Absolute quantitative analysis of fish pigment metabolites based on LC-MS / MS detection method

Abstract

The present invention discloses an absolute quantitative analysis method for fish pigment metabolites based on LC-MS / MS detection method, which belongs to the field of targeted metabolomics. The method optimizes and screens the preparation of fish pigment metabolite samples and metabolite extraction methods, as well as high-performance liquid chromatography conditions and mass spectrometry conditions, obtains the best MRM mass spectrometry analysis parameters and high-performance liquid chromatography conditions, and establishes a targeted metabolomics detection method for 16 fish pigment metabolites. The detection results show that the method has the characteristics of high throughput, high sensitivity and high stability, meets the requirements of precise targeted metabolomics research, is suitable for high-throughput detection and analysis of fish pigment-related metabolites, and helps to accelerate the research progress of fish body color.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted metabolomics, and in particular to an absolute quantitative analysis method for fish pigment metabolites based on an LC-MS / MS detection method. Background Art

[0002] Targeted Metabolomics is an important part of metabolomics research and an extension and expansion of whole metabolomics research. Targeted metabolomics is the detection of the content of specific metabolites in samples. Compared with whole metabolomics analysis, targeted metabolomics analysis has the characteristics of strong specificity, high detection sensitivity and accurate quantification. Through the enrichment and accurate quantitative and qualitative analysis of a specific metabolite in blood, urine or other body fluids and tissues, on the one hand, it can be combined with other experimental data to reveal the relevant molecular biological mechanism of action, and on the other hand, it can also provide strong support for the in-depth research and development and utilization of subsequent metabolic molecular markers.

[0003] Selective reaction / multiple reaction monitoring technology (SRM / MRM) uses standards as references to conduct targeted and specific detection and analysis of specific metabolite groups, and can obtain absolute quantitative results of target metabolites, with the characteristics of strong specificity, high sensitivity, and high accuracy. Based on known or assumed reaction ion information, SRM / MRM technology selects data for mass spectrometry signal acquisition in a targeted manner, records signals for ion pairs that meet the rules, and removes interference from ion signals that do not meet the rules. In the quantitative analysis process, this technology first screens the parent ions specific to the target metabolites, and then selectively performs collision induction on these parent ions to remove interference from other ions, and only collects mass spectrometry signals for selected MS / MS2 ions, thereby achieving a more specific, sensitive, and accurate analysis of the target metabolic molecules.

[0004] Metabolomics characterizes changes in body functions by analyzing changes in metabolite concentrations, so the ultimate goal of metabolomics is to detect as many metabolites as possible. Scientists have never stopped researching metabolomics methodology platforms, but so far, no analytical technology can detect all small molecule metabolites in organisms. At present, the mainstream research platforms of metabolomics mainly include nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) and gas chromatography-mass spectrometry (GC-MS). Among them, nuclear magnetic resonance (NMR) technology can analyze various samples without discrimination, but its sensitivity is low and it cannot effectively respond to low-concentration substances; gas chromatography-mass spectrometry (GC-MS) has high detection sensitivity, but it only detects volatile substances or substances that are volatile after derivatization, and they all have great limitations.

[0005] Previous researchers have conducted extensive research on the development and application of LC-MS / MS methods. Currently, targeted metabolomics detection based on LC-MS / MS includes medium-chain fatty acids, broken-chain fatty acids, plant hormones, flavonoids, neurotransmitters, anthocyanins, steroids, carotenoids, polyamines and sugars. During the detection process, high-throughput detection is usually only performed on a certain class of compounds with similar structures, such as medium-chain fatty acids or anthocyanins. Substances with large structural differences are difficult to extract and separate at the same time due to large differences in physical and chemical properties and thermal stability, so it is difficult to carry out targeted metabolomics research.

[0006] At present, there are no reports on metabolomics detection of fish pigment-related metabolites. In addition, due to the complex structure and huge differences in physical and chemical properties of fish pigment-related metabolites, including hormones and neurotransmitters, carotenoids and their dihydroxy derivatives, small signal molecules and nucleotides, it is difficult to perform high-throughput detection on them.

[0007] On this basis, the present application creatively conducts targeted metabolomics quantitative detection and analysis of fish pigment metabolites through LC-MS / MS technology. By screening the sample metabolite extraction method, chromatographic analysis method and mass spectrometry analysis method, an MRM method for its 16 metabolites is established to meet the requirements of targeted metabolomics detection for simultaneous quantitative detection of 16 metabolites. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide an absolute quantitative analysis method for fish pigment metabolites based on the LC-MS / MS detection method, so that an MRM method for its 16 metabolites can be established by screening its sample metabolite extraction method, chromatographic analysis method and mass spectrometry analysis method, so as to meet the targeted metabolome detection requirements for simultaneous quantitative detection of 16 metabolites, thereby overcoming the existing gaps in the detection of fish pigment metabolites.

[0009] In order to solve the above technical problems, the present invention provides an absolute quantitative analysis method for fish pigment metabolites based on LC-MS / MS detection method, characterized in that the method comprises the following steps:

[0010] (1) Metabolite extraction: Take 100 μL of fish blood or skin tissue sample, add 100-300 μL of pre-cooled water and 600-800 μL of pre-cooled 50% isopropanol-acetonitrile solution, sonicate in an ice bath for 40-80 min, incubate at -20°C for 40-80 min to precipitate protein, centrifuge at 16000g at 4°C for 15-25 min, take the supernatant, add the internal standard L-Glutamate_D5, and vacuum dry to obtain the fish pigment metabolite sample;

[0011] (2) Chromatographic separation: Take the fish pigment metabolite sample obtained in step (1), add 100 μL of 50% acetonitrile-water solution to re-dissolve, centrifuge at 16000 g and 4° C. for 15-25 min, and take the supernatant as the injection sample;

[0012] The HPLC conditions are as follows: using Shimadzu Nexera X2 LC-30AD HPLC column, column temperature 40°C, flow rate 200 μL / min, injection volume 5 μL, mobile phase: liquid A is 0.1% formic acid-water solution, liquid B is 50% isopropanol-acetonitrile solution, mobile phase liquid A and liquid B gradient conditions are as follows: 0-0.5min, liquid B is maintained at 2%; 0.5-4min, liquid B changes linearly from 2% to 60%; 4-8min, liquid B changes linearly from 60% to 98%; 8-9.9min, liquid B is maintained at 98%; 9.9-10min, liquid B changes linearly from 98% to 5%; 10.1-12min, liquid B is maintained at 5%;

[0013] (3) Mass spectrometry analysis: The sample separated by high performance liquid chromatography enters a mass spectrometer, and mass spectrometry analysis is performed in the positive / negative ion mode of the mass spectrometer. The MRM mode is used to detect the ion pairs to be tested, and the quantitative determination of 16 metabolites in the fish pigment metabolite sample is completed;

[0014] The mass spectrometer parameter conditions are as follows: ESI source parameters are as follows: positive ion mode: Source Temperature 500-600℃, Ion Source Gas1:40-50, Ion Source Gas2:40-50, Curtain Gas:20-40, Ion Spray Voltage Floating:3000-6000V; negative ion mode: Source Temperature 500-600℃, Ion Source Gas1:40-50,Ion Source Gas2:40-50,Curtain Gas:20-40,Ion Spray Voltage Floating:-2000--5500V.

[0015] Further improvement, the step (1) is specifically as follows: taking 100 μL of fish blood or skin tissue sample, adding 100 μL of pre-cooled water and 800 μL of pre-cooled 50% isopropanol-acetonitrile solution, ultrasonicating in an ice bath for 60 min, incubating at -20°C for 1 h to precipitate protein, centrifuging at 16000g at 4°C for 20 min, taking the supernatant, adding internal standard L-Glutamate_D5, and vacuum drying to obtain a fish pigment metabolite sample.

[0016] As a further improvement, in step (1), the fish blood sample is obtained by collecting blood from the fish caudal vertebrae at 4°C overnight, centrifuging at 2000 rpm for 20 minutes, and taking the supernatant.

[0017] As a further improvement, QC samples are inserted into the sample injection queue in step (2) to monitor and evaluate the stability of the system and the reliability of the experimental data.

[0018] Further improvement, the mass spectrometer parameter conditions in the step (3) are: QTRAP5500 mass spectrometer, ESI source parameters are: positive ion mode: Source Temperature 550°C, Ion Source Gas1:40, Ion Source Gas2:50, Curtain Gas:35, Ion Spray Voltage Floating:5500V; negative ion mode: Source Temperature 550°C, Ion Source Gas1:40, Ion Source Gas2:50, Curtain Gas:35, Ion Spray Voltage Floating:-4500V.

[0019] Further improved, the 16 metabolites in the fish pigment metabolite sample are: melatonin, acetylcholine, norepinephrine, progesterone, astaxanthin, lutein, α-carotene, β-carotene, canthaxanthin, zeaxanthin, tuna xanthin, epinephrine, melanin ODB-1, cyclic adenosine monophosphate, cyclic guanosine monophosphate and guanine.

[0020] After adopting such a design, the present invention has at least the following advantages:

[0021] The present invention is based on the LC-MS / MS detection method for the absolute quantitative analysis of fish pigment metabolites. By screening the high-performance liquid chromatography conditions, mass spectrometry conditions, sample preparation and metabolite extraction methods, the optimal MRM mass spectrometry analysis parameters and the optimal high-performance liquid chromatography conditions are obtained, thereby establishing a targeted metabolomics detection method. The detection results show that the targeted metabolomics detection method of this application has the characteristics of high throughput, high sensitivity and high stability, and meets the requirements of precise targeted metabolomics research. On the basis of this absolute quantitative detection method, 16 kinds of fish pigment metabolites can be detected at the same time, and each peak shape is symmetrical and the separation is good. It is suitable for high-throughput detection and analysis of fish pigment-related metabolites, which helps to accelerate the research progress of fish body color. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0023] Figure 1 It is a standard XIC spectrum of 16 metabolites in the absolute quantitative analysis method of fish pigment metabolites based on the LC-MS / MS detection method of the present invention.

[0024] Figure 2-Figure 17 They are respectively the standard curve graphs of norepinephrine, epinephrine, acetylcholine, guanine, CAMP, cGMP, melatonin, melanin, progesterone, astaxanthin, tuna xanthocyanin, canthaxanthin, zeaxanthin, lutein, β-carotene and α-carotene in the absolute quantitative analysis method of fish pigment metabolites based on LC-MS / MS detection method of the present application.

[0025] Figure 18-Figure 20 They are respectively the XIC spectra of blood samples of three different strains of koi in the absolute quantitative analysis method of fish pigment metabolites based on LC-MS / MS detection method in the present application.

[0026] Figure 21-23 They are respectively the XIC spectra of blood samples of three different strains of goldfish in the absolute quantitative analysis method of fish pigment metabolites based on LC-MS / MS detection method of the present application.

[0027] Fig.24 It is the RSD value of 16 metabolites of QC samples in the absolute quantitative analysis method of fish pigment metabolites based on LC-MS / MS detection method in this application. DETAILED DESCRIPTION

[0028] The complete metabolomics research process mainly includes steps such as sample preparation, data collection, data processing and visualization analysis, and data interpretation. Sample preparation includes sample collection and extraction and separation of metabolites. At present, there is no extraction method that can be applied to all metabolites. In actual operation, we must select a suitable extraction method according to the properties of the target metabolites and optimize it to minimize sample loss. The specific implementation example of targeted metabolomics research on fish pigment metabolites is as follows.

[0029] Example 1 Screening of HPLC Conditions and Mass Spectrometry Conditions

[0030] The 16 metabolites of fish pigment metabolites included melatonin, acetylcholine, norepinephrine, progesterone, astaxanthin, lutein, α-carotene, β-carotene, canthaxanthin, zeaxanthin, tunaxanthin, epinephrine, melanin ODB-1, cyclic adenosine monophosphate, cyclic guanosine monophosphate, and guanine.

[0031] Standard solutions of the above 16 metabolites were prepared, and their HPLC conditions and mass spectrometry conditions were screened.

[0032] The HPLC conditions used in this embodiment include:

[0033] Condition 1: Use Shimadzu Nexera X2 LC-30AD high performance liquid chromatography column, mobile phase: Liquid A is 80% methanol-water solution; Liquid B is 30% acetonitrile-water solution. After the standard is dissolved in 100μL 50% acetonitrile-water solution, it is placed in a 4℃ automatic sampler, column temperature is 40℃, flow rate is 200μL / min, and injection volume is 5μL. The relevant liquid phase gradient is as follows:

[0034]

[0035]

[0036] Condition 2: Use Shimadzu Nexera X2 LC-30AD high performance liquid chromatography column, mobile phase: Liquid A is 95% methanol-water solution; Liquid B is 70% acetonitrile-water solution. After the standard is dissolved in 100μL 50% acetonitrile-water solution, it is placed in a 4℃ automatic sampler, column temperature is 40℃, flow rate is 200μL / min, and injection volume is 5μL. The relevant liquid phase gradient is as follows:

[0037] time Liquid B ratio 0-0.5min 2％ 0.5-4min 2% linear change to 60% 4-8min 60% linear change to 98% 8-9.9min Maintain 98% 9.9-10min 98% linear change to 5% 10.1-12min Maintain 5%

[0038] Condition 3: Use Shimadzu Nexera X2 LC-30AD HPLC column, mobile phase: Liquid A is 10% formic acid-water solution; Liquid B is isopropanol / acetonitrile (5:1). After the standard is dissolved in 100μL 50% acetonitrile-water solution, it is placed in a 4℃ autosampler, column temperature is 40℃, flow rate is 200μL / min, and injection volume is 5μL. The relevant liquid phase gradient is as follows:

[0039]

[0040]

[0041] Condition 4: Use Shimadzu Nexera X2 LC-30AD high performance liquid chromatography column, mobile phase: Liquid A is 0.1% formic acid-water solution; Liquid B is isopropanol / acetonitrile (1:1). After the standard is dissolved in 100μL 50% acetonitrile-water solution, it is placed in a 4℃ automatic sampler, column temperature 40℃, flow rate 200μL / min, injection volume 5μL. The relevant liquid phase gradient is as follows:

[0042] time B fluid ratio 0-1min 2％ 1-5min 2% linear change to 60% 5-9min 60% linear change to 98% 9-11min Maintain 98% 11-11.5min 98% linear change to 5% 11.5-12min Maintain 5%

[0043] Condition 5: Use Shimadzu Nexera X2 LC-30AD high performance liquid chromatography column, mobile phase: Liquid A is 0.1% formic acid-water solution; Liquid B is isopropanol / acetonitrile (1:1). After the standard is dissolved in 100μL 50% acetonitrile-water solution, it is placed in a 4℃ automatic sampler, column temperature 40℃, flow rate 200μL / min, injection volume 5μL. The relevant liquid phase gradient is as follows:

[0044]

[0045]

[0046] Condition 6: Use Shimadzu Nexera X2 LC-30AD high performance liquid chromatography column, mobile phase: Liquid A is 0.1% formic acid-water solution; Liquid B is isopropanol / acetonitrile (1:1). After the standard is dissolved in 100μL 50% acetonitrile-water solution, it is placed in a 4℃ automatic sampler, column temperature is 40℃, flow rate is 200μL / min, and injection volume is 5μL. The relevant liquid phase gradient is as follows:

[0047] time B fluid ratio 0-0.5min 2％ 0.5-4min 2% linear change to 60% 4-8min 60% linear change to 98% 8-9.9min Maintain 98% 9.9-10min 98% linear change to 5% 10.1-12min Maintain 5%

[0048] In this embodiment, each standard product is injected in the ES I source needle pump mode, and the scanning ion mode and important parameters such as DP, EP, CEP, CE, and CXP are optimized to establish the MRM method of 16 pigment metabolites. The mass spectrometry analysis conditions are finally determined as follows: the mass spectrometer parameter conditions are: ES I source parameters are: positive ion mode: Source Temperature 500-600°C, Ion Source Gas1:40-50, Ion Source Gas2:40-50, Curtain Gas:20-40, Ion Spray Voltage Floating:3000-6000V; negative ion mode: Source Temperature 500-600°C, Ion Source Gas1:40-50, Ion Source Gas2:40-50, Curtain Gas:20-40, Ion Spray Voltage Floating:-2000--5500V.

[0049] In this embodiment, the metabolites separated by the above six chromatographic conditions are respectively introduced into the mass spectrometer, and the mass spectrometry analysis conditions are as follows: QTRAP5500 mass spectrometer (AB SCI EX) is used for mass spectrometry analysis in positive / negative ion mode. ESI source parameters are as follows: Positive ion mode: Source Temperature: 550°C, Ion Source Gas1 (GAS1): 40, Ion Source Gas2 (GAS2): 50, Curtain Gas (CUR): 35, Ion Spray Voltage Floating (ISVF): 5500V. Negative ion mode: Source Temperature: 550°C, Ion Source Gas1 (GAS1): 40, Ion Source Gas2 (GAS2): 50, Curtain Gas (CUR): 35, Ion Spray Voltage Floating (ISVF): -4500V. The MRM mode is used to detect the ion pairs to be tested.

[0050] In this example, the same standard solution was subjected to the above six HPLC conditions and mass spectrometry analysis conditions to perform chromatographic mass spectrometry separation, and XIC diagrams (extracted ion current diagrams) of 16 standards were extracted. The results showed that condition 6 was the best for separation of chromatographic peaks of each metabolite, with sharp and symmetrical peaks, and was able to perform mass spectrometry quantification of each metabolite. Figure 1 As shown. And under this condition, the retention times of 16 standards are as follows:

[0051]

[0052]

[0053] That is, this embodiment can determine that the optimal HPLC conditions for the absolute quantitative analysis method of fish pigment metabolites based on the LC-MS / MS detection method are: using Shimadzu Nexera X2LC-30AD HPLC column, mobile phase: liquid A is 0.1% formic acid-water solution; liquid B is isopropanol / acetonitrile (1:1). After the standard is dissolved with 100 μL 50% acetonitrile-water solution, it is placed in a 4°C automatic sampler, the column temperature is 40°C, the flow rate is 200 μL / min, and the injection volume is 5 μL. The relevant liquid phase gradient is: 0-0.5min, liquid B is maintained at 2%; 0.5-4min, liquid B changes linearly from 2% to 60%; 4-8min, liquid B changes linearly from 60% to 98%; 8-9.9min, liquid B is maintained at 98%; 9.9-10min, liquid B changes linearly from 98% to 5%; 10.1-12min, liquid B is maintained at 5%.

[0054] The best mass spectrometry analysis conditions are: using QTRAP5500 mass spectrometer (AB SCI EX) in positive / negative ion mode for mass spectrometry analysis. ESI source parameters are as follows: Positive ion mode: Source Temperature: 550℃, Ion Source Gas1(GAS1):40, I on Source Gas2(GAS2):50, Curtain Gas(CUR):35, Ion Spray VoltageFloating(ISVF):5500V. Negative ion mode: Source Temperature: 550℃, Ion Source Gas1(GAS1):40, Ion Source Gas2(GAS2):50, Curtain Gas(CUR):35, Ion Spray VoltageFloating(ISVF):-4500V. MRM mode was used to detect the ion pairs to be measured.

[0055] In this embodiment, standard curves are drawn for 16 kinds of standard products under the chromatographic conditions to obtain standard curves and standard equations for the 16 kinds of standard products, as shown in the attached figure. Figure 2-17 , providing the necessary conditions for calculating the loading concentration of each metabolite in subsequent samples and the final absolute quantitative value.

[0056] Example 2 Sample preparation and metabolite extraction method screening

[0057] The fish blood sample preparation method of this embodiment is as follows: 3 different strains of koi and 3 different strains of goldfish are taken, and after being anesthetized with MS222 reagent of appropriate concentration, they are placed on a sampling table, and blood is collected from the caudal vertebrae using a disposable 1mL or 2mL sterile syringe, respectively, and placed in a 1.5mL centrifuge tube. After being stored at 4°C overnight, the tube is centrifuged at 2000rpm for 20min, and the supernatant is taken as a sample for metabolomics detection, and the samples are stored at -80°C.

[0058] The metabolite extraction methods of this embodiment include the following three methods:

[0059] Method a: Thaw the samples for metabolomics testing at 4°C, take 100 μL of each sample, add 800 μL of pre-cooled acetonitrile / isopropanol (1:1, v / v), 100 μL of pre-cooled deionized water, sonicate in an ice bath for 60 min, incubate at -20°C for 1 h to precipitate protein, centrifuge at 16000g for 20 min at 4°C, and take the supernatant. Add an equal amount of internal standard L-Glutamate_D5 to each sample, and then vacuum dry. For chromatographic detection, add 100 μL of acetonitrile-water solution (1:1, v / v) for re-dissolution, centrifuge at 16000g for 20 min at 4°C, and take the supernatant as injection samples 1-6.

[0060] Method b: Thaw the samples for metabolomics testing at 4°C, take 100 μL of each sample, add 600 μL of pre-cooled acetonitrile / isopropanol (1:1, v / v), 300 μL of pre-cooled deionized water, sonicate in an ice bath for 60 min, incubate at -20°C for 1 h to precipitate protein, centrifuge at 16000g for 20 min at 4°C, and take the supernatant. Add an equal amount of internal standard L-Glutamate_D5 to each sample, and then vacuum dry. For chromatographic detection, add 100 μL of acetonitrile-water solution (1:1, v / v) to reconstitute, centrifuge at 16000g for 20 min at 4°C, and take the supernatant as injection sample 7-12.

[0061] Method c: Thaw the samples for metabolomics testing at 4°C, take 100 μL of each sample, add 600 μL of pre-cooled trichloroacetic acid-acetone (1:2, v / v), 300 μL of pre-cooled deionized water, sonicate in an ice bath for 60 min, incubate at -20°C for 1 h to precipitate protein, centrifuge at 16000g for 20 min at 4°C, and take the supernatant. Add an equal amount of internal standard L-Glutamate_D5 to each sample, and then vacuum dry. Then vacuum dry. For chromatographic detection, add 100 μL of acetonitrile-water solution (1:1, v / v) to re-dissolve, centrifuge at 16000g for 20 min at 4°C, and take the supernatant as injection sample 13-18.

[0062] By using the above-mentioned HPLC separation and mass spectrometry analysis on the above-mentioned injected samples 1-18, it is found that the peak value of each metabolite extracted by method a is higher, and the separation efficiency of each metabolite is also higher. Therefore, method a is selected as the metabolite extraction method for the fish pigment metabolite sample of this application.

[0063] Samples 1-6 of this embodiment were injected under the above-mentioned HPLC conditions and mass spectrometry conditions to obtain spectra as shown in the attached figure. Figure 18-23 , that is, the XIC graphs of blood samples from 3 different strains of koi and 3 different strains of goldfish, with sharp peaks and good separation.

[0064] Example 3 Method Evaluation

[0065] In this embodiment, the QC samples are prepared by mixing all samples in equal amounts to prepare samples for evaluating the stability and repeatability of the detection data of the absolute quantitative analysis method.

[0066] The specific evaluation method is as follows: Under the above-mentioned chromatographic and mass spectrometric conditions, a QC sample is inserted into the standard sample and each sample injection queue after a certain sample volume interval. By analyzing the QC sample injection data at different times and different times, the relative standard deviation (RSD) values ​​of the 16 metabolites in the QC sample are calculated respectively. The results are shown in the attached figure. Fig.24 .

[0067] From the attached Fig.24 It can be seen that the relative standard deviations (RSDs) of the 16 metabolites were all less than 30%, indicating that the data stability of each metabolite in the sample was high and the quantitative method was stable and reliable.

[0068] Of course, the above method is also applicable to the detection of pigment metabolites in fish skin tissue samples.

[0069] The absolute quantitative analysis method of fish pigment metabolites based on the LC-MS / MS detection method of the present invention can simultaneously detect 16 fish pigment metabolites under the above-mentioned most preferred high performance liquid chromatography conditions, mass spectrometry conditions, sample preparation and metabolite extraction method conditions, is suitable for high-throughput detection and analysis of fish pigment-related metabolites, and helps to accelerate the research progress of fish body color.

[0070] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

Claims

1. An absolute quantitative analysis method for fish pigment metabolites based on LC-MS / MS detection method, characterized in that: The method comprises the following steps: (1) Metabolite extraction: Take 100 µL of fish blood or skin tissue sample, add 100-300 µL of pre-cooled water and 600-800 µL of pre-cooled 50% isopropanol-acetonitrile solution, ultrasonicate in an ice bath for 40-80 min, incubate at -20°C for 40-80 min to precipitate protein, centrifuge at 16000g at 4°C for 15-25 min, take the supernatant, add the internal standard L-Glutamate_D5, and vacuum dry to obtain the fish pigment metabolite sample; (2) Chromatographic separation: Take the fish pigment metabolite sample obtained in step (1), add 100 μL of 50% acetonitrile-water solution to re-dissolve it, centrifuge at 16000g at 4°C for 15-25 min, and take the supernatant as the injection sample; The HPLC conditions were as follows: Shimadzu Nexera X2 LC-30AD HPLC was used, column temperature was 40°C, flow rate was 200 µL / min, injection volume was 5 µL, mobile phases were: liquid A was 0.1% formic acid-water solution, liquid B was 50% isopropanol-acetonitrile solution, mobile phase liquid A and liquid B gradient conditions were as follows: 0-0.5 min, liquid B was maintained at 2%; 0.5-4 min, liquid B changed linearly from 2% to 60%; 4-8 min, liquid B changed linearly from 60% to 98%; 8-9.9 min, liquid B was maintained at 98%; 9.9-10 min, liquid B changes linearly from 98% to 5%; 10.1-12min, maintain solution B at 5%; (3) Mass spectrometry analysis: The samples separated by high performance liquid chromatography are put into a mass spectrometer for mass spectrometry analysis in the positive / negative ion mode of the mass spectrometer. The MRM mode is used to detect the ion pairs to be tested, and the quantitative determination of 16 metabolites in the fish pigment metabolite sample is completed; The mass spectrometer parameter conditions are as follows: ESI source parameters are as follows: positive ion mode: Source Temperature 500-600°C, Ion Source Gas1:40-50, Ion Source Gas2:40-50, Curtain Gas:20-40, Ion Spray Voltage Floating:3000-6000V; negative ion mode: Source Temperature 500-600°C, Ion Source Gas1:40-50, Ion Source Gas2:40-50, Curtain Gas:20-40, Ion Spray Voltage Floating:-2000--5500V; The 16 metabolites in the fish pigment metabolite sample are: melatonin, acetylcholine, norepinephrine, progesterone, astaxanthin, lutein, α-carotene, β-carotene, canthaxanthin, zeaxanthin, tuna xanthin, epinephrine, melanin ODB-1, cyclic adenosine monophosphate, cyclic guanosine monophosphate and guanine.

2. The absolute quantitative analysis method for fish pigment metabolites based on LC-MS / MS detection method according to claim 1, characterized in that: The step (1) specifically comprises: taking 100 µL of fish blood or skin tissue sample, adding 100 µL of pre-cooled water and 800 µL of pre-cooled 50% isopropanol-acetonitrile solution, ultrasonicating in an ice bath for 60 min, incubating at -20°C for 1 h to precipitate protein, centrifuging at 16000g at 4°C for 20 min, taking the supernatant, adding internal standard L-Glutamate_D5, and vacuum drying to obtain a fish pigment metabolite sample.

3. The absolute quantitative analysis method for fish pigment metabolites based on LC-MS / MS detection method according to claim 2, characterized in that: In step (1), the fish blood sample is obtained by collecting blood from the caudal vertebrae of the fish, keeping it at 4° C. overnight, centrifuging it at 2000 rpm for 20 minutes, and taking the supernatant.

4. The absolute quantitative analysis method for fish pigment metabolites based on LC-MS / MS detection method according to claim 1, characterized in that: QC samples are inserted into the sample injection queue in step (2) to monitor and evaluate the stability of the system and the reliability of the experimental data.

5. The absolute quantitative analysis method for fish pigment metabolites based on LC-MS / MS detection method according to claim 1, characterized in that: The mass spectrometer parameter conditions in the step (3) are: QTRAP5500 mass spectrometer, ESI source parameters are: positive ion mode: Source Temperature 550°C, Ion Source Gas1:40, Ion Source Gas2:50, Curtain Gas:35, Ion Spray Voltage Floating:5500V; negative ion mode: Source Temperature 550°C, Ion Source Gas1: 40, Ion Source Gas2:50, Curtain Gas:35, Ion Spray Voltage Floating:-4500V.

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