Method for determining free fatty acid in biological sample by utilizing GC-MS (Gas Chromatography-Mass Spectrometer)

Abstract

The invention provides a method for determining free fatty acid in a biological sample by utilizing GC-MS. The method comprises the following steps: S1, taking the biological sample, adding an extracting solution containing an internal standard, performing vortex, ultrasonic uniform mixing and centrifugation, taking supernate, and concentrating and drying; s2, adding an alkaline derivatization reagent into the dried product for reaction, then adding dichloromethane-normal hexane and ultrapure water for extraction, vortex and centrifugation, and taking supernate; s3, adding an acidic derivatization reagent into the supernate for reaction; then adding an extracting agent and ultrapure water for extraction, vortex and centrifugation, and taking supernate for GC-MS detection; and S4, taking the standard substance and the internal standard substance, carrying out GC-MS detection, formulating a standard curve, and substituting the measured value in S3 into the standard curve to obtain the content of free fatty acid in the biological sample to be measured. According to the method, the operation difficulty and the sample preparation failure risk are reduced, the extraction yield of the ultra-long-chain fatty acid is improved, meanwhile, interference of C16 and C18 fatty acid impurities is avoided, and the quantitative detection accuracy is high.

Description

Technical Field

[0001] The invention belongs to the technical field of free fatty acid detection, and particularly relates to a method for measuring free fatty acids in biological samples by utilizing GC-MS. Background Art

[0002] Fatty acids are widely distributed in various biological samples and perform important metabolic functions such as oxidative energy supply, signal transduction, and hormone synthesis. Analyzing changes in fatty acid content is crucial for metabolic research. Depending on whether they are bound to other molecules such as glycerol, sugar, and protein, they can be divided into free fatty acids and esterified fatty acids. Changes in free fatty acid levels are closely linked to cardiovascular disease, liver disease, and food quality. Clinical and food research studies have shown that free fatty acids can serve as auxiliary indicators for the diagnosis of certain diseases and food quality and safety.

[0003] Methylation combined with GC-MS is currently the most thoroughly researched and widely used method for fatty acid detection. The methylation method, which uses acidic methanol as a derivatization reagent, targets all forms of fatty acids, while the alkaline methanol method targets only esterified fatty acids. Combining the two allows for the detection and analysis of free fatty acids. The free fatty acid methylation process involves first derivatizing the esterified fatty acids with alkaline methanol to form fatty acid methyl esters, which are then removed by liquid-liquid extraction. The free fatty acids are then derivatized with acidic methanol to form fatty acid methyl esters, which are then purified by liquid-liquid extraction, ultimately achieving the separation and detection of the free fatty acids.

[0004] The liquid-liquid extraction systems reported so far mainly include n-hexane / methanol / water and chloroform / methanol / water. However, these extraction systems are difficult to operate. During the liquid-liquid extraction stratification process, the target is distributed in the lower layer of the extraction system, which causes inconvenience to the subsequent separation of the target and increases the risk of impurity contamination. In addition, the above two liquid-liquid extraction systems have poor extraction effects on certain ultra-long-chain free fatty acids. In addition, the introduction of C16 and C18 fatty acid interference by plastic consumables has a huge impact on the detection and analysis of free fatty acids. Summary of the Invention

[0005] In view of this, the present invention provides a method for determining free fatty acids in a biological sample using GC-MS.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for determining free fatty acids in a biological sample using GC-MS comprises the following steps:

[0008] S1. Take the biological sample to be tested, add the extract containing the internal standard to extract, vortex, ultrasonically mix, centrifuge, take the supernatant, concentrate and dry to obtain a dry product;

[0009] S2. Add an alkaline derivatization reagent to the dried product for re-dissolution, allow to stand for reaction, and after completion of the reaction, add dichloromethane-n-hexane and ultrapure water for extraction, vortex, centrifuge, and collect the supernatant; the volume ratio of dichloromethane to n-hexane is any one of (1:0), (4:1), (7:3), (3:2), and (0:1);

[0010] S3, adding an acidic derivatization reagent to the supernatant and allowing the reaction to proceed; after the reaction is complete, adding an extractant and ultrapure water for extraction, vortexing, centrifuging, and taking the supernatant for GC-MS analysis;

[0011] S4. Take the free fatty acid standard and internal standard for GC-MS detection, develop a standard curve, substitute the GC-MS detection value in S3 into the standard curve, and the free fatty acid content in the biological sample to be tested is obtained.

[0012] Furthermore, the extract in step S1 is methanol;

[0013] The alkaline derivatization reagent in step S2 is a sodium hydroxide-methanol solution, wherein the concentration of sodium hydroxide is 0.2 mol / L;

[0014] In step S3, the acidic derivatization reagent is a hydrochloric acid-methanol solution, wherein the concentration of hydrochloric acid is 1 mol / L, and the extractant is n-hexane.

[0015] Furthermore, the internal standards in steps S1 and S4 are all undecanoic acid-d21, heptadecanoic acid-d33, and linoleic acid- 13 C18, behenic acid-d43.

[0016] Furthermore, in step S1:

[0017] The vortex conditions are as follows: rotation speed 2300-2700 rpm, time 8-12 min;

[0018] The ultrasonic conditions are as follows: power 340-380W, time 12-17 min;

[0019] The centrifugation conditions are as follows: temperature 4° C., rotation speed 10,000-14,000 rpm, time 2-4 min.

[0020] Furthermore, in step S2:

[0021] The static reaction conditions are as follows: temperature 65-75°C, time 8-12 minutes;

[0022] The vortex conditions are as follows: rotation speed 2300-2700 rpm, time 4-6 min;

[0023] The centrifugation conditions are as follows: temperature 4°C, speed 4000-4500 rpm, time 4-6 min;

[0024] In step S3:

[0025] The static reaction conditions are as follows: temperature 65-75°C, time 18-22 minutes;

[0026] The vortex conditions are as follows: rotation speed 2300-2700 rpm, time 50-70 s;

[0027] The centrifugation conditions are as follows: temperature 4° C., rotation speed 4000-4500 rpm, time 4-6 min.

[0028] In some specific embodiments, preferably, the GC-MS chromatography conditions in steps S3 and S4 are as follows: the chromatographic column is a DB-5MS capillary column, 30m×0.25mm×0.25μm; the carrier gas is helium; the column flow rate is 1mL / min; the injection volume is 2μL; the split ratio is 5:1; the injection port temperature is 250°C; the transfer line temperature is 280°C; and the column oven temperature program is shown in the following table:

[0029] gradient Heating rate ℃ / min Temperature Holding time min Running time min initial 0 50 1.5 1.5 Gradient 1 30 170 0 5.5 Gradient 2 15 200 0 7.5 Gradient 3 5 220 2 13.5 Gradient 4 8 228 0 14.5 Gradient 5 20 240 2 17.1 Gradient 6 25 265 2 20.1 Gradient 7 30 280 2 22.6

[0030] The GC-MS mass spectrometry conditions are as follows: the ion source is an electron impact ion source; the scan mode is selected ion detection; the ion source temperature is 230°C; the ionization voltage is 70 eV; the quadrupole temperature is 150°C; and the selected ion mass-to-charge ratio settings are shown in the following table:

[0031]

[0032]

[0033] In some specific embodiments, preferably, the concentration of undecanoic acid-d21 is 4 μg / mL, the concentration of heptadecanoic acid-d33 is 4 μg / mL, and the concentration of linoleic acid- 13 The concentration of C18 was 10 μg / mL, and the concentration of docosanoic acid-d43 was 10 μg / mL.

[0034] In some specific embodiments, preferably, the mass or volume ratio of the extract to the biological sample is 20:1.

[0035] In some specific embodiments, preferably, the mass or volume ratio of the alkaline derivatization reagent to the biological sample is 8:1;

[0036] The volume ratio of the dichloromethane-n-hexane to ultrapure water is 5:2.

[0037] In some specific embodiments, preferably, the mass or volume ratio of the acidic derivatization reagent to the biological sample is 6:1;

[0038] The volume ratio of n-hexane to ultrapure water is 5:3.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] In the method provided by the present invention, during the liquid-liquid extraction process of acid-base methanol derivatization products, the extraction phase is located in the upper layer of the extraction system, which greatly reduces the difficulty of operation and the risk of sample preparation failure; at the same time, the composition of the raffinate for the liquid-liquid extraction of the alkaline methanol derivatization product is optimized, effectively improving the extraction yield of very-long-chain fatty acids; during the process, plastic tubes are used for metabolite extraction (glass bottles cannot be subjected to liquid nitrogen freeze-thaw and glass bead homogenization) and glass bottles are used for sample derivatization, which is compatible with most types of samples while avoiding interference from C16 and C18 fatty acid impurities; finally, the sample pretreatment process is simulated to establish a standard curve, and the interference of C16 and C18 fatty acid impurities introduced by consumables such as gun tips in the pretreatment process is further deducted (the intercepts of the C16-0 and C18-0 standard curves are always positive), greatly improving the quantitative accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the technical solution flow of the present invention.

[0042] Figure 2 The technical schemes and detection effects formed by different volume ratios of dichloromethane and n-hexane are shown; Figure A is a schematic diagram of the scheme setting, Figure B is a graph of the detection results of mixed standard solutions represented by undecanoic acid (FFA11:0), dodecanoic acid triglyceride (TG12:0), heptadecanoic acid triglyceride (TG17:0), cis-10-nonadecenoic acid (FFA19:1n9c), heneicosanoic acid triglyceride (TG21:0), and tricosanoic acid (FFA23:0) using different schemes, and Figure C is a graph of the response values of free fatty acids in the feasibility scheme. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0044] Example 1

[0045] This embodiment provides a method for determining free fatty acids in biological samples using GC-MS, as follows:

[0046] Establishment of standard curve:

[0047] S1. Accurately weigh 48 free fatty acid external standards and 4 free fatty acid internal standards and dissolve them in methyl tert-butyl ether or methanol. Prepare the 48 external standards into a standard stock solution with a concentration of 5000 μg / mL, and the 4 internal standards into a standard stock solution with a concentration of 10000 μg / mL. Then, take an appropriate amount of each of the 48 external standard stock solutions and mix them to volume to prepare a mixed standard solution with a concentration of 100 μg / mL for each substance in methyl tert-butyl ether. Take an appropriate amount of each of the 4 internal standard stock solutions and mix them to volume to prepare an extractant solution with a concentration of 4 μg / mL undecanoic acid-d21, 4 μg / mL heptadecanoic acid-d33, 10 μg / mL linoleic acid-13C18, and 10 μg / mL docosanoic acid-d43 in methanol.

[0048] S2. Take an appropriate amount of 48 external standard mother solutions and 4 internal standard mother solutions in step S1, concentrate and dry them under nitrogen, add 300 μL of methanol solution containing 1 mol / L hydrochloric acid to carry out methyl esterification reaction, add 500 μL of n-hexane and 300 μL of ultrapure water to the derivatized product, vortex mix, centrifuge and separate, inject the upper solution into GC-MS for full scan, obtain the retention time and mass spectrum fingerprint of each substance, select the characteristic mass spectrum fragment ion of each substance according to the retention time and mass spectrum fingerprint, establish a selected ion monitoring (SIM) mode detection method, when the retention time of the two substances is close (retention time difference <0.1 min), use the characteristic fragment as the SIM ion, when the separation degree of the substances is large enough (retention time difference>0.2 min), use the universal fragment of fatty acid as the SIM ion.

[0049] S3, the 100 μg / mL mixed standard in step S1 was gradiently diluted, and the concentrations of the 48 free fatty acid mixed standards after dilution were 10 μg / mL, 7.5 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.02 μg / mL, 0.01 μg / mL, 0.005 μg / mL, and 0.002 μg / mL, and 250 μL of 4 μg / mL undecanoic acid-d21, 4 μg / mL heptadecanoic acid-d33, 10 μg / mL linoleic acid- 13 C18, 10 μg / mL docosanoic acid-d43 extractant solution and 200 μL of methanol solution containing 0.1 mol / L sodium hydroxide.

[0050] S4. The standard sample solution containing the gradient concentration external standard and the fixed concentration internal standard in step S3 was concentrated to dryness by nitrogen blowing, and 300 μL of methanol solution containing 1 mol / L hydrochloric acid was added to the dried sample for methylation reaction. 500 μL of n-hexane and 300 μL of ultrapure water were added to the derivatized product, vortexed and centrifuged to separate the layers. The upper layer solution was injected into GC-MS (the parameters were consistent with those for detecting the sample to be tested) for SIM detection to obtain the total ion chromatogram (TIC) of the sample. The TIC data was then imported into data processing software (MassHunter, Agilent Technologies) for integration to obtain the chromatographic peak areas of standard samples with different concentrations. Linear and power function fitting were performed using the internal and external standard concentration ratio and the internal and external standard peak area ratio as the horizontal and vertical coordinates to establish a material standard curve. The standard curve equation for each substance is shown in Table 1 below. As can be seen from Table 1, the correlation coefficients are all greater than 0.99.

[0051] Table 1 Standard curves of different free fatty acids

[0052]

[0053]

[0054] The GC-MS chromatographic conditions are as follows: the chromatographic column is a DB-5MS capillary column, 30 m × 0.25 mm × 0.25 μm; the carrier gas is helium; the column flow rate is 1 mL / min; the injection volume is 2 μL; the split ratio is 5:1; the injection port temperature is 250°C; the transfer line temperature is 280°C; and the column oven temperature program is shown in Table 2 below:

[0055] Table 2 Column oven heating program details

[0056] gradient Heating rate ℃ / min Temperature Holding time min Running time min initial 0 50 1.5 1.5 Gradient 1 30 170 0 5.5 Gradient 2 15 200 0 7.5 Gradient 3 5 220 2 13.5 Gradient 4 8 228 0 14.5 Gradient 5 20 240 2 17.1 Gradient 6 25 265 2 20.1 Gradient 7 30 280 2 22.6

[0057] The GC-MS mass spectrometry conditions are as follows: the ion source is an electron impact ion source; the scan mode is selected ion detection; the ion source temperature is 230°C; the ionization voltage is 70 eV; the quadrupole temperature is 150°C; and the selected ion mass-to-charge ratio settings are shown in Table 3 below:

[0058] Table 3 Details of selected ion mass-to-charge ratio settings

[0059]

[0060]

[0061]

[0062] Detection of biological samples to be tested:

[0063] S1. Take 50 mg or 50 μL of biological sample (serum or liquid nitrogen-ground liver tissue) into a centrifuge tube, add 1 mL of methanol solution containing isotope-labeled internal standards (undecanoic acid-d21 concentration of 4 μg / mL, heptadecanoic acid-d33 concentration of 4 μg / mL, linoleic acid-13C18 concentration of 10 μg / mL, and docosanoic acid-d43 concentration of 10 μg / mL); vortex at 2500 rpm for 10 min and sonicate at 360 W for 15 min; after extraction, centrifuge at 4°C and 12000 rpm for 3 min, aspirate 500 μL of the supernatant extract into a glass bottle, and concentrate to dryness using a nitrogen blower to obtain a dry product.

[0064] S2. Add 400 μL of methanol solution containing 0.1 mol / L sodium hydroxide to the above dried material, sonicate at a power of 360 W for 1 min, and further vortex at a speed of 2500 rpm for 5 min to mix; then place it in a 70°C constant temperature box and let it stand for 10 min. Take out the derivatized product solution and cool it to room temperature. Add 500 μL of dichloromethane:n-hexane (7:3, v / v) and 200 μL of ultrapure water, and vortex at a speed of 2300-2700 rpm for 5 min to mix; then centrifuge at 4°C and a speed of 4200 r / min for 5 min, separate the layers, and take 300 μL of the supernatant into a glass bottle.

[0065] S3. Add 300 μL of methanol solution containing 1 mol / L hydrochloric acid to the above supernatant, vortex at 2500 rpm for 1 min to mix, and then place it in a 70°C constant temperature box for 20 min; take out the derivatization product solution and cool it to room temperature, add 500 μL of n-hexane and 300 μL of ultrapure water, vortex for 5 min to mix; then centrifuge at 4°C and 4200 r / min for 5 min, separate the layers, and take 200 μL of the supernatant into a GC injection vial to obtain the free fatty acid derivatization product test solution.

[0066] The test solution was further subjected to GC-MS detection, wherein the GC-MS detection steps and parameters were consistent with those used in establishing the calibration curve.

[0067] Furthermore, in order to understand the influence of different volume ratios of dichloromethane and n-hexane on the results of this method, the volume ratios of dichloromethane and n-hexane were set to different ratios in the detection step S2 of the biological sample to be tested (see the setting scheme for details). Figure 2Furthermore, the selectivity of the method was verified by using mixed standard solutions of undecanoic acid (FFA11:0), dodecanoic acid triglyceride (TG12:0), heptadecanoic acid triglyceride (TG17:0), cis-10-nonadecenoic acid (FFA19:1n9c), heneicosanoic acid triglyceride (TG21:0), and tricosanoic acid (FFA23:0) as representatives using different S2 extraction schemes. The response values of esterified fatty acids are shown in Figure 1. Figure 2 B, where the response values of free fatty acids for the feasible S2 extraction scheme are shown in Figure 2 C.

[0068] Depend on Figure 2 It can be seen that when the ratio of dichloromethane to n-hexane is higher than 6:4, the signal of esterified fatty acids in the fatty acid mixture sample is close to 0, indicating that the S2 liquid-liquid extraction system successfully separates esterified fatty acids and free fatty acids ( Figure 2 B), where the response of tricosanoic acid (FA23:0) increases with the increase of dichloromethane ratio, indicating that appropriate dichloromethane is conducive to the recovery of very long chain free fatty acids ( Figure 2 C). It should be noted that in Plan_9, the derivatization reaction described in S3 was performed on the lower layer solution of the separation system, while the derivatization reaction described in S3 was performed on the upper layer solution of the separation system in all other separation systems.

[0069] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining free fatty acids in biological samples using GC-MS, characterized in that: The following steps are involved: S1. Take the biological sample to be tested, add the extract containing the internal standard to extract, vortex, ultrasonically mix, centrifuge, take the supernatant, concentrate and dry to obtain a dry product; S2. Add an alkaline derivatization reagent to the dried product for re-dissolution, allow to stand for reaction, and after completion of the reaction, add dichloromethane-n-hexane and ultrapure water for extraction, vortex, centrifuge, and collect the supernatant; the volume ratio of dichloromethane to n-hexane is any one of (1:0), (4:1), (7:3), (3:2), and (0:1); S3, adding an acidic derivatization reagent to the supernatant and allowing the reaction to proceed; after the reaction is complete, adding an extractant and ultrapure water for extraction, vortexing, centrifuging, and taking the supernatant for GC-MS analysis; S4. Take the free fatty acid standard and internal standard for GC-MS detection, develop a standard curve, substitute the GC-MS detection value in S3 into the standard curve, and the free fatty acid content in the biological sample to be tested is obtained.

2. The method according to claim 1, characterized in that The extract in step S1 is methanol; The alkaline derivatization reagent in step S2 is a sodium hydroxide-methanol solution, wherein the concentration of sodium hydroxide is 0.2 mol / L; In step S3, the acidic derivatization reagent is a hydrochloric acid-methanol solution, wherein the concentration of hydrochloric acid is 1 mol / L, and the extractant is n-hexane.

3. The method according to claim 1, characterized in that The internal standards in steps S1 and S4 are all undecanoic acid-d21, heptadecanoic acid-d33, and linoleic acid- 13 C18, behenic acid-d43.

4. The method according to claim 1, wherein In step S1: The vortex conditions are as follows: rotation speed 2300-2700 rpm, time 8-12 min; The ultrasonic conditions are as follows: power 340-380W, time 12-17 min; The centrifugation conditions are as follows: temperature 4° C., rotation speed 10,000-14,000 rpm, time 2-4 min.

5. The method according to claim 1, wherein In step S2: The static reaction conditions are as follows: temperature 65-75°C, time 8-12 minutes; The vortex conditions are as follows: rotation speed 2300-2700 rpm, time 4-6 min; The centrifugation conditions are as follows: temperature 4°C, speed 4000-4500 rpm, time 4-6 min; In step S3: The static reaction conditions are as follows: temperature 65-75°C, time 18-22 minutes; The vortex conditions are as follows: rotation speed 2300-2700 rpm, time 50-70 s; The centrifugation conditions are as follows: temperature 4° C., rotation speed 4000-4500 rpm, time 4-6 min.

6. The method according to claim 1, characterized in that The GC-MS chromatographic conditions in steps S3 and S4 are as follows: the chromatographic column is a DB-5MS capillary column, 30 m × 0.25 mm × 0.25 μm; the carrier gas is helium; the column flow rate is 1 mL / min; the injection volume is 2 μL; the split ratio is 5:1; the injection port temperature is 250°C; the transfer line temperature is 280°C; and the column oven temperature program is shown in the following table: The GC-MS mass spectrometry conditions are as follows: the ion source is an electron impact ion source; the scan mode is selected ion detection; the ion source temperature is 230°C; the ionization voltage is 70 eV; the quadrupole temperature is 150°C; and the selected ion mass-to-charge ratio settings are shown in the following table:

7. The method according to claim 3, characterized in that The concentration of the undecanoic acid-d21 is 4 μg / mL, the concentration of the heptadecanoic acid-d33 is 4 μg / mL, and the concentration of the linoleic acid- 13 The concentration of C18 was 10 μg / mL, and the concentration of docosanoic acid-d43 was 10 μg / mL.

8. The method according to claim 2, characterized in that The mass or volume ratio of the extract to the biological sample is 20:

1.

9. The method according to claim 2, characterized in that The mass or volume ratio of the alkaline derivatization reagent to the biological sample is 8:1; The volume ratio of the dichloromethane-n-hexane to ultrapure water is 5:

2.

10. The method according to claim 2, characterized in that The mass or volume ratio of the acidic derivatization reagent to the biological sample is 6:1; The volume ratio of n-hexane to ultrapure water is 5:3.

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