Detection method and application of ostrea rivularis medicinal material or decoction pieces, standard decoction and traditional Chinese medicine formula granules thereof

Abstract

The invention discloses a detection method and application of ostrea rivularis medicinal materials or decoction pieces, standard decoction and traditional Chinese medicine formula granules thereof, and relates to the technical field of analysis and detection. The detection method comprises the following steps: carrying out enzymolysis extraction on a crassostrea rivularis medicinal material or decoction pieces thereof, a standard decoction and traditional Chinese medicine formula granules to obtain a test solution; performing extraction and enzymolysis on the ostrea rivularis reference medicinal material to obtain a reference medicinal material solution; and detecting the test sample solution and the reference medicinal material solution by adopting an ultra-high performance liquid chromatograph-mass spectrometer, thereby obtaining the target product. Wherein the detection ion pair ranges from 364.21 to 307.19, the detection ion pair ranges from 364.21 to 417.25, the detection ion pair ranges from 369.71 to 512.25, and the detection ion pair ranges from 369.71 to 448.26. The detection method provided by the invention has strong specificity, can effectively identify different original oysters and counterfeit oysters through the organic matter part of the ostrea rivularis, and provides a reliable basis for quality control and evaluation of the oysters and products thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a detection method and application for Crassostrea rivularis, its cut crude drug, standard decoction, and traditional Chinese medicine formula granules. Background Art

[0002] The 2020 Edition of Chinese Pharmacopoeia stipulates that oyster is the shell of animals in the family Ostreidae, including Ostrea gigas Thunberg, Ostrea talienwhanensis Crosse, or Ostrea rivularis Gould, which has the effects of calming the mind and suppressing yang, nourishing yin, softening hardness and dissipating nodules. It is used for palpitation and insomnia, dizziness and tinnitus, scrofula and phlegm nodes, and masses and abdominal masses.

[0003] Under the item of oyster in the 2020 Edition of Chinese Pharmacopoeia, oyster is identified by microscopic and thin-layer chromatography methods, but this method has no strong specificity among different original plants, counterfeits, and other shellfish varieties. After consulting the literature and patents, most of the current research focuses on oyster meat, and little research has been done on the shell of traditional Chinese medicine oyster. The existing identification studies on oyster shells with different original plants mainly focus on characteristics and microscopy. However, due to the strong morphological plasticity of oyster shells and their susceptibility to environmental influence, it is sometimes difficult to distinguish their specific species based solely on their appearance. The material composition of oyster shells consists of two parts: inorganic matter and organic matter. The inorganic matter is mainly calcium carbonate, accounting for more than 94% of the mass of oyster shells, and the proportion of organic matter is extremely low. It is also a major difficulty to identify and distinguish oyster shells with different original plants by studying proteins and polypeptides. For the water extract products of oyster shells, such as traditional Chinese medicine standard decoctions and traditional Chinese medicine formula granules, due to the destruction of components such as proteins during water decoction, it is extremely difficult to conduct specific polypeptide identification research based on proteomics. Therefore, how to specifically identify oysters to meet the quality control requirements is an urgent technical problem to be solved, and it is also a technical problem that needs to be overcome in the field of quality control of oyster products. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a detection method for Crassostrea rivularis, its cut crude drug, standard decoction, and traditional Chinese medicine formula granules, which has strong specificity and can effectively distinguish different original plants of oysters and counterfeits of oyster products.

[0005] Another technical problem to be solved by the present invention is to provide the application of the above detection method for Crassostrea rivularis, its cut crude drug, standard decoction, and traditional Chinese medicine formula granules.

[0006] Another technical problem to be solved by the present invention is to provide a method for identifying Crassostrea rivularis and its counterfeits.

[0007] The technical problem to be further solved by the present invention is to provide a method for identifying the origin of oyster medicinal materials or their cut pieces, standard decoctions, and traditional Chinese medicine formula granules.

[0008] To solve the above technical problems, the present invention provides a detection method for Crassostrea rivularis Gould medicinal materials or their cut pieces, standard decoctions, and traditional Chinese medicine formula granules, which includes:

[0009] Enzymatically hydrolyzing and extracting the Crassostrea rivularis Gould medicinal materials or their cut pieces, standard decoctions, and traditional Chinese medicine formula granules to obtain a test solution;

[0010] Enzymatically hydrolyzing and extracting the control Crassostrea rivularis Gould medicinal materials to obtain a control medicinal material solution;

[0011] Detecting the test solution and the control medicinal material solution by an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and that's it;

[0012] Among them, the detection ion pairs are 364.21→307.19, 364.21→417.25, 369.71→512.25, and 369.71→448.26.

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

[0014] The chromatographic column is filled with octadecylsilane-bonded silica gel, the column length is 50 mm to 150 mm, the column diameter is 2 mm to 3 mm, the particle size of the filler is 1.6 to 1.9 μm, the column temperature is 25 °C to 40 °C, the flow rate is 0.2 mL / min to 0.4 mL / min, the injection volume is 1 μL to 3 μL, mobile phase A is acetonitrile, and mobile phase B is 0.05 vol% to 0.2 vol% formic acid aqueous solution; the elution curve is:

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

[0016] From 10 min to 14 min, mobile phase A changes from 20% → 80%, and mobile phase B changes from 80% → 20%.

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

[0018] The chromatographic column model is Waters ACQUITY UPLC BEH C18 chromatographic column, the column length is 100 mm, the column diameter is 2.1 mm, the particle size of the filler is 1.7 μm, the column temperature is 30 °C, the flow rate is 0.3 mL / min, the injection volume is 2 μL, mobile phase A is acetonitrile, and mobile phase B is 0.1 vol% formic acid aqueous solution; the elution curve is:

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

[0020] From 10 min to 14 min, mobile phase A changes from 20% to 80%, and mobile phase B changes 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 instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage is 1.2 kV - 1.8 kV, ion source temperature is 320 °C - 400 °C, desolvation gas flow rate is 600 L / hr - 700 L / hr, curtain gas flow rate is 5 L / hr - 15 L / hr, and ion source cone voltage is 15 V - 25 V.

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

[0023] As an improvement to the above technical solution, the steps of enzymatically hydrolyzing and extracting oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules to obtain a test solution include:

[0024] Subjecting oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules to ammonium bicarbonate solution extraction, dithiothreitol solution extraction, iodoacetamide solution treatment, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting treatment in sequence to obtain a test solution;

[0025] Among them, the dosage ratio of ammonium bicarbonate solution to oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules is 40 mL - 80 mL: 1 g - 2 g; the concentration of the ammonium bicarbonate solution is 1 wt% - 5 wt%;

[0026] The dosage ratio of the dithiothreitol solution to oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules is 100 μL - 200 μL: 1 g - 2 g; the concentration of the dithiothreitol solution is 0.5 mol / L - 3 mol / L;

[0027] The dosage ratio of the iodoacetamide solution to oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules is 300 μL - 800 μL: 1 g - 2 g; the concentration of the iodoacetamide solution is 0.2 mol / L - 2 mol / L;

[0028] The trypsin solution is prepared by adding 3 mg to 10 mg of trypsin into a 0.5 wt% to 2 wt% ammonium bicarbonate solution. The dosage ratio of the trypsin solution to the oyster medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules is 200 μL to 1000 μL: 1 g to 2 g;

[0029] The desalting treatment of the solid-phase extraction column includes:

[0030] Centrifuge the enzymolysis solution obtained by enzymolysis, add the obtained supernatant to an HLB column, elute with water, discard the eluate, and then elute successively with a 10 vol% to 30 vol% acetonitrile solution and a 30 vol% to 60 vol% acetonitrile solution, collect the eluate, and filter to obtain.

[0031] Correspondingly, the present invention also discloses the application of the above detection method for the Crassostrea rivularis medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules in the following (1) or (2):

[0032] (1) Identifying the origin of the oyster medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules;

[0033] (2) Identifying the Crassostrea rivularis medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules and their counterfeits.

[0034] As an improvement of the above technical solution, the origin of the oyster medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules includes Crassostrea gigas and Crassostrea talienwhanensis;

[0035] The counterfeits include Crassostrea angulata, Crassostrea angulata, Haliotis diversicolor, and Sepia seu Sepiellae Os.

[0036] Correspondingly, the present invention also discloses a method for identifying Crassostrea rivularis and its counterfeits, which includes: providing a test sample, and detecting the test sample by using the above detection method for the Crassostrea rivularis medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules; in the mass spectrometry diagram obtained by detecting the test solution prepared from the test sample under the preset detection ion pair, if there is no detection at the retention time of the ion peak of the Crassostrea rivularis control medicinal material solution, the test sample is a counterfeit.

[0037] Correspondingly, the present invention also discloses a method for identifying the origin of the oyster medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules, which includes: providing a test sample, and detecting the test sample by using the above detection method for the Crassostrea rivularis medicinal material or its decoction pieces, standard decoction, and traditional Chinese medicine formula granules; in the mass spectrometry diagram obtained by detecting the test solution prepared from the test sample under the preset detection ion pair, if there is no detection at the retention time of the ion peak of the Crassostrea rivularis control medicinal material solution, the origin of the test sample is Crassostrea gigas or Crassostrea talienwhanensis.

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

[0039] Based on the in-depth analysis of Crassostrea rivularis and other raw materials of oysters, a detection method for Crassostrea rivularis or its cut pieces, standard decoction, and traditional Chinese medicine formula granules has been established. This detection method has strong specificity and can effectively identify different origin oysters and counterfeits through the organic matter part of Crassostrea rivularis, providing a reliable basis for the quality control and evaluation of oysters and their products. Description of the Drawings

[0040] Figure 1 It is the mass spectrometry diagram of Crassostrea rivularis and blank solvent in the specificity investigation of Example 1;

[0041] Figure 2 It is the mass spectrometry diagram of Crassostrea rivularis at a column temperature of 28 °C in the durability investigation of different column temperatures in Example 1;

[0042] Figure 3 It is the mass spectrometry diagram of Crassostrea rivularis at a column temperature of 30 °C in the durability investigation of different column temperatures in Example 1;

[0043] Figure 4 It is the mass spectrometry diagram of Crassostrea rivularis at a column temperature of 32 °C in the durability investigation of different column temperatures in Example 1;

[0044] Figure 5 It is the mass spectrometry diagram of Crassostrea rivularis at a flow rate of 0.28 mL / min in the durability investigation of different flow rates in Example 1;

[0045] Figure 6 It is the mass spectrometry diagram of Crassostrea rivularis at a flow rate of 0.30 mL / min in the durability investigation of different flow rates in Example 1;

[0046] Figure 7 It is the mass spectrometry diagram of Crassostrea rivularis at a flow rate of 0.32 mL / min in the durability investigation of different flow rates in Example 1;

[0047] Figure 8 It is the mass spectrometry diagram of Crassostrea rivularis when using Waters ACQUITY UPLC BEH C18 in the durability investigation of different chromatographic columns in Example 1;

[0048] Figure 9 It is the mass spectrometry diagram of Crassostrea rivularis when using Agilent ZORBAX Eclipse XDBC18 in the durability investigation of different chromatographic columns in Example 1;

[0049] Figure 10 It is the mass spectrometry diagram of Crassostrea rivularis when using Agilent ZORBAX SB-C18 in the durability investigation of different chromatographic columns in Example 1;

[0050] Figure 11 It is the mass spectrometry diagram (m / z 364.21) of 17 batches of Crassostrea rivularis medicinal materials in Example 1. On the left is m / z 364.21 → 417.25, and on the right is m / z 364.21 → 307.19;

[0051] Figure 12 It is the mass spectrometry diagram (m / z 364.21) of 17 batches of Crassostrea rivularis cut pieces in Example 1. On the left is m / z 364.21 → 307.19, and on the right is m / z 364.21 → 417.25;

[0052] Figure 13 It is the mass spectrometry diagram (m / z 364.21) of 17 batches of standard decoctions of Crassostrea rivularis in Example 1. On the left is m / z364.21 → 417.25, and on the right is m / z 364.21 → 307.19;

[0053] Figure 14 It is the mass spectrometry diagram (m / z 364.21) of 6 batches of traditional Chinese medicine formula granules of Crassostrea rivularis in Example 1. On the left is m / z364.21 → 417.25, and on the right is m / z 364.21 → 307.19;

[0054] Figure 15 It is the mass spectrometry diagram (m / z 369.71) of 17 batches of Crassostrea rivularis medicinal materials in Example 1. On the left is m / z 369.71 → 512.25, and on the right is m / z 369.71 → 448.26;

[0055] Figure 16 It is the mass spectrometry diagram (m / z 369.71) of 17 batches of Crassostrea rivularis cut pieces in Example 1. On the left is m / z 369.71 → 512.25, and on the right is m / z 369.71 → 448.26;

[0056] Figure 17 It is the mass spectrometry diagram (m / z 369.71) of 17 batches of standard decoctions of Crassostrea rivularis in Example 1. On the left is m / z369.71 → 512.25, and on the right is m / z 369.71 → 448.26;

[0057] Figure 18 It is the mass spectrometry diagram (m / z 369.71) of 6 batches of traditional Chinese medicine formula granules of Crassostrea rivularis in Example 1. On the left is m / z369.71 → 512.25, and on the right is m / z 369.71 → 448.26;

[0058] Figure 19Mass spectrometry (m / z 364.21) of 1 batch of *Crassostrea talienwhanensis*, 2 batches of *Crassostrea angulata* and 6 batches of *Crassostrea angulata* in Example 2. On the left, m / z 364.21 → 417.25; on the right, m / z 364.21 → 307.19. Among them, ML is the control medicinal material of *Crassostrea rivularis*;

[0059] Figure 20 Mass spectrometry (m / z 364.21) of 6 batches of *Crassostrea gigas*, 3 batches of *Haliotis diversicolor supertexta* and 3 batches of *Sepia esculenta* in Example 2. On the left, m / z 364.21 → 417.25; on the right, m / z 364.21 → 307.19. Among them, ML is the control medicinal material of *Crassostrea rivularis*;

[0060] Figure 21 Mass spectrometry (m / z 369.71) of 1 batch of *Crassostrea talienwhanensis*, 2 batches of *Crassostrea angulata* and 6 batches of *Crassostrea angulata* in Example 2. On the left, m / z 369.71 → 512.25; on the right, m / z 369.71 → 448.26. Among them, ML is the control medicinal material of *Crassostrea rivularis*;

[0061] Figure 22 Mass spectrometry (m / z 369.71) of 6 batches of *Crassostrea gigas*, 3 batches of *Haliotis diversicolor supertexta* and 3 batches of *Sepia esculenta* in Example 2. On the left, m / z 369.71 → 512.25; on the right, m / z 369.71 → 448.26. Among them, ML is the control medicinal material of *Crassostrea rivularis*. Detailed implementation mode

[0062] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the drawings and specific implementation modes.

[0063] Detection method of *Crassostrea rivularis* in Example 1

[0064] 1. Preparation of test solution

[0065] (1) Omi Oyster medicinal material or its decoction pieces: Take 2 g of the fine powder of this product, grind it into powder, put it in a stoppered conical flask, add 50 mL of 2% ammonium bicarbonate solution, heat and reflux for 30 minutes, cool to room temperature, add 150 μL of dithiothreitol (DTT) solution (1 mol / L), mix well, and place in a 56°C water bath for 1 hour. Take it out, cool it, add 500 μL of iodoacetamide (IAA) solution (0.6 mol / L), mix well, protect it from light for 30 minutes, add trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 5 mg per 1 mL, prepare it before use). Preparation) 500μL, shake well, enzymolyze at 37℃ for 12 hours, centrifuge (speed is 4000 rpm) for 5 minutes, take the supernatant, slowly pass it through the activated and balanced HLB column [specification: 6mL (200mg), eluted with 6mL of acetonitrile and 2% ammonium bicarbonate solution in turn], elute with 2mL of purified water, discard the eluate, elute with 1.5mL of 20vol% acetonitrile solution, collect the eluate, then elute with 1.5mL of 50% acetonitrile solution, collect the eluate, combine the two eluates, filter with a microporous filter membrane (0.22μm) as the test solution.

[0066] (2) Standard decoction of Omi oyster: Take 1.5 g of this product, grind it into powder, put it in a stoppered conical flask, add 50 mL of 2% ammonium bicarbonate solution, ultrasonicate for 30 minutes, let it cool, add 150 μL of dithiothreitol (DTT) solution (1 mol / L), mix well, and place it in a 56°C water bath for 1 hour. Take it out, let it cool, add 500 μL of iodoacetamide (IAA) solution (0.6 mol / L), mix well, protect it from light for 30 minutes, and add trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 5 mg per 1 mL, prepare it before use). ) 500μL, shake well, enzymolyze at 37℃ for 12 hours, centrifuge (speed is 4000 rpm) for 5 minutes, take the supernatant, slowly pass it through the activated and balanced HLB column [specification: 6mL (200mg), eluted with 6mL of acetonitrile and 2% ammonium bicarbonate solution in turn], elute with 2mL of purified water, discard the eluate, elute with 1.5mL of 20% acetonitrile solution, collect the eluate, then elute with 1.5mL of 50% acetonitrile solution, collect the eluate, combine the two eluates, filter with a microporous filter membrane (0.22μm) as the test solution.

[0067] (3) Crassostrea ariakensis formula granule: Take 2 g of this product, grind it finely, place it in a stoppered conical flask, add 50 mL of 2% ammonium bicarbonate solution, ultrasonically treat for 30 minutes, let it cool, add 150 μL of dithiothreitol (DTT) solution (1 mol / L), mix well, place it in a water bath at 56 °C for 1 hour, take it out, let it cool, add 500 μL of iodoacetamide (IAA) solution (0.6 mol / L), mix well, protect from light for 30 minutes, add 250 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 5 mg per 1 mL, prepared before use), shake well, enzymatically digest at 37 °C for 12 hours, centrifuge (rotation speed: 4000 revolutions per minute) for 5 minutes, take the supernatant, slowly pass it through an activated and equilibrated HLB column [specification: 6 mL (200 mg), elute successively with 6 mL each of acetonitrile and 2% ammonium bicarbonate solution], elute with 2 mL of purified water, discard the eluate, then elute with 1.5 mL of 20% acetonitrile solution, collect the eluate, subsequently elute with 1.5 mL of 50% acetonitrile solution, collect the eluate, combine the two eluates, filter through a microporous membrane (0.22 μm) to obtain the test solution.

[0068] 2. Preparation of control crude drug solution

[0069] Take 2 g of the control crude drug of Crassostrea ariakensis, place it in a stoppered conical flask, add 50 mL of 2% ammonium bicarbonate solution, heat under reflux for 30 minutes, let it cool, add 150 μL of dithiothreitol (DTT) solution (1 mol / L), mix well, place it in a water bath at 56 °C for 1 hour, take it out, let it cool, add 500 μL of iodoacetamide (IAA) solution (0.6 mol / L), mix well, protect from light for 30 minutes, add 500 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 5 mg per 1 mL, prepared before use), shake well, enzymatically digest at 37 °C for 12 hours, centrifuge (rotation speed: 4000 revolutions per minute) for 5 minutes, take the supernatant, slowly pass it through an activated and equilibrated HLB column [specification: 6 mL (200 mg), elute successively with 6 mL each of acetonitrile and 2% ammonium bicarbonate solution], elute with 2 mL of purified water, discard the eluate, then elute with 1.5 mL of 20% acetonitrile solution, collect the eluate, subsequently elute with 1.5 mL of 50% acetonitrile solution, collect the eluate, combine the two eluates, filter through a microporous membrane (0.22 μm) to obtain the control crude drug solution.

[0070] 3. Chromatography and mass spectrometry conditions

[0071] 3.1 Chromatography conditions

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

[0073] Table 1 Elution program

[0074]

[0075] 3.2 Mass spectrometry conditions

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

[0077] Table 2 Mass spectrometry conditions table

[0078]

[0079] 4. Determination

[0080] Precisely pipette 2 μL of the reference crude drug solution and 2 μL of the test solution respectively, and inject them into an ultra-high performance liquid chromatography-mass spectrometry instrument (Waters UPLC-Xevo TQ-S Cronos) for determination.

[0081] 5. Methodology investigation

[0082] Prepare the test solution of Crassostrea rivularis according to the above test solution preparation method; Use different mobile phases (No. 1: 10 mmol / L ammonium formate - acetonitrile, No. 2: 0.1% formic acid - acetonitrile), investigate the influence of different mobile phases on the detection results, and select the optimal mobile phase system. The specific results are shown in Table 3:

[0083] Table 3 Mobile phase investigation table

[0084]

[0085] From the above data, it can be seen that the detection results of the mobile phase of the formic acid - acetonitrile system are better, so the mobile phase is selected as 0.1% formic acid - acetonitrile.

[0086] 6. Methodological Validation

[0087] 6.1 Exclusivity

[0088] Preparation of test solution: Take Omi oyster medicinal material and prepare the test solution according to the above test solution preparation method; take blank solvent and prepare blank solvent without sample according to the same method. Accurately pipette 2μL of each of the above solutions and inject into the LC-MS instrument. Determine according to the above chromatographic and mass spectrometric conditions. The results are as follows: Figure 1 shown.

[0089] The results showed that no characteristic ion peaks were detected in the spectrum of the blank solvent test solution without sample at the retention time corresponding to the Omi oyster, indicating that the blank solvent had no interference with the detection of characteristic ion pairs in the method and the method was specific.

[0090] 6.2 Precision

[0091] Take Omi oyster medicinal materials and prepare the test sample solution according to the above test sample preparation method. Accurately aspirate 2 μL of the test sample solution, repeat the injection 6 times according to the above chromatographic conditions and mass spectrometry conditions, record the peak area, and calculate the peak area RSD value. The results are shown in Table 4.

[0092] Table 4 Instrument precision inspection results

[0093]

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

[0095] 6.3 Stability

[0096] Omi oyster medicinal materials were taken and the test solution was prepared according to the above test solution preparation method. 2 μL was accurately aspirated and injected into the liquid chromatography-mass spectrometer at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 hours respectively. The solution stability was evaluated by the peak area of ​​the characteristic ion. The test results are shown in Table 5.

[0097] Table 5 Stability test results

[0098]

[0099] The results showed that when the test solution was placed at room temperature for 18 hours, the peak area RSD values ​​of the four pairs of characteristic ions were 1.52% to 3.55%, which were all clearly detectable. This showed that the test solution had good stability when placed at room temperature for 18 hours and did not affect the identification of characteristic ions.

[0100] 6.4 Repeatability

[0101] Take the Crassostrea rivularis medicinal material, divide it into 6 parallel portions, and prepare the test solution according to the above-mentioned test solution preparation method. Precisely pipette 2 μL of each of the above solutions and inject them into the liquid chromatography-mass spectrometry instrument. Determine according to the above-mentioned chromatographic and mass spectrometry conditions, and calculate the RSD value of the ratio of "peak area / sample weight". The results are shown in Table 6.

[0102] Table 6 Results of repeatability investigation

[0103]

[0104] The results show that when the same batch of samples is repeatedly determined 6 times, the RSD value of the ratio of "peak area / sample weight" is less than 5%, indicating that this method has good repeatability.

[0105] 6.5 Durability

[0106] ① Investigation of different column temperatures

[0107] In this subsection, the effects of different column temperatures of 28 °C, 30 °C, and 32 °C on the detection of characteristic ion peaks of the Crassostrea rivularis medicinal material are compared. Specifically, take the Crassostrea rivularis medicinal material, prepare the test solution according to the above-mentioned test solution preparation method, precisely pipette 2 μL and inject it into the liquid chromatography-mass spectrometry instrument, and conduct the determination according to the above conditions. The experimental results are shown in Figures 2 to 4 Table 7.

[0108] Table 7 Peak area results of durability investigation of Crassostrea rivularis medicinal material at different column temperatures

[0109]

[0110] The results show that the specified 4 pairs of characteristic ion pairs can be clearly detected under 3 different column temperature conditions, indicating that small adjustments in column temperature have no effect on the detection and identification of characteristic ions of the Crassostrea rivularis medicinal material.

[0111] ② Investigation of different flow rates

[0112] Compare the effects of different flow rates of 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min on the detection of characteristic ion peaks of the Crassostrea rivularis medicinal material. Prepare the test solution according to the above-mentioned test solution preparation method, precisely pipette 2 μL and inject it into the liquid chromatography-mass spectrometry instrument, and conduct the determination according to the above conditions. The experimental results are shown in Figures 5 to 7 Table 8.

[0113] Table 8 Peak area results of durability investigation of Crassostrea rivularis medicinal material at different flow rates

[0114]

[0115] The results show that the specified 4 pairs of characteristic ion pairs can be clearly detected under 3 different flow rate conditions, indicating that small adjustments in flow rate have no effect on the detection and identification of characteristic ions of the Crassostrea rivularis medicinal material.

[0116] ③Different chromatographic columns

[0117] This section compares the effects of three different brands and types of chromatographic columns, 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 ZORBAXSB-C18 (100mm×2.1mm, 1.8μm), on the detection of characteristic ion peaks of Omi oyster medicinal materials.

[0118] Take the medicinal material of Omi oyster, prepare the test solution according to the above test solution preparation method, accurately pipette 2 μL and inject it into the liquid chromatography-mass spectrometer, and measure it according to the above conditions. The experimental results are shown in Figures 8 to 10 , Table 9.

[0119] Table 9 Peak area results of different chromatographic columns for the durability of Omi oyster medicinal materials

[0120]

[0121] The results showed that the three chromatographic columns used could all 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 the characteristic ions of Omi oyster medicinal materials.

[0122] 7. Testing of Omi oyster medicinal materials, standard decoctions, and Chinese medicine granules

[0123] A total of 57 batches of samples were studied in this embodiment, including 17 batches of Omi oyster medicinal materials (batch numbers: G1-G17), 17 batches of Omi oyster decoction pieces (batch numbers: GP1-GP17), 17 batches of Omi oyster standard decoctions (batch numbers: GT1-GT18), and 6 batches of Omi oyster Chinese medicine formula granules (batch numbers: CG1-CG6). It should be noted that the Omi oyster decoction pieces here refer to the decoction pieces obtained by washing, drying and crushing the Omi oyster medicinal materials. The Omi oyster standard decoction refers to the product obtained by water decoction, solid-liquid separation, concentration and drying of the above-mentioned Omi oyster decoction pieces; the Omi oyster Chinese medicine formula granules refer to the granules formed by water heating extraction, separation, concentration, drying and granulation of the above-mentioned Omi oyster decoction pieces.

[0124] Prepare the test solution of each sample according to the method under "1", and inject the sample according to the conditions under "3". The specific test results are shown in Table 10 and Figures 11 to 18 As shown in the figure, it can be seen that in the ion flow of the Omi oyster medicinal materials, decoction pieces, standard decoctions, and formula granules, the ion peaks with the same retention time as the Omi oyster control medicinal material solution should appear at the same time, and the signal-to-noise ratio should be greater than 3:1.

[0125] Table 10 Mass spectrometry determination results of Crassostrea rivularis medicinal materials, standard decoctions, and formula granules

[0126]

[0127]

[0128] Example 2 Identification of different origins and counterfeits of oysters

[0129] In this example, a total of 7 batches of oysters with other origins were studied, including 6 batches of Crassostrea gigas (batch numbers: C1 - C6) and 1 batch of Crassostrea talienwhanensis (batch number: D1); there were 14 batches of counterfeits, including 6 batches of Ostrea angasi (batch numbers: F1 - F6), 2 batches of Crassostrea angulata (batch numbers: H1 - H2), 3 batches of Haliotis diversicolor supertexta (batch numbers: Sjm1 - Sjm3), and 3 batches of Sepia seu Sepiellae (batch numbers: Hpx1 - Hpx3).

[0130] Prepare the test solution for each sample according to the method under "1" in Example 1 and inject the sample for testing under the conditions under "3". The specific test results Figures 19 to 22 are shown as follows. It can be seen from the figure that no detections were found at the retention time of the ion peaks in the reference medicinal material solution of Crassostrea rivularis for different origins (Crassostrea gigas, Crassostrea talienwhanensis) and counterfeits (Ostrea angasi, Crassostrea angulata, Haliotis diversicolor supertexta, Sepia seu Sepiellae).

[0131] The above is the preferred implementation mode of the invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A detection method for Crassostrea rivularis medicinal materials or their cut pieces, standard decoctions, and traditional Chinese medicine formula granules, characterized in that, include: Enzymatically extract the Omi oyster medicinal material or its decoction pieces, standard decoction, and Chinese medicine formula granules to obtain a test solution; The reference medicinal material of Omi oyster is subjected to enzymatic extraction to obtain a reference medicinal material solution; The test solution and the control medicinal material solution are detected by ultra-high performance liquid spectrometry to obtain; Among them, the detected ion pairs were 364.21→307.19, 364.21→417.25, 369.71→512.25 and 369.71→448.

26.

2. The method for detecting the medicinal material of Omi oyster or its decoction pieces, standard decoction, or Chinese medicine granules as claimed in claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid spectrometer are: The chromatographic column is filled with octadecylsilane bonded silica gel, the column length is 50 mm to 150 mm, the column diameter is 2 mm to 3 mm, the particle size of the filler is 1.6 to 1.9 μm, the column temperature is 25° C. to 40° C., the flow rate is 0.2 mL / min to 0.4 mL / min, the injection volume is 1 μL to 3 μL, the mobile phase A is acetonitrile, and the mobile phase B is 0.05 vol% to 0.2 vol% formic acid aqueous solution; the elution curve is: 0min~10min, mobile phase A from 5%→20%, mobile phase B from 95%→80%; 10min~14min, mobile phase A changes from 20%→80%, and mobile phase B changes from 80%→20%.

3. The detection method of the Crassostrea rivularis medicinal material or its cut pieces, standard decoction, or traditional Chinese medicine formula granules as described in claim 1 or 2, characterized in that, The chromatographic conditions of the ultra-high performance liquid spectrometer are: The chromatographic column model is Waters ACQUITY UPLC BEH C18 chromatographic column, with a column length of 100 mm, a column diameter of 2.1 mm, a filler particle size of 1.7 μm, a column temperature of 30° C., a flow rate of 0.3 mL / min, an injection volume of 2 μL, mobile phase A is acetonitrile, and mobile phase B is 0.1 vol% formic acid aqueous solution; the elution curve is: 0min~10min, mobile phase A from 5%→20%, mobile phase B from 95%→80%; 10min~14min, mobile phase A changes from 20%→80%, and mobile phase B changes from 80%→20%.

4. The detection method of the Crassostrea rivularis medicinal material or its cut pieces, standard decoction, and traditional Chinese medicine formula granules as described in claim 1, characterized in that, The mass spectrometry conditions of the ultra-high performance liquid spectrometer are: electrospray power supply, positive ion mode, multiple reaction monitoring, 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.

5. The detection method of the Crassostrea rivularis medicinal material or its cut pieces, standard decoction, and traditional Chinese medicine formula granules as described in claim 1 or 4, characterized in that, The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometer are: 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.

6. The detection method of the Crassostrea rivularis medicinal material or its cut pieces, standard decoction, and traditional Chinese medicine formula granules as described in claim 1, characterized in that, The step of enzymatically extracting oyster medicinal materials or their decoction pieces, standard decoctions, or Chinese medicine formula granules to obtain a test solution comprises: The oyster medicinal material or its decoction pieces, standard decoction, and Chinese medicine formula granules are sequentially subjected to ammonium bicarbonate solution extraction, dithiothreitol solution extraction, iodoacetamide solution treatment, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting treatment to obtain a test solution; Among them, the dosage ratio of ammonium bicarbonate solution to oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula 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%; The dosage ratio of the dithiothreitol solution to oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules is 100 μL to 200 μL: 1 g to 2 g; the concentration of the dithiothreitol solution is 0.5 mol / L to 3 mol / L; The dosage ratio of the iodoacetamide solution to oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules is 300 μL to 800 μL: 1 g to 2 g; the concentration of the iodoacetamide solution is 0.2 mol / L to 2 mol / L; The trypsin solution is prepared by adding 3 mg to 10 mg of trypsin to a 0.5 wt% to 2 wt% ammonium bicarbonate solution, and the dosage ratio of the trypsin solution to oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules is 200 μL to 1000 μL: 1 g to 2 g; The solid-phase extraction column desalting treatment includes: Centrifuging the enzymolysis solution obtained by enzymolysis, adding the obtained supernatant to an HLB column, eluting with water, discarding the eluate, and then eluting successively with a 10 vol% to 30 vol% acetonitrile solution and a 30 vol% to 60 vol% acetonitrile solution, collecting the eluate, and filtering to obtain.

7. Use of the detection method for Crassostrea rivularis medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules according to any one of claims 1 to 6 in the following (1) or (2): (1) Identifying the origin of oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules; (2) Identifying Crassostrea rivularis medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules and their counterfeits.

8. The application according to claim 7, wherein The origin of the oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules includes Crassostrea gigas and Crassostrea talienwhanensis; The counterfeits include Crassostrea angulata, Crassostrea angulata, Haliotis diversicolor, and Sepia esculenta.

9. A method for identifying Crassostrea rivularis and its counterfeits, characterized in that Includes: Providing a test sample, and detecting the test sample by using the detection method for Crassostrea rivularis medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules according to any one of claims 1 to 6; in the mass spectrum obtained by detecting the test solution prepared from the test sample under the preset detection ion pair, if no detection is found at the retention time of the ion peak of the Crassostrea rivularis control medicinal material solution, the test sample is a counterfeit.

10. A method for identifying the origin of oyster medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules, characterized in that, Includes: Providing a test sample, and detecting the test sample by using the detection method for Crassostrea rivularis medicinal materials or their decoction pieces, standard decoctions, and traditional Chinese medicine formula granules according to any one of claims 1 to 6; in the mass spectrum obtained by detecting the test solution prepared from the test sample under the preset detection ion pair, if no detection is found at the retention time of the ion peak of the Crassostrea rivularis control medicinal material solution, the origin of the test sample is Crassostrea gigas or Crassostrea talienwhanensis.

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