A pretreatment method and a detection method of indole substances in aquatic products

Abstract

The application provides a pretreatment method and a detection method of indole substances in aquatic products, and relates to the technical field of analysis and detection. The application extracts the aquatic products to be measured by using formic acid-acetonitrile aqueous solution as an extractant, can realize the simultaneous and efficient extraction of six indole substances, i.e. indole, 3-indole acetic acid, 3-methyl indole, indole-3-methyl aldehyde, indole-4-methyl aldehyde and indole-5-methyl aldehyde; the HLB solid phase extraction purification can significantly reduce the amount of impurities in the sample liquid to be measured, and improve the accuracy of the detection result of the subsequent high performance liquid chromatography tandem mass spectrometry. The application adopts the high performance liquid chromatography tandem mass spectrometry detection and the APCI ion source, and the detection result is high in accuracy and sensitivity. Moreover, the detection method provided by the application realizes the high sensitivity detection of the six indole substances, is comprehensive in detection indexes, and can provide the basis for the rapid evaluation of the quality of the aquatic products under the cold storage condition according to the change trend of the total content of the six indole substances.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a pretreatment method and a detection method for indole-like substances in aquatic products. Background Technology

[0002] Fresh produce, especially aquatic products, is prone to spoilage and difficult to preserve, leading to decreased product quality or high losses due to deterioration. The quality and preservation of fresh aquatic products are not only a key concern for consumers but also a food safety issue that directly impacts their health.

[0003] Aquatic products are rich in various fats, proteins, and trace elements, and have a high water content. After death, they provide a favorable environment for microbial growth. Due to the hydrolytic action of these putrefactive bacteria and various proteases, the myofibrils become fragile and break, the muscle loses its inherent elasticity and gradually softens, and the aquatic product enters the autolysis stage. This autolysis is particularly pronounced in shrimp and crabs, leading to the decomposition of proteins in some tissues into amino acids, providing favorable conditions for the reproduction of putrefactive microorganisms. Due to the action of putrefactive microorganisms, amino acids are decomposed into ammonia, aldehydes, and biogenic amines, which have unpleasant flavors and give the aquatic product a putrid odor. The freshness of aquatic products is evaluated based on changes in chemical substances produced during the putrefaction process. Therefore, the chemical substances formed during the storage of aquatic products are often used as chemical indicators for evaluating meat freshness. Developing methods for rapidly establishing aquatic product quality assessment based on their internal chemical substances is of great significance.

[0004] Currently, commonly used detection methods for indole compounds include liquid chromatography, gas chromatography, and liquid chromatography-tandem mass spectrometry. Pretreatment methods mainly include direct extraction, liquid-liquid extraction, and solid-phase extraction. For example, existing technologies include "Liu Zhihang, Li Pingliang, Zhou Fei, et al. Simultaneous detection of indole-3-acetic acid and its three oxidation products in plant leaves by ultra-high performance liquid chromatography-tandem mass spectrometry [J]. Journal of Analytical Testing, 2017, 36(6):6." and "Liu Yue, Jia Man, Cui Jing, et al. Simultaneous detection of three tryptophan metabolites in cell culture medium by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry [J]. China Food and Nutrition, 2017, 36(6):6." 19,25(2):4.“Hou Jianbo, Xie Wen, Zeng Ganning, et al. Determination of indole content in shrimp meat and shrimp products by high performance liquid chromatography[J]. Physical and Chemical Testing: Chemical Section, 2015(1):4.“Simultaneous determination of indole and 3-methylindole in plasma of pregnant patients with hepatitis B by high performance liquid chromatography[J]. Chromatography, 2017,35(7):6.“Kang Le, He Jiansheng, Guo Junjun, et al. Study on method for determination of indole and two metabolites in human feces by UPLC-MS / MS[J]. Popular Standardization, 2020(17):4.” and “Verplanken K, Wauters J, Vercruysse V, et al. Development and validation of a UHPLC-HR-Orbitrap-MS method for the simultaneous determination of androstenone, skatole and indole in porcine meat and meat products[J]. Food Chemistry, 2016, 190: 944-951. However, aquatic products contain high levels of protein, and the complex matrix of aquatic product samples is subject to significant interference from biological samples. This results in current sample pretreatment methods targeting relatively single analytes, failing to achieve simultaneous and efficient extraction of multiple indole compounds such as indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde, leading to inaccurate detection results. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a pretreatment method and a detection method for indole compounds in aquatic products. The pretreatment method provided by this invention can simultaneously and efficiently extract six indole compounds, namely indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde, greatly improving the accuracy and sensitivity of the detection results of indole compounds in aquatic products.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a pretreatment method for indole compounds in aquatic products, comprising the following steps:

[0008] The aquatic product to be tested was mixed with a formic acid-acetonitrile aqueous solution for extraction to obtain the extract;

[0009] The extract was loaded into an HLB solid-phase extraction column, rinsed with a first acetonitrile aqueous solution, the HLB solid-phase extraction column was dried, and then eluted with a formic acid-acetonitrile mixture. The eluent was collected and concentrated to obtain the sample solution to be tested.

[0010] Preferably, the volume fraction of formic acid in the formic acid-acetonitrile aqueous solution is 0.1-0.2%, and the volume fraction of acetonitrile is 75-85%.

[0011] The ratio of the aquatic product to be tested to the formic acid-acetonitrile aqueous solution is 1g:4-6mL.

[0012] Preferably, the mixed extraction includes sequential homogenization extraction and oscillating extraction; the homogenization extraction speed is 10000-12000 r / min, and the time is 1-2 min; the oscillating extraction speed is 180-240 r / min, and the time is 8-15 min.

[0013] Preferably, after the mixed extraction, the extraction system is further centrifuged to obtain the supernatant as the extract.

[0014] Preferably, the volume fraction of acetonitrile in the first acetonitrile aqueous solution is 8-12%;

[0015] The volume fraction of formic acid in the formic acid-acetonitrile mixture is 0.1% to 0.2%.

[0016] Preferably, the extract is diluted with water before loading the sample to obtain a diluted extract; the volume ratio of the extract to water is 1:4 to 5.

[0017] Preferably, the concentration process further includes adjusting the volume of the resulting concentrated solution with a second acetonitrile aqueous solution;

[0018] The volume of the concentrate is less than 35% of the volume of the extract;

[0019] The volume fraction of acetonitrile in the second acetonitrile aqueous solution is preferably 40-60%.

[0020] This invention also provides a method for detecting indole compounds in aquatic products, comprising the following steps:

[0021] The aquatic product to be tested, the isotope internal standard working solution, and the formic acid-acetonitrile aqueous solution were mixed and extracted to obtain the extract;

[0022] The extract was purified by HLB solid-phase extraction column to obtain the test sample solution. The HLB solid-phase extraction column purification included rinsing with a first acetonitrile aqueous solution, drying the HLB solid-phase extraction column, eluting with a formic acid-acetonitrile mixture, collecting the eluent and concentrating it to obtain the test sample solution.

[0023] The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to obtain the detection results of indole-like substances; the mass spectrometry ion source used in the HPLC-MS / MS detection was an APCI ion source; the indole-like substances included one or more of indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde.

[0024] Preferably, the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection conditions include: a C10 column. 18 The chromatographic column was used at a temperature of 30–40℃. Mobile phase A was 0.1–0.2% formic acid aqueous solution, and mobile phase B was acetonitrile. The mobile phase flow rate was 0.25–0.35 mL / min, and the injection volume was 1–2 μL. Gradient elution was used, and the gradient elution program was as follows:

[0025] From 0 to 3 minutes, the volume fraction of the mobile phase A is 70 to 80%.

[0026] Over 3–5 minutes, the volume fraction of the mobile phase A decreased uniformly from 70–80% to 5–10%.

[0027] The volume fraction of the mobile phase A is 5-10% for 5-8 minutes.

[0028] Over 8 to 8.0 minutes, the volume fraction of the mobile phase A increases uniformly from 5 to 10% to 70 to 80%.

[0029] 8.1–12 min, the volume fraction of the mobile phase A is 70–80%.

[0030] Preferably, the mass spectrometry detection conditions of the high performance liquid chromatography-tandem mass spectrometry include: ionization mode is APCI positive ion mode, scanning mode is multiple reaction monitoring, curtain gas pressure is 25 psi, ion spray voltage is 5500 V, nebulization temperature is 400 °C, and nebulizer gas pressure is 30 psi.

[0031] This invention utilizes a formic acid-acetonitrile aqueous solution as the extractant to extract the aquatic product being tested, achieving simultaneous and efficient extraction of six indole compounds: indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde. The extract is loaded into an HLB solid-phase extraction column, washed with a first acetonitrile aqueous solution, the column is dried, and then eluted with a formic acid-acetonitrile mixture. The eluent is collected and concentrated to obtain the sample solution. This invention utilizes HLB solid-phase extraction for purification, significantly reducing the amount of other impurities in the sample solution and improving the accuracy of subsequent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection results. Furthermore, the operation is simple.

[0032] The detection method provided by this invention utilizes a formic acid-acetonitrile aqueous solution as the extractant to extract the aquatic product to be tested and the isotope internal standard during the pretreatment process. This method can simultaneously and efficiently extract six indole compounds: indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde. Purification using HLB solid-phase extraction significantly reduces the amount of other impurities in the sample solution. The addition of the isotope internal standard during pretreatment and the use of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) with an APCI source in the mass spectrometer significantly reduces the organic matter effect and interference of biological samples. This effectively improves the accuracy, precision, and sensitivity of the HPLC-MS / MS detection results of indole compounds in aquatic products, and the method is fast and simple to operate. This invention investigated the changing trends of indole compounds in black carp and prawns, finding a linear correlation between the content of indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde in aquatic products and storage time. The freshness of aquatic products can be evaluated based on changes in indole content. Detecting the total content of these six indole compounds helps in rapidly evaluating the quality of aquatic products under refrigeration conditions and can serve as a potential biomarker for characterizing the freshness of commercially available chilled aquatic products, providing a technical reference for cold chain fresh food transportation and preservation. Furthermore, compared to existing methods for detecting indole compounds, this invention can simultaneously and accurately detect all six indole compounds with high sensitivity. The detection method provided by this invention offers more comprehensive characterization indicators, higher characterization efficiency, and more accurate results.

[0033] As shown in the test results of the examples, the six indole compounds could be effectively separated within 8 minutes, with correlation coefficients (r) all exceeding 0.99. The limits of detection (LOD) for indole, 3-indoleacetic acid, and 3-methylindole were 2 μg / kg and the limits of quantitation (LOQ) were 5 μg / kg. The LODs for indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde were 1 μg / kg and the LODs were 2 μg / kg. The average recoveries ranged from 63.18% to 102.58%, and the relative standard deviations (RSDs) ranged from 2.2% to 9.5%. This indicates that the detection method provided by this invention has high recovery, simple operation, and good sensitivity. It can accurately characterize and quantify low concentrations of the six indole compounds (indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde) in aquatic products, providing an effective technical means for further accurate detection of indole content in aquatic products. After multiple experiments, it was determined that when the total indole content in blackfish is ≤100μg / kg, it is considered fresh fish meat, and when the total indole content is >100μg / kg, it is considered relatively stale fish meat; when the total indole content in prawns is ≤250μg / kg, it is considered fresh prawn meat, and when the total indole content is >250μg / kg, it is considered relatively stale prawn meat. This provides a technical reference for the preservation of fresh food in cold chain transportation. Attached Figure Description

[0034] Figure 1 The total ion chromatogram of indole compounds is shown, where 1 is indole-3-carboxaldehyde, 2 is 3-indoleacetic acid, 3 is indole-4-carboxaldehyde, 4 is indole-5-carboxaldehyde, 5 is indole, and 6 is 3-methylindole.

[0035] Figure 2 HLB column chromatography and other methods were used to analyze indole compounds. Figure showing the recovery rate results after evolution of PRiME HLB column;

[0036] Figure 3 This is a matrix effect diagram of indole compounds;

[0037] Figure 4 The graph shows the change in the concentration of indole compounds in snakehead fish samples over time.

[0038] Figure 5 The graph shows the change in the concentration of indole compounds in shrimp samples over time.

[0039] Figure 6 Sensory comparison images of fish and shrimp meat after different storage times, where a) is a black carp sample and b) is a prawn sample. Detailed Implementation

[0040] This invention provides a pretreatment method for indole compounds in aquatic products, comprising the following steps:

[0041] The aquatic product to be tested was mixed with a formic acid-acetonitrile aqueous solution for extraction to obtain the extract;

[0042] The extract was loaded into an HLB solid-phase extraction column, rinsed with a first acetonitrile aqueous solution, the HLB solid-phase extraction column was dried, and then eluted with a formic acid-acetonitrile mixture. The eluent was collected and concentrated to obtain the sample solution to be tested.

[0043] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0044] This invention involves mixing the aquatic product to be tested with a formic acid-acetonitrile aqueous solution for extraction to obtain an extract.

[0045] In this invention, the aquatic product to be tested preferably includes shrimp and / or fish, with the shrimp preferably including prawns and the fish preferably including snakehead fish. In this invention, the shrimp are preferably prepared by removing the head, tail, and shell before use, and then minced using a meat grinder. In this invention, the fish are preferably prepared by removing the skin and bones before use, and then minced using a meat grinder. In this invention, the minced aquatic product to be tested is preferably frozen and stored at -22 to -18°C.

[0046] In this invention, the volume fraction of formic acid in the formic acid-acetonitrile aqueous solution is preferably 0.1-0.2%, more preferably 0.1-0.15%, and the volume fraction of acetonitrile is preferably 75-85%, more preferably 80%.

[0047] In this invention, the preferred material-to-liquid ratio of the aquatic product to be tested to the formic acid-acetonitrile aqueous solution is 1g:4-6mL, more preferably 1g:5mL.

[0048] In this invention, the mixed extraction preferably includes sequential homogenization extraction and oscillating extraction. In this invention, the rotation speed of the homogenization extraction is preferably 10000–12000 r / min, more preferably 11000–12000 r / min; the homogenization extraction time is preferably 1–2 min, more preferably 1–1.5 min. In this invention, the oscillating extraction speed is preferably 180–240 r / min, more preferably 200–220 r / min; the oscillating extraction time is preferably 8–15 min, more preferably 9–10 min, and the oscillating extraction is preferably performed on a shaker.

[0049] The present invention preferably further includes centrifugation of the obtained extraction system after the mixed extraction, and the resulting supernatant is the extract. In the present invention, the centrifugation speed is preferably 8000-10000 r / min, more preferably 8000-9000 r / min; the centrifugation time is preferably 5-8 min, more preferably 6-7 min.

[0050] After obtaining the extract, the present invention loads the extract into an HLB solid-phase extraction column, washes it with a first acetonitrile aqueous solution, dries the HLB solid-phase extraction column, and then elutes it with a formic acid-acetonitrile mixture. The eluent is collected and concentrated to obtain the sample solution to be tested.

[0051] In this invention, the extract is preferably diluted with water before loading the sample to obtain a diluted extract. In this invention, the volume ratio of the extract to water is preferably 1:4 to 5, more preferably 1:4 to 4.5, and even more preferably 1:4.

[0052] In this invention, the HLB solid-phase extraction column is preferably activated before use. The activation is preferably organic solvent activation and rinsing with an organic solvent aqueous solution. The organic solvent preferably includes methanol and / or acetonitrile. The volume fraction of the organic solvent in the organic solvent aqueous solution is preferably 8-12%, more preferably 9-11%, and even more preferably 10%. The organic solvent in the organic solvent aqueous solution preferably includes acetonitrile.

[0053] In this invention, the flow rate of the diluted extract in the HLB solid-phase extraction column is preferably 1-2 drops / second. After the diluted extract has completely passed through, the container holding the diluted extract is rinsed with a first acetonitrile aqueous solution, and then the first acetonitrile aqueous solution used to rinse the container holding the diluted extract is used to rinse the HLB solid-phase extraction column. In this invention, the volume fraction of acetonitrile in the first acetonitrile aqueous solution is preferably 8-12%, more preferably 9-10%. In this invention, the amount of the first acetonitrile aqueous solution used is preferably 1-2 mL, more preferably 1-1.5 mL.

[0054] In this invention, the drying process is preferably carried out using nitrogen.

[0055] In this invention, the volume fraction of formic acid in the formic acid-acetonitrile mixture is preferably 0.1-0.2%, more preferably 0.1-0.15%. In this invention, the volume ratio of the extract to the formic acid-acetonitrile mixture is preferably 1:2-3, more preferably 1:2.5.

[0056] In this invention, the concentration is preferably nitrogen blowing concentration, and the concentration temperature is preferably 35-45°C, more preferably 40°C. This invention does not have a special limitation on the concentration time, and the concentration is sufficient when the volume of the resulting concentrate accounts for less than 35% of the volume of the extract.

[0057] After concentration, the present invention preferably further includes adjusting the volume of the obtained concentrate with a second acetonitrile aqueous solution to obtain the sample solution to be tested. In the present invention, the volume fraction of acetonitrile in the second acetonitrile aqueous solution is preferably 40-60%, more preferably 45-50%. In the present invention, the volume ratio of the sample solution to the extract after adjustment is preferably 0.5-1:1, more preferably 0.5-0.8:1.

[0058] After the volume is adjusted, the present invention preferably further includes subjecting the adjusted solution to ultrasonication, vortexing, and filtration using a 0.22 μm organic microporous membrane in sequence. In the present invention, the ultrasonication time is preferably 30–60 s, more preferably 30 s; the vortexing speed is preferably 360–480 r / min, more preferably 400–450 r / min, and the vortexing time is preferably 1–2 min.

[0059] This invention also provides a method for detecting indole compounds in aquatic products, comprising the following steps:

[0060] The aquatic product to be tested, the isotope internal standard working solution, and the formic acid-acetonitrile aqueous solution were mixed and extracted to obtain the extract;

[0061] The extract was purified by HLB solid-phase extraction column to obtain the test sample solution. The HLB solid-phase extraction column purification included rinsing with a first acetonitrile aqueous solution, drying the HLB solid-phase extraction column, eluting with a formic acid-acetonitrile mixture, collecting the eluent and concentrating it to obtain the test sample solution.

[0062] The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to obtain the detection results of indole-like substances; the mass spectrometry ion source used in the HPLC-MS / MS detection was an APCI ion source; the indole-like substances included one or more of indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde.

[0063] In this invention, the aquatic product to be tested, an isotope internal standard working solution, and a formic acid-acetonitrile aqueous solution are mixed and extracted to obtain an extract; the extract is purified by an HLB solid-phase extraction column to obtain a sample solution to be tested; the HLB solid-phase extraction column purification includes rinsing with a first acetonitrile aqueous solution, drying the HLB solid-phase extraction column, eluting with a formic acid-acetonitrile mixture, collecting the eluent and concentrating it to obtain the sample solution to be tested.

[0064] In this invention, the pretreatment method for the aquatic product to be tested differs from the aforementioned pretreatment methods only in the addition of an isotope internal standard working solution. Other pretreatment methods are the same as those described above and will not be repeated here. In this invention, the isotope internal standard in the isotope internal standard working solution is preferably indole-3-carboxaldehyde-13 C. The concentration of the isotope internal standard working solution is preferably 1.6–2.4 μg / mL, more preferably 1.8–2.2 μg / mL, and even more preferably 2 μg / mL. In this invention, the mass ratio of the aquatic product to be tested to the isotope internal standard is preferably 1 g: 0.2–0.3 μg, more preferably 1 g: 0.25 μg.

[0065] In this invention, the preferred high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection conditions include: a C10 column. 18 Chromatographic column, preferably Agilent EclipsePlus C 18 RRHD column or Waters CORTECS UPLCC 18 Chromatographic column; column temperature 30–40℃, more preferably 35℃; mobile phase A is 0.1–0.2% formic acid aqueous solution, more preferably 0.1–0.15% formic acid aqueous solution; mobile phase B is acetonitrile; mobile phase flow rate is 0.25–0.35 mL / min, more preferably 0.3 mL / min; injection volume is 1–2 μL, more preferably 1.5–2 μL; elution method is gradient elution, the gradient elution program is as follows: 0–3 min, the volume fraction of mobile phase A is 70–80%; 3–5 min, the volume fraction of mobile phase A decreases uniformly from 70–80% to 5–10%; 5–8 min, the volume fraction of mobile phase A… The volume fraction of mobile phase A is 5-10%; for 8-8.0 min, the volume fraction of mobile phase A increases uniformly from 5-10% to 70-80%; for 8.1-12 min, the volume fraction of mobile phase A is 70-80%; the elution program is more preferably as follows: for 0-3 min, the volume fraction of mobile phase A is 75%; for 3-5 min, the volume fraction of mobile phase A decreases uniformly from 75% to 5%; for 5-8 min, the volume fraction of mobile phase A is 5%; for 8-8.0 min, the volume fraction of mobile phase A increases uniformly from 5% to 75%; for 8.1-12 min, the volume fraction of mobile phase A is 75%.

[0066] In this invention, the preferred mass spectrometry detection conditions for the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) include: ionization mode of APCI positive ion mode, scanning mode of multiple reaction monitoring (MRM), curtain gas pressure of 25 psi, ion spray voltage of 5500 V, nebulization temperature of 400 °C, and nebulizer gas pressure of 30 psi; the curtain gas is preferably nitrogen, and the nebulizer gas is preferably nitrogen. In this invention, the preferred parent ion, daughter ion, collision voltage (CE), and declustering voltage (DP) of the indole compounds and isotopic internal standards are shown in Table 1.

[0067] Table 1. Parent ion, daughter ion, collision voltage (CE), and declustering voltage (DP) of indole compounds and isotopic internal standards.

[0068]

[0069] In this invention, the detection of the sample liquid by high performance liquid chromatography-tandem mass spectrometry preferably includes qualitative detection and quantitative detection.

[0070] In this invention, the qualitative detection step preferably includes: measuring the test sample solution and the standard curve solution according to the conditions of high performance liquid chromatography and mass spectrometry, recording the chromatographic retention time of indole substances in the test sample solution and the standard curve solution; when a chromatographic peak with the same retention time as the indole standard in a certain standard curve solution is detected in the test sample solution (with a variation range within ±2.5%), and the deviation between the relative abundance ratio of the selected monitoring ion pair in the chromatogram of the test sample solution and the relative abundance ratio (k) of the ion in the equivalent concentration standard solution does not exceed the range specified in Table 2, it can be determined that the corresponding compound is detected in the sample.

[0071] Table 2 shows the maximum permissible deviation of relative ion abundance during qualitative analysis.

[0072] Table 2. Maximum permissible deviation of relative ion abundance during qualitative analysis.

[0073] Relative ion abundance / % ＞50 20-50 10-20 ≤10 Permissible relative deviation / % ±20 ±25 ±30 ±50

[0074] In this invention, the standard curve solution is a mixed solution of indole standard, 3-indoleacetic acid standard, 3-methylindole standard, indole-3-carboxaldehyde standard, indole-4-carboxaldehyde standard, indole-5-carboxaldehyde standard, and isotope internal standard. The specific specifications of each indole standard are preferably detailed in Table 3, and the purity of each indole standard is preferably ≥98%. In this invention, the concentration of the isotopic internal standard in the standard curve solution is preferably 80–120 μg / L, more preferably 100 μg / L; the concentrations of other indole standards are, in order, 8–12 μg / L, 16–24 μg / L, 40–60 μg / L, 80–120 μg / L, and 160–240 μg / L, more preferably 9–11 μg / L, 18–22 μg / L, 45–55 μg / L, 90–110 μg / L, and 180–220 μg / L, and even more preferably 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, and 200 μg / L. In this invention, the solvent in the standard curve solution preferably includes methanol, acetonitrile, and water.

[0075] Table 3 Specific Specifications of Indole-Based Substance Standards

[0076]

[0077]

[0078] In this invention, the method for preparing the standard curve solution preferably includes the following steps:

[0079] Prepare standard stock solutions of various indole compounds with concentrations of 800–1200 μg / mL;

[0080] Prepare a mixed standard solution of indole compounds with a concentration of 1.6–2.4 μg / mL;

[0081] Prepare a mixed standard working solution of indole substances at a concentration of 160–240 μg / L;

[0082] Prepare a 1.6–2.4 μg / mL isotope internal standard working solution;

[0083] Prepare a standard curve solution.

[0084] In this invention, the preferred method for preparing the standard stock solutions of each indole substance with a concentration of 800–1200 μg / mL includes the following steps: accurately weigh 8.00–12.00 mg (accurate to 0.01 mg) of indole standard, dissolve it in methanol by ultrasonication and bring the volume to 10 mL, shake well, and obtain an indole standard stock solution with a concentration of 800–1200 μg / mL. Store at 4°C for 3 months. Prepare 3-indoleacetic acid standard stock solutions, 3-methylindole standard stock solutions, indole-3-carboxaldehyde standard stock solutions, indole-4-carboxaldehyde standard stock solutions, and indole-5-carboxaldehyde standard stock solutions according to the same method. In this invention, the concentration of indole substances in each standard stock solution is more preferably 900–1100 μg / mL, and even more preferably 1000 μg / mL.

[0085] In this invention, the preferred method for preparing the 1.6–2.4 μg / mL indole-based mixed standard solution includes the following steps: accurately transferring 0.020 mL each of indole standard stock solution, 3-indoleacetic acid standard stock solution, 3-methylindole standard stock solution, indole-3-carboxaldehyde standard stock solution, indole-4-carboxaldehyde standard stock solution, and indole-5-carboxaldehyde standard stock solution into a 10 mL volumetric flask, and diluting to volume with methanol to obtain the 1.6–2.4 μg / mL indole-based mixed standard solution. In this invention, the concentration of each indole substance in the 1.6–2.4 μg / mL indole-based mixed standard solution is more preferably 1.8–2.2 μg / mL, and even more preferably 2.0 μg / mL.

[0086] In this invention, the preparation method of the 160-240 μg / L indole-based mixed standard working solution preferably includes the following steps: accurately transferring 1.0 mL of the indole-based mixed standard solution into a 10 mL volumetric flask, dissolving and diluting with methanol to obtain a 160-240 μg / L indole-based mixed standard working solution. In this invention, the concentration of each indole substance in the indole-based mixed standard working solution is more preferably 180-220 μg / L, and even more preferably 200 μg / L.

[0087] In this invention, the preferred method for preparing the 1.6–2.4 μg / mL isotope internal standard working solution includes the following steps: accurately weighing 4.00–6.00 mg of the isotope internal standard, dissolving it ultrasonically with methanol, and diluting it to 5 mL with methanol to obtain an 800–1200 μg / mL isotope internal standard stock solution; accurately transferring 0.02 mL of the isotope internal standard stock solution and diluting it to 10 mL with methanol to obtain a 1.6–2.4 μg / mL isotope internal standard working solution. In this invention, the concentration of the isotope internal standard in the isotope internal standard stock solution is more preferably 900–1100 μg / mL, and even more preferably 1000 μg / mL. In this invention, the concentration of the isotope internal standard in the isotope internal standard working solution is more preferably 1.8–2.2 μg / mL, and even more preferably 2 μg / mL.

[0088] In this invention, the preparation of the standard curve solution preferably includes the following steps: mixing a 160–240 μg / L indole-based mixed standard working solution or a 1.6–2.4 μg / mL indole-based mixed standard solution and a 1.6–2.4 μg / mL isotope internal standard solution, and then adjusting the volume using a formic acid-acetonitrile-aqueous solution to obtain the standard curve solution. In this invention, the volume fraction of formic acid in the formic acid-acetonitrile-aqueous solution is preferably 0.1–0.2%, more preferably 0.1–0.15%, and the volume fraction of acetonitrile is preferably 40–60%, more preferably 50%. The specific preparation of the standard curve solution in this invention is shown in Table 4.

[0089] Table 4 Standard Curve Preparation Table

[0090]

[0091] In this invention, the quantitative detection preferably includes: quantification of indole-3-carboxaldehyde using an internal standard method, and quantification of indole, 3-indoleacetic acid, 3-methylindole, indole-4-carboxaldehyde, and indole-5-carboxaldehyde using an external standard method. This invention does not impose any particular limitation on the internal standard method and the external standard method; any method well-known to those skilled in the art can be used. In this invention, the preferred linear relationships of the indole-like substances are shown in Table 5:

[0092] Table 5. Linear relationships of indole compounds

[0093] compound Linear equations r Indole y = 524.71x + 2393.00 0.9931 3-Indoleacetic acid y = 2224.11x - 1709.49 0.9968 3-Methylindole y＝656.74+492.16 0.9968 Indole-3-carboxaldehyde y = 0.021x + 0.0565 0.9985 Indole-4-carboxaldehyde y = 25966.57x + 19201.38 0.9985 Indole-5-carboxaldehyde <![CDATA[y=11894.01x+1.42×10 5 ]]> 0.9951

[0094] To further illustrate the present invention, the pretreatment method and detection method for indole substances in aquatic products are described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0095] The experimental materials, reagents, and instruments are as follows:

[0096] Main chemicals: Acetonitrile, chromatographic grade, Shanghai Husheng; Formic acid, chromatographic grade, Shanghai Husheng; Methanol, chromatographic grade, Shanghai Husheng; Milli-Q Advantage A10 ultrapure water, chromatographic grade, in-house. All percentages for acetonitrile and formic acid are volume fractions.

[0097] The main standard products are shown in Table 3.

[0098] Instruments and equipment: LC-MS / MS, AB SCIEX Triple Quad 5500; HPLC, Shimadzu LC-20A; balance, XPE205; benchtop centrifuge, Multifuge X1; reciprocating shaking incubator, IKAHS260; ultrasonic cleaner, KH-500DV; vortex mixer, QT-1.

[0099] Example 1

[0100] 1. Preparation of standard solutions

[0101] Accurately weigh 10.00 mg each of indole, 3-methylindole, 3-indoleacetic acid, indole-4-carboxaldehyde, indole-3-carboxaldehyde, and indole-5-carboxaldehyde into separate 10 mL volumetric flasks. Dissolve the indole in methanol by sonication, then dilute to the mark and shake well to obtain a standard stock solution of 1000 μg / mL for each indole substance. After accurate labeling, store in a refrigerator at 4°C.

[0102] Accurately pipette 0.020 mL each of the standard stock solutions of indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a mixed standard solution of indole compounds with a concentration of 2.0 μg / mL. Then, pipette 1.0 mL of the 2.0 μg / mL mixed standard solution into a 10 mL volumetric flask and dilute to the mark with methanol to obtain a mixed standard solution of indole compounds with a concentration of 200 μg / L.

[0103] 2. Preparation of Isotope Internal Standard Working Solution

[0104] Accurately weigh indole-3-carboxaldehyde 13Dissolve 5.00 mg of C in a 5 mL volumetric flask by sonication, bring the volume up to the mark with methanol, shake well to obtain a 1000 μg / mL isotope internal standard stock solution, transfer it into a sample vial, accurately label it, and store it at 4°C.

[0105] Accurately pipette 0.02 mL of the isotope internal standard stock solution from the sample vial into a 10 mL volumetric flask, dissolve and dilute with methanol to obtain a 2 μg / mL isotope internal standard working solution.

[0106] 3. Preparation of standard curve solutions and standard curve

[0107] Accurately transfer 200 μg / L and 2 μg / mL of mixed indole standard solutions, respectively, and prepare a series of standard concentrations of 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, and 200 μg / L with 0.1% formic acid-50% acetonitrile aqueous solution. The concentration of the isotope internal standard is 100 μg / L. The preparation method of the standard curve solution is shown in Table 6.

[0108] Table 6. Preparation of Standard Curve Solutions

[0109]

[0110] 4. High Performance Liquid Chromatography Conditions

[0111] Chromatographic column: Agilent EclipsePlus C18 RRHD (2.1×100mm, 1.8μm); injection volume: 2.0μL; column temperature: 35℃; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; mobile phase flow rate: 0.3mL / min; gradient elution program is shown in Table 7.

[0112] Table 7 Gradient elution program

[0113]

[0114]

[0115] 5. Mass spectrometry conditions

[0116] Ionization mode: APCI positive ion mode; Scanning method: multiple reaction monitoring (MRM); Curtain gas (nitrogen): 25 psi; Ion spray voltage: 5500 V; Nebulization temperature: 400 °C; Nebulizing gas: 30 psi; The parent ion, daughter ion, collision voltage (CE), and declustering voltage (DP) of indole and isotopic internal standards are shown in Table 1.

[0117] Under the above LC-MS / MS detection conditions, the total ion chromatograms of the six indole compounds are as follows: Figure 1 As shown, by Figure 1It can be seen that after adjustment, the retention times of the six indole compounds are stable and the peak shapes are good. The elution order is indole-3-carboxaldehyde (1), 3-indoleacetic acid (2), indole-4-carboxaldehyde (3), indole-5-carboxaldehyde (4), indole (5) and 3-methylindole (6).

[0118] 6. Linearity, Limit of Detection, and Limit of Quantification

[0119] The standard curve solution was prepared and detected by LC-MS / MS. Linear regression was performed with the mass concentration of the six indole compounds as the x-axis and the peak area of ​​the quantitative ion pairs as the y-axis. The indole compound content corresponding to a signal-to-noise ratio of 3 (S / N=3) was used as the method limit of detection, and the indole compound content corresponding to a signal-to-noise ratio of 10 (S / N=10) was used as the method limit of quantitation. The linear relationship, limit of detection, and limit of quantitation results are shown in Table 8.

[0120] Table 8. Linearity, Limit of Detection, and Limit of Quantification of Indole Substances

[0121] compound Linear equations r Limit of detection (μg / kg) Limit of quantitation (μg / kg) Indole y = 524.71x + 2393.00 0.9931 2 5 3-Indoleacetic acid y = 2224.11x - 1709.49 0.9968 2 5 3-Methylindole y＝656.74+492.16 0.9968 2 5 Indole-3-carboxaldehyde y = 0.021x + 0.0565 0.9985 1 2 Indole-4-carboxaldehyde y = 25966.57x + 19201.38 0.9985 1 2 Indole-5-carboxaldehyde <![CDATA[y=11894.01x+1.42×10 5 ]]> 0.9951 1 2

[0122] Table 8 shows that the linear correlation coefficients (r) for the six indole compounds in the concentration range of 10–200 μg / L were all greater than 0.99, indicating good linearity. The limits of detection (LODs) for indole, 3-indoleacetic acid, and 3-methylindole were 2 μg / kg, and the limits of quantitation (LOQs) were 5 μg / kg. The LODs for indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde were 1 μg / kg, and the LODs were 2 μg / kg.

[0123] Example 2

[0124] 1. Sample pretreatment

[0125] Sample preparation: Purchase commercially available live prawns and snakehead fish. Remove the heads, tails, and shells from the prawns, and remove the skin and bones from the snakehead fish. Use a meat grinder to make meat paste, and freeze at -18℃ for storage.

[0126] Extraction: Accurately weigh 2.0 g of the meat paste sample into a 50 mL centrifuge tube, precisely add 0.25 mL of 2 μg / mL isotope internal standard working solution, then add 9.5 mL of 0.1% formic acid-80% acetonitrile-water solution, homogenize at 12000 r / min for 1 min, shake well on a shaker for 10 min, and then centrifuge at 8000 r / min for 5 min. The supernatant is the extraction solution.

[0127] HLB solid-phase extraction column purification: Accurately transfer 2.0 mL of extract into a 20 mL centrifuge tube, add 8 mL of water to dilute and mix well to obtain diluted extract. Activate the HLB solid-phase extraction column with methanol, rinse with 10% acetonitrile aqueous solution, and pass the diluted extract through the HLB column at a flow rate of 1 drop / second. After complete passage, rinse the centrifuge tube residue with 2 mL of 10% acetonitrile aqueous solution and pass it through the HLB solid-phase extraction column. After complete passage, dry the solid-phase extraction column, and dilute the remaining liquid with 5 mL of the obtained concentrated solution to 1.0 mL with 50% acetonitrile aqueous solution. Sonicate for 30 s, vortex at 420 r / min for 1 min, filter using a 0.22 μm organic microporous membrane, and collect the middle filtrate as the sample solution for LC-MS / MS analysis.

[0128] 2. Spike recovery test

[0129] Spiked recovery tests were conducted on fish and shrimp samples. Mixed standard solutions at three concentrations—equivalent to 20 μg / kg (low concentration), 100 μg / kg (medium concentration), and 250 μg / kg (high concentration)—were added, with three replicates for each concentration. The tests were performed according to the sample pretreatment method described above and the detection conditions of Example 1. The recovery rate was calculated after subtracting the sample background value from the measured results. The specific average recoveries and relative standard deviations (RSDs) of the six indole compounds in fish at the three concentrations are shown in Table 9, and those in shrimp at the three concentrations are shown in Table 10.

[0130] Table 9. Recovery rate test of fish meat samples (n=3)

[0131]

[0132]

[0133] Table 10. Spike recovery rate test of shrimp meat samples (n=3)

[0134]

[0135]

[0136] Table 9 shows that the average recoveries of the six indole compounds in fish meat ranged from 63.18% to 102.58%, with relative standard deviations (RSDs) ranging from 1.6% to 10.2%. Table 10 shows that the average recoveries of the six indole compounds in shrimp meat ranged from 70.52% to 96.57%, with relative standard deviations (RSDs) ranging from 2.1% to 9.3%. This indicates that the recovery rates of the pretreatment and detection methods provided by this invention meet the needs of analytical detection and can be used for the accurate detection of indole compound content in aquatic products.

[0137] 3. Precision test

[0138] A mixed standard solution of 100 μg / kg was added to the fish meat sample, and each level was performed in six replicates to test the precision of the method. The specific average recoveries and relative standard deviations (RSDs) of the six indole compounds in the fish meat are shown in Table 11.

[0139] Table 11 Precision Test (n = 6, 100 μg / kg)

[0140] compound Average recovery rate (%) RSD (%) Indole 71.78 9.5 3-Indoleacetic acid 68.77 2.4 3-Methylindole 69.98 2.2 Indole-3-carboxaldehyde 94.90 5.3 Indole-4-carboxaldehyde 70.75 2.6 Indole-5-carboxaldehyde 89.49 3.7

[0141] As shown in Table 11, the relative standard deviation of the recovery rates of the six indole compounds at this concentration is between 2.2% and 9.5%, indicating that the method provided by this invention has good precision and accuracy, and meets the needs of analytical detection.

[0142] Example 3

[0143] 1. Selection of mass spectrometry detection conditions

[0144] The experiment used pure standard solutions of six indole compounds (indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde) at 1 μg / mL for mass spectrometry analysis under ESI source positive ion mode. During the first-stage mass spectrometry analysis (Q1 scan), it was found that the parent ions of five indole compounds (3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde) could be detected, but their daughter ion responses were relatively low. Indole also showed a relatively low parent ion response under ESI conditions.

[0145] Therefore, the source was changed to APCI, and mass spectrometry analysis was performed in positive ion mode. A pure standard solution with a concentration of 1 μg / mL was used to optimize the mass spectrometry conditions. After parameter optimization, first-stage mass spectrometry analysis (Q1 scan) was performed to determine the quasi-molecular ion peak [M+H]. + The molecular ion was used as the parent ion, and then subjected to secondary mass spectrometry analysis (Q3 scan) to select daughter ions with strong abundance and low interference as qualitative ion pairs. Then, the collision voltage and declustering voltage mass spectrometry parameters were optimized in multiple reaction monitoring (MRM) mode. The final collision voltage (CE) and declustering voltage (DP) values ​​of the parent ion and daughter ion corresponding to the standard are shown in Table 1.

[0146] 2. Optimization of chromatographic conditions

[0147] Different chromatographic columns exhibit varying separation performance for indole compounds. This study investigated the performance of Agilent Eclipse Plus C... 18RRHD (2.1×100mm, 1.8μm), Waters CORTECS UPLCC 18 (2.1×100mm, 1.6μm), Waters BEHC 18 The separation performance of the chromatographic column (100 mm × 2.1 mm, 1.7 μm) and the Waters Atlantis@T3 column (100 mm × 2.1 mm, 5.0 pm) for indole compounds was as described in Example 1. Other LC-MS / MS detection conditions were the same as in Example 1. The results showed that the Waters BEH C... 18 Multiple components cannot be separated on the chromatographic column, affecting quantitative sensitivity; the peak shape of the Waters Atlantis@T3 column is sluggish; Waters CORTECS UPLCC 18 The chromatographic column provides good separation, but the peak shape has some tailing. (Agilent Eclipse Plus C...) 18 RRHD achieved good separation of the six components in MRM mode, with good peak shapes and minimal interference between target analytes. Agilent EclipsePlus C was selected. 18 RRHD was used as a separation chromatographic column.

[0148] The separation efficiency and sensitivity of indole compounds were investigated using multiple combinations of methanol, acetonitrile, formic acid solution (0.1% formic acid concentration), and ammonium formate solution (0.1% formic acid concentration). Other LC-MS / MS detection conditions were the same as in Example 1. The results showed that adding formic acid to the aqueous phase improved peak shape and increased the ionization efficiency of the APCI source, further improving the chromatographic peak shape. Investigations revealed that the concentrations of 0.1%–0.2% formic acid all enhanced the response of the target analytes. In the experiment, 0.1% formic acid and acetonitrile were used as the mobile phase system.

[0149] 3. Selection of Extractant

[0150] Because aquatic products contain a complex matrix rich in proteins and fats, these substances can interfere with the analysis of target analytes during experiments. Therefore, to improve the recovery rate of target analytes, the effectiveness of the extractant in denaturing and precipitating proteins should be considered when selecting the extraction solvent. In existing experiments, acidic solvents such as hydrochloric acid, perchloric acid, and trichloroacetic acid are often used as deproteinizing agents for the extraction of target analytes from aquatic products. However, using only these acidic solvents for deproteinization is not ideal, leaving many interfering impurities in the extract. Therefore, adding a solid-phase extraction step after acidic liquid deproteinization treatment can achieve better impurity removal. Furthermore, if the strong acid solution is not completely purified during the operation, it may enter the mass spectrometer after processing large batches of samples, potentially corroding the instrument and causing serious consequences.

[0151] To address the aforementioned issues, some studies have employed methanol, acetonitrile, and ethyl acetate as extraction solvents, employing the method described in Example 2. The extraction efficiency of these three solvents on indole from shrimp meat and dried shrimp was investigated. Experimental results showed that acetonitrile exhibited the highest extraction efficiency for indole compounds among the three solvents, with a recovery rate exceeding 85%; while ethyl acetate showed the lowest extraction efficiency, ranging from 60% to 83%. Therefore, to more effectively precipitate proteins from aquatic products, reduce impurities in the extract, and improve the method's recovery rate, acetonitrile was chosen as the extraction solvent.

[0152] Although acetonitrile, as a non-ionized solvent, has a certain extraction effect on indoles, it has a certain impact on the extraction effect of indoles with good water solubility. The experiment investigated the changes in the extraction efficiency of indoles using different extractants: 70% acetonitrile aqueous solution, 80% acetonitrile aqueous solution, 90% acetonitrile aqueous solution, formic acid-70% acetonitrile aqueous solution (containing 0.1%, 0.2%, 0.3%, and 0.5% formic acid, respectively), formic acid-80% acetonitrile aqueous solution (containing 0.1%, 0.2%, 0.3%, and 0.5% formic acid, respectively), and formic acid-90% acetonitrile aqueous solution (containing 0.1%, 0.2%, 0.3%, and 0.5% formic acid, respectively). Experiments showed that adding a certain proportion of water and formic acid to pure acetonitrile could improve the extraction effect, and the solution containing 80-90% acetonitrile and 0.1-0.2% formic acid had better effects. Finally, an aqueous solution containing 0.1% formic acid and 80% acetonitrile was selected as the extractant for the experiment. The results showed that the recovery rate of six indole compounds in aquatic products could reach more than 60% even at low concentrations (20 μg / kg).

[0153] 4. Selection of solid phase extraction column

[0154] Enrichment and purification of the target analytes (six indole compounds) to remove impurities from the sample will help reduce the influence of the matrix on the detection results, thereby improving sensitivity. Solid-phase extraction is commonly used for enrichment and purification in this study. Considering the polar chemical property of indole compounds, the C1O2O3 method was investigated. 18 Using column and HLB solid-phase extraction column for the purification of indole compounds, experiments showed that using C... 18 Solid-phase extraction columns retain some components relatively poorly; however, indole is retained relatively strongly in HLB solid-phase extraction columns, and indole can be eluted using pure acetonitrile with good purification effect.

[0155] Experimental investigation HLB and During the PRiME HLB analysis of substance retention, it was found that during the concentration process, nitrogen blowing concentrated the effluent to near dryness, causing some indole target substances to degrade or remain undissolved on the test tube wall, resulting in a low recovery rate. Therefore, in the experiment, the nitrogen blowing concentration was adjusted from completely dry to near dryness, and redissolution could be performed when the volume was below 0.7 mL.

[0156] After adjustment, the extraction effects of these two types of columns on six indole compounds in aquatic products are as follows: Figure 2 As shown, by Figure 2 It can be seen that the use of HLB columns and During the PRiME HLB column purification process, the recovery rates of 3-indoleacetic acid, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde were all relatively good, but during use... Even after purification with the PRiME HLB column, the recoveries of indole and 3-methylindole remained very low, failing to meet analytical detection requirements, necessitating further optimization of the conditions. The next experimental step will continue to optimize this method. Therefore, HLB was ultimately chosen as the solid-phase extraction column.

[0157] 5. Investigation of matrix effect

[0158] When analyzing drugs in biological samples using liquid chromatography-mass spectrometry (LC-MS), common extracts in the samples can enhance or inhibit the ionization efficiency of the target compound. Complete elimination during purification is difficult, and matrix effects vary between different samples using the same matrix. However, the magnitude of the matrix effect depends on the ion source used in the method. In this experiment, fish and shrimp samples were processed according to the method in Example 2 to obtain a sample matrix solution. Then, 0.75 mL of the sample matrix solution was accurately pipetted and 0.25 mL of a 200 μg / L indole-based mixed standard solution was added to obtain a matrix mixed standard solution with a concentration of 50 μg / L. Accurately pipette 0.75 mL of blank solvent and add it to 0.25 mL of a 200 μg / L indole mixed standard solution to obtain a 50 μg / L blank solvent mixed standard solution. Measure the peak areas of each target analyte in the sample matrix, the sample matrix standard solution, and the blank solvent standard solution. Multiply the difference in peak area between the sample matrix standard solution and the sample matrix indole analyte peak area by 100 to obtain the matrix effect assessment value. Results are shown in [Figure Number]. Figure 3 .

[0159] Depend on Figure 3 It was found that different matrices had little effect on indole compounds, with the matrix effect ranging from 80% to 110%, indicating a weak matrix effect. This may be related to the ion source used; compared to ESI sources, APCI sources are less susceptible to matrix effects. Therefore, a standard curve was prepared using a 0.1% formic acid-50% acetonitrile aqueous solution.

[0160] This invention establishes a complete pretreatment technology by optimizing the extractant and solid-phase extraction purification technology in the pretreatment process. This technology improves the recovery rate, accuracy, precision, and sensitivity of six indole compounds (indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde) in aquatic products, while also being simple to operate.

[0161] Example 4

[0162] Actual sample analysis and freshness determination

[0163] Under the experimental conditions established in Example 2, commercially available live prawns and snakehead fish were purchased. The prawns were deheaded, tailed, and shelled, and the snakehead fish were skinned and deboned. The mixtures were ground into a paste using a meat grinder, and each paste was divided into 10 portions and placed in sealed packaging bags (approximately 25g per portion). One portion of each paste was placed in a -60°C freezer for quick freezing to maintain its sample condition. The remaining samples were placed in a 4°C freezer, and at 4h, 8h, 24h, 36h, 2d, 3d, 5d, 7d, and 9d, one portion of each sample was immediately removed and placed in a -60°C freezer for quick freezing to maintain its sample condition. The obtained samples were analyzed according to the method (pretreatment and detection) in Example 2 to investigate the changes in the concentrations of six indole compounds over time. The determination results for the snakehead fish samples are shown in Table 12 and... Figure 4 The test results for the prawn samples are shown in Table 13 and... Figure 5 .

[0164] Table 12 Determination of indole concentrations in snakehead fish samples (μg / kg)

[0165]

[0166] From Table 12 and Figure 4 It is known that the black carp and prawn samples contain indole compounds. As the storage time increases, the types and contents of indole compounds increase over time. For example, black carp meat does not produce indole, 3-indoleacetic acid, and indole-3-carboxaldehyde within 24 hours, but gradually produces indole, 3-indoleacetic acid, and indole-3-carboxaldehyde after 24 hours, and the total amount also increases continuously.

[0167] Table 13. Determination of indole concentrations in prawn samples (μg / kg)

[0168]

[0169]

[0170] From Table 13 and Figure 5 It was found that shrimp meat initially contained small amounts of indole, indole-4-carboxaldehyde, and indole-5-carboxaldehyde. 3-Indoleacetic acid and indole-3-carboxaldehyde began to be produced with increasing storage time, and the total amount of indole compounds also increased over time. No 3-methylindole was observed in either type of sample during storage. Compared to prawns, the concentration of the six indole compounds in the snakehead fish sample was relatively low, and the total amount of indole compounds increased over time. The concentration of the six indole compounds in the prawn sample was relatively high, and the total amount of indole compounds also increased over time. Furthermore, after 2 days of storage, the rate of increase in the total amount of indole compounds gradually accelerated, indicating that indole compounds continuously accumulated in both fish and shrimp meat, and their freshness was continuously decreasing.

[0171] Sensory comparisons of fish and shrimp meat at different storage times, for example Figure 6 As shown, a) is a black carp sample and b) is a prawn sample. Observation of the samples revealed that, sensorily, the fish meat developed a stronger fishy odor after 3 days, its color faded, gradually turning yellowish, and the texture became loose with increased liquid content. The prawn meat, on the other hand, developed a stronger fishy odor and gradually turned reddish after 2 days, indicating that both fish and prawn meat were spoiling. The changes in olfactory and visual sensory perception were largely consistent with the trend of changes in the total amount of indole compounds measured in the experiment, indicating that the freshness of the fish and prawn meat began to decline after refrigeration at 4–8℃ for 2 days.

[0172] Therefore, the freshness of aquatic products can be evaluated based on changes in the content of indole compounds. After multiple experiments, it was determined that when the total content of the six indole compounds in black carp is ≤100 μg / kg, the fish meat is fresh; when the total content of the six indole compounds is >100 μg / kg, the fish meat is relatively stale. Similarly, when the total content of the six indole compounds in prawns is ≤250 μg / kg, the prawn meat is fresh; when the total content of the six indole compounds is >250 μg / kg, the prawn meat is relatively stale.

[0173] Twenty batches each of fresh fish and shrimp were randomly purchased online, totaling 40 batches of samples for analysis. The condition of the express packaging upon receipt was recorded. Samples were tested according to the method in Example 2 (pretreatment and detection). In the fish samples, the indole content was mostly between 35.7 and 125.3 μg / kg, indicating relatively freshness. One sample had a total indole content of 223.5 μg / kg, indicating poor freshness. In the shrimp samples, the indole content was mostly between 65.7 and 268.3 μg / kg, indicating good freshness.

[0174] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Other embodiments can be obtained without creative effort based on the embodiments of the present invention, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting indole compounds in aquatic products, characterized in that, Includes the following steps: The aquatic product to be tested, the isotope internal standard working solution, and the formic acid-acetonitrile aqueous solution were mixed and extracted to obtain the extract; the volume fraction of formic acid in the formic acid-acetonitrile aqueous solution was 0.1~0.2%, and the volume fraction of acetonitrile was 75~85%. The extract was purified using an HLB solid-phase extraction column to obtain the sample solution to be tested. The HLB solid-phase extraction column purification included rinsing with a first acetonitrile aqueous solution, drying the HLB solid-phase extraction column, and then eluting with a formic acid-acetonitrile mixture. The eluent was collected and concentrated to obtain the sample solution to be tested. The volume fraction of acetonitrile in the first acetonitrile aqueous solution was 8-12%, and the volume fraction of formic acid in the formic acid-acetonitrile mixture was 0.1-0.2%. The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry to obtain the detection results of indole substances; The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection conditions include: a C10 column. 18 The chromatographic column uses a mobile phase A of 0.1-0.2% formic acid aqueous solution and a mobile phase B of acetonitrile. Gradient elution is employed, with the following program: 0-3 min, mobile phase A volume fraction 70-80%; 3-5 min, mobile phase A volume fraction decreasing uniformly from 70-80% to 5-10%; 5-8 min, mobile phase A volume fraction 5-10%; 8-8.0 min, mobile phase A volume fraction increasing uniformly from 5-10% to 70-80%; 8.1-12 min, mobile phase A volume fraction 70-80%. The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection uses an APCI ion source. The indole compounds include indole, 3-indoleacetic acid, 3-methylindole, indole-3-carboxaldehyde, indole-4-carboxaldehyde, and indole-5-carboxaldehyde.

2. The detection method according to claim 1, characterized in that, The ratio of the aquatic product to be tested to the formic acid-acetonitrile aqueous solution is 1g:4~6mL.

3. The detection method according to claim 1 or 2, characterized in that, The mixed extraction includes sequential homogenization extraction and oscillation extraction; the homogenization extraction speed is 10000~12000 r / min, and the time is 1~2 min; the oscillation extraction speed is 180~240 r / min, and the time is 8~15 min.

4. The detection method according to claim 3, characterized in that, The process of mixing and extracting also includes centrifuging the resulting extraction system, with the supernatant being the extract.

5. The detection method according to claim 1, characterized in that, The extract is diluted with water before loading to obtain a diluted extract; the volume ratio of the extract to water is 1:4~5.

6. The detection method according to claim 1, characterized in that, The concentration process further includes adjusting the volume of the resulting concentrated solution with a second acetonitrile aqueous solution. The volume of the concentrate is less than 35% of the volume of the extract; The volume fraction of acetonitrile in the second acetonitrile aqueous solution is 40-60%.

7. The detection method according to claim 1, characterized in that, The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection conditions also include: column temperature of 30-40℃, mobile phase flow rate of 0.25-0.35 mL / min, and injection volume of 1-2 μL.

8. The detection method according to claim 1, characterized in that, The mass spectrometry detection conditions for the high performance liquid chromatography-tandem mass spectrometry include: ionization mode is APCI positive ion mode, scanning mode is multiple reaction monitoring, curtain gas pressure is 25 psi, ion spray voltage is 5500 V, nebulization temperature is 400 °C, and nebulizer gas pressure is 30 psi.

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