Characteristic polypeptide of Pseudohalodon and its application and method for identifying Pseudohalodon

Abstract

The present invention relates to a characteristic polypeptide of Pseudohalodon and its application and a method for identifying Pseudohalodon, belonging to the field of biotechnology detection technology. The present invention provides two characteristic polypeptides unique to Pseudohalodon, and the amino acid sequences of the characteristic polypeptides are shown in SEQ ID NO.1 and SEQ ID NO.2. The characteristic polypeptide provided by the present invention has excellent specificity and stability for Pseudohalodon, strong specificity, can be used for identifying Pseudohalodon, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology detection, and in particular to two characteristic polypeptides of Pseudohalodon and applications thereof, and a method for identifying Pseudohalodon. Background Art

[0002] Sea dragon is an important animal medicinal material in my country. It has the effects of warming the kidney and strengthening yang, dispersing nodules and reducing swelling. It has a long history of use and many sources. The Chinese Pharmacopoeia stipulates that the medicinal source of sea dragon is the dried body of the Syngnathidae animals Solenognathus hardwickii (Gray), Syngnathoides biaculeatus (Bloch) or Syngnathusa cusLinnaeus.

[0003] As a tonic medicinal material, the market demand for sea dragon is increasing year by year. As sea dragon cannot be effectively cultivated artificially, the wild sea dragon resources are decreasing, resulting in the increasing price of sea dragon in the market. In addition to being directly crushed or extracted as a raw medicinal material to make Chinese patent medicine, Pseudo-sea dragon is also made into sea dragon tonic wine as a health tonic. As the market demand for sea dragon is insufficient, the price is increasing, and there is a phenomenon of confusing it with other varieties or selling inferior products as good ones.

[0004] At present, the main methods for the identification and quality control of sea dragon species are trait identification, HPLC fingerprinting and molecular biology. The trait identification method generally requires that the sea dragon individual is complete and the appraiser has rich experience. This method has a certain degree of subjectivity; the HPLC fingerprint method has low specificity; the molecular biology method has high specificity, but is limited by the degree of preservation of the genetic material in the sample. For sea dragon medicinal wine, processed products, extracts, insect-infested and moldy samples, and samples mixed with multiple individuals, it is difficult to extract DNA and cannot be successfully experimented. In recent years, more and more methods and standards use characteristic peptides to identify or quantitatively study natural drugs. The proteins contained in each species have differences in individual amino acids. Appropriate proteases can be used to extract the difference sites as polypeptide segments with identification significance, namely characteristic peptides. The detection of characteristic peptides generally uses mass spectrometry, without considering protein activity, and the preparation method is simple and the peptide properties are relatively stable. Existing methods for identifying sea dragons have many limiting factors and cannot be applied to sea dragon powder, sea dragon medicinal wine, processed products, extracts and other derivative products. The characteristic peptides in sea dragon proteins have the potential to be used as indicator components for sea dragon species identification. At present, the protein sequence information related to Pseudohalodon is not included in various databases, and differential peptides cannot be obtained through protein sequence comparison. Therefore, there is no record of the characteristic peptides of Pseudohalodon. Summary of the invention

[0005] The purpose of the present invention is to provide two characteristic polypeptides of Pseudohalodon and their application and a method for identifying Pseudohalodon. The characteristic polypeptides provided by the present invention have excellent specificity and stability for Pseudohalodon, strong specificity, can be used for the identification of the species of halodon medicinal materials, specifically the identification of Pseudohalodon, and have good application prospects.

[0006] The present invention provides two characteristic polypeptides of Pseudohylaea, and the amino acid sequences of the characteristic polypeptides are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0007] The present invention also provides the use of the characteristic polypeptide described in the above technical solution in identifying Pseudohalodon.

[0008] Further, the use of SEQ ID NO.1 and SEQ ID NO.2 alone or together in identifying Pseudohalodon.

[0009] Preferably, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 639.6→810.2 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 639.6→753.1; a quantitative ion with a mass-to-charge ratio of m / z 852.1→1144.6 for SEQ ID NO.2, and a qualitative ion with a mass-to-charge ratio of m / z 852.1→875.4.

[0010] The present invention also provides a method for identifying Pseudohalodon, using the characteristic polypeptide shown in SEQ ID NO.1 and / or SEQ ID NO.2 as a reference substance.

[0011] The present invention also provides a method for identifying Pseudohylae based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0012] (1) Preparation of the test solution: The pretreated sea dragon powder extract or the pretreated sea dragon medicinal wine extract is mixed with trypsin to obtain a mixed solution, the mixed solution is mixed with an aqueous solution of ammonium bicarbonate, and the mixture is filtered after enzymolysis to obtain a test solution;

[0013] (2) Preparation of reference solution: using the characteristic peptide of Pseudohyla serrata described in the above technical solution as a reference substance, adding water to dissolve it to obtain a reference solution;

[0014] (3) Detection and analysis: Detection and analysis were performed using triple quadrupole mass spectrometry;

[0015] There is no time sequence limitation between the steps (1) and (2).

[0016] Preferably, in step (1), the method for preparing the pretreated sea dragon powder extract comprises the following steps: mixing sea dragon powder with water and decocting three times, the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting with an aqueous solution of ammonium bicarbonate to obtain a pretreated sea dragon powder extract;

[0017] The preparation process of the pretreated sea dragon medicinal wine extract comprises the following steps: taking out the sea dragon from the sea dragon medicinal wine, drying at low temperature, decocting with water three times, the decocting time is 4h, 3h and 2h respectively, combining the decoctions, filtering, taking the filtrate, and obtaining the pretreated sea dragon medicinal wine extract;

[0018] Preferably, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml.

[0019] Preferably, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0020] Preferably, the enzymatic hydrolysis condition is 37° C. for 2 h.

[0021] Preferably, the triple quadrupole mass spectrometry method uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute.

[0022] Preferably, the triple quadrupole mass spectrometry uses a mass spectrometer detector and electrospray positive ion mode to perform multiple reaction monitoring. SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 639.6→810.2 and a qualitative ion with a mass-to-charge ratio of m / z 639.6→753.1 as the detection ion pair, and SEQ ID NO.2 selects a quantitative ion with a mass-to-charge ratio of m / z 852.1→1144.6 and a qualitative ion with a mass-to-charge ratio of m / z 852.1→875.4 as the detection ion pair.

[0023] The present invention provides two characteristic polypeptides of Pseudohalodon. The characteristic polypeptides provided by the present invention can be used as indicator components for the identification of Pseudohalodon and can be distinguished from other species of halodon, laying a foundation for the development of identification methods for Pseudohalodon and related products. Specifically, the characteristic polypeptides of the present invention have the following beneficial effects:

[0024] (1) The present invention extracts the differential polypeptide segments in the protein sequence and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and it is not affected by the morphology and processing of the sea dragon sample. It fills the gap in the identification of the sea dragon species and the detection method of the sea dragon in the sea dragon products.

[0025] (2) The characteristic peptides and detection methods provided by the present invention provide a reference for finding characteristic peptides in closely related species that are lacking in the database;

[0026] (3) The characteristic polypeptide provided by the present invention has excellent specificity and stability for Pseudohalodon, strong specificity, can be used for the identification of Pseudohalodon medicinal materials, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1-Figure 9 The specificity map of the characteristic peptide SEQ ID NO.1 of Pseudohyla spp. provided by the present invention; wherein Figure 1 The spectrum of SEQID NO.1 reference substance m / z 639.6→810.2, 753.1, Figure 2-Figure 9 The extracted spectra of m / z 639.6→810.2, 753.1 are respectively those of Pseudohalaenopsis, Diaohalaenopsis, Shu's Halaenopsis, Diaohalaenopsis multispinosa, Baojia's Lancet Halaenopsis, Long Thick-nosed Halaenopsis, Thick-nosed Halaenopsis, and Ge's Halaenopsis.

[0028] Figure 10-Figure 18 The specificity map of the characteristic peptide SEQ ID NO.2 of Pseudohyla spp. provided by the present invention; wherein Fig.10 The spectrum of SEQ ID NO.2 reference substance m / z 852.1→1144.6, 875.4, Figure 11-Figure 18 They are the extracted m / z 852.1→1144.6 and 875.4 spectra of Pseudohalodon, Diaohalodon, Shu's Halodon, Multi-spined Diaohalodon, Baojia's Lancet Halodon, Long Thick-nosed Halodon, Thick-nosed Halodon and Ge's Halodon, respectively. DETAILED DESCRIPTION

[0029] The present invention provides two characteristic polypeptides of Pseudohalodon, and the amino acid sequences of the characteristic polypeptides are shown in SEQ ID NO.1: NAGPQGPQGAAGPR, and SEQ ID NO.2: QTGGAGSPATTGFPSGPGR. The present invention provides peptides with characteristic identification significance of Pseudohalodon. The present invention hydrolyzes water extracts of different species of halodon, such as Pseudohalodon, Diaohalodon, Polyspinous Diaohalodon, and Schuler's halodon, with trypsin, and then uses nano-liquid chromatography-high-resolution mass spectrometry to analyze and obtain a mass spectrum of the full peptide segment of halodon protein; the mass spectrum data is subjected to chemometric analysis, and then a triple quadrupole mass spectrometer is used to verify the specificity of the experimental results; and finally, 2 characteristic ion pairs are provided, corresponding to 2 characteristic peptide segments, and the specificity verification result is good.

[0030] The present invention also provides the use of the characteristic polypeptide described in the above technical solution in identifying Pseudohalodon.

[0031] In the present invention, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 639.6→810.2 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 639.6→753.1; a quantitative ion with a mass-to-charge ratio of m / z 852.1→1144.6 for SEQ ID NO.2, and a qualitative ion with a mass-to-charge ratio of m / z852.1→875.4.

[0032] The present invention also provides a method for identifying Pseudohylae based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0033] (1) mixing the pretreated sea dragon powder extract or the pretreated sea dragon medicinal wine extract with trypsin to obtain a mixed solution, mixing the mixed solution with an aqueous solution of ammonium bicarbonate, filtering after enzymolysis, and taking the filtrate to obtain a test solution;

[0034] (2) using the characteristic peptide of Pseudohyla serrata described in the above technical solution as a reference substance, adding an aqueous solution of ammonium bicarbonate to dissolve it to obtain a reference substance solution;

[0035] (3) Detection and analysis using triple quadrupole mass spectrometry;

[0036] There is no time sequence limitation between the steps (1) and (2).

[0037] The method of the present invention extracts differential polypeptide segments from the protein sequence and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and it is not affected by the morphology and processing of the sea dragon sample. It fills the gaps in the identification of Pseudohalodon species and the detection method of Pseudohalodon in sea dragon products, and provides a reference for the search for characteristic peptide segments in closely related species that are lacking in the database.

[0038] The present invention mixes the pretreated sea dragon powder extract or the pretreated sea dragon medicinal wine extract with trypsin to obtain a mixed solution, mixes the mixed solution with an aqueous solution of ammonium bicarbonate, and filters after enzymolysis to obtain a test solution. In the present invention, the preparation method of the pretreated sea dragon powder extract preferably includes the following steps: mixing the sea dragon powder with water and decocting three times, the decocting time is 4h, 3h and 2h respectively, and merging the decoction to obtain a pretreated sea dragon powder extract. The present invention preferably takes 1g of sea dragon powder, and the decoction preferably totals 50mL. Specifically, the present invention preferably takes 5mL of the decoction, places it in a 50mL measuring bottle, dilutes it to the scale with an aqueous solution of ammonium bicarbonate, shakes well, and obtains a pretreated sea dragon powder extract. In the present invention, the preparation process of the pretreated sea dragon medicinal wine extract preferably includes the following steps: taking out the sea dragon from the sea dragon medicinal wine, drying it at low temperature, decocting it with water three times, the decocting time is 4h, 3h and 2h respectively, merging the decoction, filtering, and taking the filtrate to obtain a pretreated sea dragon medicinal wine extract. After obtaining the pretreated sea dragon extract and the pretreated sea dragon medicinal wine extract, the present invention preferably measures 1 to 5 mL of sea dragon powder extract or sea dragon medicinal wine extract, preferably adds 100 μL of trypsin solution, shakes well, adds ammonium bicarbonate aqueous solution to 50 mL, shakes well, and performs enzymolysis after sealing. In the present invention, the enzymolysis conditions are preferably 37°C enzymolysis for 2 hours. After enzymolysis, the present invention is preferably cooled, more preferably cooled to room temperature, and the temperature range of room temperature in the present invention is preferably 10 to 30°C. After cooling, the present invention is filtered and the filtrate is taken as the test solution.

[0039] In the present invention, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml. In the present invention, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0040] The present invention uses the characteristic peptide of Pseudohalodon described in the above technical solution as a reference substance, adds water to dissolve it, and obtains a reference substance solution. The present invention has no special limitation on the source of the characteristic peptide of Pseudohalodon, and an artificial synthesis method can be used. For example, the two characteristic peptides of Pseudohalodon in the present invention are obtained by entrusting Nanjing Source Peptide Biotechnology Co., Ltd. to synthesize according to the specified amino acid sequence.

[0041] The present invention uses triple quadrupole mass spectrometry for detection and analysis. In the present invention, the triple quadrupole mass spectrometry preferably uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3min, 5% A→8% A; the flow rate is 0.5ml per minute. In the present invention, the triple quadrupole mass spectrometry preferably uses a mass spectrometer detector, electrospray positive ion mode, and multiple reaction monitoring. SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 639.6→810.2 and a qualitative ion with m / z 639.6→753.1; SEQ ID NO.2 selects a quantitative ion with a mass-to-charge ratio of m / z 852.1→1144.6 and a qualitative ion with m / z 852.1→875.4.

[0042] The following is a further detailed introduction to the two characteristic polypeptides of Pseudohalodon and their applications and the method for identifying Pseudohalodon in the present invention in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

[0043] Triple quadrupole mass spectrometry conditions

[0044] 0.1% formic acid acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, and gradient elution (0-3min, 5% A→8% A) was performed; the flow rate was 0.5 ml per minute. A mass spectrometer detector was used in electrospray positive ion mode (ESI+) to perform multiple reaction monitoring (MRM), and a quantitative ion with a mass-to-charge ratio of m / z 639.6→810.2 and a qualifier ion with a mass-to-charge ratio of m / z 639.6→753.1 were selected for SEQ ID NO.1; a quantitative ion with a mass-to-charge ratio of m / z 852.1→1144.6 and a qualifier ion with a mass-to-charge ratio of m / z 852.1→875.4 were selected for SEQ ID NO.2.

[0045] Example 1: Searching for characteristic peptides of Pseudohyla

[0046] (1) Preparation of test solution

[0047] Sea dragon water extract: Take 100g of the test sea dragon, grind it, put it in a conical flask, add water and boil it three times, for 4h, 3h, and 2h respectively, combine the decoctions to make a total of 500mL, boil it slightly and concentrate it until the liquid is viscous, transfer it to a silica gel bowl, and dry it in a 60℃ electric constant temperature blast drying oven until it is solid to obtain the sea dragon water extract;

[0048] Test solution: weigh 0.1 g of the water extract of the sea dragon, add 50 ml of 1% ammonium bicarbonate aqueous solution, ultrasonically treat for 30 min to dissolve, filter with a microporous filter membrane, take 100 μL of the filtrate, add 10 μL of 1 mg / ml trypsin aqueous solution, enzymatically hydrolyze at 37°C for 2 h, take out and cool to room temperature.

[0049] (2) Selection of characteristic ions and sequence inference

[0050] After the sample solution was analyzed by nano-liquid chromatography-high-resolution mass spectrometry, the mass spectrometry data was imported into PEAKS 8.5 software for de novo sequencing and sequence prediction of all peptides of the sample, and the results were analyzed. From the analysis results, peptides that were only detected in Pseudohalodon and almost not detected in other species of halodon were selected as parent ions, and their secondary spectra were analyzed one by one to select daughter ions with better responses. Further analysis obtained the mass spectrometry ion pair information representing the difference of Pseudohalodon m / z 639.6 (double charge) → 810.2, 753.1 and m / z 852.1 (double charge) → 1144.6, 875.4. Through further analysis, the characteristic peptide sequence of Pseudohalodon corresponding to the ion was inferred, SEQ ID NO.1: NAGPQGPQGAAGPR, SEQ ID NO.2: QTGGAGSPATTGFPSGPGR.

[0051] According to the inferred amino acid sequence, a characteristic peptide reference substance of Pseudohalodon was commissioned to be synthesized. The reference substance and the Pseudohalodon test solution were tested simultaneously. The retention times and secondary mass spectrometry information of the two were consistent, thus confirming the correctness of the sequence.

[0052] Example 2: Study on the specificity of the characteristic peptide of Pseudohyla

[0053] The water extracts of all species of sea dragons were tested using triple quadrupole mass spectrometry conditions. The results showed that the three batches of Pseudohalodon species all had the characteristic peptide chromatographic peak of Pseudohalodon, while the other Pseudohalodon, ... Figure 1-9 , see attached Figure 10-18 This indicates that the characteristic peptide is unique to Pseudohalodon and can be used as an indicator component to identify Pseudohalodon.

[0054] Example 3: Application of Pseudo-Haenan Peptide in Haenan Powder Extract and Haenan Medicinal Wine

[0055] (1) Sample preparation

[0056] Preparation of the test solution of sea dragon powder extract: Take 1g of the test sea dragon, grind it, put it in a conical flask, add water and boil it three times, for 4h, 3h, and 2h respectively, combine the decoctions, a total of 50mL, measure 1ml into a 5ml volumetric flask, add 100μl of 1mg / ml trypsin aqueous solution, shake well, then add 1% ammonium bicarbonate aqueous solution to the scale, shake well, seal, and hydrolyze at 37℃ for 2h. Cool, filter, and take the filtrate as the test solution.

[0057] Preparation of test solution of Hailong medicinal wine: Take 10g of Hailong in Hailong medicinal wine, dry it at low temperature, add water and boil it three times, for 4h, 3h, and 2h respectively, combine the decoctions, a total of 50mL; filter, take 1ml of the filtrate into a 5ml volumetric flask, and prepare according to the above method starting from "add 100μl of 1mg / ml trypsin aqueous solution".

[0058] Reference solution of the characteristic peptide fragment of Pseudohalodon: Weigh 10 mg of each of the characteristic peptide reference substances SEQ ID NO.1 and SEQ ID NO.2 of Pseudohalodon respectively, put them into a 50 ml volumetric flask, add water to dissolve and dilute to the scale to obtain the solution.

[0059] (2) The determination was performed using triple quadrupole mass spectrometry conditions.

[0060] (3) Sample measurement

[0061] Ten batches of Pseudohalodon powder extract samples and five batches of Pseudohalodon medicinal liquor samples were collected and tested, and the results showed that the characteristic peptides of Pseudohalodon were detected in all of them, indicating that these two characteristic peptides can be used to identify the Pseudohalodon ingredients in sea dragon products.

[0062] Example 4: Ion information with identification potential

[0063] Five ions with identification potential were screened out from 350 candidate ions. After verification by triple quadrupole mass spectrometry, it was found that not all ions could be used to identify the components of Pseudohalodon. Some ions were not specific enough for Pseudohalodon. Only ions with mass-to-nuclear ratios of 639.6 and 852.1 had good specificity. The specific results are shown in Table 1.

[0064] Table 1 Specificity verification results

[0065]

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Shandong Provincial Institute of Food and Drug Inspection <120> Characteristic polypeptide of Pseudohalodon and its application and method for identifying Pseudohalodon <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <400> 1 Asn Ala Gly Pro Gln Gly Pro Gln Gly Ala Ala Gly Pro Arg 1 5 10 <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <400> 2 Gln Thr Gly Gly Ala Gly Ser Pro Ala Thr Thr Gly Phe Pro Ser Gly 1 5 10 15 Pro Gly Arg

Claims

1. A polypeptide characteristic of Pseudohyla, characterized in that: The amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.1 and / or SEQ ID NO.

2.

2. Use of the characteristic polypeptide according to claim 1 in identifying Pseudohalodon.

3. The use according to claim 2, characterized in that: The identification is performed using mass spectrometry, and the ion pairs detected by SEQ ID NO.1 include: a quantitative ion with a mass-to-charge ratio of m / z 639.6→810.2, and a qualitative ion with a mass-to-charge ratio of m / z 639.6→753.1; the ion pairs detected by SEQ ID NO.2 include: a quantitative ion with a mass-to-charge ratio of m / z 852.1→1144.6, and a qualitative ion with a mass-to-charge ratio of m / z 852.1→875.

4.

4. A method for identifying Pseudohylae, characterized in that: The following steps are involved: (1) The pretreated sea dragon powder extract or the pretreated sea dragon medicinal wine extract is mixed with trypsin to obtain a mixed solution, the mixed solution is mixed with an ammonium bicarbonate aqueous solution, and the mixture is filtered after enzymatic hydrolysis to obtain a test solution; (2) Using the characteristic peptide of Pseudohyla spp. as a reference substance, dissolving them in water to obtain a reference substance solution; (3) Detection and analysis using triple quadrupole mass spectrometry; There is no time sequence limitation between the steps (1) and (2); The preparation method of the pretreated sea dragon powder comprises the following steps: mixing the sea dragon powder with water and decocting for three times, the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting with an aqueous solution of ammonium bicarbonate to obtain a pretreated sea dragon powder extract; The preparation process of the pretreated sea dragon medicinal wine extract comprises the following steps: taking out the sea dragon from the sea dragon medicinal wine, drying at low temperature, decocting with water three times, the decocting time is 4h, 3h and 2h respectively, combining the decoctions, filtering, taking the filtrate, and obtaining the pretreated sea dragon medicinal wine extract; The trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml; The mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%; the enzymatic hydrolysis condition is 37° C. for 2 hours; The triple quadrupole mass spectrometry method uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute; The triple quadrupole mass spectrometry method uses a mass spectrometer detector and an electrospray positive ion mode to perform multiple reaction monitoring. SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 639.6→810.2 and a qualitative ion with a mass-to-charge ratio of m / z 639.6→753.1 as the detection ion pair, and SEQ ID NO.2 selects a quantitative ion with a mass-to-charge ratio of m / z 852.1→1144.6 and a qualitative ion with m / z 852.1→875.4 as the detection ion pair.

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