CN111307976B - High-throughput detection method for carotenoid in aquatic product - Google Patents

Abstract

The invention discloses a high-flux detection method of carotenoid in aquatic products, which comprises the following steps: A. pre-treating; B. detecting a sample solution to be detected by a high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometer to obtain a high performance liquid chromatogram of the sample solution to be detected; and C, analyzing the data. The high-flux detection method for the carotenoids in the aquatic products disclosed by the invention has the following beneficial effects: 1. the determination lower limit of the carotenoid is 0.05mg/kg, and the standard curve is good in the linear range of 10 ng/mL-200 ng/mL; 2. the pretreatment combines solid phase extraction and matrix dispersion purification technologies, and the extraction and purification work of 3 carotenoids with greatly different physicochemical properties is realized through one-time pretreatment, so that the detection efficiency is improved, and the expensive time and the reagent cost consumption are reduced; 3. the method is applied to aquatic products, and 50 carotenoid species are detected in total.

Description

A high-throughput detection method for carotenoids in aquatic products

technical field

The invention relates to the field of aquatic product nutrient component detection, in particular to a high-throughput detection method for carotenoids in aquatic products.

Background technique

Carotenoids, a class of natural pigment molecules, mainly exist in water-producing objects such as fish, shrimp, crabs, and shells, and their structural composition and distribution are important indicators for evaluating the quality of aquatic products. These photosensitive carotenoids not only serve as colorants for the body and tissues to improve sensory quality, but also enhance the adaptability of aquatic organisms to the environment and some important physiological functions, including improving seedling efficiency and disease resistance. Studies have shown that aquatic organisms cannot de novo synthesize carotenoids, but can convert or directly absorb dietary carotenoids. Each organism has its own unique carotenoids, and even different tissues have their particularities. It is also one of the reasons why there are many types of carotenoids in aquatic products.

There are many kinds of carotenoids, which are divided into three categories according to their chemical structures: the first category is free carotenoids, which have no oxygen in their molecules and are a kind of highly unsaturated hydrocarbons, mostly orange-red, such as α, β - carotene, etc.; the second type is oxycarotene, which are oxidized derivatives of carotene, containing one or more oxygen molecules in the rings at both ends of the molecule, and the most common modified groups are hydroxyl, methoxy, Carbonyl groups, carboxyl groups, etc., such as lutein, astaxanthin, etc., the first two categories are collectively referred to as non-esterified carotenoids; the third category is carotene esters, including monoesters and diesters, which are free carotenoids and endogenous carotenoids. Results of the re-esterification of sexual fatty acids.

The traditional pretreatment technology for detecting carotenoids in aquatic products is mainly direct solvent extraction. This technology only uses a single solvent for extraction (mostly acetone), which has the disadvantage of selectivity, especially for small molecule free carotenoids and their metabolites, which are not compatible with low-polarity carotenoid esters at the same time; It is only applicable to the analysis method for the detection of a certain carotenoid in a fixed food matrix, and the sample processing throughput is not high. There are many types of carotenoids, and many carotenoids and their metabolites in aquatic products have not been isolated and identified. Establishing a comprehensive and efficient pretreatment method is an important prerequisite for ensuring the identification of unknown compounds. It is of great significance to explore the metabolic pathways of carotene species. Therefore, it is necessary to seek a more comprehensive and efficient method for the detection of carotenoids in aquatic products.

Commonly used instrumental analysis methods for carotenoids include: liquid phase-ultraviolet-visible absorption spectroscopy and liquid phase-triple quadrupole mass spectrometry. Most carotenoids have three maximum absorption peaks in UV, commonly known as "three-finger peaks", which can analyze the total amount of carotenoids, but cannot analyze the existing forms of carotenoids (free, esterified, oxidized) in the matrix. ) Progressive identification. At present, most of the carotenoid research uses liquid phase-triple quadrupole mass spectrometry technology, which requires known characteristic ion fragments of the compound to be tested, and obtains the data in a targeted manner through the multiple reaction ion detection mode, thereby realizing the qualitative and quantitative analysis of the known compounds. Quantitative analysis.

SUMMARY OF THE INVENTION

The purpose of the present invention is to make up for the shortcomings of the existing aquatic product carotenoid detection methods such as selectivity, few detection types, and low detection efficiency, and provides a high-throughput method suitable for carotenoids in aquatic products. Detection method.

Considering that there are many types of carotenoids covered, the polarity between different types is quite different; and the high protein and high fat matrix properties of aquatic products, which produce strong matrix effects, lead to serious deviations in the detection results. Therefore, according to the pKa value and polarity range of the extraction solvent, the present invention adopts hierarchical purification and extraction: the first stage adopts methanol vibration extraction and HLB small column purification, which can realize the purification of non-esterified carotenoids (most free carotenoids and Efficient extraction and separation of oxygenated carotenoids); the second stage adopts methanol extraction, dichloromethane assisted extraction, and neutral alumina powder purification. By combining the first and second extracts, and using high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) for data analysis, the common carotenoid in aquatic products can be analyzed. High-throughput detection of primes.

Technical solution: a high-throughput detection method for carotenoids in aquatic products, comprising the following steps:

A. Pre-processing:

(A1) Weigh 1 g of the aquatic product to be tested, add it to 10 mL of methanol solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and then rotate at 12000 r/min for 4 to Centrifuge at 10 degrees Celsius for 5-10 minutes, and obtain the organic phase and residue after completion;

(A2) Take the organic phase obtained in step (A1), add at least 4 times the volume of water to it to obtain a sample solution, and then purify the sample solution through an HLB column to obtain a purified sample solution, and then purge the sample with nitrogen. The purified sample solution was concentrated to <2.0ml, and then adjusted to 2.0ml by methanol solution containing 0.1wt% HF to obtain the purified sample solution after constant volume, wherein:

HLB column purification was carried out using methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol as eluent;

(A3) Add the remaining residue of step (A1) into 10 mL of methanol-dichloromethane solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and rotate at 12000 r/min Centrifuge at 4 to 10 degrees Celsius for 5 to 10 minutes, and obtain organic phase and residue after completion, wherein:

In the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1:1;

(A4) take the organic phase obtained in step (A3), nitrogen blowing and concentrating until the volume does not change, then dilute to 2.0ml by a methanol solution containing 0.1wt% HF, and then add 1g neutral alumina powder to it and fully shake, Centrifuge at 4 to 10 degrees Celsius for 5 to 10 minutes at a speed of 12000 r/min, and obtain supernatant and precipitate after completion;

(A5) merging the purified sample solution after constant volume obtained in step (A2) and the supernatant solution obtained in step (A4), and then passing through a filter membrane with a pore size of 0.22 μm to obtain the sample solution to be detected;

B. the sample solution to be detected obtained in step (A5) is detected by high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometer to obtain the high-efficiency liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows :

HPLC parameters:

Chromatographic column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5μm;

Mobile phase A: an aqueous solution containing 0.1% formic acid + 2 mM ammonium formate;

Mobile phase B: methanol solution containing 0.1v/v% formic acid + 15v/v% isopropanol, flow rate: 0.8mL/min, injection volume: 5.0μL, split ratio 1:4 injection, the gradient is shown in the table below:

Positive and negative ion mode gradient elution program

Mass spectrometry parameters: mass spectrometry is measured in full scan in positive/negative ion conversion mode, mass range: m/z 200～1500, resolution 70000, automatic gain control target value 5*e ⁵ ; negative ion mode 2900V, positive ion mode 3800V, ion The temperature of the transfer tube is 350°C, the flow rate of nitrogen gas for desolvation gas is 35L/h, the flow rate of nitrogen gas for auxiliary gas is 15L/h, and the temperature of the gasification chamber is 400°C; the instrument is calibrated for positive and negative ions before the sample runs; the second stage adopts automatic triggering mode, resolution 35,000, automatic gain control target value 2*e ⁵ , collision energy range 10-80%, retention time ±1.0min according to target retention time;

C data analysis

C1 draws the standard curve of the standard:

Take α-carotene, β-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combxanthin, cryptoxanthin, diacoxanthin, dihydroleaf Flavin, echinacea, fucoflavonol, neoxanthin, polydinoxanthin, polydinoxanthinol, uric acid lactone, mussel flavin, zeaxanthin, scallopone, diaoxanthin, hydroxyl Echinacea, dinoxanthin, pectoxanthin, cryptoxanthin, linoleate astaxanthin monoester, dipalmitate astaxanthin ester were prepared into mixed standard working solutions of different concentrations, which were the same as in step B The conditions are tested, and the high performance liquid chromatogram of the standard working solution is obtained. For each standard product, according to its concentration and peak area, draw a standard curve to obtain the standard curve of the above standard product;

C2 For carotenoid mono- and di-esters without standard products, according to the number of fatty acid chains, the standard curve of linoleic acid astaxanthin monoester or dipalmitate astaxanthin ester standard curve with similar structure is selected for relative quantitative analysis;

C3 according to the high-performance liquid chromatogram of the sample solution to be detected obtained in step (B), the standard curve of the corresponding standard substance, the standard curve of linoleic acid astaxanthin monoester, the standard curve of dipalmitate astaxanthin, calculate the standard curve of astaxanthin linoleate Measure the concentration of each component to be tested in the sample solution, and calculate the content of carotenoids in the aquatic product to be tested with formula (1):

X=C*V/m*1000

where:

X - the content of the analyte in the sample, the unit is mg/kg;

C - The concentration of the analyte in the sample treatment solution, calculated according to the standard curve, the standard curve of astaxanthin linoleate or the standard curve of astaxanthin dipalmitate, the unit is μg/L

V—constant volume, the unit is mL;

m - the volume or mass of the sample, in g.

Further, the aquatic product to be detected in step (A1) is one of fish, shrimp, crab, and shells.

Further, the concentrations of the mixed standard working solutions of different concentrations in step C1 are respectively 0 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL.

Further, the carotenoid esters without standard products in step C2 include astaxanthin diester-20:3/20:1, astaxanthin diester-20:3/18:2, astaxanthin diester- 22:6/18:1, Astaxanthin diester-20:4/20:5, Astaxanthin diester-20:5/20:5, Astaxanthin diester-22:6/16:0, Astaxanthin diester-22:6/16:1, Astaxanthin diester-20:5/16:0, Astaxanthin diester-22:6/14:0, Astaxanthin diester-20: 5/16:1, Astaxanthin diester-18:1/16:0, Astaxanthin diester-16:0/16:0, Astaxanthin diester-22:6/20:5, Astaxanthin diester Astaxanthin Diester-20:5/18:1, Astaxanthin Monoester-18:2, Astaxanthin Monoester-16:0, Astaxanthin Monoester-18:1, Astaxanthin Monoester-22: 6. Astaxanthin monoester-20:1, astaxanthin monoester-20:5, astaxanthin monoester-16:1, astaxanthin monoester-20:4, astaxanthin diester-22: 6/16:0, Calendula flavin diester-22:6-20:6 and Calendula flavin diester-22:6-16:0.

The difference between the high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) technique used in this application and the conventional triple quadrupole mass spectrometry is that it does not require the characteristics of the compounds to be tested to be known ion fragments, but directly use high-precision mass (resolution > 70000, m/z 200) to perform directional and non-directional full-scan detection of the sample to be tested. Some full scan data is sufficient, which is especially suitable for simultaneous screening of multiple components and qualitative and quantitative analysis of unknowns.

Beneficial effects: The high-throughput detection method of carotenoids in aquatic products disclosed by the present invention has the following beneficial effects:

1. The lower limit for the determination of carotenoids is 0.05mg/kg, and the standard curve is within the linear range of 10ng/mL to 200ng/mL (regression coefficient R2>0.99);

2. The sample pretreatment combines solid-phase extraction and matrix dispersion purification technology. Through one pretreatment, the extraction and purification of three common carotenoids with very different physical and chemical properties are realized, which greatly improves the detection efficiency and reduces the Expensive time and reagent cost consumption;

3. The method was applied to aquatic products, and 50 kinds of carotenoids were detected, involving 25 kinds of non-esterified carotenoids: 2 kinds of free carotenoids and 23 kinds of oxygenated carotenoids; and 25 kinds of esterified carotenoids Carotene: 14 types of astaxanthin diesters, 8 types of astaxanthin monoesters, 1 type of astaxanthin diesters and 2 types of calendulaxanthin diesters, all of which can be detected quickly and accurately at one time. The detected carotenoids The types and contents are better than the existing detection methods, which is of great significance for improving the types of carotenoids in aquatic products and exploring their metabolic pathways.

Description of drawings

1 is a flow chart of a high-throughput detection method for carotenoids in aquatic products disclosed in the present invention.

Detailed ways:

The specific embodiments of the present invention will be described in detail below.

Instruments and materials

Q-Exactive quadrupole electrostatic field orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific Corporation), equipped with H-ESI II source. The liquid chromatography system is UltiMate3000 high pressure liquid chromatography with automatic sampler. Chromatographic column ZORBAX Eclipse XDB C8 column (2.1mm x 100mm, particle size 3.5um). Milli-Q high-purity water generator (Millipore, USA). Freeze dryer (SIGMA, Germany). Refrigerated centrifuge (SIGMA, Germany). Vortex shaker (Heldolph, Germany). Filter membrane (DIKMA, PTFE 0.22 μm). Waters Oasis HLB cartridge (6cc/200mg, activated with methanol before loading, water equilibrated).

Ammonium formate and formic acid (chromatographically pure, Sigma-Aldrich, USA). Other reagents (chromatographically pure, Merck, Germany). The experimental water was Milli-Q ultrapure water (18.2ΩM). Carotenoid standards were purchased from Sigma and Dr. Ehrenstorfer with a purity of ≥95%. Includes: Alpha-Carotene, Beta-Carotene, Astaxanthin, Fucoxanthin, Purple Zeaxanthin, Canthaxanthin, Lutein, Comoxanthin, Cryptoxanthin, Diaoxanthin, Dihydroxanthin Lutein, Echinenone, Fucoflavonol, Neoxanthin, Dinophytin, Dinoxanthin, Uricolide, Mussel Xanthophyll, Zeaxanthin, Scallopone, Diatoxanthin, Hydroxy echinacea, dinoxanthin, pectoxanthin, cryptoxanthin; esterified carotenoids include: linoleate astaxanthin monoester, dipalmitate astaxanthin ester.

Standard stock solution: Weigh an appropriate amount of standard product, dissolve in methanol except astaxanthin dipalmitate with dichloromethane and make up to volume with methanol, and prepare a standard stock solution of 0.1 mg/mL, -20 Store at ℃ protected from light.

Standard working solution: Pipette 100 μL of the standard stock solution of each component into a 10 mL volumetric flask, and make up to the mark with methanol, the concentration is 1.0 μg/mL.

Specific Example 1

A high-throughput detection method for carotenoids in aquatic products, comprising the following steps:

A. Pre-processing:

(A1) Weigh 1g of the aquatic product to be tested, add it to 10mL of methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol, shake for 5min, extract by ultrasonic for 20min, and then rotate at 12000r/min at 4 degrees Celsius Centrifuge for 10min, and obtain the organic phase and residue after completion;

(A2) Take the organic phase obtained in step (A1), add at least 4 times the volume of water to it to obtain a sample solution, and then purify the sample solution through an HLB column to obtain a purified sample solution, and then purge the sample with nitrogen. The purified sample solution was concentrated to <2.0ml, and then adjusted to 2.0ml by methanol solution containing 0.1wt% HF to obtain the purified sample solution after constant volume, wherein:

HLB column purification was carried out using methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol as eluent;

(A3) Add the remaining residue of step (A1) into 10 mL of methanol-dichloromethane solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and rotate at 12000 r/min Centrifuge at 4 degrees Celsius for 10 minutes, and obtain the organic phase and residue after completion, wherein:

In the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1:1;

(A4) take the organic phase obtained in step (A3), nitrogen blowing and concentrating until the volume does not change, then dilute to 2.0ml by a methanol solution containing 0.1wt% HF, and then add 1g neutral alumina powder to it and fully shake, Centrifuge at 12,000 r/min for 10 min at 10 degrees Celsius, and obtain supernatant and precipitate after completion;

(A5) merging the purified sample solution after constant volume obtained in step (A2) and the supernatant solution obtained in step (A4), and then passing through a filter membrane with a pore size of 0.22 μm to obtain the sample solution to be detected;

B. the sample solution to be detected obtained in step (A5) is detected by high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometer to obtain the high-efficiency liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows :

HPLC parameters:

Chromatographic column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5μm;

Mobile phase A: an aqueous solution containing 0.1% formic acid + 2 mM ammonium formate;

Mobile phase B: methanol solution containing 0.1v/v% formic acid + 15v/v% isopropanol,

Flow rate: 0.8mL/min, injection volume: 5.0μL, injection at split ratio 1:4, gradient shown in the table below:

Positive and negative ion mode gradient elution program

Mass spectrometry parameters: mass spectrometry is measured in full scan in positive/negative ion conversion mode, mass range: m/z 200～1500, resolution 70000, automatic gain control target value 5*e ⁵ ; negative ion mode 2900V, positive ion mode 3800V, ion The temperature of the transfer tube is 350°C, the flow rate of nitrogen gas for desolvation gas is 35L/h, the flow rate of nitrogen gas for auxiliary gas is 15L/h, and the temperature of the gasification chamber is 400°C; the instrument is calibrated for positive and negative ions before the sample runs; the second stage adopts automatic triggering mode, resolution 35,000, automatic gain control target value 2*e ⁵ , collision energy range 10-80%, retention time ±1.0min according to target retention time;

C data analysis

C1 draws the standard curve of the standard:

Take α-carotene, β-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combxanthin, cryptoxanthin, diacoxanthin, dihydroleaf Flavin, echinacea, fucoflavonol, neoxanthin, polydinoxanthin, polydinoxanthinol, uric acid lactone, mussel flavin, zeaxanthin, scallopone, diaoxanthin, hydroxyl Echinacea, dinoxanthin, pectoxanthin, cryptoxanthin, linoleate astaxanthin monoester, dipalmitate astaxanthin ester were prepared into mixed standard working solutions of different concentrations, which were the same as in step B The conditions are tested, and the high performance liquid chromatogram of the standard working solution is obtained. For each standard product, according to its concentration and peak area, draw a standard curve to obtain the standard curve of the above standard product;

C2 For carotenoid mono- and di-esters without standard products, according to the number of fatty acid chains, the standard curve of linoleic acid astaxanthin monoester or dipalmitate astaxanthin ester standard curve with similar structure is selected for relative quantitative analysis;

C3 according to the high-performance liquid chromatogram of the sample solution to be detected obtained in step (B), the standard curve of the corresponding standard substance, the standard curve of linoleic acid astaxanthin monoester, the standard curve of dipalmitate astaxanthin, calculate the standard curve of astaxanthin linoleate Measure the concentration of each component to be tested in the sample solution, and calculate the content of carotenoids in the aquatic product to be tested with formula (1):

X=C*V/m*1000

where:

X - the content of the analyte in the sample, the unit is mg/kg;

C - The concentration of the analyte in the sample treatment solution, calculated according to the standard curve, the standard curve of astaxanthin linoleate or the standard curve of astaxanthin dipalmitate, the unit is μg/L

V—constant volume, the unit is mL;

m - the volume or mass of the sample, in g.

Further, the aquatic product to be detected in step (A1) is fish.

Further, the concentrations of the mixed standard working solutions of different concentrations in step C1 are respectively 0 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL.

Further, the carotenoid esters without standard products in step C2 include astaxanthin diester-20:3/20:1, astaxanthin diester-20:3/18:2, astaxanthin diester- 22:6/18:1, Astaxanthin diester-20:4/20:5, Astaxanthin diester-20:5/20:5, Astaxanthin diester-22:6/16:0, Astaxanthin diester-22:6/16:1, Astaxanthin diester-20:5/16:0, Astaxanthin diester-22:6/14:0, Astaxanthin diester-20: 5/16:1, Astaxanthin diester-18:1/16:0, Astaxanthin diester-16:0/16:0, Astaxanthin diester-22:6/20:5, Astaxanthin diester Astaxanthin Diester-20:5/18:1, Astaxanthin Monoester-18:2, Astaxanthin Monoester-16:0, Astaxanthin Monoester-18:1, Astaxanthin Monoester-22: 6. Astaxanthin monoester-20:1, astaxanthin monoester-20:5, astaxanthin monoester-16:1, astaxanthin monoester-20:4, astaxanthin diester-22: 6/16:0, Calendula flavin diester-22:6-20:6 and Calendula flavin diester-22:6-16:0.

Specific embodiment 2

A high-throughput detection method for carotenoids in aquatic products, comprising the following steps:

A. Pre-processing:

(A1) Weigh 1 g of the aquatic product to be tested, add it to 10 mL of methanol solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and then rotate at 12000 r/min at 10 degrees Celsius Centrifuge for 5min, and obtain the organic phase and residue after completion;

(A2) Take the organic phase obtained in step (A1), add at least 4 times the volume of water to it to obtain a sample solution, and then purify the sample solution through an HLB column to obtain a purified sample solution, and then purge the sample with nitrogen. The purified sample solution was concentrated to <2.0ml, and then adjusted to 2.0ml by methanol solution containing 0.1wt% HF to obtain the purified sample solution after constant volume, wherein:

HLB column purification was carried out using methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol as eluent;

(A3) Add the remaining residue of step (A1) into 10 mL of methanol-dichloromethane solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and rotate at 12000 r/min Centrifuge at 10 degrees Celsius for 5 min, and obtain the organic phase and residue after completion, wherein:

In the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1:1;

(A4) take the organic phase obtained in step (A3), nitrogen blowing and concentrating until the volume does not change, then dilute to 2.0ml by a methanol solution containing 0.1wt% HF, and then add 1g neutral alumina powder to it and fully shake, Centrifuge at 12,000 r/min for 5 min at 10 degrees Celsius, and obtain supernatant and precipitate after completion;

(A5) merging the purified sample solution after constant volume obtained in step (A2) and the supernatant solution obtained in step (A4), and then passing through a filter membrane with a pore size of 0.22 μm to obtain the sample solution to be detected;

B. the sample solution to be detected obtained in step (A5) is detected by high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometer to obtain the high-efficiency liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows :

HPLC parameters:

Chromatographic column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5μm;

Mobile phase A: an aqueous solution containing 0.1% formic acid + 2 mM ammonium formate;

Mobile phase B: methanol solution containing 0.1v/v% formic acid + 15v/v% isopropanol,

Flow rate: 0.8mL/min, injection volume: 5.0μL, injection at split ratio 1:4, gradient shown in the table below:

Positive and negative ion mode gradient elution program

Mass spectrometry parameters: mass spectrometry is measured in full scan in positive/negative ion conversion mode, mass range: m/z 200～1500, resolution 70000, automatic gain control target value 5*e ⁵ ; negative ion mode 2900V, positive ion mode 3800V, ion The temperature of the transfer tube is 350°C, the flow rate of nitrogen gas for desolvation gas is 35L/h, the flow rate of nitrogen gas for auxiliary gas is 15L/h, and the temperature of the gasification chamber is 400°C; the instrument is calibrated for positive and negative ions before the sample runs; the second stage adopts automatic triggering mode, resolution 35,000, automatic gain control target value 2*e ⁵ , collision energy range 10-80%, retention time ±1.0min according to target retention time;

C data analysis

C1 draws the standard curve of the standard:

Take α-carotene, β-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combxanthin, cryptoxanthin, diacoxanthin, dihydroleaf Flavin, echinacea, fucoflavonol, neoxanthin, polydinoxanthin, polydinoxanthinol, uric acid lactone, mussel flavin, zeaxanthin, scallopone, diaoxanthin, hydroxyl Echinacea, dinoxanthin, pectoxanthin, cryptoxanthin, linoleate astaxanthin monoester, dipalmitate astaxanthin ester were prepared into mixed standard working solutions of different concentrations, which were the same as in step B The conditions are tested, and the high performance liquid chromatogram of the standard working solution is obtained. For each standard product, according to its concentration and peak area, draw a standard curve to obtain the standard curve of the above standard product;

C2 For carotenoid mono- and di-esters without standard products, according to the number of fatty acid chains, the standard curve of linoleic acid astaxanthin monoester or dipalmitate astaxanthin ester standard curve with similar structure is selected for relative quantitative analysis;

C3 according to the high-performance liquid chromatogram of the sample solution to be detected obtained in step (B), the standard curve of the corresponding standard substance, the standard curve of linoleic acid astaxanthin monoester, the standard curve of dipalmitate astaxanthin, calculate the standard curve of astaxanthin linoleate Measure the concentration of each component to be tested in the sample solution, and calculate the content of carotenoids in the aquatic product to be tested with formula (1):

X=C*V/m*1000

where:

X - the content of the analyte in the sample, the unit is mg/kg;

C - The concentration of the analyte in the sample treatment solution, calculated according to the standard curve, the standard curve of astaxanthin linoleate or the standard curve of astaxanthin dipalmitate, the unit is μg/L

V—constant volume, the unit is mL;

m - the volume or mass of the sample, in g.

Further, the aquatic product to be detected in step (A1) is crab. Of course, in another embodiment, the aquatic product to be detected in step (A1) is a shell.

Further, the concentrations of the mixed standard working solutions of different concentrations in step C1 are respectively 0 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL.

Further, the carotenoid esters without standard products in step C2 include astaxanthin diester-20:3/20:1, astaxanthin diester-20:3/18:2, astaxanthin diester- 22:6/18:1, Astaxanthin diester-20:4/20:5, Astaxanthin diester-20:5/20:5, Astaxanthin diester-22:6/16:0, Astaxanthin diester-22:6/16:1, Astaxanthin diester-20:5/16:0, Astaxanthin diester-22:6/14:0, Astaxanthin diester-20: 5/16:1, Astaxanthin diester-18:1/16:0, Astaxanthin diester-16:0/16:0, Astaxanthin diester-22:6/20:5, Astaxanthin diester Astaxanthin Diester-20:5/18:1, Astaxanthin Monoester-18:2, Astaxanthin Monoester-16:0, Astaxanthin Monoester-18:1, Astaxanthin Monoester-22: 6. Astaxanthin monoester-20:1, astaxanthin monoester-20:5, astaxanthin monoester-16:1, astaxanthin monoester-20:4, astaxanthin diester-22: 6/16:0, Calendula flavin diester-22:6-20:6 and Calendula flavin diester-22:6-16:0.

Specific embodiment 3 (method one)

A high-throughput detection method for carotenoids in aquatic products, comprising the following steps:

A. Pre-processing:

(A1) Weigh 1g of the aquatic product to be tested, add it to 10mL of methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol, shake for 5min, extract by ultrasonic for 20min, and then rotate at 12000r/min at 5 degrees Celsius Centrifuge for 8min, obtain organic phase and residue after completion;

(A2) Take the organic phase obtained in step (A1), add at least 4 times the volume of water to it to obtain a sample solution, and then purify the sample solution through an HLB column to obtain a purified sample solution, and then purge the sample with nitrogen. The purified sample solution was concentrated to <2.0ml, and then adjusted to 2.0ml by methanol solution containing 0.1wt% HF to obtain the purified sample solution after constant volume, wherein:

HLB column purification was carried out using methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol as eluent;

(A3) Add the remaining residue of step (A1) into 10 mL of methanol-dichloromethane solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and rotate at 12000 r/min Centrifuge at 6 degrees Celsius for 7 minutes, and obtain the organic phase and residue after completion, wherein:

In the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1:1;

(A4) take the organic phase obtained in step (A3), nitrogen blowing and concentrating until the volume does not change, then dilute to 2.0ml by a methanol solution containing 0.1wt% HF, and then add 1g neutral alumina powder to it and fully shake, Centrifuge at 12000r/min at 6 degrees Celsius for 8min, and obtain supernatant and precipitate after completion;

(A5) merging the purified sample solution after constant volume obtained in step (A2) and the supernatant solution obtained in step (A4), and then passing through a filter membrane with a pore size of 0.22 μm to obtain the sample solution to be detected;

B. the sample solution to be detected obtained in step (A5) is detected by high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometer to obtain the high-efficiency liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows :

HPLC parameters:

Chromatographic column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5μm;

Mobile phase A: an aqueous solution containing 0.1% formic acid + 2 mM ammonium formate;

Mobile phase B: methanol solution containing 0.1v/v% formic acid + 15v/v% isopropanol, flow rate: 0.8mL/min, injection volume: 5.0μL, split ratio 1:4 injection, the gradient is shown in the table below:

Positive and negative ion mode gradient elution program

Mass spectrometry parameters: mass spectrometry is measured in full scan in positive/negative ion conversion mode, mass range: m/z 200～1500, resolution 70000, automatic gain control target value 5*e ⁵ ; negative ion mode 2900V, positive ion mode 3800V, ion The temperature of the transfer tube is 350°C, the flow rate of nitrogen gas for desolvation gas is 35L/h, the flow rate of nitrogen gas for auxiliary gas is 15L/h, and the temperature of the gasification chamber is 400°C; the instrument is calibrated for positive and negative ions before the sample runs; the second stage adopts automatic triggering mode, resolution 35,000, automatic gain control target value 2*e ⁵ , collision energy range 10-80%, retention time ±1.0min according to target retention time;

C data analysis

C1 draws the standard curve of the standard:

Take α-carotene, β-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combxanthin, cryptoxanthin, diacoxanthin, dihydroleaf Flavin, echinacea, fucoflavonol, neoxanthin, polydinoxanthin, polydinoxanthinol, uric acid lactone, mussel flavin, zeaxanthin, scallopone, diaoxanthin, hydroxyl Echinacea, dinoxanthin, pectoxanthin, cryptoxanthin, linoleate astaxanthin monoester, dipalmitate astaxanthin ester were prepared into mixed standard working solutions of different concentrations, which were the same as in step B The conditions are tested, and the high performance liquid chromatogram of the standard working solution is obtained. For each standard product, according to its concentration and peak area, draw a standard curve to obtain the standard curve of the above standard product;

C2 For carotenoid mono- and di-esters without standard products, according to the number of fatty acid chains, the standard curve of linoleic acid astaxanthin monoester or dipalmitate astaxanthin ester standard curve with similar structure is selected for relative quantitative analysis;

C3 according to the high-performance liquid chromatogram of the sample solution to be detected obtained in step (B), the standard curve of the corresponding standard substance, the standard curve of linoleic acid astaxanthin monoester, the standard curve of dipalmitate astaxanthin, calculate the standard curve of astaxanthin linoleate Measure the concentration of each component to be tested in the sample solution, and calculate the content of carotenoids in the aquatic product to be tested with formula (1):

X=C*V/m*1000

where:

X - the content of the analyte in the sample, the unit is mg/kg;

C - The concentration of the analyte in the sample treatment solution, calculated according to the standard curve, the standard curve of astaxanthin linoleate or the standard curve of astaxanthin dipalmitate, the unit is μg/L

V—constant volume, the unit is mL;

m - the volume or mass of the sample, in g.

Further, the aquatic product to be detected in step (A1) is shrimp.

Further, the concentrations of the mixed standard working solutions of different concentrations in step C1 are respectively 0 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL.

Further, the carotenoid esters without standard products in step C2 include astaxanthin diester-20:3/20:1, astaxanthin diester-20:3/18:2, astaxanthin diester- 22:6/18:1, Astaxanthin diester-20:4/20:5, Astaxanthin diester-20:5/20:5, Astaxanthin diester-22:6/16:0, Astaxanthin diester-22:6/16:1, Astaxanthin diester-20:5/16:0, Astaxanthin diester-22:6/14:0, Astaxanthin diester-20: 5/16:1, Astaxanthin diester-18:1/16:0, Astaxanthin diester-16:0/16:0, Astaxanthin diester-22:6/20:5, Astaxanthin diester Astaxanthin Diester-20:5/18:1, Astaxanthin Monoester-18:2, Astaxanthin Monoester-16:0, Astaxanthin Monoester-18:1, Astaxanthin Monoester-22: 6. Astaxanthin monoester-20:1, astaxanthin monoester-20:5, astaxanthin monoester-16:1, astaxanthin monoester-20:4, astaxanthin diester-22: 6/16:0, Calendula flavin diester-22:6-20:6 and Calendula flavin diester-22:6-16:0.

Method Two:

A. Pre-processing steps are as follows:

(1) Weigh 1.00 g of sample, add 10 mL of methanol solution (containing 0.1% BHT), shake for 5 min, ultrasonically extract for 20 min, and centrifuge at low temperature and high speed at 12000 r/min to obtain the organic layer and residue; (2) Take out the supernatant, nitrogen Blow and concentrate to <2.0ml, and dilute to 2.0ml with methanol (containing 0.1wt% HF); (3) add 10mL methanol-dichloromethane (1:1, v:v, containing 0.1wt% BHT) solution to the remaining residue, Shake for 5 min, ultrasonically extract for 20 min, and centrifuge at 12,000 r/min at low temperature and high speed to obtain the organic layer and residue; (4) Take the upper organic phase, concentrate it to dryness with nitrogen blowing, and dilute to 2.0 ml with methanol (containing 0.1 wt% HF). 12000r/min low-temperature high-speed centrifugation to obtain supernatant and precipitate; (5) Combine (2) and (4) supernatant, and pass through 0.22 μm filter membrane.

Step B and Step C are the same as method 1 (specific embodiment 3), and will not be repeated here.

Method three:

A. Pre-processing steps are as follows:

(1) Weigh 1.00 g of sample, add 10 mL of acetone solution (containing 0.1 wt% BHT), shake for 5 min, extract ultrasonically for 20 min, and centrifuge at 12,000 r/min at low temperature and high speed to obtain the organic layer and residue; (2) Repeat the extraction, combine the The supernatant was concentrated to <4.0ml by nitrogen blowing, and methanol (containing 0.1% wtHF) was made up to 4.0ml, and passed through a 0.22 μm filter membrane.

Step B and Step C are the same as method 1 (specific embodiment 3), and will not be repeated here.

Method four:

A. Pre-processing steps are as follows:

(1) Weigh 1.00 g of sample, add 10 mL of ethyl acetate (containing 0.1 wt% BHT) solution, shake for 5 min, ultrasonically extract for 20 min, and centrifuge at low temperature and high speed at 12000 r/min to obtain an organic layer and residue; (2) Repeat the extraction, The supernatants were combined, concentrated to near dryness by nitrogen blowing, and methanol (containing 0.1 wt% HF) was made up to 4.0 ml, and passed through a 0.22 μm filter membrane.

Step B and Step C are the same as method 1 (specific embodiment 3), and will not be repeated here.

Verification test:

The pretreatment method established above was used to screen the carotenoids in common aquatic products, black tiger shrimp. The results show (see table below), where:

Method 1: Use fractional extraction (methanol solution and methanol-dichloromethane solution sequentially extract), step-by-step purification (purify according to the matrix properties of the extract, combined with HLB column purification and neutral alumina powder degreasing), a total of 50 carotenoids were detected, including 25 non-esterified carotenoids and 25 esterified carotenoids, with a total content value of 2055±199 mg/kg, which fully verified the feasibility of the extraction and purification method.

Method 2: Due to the lack of corresponding purification steps, the extract has a strong matrix inhibitory effect, resulting in the failure to detect low-content carotenoids. A total of 24 carotenoids were detected, including 10 non-esterified carotenoids and 10 esterified carotenoids. There are 14 kinds of carotene, and the total content value is 1052±102mg/kg.

Method 3: Medium-polarity acetone was used as the extraction solution for pretreatment, which could not guarantee the extraction rate of low-polarity carotenoid esters. A total of 22 carotenoids were detected, including 16 non-esterified carotenoids and 16 esterified carotenoids. There are 6 kinds of elements, and the total content value is 716±80.3mg/kg; the same.

Method 4: The pretreatment uses low-polarity ethyl acetate as the extraction solution. The simultaneously extracted oils and fats have a strong ionization inhibitory effect, resulting in the reduction or failure to detect the low content of carotenoid esters. The extraction rate of medium-polar non-esterified carotenoids and their metabolites cannot be guaranteed. A total of 19 carotenoids were detected, including 5 non-esterified carotenoids and 14 esterified carotenoids, with a total content value of 346± 29.6 mg/kg.

To sum up, the method has strong compatibility with compounds of different aquatic product matrices and different physicochemical properties. The carotenoids detected by this method are more abundant and the content values are more accurate. The comparison (method 3 and method 4) fully verified the high throughput of the detection method (see the table below).

Types and contents of carotenoids detected under 4 treatments (n=6)

"-" means content <0.05mg/kg

The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above-mentioned embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims (3)

1. a high-throughput detection method of carotenoid in aquatic products, is characterized in that, comprises the following steps: A. Pre-processing: (A1) Weigh 1 g of the aquatic product to be tested, add it to 10 mL of methanol solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and then rotate at 12000 r/min for 4 to Centrifuge at 10 degrees Celsius for 5-10 minutes, and obtain organic phase and residue after completion; (A2) Take the organic phase obtained in step (A1), add at least 4 times the volume of water to it to obtain a sample solution, and then purify the sample solution through an HLB column to obtain a purified sample solution, and then purge the sample with nitrogen. The purified sample solution was concentrated to <2.0ml, and then adjusted to 2.0ml by methanol solution containing 0.1wt% HF to obtain the purified sample solution after constant volume, wherein: HLB column purification was carried out using methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol as eluent; (A3) Add the remaining residue of step (A1) into 10 mL of methanol-dichloromethane solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, extract by ultrasonic for 20 min, and rotate at 12000 r/min Centrifuge at 4 to 10 degrees Celsius for 5 to 10 minutes, and obtain organic phase and residue after completion, wherein: In the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1:1; (A4) take the organic phase obtained in step (A3), nitrogen blowing and concentrating until the volume does not change, then dilute to 2.0ml by a methanol solution containing 0.1wt% HF, and then add 1g neutral alumina powder to it and fully shake, Centrifuge at 4 to 10 degrees Celsius for 5 to 10 minutes at a speed of 12000 r/min, and obtain supernatant and precipitate after completion; (A5) merging the purified sample solution after constant volume obtained in step (A2) and the supernatant solution obtained in step (A4), and then passing through a filter membrane with a pore size of 0.22 μm to obtain the sample solution to be detected; B. the sample solution to be detected obtained in step (A5) is detected by high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometer to obtain the high-efficiency liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows : HPLC parameters: Chromatographic column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5μm; Mobile phase A: an aqueous solution containing 0.1% formic acid + 2 mM ammonium formate; Mobile phase B: methanol solution containing 0.1v/v% formic acid + 15v/v% isopropanol, flow rate: 0.8mL/min, injection volume: 5.0μL, split ratio 1:4 injection, the gradient is shown in the table below: Positive and negative ion mode gradient elution program

Mass spectrometry parameters: mass spectrometry is measured in full scan in positive/negative ion conversion mode, mass range: m/z 200～1500, resolution 70000, automatic gain control target value 5*e ⁵ ; negative ion mode 2900V, positive ion mode 3800V, ion The temperature of the transfer tube is 350°C, the flow rate of nitrogen gas for desolvation gas is 35L/h, the flow rate of nitrogen gas for auxiliary gas is 15L/h, and the temperature of the gasification chamber is 400°C; the instrument is calibrated for positive and negative ions before the sample runs; the second stage adopts automatic triggering mode, resolution 35,000, automatic gain control target value 2*e ⁵ , collision energy range 10-80%, retention time ±1.0min according to target retention time; C data analysis C1 draws the standard curve of the standard: Take α-carotene, β-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combxanthin, cryptoxanthin, diacoxanthin, dihydroleaf Flavin, echinacea, fucoflavonol, neoxanthin, polydinoxanthin, polydinoxanthinol, uric acid lactone, mussel flavin, zeaxanthin, scallopone, diaoxanthin, hydroxyl Echinacea, dinoxanthin, pectoxanthin, cryptoxanthin, linoleate astaxanthin monoester, dipalmitate astaxanthin ester were prepared into mixed standard working solutions of different concentrations, which were the same as in step B The conditions are tested, and the high performance liquid chromatogram of the standard working solution is obtained. For each standard product, according to its concentration and peak area, draw a standard curve to obtain the standard curve of the above standard product; C2 For carotenoid mono- and di-esters without standard products, according to the number of fatty acid chains, the standard curve of linoleic acid astaxanthin monoester or dipalmitate astaxanthin ester standard curve with similar structure is selected for relative quantitative analysis; C3 according to the high-performance liquid chromatogram of the sample solution to be detected obtained in step (B), the standard curve of the corresponding standard substance, the standard curve of linoleic acid astaxanthin monoester, the standard curve of dipalmitate astaxanthin, calculate the standard curve of astaxanthin linoleate Measure the concentration of each component to be tested in the sample solution, Calculate the content of carotenoids in the aquatic product to be detected with formula (1): X=C*V/m*1000 where: X - the content of the analyte in the sample, the unit is mg/kg; C - The concentration of the analyte in the sample treatment solution, calculated according to the standard curve, the standard curve of astaxanthin linoleate or the standard curve of astaxanthin dipalmitate, the unit is μg/L V—constant volume, the unit is mL; m - the volume or mass of the sample, in g, where: Carotenoid esters without standard in step C2 include astaxanthin diester-20:3/20:1, astaxanthin diester-20:3/18:2, astaxanthin diester-22:6 /18:1, Astaxanthin diester-20:4/20:5, Astaxanthin diester-20:5/20:5, Astaxanthin diester-22:6/16:0, Astaxanthin Diester-22:6/16:1, Astaxanthin diester-20:5/16:0, Astaxanthin diester-22:6/14:0, Astaxanthin diester-20:5/16 :1, Astaxanthin diester-18:1/16:0, Astaxanthin diester-16:0/16:0, Astaxanthin diester-22:6/20:5, Astaxanthin diester -20:5/18:1, Astaxanthin Monoester-18:2, Astaxanthin Monoester-16:0, Astaxanthin Monoester-18:1, Astaxanthin Monoester-22:6, Shrimp Astaxanthin Monoester-20:1, Astaxanthin Monoester-20:5, Astaxanthin Monoester-16:1, Astaxanthin Monoester-20:4, Astaxanthin Diester-22:6/16 : 0, Calendula officinalis diester-22:6-20:6 and Calendula officinalis diester-22:6-16:0. 2. the high-throughput detection method of carotenoid in a kind of aquatic product as claimed in claim 1, is characterized in that, the aquatic product to be detected in step (A1) is a kind of in fish, shrimp, crab, shell . 3. the high-throughput detection method of carotenoid in a kind of aquatic product as claimed in claim 1, is characterized in that, the concentration of the mixed standard working solution of different concentrations in step C1 is respectively 0ng/mL, 10ng/mL mL, 20ng/mL, 50ng/mL, 100ng/mL and 200ng/mL.

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