CN116908354A

CN116908354A - Fatty acid derivative, derivative method thereof, method for simultaneously detecting multiple fatty acids and kit

Info

Publication number: CN116908354A
Application number: CN202310970071.3A
Authority: CN
Inventors: 宋征奇; 曾永福; 张浩然; 孟庆江; 杨彩燕
Original assignee: Hebei Qianye Biotechnology Co ltd
Current assignee: Hebei Qianye Biotechnology Co ltd
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2023-10-20
Anticipated expiration: 2043-08-03
Also published as: CN116908354B

Abstract

The invention relates to the technical field of substance detection, in particular to a fatty acid derivative, a derivative method thereof, a method for simultaneously detecting multiple fatty acids and a kit. The method for derivatizing fatty acid comprises the following steps: (1) Mixing the biological sample with the internal standard solution, and standing for 4-6 min to obtain a mixed solution; (2) Mixing the mixed solution with ethyl acetate for 4-6 min, centrifuging, taking supernatant, and drying with nitrogen to obtain a residue; (3) Adding N, N' -carbonyl imidazole solution into the residue, and incubating for 15-20 min to obtain intermediate solution; (4) And adding a 4- (dimethylamino) -benzyl amine solution into the intermediate solution, and incubating for 30-40 min to obtain the fatty acid derivative. The product obtained by the derivatization method is used for mass spectrometry, and 11 fatty acids can be detected rapidly and accurately.

Description

Fatty acid derivative, derivative method thereof, method for simultaneously detecting multiple fatty acids and kit

Technical Field

The invention relates to the technical field of substance detection, in particular to a fatty acid derivative, a derivative method thereof, a method for simultaneously detecting multiple fatty acids and a kit.

Background

Free Fatty Acid (FFA) is a class of carboxyl compounds with Fatty chains, and is also a product of fat tissue metabolism, consisting of linoleic acid, oleic acid, palmitic acid, and the like, and is a hydrolysate of triglyceride; wherein most of the free fatty acids are present in the blood in combination with serum albumin. Fatty acids have important roles in organisms, for example, fatty acids are important components of various functional lipid molecules of biological membranes; can oxidize energy supply, is an important energy supply substance for organisms; participating in molecular signaling and regulating physiological functions; closely related to diseases such as tumor, type 2 diabetes, heart disease, hypertension, insulin resistance, metabolic syndrome, and drug induced liver injury; meanwhile, serum free fatty acid has been confirmed to be closely related to the occurrence and development of cardiovascular and cerebrovascular diseases, respiratory diseases, digestive diseases, endocrine diseases, immune diseases and other system diseases, traumatic stress and other energy metabolism. Therefore, the method has important significance for qualitative and quantitative detection of free fatty acid in human serum, medical basic research and clinical examination.

Currently, there are many methods for determining fatty acids in serum, such as an enzymatic method, a titration method, a colorimetric method, an atomic spectrophotometry method, a gas chromatography method, etc., however, most methods for determining fatty acids are by gas chromatography for determining fatty acid methyl vinegar. The free fatty acid determination kit in the current market has the defects of poor stability, inconsistent determination range, narrow linear range and the like, and is not beneficial to the development of clinical detection work. Therefore, a method for rapidly and accurately detecting free fatty acids is urgently needed.

Disclosure of Invention

The invention aims to provide a fatty acid derivative, a derivative method thereof, a method for simultaneously detecting multiple fatty acids and a kit.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for derivatizing fatty acid, which comprises the following steps:

(1) Mixing the biological sample with the internal standard solution, and standing for 4-6 min to obtain a mixed solution;

(2) Mixing the mixed solution with ethyl acetate for 4-6 min, centrifuging, taking supernatant, and drying with nitrogen to obtain a residue;

(3) Adding N, N' -carbonyl imidazole solution into the residue, and incubating for 15-20 min to obtain intermediate solution;

(4) And adding a 4- (dimethylamino) -benzyl amine solution into the intermediate solution, and incubating for 30-40 min to obtain the fatty acid derivative.

Preferably, the biological sample is serum;

the internal standard liquid is a fatty acid isotope;

the fatty acid isotopes are arachidonic acid-d 8, oleic acid-13C 18, linoleic acid-13C 18, docosahexaenoic acid-d 5, alpha-linolenic acid-d 14 and eicosapentaenoic acid-d 5;

the volume ratio of the biological sample to the internal standard solution is 5-10:1-5.

Preferably, the volume ratio of the biological sample to the ethyl acetate is 5-10:60-80;

the mixing in the step (2) is vortex mixing;

the rotational speed of the centrifugation in the step (2) is 14000-15000 rpm;

and (3) centrifuging for 4-6 min.

Preferably, the volume ratio of the biological sample to the N, N' -carbonyl imidazole solution is 5-10:20-30;

the preparation method of the N, N' -carbonyl imidazole solution comprises the following steps: mixing N, N '-carbonyl imidazole with acetonitrile to obtain an N, N' -carbonyl imidazole solution;

the mass volume ratio of the N, N' -carbonyl imidazole to acetonitrile is 0.5-1.5 mg/1 mL;

the incubation temperature in the step (3) is 20-25 ℃.

Preferably, the volume ratio of the biological sample to the 4- (dimethylamino) -benzyl amine solution is 5-10:20-30;

the preparation method of the 4- (dimethylamino) -benzyl amine solution comprises the following steps: mixing 4- (dimethylamino) -benzyl amine with acetonitrile to obtain 4- (dimethylamino) -benzyl amine solution;

the mass volume ratio of the 4- (dimethylamino) -benzyl amine to the acetonitrile is as follows: 0.15-0.3 mg:1mL;

the incubation temperature in the step (4) is 40-50 ℃.

The invention also provides the fatty acid derivative derived by the derivation method, and the fatty acid derivative is used for simultaneously detecting various fatty acids in a sample.

The invention also provides a method for simultaneously detecting multiple fatty acids, which comprises the following steps:

detecting fatty acid derivatives by utilizing a liquid chromatography tandem mass spectrometry, recording the peak area of the fatty acid derivatives and the peak area of an internal standard liquid, calculating the ratio of the peak area of the fatty acid to the peak area of the internal standard liquid, substituting the ratio into a standard curve, and calculating the concentration of the fatty acid;

the fatty acid derivative is a fatty acid derivative derived by the derivation method or the fatty acid derivative;

the liquid chromatography conditions were:

chromatographic column: waters ACOUITYHSS T3 1.8μm2.1×50mm；

Flow rate: 0.4-0.6 mL/min;

column temperature: 54-56 ℃;

sample injection amount: 1-3 mu L;

the mobile phase A is: 0.05vt% formic acid water;

the mobile phase B is: 0.05vt% acetonitrile formate;

the elution gradient is: 0 to 1.7min,10 to 20 percent of mobile phase B;1.7 to 1.8min,20 to 60 percent of mobile phase B;1.8 to 6.0min,60 to 100 percent of mobile phase B;6.0 to 6.5min,100% mobile phase B;6.5 to 6.51min,100 to 10 percent of mobile phase B; 6.51-7.00 min,10% mobile phase B.

Preferably, the mass spectrometry conditions are as follows:

electrospray ionization source, positive ion scanning mode; a multi-reaction monitoring acquisition mode; electrospray ionization source parameters: spray voltage: 5000-6000V; ion source temperature: 490-510 ℃; atomizing gas: 49-51 psi; auxiliary heating gas: 49-51 psi; air curtain gas: 39-41 psi.

Preferably, the fatty acid comprises palmitoleic acid, oleic acid, linoleic acid, eicosapentaenoic acid, arachidonic acid, docosahexaenoic acid, omega 3-docosapentaenoic acid, omega 6-docosapentaenoic acid, gamma-linolenic acid and alpha-linolenic acid.

The invention also provides a kit for detecting various fatty acids simultaneously, which comprises an N, N' -carbonyl imidazole solution and a 4- (dimethylamino) -benzyl amine solution;

the fatty acids include palmitoleic acid, oleic acid, linoleic acid, eicosapentaenoic acid, arachidonic acid, docosahexaenoic acid, omega 3-docosapentaenoic acid, omega 6-docosapentaenoic acid, gamma-linolenic acid, and alpha-linolenic acid.

The invention provides a fatty acid derivative, a derivative method thereof, a method for simultaneously detecting multiple fatty acids and a kit.

The first step of the invention: the N, N' -carbonyl imidazole reacts with the carboxyl of the fatty acid to obtain an intermediate acyl imidazole compound with very high reactivity; and a second step of: the generated acyl imidazole compound can be quickly combined with amino in 4- (dimethylamino) -benzyl amine to generate a corresponding fatty acid acylated product, and meanwhile, as a plurality of nitrogen atoms exist in the 4- (dimethylamino) -benzyl amine structure, the ionization efficiency of the fatty acid modified by the 4- (dimethylamino) -benzyl amine is obviously increased under an electrospray ionization source, so that the detection sensitivity of the fatty acid in mass spectrum is greatly improved. The derivatization mechanism of N, N' -carbonylimidazole and 4- (dimethylamino) -phenylmethylamine is shown in FIG. 1. The fatty acid can be indirectly quantitatively analyzed by mass spectrometry of the derivative product. Fatty acids derived from N, N' -carbonylimidazole and 4- (dimethylamino) -benzylamine can generate characteristic daughter ions of m/z134 and m/z120 under the action of collision energy, and have strong response in a mass spectrometer. Derivatization of the fatty acid isotope internal standard with the fatty acid can reduce the matrix effect of the fatty acid in the sample.

The invention comprises at least the following advantages: (1) The derivatization condition is mild, the reaction temperature of the derivatization reagent N, N' -carbonyl imidazole can be carried out at room temperature, the reaction temperature of the 4- (dimethylamino) -benzyl amine is not more than 50 ℃, and the total reaction time is not more than 50min; (2) The derivatization efficiency is high, and the derivatization efficiency of the N, N' -carbonyl imidazole and 4- (dimethylamino) -benzyl amine can reach 99.9 percent; (3) By introducing a structure which is easy to ionize into a fatty acid structure, the sensitivity in mass spectrometry detection is greatly improved after the derivative of N, N' -carbonyl imidazole and 4- (dimethylamino) -benzyl amine; (4) The characteristics are enhanced, and after the fatty acid is derived from N, N' -carbonyl imidazole and 4- (dimethylamino) -benzyl amine, high-abundance sub-ion fragments can be obtained, so that the problem of insufficient specificity in a multi-reaction monitoring mode can be effectively avoided; (5) The matrix effect of fatty acid mass spectrometry detection is reduced, endogenous fatty acid is derived into components which are not contained in serum through a derivation means, and meanwhile, the matrix effect of fatty acid in the mass spectrometry detection can be obviously reduced by matching with a fatty acid isotope internal standard, so that the accuracy and precision of fatty acid detection are obviously improved. (6) The invention also realizes the accurate quantitative analysis of 11 free fatty acids in serum at the same time, wherein the fatty acids comprise 2 pairs of isomers, and can be widely applied to clinical detection and metabonomics research.

Drawings

FIG. 1 shows the derivatization mechanism of N, N' -carbonylimidazole and 4- (dimethylamino) -phenylmethylamine.

FIG. 2 is a flow chart of fatty acid derivatization.

FIG. 3 is a chromatogram of a fatty acid derivative.

Detailed Description

In the present invention, the standard curve of palmitoleic acid is y=1.23428 x+0.43392, r=0.9995;

the standard curve of oleic acid is y=0.08082x+0.14685, r=0.9987;

the standard curve of linoleic acid is y=0.06380x+0.14367, r=0.9999;

the standard curve of eicosapentaenoic acid is y=1.42352x+0.02732, r=0.9997;

the standard curve of the arachidonic acid is y= 0.28883x-6.33729e ^-4 ,r＝0.9983；

The standard curve of arachidonic acid is y=2.26137x+0.55088, r= 0.9988;

the standard curve of docosahexaenoic acid is y=2.92393x+0.04462, r=0.9998;

the standard curve of the omega 3-docosapentaenoic acid is y= 0.61281x-0.01596, r=0.9984;

the standard curve of the alpha-linolenic acid is y=0.35269x+0.00156, and r=0.9989;

the standard curve of the omega 6-docosapentaenoic acid is y= 2.92788x-0.03010, r=0.9997;

the standard curve for gamma-linolenic acid is y=0.28354 x+0.00203, r= 0.9991.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The fatty acid in the embodiment of the invention is palmitoleic acid (C16:1), oleic acid (C18:1), linoleic acid (C18:2), eicosapentaenoic acid (C20:5), arachidonic acid (C20:1),Arachidonic acid (C20:4), docosahexaenoic acid (C22:6), omega 3-docosapentaenoic acid (omega) ₃ -C22:5), omega 6-docosapentaenoic acid (omega) ₆ -C22:5), gamma-linolenic acid (gamma-C18:3), alpha-linolenic acid (alpha-C18:3).

The fatty acid isotope is arachidonic acid-d ₈ (C20:4-d ₈ ) Oleic acid (C18:1-13C 18), linoleic acid-13C 18 (C18:2-13C 18), docosahexaenoic acid-d ₅ (C22:6-d ₅ ) Alpha-linolenic acid-d ₁₄ (α-C18:3-d ₁₄ ) Eicosapentaenoic acid-d ₅ (C20:5-d ₅ )。

Serum as described in the examples of the present invention was collected from healthy volunteers at the medical facility.

Example 1

C16:1 was diluted to 0.4. Mu.g/mL, 0.8. Mu.g/mL, 2. Mu.g/mL, 8. Mu.g/mL, 20. Mu.g/mL with 1% aqueous BSA; c18:1 was diluted to 2.4. Mu.g/mL, 4.8. Mu.g/mL, 12. Mu.g/mL, 24. Mu.g/mL, 48. Mu.g/mL, 120. Mu.g/mL; c18:2 was diluted to 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 100. Mu.g/mL; c20:5 was diluted to 0.08. Mu.g/mL, 0.16. Mu.g/mL, 0.4. Mu.g/mL, 0.8. Mu.g/mL, 1.6. Mu.g/mL, 4. Mu.g/mL; c20:1 was diluted to 0.08. Mu.g/mL, 0.16. Mu.g/mL, 0.4. Mu.g/mL, 0.8. Mu.g/mL, 1.6. Mu.g/mL, 4. Mu.g/mL; c20:4 was diluted to 0.4. Mu.g/mL, 0.8. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 8. Mu.g/mL, 20. Mu.g/mL; c22:6 was diluted to 0.2 μg/mL, 0.4 μg/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, and 40 μg/mL; will omega ₃ C22:5 was diluted to 0.08. Mu.g/mL, 0.16. Mu.g/mL, 0.4. Mu.g/mL, 0.8. Mu.g/mL, 1.6. Mu.g/mL, 4. Mu.g/mL; will omega ₆ C22:5 was diluted to 0.08. Mu.g/mL, 0.16. Mu.g/mL, 0.4. Mu.g/mL, 0.8. Mu.g/mL, 1.6. Mu.g/mL, 4. Mu.g/mL; gamma-C18:3 was diluted to 0.08. Mu.g/mL, 0.16. Mu.g/mL, 0.4. Mu.g/mL, 0.8. Mu.g/mL, 1.6. Mu.g/mL, 4. Mu.g/mL; alpha-C18:3 was diluted to 0.2. Mu.g/mL, 0.4. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, and 10. Mu.g/mL. Then 0.4 mug/mL of C16:1, 2.4 mug/mL of C18:1, 2 mug/mL of C18:2, 0.08 mug/mL of C20:5, 0.08 mug/mL of C20:1, 0.4 mug/mL of C20:4, 0.2 mug/mL of C22:6, 0.08 mug/mL of omega are added ₃ Omega of-C22:5, 0.08. Mu.g/mL ₆ -C22:5, 0.08. Mu.g/mL of gamma-C18:3, 0.2. Mu.g/mL of alpha-C18:3 to obtain a first standard working fluid (std-1); then 0.8 mug/mL of CC18:1, C18:2, C20:5, C20:1, C20:4, C22:6, omega. 6, 0.16. Mu.g/mL, C18:1, C18:4.8. Mu.g/mL, C20:5, C20:1, C20:4, C20:6, omega. 0.16. Mu.g/mL ₃ Omega of-C22:5, 0.16. Mu.g/mL ₆ -C22:5, 0.16. Mu.g/mL of gamma-C18:3, 0.4. Mu.g/mL of alpha-C18:3 to obtain a second standard working fluid (std-2); c16:1 at 2. Mu.g/mL, C18:1 at 12. Mu.g/mL, C18:2 at 10. Mu.g/mL, C20:5 at 0.4. Mu.g/mL, C20:1 at 0.4. Mu.g/mL, C20:4 at 2. Mu.g/mL, C22:6 at 1. Mu.g/mL, omega. At 0.4. Mu.g/mL ₃ C22:5, 0.4. Mu.g/mL omega. Of omega ₆ -C22:5, 0.4. Mu.g/mL of gamma-C18:3, 1. Mu.g/mL of alpha-C18:3 to obtain a third standard working fluid (std-3); c16:1 at 4. Mu.g/mL, C18:1 at 24. Mu.g/mL, C18:2 at 20. Mu.g/mL, C20:5 at 0.8. Mu.g/mL, C20:1 at 0.8. Mu.g/mL, C20:4 at 4. Mu.g/mL, C22:6 at 2. Mu.g/mL, omega. At 0.8. Mu.g/mL ₃ C22:5, 0.8. Mu.g/mL omega. Of omega ₆ -C22:5, 0.8. Mu.g/mL of gamma. -C18:3, 2. Mu.g/mL of alpha. -C18:3 to obtain a fourth standard working fluid (std-4); 8. Mu.g/mL of C16:1, 48. Mu.g/mL of C18:1, 40. Mu.g/mL of C18:2, 1.6. Mu.g/mL of C20:5, 1.6. Mu.g/mL of C20:1, 8. Mu.g/mL of C20:4, 4. Mu.g/mL of C22:6, 1.6. Mu.g/mL of omega ₃ Omega of-C22:5, 1.6. Mu.g/mL ₆ -C22:5, 1.6 μg/mL γ -C18:3, 4 μg/mL α -C18:3 to obtain fifth standard working fluid (std-5); 20. Mu.g/mL of C16:1, 120. Mu.g/mL of C18:1, 100. Mu.g/mL of C18:2, 4. Mu.g/mL of C20:5, 4. Mu.g/mL of C20:1, 20. Mu.g/mL of C20:4, 40. Mu.g/mL of C22:6, 4. Mu.g/mL of omega ₃ Omega of-C22:5, 4. Mu.g/mL ₆ Gamma-C18:3 of 5 and 4 mug/mL and alpha-C18:3 of 10 mug/mL are mixed to obtain a sixth standard working solution (std-6). Specifically, the results are shown in Table 1.

Table 1 standard working fluids and labeled concentrations of fatty acids

Respectively taking std-1-std-6, a serum sample and 100 mu L of a quality control product in a 1.5mLEP tube, adding 20 mu L of a mixed fatty acid isotope, standing for 5min, adding 600 mu L of ethyl acetate, uniformly mixing by vortex for 5min, centrifuging at 15000rpm for 5min, respectively taking 400 mu L of supernatant, and blowing water bath nitrogen to be nearly dry to obtain a residue.

The quality control product is as follows: preparation of a 1% BSA solution containing palmitoleic acid (C16:1), oleic acid (C18:1), linoleic acid (C18:2), eicosapentaenoic acid (C20:5), arachidonic acid (C20:1), arachidonic acid (C20:4), docosahexaenoic acid (C22:6), omega 3-docosapentaenoic acid (omega) ₃ -C22:5), omega 6-docosapentaenoic acid (omega) ₆ -C22:5), gamma-linolenic acid (gamma-C18:3), low value quality control and high value quality control of alpha-linolenic acid (alpha-C18:3), the specific indicated concentrations are shown in Table 2.

TABLE 2 labeling concentration of fatty acid quality control

Compounds of formula (I)	Low value quality control (μg/mL) product	High quality control product (mug/mL)
			C16:1	1.2	12
C18:1	7.2	72
			C18:2	6	60
C20:5	0.24	2.4
			C20:1	0.24	2.4
C20:4	1.2	12
			C22:6	0.6	6
ω6-C22:5	0.24	2.4
			α-C18:3	0.24	2.4
ω3-C22:5	0.24	2.4
			γ-C18:3	0.6	6

The fatty acid isotope is arachidonic acid-d ₈ (C20:4-d ₈ ) Oleic acid (C18:1-13C 18), linoleic acid-13C 18 (C18:2-13C 18), docosahexaenoic acid-d ₅ (C22:6-d ₅ ) Alpha-linolenic acid-d ₁₄ (α-C18:3-d ₁₄ ) Eicosapentaenoic acid-d ₅ (C20:5-d ₅ ) The method comprises the steps of carrying out a first treatment on the surface of the The preparation method of the mixed fatty acid isotope internal standard working solution comprises the following steps: the acetonitrile solution contains arachidonic acid-d ₈ Linoleic acid-13C 18, docosahexaenoic acid-d ₅ Alpha-linolenic acid-d ₁₄ Eicosapentaenoic acid-d ₅ Is diluted into a mixed fatty acid isotope working solution with the concentration of 2 mug/mL.

200. Mu.L of an N, N' -carbonylimidazole solution was added to the residue, vortexed and mixed, and incubated at 25℃for 15 minutes to obtain an intermediate solution, and 200. Mu.L of a 4- (dimethylamino) -benzylamine solution was added to the intermediate solution, and incubated at 45℃for 35 minutes to obtain a fatty acid derivative. The fatty acid derivatization mechanism is shown in fig. 1, and the derivatization flow chart is shown in fig. 2.

The preparation method of the N, N' -carbonyl imidazole solution comprises the following steps: mixing N, N '-carbonyl imidazole with acetonitrile to obtain an N, N' -carbonyl imidazole solution; the mass volume ratio of the N, N' -carbonyl imidazole to the acetonitrile is 1mg to 1mL; the preparation method of the 4- (dimethylamino) -benzyl amine solution comprises the following steps: mixing 4- (dimethylamino) -benzyl amine with acetonitrile to obtain 4- (dimethylamino) -benzyl amine solution; the mass volume ratio of the 4- (dimethylamino) -benzyl amine to the acetonitrile is as follows: 0.2mg:1mL.

Example 2

LC-MS/MS system for detection and analysis

1. Chromatographic conditions

Chromatographic column: waters ACOUITYHSS T3 1.8μm2.1×50mm；

Flow rate: 0.5mL/min;

column temperature: 55 ℃;

sample injection amount: 2. Mu.L;

the mobile phase A is: 0.05vt% formic acid water;

the mobile phase B is: 0.05vt% acetonitrile formate;

the elution gradient is: 0 to 1.7min,15% mobile phase B;1.7 to 1.8min,30 percent of mobile phase B;1.8 to 6.0min,70 percent of mobile phase B;6.0 to 6.5min,100% mobile phase B;6.5 to 6.51min,50% mobile phase B; 6.51-7.00 min,10% mobile phase B.

2. Mass spectrometry conditions: positive ion scan mode (ESI) using electrospray ionization source ⁺ ) The method comprises the steps of carrying out a first treatment on the surface of the Acquisition mode (MRM) of multiple response monitoring; electrospray ionization source parameters: spray voltage (IS): 5500V; ion source Temperature (TEM): 500 ℃; atomizing Gas (Gas 1): 50psi; auxiliary heating Gas (Gas 2): 50psi; curtain gas (CUR): 40psi. The ion channels are shown in table 3, where Q1 is the precursor ion mass to charge ratio and Q3 is the fragment ion mass to charge ratio; the fatty acid derivative chromatogram is shown in FIG. 3 (1 represents alpha-C18:3, 2 represents gamma-C18:3, 3 represents C20:5, 4 represents C16:1, 5 represents C20:4, 6 represents C18:2, 7 represents omega) ₃ C22:5, 8 represents ω ₆ C22:5, 9 denotes C18:1, 10 denotes C20:1, 11 denotes C22:6).

Table 3 mass spectral parameters

3. Quantitative analysis

The drawing method of the standard curve comprises the following steps: and drawing a standard curve by taking the marked concentration of each standard substance as an abscissa and taking the ratio of the actual detection peak area of the standard substance to the peak area of each internal standard substance as an ordinate. Fitting of the standard curve equation linear regression was performed on the indicated concentrations with the peak area ratio of the standard. Regression equation y=ax+b can be obtained, where y is the ordinate, x is the abscissa, b is the intercept, and a is the slope.

4. Methodology investigation

The Matrix Effect (ME) is a phenomenon that other components in a sample except a target compound interfere with analysis of the target compound, and ionization efficiency of the target compound is different from one matrix to another, and there are two cases of matrix enhancement and matrix inhibition in general. The experiment is carried out by comparing 4 levels of serum matrix matching fatty acid isotope standard solution of 0.2, 0.4, 1 and 2 mug/mL with pure solvent fatty acid isotope standard solution with corresponding mass concentration. The peak area differences of the fatty acid isotope internal standard in serum and acetonitrile were compared. The results are shown in Table 4.

TABLE 4 matrix Effect of fatty acid isotope internal standard derivatives in serum

Table 4 shows that the fatty acid isotope internal standard derivatives have small differences in peak areas between serum and fatty acids, indicating that the fatty acids do not have significant matrix interference in serum.

The average recovery and relative standard deviation of each fatty acid were calculated by processing serum samples at 3 additive levels, low, medium, and high, in parallel 6 times, as shown in table 5.

TABLE 5 accuracy and precision of fatty acids

Compounds of formula (I)	Addition level/(μg/mL) (n=6)	Average recovery/% (n=6)	RSD/％
				C16:1	0.8/4/10	95.6/97.1/99.1	5.1/6.2/4.1
C18:1	4.8/24/96	96.8/97.6/94.6	3.3/4.5/2.6
				C18:2	4/20/80	101.3/96.7/96.1	7.6/4.7/6.2
C20:5	0.16/0.8/3.2	100.2/98.2/94.3	5.4/5.0/6.3
				C20:1	0.16/0.8/3.2	91.3/95.1/93.3	6.0/5.4/7.1
C20:4	0.8/4/10	102.4/105.1/99.4	2.3/4.6/1.5
				C22:6	0.4/2/32	94.2/99.8/101.3	2.9/6.5/4.2
ω3-C22:5	0.16/0.8/3.2	96.4/93.1/94.6	7.0/4.6/5.8
				ω6-C22:5	0.16/0.8/3.2	94.3/95.4/98.8	6.2/4.3/7.7
γ-C18:3	0.16/0.8/3.2	96.6/93.3/95.6	3.3/5.2/6.7
				α-C18:3	0.4/2/32	97.5/95.6/96.1	2.9/7.3/4.8

Table 5 shows that the average recovery rate of 11 fatty acids is 85-110%, and RSD is less than 10%, which shows that the accuracy and precision of the method are good.

The standard curve was regressed according to the method described above, and the linear ranges and detection limits of 11 fatty acids are shown in table 6.

TABLE 6 Linear Range and detection Limit of 11 fatty acids

Compounds of formula (I)	Linear range (μg/mL)	r ²	LOD/(μg/mL)	LOQ/(μg/mL)
					C16:1	0.4～20	0.9995	0.04	0.08
C18:1	2.4～120	0.9987	0.24	0.48
					C18:2	2～100	0.9999	0.025	0.05
α-C18:3	0.2～10	0.9989	0.0025	0.02
					γ-C18:3	0.08～4	0.9991	0.008	0.016
C20:1	0.08～4	0.9983	0.001	0.016
					C20:4	0.4～20	0.9988	0.005	0.01
C20:5	0.08～4	0.9997	0.001	0.002
					ω ₃ -C22:5	0.08～4	0.9984	0.001	0.002
ω ₆ -C22:5	0.08～4	0.9997	0.001	0.002
					C22:6	0.2～40	0.9998	0.00025	0.0025

Table 6 shows that the correlation of the curves of 11 fatty acids in the corresponding linear range is high (r ² >0.995 Good sensitivity and high accuracy of detection results.

The standard curve obtained is that of palmitoleic acid, y=1.23428 x+0.43392, r=0.9995;

the standard curve of oleic acid is y=0.08082x+0.14685, r=0.9987;

the standard curve of linoleic acid is y=0.06380x+0.14367, r=0.9999;

the standard curve of eicosapentaenoic acid is y=1.42352x+0.02732, r=0.9997;

The standard curve of arachidonic acid is y=2.26137x+0.55088, r= 0.9988;

the standard curve of docosahexaenoic acid is y=2.92393x+0.04462, r=0.9998;

the standard curve for gamma-linolenic acid is y=0.28354 x+0.00203, r= 0.9991.

Example 3

The serum samples of 12 persons were subjected to fatty acid derivatization as in example 1 to obtain fatty acid derivatives.

The fatty acid derivative was detected as in example 2, and the peak area ratio obtained was substituted into the corresponding standard curve to calculate the concentration. The test results are shown in Table 7.

TABLE 7 concentration of free fatty acids in human serum samples

From the above examples, the present invention provides a fatty acid derivative and its derivative method, and a method and kit for simultaneously detecting multiple fatty acids. The method can be used for rapidly and accurately measuring 11 free fatty acids in human serum.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A method of derivatizing fatty acids, comprising the steps of:

2. The derivatization method according to claim 1, wherein the biological sample is serum;

the internal standard liquid is a fatty acid isotope;

3. The derivatization method of claim 2, wherein the volume ratio of biological sample to ethyl acetate is 5-10:60-80;

the mixing in the step (2) is vortex mixing;

the rotational speed of the centrifugation in the step (2) is 14000-15000 rpm;

and (3) centrifuging for 4-6 min.

4. The method of claim 3, wherein the volume ratio of the biological sample to the N, N' -carbonylimidazole solution is 5-10:20-30;

the incubation temperature in the step (3) is 20-25 ℃.

5. The method according to claim 4, wherein the volume ratio of the biological sample to the 4- (dimethylamino) -benzylamine solution is 5-10:20-30;

the incubation temperature in the step (4) is 40-50 ℃.

6. The fatty acid derivative derived by the method according to any one of claims 1 to 5, wherein the fatty acid derivative is used for simultaneous detection of a plurality of fatty acids in a sample.

7. A method for simultaneously detecting a plurality of fatty acids, comprising the steps of:

the fatty acid derivative is a fatty acid derivative derived by the derivation method according to any one of claims 1 to 5 or a fatty acid derivative according to claim 6;

the liquid chromatography conditions were:

chromatographic column: watersACCOUITYHSS T3 1.8μm 2.1×50mm；

Flow rate: 0.4-0.6 mL/min;

column temperature: 54-56 ℃;

sample injection amount: 1-3 mu L;

the mobile phase A is: 0.05vt% formic acid water;

the mobile phase B is: 0.05vt% acetonitrile formate;

8. The method of claim 7, wherein mass spectrometry conditions are as follows:

9. The method of claim 7 or 8, wherein the fatty acid comprises palmitoleic acid, oleic acid, linoleic acid, eicosapentaenoic acid, arachidonic acid, docosahexaenoic acid, omega 3-docosapentaenoic acid, omega 6-docosapentaenoic acid, gamma-linolenic acid, and alpha-linolenic acid.

10. A kit for simultaneous detection of a plurality of fatty acids, comprising an N, N' -carbonylimidazole solution and a 4- (dimethylamino) -benzylamine solution;