Detection methods and applications of Oyster shell medicinal materials or its processed slices, standard decoctions, and traditional Chinese medicine granules

Abstract

This invention discloses a detection method and application for *Crassostrea gigas* medicinal material or its processed slices, standard decoctions, and traditional Chinese medicine granules, belonging to the field of analytical detection technology. The detection method includes: enzymatic extraction of *Crassostrea gigas* medicinal material or its processed slices, standard decoctions, and traditional Chinese medicine granules to obtain a test solution; enzymatic extraction and enzymatic digestion of *Crassostrea gigas* reference material to obtain a reference solution; and detection of the test solution and the reference solution using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). The detected ion pairs are 364.21→307.19, 364.21→417.25, 369.71→512.25, and 369.71→448.26. The detection method of this invention has strong specificity and can effectively identify different oyster origins and adulterants through the organic matter of *Crassostrea gigas*, providing a reliable basis for the quality control and evaluation of oysters and their products.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a method and application for detecting Oyster shell or its processed slices, standard decoctions, and traditional Chinese medicine granules. Background Technology

[0002] The 2020 edition of the Chinese Pharmacopoeia defines oyster as the shell of the oyster species *Ostrea gigas* Thunberg, *Ostrea talienwhanensis* Crosse, or *Ostrea rivularis* Gould, belonging to the family Ostreidae. It is believed to have calming and sedative effects, nourishing Yin and suppressing Yang, and softening and dispersing masses. It is used for palpitations, insomnia, dizziness, tinnitus, scrofula, phlegm nodules, and abdominal masses.

[0003] The 2020 edition of the Chinese Pharmacopoeia identifies oysters using microscopic and thin-layer chromatography methods. However, these methods lack strong specificity for different origins, adulterants, and other shellfish species. A review of literature and patents reveals that current research largely focuses on oyster meat, with very little research on oyster shells used in traditional Chinese medicine. Existing studies on the identification of oyster shells from different origins primarily focus on morphology and microscopic aspects. However, due to the high morphological flexibility of oyster shells and their susceptibility to environmental influences, it is sometimes difficult to distinguish specific species based solely on appearance. Oyster shells are composed of both inorganic and organic matter. The inorganic matter, primarily calcium carbonate, accounts for over 94% of the shell's mass, while the organic matter constitutes a very small percentage. Identifying and distinguishing oyster shells from different origins using protein and peptide studies is also a significant challenge. Furthermore, for water-extracted oyster shell products, such as standard decoctions and granules of traditional Chinese medicine, the decoction process destroys proteins and other components, making specific peptide identification based on proteomics extremely difficult. Therefore, how to achieve highly specific identification of oysters to meet the needs of quality control is an urgent technical problem to be solved, and a technical challenge that urgently needs to be overcome in the field of oyster product quality control. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a detection method for oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine granules, which has strong specificity and can effectively distinguish different oyster raw materials and counterfeit oyster products.

[0005] The technical problem that this invention also needs to solve is to provide the application of the detection method for the above-mentioned Oyster shell medicinal material or its processed slices, standard decoctions, and traditional Chinese medicine formula granules.

[0006] The technical problem that this invention also aims to solve is to provide a method for identifying the Os oyster and its adulterants.

[0007] The technical problem that this invention also aims to solve is to provide a method for identifying the origin of oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine granules.

[0008] To address the aforementioned technical problems, this invention provides a method for detecting *Ostrea gigas* medicinal material or its processed slices, standard decoctions, and traditional Chinese medicine granules, comprising:

[0009] The test solution was obtained by enzymatic extraction of the raw material of Oyster of Jinjiang or its processed slices, standard decoction, and traditional Chinese medicine formula granules.

[0010] The reference herb, *Crassostrea gigas*, was enzymatically extracted to obtain a reference herb solution.

[0011] The test solution and the control medicinal material solution were analyzed using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) instrument to obtain the results.

[0012] The detected ion pairs were 364.21→307.19, 364.21→417.25, 369.71→512.25 and 369.71→448.26.

[0013] As an improvement to the above technical solution, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows:

[0014] The chromatographic column was packed with octadecylsilane-bonded silica gel, with a column length of 50 mm to 150 mm, a column diameter of 2 mm to 3 mm, a packing particle size of 1.6 to 1.9 μm, a column temperature of 25℃ to 40℃, a flow rate of 0.2 mL / min to 0.4 mL / min, and an injection volume of 1 μL to 3 μL. Mobile phase A was acetonitrile, and mobile phase B was a 0.05 vol% to 0.2 vol% aqueous formic acid solution. The elution curve was as follows:

[0015] From 0 min to 10 min, mobile phase A decreased from 5% to 20%, and mobile phase B decreased from 95% to 80%.

[0016] Between 10 and 14 minutes, mobile phase A decreased from 20% to 80%, while mobile phase B decreased from 80% to 20%.

[0017] As an improvement to the above technical solution, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows:

[0018] The chromatographic column used was a Waters ACQUITY UPLC BEH C18 column, 100 mm long, 2.1 mm in diameter, with a packing particle size of 1.7 μm. The column temperature was 30 °C, the flow rate was 0.3 mL / min, the injection volume was 2 μL, mobile phase A was acetonitrile, and mobile phase B was 0.1 vol% formic acid aqueous solution. The elution curve was as follows:

[0019] From 0 min to 10 min, mobile phase A decreased from 5% to 20%, and mobile phase B decreased from 95% to 80%.

[0020] Between 10 and 14 minutes, mobile phase A decreased from 20% to 80%, while mobile phase B decreased from 80% to 20%.

[0021] As an improvement to the above technical solution, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1.2kV to 1.8kV, ion source temperature of 320℃ to 400℃, desolvation gas flow rate of 600L / hr to 700L / hr, curtain gas flow rate of 5L / hr to 15L / hr, and ion source cone voltage of 15V to 25V.

[0022] As an improvement to the above technical solution, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1.5 kV, ion source temperature of 350 °C, desolvation gas flow rate of 650 L / hr, curtain gas flow rate of 10 L / hr, and ion source cone voltage of 20 V.

[0023] As an improvement to the above technical solution, the step of enzymatically extracting oyster medicinal materials or their processed slices, standard decoctions, or traditional Chinese medicine formula granules to obtain the test sample solution includes:

[0024] The test solution was obtained by sequentially extracting oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine formula granules with ammonium bicarbonate solution, dithiothreitol solution, iodoacetamide solution, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting.

[0025] The ratio of ammonium bicarbonate solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 40 mL to 80 mL: 1 g to 2 g; the concentration of the ammonium bicarbonate solution is 1 wt% to 5 wt%.

[0026] The ratio of the dithiothreitol solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 100μL~200μL:1g~2g; the concentration of the dithiothreitol solution is 0.5mol / L~3mol / L.

[0027] The ratio of the iodoacetamide solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 300μL~800μL:1g~2g; the concentration of the iodoacetamide solution is 0.2mol / L~2mol / L.

[0028] The trypsin solution is prepared by adding 3 mg to 10 mg of trypsin to 0.5 wt% to 2 wt% ammonium bicarbonate solution. The ratio of the trypsin solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 200 μL to 1000 μL: 1 g to 2 g.

[0029] The solid-phase extraction column desalting process includes:

[0030] Centrifuge the enzymatic hydrolysate obtained by enzymatic hydrolysis, add the supernatant to an HLB column, elute with water, discard the eluent, and then elute sequentially with 10 vol%–30 vol% acetonitrile solution and 30 vol%–60 vol% acetonitrile solution. Collect the eluent, filter, and obtain the final product.

[0031] Accordingly, the present invention also discloses the application of the above-mentioned detection methods for Ostrea gigas medicinal material or its processed slices, standard decoctions, and traditional Chinese medicine formula granules in the following (1) or (2):

[0032] (1) Identify the origin of oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine granules;

[0033] (2) Identify the medicinal materials of Oyster roe or its processed slices, standard decoctions, Chinese medicine formula granules and their counterfeits.

[0034] As an improvement to the above technical solution, the oyster medicinal material or its processed slices, standard decoctions, and traditional Chinese medicine formula granules include the Pacific oyster and the Dalian Bay oyster.

[0035] The counterfeit products include Fujian oysters, Portuguese oysters, abalone shells, and cuttlebone.

[0036] Accordingly, the present invention also discloses a method for identifying oyster shell and its adulterants, comprising: providing a sample to be tested; testing the sample to be tested using the above-mentioned detection methods for oyster shell medicinal materials or its processed slices, standard decoctions, and traditional Chinese medicine granules; if the mass spectrum obtained by detecting the test solution prepared from the sample under a preset detection ion pair does not show any detection at the retention time of the ion peak of the oyster shell reference medicinal material solution, then the sample to be tested is an adulterant.

[0037] Accordingly, the present invention also discloses a method for identifying the origin of oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine formula granules, which includes: providing a sample to be tested; testing the sample to be tested using the above-mentioned detection method for oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine formula granules; if the mass spectrum obtained by detecting the test solution prepared from the sample to be tested under a preset detection ion pair does not show any detection at the ion peak retention time of the oyster reference medicinal material solution, then the origin of the sample to be tested is oyster or Dalian Bay oyster.

[0038] Implementing this invention has the following beneficial effects:

[0039] Based on in-depth analysis of oyster medicinal materials and other oyster-based medicinal materials, this invention establishes a detection method for oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine granules. This detection method has strong specificity and can effectively identify different oysters and counterfeit products through the organic matter of oysters, providing a reliable basis for the quality control and evaluation of oysters and their products. Attached Figure Description

[0040] Figure 1 These are the mass spectra of the *Crassostrea gigas* medicinal material and the blank solvent in the specificity investigation of Example 1;

[0041] Figure 2 This is the mass spectrum of Oyster shell at a column temperature of 28°C during the durability test at different column temperatures in Example 1.

[0042] Figure 3 This is the mass spectrum of Oyster shell at a column temperature of 30°C during the durability test at different column temperatures in Example 1;

[0043] Figure 4 This is the mass spectrum of Oyster shell at a column temperature of 32°C during the durability test at different column temperatures in Example 1;

[0044] Figure 5 This is the mass spectrum of the Oyster shell at a flow rate of 0.28 mL / min during the durability study at different flow rates in Example 1;

[0045] Figure 6 This is the mass spectrum of the *Crassostrea gigas* herb at a flow rate of 0.30 mL / min during the durability study at different flow rates in Example 1.

[0046] Figure 7 This is the mass spectrum of the Oyster shell at a flow rate of 0.32 mL / min during the durability study at different flow rates in Example 1;

[0047] Figure 8 This is the mass spectrum of Oyster shell from the Ōmi River when Waters ACQUITY UPLC BEH C18 was used in the robustness test of different chromatographic columns in Example 1.

[0048] Figure 9 This is the mass spectrum of Oyster shell material when using Agilent ZORBAX Eclipse XDBC18 in the robustness test of different chromatographic columns in Example 1.

[0049] Figure 10 This is the mass spectrum of Oyster shell material when using Agilent ZORBAX SB-C18 in the robustness test of different chromatographic columns in Example 1.

[0050] Figure 11 The mass spectrum (m / z 364.21) of 17 batches of Oyster shell medicinal materials in Example 1 is shown on the left, from m / z 364.21 to 417.25, and on the right, from m / z 364.21 to 307.19.

[0051] Figure 12 The mass spectrum (m / z 364.21) of 17 batches of Oyster shell slices from Example 1 is shown on the left, from m / z 364.21 to 307.19, and on the right, from m / z 364.21 to 417.25.

[0052] Figure 13 The mass spectrum (m / z 364.21) of the 17 batches of Oyster shell standard decoction in Example 1 is shown on the left, with m / z 364.21→417.25 and m / z 364.21→307.19 on the right.

[0053] Figure 14 The mass spectrum (m / z 364.21) of the 6 batches of Oyster shell traditional Chinese medicine formula granules in Example 1 is shown on the left, with m / z 364.21→417.25 and m / z 364.21→307.19 on the right.

[0054] Figure 15 The mass spectrum (m / z 369.71) of 17 batches of Oyster shell medicinal materials in Example 1 is shown on the left, with m / z 369.71→512.25 and m / z 369.71→448.26 on the right.

[0055] Figure 16 The mass spectrum (m / z 369.71) of 17 batches of Oyster shell slices from Example 1 is shown on the left, with m / z 369.71→512.25 and m / z 369.71→448.26 on the right.

[0056] Figure 17 The mass spectrum (m / z 369.71) of the 17 batches of Oyster shell standard decoction in Example 1 is shown on the left, with m / z 369.71→512.25 and m / z 369.71→448.26 on the right.

[0057] Figure 18 The mass spectrum (m / z 369.71) of the 6 batches of Oyster shell traditional Chinese medicine formula granules in Example 1 is shown on the left, with m / z 369.71→512.25 and m / z 369.71→448.26 on the right.

[0058] Figure 19The mass spectra (m / z 364.21) of one batch of Dalian Bay oysters, two batches of Portuguese oysters, and six batches of Fujian oysters in Example 2 are shown. The left side shows m / z 364.21→417.25, and the right side shows m / z 364.21→307.19. Among them, ML represents the reference material of Ostrinia roxburghii.

[0059] Figure 20 The mass spectra (m / z 364.21) of 6 batches of *Crassostrea gigas*, 3 batches of *Abalone shell*, and 3 batches of cuttlebone in Example 2 are shown. The left side shows m / z 364.21→417.25, and the right side shows m / z 364.21→307.19. Among them, ML represents the *Crassostrea gigas* reference material.

[0060] Figure 21 The mass spectra (m / z 369.71) of one batch of Dalian Bay oysters, two batches of Portuguese oysters, and six batches of Fujian oysters in Example 2 are shown. The left side shows m / z 369.71→512.25, and the right side shows m / z 369.71→448.26. Among them, ML represents the reference material of Ostrinia roxburghii.

[0061] Figure 22 The mass spectra (m / z 369.71) of 6 batches of oyster shell, 3 batches of abalone shell, and 3 batches of cuttlebone in Example 2 are shown. The left side is m / z 369.71→512.25, and the right side is m / z 369.71→448.26. Among them, ML is the reference material of oyster shell. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0063] Example 1: Detection method for Omi oyster

[0064] 1. Preparation of the test solution

[0065] (1) Oyster shell or its processed slices: Take 2g of the fine powder of this product, grind it finely, place it in a stoppered conical flask, add 50mL of 2% ammonium bicarbonate solution, heat under reflux for 30 minutes, cool to room temperature, add 150μL of dithiothreitol (DTT) solution (1mol / L), mix well, incubate in a water bath at 56℃ for 1 hour, remove, cool, add 500μL of iodoacetamide (IAA) solution (0.6mol / L), mix well, protect from light for 30 minutes, add trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 5mg per 1mL, prepare immediately before use). Prepare 500 μL of the solution, shake well, and incubate at 37°C for 12 hours. Centrifuge (4000 rpm) for 5 minutes, collect the supernatant, and slowly pass it through an activated and equilibrated HLB column [specification: 6 mL (200 mg), eluted sequentially with 6 mL each of acetonitrile and 2% ammonium bicarbonate solution]. Elute with 2 mL of purified water, discard the eluent, and then elute with 1.5 mL of 20 vol% acetonitrile solution. Collect the eluent, followed by elution with 1.5 mL of 50% acetonitrile solution. Collect the eluent, combine the two eluents, and filter through a microporous membrane (0.22 μm) to obtain the test solution.

[0066] (2) Standard decoction of Oyster shell: Take 1.5g of this product, grind it into a fine powder, place it in a stoppered conical flask, add 50mL of 2% ammonium bicarbonate solution, sonicate for 30 minutes, cool, add 150μL of dithiothreitol (DTT) solution (1mol / L), mix well, incubate in a 56℃ water bath for 1 hour, remove, cool, add 500μL of iodoacetamide (IAA) solution (0.6mol / L), mix well, protect from light for 30 minutes, add trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 5mg per 1mL, prepare immediately before use). 500 μL, shake well, incubate at 37℃ for 12 hours, centrifuge (4000 rpm) for 5 minutes, collect the supernatant, and slowly pass it through an activated and equilibrated HLB column [specification: 6 mL (200 mg), eluted sequentially with 6 mL each of acetonitrile and 2% ammonium bicarbonate solution]. Elute with 2 mL of purified water, discard the eluent, elute again with 1.5 mL of 20% acetonitrile solution, collect the eluent, and then elute with 1.5 mL of 50% acetonitrile solution, collect the eluent, combine the two eluents, and filter through a microporous membrane (0.22 μm) to obtain the test solution.

[0067] (3) Oyster shell granules: Take 2g of this product, grind it into a fine powder, place it in a stoppered conical flask, add 50mL of 2% ammonium bicarbonate solution, sonicate for 30 minutes, cool, add 150μL of dithiothreitol (DTT) solution (1mol / L), mix well, incubate in a 56℃ water bath for 1 hour, remove, cool, add 500μL of iodoacetamide (IAA) solution (0.6mol / L), mix well, protect from light for 30 minutes, add trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 5mg per 1mL, prepare immediately before use) 250 μL, shake well, incubate at 37℃ for 12 hours, centrifuge (4000 rpm) for 5 minutes, collect the supernatant, and slowly pass it through an activated and equilibrated HLB column [specification: 6 mL (200 mg), eluted sequentially with 6 mL each of acetonitrile and 2% ammonium bicarbonate solution]. Elute with 2 mL of purified water, discard the eluent, elute again with 1.5 mL of 20% acetonitrile solution, collect the eluent, and then elute with 1.5 mL of 50% acetonitrile solution, collect the eluent, combine the two eluents, and filter through a microporous membrane (0.22 μm) to obtain the test solution.

[0068] 2. Preparation of the control herbal solution

[0069] Take 2g of *Crassostrea gigas* reference material and place it in a stoppered conical flask. Add 50mL of 2% ammonium bicarbonate solution, heat under reflux for 30 minutes, cool, add 150μL of dithiothreitol (DTT) solution (1mol / L), mix well, incubate at 56℃ for 1 hour, remove, cool, add 500μL of iodoacetamide (IAA) solution (0.6mol / L), mix well, incubate in the dark for 30 minutes, and add 500μL of trypsin solution (prepare a solution containing 5mg per mL with 1% ammonium bicarbonate solution for sequence analysis, and prepare immediately before use). Shake well, incubate at 37℃ for 12 hours, centrifuge (4000 rpm) for 5 minutes, collect the supernatant, and slowly pass it through an activated and equilibrated HLB column [specification: 6 mL (200 mg), eluted sequentially with 6 mL each of acetonitrile and 2% ammonium bicarbonate solution]. Elute with 2 mL of purified water, discard the eluent, elute again with 1.5 mL of 20% acetonitrile solution, collect the eluent, and then elute with 1.5 mL of 50% acetonitrile solution, collect the eluent, combine the two eluents, and filter through a microporous membrane (0.22 μm) as the control drug solution.

[0070] 3. Chromatographic and mass spectrometric conditions

[0071] 3.1 Chromatographic conditions

[0072] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm); mobile phase: acetonitrile (A) - 0.1 vol% formic acid solution (B); elution mode: gradient elution; flow rate: 0.3 mL / min; column temperature: 30 °C. The gradient elution program is shown in Table 1.

[0073] Table 1 Elution Procedure

[0074]

[0075] 3.2 Mass Spectrometry Conditions

[0076] An electrospray positive ion source (ESI+) was used, with a capillary voltage of 1.5 kV, an ion source temperature of 350 °C, a desolvation gas flow rate of 650 L / hr, and a curtain gas flow rate of 10 L / hr. Multiple reaction monitoring (MRM) was performed using the following ion pairs: m / z 364.21 (double charge) → 307.19, m / z 364.21 (double charge) → 417.25, m / z 369.71 (double charge) → 512.25, and m / z 369.71 (double charge) → 448.26. The cone voltage and collision voltage of each ion pair are shown in Table 2. The signal-to-noise ratio of the MRM ion peaks measured using the above ion pairs should be greater than 3:1.

[0077] Table 2 Mass Spectrometry Conditions

[0078]

[0079] 4. Measurement

[0080] Accurately pipette 2 μL of the reference herb solution and 2 μL of the test sample solution into an ultra-high performance liquid chromatography-mass spectrometry (WatersUPLC-Xevo TQ-S Cronos) instrument for determination.

[0081] 5. Methodological Examination

[0082] The test solution of Crassula ovata was prepared according to the above-described method. Different mobile phases (1#: 10 mmol / L ammonium formate-acetonitrile, 2#: 0.1% formic acid-acetonitrile) were used to investigate the effect of different mobile phases on the detection results. The optimal mobile phase system was selected, and the specific results are shown in Table 3.

[0083] Table 3 Mobile Phase Analysis Table

[0084]

[0085] The data above shows that the mobile phase of the formic acid-acetonitrile system yields better detection results, therefore, the mobile phase of 0.1% formic acid-acetonitrile was selected.

[0086] 6. Methodological Validation

[0087] 6.1 Exclusivity

[0088] Preparation of the test solution: Take *Crassostrea gigas* (Japanese oyster shell) and prepare the test solution according to the above method; take the blank solvent and prepare a blank solvent for the missing sample using the same method. Accurately pipette 2 μL of each of the above solutions and inject them into the liquid chromatography-mass spectrometry (LC-MS) instrument. Analyze the results according to the above chromatographic and mass spectrometric conditions. The results are as follows: Figure 1 As shown.

[0089] The results showed that the blank solvent test solution spectrum lacked the sample and did not detect characteristic ion peaks at the retention times corresponding to those of Omi oyster, indicating that the blank solvent did not interfere with the detection of characteristic ion pairs in the method, and that the method is specific.

[0090] 6.2 Precision

[0091] Take oyster shell (Crassostrea gigas) and prepare the test solution according to the above-described test solution preparation method. Precisely inhale 2 μL of the test solution, and repeat the injection 6 times under the above-described chromatographic and mass spectrometric conditions. Record the peak area and calculate the peak area RSD value. The results are shown in Table 4.

[0092] Table 4. Results of Instrument Precision Evaluation

[0093]

[0094] The results showed that when the same sample solution was injected six times consecutively, the peak area RSD of the characteristic ion pairs was less than 3.0%, indicating that the instrument had good precision.

[0095] 6.3 Stability

[0096] Take the oyster shell material and prepare the test solution according to the above-mentioned test solution preparation method. Accurately pipette 2 μL into the liquid chromatography-mass spectrometry instrument at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 hours respectively, and perform the determination according to the above-mentioned chromatographic and mass spectrometric conditions. The stability of the solution is evaluated by the peak area of ​​characteristic ions. The determination results are shown in Table 5.

[0097] Table 5. Results of the stability test

[0098]

[0099] The results showed that after the test solution was left at room temperature for 18 hours, the peak area RSD values ​​of the four characteristic ions ranged from 1.52% to 3.55%, and all were clearly detectable. This indicates that the test solution has good stability after being left at room temperature for 18 hours and does not affect the identification of characteristic ions.

[0100] 6.4 Repeatability

[0101] Take six parallel portions of oyster shell (Crassostrea gigas) and prepare test solutions according to the above-described method. Accurately pipette 2 μL of each solution and inject it into a liquid chromatography-mass spectrometry (LC-MS) instrument. Measure the solution under the described chromatographic and mass spectrometric conditions and calculate the RSD value of the peak area / sample weight ratio. The results are shown in Table 6.

[0102] Table 6 Results of Repeatability Testing

[0103]

[0104] The results showed that when the same batch of samples was measured repeatedly 6 times, the RSD value of the "peak area / sample weight" ratio was less than 5%, indicating that the method had good repeatability.

[0105] 6.5 Durability

[0106] ① Investigation at different column temperatures

[0107] This section compares the effects of different column temperatures (28℃, 30℃, and 32℃) on the detection of characteristic ion peaks in *Crassostrea gigas* medicinal materials. Specifically, *Crassostrea gigas* medicinal materials were used, and a test solution was prepared according to the above-described method. 2 μL of the solution was accurately pipetted into the liquid chromatography-mass spectrometry (LC-MS) instrument, and the determination was performed under the conditions described above. The experimental results are shown in […]. Figures 2-4 Table 7.

[0108] Table 7. Peak area results of durability study of Oyster shell at different column temperatures.

[0109]

[0110] The results showed that the four specified characteristic ion pairs could be clearly detected under three different column temperature conditions, indicating that small-scale adjustments to the column temperature had no effect on the detection and identification of characteristic ions in Oyster shell.

[0111] ② Investigation of different flow velocities

[0112] The effects of flow rates of 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min on the detection of characteristic ion peaks in *Crassostrea gigas* were compared. The test solution was prepared according to the above-described method, and 2 μL was accurately pipetted into the liquid chromatography-mass spectrometry (LC-MS) instrument. The analysis was performed under the conditions described above, and the results are shown in the figure. Figures 5-7 Table 8.

[0113] Table 8. Peak area results of durability study of Oyster shell medicinal material at different flow velocities.

[0114]

[0115] The results showed that the four specified characteristic ion pairs could be clearly detected under three different flow rate conditions, indicating that small-scale adjustments to the flow rate had no effect on the detection and identification of characteristic ions in Oyster shell.

[0116] ③ Different chromatographic columns

[0117] This section compares the effects of three different brands and types of chromatographic columns on the detection of characteristic ion peaks in Ostrea gigas medicinal materials: 1#: Waters ACQUITY UPLC BEH C18 column (100mm×2.1mm, 1.7μm), 2#: Agilent ZORBAX Eclipse XDB C18 column (100mm×2.1mm, 1.8μm), and 3#: Agilent ZORBAX SB-C18 column (100mm×2.1mm, 1.8μm).

[0118] Take the oyster shell (Crassostrea gigas) and prepare the test solution according to the above-described method. Accurately pipette 2 μL and inject it into the liquid chromatography-mass spectrometry (LC-MS) instrument. Perform the analysis under the conditions described above. The experimental results are shown in [Figure 1]. Figures 8-10 Table 9.

[0119] Table 9. Peak area results of column durability testing for Oyster shell medicinal materials.

[0120]

[0121] The results showed that all three chromatographic columns used could clearly detect the four specified characteristic ion pairs, indicating that different brands and models of chromatographic columns had no effect on the detection and identification of characteristic ions in Oyster shell.

[0122] 7. Detection of Oyster Shell (Oyster Mukorossi) medicinal materials, standard decoctions, and traditional Chinese medicine granules.

[0123] This embodiment studied a total of 57 batches of samples, including 17 batches of raw *Ostrea gigas* (batch numbers: G1-G17), 17 batches of processed *Ostrea gigas* (batch numbers: GP1-GP17), 17 batches of standard *Ostrea gigas* decoction (batch numbers: GT1-GT18), and 6 batches of *Ostrea gigas* herbal formula granules (batch numbers: CG1-CG6). It should be noted that processed *Ostrea gigas* refers to the raw *Ostrea gigas* material obtained by washing, drying, and crushing. Standard *Ostrea gigas* decoction refers to the product obtained by decocting, separating solids and liquids, concentrating, and drying the aforementioned processed *Ostrea gigas*. Herbal formula granules refer to the granules obtained by extracting, separating, concentrating, drying, and granulating the aforementioned processed *Ostrea gigas*.

[0124] Prepare test solutions for each sample according to the method described in section "1", and inject and test them according to the conditions described in section "3". Specific test results are shown in Table 10 and... Figures 11-18 As shown in the figure, the ion currents of the tested samples of Oyster shell medicinal material, processed slices, standard decoction, and formula granules should simultaneously exhibit ion peaks with retention times consistent with those of the Oyster shell control solution, and the signal-to-noise ratio should be greater than 3:1.

[0125] Table 10 Mass Spectrometry Results of Oyster Shell Herbs, Standard Decoctions, and Formula Granules

[0126]

[0127]

[0128] Example 2: Identification of different oyster origins and adulterants

[0129] This embodiment studied a total of 7 batches of other original oysters, including 6 batches of Pacific oyster (batch numbers: C1~C6) and 1 batch of Dalian Bay oyster (batch number: D1); a total of 14 batches of counterfeit products were studied, including 6 batches of Fujian oyster (batch numbers: F1~F6), 2 batches of Portuguese oyster (batch numbers: H1~H2), 3 batches of abalone shell (batch numbers: Sjm1~Sjm3), and 3 batches of cuttlebone (batch numbers: Hpx1~Hpx3).

[0130] Prepare test solutions for each sample according to the method described in section "1" of Example 1, and inject and test them according to the conditions described in section "3". Specific test results are as follows. Figures 19-22 As shown in the figure, different original substances (Oyster caryophyllum, Oyster dalianense) and adulterants (Oyster fusiforme, Oyster dalianense, Abalone shell, Cuttlebone) were not detected at the retention time of the ion peak in the Oyster caryophyllum reference solution.

[0131] The above description is a preferred embodiment of the invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. The application of the detection methods for Oyster shell or its processed slices, standard decoctions, and traditional Chinese medicine granules in the following (1) or (2): (1) Identify the origin of oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine granules; among which, The oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine formula granules include the oyster of the Yangtze River, the long oyster, and the Dalian Bay oyster; (2) Identify the raw materials of Oyster dauricum or its processed slices, standard decoctions, Chinese medicine formula granules and their counterfeits; wherein the counterfeits include Oyster dauricum fruticosa, Oyster dauricum var. dauricum, and cuttlebone. The detection methods for Oyster shell (Oyster roe) or its processed slices, standard decoctions, and traditional Chinese medicine granules include: The test solution was obtained by sequentially extracting oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine formula granules with ammonium bicarbonate solution, dithiothreitol solution, iodoacetamide solution, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting. The reference herb, *Crassostrea gigas*, was enzymatically extracted to obtain a reference herb solution. The test solution and the control medicinal material solution were analyzed using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) instrument to obtain the results. The chromatographic conditions for the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, 100 mm long and 2.1 mm in diameter, with a packed particle size of 1.7 μm. Mobile phase A was acetonitrile, and mobile phase B was 0.1 vol% formic acid aqueous solution. The elution curve was as follows: From 0 min to 10 min, mobile phase A decreased from 5% to 20%, and mobile phase B decreased from 95% to 80%. From 10 to 14 minutes, mobile phase A decreased from 20% to 80%, and mobile phase B decreased from 80% to 20%. The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring (MRM), capillary voltage of 1.2 kV to 1.8 kV, ion source temperature of 320°C to 400°C, desolvation gas flow rate of 600 L / hr to 700 L / hr, curtain gas flow rate of 5 L / hr to 15 L / hr, and ion source cone voltage of 15 V to 25 V; the detected ion pairs are 364.21→307.19, 364.21→417.25, 369.71→512.25, and 369.71→448.

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2. The application as described in claim 1, characterized in that, The column temperature is 25℃~40℃, the flow rate is 0.2mL / min~0.4mL / min, and the injection volume is 1μL~3μL.

3. The application as described in claim 1 or 2, characterized in that, The column temperature was 30℃, the flow rate was 0.3 mL / min, and the injection volume was 2 μL.

4. The application as described in claim 1, characterized in that, The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1.5 kV, ion source temperature of 350 °C, desolvation gas flow rate of 650 L / hr, curtain gas flow rate of 10 L / hr, and ion source cone voltage of 20 V.

5. The application as described in claim 1, characterized in that, In the step of sequentially extracting oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine granules with ammonium bicarbonate solution, dithiothreitol solution, iodoacetamide solution, trypsin solution hydrolysis, and solid-phase extraction column desalting to obtain the test solution: The ratio of ammonium bicarbonate solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 40mL~80mL:1g~2g; the concentration of the ammonium bicarbonate solution is 1wt%~5wt%. The ratio of the dithiothreitol solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 100μL~200μL:1g~2g; the concentration of the dithiothreitol solution is 0.5mol / L~3mol / L. The ratio of the iodoacetamide solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 300μL~800μL:1g~2g; the concentration of the iodoacetamide solution is 0.2mol / L~2mol / L. The trypsin solution is prepared by adding 3mg~10mg of trypsin to 0.5wt%~2wt% ammonium bicarbonate solution. The ratio of the trypsin solution to oyster shell or its processed slices, standard decoction, or Chinese medicine formula granules is 200μL~1000μL:1g~2g. The solid-phase extraction column desalting process includes: Centrifuge the enzymatic hydrolysate obtained by enzymatic hydrolysis, add the supernatant to an HLB column, elute with water, discard the eluent, and then elute sequentially with 10 vol%~30 vol% acetonitrile solution and 30 vol%~60 vol% acetonitrile solution. Collect the eluent, filter, and obtain the final product.

6. A method for identifying the oyster *Crassostrea gigas* and its adulterants, characterized in that, include: Provide the sample to be tested, and test the sample using the detection methods for Oyster shell or its processed slices, standard decoctions, and traditional Chinese medicine granules; If the mass spectrum obtained by detecting the test solution prepared from the sample under the preset detection ion pair does not show any ion peak retention time with the reference herb solution of Ostrea gigas, then the test sample is a counterfeit; wherein, the counterfeit includes Ostrea gigas from Fujian, Ostrea gigas from Portugal, Abalone shell, and Cuttlebone. The detection methods for Oyster shell (Oyster roe) or its processed slices, standard decoctions, and traditional Chinese medicine granules include: The test solution was obtained by sequentially extracting oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine formula granules with ammonium bicarbonate solution, dithiothreitol solution, iodoacetamide solution, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting. The reference herb, *Crassostrea gigas*, was enzymatically extracted to obtain a reference herb solution. The test solution and the control medicinal material solution were analyzed using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) instrument to obtain the results. The chromatographic conditions for the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, 100 mm long and 2.1 mm in diameter, with a packed particle size of 1.7 μm. Mobile phase A was acetonitrile, and mobile phase B was 0.1 vol% formic acid aqueous solution. The elution curve was as follows: From 0 min to 10 min, mobile phase A decreased from 5% to 20%, and mobile phase B decreased from 95% to 80%. From 10 to 14 minutes, mobile phase A decreased from 20% to 80%, and mobile phase B decreased from 80% to 20%. The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring (MRM), capillary voltage of 1.2 kV to 1.8 kV, ion source temperature of 320°C to 400°C, desolvation gas flow rate of 600 L / hr to 700 L / hr, curtain gas flow rate of 5 L / hr to 15 L / hr, and ion source cone voltage of 15 V to 25 V; the detected ion pairs are 364.21→307.19, 364.21→417.25, 369.71→512.25, and 369.71→448.

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7. A method for identifying the origin of oyster medicinal material or its processed slices, standard decoctions, and traditional Chinese medicine granules, characterized in that, include: Provide the sample to be tested, and test the sample using the detection methods for Oyster shell or its processed slices, standard decoctions, and traditional Chinese medicine granules; If the mass spectrum obtained by detecting the test solution prepared from the sample under the preset detection ion pair does not show any ion peak retention time with the reference medicinal material solution of Ostrea gigas, then the origin of the test sample is Ostrea gigas or Ostrea daliani. The detection methods for Oyster shell (Oyster roe) or its processed slices, standard decoctions, and traditional Chinese medicine granules include: The test solution was obtained by sequentially extracting oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine formula granules with ammonium bicarbonate solution, dithiothreitol solution, iodoacetamide solution, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting. The reference herb, *Crassostrea gigas*, was enzymatically extracted to obtain a reference herb solution. The test solution and the control medicinal material solution were analyzed using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) instrument to obtain the results. The chromatographic conditions for the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, 100 mm long and 2.1 mm in diameter, with a packed particle size of 1.7 μm. Mobile phase A was acetonitrile, and mobile phase B was 0.1 vol% formic acid aqueous solution. The elution curve was as follows: From 0 min to 10 min, mobile phase A decreased from 5% to 20%, and mobile phase B decreased from 95% to 80%. From 10 to 14 minutes, mobile phase A decreased from 20% to 80%, and mobile phase B decreased from 80% to 20%. The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring (MRM), capillary voltage of 1.2 kV to 1.8 kV, ion source temperature of 320°C to 400°C, desolvation gas flow rate of 600 L / hr to 700 L / hr, curtain gas flow rate of 5 L / hr to 15 L / hr, and ion source cone voltage of 15 V to 25 V; the detected ion pairs are 364.21→307.19, 364.21→417.25, 369.71→512.25, and 369.71→448.

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8. The identification method as described in claim 6 or 7, characterized in that, The column temperature is 25℃~40℃, the flow rate is 0.2mL / min~0.4mL / min, and the injection volume is 1μL~3μL.

9. The identification method as described in claim 6 or 7, characterized in that, The column temperature was 30℃, the flow rate was 0.3 mL / min, and the injection volume was 2 μL.

10. The identification method as described in claim 6 or 7, characterized in that, The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (ULCS) instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1.5 kV, ion source temperature of 350 °C, desolvation gas flow rate of 650 L / hr, curtain gas flow rate of 10 L / hr, and ion source cone voltage of 20 V.

11. The identification method as described in claim 6 or 7, characterized in that, In the step of sequentially extracting oyster medicinal materials or their processed slices, standard decoctions, and traditional Chinese medicine granules with ammonium bicarbonate solution, dithiothreitol solution, iodoacetamide solution, trypsin solution hydrolysis, and solid-phase extraction column desalting to obtain the test solution: The ratio of ammonium bicarbonate solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 40mL~80mL:1g~2g; the concentration of the ammonium bicarbonate solution is 1wt%~5wt%. The ratio of the dithiothreitol solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 100μL~200μL:1g~2g; the concentration of the dithiothreitol solution is 0.5mol / L~3mol / L. The ratio of the iodoacetamide solution to oyster shell or its processed slices, standard decoction, or traditional Chinese medicine granules is 300μL~800μL:1g~2g; the concentration of the iodoacetamide solution is 0.2mol / L~2mol / L. The trypsin solution is prepared by adding 3mg~10mg of trypsin to 0.5wt%~2wt% ammonium bicarbonate solution. The ratio of the trypsin solution to oyster shell or its processed slices, standard decoction, or Chinese medicine formula granules is 200μL~1000μL:1g~2g. The solid-phase extraction column desalting process includes: Centrifuge the enzymatic hydrolysate obtained by enzymatic hydrolysis, add the supernatant to an HLB column, elute with water, discard the eluent, and then elute sequentially with 10 vol%~30 vol% acetonitrile solution and 30 vol%~60 vol% acetonitrile solution. Collect the eluent, filter, and obtain the final product.

Images