A rapid and accurate method for the determination of 48 free fatty acids in biological samples

Abstract

The present invention utilizes the GC-MS / MS method to separate and extract samples, and can detect 48 free fatty acids in human serum in 25 minutes; the multi-reaction detection mode library is built to eliminate false positive interference, which is more accurate and highly sensitive.

Description

Technical Field

[0001] The present application relates to the detection of markers in samples, specifically to the detection of 48 free fatty acids, and belongs to the field of biological detection. Background Art

[0002] Fatty acid (FA) is composed of three elements: C, H, and O. It is an aliphatic hydrocarbon chain with a carboxyl group at one end. Fatty acids can be divided into two categories: one is saturated fatty acids without carbon-carbon double bonds in the molecule, such as stearic acid and palmitic acid; the other is unsaturated fatty acids with one or more carbon-carbon double bonds in the molecule. The most common one is oleic acid. Oleic acid has only one carbon-carbon double bond in its carbon chain, so it is also called monounsaturated fatty acid. Generally, the carbon chain of fatty acid compounds is short, and its length is generally 18-36 carbon atoms. The shortest is 12 carbon atoms, such as lauric acid. Regardless of saturated or unsaturated, the number of carbon atoms in fatty acids in organisms is mostly even, and rarely contains odd carbon atoms, especially in higher animals and plants, where higher fatty acids with more than 12 carbons are mainly present, generally 14-24 carbons, with 16 and 18 carbon fatty acids being the most common.

[0003] Free fatty acids are also called nonestesterified fatty acids (NEFA). The lipids present in the human body can be roughly divided into three types: cholesterol, neutral fat (triglycerides), and phospholipids. Free fatty acids are one of the substances broken down from neutral fat. When the energy source required for muscle activity, glycogen, is exhausted, adipose tissue will break down neutral fat into free fatty acids to be used as energy. Therefore, free fatty acids can be said to be substances required for sustained activity. Studies have shown that insulin, adiponectin, pantothenic acid, acylation-stimulating protein, etc. will cause a decrease in NEFA levels in the blood, while tumor necrosis factor-α, catecholamine hormones, interleukin-6, leptin, etc. will cause an increase in NEFA levels. In addition, the increase in NEFA levels in the blood is closely related to cardiovascular disease, liver disease, diabetes and other related diseases. Therefore, the determination of NEFA levels in the blood has been used clinically as an auxiliary indicator for evaluating and diagnosing the above diseases.

[0004] Gas chromatography–mass spectrometry (GC-MS) is an analytical method that combines the properties of gas chromatography and mass spectrometry to identify different substances in a sample. Uses of GC-MS include drug testing (primarily for monitoring drug abuse), fire investigations, environmental analysis, explosion investigations, and the determination of unknown samples. GC-MS is also used to determine substances in luggage and the human body for airport security. In addition, GC-MS can be used to identify trace elements in a substance that were previously thought to have been transformed before they were identified.

[0005] GC-MS has been widely hailed as the gold standard method for forensic substance identification because it is used to conduct "specificity testing". The so-called "specificity test" is to be able to identify the actual presence of a substance in a given sample with great certainty. Non-specific tests can only indicate what type of substance is present in the sample. Although non-specific tests can use statistical methods to indicate what kind of substance the substance is, there is a positive bias in identification. Liu Peishan et al. used the GC-MS method to detect free fatty acids in serum, and 31 types can be detected (Liu Peishan et al., Determination of the content of 31 free fatty acids in plasma by gas chromatography-mass spectrometry, Journal of Analysis and Testing, Issue 8, 2020). The present invention will overcome the defects in the prior art and detect more types of free fatty acids in a shorter time. Summary of the invention

[0006] The present invention discloses a method for accurately qualitatively and quantitatively analyzing 48 kinds of free fatty acids in biological samples within 25 minutes using a gas chromatography-mass spectrometer, comprising the following steps:

[0007] (1) Sample extraction;

[0008] (2) adding an isotope n-heptadecanoic acid internal standard to the extract, and adding boron trifluoride methanol solution to carry out a derivatization reaction;

[0009] (3) After derivatization, the solution was extracted and GC-MS / MS was used to accurately identify and quantify 48 free fatty acids in biological samples within 25 minutes.

[0010] In a specific embodiment, step (1) is specifically as follows: adding an extracting solution to the serum sample, wherein the extracting solution is composed of methanol: MEBE: 36% phosphoric acid in a volume ratio of 3:4:1; shaking for 3 minutes, blowing the supernatant to dryness, adding 200 μL of 15% boron trifluoride methanol solution, shaking for 2 minutes, and keeping in an oven at 60°C for 30 minutes.

[0011] In another specific embodiment, the extraction in step (3) refers to: cooling the solution obtained in step (1) to room temperature, adding 500 μL of n-hexane, 200 μL of saturated sodium chloride solution, shaking for 3 minutes, centrifuging at 12000 r / min for 2 minutes at 4°C, taking 200 μL of the upper solution, and transferring it to a glass lined tube in a brown injection bottle for GC-MS / MS analysis.

[0012] In another specific embodiment, in the GS-MS / MS analysis of step (3), the gas chromatograph conditions mainly include: 1) chromatographic column: DB-5MS capillary column (30m×0.25mm×0.25μm, Agilent); 2) carrier gas: high-purity helium (purity>99.999%); 3) heating program: 40°C (maintained for 2min), increased to 200°C (maintained for 1min) at 30°C / min, increased to 240°C (maintained for 1min) at 10°C / min, increased to 285°C (maintained for 3min) at 5°C / min; 4) flow rate: 1.0mL / min; injection port temperature: 230°C; injection volume: 1.0μL.

[0013] In another specific embodiment, in the GS-MS / MS analysis in step (3), the mass spectrometry conditions are as follows: electron impact ion source (EI) temperature 230°C; ionization voltage: 70Ev; transmission line temperature: 240°C; quadrupole temperature: 150°C; solvent delay: 4.0min; scanning mode: SIM.

[0014] In another specific embodiment, the GS-MS / MS analysis in step (3) further includes a data processing step; preferably, the data processing is: processing mass spectrometry data using the software Qualitative Navigator; more preferably, the mass spectrometry peak of each free fatty acid detected in the sample is calibrated according to the retention time and ion pair information of each substance to ensure the accuracy of qualitative and quantitative analysis; more preferably, the peak area (Peak Area) of each chromatographic peak represents the relative content of the corresponding free fatty acid, and finally the qualitative and quantitative analysis results of the free fatty acids in the sample are obtained.

[0015] In another specific embodiment, the data processing includes: (1) sample quality control analysis: by overlapping and displaying the total ion current diagram (TIC diagram) of the fixed concentration mixed standard mass spectrometry detection analysis, the repeatability of free fatty acid extraction and detection is determined; (2) standard curve drawing: preparing free fatty acid standard solutions of different concentrations, and obtaining the corresponding quantitative signal mass spectrum peak intensity data of 48 free fatty acid standards of each concentration; using the standard concentration (μg / mL) as the horizontal coordinate and the peak area (Peak Area) of the mass spectrum peak as the vertical coordinate, 48 free fatty acid standard curves are drawn; (3) absolute quantification: the integrated peak areas of all free fatty acids detected in all samples are respectively substituted into the linear equation of the standard curve for calculation, and the calculation results are further substituted into the content calculation formula to obtain the absolute content of each free fatty acid in the actual sample.

[0016] The second aspect of the present invention is to provide the application of the above method for detecting the free amino acid content in a serum sample; preferably, the application is a non-diagnostic method; more preferably, the detection sample is a human sample; most preferably, the sample is a human serum sample.

[0017] The beneficial effects of the present invention include: short separation time, most separation times are more than 30 minutes, and this method is 25 minutes; many detectable substances, most of which are within 40, and this method has 48 substances; multi-reaction detection mode library construction eliminates false positive interference, is more accurate, and has high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Overlapping diagram of TIC of 48 free fatty acids. DETAILED DESCRIPTION

[0019] 1. Liquid sample extraction method:

[0020] (1) Take 50 μL of serum sample and add 400 μL of extraction solution (methanol: methyl tert-butyl ether: 36% phosphoric acid = 3:4:1, V / V / V). Add 20 μL (10 ppm) isotope n-heptadecanoic acid internal standard to each sample and shake for 3 min. Dry the supernatant and add 200 μL of 15% boron trifluoride methanol solution. Shake for 2 min and keep in a 60°C oven for 30 min.

[0021] (2) Cool to room temperature, add 500 μL of n-hexane and 200 μL of saturated sodium chloride solution, shake for 3 min, centrifuge at 12,000 r / min at 4°C for 2 min, take 200 μL of the upper layer solution and transfer it to a glass-lined tube in a brown injection bottle for GC-MS / MS analysis.

[0022] 2. Instrument parameter conditions:

[0023] The data acquisition instrument system used for GC-MS / MS detection mainly includes gas chromatograph (Agilent7890B) and tandem mass spectrometer MS / MS (7000D). Among them, the gas chromatograph conditions mainly include:

[0024] 1) Chromatographic column: DB-5MS capillary column (30m×0.25mm×0.25μm, Agilent);

[0025] 2) Carrier gas: high purity helium (purity>99.999%);

[0026] 3) Heating program: 40°C (hold for 2 min), increase to 200°C at 30°C / min (hold for 1 min), increase to 240°C at 10°C / min (hold for 1 min), increase to 285°C at 5°C / min (hold for 3 min);

[0027] 4) Flow rate: 1.0 mL / min; Inlet temperature: 230°C; Injection volume: 1.0 μL.

[0028] 3. Mass spectrometry conditions are as follows:

[0029] The electron impact ion source (EI) temperature was 230°C; the ionization voltage was 70 Ev; the transfer line temperature was 240°C; the quadrupole temperature was 150°C; the solvent delay was 4.0 min; and the scanning mode was SIM.

[0030] 4. Data processing of free fatty acids

[0031] The mass spectrometry data were processed using the Qualitative Navigator software. Based on the retention time and ion pair information of each substance, the mass spectrometry peaks detected for each free fatty acid in the sample were calibrated to ensure the accuracy of qualitative and quantitative analysis. The peak area of ​​each chromatographic peak represents the relative content of the corresponding free fatty acid, and finally the qualitative and quantitative analysis results of the free fatty acids in the sample were obtained.

[0032] The ion pair information is shown in the following table:

[0033]

[0034]

[0035] (1) Sample quality control analysis: The total ion current (TIC) diagram of the fixed concentration mixed standard mass spectrometry analysis was overlapped to determine the repeatability of free fatty acid extraction and detection. The TIC overlap diagrams of 48 free fatty acids are shown in Figure 1. Figure 1 shown.

[0036] (2) Standard curve drawing: Prepare free fatty acid standard solutions of different concentrations, and obtain the corresponding quantitative signal mass spectrometry peak intensity data of the 48 free fatty acid standards at each concentration; draw the 48 free fatty acid standard curves with the standard concentration (μg / mL) as the horizontal axis and the peak area (Peak Area) of the mass spectrometry peak as the vertical axis.

[0037] The standard curve is shown in the following table:

[0038]

[0039]

[0040] (3) Absolute quantification: The integrated peak areas of all free fatty acids in all samples detected are substituted into the linear equation of the standard curve for calculation. The calculation results are further substituted into the content calculation formula to obtain the absolute content of each free fatty acid in the actual sample.

[0041] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with the technology may make various changes and modifications without departing from the spirit and scope of the present invention. All changes and modifications obtained by those skilled in the art in combination with conventional techniques in the field are within the scope of the present invention.

Claims

1. A method for accurately identifying and quantitatively analyzing 48 free fatty acids in biological samples by gas chromatography-mass spectrometry, comprising the following steps: (1) extracting a sample, wherein the sample is a human serum sample; (2) adding an isotope n-heptadecanoic acid internal standard to the extract, and adding boron trifluoride methanol solution to carry out a derivatization reaction; (3) After derivatization, the solution was extracted and GC-MS / MS was used to accurately identify and quantify 48 free fatty acids in biological samples within 25 min; The step (1) specifically comprises: adding an extracting solution to the serum sample, wherein the extracting solution is composed of methanol: MEBE: 36% phosphoric acid in a volume ratio of 3:4:1; shaking for 3 minutes, blowing the supernatant to dryness, adding 200 μL of 15% boron trifluoride methanol solution, shaking for 2 minutes, and keeping in an oven at 60° C. for 30 minutes; The GC-MS / MS analysis in step (3) further includes a data processing step; the data processing comprises: using the software Qualitative Navigator to process the mass spectrometry data, and correcting the mass spectrometry peaks of each free fatty acid detected in the sample according to the retention time and ion pair information of each substance to ensure the accuracy of qualitative and quantitative analysis; the peak area of ​​each chromatographic peak represents the relative content of the corresponding free fatty acid, and finally obtaining the qualitative and quantitative analysis results of the free fatty acids in the sample; The ion pair information is shown in the following table:

2. According to the method of claim 1, the extraction in step (3) refers to: cooling the solution obtained in step (2) to room temperature, adding 500 μL of n-hexane and 200 μL of saturated sodium chloride solution, shaking for 3 minutes, centrifuging at 12000 r / min for 2 minutes at 4°C, taking 200 μL of the upper solution, and transferring it to a glass lined tube in a brown injection bottle for GC-MS / MS analysis.

3. The method according to claim 1, in the GC-MS / MS analysis of step (3), the gas chromatograph conditions mainly include: 1) Chromatographic column: DB-5MS capillary column, 30m×0.25mm×0.25μm, Agilent; 2) Carrier gas: high purity helium with purity >99.999%; 3) Heating program: hold at 40℃ for 2min, increase to 200℃ at 30℃ / min, hold for 1min, increase to 240℃ at 10℃ / min, hold for 1min, increase to 285℃ at 5℃ / min, hold for 3min; 4) Flow rate: 1.0mL / min; Inlet temperature: 230℃; Injection volume: 1.0μL.

4. According to the method of claim 1, in the GC-MS / MS analysis in step (3), the mass spectrometry conditions are as follows: electron bombardment ion source temperature: 230°C; ionization voltage: 70Ev; transfer line temperature: 240°C; quadrupole temperature: 150°C; solvent delay: 4.0min; scanning mode: SIM.

5. The method according to claim 1, wherein the data processing comprises: a. Sample quality control analysis: The repeatability of free fatty acid extraction and detection is determined by overlapping the total ion flow chart of the fixed concentration mixed standard mass spectrometry analysis; b. Drawing of standard curve: prepare free fatty acid standard solutions of different concentrations, obtain the corresponding quantitative signal mass spectrum peak intensity data of 48 free fatty acid standards of each concentration; draw the standard curve of 48 free fatty acids with the standard concentration μg / mL as the abscissa and the peak area of ​​the mass spectrum peak as the ordinate; c. Absolute quantification: Substitute the integrated peak areas of all free fatty acids in all samples detected into the linear equation of the standard curve for calculation, and further substitute the calculation results into the content calculation formula to obtain the absolute content of each free fatty acid in the actual sample.

6. The method according to claim 5, wherein the standard curve equation of step b. is as follows:

7. Use of the method according to any one of claims 1 to 6 for detecting the content of free amino acids in serum samples, wherein the application is a non-diagnostic method.

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