Method for identifying zzyzania villosa and its products and application

Abstract

The present application relates to the technical field of quality analysis and detection of traditional Chinese medicine, and particularly relates to a method for identifying Trimeresurus and products thereof and application. The method for identifying Trimeresurus and products thereof comprises the following steps: preparing a control medicinal material solution and a test solution; determining the control medicinal material solution and the test solution by high performance liquid chromatography-mass spectrometry, gradient elution is performed with acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B. The present application establishes a specific identification method for Trimeresurus and products thereof by high performance liquid chromatography-mass spectrometry, solves the problem that it is difficult to identify Trimeresurus standard decoction, formula granules and Chinese patent medicine containing Trimeresurus due to the loss of appearance characteristics, and the identification method has strong specificity, and provides a basis for quality control and evaluation of Trimeresurus and products thereof, especially Trimeresurus water extract products.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality analysis and testing technology, and in particular to a method and application for the identification of Zaocys dhumnades and its products. Background Technology

[0002] The Chinese Pharmacopoeia (2020 edition) specifies that the origin of the black-striped snake (Zaocys dhumnades) is the dried body of the snake, a member of the Colubridae family. The black-striped snake was first recorded in *Lei Gong Pao Zhi Lun*, which states, "The Qizhou black-striped snake weighs only one-third to one-fifth of a tael; it is excellent, with its head and tail intact, its eyes not closed as if it were alive. There is a two-inch-long strip of reverse-flowing hair on its head, which can grow to about half an inch long, with its head and tail facing each other." The black-striped snake has the effects of dispelling wind, unblocking meridians, and stopping spasms. Clinically, it is used for stubborn rheumatism, numbness and contractures, stroke with facial paralysis, hemiplegia, convulsions, tetanus, leprosy, and scabies. Modern pharmacological studies have shown that the black-striped snake has anti-inflammatory and analgesic effects.

[0003] As a medicinal snake, the black-banded rat snake is highly valued due to its significant therapeutic effects, limited resources, and high price. It is often sold in the market as dried body or powder, losing its original characteristics and making it difficult to distinguish genuine from counterfeit. This has led to rampant counterfeiting by unscrupulous individuals, seriously affecting the efficacy and safety of the medicine. Therefore, traditional identification methods are difficult and inaccurate, especially after water extraction to prepare standard decoctions or other dosage forms, making effective identification even more challenging.

[0004] With the development of proteomics and molecular biology, breakthroughs have been made in the specific identification of peptides and nucleic acids. Identification techniques for various animal-derived traditional Chinese medicines are becoming increasingly sophisticated, and many researchers have used PCR (polymerase chain reaction) to identify the medicinal material *Zaocys dhumnades*. However, there is limited research on the specific identification and characteristic ion pair identification of water extracts of *Zaocys dhumnades*, such as standard decoctions, traditional Chinese medicine granules, or other proprietary Chinese medicines containing *Zaocys dhumnades*.

[0005] Therefore, how to achieve highly specific identification of Zaocys dhumnades and its products to meet the needs of quality control is an urgent technical problem to be solved, and a technical challenge that needs 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 the black-tailed snake and its products, comprising the following steps:

[0007] Preparation of reference herb solution and test sample solution;

[0008] The reference herb solution and the test sample solution were determined by high performance liquid chromatography-mass spectrometry, with acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution.

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

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

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

[0012] In some embodiments, the formic acid aqueous solution contains formic acid at a volume percentage of 0.05% to 0.15%.

[0013] In some embodiments, the high-performance liquid chromatography (HPLC) conditions include a column packed with octadecylsilane-bonded silica gel.

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

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

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

[0017] In some embodiments, the mass spectrometry conditions include using mass-to-charge ratios of 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.

[0018] In some embodiments, the mass spectrometry conditions include: employing an electrospray positive ion mode.

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

[0020] In some embodiments, the mass spectrometry conditions include a nebulizer flow rate of 2 L / min to 3 L / min.

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

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

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

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

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

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

[0027] In some embodiments, the preparation of the control herbal solution includes the following steps:

[0028] The reference herb *Zaocys dhumnades* was mixed with a first ammonium bicarbonate aqueous solution for the first extraction. The filtrate was then mixed with a first trypsin solution for the first enzymatic hydrolysis.

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

[0030] In some embodiments, the first extraction method is heating reflux extraction or ultrasonic extraction.

[0031] In some embodiments, the first trypsin solution is a mixture of 1 mg of trypsin and 1 mL of an aqueous solution of 0.5% to 1.5% ammonium bicarbonate.

[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 hours to 14 hours.

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

[0034] The test sample is mixed with a second ammonium bicarbonate aqueous solution for a second extraction, and the filtrate is mixed with a second trypsin solution for a second enzymatic hydrolysis.

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

[0036] In some embodiments, the second extraction method is heating reflux extraction or ultrasonic extraction.

[0037] In some embodiments, the second trypsin solution is a mixture of 1 mg of trypsin and 1 mL of an aqueous solution of 0.5% to 1.5% ammonium bicarbonate.

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

[0039] In some embodiments, the test sample includes one or more of the following: Chinese black snake medicinal material, Chinese black snake standard decoction, Chinese black snake formula granules, Chinese black snake slices, Chinese black snake slices in wine, Chinese black snake standard decoction in wine, Chinese black snake formula granules in wine, Chinese patent medicine containing Chinese black snake, and counterfeit products.

[0040] In some embodiments, the counterfeit snakes include one or more of the following: king cobra, black-browed rat snake, flower cobra, gray rat snake, slippery rat snake, Agkistrodon halys, and cobra.

[0041] This invention also provides the application of the above-mentioned identification method in the identification and detection of the authenticity of Zaocys dhumnades medicinal materials, processed slices, standard decoctions and their formula granules.

[0042] This invention also provides the application of the above-mentioned identification method in the identification and detection of the authenticity of processed black snake slices, standard decoctions and their formula granules.

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

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

[0045] This invention establishes a specific identification method for Zaocys dhumnades and its products using high performance liquid chromatography-mass spectrometry (HPLC-MS). This method solves the problem of difficulty in identifying Zaocys dhumnades standard decoctions, formula granules, and traditional Chinese medicine products containing Zaocys dhumnades due to the lack of visual characteristics. The identification method of this invention has high specificity and provides a basis for the quality control and evaluation of Zaocys dhumnades and its products, especially water extracts of Zaocys dhumnades. Attached Figure Description

[0046] Figure 1 This is a secondary mass spectrum of characteristic ions from the black-striped snake.

[0047] Figure 2 The mass spectrometry identification diagram of the medicinal material *Zaocys dhumnades* (m / z 674.00) (a) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96.

[0048] Figure 3 The mass spectrometry identification diagram of the medicinal material *Zaocys dhumnades* (m / z 674.00) (b) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96.

[0049] Figure 4 The mass spectrometry identification chromatogram of the processed slices of Zaocys dhumnades (m / z 674.00) (a) shows that, in the left chromatogram, m / z 674.00→747.37 and in the right chromatogram, m / z 674.00→917.96.

[0050] Figure 5 The mass spectrometry identification chromatogram of the processed slices of Zaocys dhumnades (m / z 674.00) (b) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96.

[0051] Figure 6 The mass spectrometry identification chromatogram of the standard decoction of Zaocys dhumnades (m / z 674.00) (a) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96;

[0052] Figure 7 The mass spectrometry identification chromatogram of the standard decoction of Zaocys dhumnades (m / z 674.00) (b) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96.

[0053] Figure 8 The mass spectrometry identification chromatogram of the formula granules of *Zaocys dhumnades* (m / z 674.00) shows that, in the left chromatogram, m / z 674.00 → 747.37, and in the right chromatogram, m / z 674.00 → 917.96.

[0054] Figure 9 The mass spectrometry identification diagram of the medicinal material *Zaocys dhumnades* (m / z 700.33) (a) shows that, in the left image: m / z 700.33→607.29, and in the right image: m / z 700.33→869.44.

[0055] Figure 10 The mass spectrometry identification diagram of the medicinal material *Zaocys dhumnades* (m / z 700.33) (b) shows that, in the left image: m / z 700.33→607.29, and in the right image: m / z 700.33→869.44.

[0056] Figure 11 The mass spectrometry identification chromatogram of the processed slices of Zaocys dhumnades (m / z 700.33) (a) shows that, in the left chromatogram, m / z 700.33→607.29 and in the right chromatogram, m / z 700.33→869.44.

[0057] Figure 12 The mass spectrometry identification chromatogram of the processed slices of Zaocys dhumnades (m / z 700.33) (b) shows that, in the left image: m / z 700.33→607.29, and in the right image: m / z 700.33→869.44.

[0058] Figure 13 The mass spectrometry identification chromatogram of the standard decoction of Zaocys dhumnades (m / z 700.33) (a) shows that, in the left chromatogram, m / z 700.33→607.29 and in the right chromatogram, m / z 700.33→869.44.

[0059] Figure 14 The mass spectrometry identification chromatogram of the standard decoction of Zaocys dhumnades (m / z 700.33) (b) shows that, in the left image: m / z 700.33→607.29, and in the right image: m / z 700.33→869.44.

[0060] Figure 15 The mass spectrometry identification chromatogram of the formula granules of *Zaocys dhumnades* (m / z 700.33) shows that, in the left chromatogram, m / z 700.33 → 607.29, and in the right chromatogram, m / z 700.33 → 869.44.

[0061] Figure 16 The mass spectrometry identification chromatogram of the processed black snake slices (m / z 674.00) (a) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96;

[0062] Figure 17 The mass spectrometry identification chromatogram of the processed black snake slices (m / z 674.00) (b) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96;

[0063] Figure 18 The mass spectrometry identification chromatogram of the standard decoction of *Zaocys dhumnades* (m / z 674.00) (a) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96.

[0064] Figure 19 The mass spectrometry identification chromatogram of the standard decoction of *Zaocys dhumnades* (m / z 674.00) (b) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96.

[0065] Figure 20 The mass spectrometry identification chromatogram of the formula granules of black snake in wine (m / z 674.00) shows that, in the left image: m / z 674.00→747.37, and in the right image: m / z 674.00→917.96.

[0066] Figure 21 The mass spectrometry identification chromatogram of the processed black snake (m / z 700.33) (a) shows that, in the left image: m / z 700.33→607.29, and in the right image: m / z 700.33→869.44.

[0067] Figure 22The mass spectrometry identification chromatogram of the processed black snake slices (m / z 700.33) (b) shows that, in the left image: m / z 700.33→607.29, and in the right image: m / z 700.33→869.44.

[0068] Figure 23 The mass spectrometry identification chromatogram of the standard decoction of *Zaocys dhumnades* (m / z 700.33) (a) shows that, in the left chromatogram, m / z 700.33 → 607.29, and in the right chromatogram, m / z 700.33 → 869.44.

[0069] Figure 24 The mass spectrometry identification chromatogram of the standard decoction of *Zaocys dhumnades* (m / z 700.33) (b) shows that, in the left image: m / z 700.33→607.29, and in the right image: m / z 700.33→869.44.

[0070] Figure 25 The mass spectrometry identification chromatogram of the formula granules for the wine-processed black snake is shown (m / z 700.33). In the left chromatogram, m / z 700.33→607.29, and in the right chromatogram, m / z 700.33→869.44.

[0071] Figure 26 Mass spectrometry diagrams for differentiating Zaocys dhumnades from other adulterants (m / z 674.00); Left image: m / z 674.00→747.37; Right image: m / z 674.00→917.96;

[0072] Figure 27 Mass spectrometry diagrams for differentiating Zaocys dhumnades from other adulterants (m / z 700.33), where, left image: m / z 700.33→607.29, right image: m / z 700.33→869.44;

[0073] Figure 28 The mass spectrometry identification diagram of traditional Chinese medicine containing Zaocys dhumnades (m / z 674.00) shows that the left image is m / z 674.00→747.37 and the right image is m / z 674.00→917.96.

[0074] Figure 29 The mass spectrometry identification diagram of traditional Chinese medicine containing Zaocys dhumnades (m / z 700.33) shows that the left image is m / z 700.33→607.29 and the right image is m / z 700.33→869.44. Detailed Implementation

[0075] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0077] Medicinal snakes are an important part of traditional Chinese medicine. The black-banded snake (Zaocys dhumnades) is one such medicinal snake. Due to its significant efficacy, limited resources, and high price, it is often sold in the market as dried body or powder, losing its original characteristics and making it difficult to distinguish genuine from counterfeit. This has led to rampant counterfeiting, seriously affecting the efficacy and safety of the medicine. Currently, many counterfeit or adulterated products are available on the market, including common ones such as the king cobra, black-browed rat snake, hundred-flowered cobra, gray rat snake, silk rat snake, Qi snake, and cobra. In terms of identification, snake medicinal materials are mainly distinguished by the scales of the head, back, abdomen, and tail. However, some commercially available black-banded snakes and their counterfeits have had their heads, skins, and scales removed, significantly obliterating these characteristics. Traditional identification methods are difficult and inaccurate, especially after water extraction to prepare standard decoctions or other dosage forms, making effective identification even more challenging, particularly for detecting adulteration with similar morphological species. To ensure the quality and efficacy of black-banded snake medicinal materials, there is an urgent need for an accurate and rapid method to distinguish genuine black-banded snake from its counterfeits.

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

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

[0080] A method for identifying the black-banded snake and its products includes the following steps:

[0081] Preparation of reference herb solution and test sample solution;

[0082] The reference herb solution and the test sample solution were determined by high performance liquid chromatography-mass spectrometry, with acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution.

[0083] In some of these examples, the gradient elution procedure is as follows:

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

[0085] Between 5 and 8 minutes, the volume percentage of the mobile phase A increased from 15% to 40%.

[0086] In some of these examples, the volume percentage of formic acid in the aqueous formic acid solution is 0.05% to 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 of these examples, the high-performance liquid chromatography (HPLC) conditions include a column packed with octadecylsilane-bonded silica gel.

[0088] Specifically, the chromatographic column has a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm to 1.9 μm.

[0089] In some examples, the high-performance liquid chromatography (HPLC) conditions include a flow rate of 0.25 mL / min to 0.35 mL / min, for example, 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 0.3 mL / min.

[0090] In some of these examples, the high-performance liquid chromatography (HPLC) conditions include a column temperature of 25°C to 35°C. For example, these temperatures 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, with a preferred column temperature of 30°C.

[0091] In some of these examples, the high-performance liquid chromatography (HPLC) conditions include an injection volume of 1 μL to 3 μL, for example, 1 μL, 2 μL or 3 μL, preferably 2 μL.

[0092] In some of these examples, the mass spectrometry conditions included using mass-to-charge ratios of 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 detection ion pairs.

[0093] In the ion chromatography of the test sample extracted using ion pairs with 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, the following chromatographic peaks should simultaneously appear in the chromatographic retention time of the reference medicinal material, including *Zaocys dhumnades* medicinal material, processed slices, standard decoctions and their formulation granules, and traditional Chinese medicine containing *Zaocys dhumnades*, as well as in the case of wine-processed *Zaocys dhumnades* processed slices, standard decoctions and their formulation granules: chromatographic peaks should not appear in the counterfeit product.

[0094] This invention utilizes characteristic ion pairs to specifically identify the *Zaocys dhumnades* component in *Zaocys dhumnades* and its products, solving the problem of difficulty in identification due to the lack of appearance characteristics in *Zaocys dhumnades* standard decoctions, formula granules, and traditional Chinese medicine products containing *Zaocys dhumnades*.

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

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

[0097] In some of these examples, the conditions for mass spectrometry detection include: a nebulizer flow rate of 2 L / min to 3 L / min, for example, 2 L / min, 2.5 L / min and 3 L / min, preferably 3 L / min.

[0098] In some of these examples, the conditions for mass spectrometry detection include a heating gas flow rate of 8 L / min to 10 L / min, for example, 8 L / min, 9 L / min and 10 L / min, preferably 10 L / min.

[0099] In some of these examples, the conditions for mass spectrometry detection include an interface temperature of 300°C to 400°C, for example, 300°C, 320°C, 350°C, 380°C, and 400°C, with the interface temperature preferably being 300°C.

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

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

[0102] In some examples, the conditions for mass spectrometry detection include a heating block temperature of 350°C to 450°C, for example, 350°C, 360°C, 380°C, 400°C, 420°C and 450°C, preferably 400°C.

[0103] In some of these examples, the conditions for mass spectrometry detection include a dry gas flow rate of 8 L / min to 10 L / min, for example, 8 L / min, 9 L / min and 10 L / min, preferably 10 L / min.

[0104] In some examples, the preparation of the control herbal solution includes the following steps:

[0105] The reference herb *Zaocys dhumnades* was mixed with a first ammonium bicarbonate aqueous solution for the first extraction. The filtrate was then mixed with a first trypsin solution for the first enzymatic hydrolysis.

[0106] In some specific examples, the mass fraction of ammonium bicarbonate in the first aqueous solution 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, the mass fraction of ammonium bicarbonate in the first aqueous solution is 1%.

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

[0108] In some specific examples, the first extraction method is heating reflux extraction or ultrasonic extraction, preferably heating reflux.

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

[0110] In some specific examples, the first trypsin solution is a mixture of 1 mg of trypsin and 1 mL of an aqueous solution of 0.5% to 1.5% ammonium bicarbonate.

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

[0112] In some specific examples, in the step of preparing the control herbal 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℃~40℃, and the time of the first enzymatic hydrolysis is 6h~14h. Preferably, the temperature of the first enzymatic hydrolysis is 37℃, and the time of the first enzymatic hydrolysis is 12h.

[0114] Understandably, the first enzymatic hydrolysis of the present invention is carried out under isothermal conditions.

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

[0116] The test sample is mixed with a second ammonium bicarbonate aqueous solution for a second extraction, and the filtrate is mixed with a second trypsin solution for a second enzymatic hydrolysis.

[0117] In some examples, the mass fraction of ammonium bicarbonate in the second aqueous solution 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, the mass fraction of ammonium bicarbonate in the second aqueous solution is 1%.

[0118] In some specific examples, the second trypsin solution is a mixture of 1 mg of trypsin and 1 mL of an aqueous solution of 0.5% to 1.5% ammonium bicarbonate.

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

[0120] In some specific examples, in the step of preparing the test 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℃~40℃, and the hydrolysis time is 6h~14h. Preferably, the temperature of the second enzymatic hydrolysis is 37℃, and the hydrolysis time is 12h.

[0122] Understandably, the second enzymatic hydrolysis of the present invention is carried out under isothermal conditions.

[0123] In some specific examples, the test sample is one or more of the following: *Zaocys dhumnades* medicinal material, *Zaocys dhumnades* processed slices, wine-processed *Zaocys dhumnades* processed slices, or counterfeit products; the mass-to-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 the following: *Zaocys dhumnades* medicinal material, *Zaocys dhumnades* processed slices, wine-processed *Zaocys dhumnades* processed slices, or counterfeit products; the second extraction method is heating reflux extraction or ultrasonic extraction, preferably heating reflux extraction.

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

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

[0127] In some specific examples, the test sample is one or more of the standard decoction of Zaocys dhumnades or the standard decoction of Zaocys dhumnades in wine, and the second extraction method is heating reflux extraction or ultrasonic extraction, preferably ultrasonic extraction.

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

[0129] In some specific examples, the test sample is one or more of the following: *Zaocys dhumnades* formula granules or *Zaocys dhumnades* wine formula granules, and the mass-to-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 the following: *Zaocys dhumnades* formula granules or *Zaocys dhumnades* wine formula granules. The second extraction method is heating reflux extraction or ultrasonic extraction. Preferably, ultrasonic extraction is used.

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

[0132] In some specific examples, the test sample is a traditional Chinese medicine containing black shad, and the mass-to-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 traditional Chinese medicine containing Zaocys dhumnades, and the second extraction method is heating reflux extraction or ultrasonic extraction. Preferably, heating reflux extraction is used.

[0134] In some specific examples, the reflux temperature is 90℃~100℃ and the reflux time is 0.5h~5h. Preferably, the reflux temperature is 100℃ and the reflux time is 1h.

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

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

[0137] In some specific examples, the test samples include one or more of the following: Chinese black snake medicinal material, Chinese black snake standard decoction, Chinese black snake formula granules, Chinese black snake slices, Chinese black snake slices in wine, Chinese black snake standard decoction in wine, Chinese black snake formula granules in wine, Chinese patent medicines containing Chinese black snake, and counterfeit products.

[0138] In some specific examples, the counterfeit snakes include one or more of the following: king cobra, black-browed rat snake, flower cobra, gray rat snake, slippery rat snake, Agkistrodon halys, and cobra.

[0139] This invention also provides the application of the above-mentioned identification method in the identification and detection of the authenticity of Zaocys dhumnades medicinal materials, processed slices, standard decoctions and their formula granules.

[0140] This invention also provides the application of the above-mentioned identification method in the identification and detection of the authenticity of processed black snake slices, standard decoctions and their formula granules.

[0141] This invention also provides the application of the above-mentioned identification method in the identification and detection of authenticity of traditional Chinese medicines containing Zaocys dhumnades.

[0142] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0143] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[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 LC-MS / MS (Shimadzu Corporation); Waters Xevo TQ-S Cronos triple quadrupole LC-MS / MS (Waters Corporation); Waters BEH C18 column (2.1mm × 100mm, 1.7μm); electric thermostatic water bath (HWS-28 type, Shanghai Yiheng Scientific Instruments Co., Ltd.); CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.); pharmaceutical stability test chamber (SHH-250SD, Chongqing Yongsheng Experimental Instrument Factory); 0.001 g balance (ME204E, Mettler Toledo); 0.1 mg balance (Mettler Toledo, XP26); ultrapure water system (Milli-Q Direct, Merck KGaA).

[0147] 2. Drug Trial

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

[0149] Example 1

[0150] 1. Characteristic ions

[0151] A set of characteristic ions for identifying *Zaocys dhumnades* components in *Zaocys dhumnades* and its products; the secondary mass spectra of these characteristic ions are shown below. Figure 1The mass-to-charge ratios of the parent ion and the daughter ion 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. Study samples

[0153] This study included 93 batches of samples, including 1 batch of *Zaocys dhumnades* (Chinese black-striped snake) control material, 15 batches each of *Zaocys dhumnades* raw material (batch numbers: G1~G15), *Zaocys dhumnades* sliced ​​pieces (batch numbers: GP1~GP15), and standard decoction (batch numbers: GT1~GT15), 3 batches of *Zaocys dhumnades* formula granules (batch numbers: CG1~CG3), 15 batches each of wine-processed *Zaocys dhumnades* sliced ​​pieces (batch numbers: GP16~GP30) and standard decoction (batch numbers: GT16~GT30), and 3 batches of wine-processed *Zaocys dhumnades* formula granules (batch numbers: CG4~CG6). The traditional Chinese medicine preparations containing *Zaocys dhumnades* included Tongbi tablets, Musk Rheumatism Capsules, Vitiligo Capsules, and Yixian Pills. Counterfeit products included *King Rat Snake*, *Black-browed Rat Snake*, *Hundred-flower Rat Snake*, *Grey Rat Snake*, *Slippery Rat Snake*, *Agkistrodon halys*, and *Cobra*. See Table 1 for details.

[0154] Table 1 Sample Information Table

[0155]

[0156] 3. Preparation of reference herb solution and test sample solution

[0157] 3.1 Preparation of control herbal solution

[0158] Take about 0.2g of the reference herb *Zaocys dhumnades*, add 25mL of 1% ammonium bicarbonate solution, heat under reflux for 30 minutes, filter through a microporous membrane, take 1mL of the filtrate, place it in a sample vial, add 200μL of trypsin solution (prepare a solution containing 1mg per 1mL of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and incubate at 37℃ for 12 hours to obtain the final product.

[0159] 3.2 Preparation of test solution

[0160] 3.2.1 Chinese black snake medicinal materials, Chinese black snake slices, Chinese black snake slices made with wine, and counterfeit products (such as king snake, black-browed rat snake, hundred-flower rat snake, gray rat snake, smooth rat snake, Agkistrodon halys, and cobra).

[0161] Take about 0.2 g of the powder (passed through a No. 3 sieve), add 25 mL of 1% ammonium bicarbonate solution, and heat under reflux for 2 hours. Filter through a microporous membrane, take 1 mL of the filtrate, place it in a sample vial, add 200 μL of trypsin solution (prepare a solution containing 1 mg per mL of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and incubate at 37°C for 12 hours to obtain the final product.

[0162] 3.2.2 Standard decoction of Zaocys dhumnades and standard decoction of Zaocys dhumnades in wine

[0163] Take an appropriate amount of this product, grind it into a fine powder, take about 0.1g, add 50mL of 1% ammonium bicarbonate solution, sonicate for 30 minutes, cool, and shake well. Filter through a microporous membrane, take 1mL of the filtrate, place it in a sample vial, add 200μL of trypsin solution (prepare a solution containing 1mg per 1mL of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and incubate at 37℃ for 12 hours to obtain the final product.

[0164] 3.2.3 *Zaocys dhumnades* formula granules, *Zaocys dhumnades* wine formula granules

[0165] Take an appropriate amount of this product, grind it into a fine powder, take about 0.1g, add 25mL of 1% ammonium bicarbonate solution, sonicate for 30 minutes, cool, and shake well. Filter through a microporous membrane, take 1mL of the filtrate, place it in a sample vial, add 200μL of trypsin solution (prepare a solution containing 1mg per 1mL of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and incubate at 37℃ for 12 hours to obtain the final product.

[0166] 3.2.4 Traditional Chinese medicines containing Zaocys dhumnades

[0167] Take an appropriate amount of this product, grind it into a fine powder, take about 1g, place it in an Erlenmeyer flask, add 20mL of 1% ammonium bicarbonate solution, heat under reflux for 1 hour, cool, shake well, centrifuge (at a speed of 12000 rpm) for 5 minutes, take the supernatant, filter it through a microporous membrane, take 1mL of the filtrate, place it in a sample vial, add 200μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1mg per 1mL, prepare it immediately before use), shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the product.

[0168] 4. Chromatographic and mass spectrometric conditions

[0169] 4.1 Chromatographic conditions

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

[0171] Table 2 Gradient Elution Table

[0172]

[0173] 4.2 Mass Spectrometry Conditions

[0174] Mass spectrometry was used with an electrospray ionization (ESI+) detector in positive ion mode. The nebulizer gas flow rate was 3 L / min; the heating gas flow rate was 10 L / min; the interface temperature was 300℃; the desolventizing temperature was 526℃; the DL temperature was 250℃; the heating block temperature was 400℃; and the drying gas flow rate was 10 L / min. Collision energies and Q1 and Q3 deviations are shown in Table 3. Multiple reaction monitoring (MRM) was performed, and the mass-to-charge ratios m / z 674.00 (triple charge) → 747.37 and m / z 674.00 (triple charge) → 917.96, and m / z 700.33 (double charge) → 607.29 and m / z 700.33 (double charge) → 869.44 were selected as detection ion pairs. 2 μl of the *Zaocys dhumnades* reference herb solution was injected. The signal-to-noise ratio of the MRM chromatographic peaks determined according to the above detection ion pairs should be greater than 3:1. Take 2 μl of the test solution and inject it into the high performance liquid chromatography-mass spectrometry instrument for determination.

[0175] Table 3 Collision Energy and Q1, Q3 Deviations

[0176]

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

[0178] 5.1 Investigation on the pre-processing methods of *Zaocys dhumnades* medicinal materials, *Zaocys dhumnades* processed slices, wine-processed *Zaocys dhumnades* slices, and counterfeit products (such as king cobra, black-browed rat snake, hundred-flowered cobra, gray rat snake, silk rat snake, Agkistrodon halys, and cobra) (taking *Zaocys dhumnades* medicinal materials as an example)

[0179] (1) Examination of extraction methods

[0180] The study mainly examined two extraction methods: reflux and ultrasonic extraction. The effects of different extraction methods on the spectral identification of *Zaocys dhumnades* were compared by calculating the "peak area / sample weight" of four characteristic ion pairs, and the optimal extraction method was selected.

[0181] Take an appropriate amount of *Zaocys dhumnades* (passed through a No. 3 sieve, batch number: G7), approximately 0.2 g, accurately weighed, and perform two parallel groups, two portions per group. Add 25 mL of 1% ammonium bicarbonate solution to each group, weigh them, and extract one group by sonication (300 W power, 40 kHz frequency) for 30 minutes, and extract the other group by reflux for 30 minutes. After cooling, replenish the weight loss with 1% ammonium bicarbonate, shake well, filter through a microporous membrane, transfer 1 mL to a sample vial, add 100 μL of trypsin solution (prepare a solution containing 1 mg per mL of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and incubate at 37℃ for 12 hours for enzymatic hydrolysis. The experimental results are shown in Table 4.

[0182] Table 4. Mass spectrometry identification results of different extraction methods

[0183]

[0184] The results showed that the peak area / sample weight ratio of reflux extraction was significantly higher than that of ultrasonic extraction, indicating that the reflux extraction method was more effective. Therefore, reflux extraction was chosen.

[0185] (2) Examination of extraction time

[0186] The study mainly examined different extraction times and compared the effects of different extraction times on the spectral identification of *Zaocys dhumnades* medicinal materials by calculating the "peak area / sample weight" of four characteristic ion pairs, and selected the optimal extraction time.

[0187] Take an appropriate amount of *Zaocys dhumnades* (passed through a No. 3 sieve, batch number: G7), approximately 0.2 g, accurately weighed, and divided into 4 parallel groups, 2 portions per group. Add 25 ml of 1% ammonium bicarbonate solution to each group, weigh them, and reflux for 0.5 hours, 1 hour, 2 hours, and 4 hours respectively. After cooling, replenish the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22 μm microporous membrane, transfer 1 ml to a sample vial, add 100 μl of trypsin solution (prepare a solution containing 1 mg per ml of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and incubate at 37℃ for 12 hours. The experimental results are shown in Table 5.

[0188] Table 5. Mass spectrometry identification results at different extraction times

[0189]

[0190] The results showed that as the extraction time increased, the ratio of "peak area / sample weight" of the four characteristic ion pairs increased. When the extraction time reached 2 hours, the ratio of "peak area / sample weight" basically stopped increasing, indicating that the extraction was basically complete after 2 hours. Therefore, the extraction time was selected as 2 hours.

[0191] (3) Investigation on the dosage of trypsin

[0192] The main purpose of this study was to investigate the effect of different amounts of trypsin on the spectral identification of *Zaocys dhumnades* medicinal material. The effect of different amounts of trypsin on the spectral identification of *Zaocys dhumnades* medicinal material was compared by calculating the "peak area / sample weight * dilution factor" of four characteristic ion pairs, and the optimal amount of trypsin was selected to achieve the best enzymatic hydrolysis effect.

[0193] Take an appropriate amount of *Zaocys dhumnades* (passed through a No. 3 sieve, batch number: G7), approximately 0.2 g, accurately weighed, and perform 4 parallel groups (2 portions per group). Add 25 mL of 1% ammonium bicarbonate solution to each group, weigh, reflux for 2 hours, cool, and replenish the lost weight with 1% ammonium bicarbonate. Shake well, filter through a 0.22 μm microporous membrane, transfer 1 mL to a sample vial, and add 50 μL, 100 μL, 200 μL, and 400 μL of trypsin solution respectively (prepare a solution containing 1 mg per ml of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use). Shake well, and incubate at 37℃ for 12 hours for enzymatic hydrolysis. Inject the sample for analysis, record the peak area, and calculate "peak area / sample weight * dilution factor". The experimental results are shown in Table 6.

[0194] Table 6. Mass spectrometry identification results of different amounts of trypsin added

[0195]

[0196] The results showed that when the amount of trypsin added was 200 μL and 400 μL, the "peak area / sample weight * dilution factor" value of the detected ions was relatively large. In order to ensure complete enzymatic digestion and avoid excessive enzyme and inhibition, 200 μL was selected as the amount of trypsin added for the method.

[0197] Based on the results of the sample pretreatment investigation, the preparation method of the *Zaocys dhumnades* medicinal material test sample can be determined as follows: Take about 0.2 g of the powder (passed through a No. 3 sieve), add 25 ml of 1% ammonium bicarbonate solution, and heat under reflux for 2 hours. Filter through a microporous membrane, take 1 ml of the filtrate, place it in a sample bottle, add 200 μl of trypsin solution (prepare a solution containing 1 mg per ml of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the final product.

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

[0199] (1) Examination of extraction methods

[0200] The study mainly examined two extraction methods: reflux and ultrasonic extraction. The effects of different extraction methods on the mass spectrometry identification of the standard decoction of Zaocys dhumnades were compared by calculating the "peak area / sample weight" of four characteristic ion pairs, and the optimal extraction method was selected.

[0201] Take an appropriate amount of the standard decoction of *Zaocys dhumnades* (batch number: GT2), grind it into a fine powder, and accurately weigh about 0.1g. Perform two parallel groups, two portions per group. Add 50ml of 1% ammonium bicarbonate solution to each group and weigh them. Extract one group by sonication (300W power, 40kHz frequency) for 30 minutes, and extract the other group by reflux for 30 minutes. After cooling, replenish the lost weight with 1% ammonium bicarbonate, shake well, filter through a 0.22μm microporous membrane, transfer 1mL to a sample vial, add 100μL of trypsin solution (prepare a solution containing 1mg per ml of trypsin for sequence analysis, prepared immediately before use), shake well, and incubate at 37℃ for 12 hours. The experimental results are shown in Table 7.

[0202] Table 7. Mass spectrometry identification results of different extraction methods

[0203]

[0204] The results showed that the peak area / sample weight ratio of ultrasonic extraction and reflux extraction was not significantly different. For ease of operation, ultrasonic extraction was chosen.

[0205] (2) Examination of extraction time

[0206] The study mainly examined three extraction times: 15 minutes, 30 minutes, and 45 minutes. The effect of different extraction times on the mass spectrometry identification of the standard decoction of *Zaocys dhumnades* was compared by calculating the "peak area / sample weight" of four characteristic ion pairs, and the optimal extraction time was selected.

[0207] Take an appropriate amount of the standard decoction of *Zaocys dhumnades* (batch number: GT2), grind it into a fine powder, take about 0.1g, accurately weigh it, and divide it into 3 parallel groups, 2 portions per group. Add 50ml of 1% ammonium bicarbonate solution to each group, weigh them, and sonicate them (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. After cooling, make up the weight loss with 1% ammonium bicarbonate, shake well, filter through a 0.22μm microporous membrane, transfer 1ml to a sample bottle, add 100μl of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1mg per ml, prepare it immediately before use), shake well, and enzymatically hydrolyze at 37℃ for 12 hours. The experimental results are shown in Table 8.

[0208] Table 8. Mass spectrometry identification results at different extraction times

[0209]

[0210] The results showed that different extraction times did not significantly affect the peak area / sample weight ratio of the monitored ions. To ensure complete extraction, an ultrasonic time of 30 minutes was selected.

[0211] (3) Investigation on the dosage of trypsin

[0212] This study primarily investigated the relationship between the amount of trypsin used and the sample size of the *Zaocys dhumnades* standard decoction, aiming to determine the optimal trypsin dosage for a given sample size of *Zaocys dhumnades* standard decoction to achieve the best enzymatic hydrolysis effect. The study involved adding 50 μl, 100 μl, 200 μl, 400 μl, and 600 μl of trypsin to a 0.1 g sample size of *Zaocys dhumnades* standard decoction for enzymatic hydrolysis. The optimal trypsin dosage was selected by calculating the ratio of peak area to sample size multiplied by the dilution factor for four pairs of detected ion pairs.

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

[0214] Table 9. Mass spectrometry identification results of different amounts of trypsin added.

[0215]

[0216] The results showed that adding different amounts of trypsin had little effect on the ratio of "peak area / sample weight * dilution factor" of the detected ions. In order to ensure complete enzymatic digestion and avoid excessive enzyme to prevent inhibitory reaction, 200 μL was selected as the amount of trypsin added for the method.

[0217] Based on the results of the sample pretreatment investigation, the preparation method of the standard decoction of *Zaocys dhumnades* can be determined as follows: Take an appropriate amount of the product, grind it into a fine powder, take about 0.1g, add 50ml of 1% ammonium bicarbonate solution, sonicate for 30 minutes, cool, and shake well. Filter through a microporous membrane, take 1ml of the filtrate, place it in a sample bottle, add 200μl of trypsin solution (prepare a solution containing 1mg per ml of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the final product.

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

[0219] (1) Examination of extraction methods

[0220] The study mainly examined two extraction methods: reflux and ultrasonic extraction. The effects of different extraction methods on the mass spectrometry identification of *Zaocys dhumnades* particles were compared by calculating the "peak area / sample weight" of four characteristic ion pairs, and the optimal extraction method was selected.

[0221] Take an appropriate amount of *Zaocys dhumnades* formula granules (batch number: CG2), grind them finely, take about 0.1g, accurately weigh it, and place it in an Erlenmeyer flask. Perform two parallel groups, two portions per group. Add 25ml of 1% ammonium bicarbonate solution to each group, weigh them, and extract one group by ultrasonic treatment (300W power, 40kHz frequency) for 30 minutes. Extract the other group by reflux for 30 minutes. Remove the extracts, cool them, and replenish the lost weight with 1% ammonium bicarbonate. Shake well, filter through a 0.22μm microporous membrane, transfer 1ml of the filtrate to a micro-volume injection bottle, add 100μl of trypsin solution (prepare a solution containing 1mg per ml of trypsin for sequence analysis, prepared with 1% ammonium bicarbonate solution, and ready for use), shake well, and incubate at 37℃ for 12 hours. The experimental results are shown in Table 10.

[0222] Table 10 Mass spectrometry identification results of different extraction methods

[0223]

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

[0225] (2) Examination of extraction time

[0226] This experiment investigated the effect of different extraction times on the mass spectrometry identification of *Zaocys dhumnades* formula granules, mainly focusing on three extraction times: 15 minutes, 30 minutes, and 45 minutes. The effect of different extraction times on the mass spectrometry identification of *Zaocys dhumnades* formula granules was compared by calculating the "peak area / sample weight" of four characteristic ion pairs, and the optimal extraction time was selected.

[0227] Take an appropriate amount of *Zaocys dhumnades* formula granules (batch number: CG2), grind them finely, take about 0.1g, accurately weigh it, and place it in an Erlenmeyer flask. Perform three parallel groups, two portions per group, adding 25ml of 1% ammonium bicarbonate solution to each group, weighing them, and ultrasonically treating them (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Remove them, cool them, and replenish the lost weight with 1% ammonium bicarbonate. Shake well, filter through a 0.22μm microporous membrane, transfer 1ml of the filtrate to a micro-volume injection bottle, add 100μL of trypsin solution (prepare a solution containing 1mg per mL of trypsin for sequence analysis, prepared with 1% ammonium bicarbonate solution, and ready for use), shake well, and incubate at 37℃ for 12 hours. The experimental results are shown in Table 11.

[0228] Table 11 Mass spectrometry identification results at different extraction times

[0229]

[0230] The results showed that different extraction times did not significantly affect the peak area / sample weight ratio of the monitored ions. Extraction for 15 minutes was basically complete. To ensure the robustness of the method, an ultrasonic extraction time of 30 minutes was selected.

[0231] (3) Investigation on the dosage of trypsin

[0232] This study primarily investigated the relationship between trypsin dosage and the sample size of *Zaocys dhumnades* formula granules, aiming to determine the optimal trypsin dosage for a given sample size of *Zaocys dhumnades* formula granules to achieve the best enzymatic hydrolysis effect. The experiment involved adding 100 μL, 200 μL, and 400 μL of trypsin to a sample size of 0.1 g of *Zaocys dhumnades* formula granules for enzymatic hydrolysis. The optimal trypsin dosage was selected by calculating the ratio of peak area to sample size multiplied by the dilution factor for four pairs of detected ion pairs.

[0233] Take an appropriate amount of *Zaocys dhumnades* formula granules (batch number: CG2), grind them finely, take about 0.1g, accurately weigh it, place it in an Erlenmeyer flask, add 25ml of 1% ammonium bicarbonate solution, weigh it, sonicate (power 300W, frequency 40kHz) for 30 minutes, remove it, cool it, replenish the lost weight with 1% ammonium bicarbonate, shake it well, filter it through a 0.22μm microporous membrane, transfer 1ml of the filtrate to a micro-volume injection bottle, repeat 3 groups, 2 portions for each group, add 100μl, 200μl and 400μl of trypsin solution respectively (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 1mg per ml, prepare it immediately before use), shake it well, and enzymatically hydrolyze it at 37℃ for 12 hours. Inject the sample for analysis, record the peak area, and calculate "peak area / sample weight × dilution factor". The experimental results are shown in Table 12.

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

[0235]

[0236] The results showed that different amounts of trypsin added did not significantly affect the ratio of "peak area / sample weight × dilution factor" of the monitored ions. In order to better ensure that the sample can be completely digested, 200 μl was selected as the amount of trypsin added for the method.

[0237] Based on the results of the sample pretreatment investigation, the preparation method of the *Zaocys dhumnades* formula granules was determined as follows: Take an appropriate amount of the product, grind it finely, take about 0.1g, add 25ml of 1% ammonium bicarbonate solution, sonicate for 30 minutes, cool, and shake well. Filter through a microporous membrane, take 1ml of the filtrate, place it in a sample bottle, add 200μl of trypsin solution (prepare a solution containing 1mg per ml of trypsin for sequence analysis with 1% ammonium bicarbonate solution, and prepare immediately before use), shake well, and enzymatically hydrolyze at 37℃ for 12 hours to obtain the final product.

[0238] Example 2: Application of Characteristic Ion Pair Identification

[0239] (1) Application of the identification of black-striped snake, wine-processed black-striped snake and counterfeit products

[0240] Take powdered *Zaocys dhumnades* (batch numbers: G1~G15), *Zaocys dhumnades* sliced ​​herbs (batch numbers: GP1~GP15), *Zaocys dhumnades* sliced ​​herbs (batch numbers: GP16~GP30), and adulterant (batch numbers: X1~X7) respectively (passed through a No. 3 sieve), and prepare test solutions of *Zaocys dhumnades*, *Zaocys dhumnades* sliced ​​herbs, *Zaocys dhumnades* sliced ​​herbs (batch numbers: GP16~GP30), and adulterant (batch numbers: X1~X7) respectively according to the test solution preparation method under "3.2.1" in Example 1.

[0241] Take appropriate amounts of the standard decoction of Zaocys dhumnades (batch numbers: GT1~GT15) and the standard decoction of Zaocys dhumnades in alcohol (batch numbers: GT16~GT30), grind them into fine powder, and prepare the test solution of the standard decoction of Zaocys dhumnades and the test solution of the standard decoction of Zaocys dhumnades in alcohol according to the test solution preparation method under item "3.2.2" in Example 1.

[0242] Take appropriate amounts of *Zaocys dhumnades* formula granules (batch numbers: CG1~CG3) and *Zaocys dhumnades* formula granules (batch numbers: CG4~CG6) respectively, grind them into fine powder, and prepare *Zaocys dhumnades* formula granule test solutions and *Zaocys dhumnades* formula granule test solutions respectively according to the test solution preparation method under "3.2.3" in Example 1.

[0243] Take the black-striped snake reference material (batch number: STD) and prepare the black-striped snake reference material solution according to the preparation method of reference material solution under item "3.1" in Example 1.

[0244] Accurately pipette 2 μL of the above-mentioned test sample solution and the *Zaocys dhumnades* reference herb solution, respectively, and inject them into the liquid chromatograph. Using a mass spectrometer detector and electrospray ionization positive ion mode (ESI+), perform multiple reaction monitoring (MRM). In the ion chromatograms of the *Zaocys dhumnades* herb, processed slices, standard decoctions and their formulations, and wine-processed *Zaocys dhumnades* processed slices, standard decoctions and their formulations extracted under the chromatographic conditions described in section "4.1" of Example 1 and the mass spectrometric conditions described in section "4.2" of Example 1, the chromatograms should simultaneously show chromatographic peaks with the same retention time as the *Zaocys dhumnades* reference herb solution. However, in the ion chromatograms of the test samples of counterfeit products (such as *Elaphe carinata*, *Synonymus alatus*, *Elaphe yunnanensis*, *Synonymus alatus*, *Synonymus latae*, *Synonymus latae*, *Synonymus alatus*, *Synonymus latae*, *Synonymus alatus*, *Synonymus latae*, and *Synonymus latae*), no detection should be found. See Tables 13-20 for details. Figures 2-27 .in, Figures 2-3 The mass spectrometry identification diagram of the medicinal material *Zaocys dhumnades* (m / z 674.00). Figures 4-5 The mass spectrometry chromatogram for identifying slices of Zaocys dhumnades (m / z 674.00) is shown. Figures 6-7 The mass spectrometry identification chromatogram of the standard decoction of Zaocys dhumnades (m / z 674.00). Figure 8 The mass spectrometry identification chromatogram of the formula granules of Zaocys dhumnades (m / z 674.00). Figures 9-10 The mass spectrometry identification diagram of the medicinal material *Zaocys dhumnades* (m / z 700.33). Figures 11-12 The mass spectrometry chromatogram of processed black snake (m / z 700.33) is shown. Figures 13-14 The mass spectrometry identification chromatogram of the standard decoction of Zaocys dhumnades (m / z 700.33). Figure 15 The mass spectrometry identification chromatogram of the formula granules of Zaocys dhumnades (m / z 700.33). Figures 16-17 Mass spectrometry chromatogram for identifying processed black snake (Zaocys dhumnades) in wine (m / z 674.00). Figures 18-19 The mass spectrometry identification chromatogram of the standard decoction of *Zaocys dhumnades* (m / z 674.00). Figure 20 Mass spectrometry identification chromatogram of the formula granules for black snake in wine (m / z 674.00). Figures 21-22 Mass spectrometry chromatogram for identifying processed black snake (Zaocys dhumnades) in wine (m / z 700.33). Figures 23-24 The mass spectrometry identification chromatogram of the standard decoction of *Zaocys dhumnades* (m / z 700.33). Figure 25 Mass spectrometry identification chromatogram of the formula granules for the wine-processed black snake (m / z 700.33). Figure 26 Mass spectrometry diagram for differentiating Zaocys dhumnades from other adulterants (m / z 674.00). Figure 27 Mass spectrometry diagram for differentiating Zaocys dhumnades from other adulterants (m / z 700.33).

[0245] Table 13 Mass Spectrometry Results of Samples (Black-striped Snake Medicinal Material)

[0246]

[0247] Table 14 Mass Spectrometry Results of Samples (Black-striped Snake Slices)

[0248]

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

[0250]

[0251] Table 16 Mass Spectrometry Results of Samples (Black-striped Snake Formula Granules)

[0252]

[0253] Table 17 Mass Spectrometry Results of Samples (Wine-processed Black-striped Snake Slices)

[0254]

[0255] Table 18 Mass Spectrometry Results of Samples (Standard Decoction of Zaocys dhumnades)

[0256]

[0257] Table 19 Mass Spectrometry Results of Samples (Wine-processed Black Snake Formula Granules)

[0258]

[0259] Table 20 Mass Spectrometry Results of Samples (Other Counterfeit Products)

[0260]

[0261] (2) Detection of Chinese patent medicines containing Zaocys dhumnades

[0262] Take appropriate amounts of Tongbi tablets (batch number: C1), Musk Rheumatism Capsules (batch number: C2), Vitiligo Capsules (batch number: C3), and Yixian Pills (batch number: C4), grind them into a fine powder, and prepare test solutions containing Zaocys dhumnades according to the test solution preparation method under section "3.2.4".

[0263] Take the reference herb *Zaocys dhumnades* (batch number: STD) and prepare the reference herb solution according to the method for preparing the reference herb solution in section “3.1”.

[0264] Accurately pipette 2 μL of the above-mentioned test solution of the prepared Chinese medicine containing *Zaocys dhumnades* and the reference medicinal material solution of *Zaocys dhumnades*, respectively, and inject them into the liquid chromatograph. Using a mass spectrometer with a positive ion spray mode (ESI+), perform multiple reaction monitoring (MRM). The ion chromatogram of the test sample of the prepared Chinese medicine containing *Zaocys dhumnades* extracted under the above chromatographic and mass spectrometric conditions should simultaneously show a chromatographic peak with the same retention time as the reference medicinal material solution of *Zaocys dhumnades*. See Table 21 for details. Figures 28-29 ,in, Figure 28 The mass spectrometry identification diagram of traditional Chinese medicine containing Zaocys dhumnades (m / z 674.00) is shown. Figure 29 The mass spectrometry identification diagram of traditional Chinese medicine containing Zaocys dhumnades (m / z 700.33).

[0265] Table 21 Mass spectrometry results of traditional Chinese medicines containing Zaocys dhumnades

[0266]

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

[0268] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for identifying the black-tailed snake and its products, characterized in that, Includes the following steps: Preparation of reference herb solution and test sample solution; The reference medicinal material solution and the test solution were determined by high performance liquid chromatography-mass spectrometry, using a Waters BEH C18 column as the chromatographic column, acetonitrile as mobile phase A, and formic acid aqueous solution as mobile phase B for gradient elution. The Waters BEH C18 column has dimensions of 2.1 mm × 100 mm and a diameter of 1.7 μm. The gradient elution procedure is as follows: From 0 to 5 minutes, the volume percentage of the mobile phase A increased from 8% to 15%. Within 5 to 8 minutes, the volume percentage of the mobile phase A increased from 15% to 40%. In the formic acid aqueous solution, the volume percentage of formic acid is 0.05% to 0.15%; Mass spectrometry conditions include: The 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 were used as the detection ion pairs; The preparation of the reference herb solution includes the following steps: The reference herb *Zaocys dhumnades* was mixed with a first ammonium bicarbonate aqueous solution for the first extraction, and the filtrate was mixed with a first trypsin solution for the first enzymatic hydrolysis. Satisfy the following (1)~(4): (1) In the first aqueous solution of ammonium bicarbonate, the mass fraction of ammonium bicarbonate is 0.5%~1.5%; (2) The first extraction method is either heating and reflux extraction or ultrasonic extraction; (3) The first trypsin solution is made by mixing 1 mg of trypsin with 1 mL of 0.5%~1.5% ammonium bicarbonate aqueous solution; (4) The temperature of the first enzymatic hydrolysis is 35℃~40℃, and the time of the first enzymatic hydrolysis is 6h~14h; The preparation of the test solution includes the following steps: The test sample is mixed with a second ammonium bicarbonate aqueous solution for a second extraction, and the filtrate is mixed with a second trypsin solution for a second enzymatic hydrolysis. Satisfy 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 made by mixing 1 mg of trypsin with 1 mL of 0.5%~1.5% ammonium bicarbonate aqueous solution; (4) The temperature of the second enzymatic hydrolysis is 35℃~40℃, and the time of the second enzymatic hydrolysis is 6h~14h; The test samples include one or more of the following: Chinese black snake medicinal material, Chinese black snake standard decoction, Chinese black snake formula granules, Chinese black snake slices, Chinese black snake slices with wine, Chinese black snake standard decoction with wine, Chinese black snake formula granules with wine, Chinese patent medicine containing Chinese black snake, and counterfeit products. The counterfeit products include one or more of the following: king cobra, black-browed rat snake, flower cobra, gray rat snake, smooth rat snake, Agkistrodon halys, and cobra.

2. The identification method according to claim 1, characterized in that, The conditions for high performance liquid chromatography include one or more of the following (1) to (3): (1) The flow rate is 0.25 mL / min to 0.35 mL / min; (2) The column temperature is 25℃~35℃; (3) The injection volume is 1 μL to 3 μL.

3. The identification method according to claim 1 or 2, characterized in that, Mass spectrometry conditions include one or more of the following (1) to (9): (1) Electrospray positive ion mode is adopted; (2) Multiple reaction monitoring scan; (3) The atomizing gas flow rate is 2L / min ~ 3L / min; (4) The heating gas flow rate is 8L / min ~ 10L / min; (5) The interface temperature is 300℃~400℃; (6) The solvent removal temperature is 526℃; (7) The DL temperature is 150℃~250℃; (8) The temperature of the heating block is 350℃~450℃; (9) The flow rate of the drying gas is 8L / min ~10L / min.

4. The application of the identification method according to any one of claims 1 to 3 in the identification and detection of the authenticity of Zaocys dhumnades medicinal materials, processed slices, standard decoctions and their formula granules.

5. The application of the identification method according to any one of claims 1 to 3 in the identification and detection of the authenticity of processed black snake slices, standard decoctions and their formula granules.

6. The application of the identification method according to any one of claims 1 to 3 in the identification and detection of authenticity of traditional Chinese medicines containing Zaocys dhumnades.

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