A method for detecting the swim bladder of yellow croaker by fingerprint spectrum

Abstract

The application discloses a method for detecting yellow croaker swim bladder by fingerprint spectrum, comprising the following steps: step 1, preparation of yellow croaker swim bladder sample solution; step 2, precise quantitative sample solution is sucked and injected into a liquid chromatograph, and a chromatogram is recorded; step 3, the yellow croaker swim bladder fingerprint spectrum obtained in step 2 is introduced into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, common peaks existing in chromatograms of different batches of yellow croaker swim bladder sample are selected as common peaks, and an average value calculation method is used to generate a control fingerprint spectrum of the yellow croaker swim bladder; step 4, typical common peaks in the yellow croaker swim bladder fingerprint spectrum are separated and purified by liquid phase preparation, and representative polypeptide components are obtained; and step 5, the representative polypeptide is analyzed based on liquid chromatography-mass spectrometry, and according to mass spectrometry fragmentation characteristics and fragment ion information, the sample is characterized by comparing with a database. The yellow croaker swim bladder fingerprint spectrum can comprehensively and objectively characterize the quality of the yellow croaker swim bladder, and can be used for identifying the authenticity of the swim bladder.

Description

Technical Field

[0001] This invention belongs to the field of animal polypeptide detection technology, and relates to a detection method for traditional Chinese medicine materials, particularly to a method for establishing a swim bladder fingerprint spectrum of yellow croaker and a method for detection using its standard fingerprint spectrum. Background Technology

[0002] Fish swim bladders are believed to have various health benefits, including nourishing the liver and kidneys, replenishing essence and yang, moistening the lungs and strengthening the spleen, replenishing qi and blood, promoting blood circulation and stopping bleeding, dispersing blood stasis and reducing swelling, calming wind and relieving spasms, and detoxifying and astringing sores. They are considered one of the "Eight Treasures of the Ocean," alongside bird's nest and shark fin, and are often referred to as "marine ginseng." Currently, they are included in the standards for traditional Chinese medicine in Beijing, Shandong, Hunan, Gansu, and other regions. Medicinal fish swim bladders are those of species such as the large yellow croaker, small yellow croaker, yellow croaker, croaker, Chinese sturgeon, beluga sturgeon, or paddlefish. The yellow croaker swim bladder is currently the most widely circulated in the market. However, the quality control of fish swim bladders in these local standards only includes morphological identification, moisture content, total ash content testing, and total nitrogen content determination, which is insufficient to effectively control the quality of fish swim bladders.

[0003] Animal-derived drugs have complex compositions, and qualitative and quantitative analysis of individual components is insufficient to comprehensively reflect the overall information of the drug. Furthermore, there are currently no research reports on the chemical composition and pharmacodynamic material basis of the fish swim bladder system. Therefore, establishing a fingerprint spectrum that can comprehensively reflect the quality of fish swim bladders and identifying representative components is of great significance for the quality control of fish swim bladders. Fish swim bladders are rich in protein, and peptide fragments obtained after enzymatic hydrolysis or chemical degradation of proteins under specific conditions possess characteristic and specific properties. The peptide spectra obtained by separating and analyzing these fragments can provide detailed information about the proteins. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a method for detecting yellow croaker swim bladder using fingerprint spectroscopy, which has high precision, repeatability, and stability. The fingerprint spectroscopy established by this invention can quickly and accurately identify the quality of yellow croaker swim bladder, and further, by accurately analyzing the representative polypeptide components of yellow croaker swim bladder, it provides an important means for effectively controlling swim bladder quality.

[0005] To achieve the above objectives, this invention optimizes the sample pretreatment method and chromatographic conditions, establishes an HPLC-ELSD fingerprint of yellow croaker swim bladder, and further accurately analyzes the representative polypeptide components of yellow croaker swim bladder, which can be used to comprehensively and objectively evaluate the quality of yellow croaker swim bladder.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] A method for detecting the swim bladder of yellow croaker using fingerprint spectroscopy, characterized by comprising the following steps:

[0008] Step 1: Preparation of Yellow Croaker Swim Bladder Test Solution: Take different batches of yellow croaker swim bladder test samples, weigh a certain weight of the test sample and place it in a container, add water and soak at 4℃. The mass-to-volume ratio of test sample to water is 1:100 to 1:200. Homogenize with a wall-breaking machine, adjust the pH to 7 to 7.5 with sodium hydroxide, add trypsin for enzymatic hydrolysis, the enzyme activity is 3 to 5 U / g, and hydrolyze at 37℃ and 100 to 200 r / min for 3 hours with shaking. Boil the hydrolysate for 10 minutes to inactivate the enzyme, centrifuge the hydrolysate, take the supernatant, concentrate under reduced pressure, transfer with 0.05 to 0.15% formic acid water, and desalt using a C18 solid-phase extraction column.

[0009] Step 2: Accurately pipette the test solution from Step 1 and inject it into the liquid chromatograph, then record the chromatogram.

[0010] Step 3: Export the fingerprint chromatogram of the yellow croaker swim bladder obtained in Step 2 and import it into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; select the chromatographic peaks present in the chromatograms of different batches of yellow croaker swim bladder as common peaks; generate the reference fingerprint chromatogram of yellow croaker swim bladder using the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak; the results should meet the following standards: there are 18 common peaks in the fingerprint chromatogram of the test sample, the fingerprint chromatogram of the test sample is consistent with the reference fingerprint chromatogram, and the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram calculated by the similarity calculation software according to the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System is not less than 0.9.

[0011] Furthermore, the present invention also includes the following steps:

[0012] Step 4: The typical common peaks in the swim bladder fingerprint of yellow croaker, peaks 4 and 17, were separated and purified by liquid chromatography under the same conditions as in step 2 to obtain representative polypeptide components.

[0013] Step 5: Based on the representative peptide components obtained in Step 4, analyze them using liquid chromatography-mass spectrometry (LC-MS) with triple quadrupole mass spectrometry (CMS) as the detector and electrospray ionization (ESI) as the source. + Multiple reaction monitoring was performed using electrospray positive ion mode. Based on the fragmentation characteristics of mass spectrometry and the fragment ion information, the data was compared with the database to characterize the peptide components of the sample.

[0014] The desalting process of the C18 solid-phase extraction column in the preparation method of the test solution in step 1 is as follows: Activate with acetonitrile, equilibrate with a trifluoroacetic acid aqueous solution of 0.05%–0.15% (v / v), load the sample, wash with two column volumes of a trifluoroacetic acid aqueous solution of 0.05%–0.15% (v / v), elute with three column volumes of a 30% acetonitrile aqueous solution (containing 0.05%–0.15% trifluoroacetic acid), collect the eluent, concentrate to dryness under reduced pressure, redissolve the residue in a 30% acetonitrile aqueous solution (containing 0.05%–0.15% formic acid), centrifuge, and take the supernatant to obtain the test solution.

[0015] In step 2, the liquid chromatography conditions are as follows: octadecylsilane-bonded silica gel as the packing material; column temperature 30–40℃; mobile phase A: formic acid acetonitrile-B: formic acid water, gradient elution, elution gradient 0–67 min, 0%–100% A; flow rate 0.9–1.1 mL / min. -1 The injection volume was 1–20 μL, and the analysis was performed using an Alltech 2000ES evaporative light scattering detector. The detector parameters were: drift tube temperature 110–120 °C, carrier gas flow rate 2.5–3.5 mL / min. -1 Alternatively, use the Agilent 1260 evaporative light scattering detector. Detector parameters: drift tube temperature: 40–45°C, carrier gas pressure: 3.0–3.5 bar, gain: 6–7.

[0016] As a preferred embodiment, the volume ratio of formic acid in the A: formic acid / acetonitrile-B: formic acid / water mobile phase is 0.05% to 0.15%; further, the volume ratio of formic acid in the A: formic acid / acetonitrile-B: formic acid / water mobile phase is 0.1%.

[0017] In step 3, the fingerprint chromatogram of the test sample contains 18 common peaks. Taking the chromatographic peak corresponding to reference peak 4 as the S peak, the relative retention times of each common peak should meet the following requirements: peak 1 0.561–0.563, peak 2 0.630–0.632, peak 3 0.772–0.775, S peak 1.000, peak 5 1.579–1.586, peak 6 2.738–2.749, peak 7 4.076–4.095, peak 8… Peak 1: 4.275–4.296; Peak 9: 4.464–4.489; Peak 10: 4.608–4.747; Peak 11: 5.238–5.260; Peak 12: 5.482–5.508; Peak 13: 8.326–8.366; Peak 14: 8.591–8.628; Peak 15: 8.780–8.822; Peak 16: 9.058–9.100; Peak 17: 9.274–9.319. Peak 18: 10.528–10.580; the relative peak areas after taking the logarithmic values ​​of each common peak and peak S should conform to the following: Peak 1: 0.796–0.869, Peak 2: 0.846–0.950, Peak 3: 0.885–0.973, Peak S: 1.000, Peak 5: 0.831–0.904, Peak 6: 0.914–1.006, Peak 7: 0.991–1.073, Peak 8: 0.949–1.007, Peak 9 ... Peak 0.960–1.044, Peak 10 0.956–1.029, Peak 11 0.988–1.073, Peak 12 0.973–1.051, Peak 13 1.059–1.135, Peak 14 1.097–1.177, Peak 15 1.042–1.141, Peak 16 0.970–1.033, Peak 17 1.051–1.124, Peak 18 0.889–0.968.

[0018] In step 5, the representative peptides are characterized by targeted secondary fragmentation. The liquid chromatography-mass spectrometry (LC-MS) conditions are as follows:

[0019] An EASY-nLC™ 1200 nano-scale UHPLC system was used with octadecylsilane-bonded silica gel as the packing material (15 cm × 150 μm, 1.9 μm), and the column temperature was 35 °C. Mobile phase A consisted of 80% acetonitrile containing 0.1% formic acid, and mobile phase B consisted of 0.1% formic acid in water. Gradient elution was used, with the following gradients: 0–2 min, 6%–10% A; 2–45 min, 10%–30% A; 45–48 min, 30%–35% A; 48–50 min, 35%–50% A; 50–51 min, 50%–100% A. The flow rate was 2 μL·min⁻¹, and the injection volume was 1 μL. A QExactive™ HF-X mass spectrometer with Nanospray Flex was used. TMThe ion source was an (ESI) system with an ion spray voltage of 2.1 kV and an ion transmission tube temperature of 320 °C. The mass spectrometer employed a data-dependent acquisition mode with a full scan range of m / z 350–1500. The primary mass spectrometer resolution was set to 60,000 (200 m / z), and the maximum C-trap capacity was 3 × 10⁻⁶. 6 The maximum C-trap injection time was 20 ms; fragmentation was performed using high-energy collisional fragmentation (HCD), followed by secondary mass spectrometry detection at a resolution of 15000 (200 m / z). The maximum C-trap capacity was 1 × 10⁻⁶. 5 The maximum C-trap injection time was 45 ms, the peptide fragmentation collision energy was set to 27%, and the threshold intensity was set to 2.2 × 10⁻⁶. 4 The dynamic resistance range is set to 20s.

[0020] The spectral data were searched using Proteome Discoverer 2.2 (PD2.2, Thermo) based on the protein database. Search parameters were set as follows: precursor ion mass tolerance was 10 ppm, and fragment ion mass tolerance was 0.02 Da. Immobilization modification was alkylation of cysteine, variable modification was methionine oxidation, and N-terminal acetylation was allowed, with a maximum of two missed cleavage sites permitted.

[0021] Optimization of fingerprint detection conditions:

[0022] 1. Optimization of test solution preparation:

[0023] By comparing different sample pretreatment methods (pepsin combined with pancreatic enzyme digestion, pancreatic enzyme digestion), it was found that the size and type of peptides obtained by pancreatic enzyme digestion were suitable, so pancreatic enzyme digestion was adopted. In the investigation of the addition volume of trifluoroacetic acid (0.05%, 0.1%, 0.15%, 0.2%) of the eluent and acetonitrile in the solid phase extraction desalting process, it was found that the addition volume of trifluoroacetic acid in the eluent and elution was 0.05-0.15%, with 0.1% being the optimal value. When the addition volume of acetonitrile in the elution was 30%, the chromatogram obtained had the most information and the highest component content.

[0024] 2. Optimization of chromatographic conditions:

[0025] This invention compared ultraviolet (UV) detectors with evaporative light scattering (ELS) detectors and found that UV detectors were unsatisfactory at wavelengths of 210 nm, 254 nm, 280 nm, and 360 nm, exhibiting baseline drift during gradient elution and poor peak resolution, failing to fully reflect the characteristic information of the sample. In contrast, ELS detectors showed virtually no baseline drift, produced a large number of peaks, represented a greater amount of information, and exhibited better peak resolution and shape. Therefore, ELS was chosen to establish the swim bladder fingerprint of yellow croaker.

[0026] This invention compares the elution ratios and time conditions of multiple formic acid-acetonitrile-formic acid aqueous mobile phases. After optimization, it was found that the separation effect was better when the volume ratio of formic acid added was in the range of 0.05% to 0.15%, and the optimal volume ratio of formic acid added was 0.1%. The elution gradient was determined through optimization.

[0027] The beneficial effects of this invention are:

[0028] 1. Based on the fact that yellow croaker swim bladder is rich in protein and its active components are polypeptides, this invention establishes a fingerprint spectrum for the polypeptide components of yellow croaker swim bladder. Through extensive experiments, the optimal sample enzymatic hydrolysis and desalting pretreatment method, the optimal mobile phase composition, gradient elution program, flow rate, detector type and corresponding optimal operating parameters, chromatographic column, flow rate, column temperature and other analytical conditions were screened. Multiple experiments have verified that the yellow croaker swim bladder fingerprint detection method provided by this invention can comprehensively, objectively and accurately detect and evaluate the quality of yellow croaker swim bladder.

[0029] 2. The swim bladder fingerprint spectrum established by the method provided by the present invention can effectively characterize the quality of the swim bladder of yellow croaker, objectively reflect the order and interrelationship of each fingerprint characteristic peak, and focus on the overall appearance characteristics. This can avoid the one-sidedness of judging the quality of the swim bladder by measuring individual chemical components, and reduce the possibility of artificial processing to meet quality standards. It is suitable for the quality control of swim bladder.

[0030] 3. This invention establishes a standard fingerprint spectrum for yellow croaker swim bladder and further identifies representative polypeptide components, which helps to understand the pharmacological activity of yellow croaker swim bladder, provides a scientific basis for pharmacological research and adulteration prevention, and is applicable to the quality control of yellow croaker swim bladder in future processing.

[0031] 4. The universal technical system for identifying the quality of yellow croaker swim bladder using fingerprint spectrum established by this invention can be applied to other fields such as animal medicine, microbiology, and food safety. Attached Figure Description

[0032] Figure 1 HPLC-ELSD fingerprints of swim bladders from ten batches of yellow croaker.

[0033] Figure 2 The standard fingerprint of the swim bladder of yellow croaker is obtained by HPLC-ELSD.

[0034] Figure 3 This is a secondary mass spectrum of GYTGLDGR, a representative polypeptide from the swim bladder of yellow croaker.

[0035] Figure 4 This is a secondary mass spectrum of LLAESVTEVTSFNK, a representative polypeptide from the swim bladder of yellow croaker.

[0036] Figure 5 Precision fingerprint spectrum of yellow croaker swim bladder for batch number NO.1:20210801-HY.

[0037] Figure 6 The stability fingerprint spectrum of the swim bladder of yellow croaker with batch number NO.1:20210801-HY.

[0038] Figure 7 The repeatability fingerprint of the swim bladder of yellow croaker with batch number NO.1:20210801-HY.

[0039] Figure 8 HPLC-ELSD chromatogram for identifying genuine and counterfeit yellow croaker swim bladders. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Instruments and reagents used in the examples:

[0042] 1. Instruments and reagents

[0043] Thermo U3000 HPLC system, DAD UV detector, Alltech 2000ES evaporative light scattering detector; Agilent 1260 HPLC system, Agilent 1260 evaporative light scattering detector; Newclassis ME MS105DU electronic analytical balance (Mettler-Toledo); SHA-B thermostatic shaker (Guohua Enterprise Group Co., Ltd.);

[0044] H1650-W benchtop high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); Mili-QⅡ pure water system (Millipore, USA); PHS-3E pH meter (Shanghai Instrument & Electronics Scientific Instruments Co., Ltd.); Cleanert SC18 solid-phase extraction column (1g / 6mL, Bona Instruments Technology Co., Ltd.); trypsin (enzyme activity ≥4000U / mg, batch number: 20201026, Beijing Solarbio Science & Technology Co., Ltd.); pepsin (enzyme activity ≥400U / mg, batch number: 20201026, Beijing Solarbio Science & Technology Co., Ltd.); acetonitrile (chromatographic grade, Merck KGaA); trifluoroacetic acid and formic acid (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.). All other reagents were of analytical grade.

[0045] 2. Sample Source

[0046] The swim bladders of yellow croaker used in this invention comprise a total of 10 batches (NO.1: 20210801-HY, NO.2: 20210802-HY, NO.3: 20210803-HY, NO.4: 20210901-HY, NO.5: 20210902-HY, NO.6: 20210903-HY, NO.7: 20210904-HY, NO.8: 20 210905-HY, NO.9: 20211001-HY, NO.10: 20211002-HY), two batches of croaker swim bladders (NO.1: 20210801-MY, NO.2: 20210901-MY), and two batches of cod swim bladders (NO.1: 20210801-XY, NO.2: 20210901-XY) are all mainstream products sold in the market.

[0047] Example 1:

[0048] Step 1: Preparation of Yellow Croaker Swim Bladder Test Solution: Accurately weigh approximately 3g of 10 batches of yellow croaker swim bladder test sample, add 500mL of water and soak overnight at 4℃, homogenize using a high-speed blender, adjust the pH to 7.25 with 6M sodium hydroxide, add trypsin for enzymatic hydrolysis (enzyme activity 4U / g), and react with shaking at 37℃ and 150r / min for 3h. Boil the hydrolysate for 10min to inactivate the enzyme, ending the reaction. Centrifuge at 10000r / min for 10min, collect the supernatant, concentrate under reduced pressure, and dilute to 10mL. Transfer 0.1% formic acid to aqueous solution; take 2 mL for C18 solid-phase extraction to remove salt. The process is as follows: activate with acetonitrile, equilibrate with 0.1% trifluoroacetic acid aqueous solution, load the sample, rinse with two column volumes of 0.1% trifluoroacetic acid aqueous solution, elute with three column volumes of 30% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid), collect the eluent, concentrate to dryness under reduced pressure, redissolve the residue with 30% acetonitrile aqueous solution (containing 0.1% formic acid), centrifuge, and take the supernatant.

[0049] Step 2: Accurately pipette the test solution from Step 1 and inject it into the liquid chromatograph. Record the chromatogram after 67 minutes. Specific chromatographic conditions: Aglient ZORBAX SB-Aq C18 (4.6 mm × 250 mm, 5 μm) column, column temperature 35℃; mobile phase: A: 0.1% formic acid / acetonitrile - B: 0.1% formic acid / water, gradient elution. Elution gradient: 0–5 min, 0% A; 5–18 min, 0%–5% A; 18–25 min, 5%–6.5% A; 25–28 min, 6.5%–9% A; 28–32 min, 9%–12% A; 32–36 min, 12% A. A; 36~42min, 12%~13.5%A; 42~45min, 13.5%~16%A; 45~55min, 16%~20%A; 55~65min, 20%~25%A; 65~67min; flow rate 1.0mL·min -1 The injection volume was 10 μL, and the analysis was performed using an Alltech 2000ES evaporative light scattering detector. The detector parameters were: drift tube temperature 115℃, carrier gas flow rate 3.2 mL / min. -1 .

[0050] Step 3: Export the swim bladder fingerprint spectra of the 10 batches of yellow croaker obtained in Step 2 (see...). Figure 1 The data was imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System A. Eighteen chromatographic peaks present in all ten batches of yellow croaker swim bladder fingerprints were selected as common peaks. A control fingerprint chromatogram against yellow croaker swim bladder was generated using the average value calculation method. (See attached image). Figure 2The relative retention time, relative peak area after taking the logarithm, and similarity of each common peak were calculated, and the results are shown in Table 1-2.

[0051] Selection of reference peak: In this invention, peak No. 4, which has a high content, moderate retention time and good separation, is selected as the reference peak.

[0052] Table 1. Relative retention time of common peaks and relative peak area limits after LOG collection for 10 batches of yellow croaker swim bladders.

[0053]

[0054]

[0055] Table 2. Similarity results of swim bladder fingerprints from 10 batches of yellow croaker

[0056] batch Similarity batch Similarity NO.1 0.952 NO.6 0.948 NO.2 0.952 NO.7 0.925 NO.3 0.950 NO.8 0.916 NO.4 0.952 NO.9 0.948 NO.5 0.902 NO.10 0.925

[0057] Regarding the determination of fingerprint chromatographic technical parameters, the experimental results above show that the similarity between the fingerprint chromatograms of ten batches of yellow croaker swim bladders and the control fingerprint chromatograms is greater than 0.9. Therefore, the provisional standard for yellow croaker swim bladder fingerprint chromatograms is: the fingerprint chromatogram of the test sample should be consistent with the control fingerprint chromatogram. According to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the control fingerprint chromatogram, calculated by similarity calculation software, should not be lower than 0.9. Furthermore, the relative retention time and relative peak area of ​​each common peak in the ten batches of fingerprint chromatograms should meet the limits specified in Table 1.

[0058] Further, in step 4, the typical common peaks 4 and 17 in the swim bladder fingerprint spectrum of yellow croaker are separated and purified by liquid phase preparation to obtain representative polypeptide components.

[0059] Step 5: Based on the representative peptides obtained in Step 4, analyze them using a EASY-nLC™ 1200 nano-scale UHPLC system with a C18 column (15cm × 150μm, 1.9μm) at 35℃. Mobile phase A: 80% acetonitrile containing 0.1% formic acid; Mobile phase B: 0.1% formic acid in water. Gradient elution was used, with the following gradients: 0–2 min, 6%–10% A; 2–45 min, 10%–30% A; 45–48 min, 30%–35% A; 48–50 min, 35%–50% A; 50–51 min, 50%–100% A. The flow rate was 2 μL·min⁻¹, and the injection volume was 1 μL. The system was analyzed using a Q Exactive™ HF-X mass spectrometer with Nanospray Flex. TMThe ion source was an (ESI) system with an ion spray voltage of 2.1 kV and an ion transmission tube temperature of 320 °C. The mass spectrometer employed a data-dependent acquisition mode with a full scan range of m / z 350–1500. The primary mass spectrometer resolution was set to 60,000 (200 m / z), and the maximum C-trap capacity was 3 × 10⁻⁶. 6 The maximum C-trap injection time was 20 ms; fragmentation was performed using high-energy collisional fragmentation (HCD), followed by secondary mass spectrometry detection at a resolution of 15000 (200 m / z). The maximum C-trap capacity was 1 × 10⁻⁶. 5 The maximum C-trap injection time was 45 ms, the peptide fragmentation collision energy was set to 27%, and the threshold intensity was set to 2.2 × 10⁻⁶. 4 The dynamic resistance range is set to 20s.

[0060] The spectral data were searched using Proteome Discoverer 2.2 (PD2.2, Thermo) based on the protein database. Search parameters were set as follows: precursor ion mass tolerance was 10 ppm, and fragment ion mass tolerance was 0.02 Da. Immobilization modification was alkylation of cysteine, variable modification was methionine oxidation, and N-terminal acetylation was allowed, with a maximum of two missed cleavage sites permitted.

[0061] This experiment involved liquid chromatography-mass spectrometry (LC-MS) analysis of the isolated differentially expressed peptide components. Peaks 4 and 17 among the typical common peaks were identified as GYTGLDGR and LLAESSVTEVTSFNK, respectively. The secondary mass spectra of these two representative peptide components are shown below. Figure 3 and Figure 4 .

[0062] 3. Optimization of chromatographic conditions:

[0063] 3.1 Detector Selection

[0064] The combination of a UV detector and an Alltech evaporative light dispersive detector revealed that the UV detector performed poorly at wavelengths of 210 nm, 254 nm, 280 nm, and 360 nm, exhibiting baseline drift during gradient elution and a limited number of fingerprint peaks, failing to comprehensively reflect the sample's characteristic information. In contrast, the evaporative light dispersive detector showed virtually no baseline drift, produced a large number of chromatographic peaks, represented a greater amount of information, and demonstrated superior peak resolution and shape. Therefore, the enhanced light dispersive detector was chosen to establish the swim bladder fingerprint of yellow croaker.

[0065] 3.2 Selection of mobile phase

[0066] The effects of formic acid concentrations of 0.05%, 0.1%, and 0.15% on the separation efficiency were investigated. The results showed that formic acid concentrations of 0.05%, 0.1%, and 0.15% all exhibited better separation efficiency. Considering the separation efficiency, peak shape, and column wear, the optimal formic acid addition ratio was 0.1%.

[0067] 3.3 Investigation of column temperature

[0068] The effects of column temperatures of 30℃, 35℃ and 40℃ on the separation effect were investigated. The results showed that all 18 common peaks could be well separated under the three column temperature conditions. The separation degree and peak shape of each peak were the best when the column temperature was 35℃. Therefore, the column temperature of 35℃ is preferred.

[0069] 3.4 Examination of Flow Velocity

[0070] The effects of flow rates of 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, and 1.2 mL / min on the separation effect were investigated. The results showed that when the flow rate was 0.9 to 1.1 mL / min, all 18 common peaks could be separated well. The flow rate of 1.0 mL / min showed the best separation degree and peak shape, and the retention time was appropriate. Therefore, the preferred flow rate was 1.0 mL / min.

[0071] 3.5 Examination of the chromatographic column

[0072] Different brands of chromatographic columns were investigated, including Aglient ZORBAX SB-Aq C18 (4.6mm×250mm, 5μm), COSMOSIL Packed 5C18-PAQ (4.6mm×250mm, 5μm), COSMOSIL Packed 5C18-PAQ (4.6mm×150mm, 5μm), and Ultimate XB-C18 (4.6mm×250mm, 5μm). The results showed that Aglient ZORBAX SB-Aq C18 and COSMOSIL Packed 5C18-PAQ both performed well in separating the samples. Among them, the Aglient ZORBAX SB-Aq C18 column had the best peak resolution and peak shape. Therefore, the Aglient ZORBAX SB-Aq C18 (4.6mm×250mm, 5μm) column was selected as the preferred choice.

[0073] 3.6 Preparation of the test solution

[0074] Method 1: Accurately weigh approximately 3g of this product, add 500mL of water and soak overnight at 4℃. Homogenize using a high-speed blender, adjust the pH to 2.5 with dilute hydrochloric acid, add 5mg of pepsin, and react with shaking at 37℃ and 150r / min for 3 hours. Adjust the pH to 7.0-7.5 to stop the reaction. Then add 3mg of trypsin and hydrolyze at 37℃ and 150r / min for 3 hours. Boil the hydrolysate for 10 minutes to inactivate the enzyme. Centrifuge the hydrolysate, collect the supernatant, concentrate under reduced pressure, and transfer with 0.1% formic acid solution. 18. The solid-phase extraction column was desalted. The solid-phase extraction process was as follows: acetonitrile activation, equilibration with 0.1% trifluoroacetic acid aqueous solution, sample loading, rinsing with two column volumes of 0.1% trifluoroacetic acid aqueous solution, elution with three column volumes of 50% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid), collection of eluent, concentration under reduced pressure to dryness, redissolving the residue in 50% acetonitrile aqueous solution (containing 0.1% formic acid), centrifugation, and collection of the supernatant.

[0075] Method 2: Take approximately 3g of this product, accurately weigh it, add 500mL of water and soak overnight at 4℃. Homogenize the mixture using a high-speed blender, adjust the pH to 7-7.5 with sodium hydroxide, add trypsin for enzymatic hydrolysis (enzyme activity 4U / g), and hydrolyze at 37℃ and 150r / min for 3 hours with shaking. Boil the hydrolysate for 10 minutes to inactivate the enzyme. Centrifuge the hydrolysate, collect the supernatant, concentrate under reduced pressure, and transfer to a dialysis bag (MD31: 0.1-0.5kDa) with 0.1% formic acid solution to remove salts. Concentrate the dialysis solution under reduced pressure, redissolve in 0.1% formic acid solution, centrifuge, and collect the supernatant.

[0076] Method 3: Accurately weigh approximately 3g of this product, add 500mL of water and soak overnight at 4℃. Homogenize using a high-speed blender, adjust the pH to 7-7.5 with sodium hydroxide, add trypsin for enzymatic hydrolysis (enzyme activity 4U / g), and hydrolyze at 37℃ and 150r / min for 3 hours with shaking. Inactivate the enzyme by boiling for 10 minutes. Centrifuge the hydrolysate, collect the supernatant, concentrate under reduced pressure, transfer with 0.1% formic acid-water mixture, and desalt using a C18 solid-phase extraction column. The procedure is as follows: Activate with acetonitrile, equilibrate with 0.1% trifluoroacetic acid aqueous solution, load the sample, elute with two column volumes of 0.1% trifluoroacetic acid aqueous solution, elute with three column volumes of 30% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid), collect the eluent, concentrate to dryness under reduced pressure, redissolve the residue with 30% acetonitrile aqueous solution (containing 0.1% formic acid), centrifuge, and collect the supernatant.

[0077] Upon investigation, the sample solution prepared by Method 1 showed low signal response and was highly mixed, lacking major components and failing to fully reflect the characteristic information of the sample. Method 2, which involved dialysis desalination, resulted in high viscosity after the dialysate was concentrated under reduced pressure and then reconstituted with a small volume of 0.1% formic acid solution, making liquid chromatography detection impossible. Increasing the reconstitution volume resulted in an excessively low sample detection signal response. Method 3 effectively removed interference from highly polar components without causing loss of medium and low polar components, and maintained a stable baseline. Therefore, Method 3 was chosen to prepare the sample solution.

[0078] 4. Methodological Validation

[0079] 4.1 Precision Test

[0080] Yellow croaker swim bladder with batch number NO1:20210801-HY was used. A test solution was prepared according to the method for preparing the test solution. 10 μL of the test solution was accurately pipetted and injected repeatedly 6 times. Chromatograms were recorded and imported into the Traditional Chinese Medicine Chromatogram Fingerprint Similarity Evaluation System A to examine the similarity between chromatograms. The results are shown in Table 3. Figure 5 .

[0081] Table 3. Precision Similarity

[0082]

[0083]

[0084] The fingerprint spectrum similarity was greater than 0.98 for each of the six consecutive injections of the test solution, indicating that the method has good precision.

[0085] 4.2 Stability Test

[0086] The same test solution (batch number NO1:20210801-HY) was injected at 0h, 4h, 8h, 12h and 24h, and the chromatograms were recorded. The chromatograms were then imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System A to examine the similarity between the chromatograms. The results are shown in Table 4. Figure 6 .

[0087] Table 4. Stability Similarity

[0088] Time / h Similarity 0 0.966 4 0.977 8 0.971 12 0.962 24 0.969

[0089] After the test solution was left at room temperature for 24 hours, the fingerprint similarity was greater than 0.96, indicating that the test solution had good stability within 24 hours.

[0090] 4.3 Repeatability

[0091] Yellow croaker swim bladder sample with batch number NO1:20210801-HY was taken. A test solution was prepared according to the same method. Six test solutions were prepared in the same manner, and the results were determined according to the prescribed method. Chromatograms were recorded and imported into the Traditional Chinese Medicine Chromatogram Fingerprint Similarity Evaluation System A to examine the similarity between chromatograms. The results are shown in Table 5. Figure 7 .

[0092] Table 5. Repeatability Similarity

[0093] serial number Similarity 1 0.946 2 0.951 3 0.947 4 0.956 5 0.958 6 0.943

[0094] Six test solutions were prepared in parallel, and the fingerprint similarity was determined to be greater than 0.94 in all cases, indicating that the method has good repeatability.

[0095] The above results indicate that the yellow croaker swim bladder fingerprint detection method provided by this invention has good stability, high precision, and good repeatability. It can comprehensively and objectively evaluate the quality of yellow croaker swim bladders, which is of great significance for ensuring clinical efficacy.

[0096] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its spirit and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

[0097] Example 2:

[0098] The swim bladder fingerprint detection method for yellow croaker established according to this invention was used to detect the swim bladders of three types of fish: yellow croaker swim bladder, croaker swim bladder, and cod swim bladder, to verify the accuracy of the established yellow croaker swim bladder fingerprint detection method in identifying authenticity.

[0099] Step 1: Preparation of test solutions: Take approximately 3g of each of the following test samples: yellow croaker swim bladder (NO.9: 20211001-HY, NO.10: 20211002-HY), croaker swim bladder (NO.1: 20210801-MY, NO.2: 20210901-MY), and cod swim bladder (NO.1: 20210801-XY, NO.2: 20210901-XY). Accurately weigh the samples and prepare the test solutions of yellow croaker swim bladder, croaker swim bladder, and cod swim bladder according to the method for preparing test solutions in Example 1.

[0100] Step 2: Accurately pipette each test solution from Step 1 and inject it into the liquid chromatograph, recording the chromatogram over 67 minutes; refer to Example 1 for specific chromatographic conditions.

[0101] Step 3: Export the swim bladder fingerprints of yellow croaker, croaker, and cod obtained in Step 2 (see [link to Step 2]). Figure 8 ), and imported into the Chinese herbal chromatographic fingerprint similarity evaluation system A.

[0102] Table 6. Relative retention time and relative peak area after LOG extraction of the common peak of yellow croaker swim bladder in batches NO.9: 20211001-HY and NO.10: 20211002-HY

[0103]

[0104]

[0105] The fingerprint chromatograms of the swim bladders of two batches of yellow croaker (NO.9:20211001-HY and NO.10:20211002-HY) were determined to contain 18 common peaks. The fingerprint chromatograms of the test samples were consistent with those of the yellow croaker swim bladder control. Further, according to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatograms of the test samples and those of the yellow croaker swim bladder control was calculated using similarity calculation software, and the similarity scores were 0.948 and 0.925 (both greater than 0.9), respectively. Moreover, the 18 common peaks in the fingerprint chromatograms of the test samples, with the chromatographic peak corresponding to the peak of reference material No. 4 as the S peak, and the relative retention times of each common peak, as well as the relative peak areas of the common peaks and the S peaks after taking the LOG value, all met the requirements (see Table 6). The fingerprint spectra of croaker swim bladders from batches NO.1: 20210801-MY and NO.2: 20210901-MY contained 22 common peaks, and the fingerprint spectrum of the croaker swim bladder sample was inconsistent with the control fingerprint spectrum of yellow croaker swim bladder. The fingerprint spectra of cod swim bladders from batches NO.1: 20210801-XY and NO.2: 20210901-XY contained 31 common peaks, and the fingerprint spectrum of the cod swim bladder sample was inconsistent with the control fingerprint spectrum of yellow croaker swim bladder. Neither the croaker nor cod swim bladder samples met the fingerprint spectrum standards for yellow croaker swim bladder. Therefore, the yellow croaker swim bladder fingerprint detection method established in this application can accurately distinguish between yellow croaker swim bladder, croaker swim bladder, and cod swim bladder, proving that this method can be used for the identification of genuine and counterfeit yellow croaker swim bladder, providing a scientific basis for preventing adulteration.

Claims

1. A method for detecting the swim bladder of yellow croaker using fingerprinting, characterized in that, Includes the following steps: Step 1: Preparation of Yellow Croaker Swim Bladder Test Solution: Take different batches of yellow croaker swim bladder test samples, weigh a certain weight of each sample and place it in a container. Add water and soak at 4℃. The mass-to-volume ratio of test sample to water is 1:100 to 1:

200. Homogenize using a high-speed blender. Adjust the pH to 7 to 7.5 with sodium hydroxide. Add trypsin for enzymatic hydrolysis. The enzyme activity is 3 to 5 U / g. Hydrolyze at 37℃ and 100 to 200 r / min for 3 hours with shaking. Boil the hydrolysate for 10 minutes to inactivate the enzyme. Centrifuge the hydrolysate, collect the supernatant, concentrate under reduced pressure, and add 0.05 to 0.15% (v / v) methanesulfonic acid. Acid-water transfer and desalting treatment using a C18 solid-phase extraction column are performed as follows: acetonitrile activation, equilibration with a 0.05%–0.15% (v / v) trifluoroacetic acid aqueous solution, sample loading, elution with two column volumes of a 0.05%–0.15% (v / v) trifluoroacetic acid aqueous solution, elution with three column volumes of a 30% (v / v) acetonitrile aqueous solution containing 0.05%–0.15% (v / v) trifluoroacetic acid, collection of the eluent, concentration under reduced pressure to dryness, redissolving the residue in a 30% (v / v) acetonitrile solution containing 0.05%–0.15% (v / v) formic acid, centrifugation, and collection of the supernatant. Step 2, precisely pipette the sample solution in step 1 into the liquid chromatograph, and record the chromatogram; the liquid chromatography conditions are: octadecylsilane-bonded silica gel as the filler, column temperature 30-40℃, A: formic acid / acetonitrile-B: formic acid / water as the mobile phase, gradient elution, elution gradient is 0-5min, 0% A; 5-18min, 0%-5% A; 18-25min, 5%-6.5% A; 25-28min, 6.5%-9% A; 28-32min, 9%-12% A; 32-36min, 12% A; 36-42min, 12%-13.5% A; 42-45min, 13.5%-16% A; 45-55min, 16%-20% A; 55-65min, 20%-25% A; flow rate 0.9-1.1 mL·min -1 , injection volume is 1-20μL, Alltech 2000ES evaporative light scattering detector for detection, evaporative light scattering detector parameters: drift tube temperature 110-120℃, carrier gas flow rate 2.5-3.5mL·min -1 ; or Agilent 1260 evaporative light scattering detector, evaporative light scattering detector parameters: drift tube temperature: 40-45℃, carrier gas pressure: 3.0-3.5bar, Gain: 6-7; Step 3: Import the fingerprint chromatogram of the yellow croaker swim bladder obtained in Step 2 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; select the chromatographic peaks present in the chromatograms of different batches of yellow croaker swim bladder as common peaks; generate the reference fingerprint chromatogram of yellow croaker swim bladder using the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak; the results should meet the following standards: there are 18 common peaks in the fingerprint chromatogram of the test sample, the fingerprint chromatogram of the test sample is consistent with the reference fingerprint chromatogram, and according to the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram calculated by the similarity calculation software is not less than 0.9, and the test sample... The fingerprint chromatogram contains 18 common peaks. Using the chromatographic peak corresponding to reference peak 4 as the S peak, the relative retention times of each common peak should meet the following requirements: Peak 1: 0.561–0.563; Peak 2: 0.630–0.632; Peak 3: 0.772–0.775; S peak: 1.000; Peak 5: 1.579–1.586; Peak 6: 2.738–2.749; Peak 7: 4.076–4.095; Peak 8: 4.275–4.296; Peak 9: 4.464–4.489; Peak 10: 4.608–4.747; Peak 11: 5.238–5.

26. Peaks 0 and 12: 5.482–5.508; Peak 13: 8.326–8.366; Peak 14: 8.591–8.628; Peak 15: 8.780–8.822; Peak 16: 9.058–9.100; Peak 17: 9.274–9.319; Peak 18: 10.528–10.

580. The relative peak areas after taking the logarithmic values ​​for each common peak and the S-peak should conform to the following: Peak 1: 0.796–0.869; Peak 2: 0.846–0.950; Peak 3: 0.885–0.973; S-peak: 1.000; Peak 5: 0.831–0.

90. Peaks 4 and 6: 0.914–1.006; Peak 7: 0.991–1.073; Peak 8: 0.949–1.007; Peak 9: 0.960–1.044; Peak 10: 0.956–1.029; Peak 11: 0.988–1.073; Peak 12: 0.973–1.051; Peak 13: 1.059–1.135; Peak 14: 1.097–1.177; Peak 15: 1.042–1.141; Peak 16: 0.970–1.033; Peak 17: 1.051–1.124; Peak 18: 0.889–0.

968.

2. The method according to claim 1, characterized in that, It further includes the following step 4: using the same liquid chromatography conditions as step 2, to perform liquid phase preparation separation and purification on the typical common peaks in the swim bladder fingerprint of yellow croaker: peak 4 and peak 17, to obtain representative polypeptide components; Step 5: Based on the representative peptide components obtained in Step 4, analyze them using liquid chromatography-mass spectrometry (LC-MS) with triple quadrupole mass spectrometry (CMS) as the detector and electrospray ionization (ESI) as the source. + Multiple reaction monitoring (MRM) was performed using electrospray positive ion mode. The fragmentation characteristics and fragment ion information of the mass spectrometer were compared with the database to achieve sample characterization.

3. The method according to claim 2, characterized in that, The liquid chromatography conditions for liquid chromatography-mass spectrometry (LC-MS) in step 5 are as follows: octadecylsilane-bonded silica gel as the packing material; column temperature 35℃; mobile phase A: 80% acetonitrile containing 0.1% formic acid, mobile phase B: 0.1% formic acid in water; flow rate 2 μL / min. -1 The injection volume was 1 μL.

4. The method according to claim 2, characterized in that, The amino acid sequences of the representative polypeptide components obtained in step 4 are: GYTGLDGR and LLAESSVTEVTSFNK.

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