected as 30 minutes.

[0231] (3)Investigation of the amount of trypsin used

[0232] The relationship between the amount of trypsin used and the sampling amount of Zaocys dhumnades formula granules was mainly investigated, and the amount of trypsin that could achieve the best enzymatic hydrolysis effect under a certain sampling amount of Zaocys dhumnades formula granules was investigated. It was designed to add 100 μL, 200 μL, and 400 μL of trypsin to 0.1 g of Zaocys dhumnades formula granules for enzymatic hydrolysis reaction. Finally, the "peak area / sample weight × dilution factor" of 4 pairs of detected ion pairs was calculated for comparison to select the optimal amount of trypsin.

[0233] Appropriately take Zaocys dhumnades formula granules (batch number: CG2), grind them finely, take about 0.1 g, weigh accurately, place them in a conical flask, add 25 ml of 1% ammonium bicarbonate solution, weigh, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, take out, let it cool, make up the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22 μm microporous membrane, transfer 1 ml of the continued filtrate to a micro-sampling vial, in parallel for 3 groups, 2 portions in each group, and add 100 μl, 200 μl, and 400 μl of trypsin solution respectively (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, enzymatically hydrolyze at 37 °C for 12 hours to obtain. Inject for analysis, record the peak area, and calculate the "peak area / sample weight × dilution factor". The experimental results are shown in Table 12.

[0234] Table 12 Mass spectrometry identification and determination results with different amounts of trypsin added

[0235]

[0236] The results showed that there was no obvious difference in the ratio of "peak area / sample weight × dilution factor" of the monitored ions with different amounts of trypsin added. To better ensure complete enzymatic hydrolysis of the sample, 200 μl was selected as the amount of trypsin added in the method.

[0237] According to the experimental results of sample pretreatment, the preparation method of the test sample of Zaocys dhumnades formula granules was determined as follows: Take an appropriate amount of this product, grind it finely, take about 0.1 g, add 25 ml of 1% ammonium bicarbonate solution, ultrasonicate for 30 minutes, cool, and shake well. Filter through a microporous membrane, take 1 ml of the subsequent filtrate, place it in an injection vial, add 200 μl of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, prepared before use), shake well, and enzymatically hydrolyze at a constant temperature of 37 °C for 12 hours to obtain the product.

[0238] Example 2 Identification Application of Characteristic Ion Pairs

[0239] (1)Identification Application of Zaocys dhumnades, Stir-Fried Zaocys dhumnades with Wine and Counterfeits

[0240] Respectively take the powders of Zaocys dhumnades herbs (batch numbers: G1 - G15), Zaocys dhumnades cut pieces (batch numbers: GP1 - GP15), Stir-Fried Zaocys dhumnades with Wine cut pieces (batch numbers: GP16 - GP30), and counterfeits (batch numbers: X1 - X7) (passed through No. 3 sieve), and prepare the test sample solutions of Zaocys dhumnades herbs, Zaocys dhumnades cut pieces, Stir-Fried Zaocys dhumnades with Wine cut pieces, and counterfeits respectively according to the test sample solution preparation method under "3.2.1" in Example 1.

[0241] Respectively take appropriate amounts of the standard decoctions of Zaocys dhumnades (batch numbers: GT1 - GT15) and the standard decoctions of Stir-Fried Zaocys dhumnades with Wine (batch numbers: GT16 - GT30), grind them finely, and prepare the test sample solutions of the standard decoctions of Zaocys dhumnades and the standard decoctions of Stir-Fried Zaocys dhumnades with Wine respectively according to the test sample solution preparation method under "3.2.2" in Example 1.

[0242] Respectively take appropriate amounts of Zaocys dhumnades formula granules (batch numbers: CG1 - CG3) and Stir-Fried Zaocys dhumnades with Wine formula granules (batch numbers: CG4 - CG6), grind them finely, and prepare the test sample solutions of Zaocys dhumnades formula granules and Stir-Fried Zaocys dhumnades with Wine formula granules respectively according to the test sample solution preparation method under "3.2.3" in Example 1.

[0243] Take the reference herb of Zaocys dhumnades (batch number: STD), and prepare the reference herb solution of Zaocys dhumnades according to the reference herb solution preparation method under "3.1" in Example 1.

[0244] Precisely pipette 2 μL of the above-mentioned test solution and the control medicinal material solution of Zaocys dhumnades into a liquid chromatograph, and use a mass spectrometry detector in the electrospray positive ion mode (ESI+) for multiple reaction monitoring (MRM). In the ion current chromatograms of the test samples of Zaocys dhumnades medicinal materials, cut pieces, standard decoctions and their formula granules, stir-fried Zaocys dhumnades cut pieces, standard decoctions and their formula granules extracted under the chromatographic conditions described in item "4.1" of Example 1 and the mass spectrometry conditions described in item "4.2" of Example 1, chromatographic peaks consistent with the chromatographic retention time of the control medicinal material solution of Zaocys dhumnades should appear simultaneously. However, no detection should be found in the ion current chromatograms of the test samples of counterfeits (Elaphe carinata, Elaphe taeniura, Elaphe moellendorffi, Ptyas korros, Ptyas mucosus, Agkistrodon acutus, Naja naja). See Tables 13 to 20 and Figures 2 to 27 . Among them, Figures 2 to 3 is the mass spectrometry identification diagram of Zaocys dhumnades medicinal materials (m / z 674.00), Figures 4 to 5 is the mass spectrometry identification diagram of Zaocys dhumnades cut pieces (m / z 674.00), Figures 6 to 7 is the mass spectrometry identification diagram of Zaocys dhumnades standard decoctions (m / z 674.00), Figure 8 is the mass spectrometry identification diagram of Zaocys dhumnades formula granules (m / z 674.00), Figures 9 to 10 is the mass spectrometry identification diagram of Zaocys dhumnades medicinal materials (m / z700.33), Figures 11 to 12 is the mass spectrometry identification diagram of Zaocys dhumnades cut pieces (m / z 700.33), Figures 13 to 14 is the mass spectrometry identification diagram of Zaocys dhumnades standard decoctions (m / z 700.33), Figure 15 is the mass spectrometry identification diagram of Zaocys dhumnades formula granules (m / z 700.33), Figures 16 to 17 is the mass spectrometry identification diagram of stir-fried Zaocys dhumnades cut pieces (m / z 674.00), Figures 18 to 19 is the mass spectrometry identification diagram of stir-fried Zaocys dhumnades standard decoctions (m / z 674.00), Figure 20 is the mass spectrometry identification diagram of stir-fried Zaocys dhumnades formula granules (m / z 674.00), Figures 21 to 22 is the mass spectrometry identification diagram of stir-fried Zaocys dhumnades cut pieces (m / z 700.33), Figures 23 to 24 is the mass spectrometry identification diagram of stir-fried Zaocys dhumnades standard decoctions (m / z 700.33), Figure 25 is the mass spectrometry identification diagram of stir-fried Zaocys dhumnades formula granules (m / z 700.33), Figure 26 is the mass spectrometry identification diagram of Zaocys dhumnades and other counterfeits (m / z 674.00), Figure 27 is the mass spectrometry identification diagram of Zaocys dhumnades and other counterfeits (m / z700.33).

[0245] Table 13 Results of Mass Spectrometry Determination of Samples (Zaocys dhumnades Medicinal Materials)

[0246]

[0247] Table 14 Sample Mass Spectrometry Determination Results Table (Zaocys dhumnades Slices)

[0248]

[0249] Table 15 Sample Mass Spectrometry Determination Results Table (Standard Decoction of Zaocys dhumnades)

[0250]

[0251] Table 16 Sample Mass Spectrometry Determination Results Table (Formula Granules of Zaocys dhumnades)

[0252]

[0253] Table 17 Sample Mass Spectrometry Determination Results Table (Stir-Fried Zaocys dhumnades with Wine Slices)

[0254]

[0255] Table 18 Sample Mass Spectrometry Determination Results Table (Standard Decoction of Stir-Fried Zaocys dhumnades with Wine)

[0256]

[0257] Table 19 Sample Mass Spectrometry Determination Results Table (Formula Granules of Stir-Fried Zaocys dhumnades with Wine)

[0258]

[0259] Table 20 Sample Mass Spectrometry Determination Results Table (Other Counterfeits)

[0260]

[0261] (2) Detection of Chinese Patent Medicines Containing Zaocys dhumnades

[0262] Appropriate amounts of Tongbi Tablets (batch number: C1), Shexiang Fengshi Capsules (batch number: C2), Vitiligo Capsules (batch number: C3), and Yixian Pills (batch number: C4) were taken and ground finely. The test solution of Chinese patent medicines containing Zaocys dhumnades was prepared respectively according to the preparation method of the test solution under item "3.2.4".

[0263] Take the reference medicinal material of Zaocys dhumnades (batch number: STD), and prepare the reference medicinal material solution of Zaocys dhumnades according to the preparation method of the reference medicinal material solution under item "3.1".

[0264] 2 μL of the above test solution of Chinese patent medicines containing Zaocys dhumnades and the reference medicinal material solution of Zaocys dhumnades were accurately pipetted respectively and injected into the liquid chromatograph, and a mass spectrometry detector was used. In the positive electrospray ionization mode (ESI+), multiple reaction monitoring (MRM) was carried out. In the ion current chromatogram of the test solution of Chinese patent medicines containing Zaocys dhumnades extracted under the above chromatographic and mass spectrometric conditions, chromatographic peaks consistent with the chromatographic retention time of the reference medicinal material solution of Zaocys dhumnades should be presented simultaneously. See Table 21 andFigures 28 to 29 , wherein, Figure 28 is the mass spectrometry identification diagram of the Chinese patent medicine containing Zaocys dhumnades (m / z 674.00), Figure 29 is the mass spectrometry identification diagram of the Chinese patent medicine containing Zaocys dhumnades (m / z 700.33).

[0265] Table 21 Mass spectrometry determination results of Chinese patent medicine containing Zaocys dhumnades

[0266]

[0267] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0268] The above-described embodiments only represent several implementation manners of the present invention, which are convenient for understanding the technical solutions of the present invention specifically and in detail, but should not be construed as a limitation on the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention patent should be subject to the content of the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A method for identifying Zaocys dhumnades and its products, characterized in that It includes the following steps: Prepare a reference medicinal material solution and a test sample solution; Determine the reference medicinal material solution and the test sample solution by high performance liquid chromatography - mass spectrometry (HPLC - MS). Use acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution.

2. The identification method according to claim 1, wherein The program of the gradient elution is as follows: From 0 to 5 minutes, the volume percentage of mobile phase A increases from 8% to 15%; From 5 minutes to 8 minutes, the volume percentage of mobile phase A increases from 15% to 40%.

3. The authentication method according to claim 1, wherein In the formic acid aqueous solution, the volume percentage of formic acid is 0.05% - 0.15%.

4. The authentication method according to claim 1, characterized in that The conditions of high performance liquid chromatography include one or more of the following (1) - (4): (1) A chromatographic column filled with octadecylsilyl silica gel; (2) The flow rate is 0.25 mL / min - 0.35 mL / min; (3) The column temperature is 25°C - 35°C; (4) The injection volume is 1 μL - 3 μL.

5. The identification method of Zaocys dhumnades and its products according to claim 1, characterized in that, The mass spectrometry conditions include: Use m / z 674.00→747.37, m / z 674.00→917.96, m / z 700.33→607.29 and m / z 700.33→869.44 as the detected ion pairs.

6. The identification method according to any one of claims 1 to 5, characterized in that The mass spectrometry conditions include one or more of the following (1) - (9): (1) Adopt the electrospray positive ion mode; (2) Multiple reaction monitoring scan; (3) The nebulizing gas flow rate is 2 L / min - 3 L / min; (4) The heating gas flow rate is 8 L / min - 10 L / min; (5) The interface temperature is 300°C - 400°C; (6) The desolvation temperature is 526°C; (7) The DL temperature is 150°C - 250°C; (8) The heating block temperature is 350°C - 450°C; (9) The drying gas flow rate is 8 L / min - 10 L / min.

7. The identification method according to any one of claims 1 to 5, characterized in that, Preparing the reference medicinal material solution includes the following steps: Mix the reference medicinal material of Zaocys dhumnades with the first ammonium bicarbonate aqueous solution, conduct the first extraction, and take the filtrate and mix it with the first trypsin solution for the first enzymatic hydrolysis.

8. The discrimination method according to claim 7, characterized in that, Meet one or more of the following (1) - (4): (1) In the first ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 0.5% - 1.5%; (2) The first extraction method is heating reflux extraction or ultrasonic extraction; (3) The first trypsin solution is prepared by mixing 1 mg of trypsin with 1 mL of ammonium bicarbonate aqueous solution with a mass fraction of 0.5% - 1.5%; (4) The temperature of the first enzymatic hydrolysis is 35°C - 40°C, and the time of the first enzymatic hydrolysis is 6 h - 14 h.

9. The identification method according to any one of claims 1 to 5, characterized in that Preparing the test sample solution includes the following steps: Mix the test sample with the second ammonium bicarbonate aqueous solution, conduct the second extraction, and take the filtrate and mix it with the second trypsin solution for the second enzymatic hydrolysis.

10. The identification method according to claim 9, wherein Meet one or more of the following (1) - (4): (1) In the second ammonium bicarbonate aqueous solution, the mass fraction of ammonium bicarbonate is 0.5% - 1.5%; (2) The second extraction method is heating reflux extraction or ultrasonic extraction; (3) The second trypsin solution is prepared by mixing 1 mg of trypsin with 1 mL of ammonium bicarbonate aqueous solution with a mass fraction of 0.5% - 1.5%; (4)The temperature of the second enzymolysis is 35°C to 40°C, and the time of the second enzymolysis is 6h to 14h.

11. The authentication method according to claim 9, characterized in that, The test samples include one or more of Zaocys dhumnades medicinal materials, Zaocys dhumnades standard decoctions, Zaocys dhumnades formula granules, Zaocys dhumnades cut pieces, stir-fried Zaocys dhumnades cut pieces with wine, stir-fried Zaocys dhumnades standard decoctions with wine, stir-fried Zaocys dhumnades formula granules with wine, Chinese patent medicines containing Zaocys dhumnades, and counterfeits.

12. The identification method according to claim 11, wherein The counterfeits include one or more of Elaphe carinata, Elaphe taeniura, Elaphe moellendorffi, Ptyas korros, Ptyas mucosus, Agkistrodon acutus, and Naja naja.

13. Application of the identification method according to any one of claims 1 to 12 in the authenticity identification and detection of Zaocys dhumnades medicinal materials, cut pieces, standard decoctions, and their formula granules.

14. Application of the identification method according to any one of claims 1 to 12 in the authenticity identification and detection of stir-fried Zaocys dhumnades cut pieces with wine, stir-fried Zaocys dhumnades standard decoctions with wine, and their formula granules.

15. Application of the identification method according to any one of claims 1 to 12 in the authenticity identification and detection of Chinese patent medicines containing Zaocys dhumnades.

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