CN115326909A - Method for detecting free fatty acid in human serum - Google Patents
Method for detecting free fatty acid in human serum Download PDFInfo
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- CN115326909A CN115326909A CN202210745599.6A CN202210745599A CN115326909A CN 115326909 A CN115326909 A CN 115326909A CN 202210745599 A CN202210745599 A CN 202210745599A CN 115326909 A CN115326909 A CN 115326909A
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Abstract
The invention provides a detection method of free fatty acid in human serum, which comprises the following steps: adding various fatty acid standard substances and a condensing agent into a solvent to obtain a fatty acid solution for activating carboxyl; adding an isotope compound into the fatty acid solution, and performing condensation reaction on carboxyl of fatty acid and amino of the isotope compound to obtain a derivatized isotope internal standard solution; adding a condensing agent and a non-isotopic compound which is the same as the isotopic compound into a serum sample, wherein carboxyl of fatty acid in the serum sample and amino of the non-isotopic compound are subjected to condensation reaction to obtain a reacted solution; adding the isotope internal standard solution, and sending the isotope internal standard solution to a mass spectrometer for detection, thereby obtaining the content of the fatty acid in the serum sample. The invention has the advantages of low detection difficulty, high precision and the like.
Description
Technical Field
The invention relates to fatty acid, in particular to a method for detecting free fatty acid in human serum.
Background
Fatty acids are carboxylic acid compounds having a fatty chain, and are classified into short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids, and ultra-long-chain fatty acids according to the length of the carbon chain; depending on whether the chemical structure contains double bonds, saturated fatty acids and unsaturated fatty acids can be distinguished. Unsaturated fatty acid is a fatty acid constituting body fat, and is essential to the human body. Polyunsaturated fatty acids are essential components for the formation of cell membranes, participate in the synthesis of phospholipids, and are present in mitochondria and cell membranes. The unsaturated free fatty acid is closely related to the occurrence and development of cardiovascular, respiratory, digestive and endocrine system diseases and the energy metabolism of tumor diseases, and has the effects of regulating blood fat, resisting arrhythmia, resisting hypertension, regulating carbohydrate metabolism and the like. However, unsaturated fatty acids also have certain side effects on the human body. The trans-fatty acid has certain inhibition effect on the growth and development of young glumes: trans fatty acids can interfere with the metabolism of essential fatty acids; trans fatty acids can bind to lipids in the brain and inhibit the synthesis of long chain polyunsaturated fatty acids, thereby affecting the normal development of the central nervous system of infants; trans fatty acids inhibit the synthesis of prostaglandins in the mother and adversely affect the growth and development of infants.
For the detection of fatty acids, the following methods are known in the art:
1. at present, a great deal of literature reports that GC-MS is used for directly measuring fatty acid, but the application field is more to measure a plurality of common fatty acids in food, and a few patents report that GC-MS is used for measuring fatty acid in serum, but only a few short-chain fatty acids can be measured, and derivatization treatment may be needed. In addition, with GC-MS, the injection time is relatively long. Therefore, the measurement mode of GC-MS is limited in clinical application.
2. In addition, the method is reported in literature that GC-FID is used for determining fatty acid, the method is almost applied to detection in the food industry, derivatization treatment such as methyl esterification is needed, and the effect difference of different derivatization modes is large; meanwhile, the sensitivity is not enough, the pretreatment time is complicated, the sample injection time is long, and the data of the detection applied to clinic is rarely reported;
3. relevant data report that LC-MS derivatization is used for treating fatty acid, a plurality of pretreatment types are adopted, and the method is also a detection method which is applied to clinic more at present, however, due to the chemical structure characteristics of fatty acid, the ionization efficiency of the fatty acid in an electrospray ion source is poor, and the mass spectrum detection sensitivity of the fatty acid is poor. Therefore, a fatty acid detection pretreatment requires derivatization. However, different pretreatment and derivatization methods lead to very different detection effects.
Disclosure of Invention
In order to overcome the defects in the prior art scheme, the invention provides a method for detecting fatty acid.
The purpose of the invention is realized by the following technical scheme:
the method for detecting the free fatty acid in the human serum comprises the following steps:
adding various fatty acid standard substances and a condensing agent into a solvent to obtain a fatty acid solution for activating carboxyl;
adding an isotope compound into the fatty acid solution, and carrying out condensation reaction on carboxyl of fatty acid and amino of the isotope compound to obtain a derivative isotope internal standard solution;
adding a condensing agent and a non-isotopic compound which is the same as the isotopic compound into a serum sample, wherein carboxyl of fatty acid in the serum sample and amino of the non-isotopic compound are subjected to condensation reaction to obtain a reaction solution;
and adding the derivative isotope internal standard solution into the reaction solution, and sending the solution to a mass spectrometer for detection, thereby obtaining the content of free fatty acid in the serum sample.
Compared with the prior art, the invention has the beneficial effects that:
1. the detection process is simple;
the instability of a derivatization reagent system and the difficulty of a reaction system are reduced, so that unnecessary pretreatment steps are reduced, the process is simple, and the problems of easy interference and easy deterioration are solved;
2. the detection cost is low;
by using a cheap isotope compound, isotope internal standards corresponding to all analytes are prepared at the same time, the difficulty in purchasing the isotope internal standards is reduced, and the problems of inconsistent reaction efficiency and poor experimental reproducibility of substituting the isotope internal standards are solved;
3. the detection requirement is low;
the derivatization reagent is preferential, is not easily influenced by a derivatization system, such as water is avoided, side reaction salification is avoided, so that the derivatization efficiency is reduced, multi-step derivatization is not needed, the reaction system is mild, the pretreatment process is greatly simplified, and the detection requirement is reduced;
4. the detection precision is high;
the reaction system based on the preparation of the isotope internal standard can be used as a derivative system to change the physical and chemical characteristics of fatty acid, optimize the chromatographic behavior, enhance the ionization efficiency in the mass spectrum detection process and reduce the difficulty of the mass spectrum detection of the fatty acid.
Drawings
The disclosure of the present invention will become more readily understood with reference to the accompanying drawings. As is readily understood by those skilled in the art: these drawings are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of the present invention. In the figure:
fig. 1 is a schematic diagram of a method for detecting fatty acids according to an embodiment of the present invention.
Detailed Description
Fig. 1 and the following description depict alternative embodiments of the invention to teach those skilled in the art how to make and reproduce the invention. Some conventional aspects have been simplified or omitted for the purpose of teaching the present invention. Those skilled in the art will appreciate variations or substitutions from these embodiments that will be within the scope of the invention. Those skilled in the art will appreciate that the features described below can be combined in various ways to form multiple variations of the invention. Thus, the present invention is not limited to the following alternative embodiments, but is only limited by the claims and their equivalents.
Example 1:
fig. 1 is a schematic diagram of a method for detecting free fatty acids in human serum according to an embodiment of the present invention, and as shown in fig. 1, the method for detecting free fatty acids includes:
adding various fatty acid standard substances and a condensing agent into a solvent to obtain a fatty acid solution for activating carboxyl;
adding an isotope compound into the fatty acid solution, and performing condensation reaction on carboxyl of fatty acid and amino of the isotope compound to obtain a derivative isotope internal standard solution;
adding a condensing agent and a non-isotopic compound which is the same as the isotopic compound into a serum sample, wherein carboxyl of fatty acid in the serum sample and amino of the non-isotopic compound are subjected to condensation reaction to obtain a reaction solution;
and adding the derivative isotope internal standard solution into the reaction solution, and sending the solution to an analyzer for detection, thereby obtaining the content of free fatty acid in the serum sample.
To reduce the reaction requirements, further, the condensing agent is BOP.
To reduce detection requirements and cost, further, the isotopic compound is 3 picolylamine- β, β is an isotope, the non-isotopic compound is 3 picolylamine, and the isotope is an isotope of carbon, nitrogen, or hydrogen.
In order to reduce interference and improve detection accuracy, the derivatized internal standard solution is further purified by a separation column, and then the reaction solution is added.
In order to improve the detection precision, further, after the internal standard solution is added to the reaction solution, protein precipitation occurs, the reaction solution is vortexed and centrifuged, and the supernatant is aspirated and sent to the analyzer.
In order to improve the detection accuracy, further, the analyzer is a liquid chromatography tandem mass spectrometry system.
In order to improve the reaction efficiency and reaction effect, furthermore, in the fatty acid solution for obtaining the activated carboxyl, the reaction time is less than 30 minutes, and the reaction temperature is 30-60 ℃.
In order to improve the reaction efficiency and the reaction effect, further, the reaction temperature is 20-40 ℃ in the derivation internal standard solution and/or the reaction solution.
Example 2:
an application example of the method for detecting free fatty acids in human serum according to example 1 of the present invention.
In this application example, as shown in fig. 1, the method for detecting free fatty acids in this example is:
1. preparing an isotope internal standard;
(A) Preparing a standard solution mother solution by taking anhydrous acetonitrile as an organic solvent: hexadecenoic acid 800. Mu. Mol/L, octadecenoic acid 10mmol/L, octadecadienoic acid 6mmol/L, a-octadecatrienoic acid 1mmol/L, gamma-octadecatrienoic acid 200. Mu. Mol/L, eicosenoic acid 200. Mu. Mol/L, eicosatetraenoic acid 800. Mu. Mol/L, eicosapentaenoic acid 200. Mu. Mol/L, omega 3-docosapentaenoic acid 200. Mu. Mol/L, omega 6-docosapentaenoic acid 100. Mu. Mol/L, docosahexaenoic acid 800. Mu. Mol/L.
(B) And (2) taking anhydrous acetonitrile as a diluting solvent, diluting the standard solution mother liquor by 20 times to obtain a fatty acid mixed standard substance, wherein the concentrations are as follows in sequence: 40 mu mol/L of hexadecenoic acid, 5000 mu mol/L of octadecenoic acid, 300 mu mol/L of octadecadienoic acid, 50 mu mol/L of a-octadecatrienoic acid, 10 mu mol/L of gamma-octadecatrienoic acid, 10 mu mol/L of eicosenoic acid, 40 mu mol/L of arachidonic acid, 10 mu mol/L of eicosapentaenoic acid, 10 mu mol/L of Omega 3-docosapentaenoic acid, 5 mu mol/L of Omega 6-docosapentaenoic acid and 40 mu mol/L of docosahexaenoic acid.
(C) Kate condensing agent BOP (amide condensing agent), 500PPM, shaking reacting at 40 deg.C for 5min, and adding 3-picolylamine-15 N (isotope compound) 20.3mmol/L, shaking reacting at 40 ℃ for 30min to obtain the isotope internal standard of the fatty acid derivative mixture.
(D) And (3) passing the obtained standard solution through an SPE column, diluting the solution by 10 times with acetonitrile, transferring a small amount of the solution to a sample injection vial by using a liquid transfer device, and detecting by a higher-level mass spectrometry to confirm that the reaction is complete for later use.
2. Preparing a serum calibrator solution and a quality control solution;
(A) Serum without fatty acid background is used as a substrate, and the standard solution mother liquor is diluted into standard solutions with the following concentration ratios:
unit: mu mol/L
(B) Serum without fatty acid background is used as a substrate, standard solution mother liquor with the same concentration is prepared again, and the high-value quality control product and the low-value quality control product are obtained by respectively diluting by 33.3 times and 333 times.
3. Preparing a derivatization reagent and an amide condensation reagent;
(A) Preparing 50mmol/L of 3-picolylamine and 50mmol/L of amide condensation reagent by using anhydrous acetonitrile respectively for later use;
4. quality control product assignment and on-machine detection;
(A) Respectively taking 100 mu L of calibrator, quality control serum and to-be-detected sample serum, adding 20 mu L of amide condensing agent BOP, reacting for 5min at room temperature, adding 3 picolylamine, reacting for 30min at 40 ℃, finally adding 280 mu L of fatty acid derivative internal standard solution, precipitating protein, whirling for 1min, centrifuging for 5min at 13000g, sucking supernatant, feeding sample, and detecting.
(B) And (3) data processing, wherein the concentration of the quality control material is assigned as follows:
unit: mu mol/L
(B) LC-MS parameters
The mass spectrometer is a PreMed5200 liquid chromatogram tandem mass spectrum system produced by Hangzhou spectral aggregation medical technology limited company;
conditions of liquid chromatography
And (3) chromatographic column: agilent, zoRBAX Eclipse Plus C18
100X 3.0mm,3.5 μm column
A mobile phase A: (ultrapure water containing 0.01% of fatty acid mobile phase additive A and 0.1% of fatty acid mobile phase additive B: acetonitrile = 95) in the preparation process, namely, respectively adding the fatty acid mobile phase additive A and the fatty acid mobile phase additive B into the ultrapure water according to the proportion, uniformly mixing, carrying out suction filtration, and carrying out ultrasound treatment for 10min for later use.
Mobile phase B: ( Ultrapure water of 0.01% fatty acid mobile phase additive a and 0.1% fatty acid mobile phase additive B: acetonitrile =5:95 )
The preparation process comprises the following steps: adding ultrapure water, a fatty acid mobile phase additive A and a fatty acid mobile phase additive B into acetonitrile according to a certain proportion, uniformly mixing, carrying out suction filtration, and carrying out ultrasonic treatment for 10min for later use.
Column temperature: 40 deg.C
Sample introduction amount: 10 μ l (100. Mu.l quantitative loop, microliter sample mode, pipette tip sampling height 2 mm)
Gradient elution:
| elution procedure | Flow rate of flow | Elution solution | Time of the program |
| 1 | 0.4mL/min | Mobile phase B70% | 0min |
| 2 | 0.4mL/min | Mobile phase B100% | 8min |
| 3 | 0.4mL/min | Mobile phase B100% | 10min |
| 4 | 0.4mL/min | Mobile phase B70% | 10.1min |
| 5 | 0.4mL/min | Mobile phase B70% | 12min |
Mass spectrum conditions:
electrospray ion source (ESI), positive ion, MRM scan mode.
Ion source parameters:
| parameter name | Numerical value | Parameter name | Numerical value |
| Capillary high pressure | 5.0KV | Desolventizing air flow rate | 5.0L/min |
| Temperature of desolventizing gas | 450℃ | Reverse blowing air flow rate | 1.5L/min |
| Velocity of atomizing gas | 1.5L/min | Collision air flow velocity | 0.5mL/min |
Mass spectrum MRM parameters:
Claims (10)
1. the detection method of the free fatty acid in the human serum comprises the following steps:
adding various fatty acid standard substances and a condensing agent into a solvent to obtain a fatty acid solution for activating carboxyl;
adding an isotope compound into the fatty acid solution, and carrying out condensation reaction on carboxyl of fatty acid and amino of the isotope compound to obtain a derivative isotope internal standard solution;
adding a condensing agent and a non-isotopic compound which is the same as the isotopic compound into a serum sample, wherein carboxyl of fatty acid in the serum sample and amino of the non-isotopic compound are subjected to condensation reaction to obtain a reaction solution;
and adding the derivative isotope internal standard solution into the reaction solution, and sending the solution to a mass spectrometer for detection, thereby obtaining the content of free fatty acid in the serum sample.
2. The method for detecting free fatty acids according to claim 1, wherein the condensing agent is BOP.
3. The method for detecting a free fatty acid as claimed in claim 1, wherein the isotopic compound is 3 picolylamine- β, β is an isotope, and the non-isotopic compound is 3 picolylamine.
4. The method for detecting free fatty acids according to claim 1 or 3, wherein the isotope is an isotope of carbon, nitrogen or hydrogen.
5. The method of claim 1, wherein the derivatized internal standard solution is purified by a separation column, and then added to the reaction solution.
6. The method for detecting free fatty acids according to claim 1, wherein protein precipitation occurs after the internal standard solution is added to the reaction solution, and the reaction solution is vortexed and centrifuged, and the supernatant is aspirated and sent to the analyzer.
7. The method of claim 1 or 6, wherein the analyzer is a liquid chromatography tandem mass spectrometry system.
8. The method for detecting free fatty acids according to claim 1, wherein the solvent is anhydrous acetonitrile.
9. The method for detecting free fatty acids according to claim 1, wherein the reaction time is less than 30 minutes and the reaction temperature is 30 to 60 degrees in the fatty acid solution to obtain activated carboxyl groups.
10. The method for detecting free fatty acids according to claim 1, wherein the reaction temperature in obtaining the internal standard solution for derivatization and/or the reaction solution is 20 to 40 degrees.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116908354A (en) * | 2023-08-03 | 2023-10-20 | 河北乾业生物科技有限公司 | Fatty acid derivative, derivative method thereof, method for simultaneously detecting multiple fatty acids and kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116908354A (en) * | 2023-08-03 | 2023-10-20 | 河北乾业生物科技有限公司 | Fatty acid derivative, derivative method thereof, method for simultaneously detecting multiple fatty acids and kit |
| CN116908354B (en) * | 2023-08-03 | 2024-05-17 | 河北乾业生物科技有限公司 | Fatty acid derivative, derivative method thereof, method for simultaneously detecting multiple fatty acids and kit |
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