Method for detecting free fatty acids in human serum

Abstract

This invention provides a method for detecting free fatty acids in human serum. The method comprises: adding multiple fatty acid standards and a condensing agent to a solvent to obtain a fatty acid solution with activated carboxyl groups; adding an isotopic compound to the fatty acid solution, where the carboxyl groups of the fatty acids undergo a condensation reaction with the amino groups of the isotopic compound to obtain a derivatized isotopic internal standard solution; adding the condensing agent and a non-isotopic compound of the same type as the isotopic compound to a serum sample, where the carboxyl groups of the fatty acids in the serum sample undergo a condensation reaction with the amino groups of the non-isotopic compound to obtain a reacted solution; adding the isotopic internal standard solution and sending the sample to a mass spectrometer for detection to obtain the fatty acid content in the serum sample. This invention has the advantages of low detection difficulty and high accuracy.

Description

Technical Field

[0001] This invention relates to fatty acids, and more particularly to a method for detecting free fatty acids in human serum. Background Technology

[0002] Fatty acids are carboxylic acid compounds with fatty chains. Based on carbon chain length, they can be classified into short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids, and very long-chain fatty acids; based on whether their chemical structure contains double bonds, they can be classified into saturated fatty acids and unsaturated fatty acids. Unsaturated fatty acids are a type of fatty acid that constitutes body fat and are essential fatty acids for the human body. Polyunsaturated fatty acids are essential components of cell membranes, participate in phospholipid synthesis, and are found in mitochondria and cell membranes. Unsaturated free fatty acids are closely related to the occurrence and development of diseases of the cardiovascular, respiratory, digestive, and endocrine systems, as well as energy metabolism in diseases such as tumors. They have functions such as regulating blood lipids, anti-arrhythmia, anti-hypertensive, and regulating glucose metabolism. However, unsaturated fatty acids also have certain side effects on the human body. Trans fatty acids have a certain inhibitory effect on the growth and development of infants and young children: trans fatty acids can interfere with the metabolism of essential fatty acids; trans fatty acids can bind to lipids in the brain, inhibiting the synthesis of long-chain polyunsaturated fatty acids, thereby affecting the normal development of the central nervous system in infants and young children; trans fatty acids can inhibit the synthesis of prostaglandins in the mother, adversely affecting the growth and development of infants.

[0003] For the detection of fatty acids, the following methods exist in the prior art:

[0004] 1. Currently, numerous studies report the direct determination of fatty acids using GC-MS, but its application is primarily limited to the determination of a few common fatty acids in food. A few patents report the use of GC-MS to determine fatty acids in serum, but this can only detect a limited number of short-chain fatty acids and may require derivatization. Furthermore, the injection time for GC-MS is relatively long. Therefore, the clinical application of GC-MS has significant limitations.

[0005] 2. In addition, there are reports in the literature on the use of GC-FID to determine fatty acids, which is almost used in the food industry. It requires derivatization treatment such as methyl esterification, and the effects of different derivatization methods vary greatly. At the same time, the sensitivity is insufficient, the pretreatment time is cumbersome, and the injection time is long. There are few reports on the clinical application of this type of detection.

[0006] 3. Relevant literature reports the use of LC-MS derivatization for fatty acid pretreatment, with various methods available. This is currently a widely used clinical detection method. However, due to the chemical structure of fatty acids, their ionization efficiency in electrospray ionization sources is poor, resulting in low mass spectrometry detection sensitivity. Therefore, derivatization is necessary for fatty acid detection pretreatment. However, different pretreatment methods and derivatization techniques lead to significant differences in detection results. Summary of the Invention

[0007] To address the shortcomings of the existing technical solutions, the present invention provides a method for detecting fatty acids.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for detecting free fatty acids in human serum, wherein the method for detecting free fatty acids is as follows:

[0010] A solution of fatty acids with activated carboxyl groups is obtained by adding various fatty acid standards and condensing agents to a solvent.

[0011] An isotope compound is added to the fatty acid solution, and the carboxyl group of the fatty acid undergoes a condensation reaction with the amino group of the isotope compound to obtain a derivatized isotope internal standard solution.

[0012] A condensing agent and a non-isotopic compound of the same type as the isotopic compound are added to a serum sample, and the carboxyl groups of fatty acids in the serum sample undergo a condensation reaction with the amino groups of the non-isotopic compound to obtain a reaction solution.

[0013] The derivatized isotope internal standard solution is added to the reaction solution and sent to a mass spectrometer for detection, thereby obtaining the content of free fatty acids in the serum sample.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1. The testing process is simple;

[0016] This reduces the instability of the derivatization reagent system and the complexity of the reaction system, thereby reducing unnecessary pretreatment steps. The process is simple and solves the problems of easy interference and easy deterioration.

[0017] 2. Low testing cost;

[0018] By using a low-cost isotope compound, isotope internal standards corresponding to all analytes can be prepared simultaneously, reducing the difficulty of purchasing isotope internal standards and solving the problems of inconsistent reaction efficiency and poor experimental reproducibility of alternative isotope internal standards.

[0019] 3. Low testing requirements;

[0020] The derivatization reagents are preferred and less affected by the derivatization system, such as avoiding moisture and side reactions that form salts and reduce derivatization efficiency. They do not require multi-step derivatization, the reaction system is mild, the pretreatment process is greatly simplified, and the detection requirements are reduced.

[0021] 4. High detection accuracy;

[0022] This invention provides a reaction system based on the preparation of isotope internal standards, which can also be used as a derivatization system to modify the physicochemical characteristics of fatty acids, optimize their chromatographic behavior, enhance ionization efficiency in the mass spectrometry detection process, and reduce the difficulty of mass spectrometry detection of fatty acids. Attached Figure Description

[0023] The disclosure of this invention will become more readily understood with reference to the accompanying drawings. It will be readily understood by those skilled in the art that these drawings are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention. In the drawings:

[0024] Figure 1 This is a schematic diagram of a fatty acid detection method according to an embodiment of the present invention. Detailed Implementation

[0025] Figure 1 The following description illustrates optional embodiments of the invention to teach those skilled in the art how to implement and reproduce the invention. Some conventional aspects have been simplified or omitted for the purpose of teaching the invention. Those skilled in the art should understand that variations or substitutions derived from these embodiments will be within the scope of the invention. Those skilled in the art should understand that the following features can be combined in various ways to form multiple variations of the invention. Therefore, the invention is not limited to the following optional embodiments, but is defined only by the claims and their equivalents.

[0026] Example 1:

[0027] Figure 1 A schematic diagram of the method for detecting free fatty acids in human serum according to an embodiment of the present invention is provided, as follows: Figure 1 As shown, the method for detecting the free fatty acids is as follows:

[0028] A solution of fatty acids with activated carboxyl groups is obtained by adding various fatty acid standards and condensing agents to a solvent.

[0029] An isotope compound is added to the fatty acid solution, and the carboxyl group of the fatty acid undergoes a condensation reaction with the amino group of the isotope compound to obtain a derivatized isotope internal standard solution.

[0030] A condensing agent and a non-isotopic compound of the same type as the isotopic compound are added to a serum sample, and the carboxyl groups of fatty acids in the serum sample undergo a condensation reaction with the amino groups of the non-isotopic compound to obtain a reaction solution.

[0031] The derivatized isotope internal standard solution is added to the reaction solution and sent to an analyzer for detection, thereby obtaining the content of free fatty acids in the serum sample.

[0032] To further reduce reaction requirements, the condensing agent is BOP.

[0033] To reduce detection requirements and costs, the isotopic compound is 3-pyridinemethylamine-β, where β is an isotope, and the non-isotopic compound is 3-pyridinemethylamine, where the isotope is a carbon, nitrogen, or hydrogen isotope.

[0034] To reduce interference and improve detection accuracy, the derivatized internal standard solution is further purified by a separation column before being added to the reaction solution.

[0035] To improve detection accuracy, the internal standard solution is added to the reaction solution, resulting in protein precipitation. The solution is then vortexed and centrifuged, and the supernatant is collected and sent to the analyzer.

[0036] To further improve detection accuracy, the analyzer is a liquid chromatography-tandem mass spectrometry system.

[0037] To further improve reaction efficiency and effect, the reaction time in the fatty acid solution with activated carboxyl groups is less than 30 minutes, and the reaction temperature is between 30 and 60 degrees Celsius.

[0038] To improve reaction efficiency and effect, the reaction temperature is further set at 20-40 degrees Celsius in the preparation of the derivatized internal standard solution and / or reaction solution.

[0039] Example 2:

[0040] An application example of the method for detecting free fatty acids in human serum according to Embodiment 1 of the present invention.

[0041] In this application example, such as Figure 1 As shown, the method for detecting free fatty acids in this embodiment is as follows:

[0042] 1. Preparation of isotope internal standards;

[0043] (A) Using anhydrous acetonitrile as an organic solvent, prepare the following stock solutions: hexadecanoic acid 800 μmol / L, octadecanoic acid 10 mmol / L, octadecadienoic acid 6 mmol / L, α-octadecanetrienoic acid 1 mmol / L, γ-octadecanetrienoic acid 200 μmol / L, eicosanoic acid 200 μmol / L, eicosatetraenoic acid 800 μmol / L, eicosatepentanoic acid 200 μmol / L, Omega3-docosapentaenoic acid 200 μmol / L, Omega6-docosapentaenoic acid 100 μmol / L, and docosahexaenoic acid 800 μmol / L.

[0044] (B) Using anhydrous acetonitrile as a diluent, the standard solution stock solution was diluted 20 times to obtain a mixed standard of fatty acids with the following concentrations in descending order: hexadecanoic acid 40 μmol / L, octadecanoic acid 5000 μmol / L, octadecadienoic acid 300 μmol / L, α-octadecanetrienoic acid 50 μmol / L, γ-octadecanetrienoic acid 10 μmol / L, eicosanoic acid 10 μmol / L, eicosapentaenoic acid 40 μmol / L, eicosapentaenoic acid 10 μmol / L, Omega 3-docosapentaenoic acid 10 μmol / L, Omega 6-docosapentaenoic acid 5 μmol / L, and docosahexaenoic acid 40 μmol / L.

[0045] (C) Carter's condensing agent BOP (amide condensing agent), 500 PPM, 40℃, shaken reaction for 5 min, followed by the addition of 3-pyridinemethylamine-15 N (Isotopic compound) 20.3 mmol / L, shaken at 40℃ for 30 min to obtain a mixture of fatty acid derivatives isotopic internal standard.

[0046] (D) Pass the standard solution obtained above through an SPE column, dilute it 10 times with acetonitrile, transfer a small amount to a vial using a pipette, and detect it with a mass spectrometer to confirm that the reaction is complete. Set aside for use.

[0047] 2. Prepare serum calibrator and quality control solutions;

[0048] (A) Using serum without fatty acid background as the matrix, the standard solution stock solution was diluted to the following concentration ratio:

[0049] Unit: μmol / L

[0050]

[0051] (B) Using serum without fatty acid background as matrix, standard solution stock solution of the same concentration was prepared again and diluted by 33.3 times and 333 times respectively to obtain high value quality control product and low value quality control product.

[0052] 3. Prepare derivatization reagents and amide condensation reagents;

[0053] (A) Prepare 50 mmol / L 3-pyridinemethylamine and 50 mmol / L amide condensation reagent separately with anhydrous acetonitrile, and set aside for later use;

[0054] 4. Quality control product value assignment and on-machine testing;

[0055] (A) Take 100 μL of calibrator serum, quality control serum and test sample serum respectively, add 20 μL of amide condensing agent BOP, react at room temperature for 5 min, then add 3-pyridine methylamine, react at 40℃ for 30 min, finally add 280 μL of fatty acid derivative internal standard solution, precipitate protein, vortex for 1 min, centrifuge at 13000g for 5 min, aspirate the supernatant, inject into the instrument and detect.

[0056] (B) Data processing, the concentration of the quality control sample is assigned as follows:

[0057] Unit: μmol / L

[0058]

[0059] (B) LC-MS parameters

[0060] The mass spectrometer is a PreMed5200 liquid chromatography-tandem mass spectrometry system manufactured by Hangzhou Spectrum Medical Technology Co., Ltd.

[0061] Liquid chromatography conditions

[0062] Column: Agilent ZoRBAX Eclipse Plus C18

[0063] 100×3.0mm, 3.5μm chromatographic column

[0064] Mobile phase A: (0.01% fatty acid mobile phase additive A and 0.1% fatty acid mobile phase additive B in ultrapure water: acetonitrile = 95:5) Preparation process: Add fatty acid mobile phase additive A and fatty acid mobile phase additive B to ultrapure water according to the ratio, mix well, filter, sonicate for 10 min, and set aside.

[0065] Mobile phase B: (Ultrapure water:acetonitrile = 5:95 of 0.01% fatty acid mobile phase additive A and 0.1% fatty acid mobile phase additive B)

[0066] Preparation process: Add ultrapure water, fatty acid mobile phase additive A, and fatty acid mobile phase additive B to acetonitrile according to the proportion, mix well, filter, and sonicate for 10 minutes.

[0067] Column temperature: 40℃

[0068] Injection volume: 10 μl (100 μL quantitative loop, micro-injection mode, sampling needle height 2 mm)

[0069] Gradient elution:

[0070] Washing process Flow rate Elution solution Program time 1 0.4 mL / min Mobile phase B 70% 0min 2 0.4 mL / min Mobile phase B 100% 8min 3 0.4 mL / min Mobile phase B 100% 10min 4 0.4 mL / min Mobile phase B 70% 10.1min 5 0.4 mL / min Mobile phase B 70% 12min

[0071] Mass spectrometry conditions:

[0072] Electrospray ionization (ESI) source, positive ions, MRM scan mode.

[0073] Ion source parameters:

[0074] Parameter name numerical values Parameter name numerical values Capillary high pressure 5.0KV Desolvent gas flow rate 5.0L / min Desolvent gas temperature 450℃ Backflush airflow velocity 1.5L / min Atomizing airflow rate 1.5L / min Collision airflow velocity 0.5 mL / min

[0075] Mass spectrometry MRM parameters:

[0076]

[0077]

Claims

1. A method for detecting free fatty acids in human serum, the method comprising: adding a plurality of fatty acid standards and a condensing agent to a solvent to obtain a solution of fatty acids with activated carboxyl groups; adding an isotopic compound to the solution of fatty acids, wherein the carboxyl groups of the fatty acids condense with the amino groups of the isotopic compound to obtain a solution of derivatized isotopic internal standards; adding a condensing agent and a non-isotopic compound of the same class as the isotopic compound to a serum sample, wherein the carboxyl groups of the fatty acids in the serum sample condense with the amino groups of the non-isotopic compound to obtain a reaction solution; the isotopic compound is 3-pyridylmethylamine-β,β is an isotope, and the non-isotopic compound is 3-pyridylmethylamine; adding the solution of derivatized isotopic internal standards to the reaction solution and sending the solution to a mass spectrometer to obtain the content of free fatty acids in the serum sample. The condensing agent is BOP. The isotope is an isotope of carbon, nitrogen or hydrogen. The solution of derivatized isotopic internal standards is purified by a separation column before being added to the reaction solution. Protein precipitates appear after the solution of internal standards is added to the reaction solution, and the solution is vortexed and centrifuged, and the supernatant is aspirated and sent to the analyzer.

2. The method of detecting free fatty acids according to claim 1, characterized in that, The analyzer is a liquid chromatography tandem mass spectrometry system.

3. The method of detecting free fatty acids according to claim 1, characterized in that, The solvent is anhydrous acetonitrile.

4. The method of detecting free fatty acids according to claim 1, characterized in that, In obtaining the solution of fatty acids with activated carboxyl groups, the reaction time is less than 30 minutes, and the reaction temperature is 30-60 degrees.

5. The method of detecting free fatty acids according to claim 1, wherein In obtaining the solution of derivatized isotopic internal standards and / or the reaction solution, the reaction temperature is 20-40 degrees.

6. The method for detecting free fatty acid according to claim 1 or 5, characterized by, ​ 7. The method of detecting free fatty acids according to claim 1, wherein ​ 8. The method of detecting free fatty acids according to claim 1, wherein ​ 9. The method of detecting free fatty acids according to claim 1, wherein, ​

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