CN106706814B - The method of random measurement chain compound containing EPA - Google Patents

Abstract

, sample pre-treatments the invention discloses a kind of random methods of measurement chain compound containing EPA, comprising the following steps: 1): carassias auratus muscLe tissue being carried out pre-treatment, obtains lipid crude extract；2), sample introduction: lipid crude extract organic solvent constant volume, filtering are injected into mass spectrum using needle pump syringe；3), Mass Spectrometer Method: negative ion mode is used, all parent ions containing the daughter ion are found according to feature daughter ion, use Analyst software collection data；4), data are analyzed: spectrogram obtained by step 3) Mass Spectrometer Method being analyzed, the structural information of each quasi-molecular ions is obtained.All compounds containing EPA fatty acid chain in sample can be known by non-directional at random using of the invention.

Description

Method for random determination of EPA chain-containing compounds

technical field

The invention belongs to the field of instrument analysis, and relates to a method for randomly measuring compounds containing EPA chains, in particular to a detection and analysis method for randomly scanning compounds containing EPA fatty acid chains from complex samples.

Background technique

EPA (Eicosapentaenoic Acid), namely eicosapentaenoic acid, is the main component of fish oil. EPA belongs to the omega-3 series of polyunsaturated fatty acids, that is, polyunsaturated fatty acids whose first double bond position is at the third carbon position from the methyl end. EPA is an important nutrient element that is indispensable to the human body but cannot be synthesized by itself, so it is called an essential fatty acid for the human body. Although linolenic acid can be converted into EPA in the human body, this reaction is very slow in the human body and the amount of conversion is very small, which is far from meeting the needs of the human body for EPA, so it must be supplemented directly from food.

Medical and nutritional studies have shown that EPA has the functions of inhibiting platelet aggregation, antithrombosis, relaxing blood vessels, adjusting blood lipids, etc., and preventing cardiovascular and cerebrovascular diseases. EPA peroxides and free radicals can inhibit the expression of tumor cells and shorten the telomeres of chromosomes. Promote apoptosis of tumor cells. EPA reduces harmful immune responses and is effective in treating inflammation caused by autoimmune deficiencies. Omega-3 fatty acids, including EPA, play a positive role in the treatment of lung disease, kidney disease, type 2 diabetes, colorectal ulcers and Crohn's disease. Recently, it has been found that it also has the functions of promoting the growth and development of brain cells, improving brain function, improving memory and learning ability, enhancing retinal reflection ability, and preventing and treating senile dementia, etc. to improve the quality of life. Therefore, the extraction and separation of high-purity EPA has become a hot research topic in developed countries.

Commercial sources of EPA are mainly marine fish with high fat content, and the mass fraction of EPA can reach 20% to 30%. With the change of sea conditions and the continuous increase of sea fishing intensity, the structure of sea fishery resources has undergone major changes. Factors such as environmental pollution and overfishing have caused resource shortages, prompting people to actively seek and develop new resources rich in EPA.

At present, the relevant detection methods of EPA are mainly based on gas chromatography-mass spectrometry, which mainly detects free EPA and methylated or ethylated EPA. The detection method of EPA-containing compounds is mainly liquid chromatography-mass spectrometry. The currently reported methods are all directional screening methods, that is, it is known that compound A has an EPA chain, and compound A is directional screened from samples according to the chemical or physical characteristics of A. The random non-directional screening method has not been reported.

The establishment of a method for the random determination of EPA chain-containing compounds, rapid scanning of compounds containing EPA fatty acid chains in complex samples, and structural identification and content determination is conducive to the rapid screening of EPA raw material resources and the promotion of the development of the EPA health care product industry.

Contents of the invention

The technical problem to be solved by the invention is to provide a simple and easy-to-operate method for randomly measuring EPA chain-containing compounds in a complex matrix.

In order to solve the problems of the technologies described above, the present invention provides a method for random determination of compounds containing EPA chains, comprising the following steps:

1), sample pretreatment:

Wash the crucian carp muscle tissue and grind it into mince, add organic solvent Ⅰ to the mince and extract by shaking, after the extraction, add pure water and shake to mix;

The resulting mixture was centrifuged with a refrigerated centrifuge, the lower phase solution was removed, and the lower phase solution was dried (low-temperature blow-drying with nitrogen gas) to obtain a crude lipid extract;

The organic solvent I is a mixture of methanol: chloroform = 1:2 volume ratio or a mixture of methanol: dichloromethane = 1:2 volume ratio, and the ratio of solid to organic solvent I is 1g/15 ~ 30ml; The amount of water added is 0.5 volume times of the organic solvent I;

2), sample injection:

Dilute the crude lipid extract to 100 mL with an organic solvent with a volume ratio of methanol:dichloromethane=1:2 as a sample;

Pipette 1 mL of the sample through a 0.22 μm filter membrane as the sample solution to be tested, and inject it into the mass spectrometer using a needle pump syringe;

3), mass spectrometry detection:

Use the negative ion mode to find all precursor ions containing the product ion according to the characteristic product ion. The scanning range is 400-1000Da, the dwell time is 2 seconds, the curtain gas is 10psi, the accelerating voltage is -4500ev, the auxiliary gas I is 20psi, and the auxiliary gas is 20psi. The gas II is 30psi, and the spectrum is accumulated 100 to 200 times; use Analyst software to collect data;

4), data analysis:

The spectrogram obtained in the step 3) mass spectrometry detection is analyzed to obtain the structural information of each ion peak. That is, the type and amount of compounds containing EPA chains in the sample can be known.

As the improvement of the method for stochastic determination of the present invention containing EPA chain compound:

Described step 4) is:

The spectrogram obtained in step 3) mass spectrometry detection is subjected to noise filtering (the minimum peak width is 0.2Da), the baseline is subtracted by 4Da, and the isotope peak is removed to obtain the processed spectrogram; the processed spectrogram is exported as a txt file and then used Lipid MassSpec. The structural information of each ion peak can be obtained after identification by Prediction software.

Remarks: Take PE 18:1/20:5 as an example, the structure includes 3 parts of information, PE refers to phosphatidylethanolamines, 18:1 refers to one of the fatty acid chains is 18 carbons and 1 double bond, 20:5 refers to another chain.

As a further improvement of the method for stochastic determination of the present invention containing EPA chain compounds:

Described step 4) also comprises:

The concentration of each parent ion is calculated by the formula: Calculate, where Abundance _i is the abundance of the compound, A _i is the peak area of the specific ion peak in the spectrum (step 3) mass spectrometry detection spectrum), and A ₁ ~ A _n are the peaks of all ion peaks obtained in the spectrum area.

The approximate content of the EPA chain-containing compound in the sample can be known by using the above formula.

As a further improvement of the method for stochastic determination of the present invention containing EPA chain compounds:

In the step 1), replace the aquatic product tissue with the centrifuged product after removing the lower phase solution (that is, including the upper phase solution and solids), and replace the organic solvent I with dichloromethane or chloroform in the organic solvent I; The extraction was repeated 1 to 3 times; all the lower phase solutions obtained after extraction were combined for subsequent drying and reconstitution.

Remarks: After centrifugation, the solution in the centrifuge tube is layered up and down, and the salmon muscle tissue sample is in the middle of the two phases in a cake shape.

Methanol, chloroform, and water are immiscible when they are together, and will be divided into upper and lower phases. Methanol is more polar and miscible with water. Therefore, in a centrifuge tube, a small amount of methanol is miscible with dichloromethane or chloroform in the lower phase. , a large amount of methanol is water-miscible in the upper phase; since most of the methanol is still in the upper phase after removing the lower phase, it is only necessary to add dichloromethane or chloroform during repeated extraction. That is, the volume consumption of dichloromethane or chloroform=the volume consumption of dichloromethane or chloroform in the machine solvent I during repeated extraction. Dichloromethane and chloroform have similar extraction efficiencies, and dichloromethane is less toxic than chloroform.

As a further improvement of the method for stochastic determination of the present invention containing EPA chain compounds:

In the step 1), centrifuging with a refrigerated centrifuge is as follows: centrifuging at 4° C., 8000-12000 rpm for 5-15 minutes.

As a further improvement of the method for stochastic determination of the present invention containing EPA chain compounds:

In the step 2), the flow rate of the needle pump syringe is 10-20 μL/min.

As a further improvement of the method for stochastic determination of the present invention containing EPA chain compounds:

In the step 3), the scanning method used is Precursor ion scan, the characteristic product ion is m/z 301, the declustering voltage is -80～-100ev, and the collision voltage is -30～-40ev .

To sum up, the present invention uses m/z 301 as the characteristic product ion to scan complex matrix samples containing EPA chain compounds through the negative ion mode to scan the complex matrix sample, and after spectral processing and data analysis, all the EPA-containing Compounds of fatty acid chains. The invention has the steps of sample pretreatment, sample injection, mass spectrometry detection and data analysis, simple operation, easy training, strong qualitative and quantitative ability, strong practicability, and is suitable for wide use in laboratories.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is the mass spectrum of crucian carp tissue samples scanned by the son-to-mother scanning method in negative ion mode.

Detailed ways

The specific embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that those skilled in the art can more clearly understand how to practice the present invention. It should be understood that while the invention has been described in connection with preferred specific embodiments thereof, these embodiments are illustrative only and are not intended to limit the scope of the invention.

Embodiment 1, a kind of method of randomly measuring the compound containing EPA chain, carries out the following steps successively:

1), sample pretreatment:

Wash 100g of crucian carp muscle tissue and grind it into mince. Take 1g of minced meat and add 21mL of organic solvent (methanol: dichloromethane = 1:2 volume ratio) and shake to extract. After the extraction, add 10.5mL of pure water and shake to mix;

The resulting mixture was centrifuged in a refrigerated centrifuge at 12000 rpm at 4°C for 5 minutes. Remove the lower phase solution, add 14mL dichloromethane to the remaining upper phase and solids, and repeat the extraction twice; combine all the lower phase solutions obtained after extraction, and dry (blow dry with nitrogen at low temperature) to obtain lipid crude extract;

2), sample injection:

Dilute the crude lipid extract to 100 mL with methanol:dichloromethane=1:2 volume ratio organic solvent as a sample. Pipette 1 mL of the sample through a 0.22 μm filter membrane and inject it into the mass spectrometer using a needle pump syringe with a flow rate of 20 μL/min.

3), mass spectrometry detection:

Using the negative ion mode, the scanning method used is Precursor ion scan, the characteristic product ion is m/z 301, the declustering voltage is -100ev, and the collision voltage is -40ev. Find all parent ions containing the product ion according to the characteristic product ion, the scanning range is 400-1000Da, the dwell time is 2 seconds, the curtain gas is 10psi, the accelerating voltage is -4500ev, the auxiliary gas I is 20psi, and the auxiliary gas II is 30psi , and the spectrum is accumulated 100 times. Data were collected using Analyst software.

4), data analysis:

The spectrogram obtained in step 3) was subjected to noise filtering (the minimum peak width was 0.2 Da), the baseline was subtracted by 4 Da, and the isotope peak was removed to obtain the processed spectrogram (Figure 1). Export the processed spectrum as a txt file and use Lipid MassSpec.Prediction software to identify the structure information of each ion peak. The concentration of each parent ion is calculated by the formula: Calculate, where Abundance _i is the abundance of the compound, A _i is the peak area of the specific ion peak in the spectrum (step 3) mass spectrometry detection spectrum), and A ₁ ~ A _n are the peaks of all ion peaks obtained in the spectrum area.

The result is as follows:

Crucian carp tissue samples are rich in lipid types, and there are many compounds containing EPA chains, all of which were found to be phospholipids after structural identification; a total of 31 phospholipid molecular species were identified, including 8 phosphatidylcholines, 14 phosphatidylethanolamines, and 1 Phosphatidylinositol, 4 phosphatidylserine, 3 phosphatidic acid, 1 phosphatidylglycerol. The strongest signal was at m/z 764.9, which was identified as an overlap of [PCo-16:0/20:5-H] ^- and [PE18:0/20:5-H] ^- . Specifically as described in Table 1.

Table 1. Structure and abundance information of compounds containing EPA chains in crucian carp tissue samples

Note: a:b/c:d represent the two fatty acid chain structures of the compound, one of which is 20:5, that is, the fatty acid chain with 20 carbon atoms contains 5 unsaturated double bonds; the prefix o- represents the alkyl chain, and the rest for the acyl chain.

Verification experiment: According to liquid chromatography tandem mass spectrometry, the sample crucian carp described in Example 1 is detected, and the obtained result is: a total of 31 kinds of phospholipid molecular species containing EPA fatty acid chains have been identified, including 8 phosphatidylcholines, 14 Phosphatidylethanolamine, 1 phosphatidylinositol, 4 phosphatidylserine, 3 phosphatidic acid, 1 phosphatidylglycerol, the relative standard deviation RSD of the abundance of each ion peak was ≤7%.

Comparative Example 1-1, changing the repeated extraction twice in Step 1) of Example 1 to 0 repeated extraction; the rest is the same as that of Example 1.

Comparative example 2-1, the refrigerated centrifuge condition in step 1) of embodiment 1 is 8000rpm, temperature, time are constant; All the other are equal to embodiment 1.

Comparative Example 3-1, the flow rate of the needle pump syringe in step 2) of Example 1 was set to 5 μL/min; the rest was the same as that of Example 1.

Comparative example 3-2, set the flow rate of the needle pump syringe in step 2) of Example 1 to 30 μL/min; the rest is the same as Example 1.

Comparative Example 4-1. The scanning method in step 3) of Example 1 was changed to product ion scanning (Product ionscan), and the characteristic product ion was m/z 764.9; the rest was the same as that of Example 1.

Comparative Example 4-2. The scanning method in step 3) of Example 1 is set to full scan (Q1scan), and the rest is the same as that of Example 1.

Comparative Example 4-3, changing the characteristic product ion m/z 301 in step 3) of Example 1 to m/z 300; the rest is the same as Example 1.

Comparative Example 4-4, changing the characteristic product ion m/z 301 in step 3) of Example 1 to m/z 302; the rest are the same as in Example 1.

Comparative Example 4-5, changing the declustering voltage -100ev in Step 3) of Example 1 to -60ev; the rest is the same as Example 1.

Comparative Examples 4-6, changing the declustering voltage -100ev in Step 3) of Example 1 to -120ev; the rest is the same as Example 1.

Comparative Examples 4-7, changing the collision voltage -40ev in Step 3) of Example 1 to -20ev; the rest is the same as Example 1.

Comparative Examples 4-8, changing the collision voltage -40ev in Step 3) of Example 1 to -50ev; the rest is the same as Example 1.

The EPA-containing compounds and their abundance obtained by the method described in the above comparative example are described in Table 2 below.

Table 2

In summary, a method for randomly measuring EPA chain-containing compounds of the present invention can quickly and effectively non-directionally scan and measure compounds containing EPA chains in aquatic product tissue samples such as crucian carp, and solve the problem of directional scanning in the prior art. Only target compounds, EPA raw material resource screening efficiency and other problems.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (4)

1. the method for measurement chain compound containing EPA at random, it is characterized in that the following steps are included:

1), sample pre-treatments:

It is ground into gruel after carassias auratus muscLe tissue is cleaned, extraction is shaken after organic solvent I is added in gruel, is added after extraction Pure water simultaneously shakes mixing；

Resulting mixture is centrifuged with refrigerated centrifuge, pipettes lower phase solution, and the lower phase solution is dry, obtains lipid crude extract；

Refrigerated centrifuge centrifugation are as follows: be centrifuged 5~15 minutes in 4 DEG C, 8000~12000rpm；

Organic solvent I is methanol: chloroform=1:2 volume ratio mixed liquor is methanol: methylene chloride=1:2 volume ratio is mixed Liquid is closed, the rotten solid-liquid ratio with organic solvent I is 1g/15~30ml；The amount of water be added is 0.5 volume times of organic solvent I；

2), sample introduction:

Using lipid crude extract methanol: methylene chloride=1:2 volume ratio organic solvent is settled to 100mL as sample；

It pipettes 1mL sample to cross after 0.22 μm of filter membrane as the sample solution to be tested, is injected into mass spectrum using needle pump syringe；

It is 10~20 μ L/min that needle, which pumps syringe flow velocity,；

3), Mass Spectrometer Method:

Using negative ion mode, all parent ions containing the daughter ion, scanning range 400- are found according to feature daughter ion 1000Da, residence time are 2 seconds, and gas curtain gas is 10psi, and acceleration voltage is -4500ev, and auxiliary gas I is 20psi, assist gas II For 30psi, map adds up 100~200 times；Use Analyst software collection data；

Used scan method is that son looks for female scanning, and feature daughter ion is m/z 301, and removing cluster voltage is -80~-100ev, is touched Hitting voltage is -30~-40ev；

4), data are analyzed:

Spectrogram obtained by step 3) Mass Spectrometer Method is analyzed, the structural information of each quasi-molecular ions is obtained.

2. the method for random measurement chain compound containing EPA according to claim 1, it is characterized in that:

The step 4) are as follows:

Spectrogram obtained by step 3) Mass Spectrometer Method is passed through into noise filtering, baseline deduction 4Da, after removing isotopic peak, after obtaining processing Spectrogram；It uses Lipid Mass Spec.Prediction software to identify after spectrogram after processing is exported as txt file, can obtain To the structural information of each quasi-molecular ions.

3. the method for random measurement chain compound containing EPA according to claim 2, it is characterized in that:

The step 4) further include:

Each parent ion concentration is through formula:It calculates, wherein Abundance_iFor Compound abundance, A_iFor the peak area at the specific ion peak in spectrogram, A₁~A_nTo own obtained in spectrogram The peak area of quasi-molecular ions.

4. the method for any random measurement chain compound containing EPA according to claim 1~3, it is characterized in that:

In the step 1), to pipette the substitution aquatic products tissue of the centrifugation gains after lower phase solution, in organic solvent I Methylene chloride or chloroform substitute organic solvent I；It repeats extraction 1~3 time；Lower phase solution resulting after all extractions is closed Subsequent drying and redissolution are carried out after and.