Anadara, tanner and mud clam anadara species characteristic peptide and application thereof

Abstract

The application discloses clam species characteristic peptides of Arca inflata, Cyclotellanae and Tegillarca granosa and application thereof. The clam species characteristic peptides comprise Arca inflata characteristic peptides PSVQGAAAQLTADVK, Cyclotellanae characteristic peptides VAELANAVVSNADQK and Tegillarca granosa characteristic peptides DSWAALGSDK, which can be used for identifying Arca inflata, Cyclotellanae and Tegillarca granosa and evaluating the quality of medicinal materials. The application further provides a detection method based on the three clam species characteristic peptides and liquid chromatography-triple quadrupole mass spectrometry, which is used for component detection in clam medicinal materials or other related products, so that efficient and accurate identification of Arca inflata, Cyclotellanae and Tegillarca granosa can be realized. The method is simple in operation, strong in specificity, high in sensitivity and good in stability, and provides a reliable quality detection method for safe and effective use of clams in the fields of food and medicine, and has a good application prospect in the quality control of clam medicinal materials and related products.

Description

Technical Field

[0001] This invention belongs to the field of cockle species identification technology, specifically involving the characteristic peptides of cockle species such as the giant cockle, hairy cockle, and mud cockle, and their applications. Background Technology

[0002] The Chinese medicinal herb *Scapharcae et tegillarcae musculus* is the guar beetle, an animal belonging to the family Scapharcaceae. Scapharca broughtonii (Schrenck), mud clam Tegillarca granosa (Linnaeus) and blood cockles Scapharca subcrenata The meat of cockles (Lischke) and other species has the effects of replenishing qi and blood, strengthening the stomach and warming the middle jiao, and promoting digestion. Modern pharmacological studies have further shown that it has significant biological activities such as anti-oxidation, anti-inflammation, antibacterial and anti-tumor effects. At the same time, cockle meat, as a high-protein seafood, is rich in hemoglobin, iron and a variety of essential amino acids, making it a natural iron supplement and possessing nutritional and therapeutic value.

[0003] As a food and medicinal material with economic value, the quality control of cockles still faces challenges. The high similarity in morphological characteristics among species makes original identification difficult. Identification methods based on phenotypic differences such as shell morphology and the number of radial ribs are easily affected by individual developmental stages and environmental factors, leading to misjudgments. Although DNA barcoding technologies (such as COI gene sequencing) are highly specific, they have limitations such as the easy degradation of DNA in samples processed at high temperatures, making accurate measurement impossible and failing to meet the needs of rapid testing at the grassroots level. In recent years, adulteration with closely related species has been found in commercially available cockle products, directly affecting their medicinal efficacy and food safety. Summary of the Invention

[0004] The purpose of this invention is to provide characteristic peptides of cockle species such as cockle, hairy cockle, and mud cockle, and to provide specific applications of these characteristic peptides to overcome the shortcomings of the prior art.

[0005] Clams are among the very few bivalves possessing a hemoglobin oxygen-carrying system. It is hypothesized that using characteristic peptides from clam hemoglobin for species identification could rapidly and accurately identify different species, overcoming the limitations of traditional methods. This invention, by screening and identifying characteristic peptides in the hemoglobin of *Clamus spp.*, *Clamus davidii*, and *Clamus trichotiliforme*, provides an effective supplement to existing quality control systems and offers an effective solution for clam identification and quality control.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The clam species characteristic peptides of cockle, hairy clam, and mud clam include:

[0008] The sequence of the characteristic peptide of cockle is: PSVQGAAAQLTADVK (PSV);

[0009] The sequence of the characteristic peptide of blood clam is: VAELANAVVSNADQK (VAE);

[0010] The sequence of the characteristic peptide of mud clam is: DSWAALGSDK (DSW).

[0011] Application of the characteristic peptides of the three original species of cockle (Gnaphalium affine, Cervus nipponense, and Cervus tigrinum) in the identification of cockle medicinal materials.

[0012] A method for identifying three original species of cockle medicinal material involves detecting whether the sample contains the characteristic peptides of the aforementioned cockle species, namely PSV, VAE, and DSW; the judgment principle is as follows:

[0013] If PSV is detected in the sample but VAE and DSW are not detected, the sample is determined to be cockle.

[0014] If VAE is detected in the sample, but DSW and PSV are not detected, the sample is determined to be blood clam.

[0015] If DSW is detected in the sample but VAE and PSV are not detected, the sample is determined to be mud clam.

[0016] If PSV, VAE, and DSW are not detected in the sample, the sample is determined not to be any of the following: cockle, blood cockle, or mud cockle.

[0017] A method for identifying the original species of cockles (Gnaphalium affine, Cervus nipponense, and Cervus tigrinum) based on characteristic peptides of the aforementioned cockle species includes the following steps:

[0018] (1) Preparation of test solution: Take the powder of cockle medicinal material sample, enzymatically hydrolyze it, centrifuge it, collect the supernatant, freeze dry it to obtain the freeze-dried powder of cockle enzymatic hydrolysate; take the freeze-dried powder of cockle enzymatic hydrolysate, prepare the cockle enzymatic hydrolysate solution, desalt it, and then reconstitute it to obtain the test solution.

[0019] (2) Preparation of reference solution: PSV, VAE and DSW, three characteristic peptides of cockle species, were used as references and dissolved in 0.1% formic acid water to prepare reference solution;

[0020] (3) Detection and analysis: Detection and analysis were performed using liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS);

[0021] (4) If PSV is detected in the sample to be tested, and VAE and DSW are not detected, the sample to be tested is determined to be cockle; if VAE is detected in the sample to be tested, and DSW and PSV are not detected, the sample to be tested is determined to be cockle; if DSW is detected in the sample to be tested, and VAE and PSV are not detected, the sample to be tested is determined to be cockle; if PSV, VAE and DSW are not detected in the sample to be tested, the sample to be tested is determined to be neither cockle, cockle, nor cockle.

[0022] Furthermore, in step (1): the enzymatic hydrolysis conditions are as follows: take the powder of cockle medicinal material sample, add PBS buffer at a material-to-liquid ratio of 1:10 (w / v, g / mL), add trypsin (final concentration 800 U / mL), and enzymatically hydrolyze for 5 hours at 50℃ and pH 8.0; the desalting treatment is to desalt the sample using a desalting column; the reconstitution is to reconstitute with 50 μL of 0.1% formic acid aqueous solution.

[0023] Further, the chromatographic conditions in step (3) are as follows: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, and the flow rate is set to 0.3 mL / min. The elution gradient is as follows: 0-1 min, 95% A, 1-2 min, 95-80% A, 2-8 min, 80-60% A, 8-10 min, 60-10% A, 10-11.5 min, 10% A, 11.5-11.6 min, 10-95% A, 11.6-14 min, 95% A. The monitored ion pairs include VAE as... m / z 765.3→171.1; DSW is m / z 525.5→661.4; PSV is m / z 728.9→185.1.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects:

[0025] The characteristic peptides provided by this invention exhibit excellent specificity and stability for three different cockle origins, demonstrating high specificity and potential for identification of cockle medicinal materials. This invention can be widely applied to the component detection of cockle medicinal materials or other related products, accurately distinguishing whether a product contains cockle components. It is particularly suitable for identifying the three cockle origins—*Cynodon dactylon*, *Cynodon dactylon*, and *Cynodon dactylon*. It effectively prevents counterfeiting and adulteration, providing a reliable technical means for the quality control of cockle products.

[0026] This invention also provides an identification method based on three characteristic peptides of cockle species. This method exhibits significant specificity, sensitivity, and stability, enabling efficient and accurate identification of three cockle precursors—*Cynodon dactylon*, *Cynodon dactylon*, and *Cynodon dactylon*. Compared with existing technologies, this invention effectively overcomes the common problems of cross-contamination and misjudgment in traditional methods, ensuring the reliability and accuracy of the identification results. Furthermore, the method is simple to operate, simplifies sample processing, and can complete efficient identification in a short time, demonstrating strong practicality. This method effectively fills the gap in existing technologies that cannot accurately identify cockle precursors, and its broad application potential makes it of significant value in research and industrialization in pharmaceuticals, food, and related fields. Attached Figure Description

[0027] Figure 1 These are the mass spectra of secondary fragments from PSV, VAE, and DSW.

[0028] Figure 2 This is an ion chromatogram of standards (50 ng / mL) for the characteristic peptides of cockle (PSV), hairy cockle (VAE), and mud cockle (DSW).

[0029] Figure 3 This is the extraction ion chromatogram of PSV, VAE, and DSW in cockles.

[0030] Figure 4 This is the extracted ion chromatogram of PSV, VAE and DSW in blood cockles.

[0031] Figure 5 This is the extracted ion chromatogram of PSV, VAE and DSW in mud clams.

[0032] Figure 6 This is the extracted ion chromatogram of PSV, VAE, and DSW in sample HWZ05.

[0033] Figure 7 This is the extracted ion chromatogram of PSV, VAE, and DSW in sample HLYG06.

[0034] Figure 8 This is the extracted ion chromatogram of PSV, VAE, and DSW in the HLYG11 sample.

[0035] Figure 9 This is the extracted ion chromatogram of PSV, VAE, and DSW in the HYT20 sample. Detailed Implementation

[0036] The invention will be further explained and illustrated below with reference to specific embodiments and accompanying drawings.

[0037] Example 1: Screening for characteristic peptides from three clam species: cockle, hairy clam, and mud clam.

[0038] Preparation of test solution

[0039] Take 0.1 g of powder from three species of cockles—*Gnaphalium affine*, *Gnaphalium affine*, and *Gnaphalium affine*—and add 1 mL of PBS buffer and 800 U of trypsin. Incubate at 50°C and pH 8.0 for 5 hours, centrifuge at 8000 rpm for 10 minutes at 4°C, collect the supernatant, and freeze-dry to obtain lyophilized cockle hydrolysate powder. Dissolve the prepared lyophilized cockle hydrolysate powder in water to prepare a 10 mg / mL solution. Desalt the sample using a Millipore ZIPTIP C18 desalting column, and reconstitute with 50 μL of 0.1% formic acid aqueous solution to obtain the test solution. Three batches of samples from each of the three species were replicated.

[0040] (2) Selection of characteristic ions and sequence deduction

[0041] LC-MS / MS parameters: Analysis was performed using an EASY-nLC1000 nanoliter ultra-high performance liquid chromatography system equipped with a nanoliter Acclaim PepMap C18 peptide trapping column (100 μm × 20 mm, 3 μm) and a nanoliter Acclaim PepMap C18 peptide analysis column (75 μm × 250 mm, 3 μm). Column temperature: 50 ℃; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution, with gradient elution; injection volume was 2 μL. Data-dependent acquisition (DDA) was performed on a Q-Exactive mass spectrometer with electrospray ionization (ESI) in positive ion mode to acquire mass spectrometry information.

[0042] Database retrieval and screening of characteristic peptides: The mass spectrometry data analysis and retrieval software used was MaxQuant 2.4.3.0, and the database used was the Uniprot protein database. Arcidae ) database, including cockles ( Scapharca broughtonii ), hairy cockles ( Scapharca subcrenata ), mud clam ( Tegillarca granosa Three species and other genera of cockles.

[0043] Characteristic peptides of *Cynodon dactylon*, *Cynodon dactylon*, and *Cynodon dactylon* were screened using MaxQuant analysis, protein-protein BLAST analysis, and chemometric methods. Specific requirements were as follows: (1) Elimination of missing peptides: Proteins / peptides with a deletion value greater than 2 in a species were eliminated; (2) The peptide must be stably detected and have a high response in at least one of the three species; (3) The peptide originated from hemoglobin of the *Cynodon dactylon* species; (4) VIP>1 in the OPLS-DA model; (5) The fold change was at least 2 (Ratio>2), and P<0.01 was satisfied. The final characteristic peptides of *Cynodon dactylon* were obtained as follows: Figure 1 As shown, PSVQGAAAQLTADVK (PSV), characteristic peptide of hairy cockle: VAELANAVVSNADQK (VAE), characteristic peptide of mud cockle: DSWAALGSDK (DSW).

[0044] Based on the predicted amino acid sequence, a characteristic peptide reference standard was synthesized. The reference standard was simultaneously detected with the test solutions of blood clam, cockle, and mud clam. The retention times and secondary mass spectrometry information of both were consistent, thus confirming the correctness of the sequence.

[0045] Example 2: Detection method of characteristic peptides of three cockle species: cockle davidii, cockle hairy cockle, and cockle mud cockle.

[0046] Three characteristic peptides of cockle species in the cockle enzymatic hydrolysate solution were detected by LC-MS / MS.

[0047] (1) Sample preparation

[0048] Preparation of test solution: Take 0.1 g of powdered cockles (including hairy cockles and mud cockles), add 1 mL of PBS buffer, add 800 U of trypsin, and enzymatically hydrolyze for 5 hours at 50℃ and pH 8.0. Centrifuge at 8000 rpm for 10 minutes at 4℃, collect the supernatant, and lyophilize to obtain lyophilized cockle hydrolysate powder. Dissolve the lyophilized cockle hydrolysate powder in water to prepare a 10 mg / mL solution. Desalt the sample using a ZIPTIP C18 desalting column (Millipore), and reconstitute with 50 μL of 0.1% formic acid aqueous solution to obtain the test solution.

[0049] Preparation of reference solutions: Accurately weigh three characteristic peptide reference standards of cockle species, dissolve them in 0.1% formic acid water to obtain a stock solution with a concentration of 1 mg / mL, and store at -20 ℃ for later use. Accurately measure the cockle species characteristic peptide reference standard stock solution, and serially dilute it with 0.1% formic acid aqueous solution, adding an internal standard to obtain a series of reference solution with concentrations of 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 500 ng / mL, and 600 ng / mL, with an internal standard concentration of 250 ng / mL.

[0050] Spiked matrix samples: Take the cockle species characteristic peptide reference standard, add the enzymatic digestion system matrix and internal standard to prepare cockle species characteristic peptide spiked matrix samples with concentrations of 10 ng / mL, 300 ng / mL and 500 ng / mL, and the internal standard concentration is 250 ng / mL.

[0051] Quality control samples: Take the stock solution of cockle species characteristic peptide reference standard, dilute it stepwise with 0.1% formic acid aqueous solution, add internal standard, and prepare three concentrations of cockle species characteristic peptide quality control solutions: 10 ng / mL (LQC), 300 ng / mL (MQC), and 500 ng / mL (HQC). The concentration of internal standard is 250 ng / mL.

[0052] (2) LC-MS / MS detection method

[0053] An UPLC H-Class PLUS ultra-high performance liquid chromatography system equipped with a Xevo TQ-XS mass spectrometer detector was used. The chromatographic column was an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), with a column temperature of 40°C. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution, with gradient elution according to Table 1. The injection volume was 4 μL. An ESI mass spectrometer was used, with multiple reaction monitoring (MRM) in positive ion mode. The capillary voltage was 3.0 kV, and the desolvation gas temperature was 500°C. Nitrogen was used as the desolvation gas (1000 L / Hr) and the cone gas (150 L / Hr), and argon was used as the collision gas (0.15 mL / min). The monitored ion pairs are shown in Table 2.

[0054] Table 1. Liquid chromatography conditions for the detection of characteristic peptides in cockle species

[0055]

[0056] Table 2. Mass spectrometry MRM conditions for the detection of characteristic peptides in cockle species.

[0057]

[0058] Note: TTLLEDDIAK ( 13 C6, 15 N2) is an isotope-labeled peptide used as an internal standard.

[0059] (3) Exclusivity

[0060] Extraction ion chromatogram of cockle sample ( Figure 3 In ), at the retention time position corresponding to the PSV reference solution of the characteristic peptide of cockle (e.g. Figure 2 As shown), all exhibited chromatographic peaks with consistent retention times, and none showed corresponding chromatographic peaks for the mud clam characteristic peptide DSW or the hairy clam characteristic peptide VAE reference solutions; the extraction ion chromatogram of the hairy clam sample ( Figure 4 In ), at the retention time positions corresponding to the VAE reference solution of the blood clam characteristic peptide (e.g. Figure 2 As shown), all exhibited chromatographic peaks with consistent retention times, and none showed corresponding chromatographic peaks for the mud clam characteristic peptide DSW or the clams characteristic peptide PSV reference solutions; the extraction ion chromatogram of the mud clam sample ( Figure 5 In ), at the retention time positions corresponding to the DSW reference solution of the mud clam characteristic peptide (e.g. Figure 2 As shown in the figure, all three peaks exhibited consistent retention times and lacked the corresponding peaks of the blood clam characteristic peptide VAE and the ark clam characteristic peptide PSV reference solutions. This indicates that the three clam species characteristic peptides are detected only in their respective species, demonstrating strong specificity.

[0061] (4) Linearity and detection limit, quantitation limit

[0062] Standard curves were plotted using the ratio of the peak area of ​​the quantitative ion pair to the peak area of ​​the internal standard for VAE, DSW, and PSV and their corresponding mass concentrations. The limits of detection (LOD, S / N≥3), limits of quantitation (LOQ, S / N≥10), and regression equations were calculated and are shown in Table 3. The three characteristic peptides of cockle species showed good linearity in the range of 5–600 ng / mL.

[0063] Table 3. Sensitivity and linearity of the method for detecting characteristic peptides of cockle species

[0064]

[0065] (5) Matrix effect

[0066] The matrix effect of each concentration of sample was calculated by comparing the peak areas of the three cockle species characteristic peptide reference solutions and the spiked matrix samples. The results showed that the matrix effects of VAE, DSW, and PSV were 100.4%–109.1%, 94.8%–106.5%, and 96.1%–99.9%, respectively. This indicates that there was no significant matrix ion inhibition or enhancement effect interfering with the determination of the three cockle species characteristic peptides.

[0067] (6) Recovery rate

[0068] Recovery rates were calculated by comparing the peak areas of LQC, MQC, and HQC with those of the standards after preparation and processing according to the sample solution. As shown in Table 4, the recoveries of VAE, DSW, and PSV were 107.5%–110.3%, 88.1%–104.7%, and 95.0%–102.9%, respectively, indicating that the method has good accuracy and is not affected by the sample processing.

[0069] Table 4. Matrix effect and recovery rate of the method for detecting characteristic peptides of cockle species (n=6)

[0070]

[0071] (7) Accuracy and precision

[0072] Accuracy and precision were analyzed through intra-day and inter-day experiments on four concentrations: limit of quantitation (LLOQ), limit of quality (LQC), limit of quantity (MQC), and limit of quality (HQC). As shown in Table 5, the intra-day and inter-day precision (RSD) of the VAE, DSW, and PSV determination results were 2.1%–9.3%, 2.7%–9.3%, and 1.4%–9.3%, respectively; the accuracy (RE) were -5.7%–11.3%, -2.9%–13.3%, and -5.5%–3.0%, respectively, indicating that the detection methods are accurate, reliable, and have good reproducibility.

[0073] Table 5. Precision and accuracy of the method for detecting characteristic peptides of cockle species (n=6)

[0074]

[0075] (8) Stability

[0076] After being placed at the autosampler temperature for 72 h, subjected to three freeze-thaw cycles, and stored at -40 °C for 7 days, the accuracy (RE) of the detection results for VAE, DSW, and PSV was -7.4%~7.9%, -1.8%~7.1%, and -9.5%~10.4%, respectively (Table 6), indicating that the three characteristic peptides of cockle species are stable under storage and detection conditions.

[0077] Table 6. Stability of the method for detecting characteristic peptides of cockle species (n=6)

[0078]

[0079] Example 3: Detection and application of characteristic peptides of cockle species

[0080] Twenty-seven batches of cockle samples were collected. The origin was identified by DNA barcoding. There were 6 batches of hairy cockles, 12 batches of broad cockles, 5 batches of mud cockles, and 4 batches failed the test. The samples were prepared according to the method under the preparation of test solution in Example 2 (1), and analyzed according to the LC-MS / MS method in Example 2.

[0081] The results are shown in Tables 7, 8, and 9. In all six batches of blood clams, only the characteristic peptide VAE peak was observed, with an average content of 0.42 μg / g; in all five batches of mud clams, only the characteristic peptide DSW peak was observed, with an average content of 0.10 μg / g; and in all twelve batches of large clams, only the characteristic peptide PSV peak was observed, with an average content of 0.27 μg / g. Figure 6 , Figure 7 , Figure 8 As shown in Figure 9, among the four batches of samples that failed DNA barcoding detection, HWZ05 and HLYG06 were found to contain the characteristic peptide DSW of the mud clam, HLYG11 was found to contain the characteristic peptide PSV of the blood clam, while HYT20 did not contain DSW, PSV, or VAE. This indicates that HWZ05 and HLYG06 are mud clams, HLYG11 is blood clam, and HYT20 is neither mud clam, blood clam, nor hairy clam.

[0082] Table 7. Content of VAE, PSV and DSW in blood clam samples (n = 3)

[0083]

[0084] Table 8. Content of VAE, PSV and DSW in mud clam samples (n = 3)

[0085]

[0086] Table 9. Content of VAE, PSV and DSW in mud clam samples (n = 3)

[0087]

[0088] The above results indicate that the VAE, DSW, and PSV characteristic peptides obtained by the present invention can be used as characteristic indicator components to accurately identify the three origins of blood clam, giant clam, and mud clam.

[0089] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Species-specific peptides of cockle, hairy cockle, and mud cockle, characterized in that, The characteristic peptides of this clam species are: PSV (characteristic peptide of cockle), VAE (characteristic peptide of hairy clam), and DSW (characteristic peptide of mud clam); the sequence of PSV (characteristic peptide of cockle) is: PSVQGAAAQLTADVK; The sequence of the characteristic peptide VAE of the hairy clam is: VAELANAVVSNADQK; the sequence of the characteristic peptide DSW of the mud clam is: DSWALGSDK.

2. The application of the characteristic peptides of the cockle species described in claim 1 (such as the blood cockle, the mud cockle, and the mud cockle) in the identification of the original species of cockle medicinal materials.

3. The application of the clam species characteristic peptides of *Clamus spp.*, *Clamus vulgaris*, and *Clamus hyacinthus* as described in claim 1 in the method for identifying the original species of clam medicinal materials, wherein the identification method involves detecting whether the sample contains the clam species characteristic peptides of *Clamus spp.*, *Clamus vulgaris*, *Clamus vulgaris*, and *Clamus hyacinthus*, namely PSV, VAE, and DSW; the determination principle is: If PSV is detected in the sample but VAE and DSW are not detected, the sample is determined to be cockle. If VAE is detected in the sample, but DSW and PSV are not detected, the sample is determined to be blood clam. If DSW is detected in the sample but VAE and PSV are not detected, the sample is determined to be mud clam. If PSV, VAE, and DSW are not detected in the sample, the sample is determined not to be any of the following: cockle, blood cockle, or mud cockle.

4. The method for identifying the original species of cockle based on the characteristic peptides of cockle species as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the powder of cockle medicinal material sample, enzymatically hydrolyze it, centrifuge it, collect the supernatant, freeze dry it to obtain the freeze-dried powder of cockle enzymatic hydrolysate; take the freeze-dried powder of cockle enzymatic hydrolysate, prepare the cockle enzymatic hydrolysate solution, desalt it, and then reconstitute it to obtain the test solution. (2) Preparation of reference solution: The three characteristic peptides of the cockle species PSV, VAE and DSW are used as references. They are dissolved in 0.1% formic acid water to prepare the reference solution. (3) Detection and analysis: Detection and analysis were performed using liquid chromatography-triple quadrupole mass spectrometry; (4) If PSV is detected in the sample to be tested, and VAE and DSW are not detected, the sample to be tested is determined to be cockle; if VAE is detected in the sample to be tested, and DSW and PSV are not detected, the sample to be tested is determined to be cockle; if DSW is detected in the sample to be tested, and VAE and PSV are not detected, the sample to be tested is determined to be cockle; if PSV, VAE and DSW are not detected in the sample to be tested, the sample to be tested is determined to be neither cockle, cockle, nor cockle.

5. The method as described in claim 4, characterized in that, In step (1), the enzymatic hydrolysis conditions are as follows: take the powder of cockle medicinal material sample, add PBS buffer at a material-to-liquid ratio of 1:10, add trypsin with a final concentration of 800 U / mL, and enzymatically hydrolyze for 5 hours at 50℃ and pH 8.0; the desalting treatment is to desalt the sample using a desalting column; the resolution is to resolution with 50 μL of 0.1% formic acid aqueous solution.

6. The method as described in claim 4, characterized in that, The chromatographic conditions in step (3) are as follows: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, and the flow rate is set to 0.3 mL / min; the elution gradient is as follows: 0-1 min, 95% A, 1-2 min, 95-80% A, 2-8 min, 80-60% A, 8-10 min, 60-10% A, 10-11.5 min, 10% A, 11.5-11.6 min, 10-95% A, 11.6-14 min, 95% A; the monitored ion pairs include VAE as... m / z 765.3→171.1; DSW is m / z 525.5→661.4; PSV is m / z 728.9→185.1.

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