CN109828047B - Kit for identifying accurate structure of unsaturated fatty acid in grease - Google Patents
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Abstract
The invention discloses a kit for accurately identifying the structure of unsaturated fatty acid in grease, which has the advantages of high specificity, capability of realizing accurate structure identification and the like. The kit comprises alkaline hydrolysis liquid, a hydrolysis reaction quenching agent, an extracting solution, P-B reaction liquid, a DEEA (dehydroepiandrosterone) reactant, a salt cleaning agent and a complex solution, and the positions of fatty acid and double bonds thereof are analyzed by using a mass spectrometry technology, so that the food detection quality is improved. The invention has the advantages of high sensitivity, accurate structure identification and the like, can avoid operation errors caused by corresponding self-prepared reagents, and can improve the simplicity of operation and the stability of the structure.
Description
Technical Field
The invention relates to a kit, in particular to an unsaturated fatty acid determination kit in the field of agricultural products such as grease and the like or foods.
Background
Edible oil and fat are important sources of dietary lipids and play an important role in various biological functions of human beings, and are energy sources, and contain a large amount of essential fatty acids (such as linoleic acid and linolenic acid) and fat-soluble vitamins (A, D, E and K), and the main components (accounting for 95% -98%) of triglyceride (triacylglycerol, TAGs) edible oil, wherein triglyceride is the most important existing form of fatty acid, and the carbon chain length, the double bond number, the cis/trans configuration of double bonds and the like of the fatty acid have important influences on the physicochemical properties and the nutritional properties of the oil and fat.
The position of the double bond of unsaturated fatty acid is closely related to its function, and fatty acid can be divided into n-3, n-6, n-7 and n-9 series according to the position of the first double bond from the methyl end. Of which groups n-3 and n-6 are of wide interest to researchers. The n-6 series includes linoleic acid (linoleic acid C18:2n-6, LA), gamma-linolenic acid (gamma-linolenic acid C18:3n-6, GLA), arachidonic acid (arachidonic acid C20:4n-6, AA), etc., and the n-3 series mainly includes long chain PUFAs such as alpha-linolenic acid (alfa-linolenic acid C18:3n-3, LNA), eicosapentaenoic acid (eicosapentaenoic acid C20:5n-3, EPA), and docosahexaenoic acid (docosahexaenoic acid C22:6n-3, DHA). Since humans and other mammals cannot synthesize these fatty acids themselves and need to be supplied with food, these two groups of PUFAs are functionally coordinated with each other in many cases to regulate the living activities of the living bodies. However, these two groups of fatty acids differ in many functions, for example, too much n-6PUFAs leads to platelet aggregation and thrombosis, while n-3PUFAs have the opposite effect. Compared with n-6PUFAs, n-3PUFAs can maintain higher insulin sensitivity, thereby exerting stronger blood sugar reducing function. More and more studies show that the precise structure and levels of fatty acids are closely related to abnormal carbohydrate and lipid metabolism and other risk factors of cardiovascular diseases, such as obesity, hypertension, etc. Because of the important role of polyunsaturated fatty acids in human health, many new sources of unsaturated fatty acid-containing oils and fats are continuously discovered and mined, including animal oils and fats, vegetable oils and microbial oils. With the emergence of new products, higher requirements are also put forward on the analysis and identification method of fatty acid in oil.
The traditional mass spectrometric identification method of the position of double bonds of fatty acid comprises a gas chromatography-tandem mass spectrometric method which can realize the differentiation of each fatty acid, but the method needs to carry out complex derivatization reaction on a sample and can only accurately identify 1 double bond. For unsaturated fatty acids with a large number of double bonds, the phenomenon of co-elution often occurs in gas chromatography, and the accurate judgment of the position of the double bond cannot be realized.
Disclosure of Invention
The invention aims to solve the technical problem of providing a kit for accurately identifying the mass spectrum structure of unsaturated fatty acid in grease aiming at the defects of the prior art, which realizes the accurate qualitative of the unsaturated fatty acid under the condition of no standard substance and is convenient to use.
In order to solve the technical problems, the invention adopts the technical scheme that:
a kit for accurately identifying the structure of unsaturated fatty acid in grease comprises alkaline hydrolysis liquid, a hydrolysis reaction quenching agent, an extracting solution, P-B reaction liquid, a DEEA (dehydroepiandrosterone) reaction agent, a salt cleaning agent and a complex solution, wherein the specific components are as follows:
alkali hydrolysate: 0.3-0.4mol/L potassium hydroxide solution, 100-;
② a quenching agent for hydrolysis reaction: 0.4-0.6mol/L hydrochloric acid, 400-;
③ extracting solution: n-hexane, 5-15 mL;
fourthly patent OA-Buchi (P-B) reaction solution: the volume ratio of the acetone/water/ethanol mixed solution is (550-: (350-400): (40-60), 0.5-1.5 mL;
fifthly, DEEA reactant: n, N-diethyl ethylenediamine (N, N-diethyl-1, 2-ethylenediamine, DEEA) solution: 15-25 mu mol/mL, 40-60 mu L; 2-chloro-1-methylpyridinium iodide (2-chloro-1-methylpyridinium iodide, CMPI) solution: 15-25 mu mol/mL, 40-60 mu L; triethylamine (triethylamine, TEA) solution: 15-25 mu mol/mL, 20-40 mu L;
sixthly, salt cleaning agent: 2-10mL of formic acid aqueous solution with volume fraction of 85-95%;
and seventh, recovering the solution 1: 0.5-2mL of chloroform;
and (2): acetonitrile, 0.5-2 mL.
According to the scheme, when the kit is used, the dosage of a sample to be tested for the grease is 1-6 mg, and the sample to be tested for the grease is diluted to 0.3-2mg/mL by using a chloroform/methanol mixed solution in advance.
According to the scheme, the potassium hydroxide solution is prepared by taking ethanol/water (95:5, v/v) as a solvent, and the hydrochloric acid is prepared by taking ultrapure water as a solvent.
According to the scheme, the DEEA solution, the CMPI solution and the TEA solution are prepared by taking chromatographic grade acetonitrile as a solvent.
The kit is mainly suitable for unsaturated fatty acids in vegetable oil, bacterial oil, algae oil and the like, and the specific use method is as follows:
1) weighing 1-6 mg of grease, mixing and diluting the grease with chloroform/methanol, and blowing the grease by nitrogen; then adding alkaline hydrolysis liquid for ultrasonic dispersion, carrying out water bath at the temperature of 75-85 ℃ for 1.5-2.5 hours, adding a hydrolysis quenching agent for quenching reaction, extracting fatty acid by using extracting solution n-hexane, and drying by nitrogen;
2) photochemical derivatization of unsaturated fatty acids
Re-dissolving the fatty acid extract obtained in the step 1) by nitrogen drying with a P-B reaction solution, and reacting for 55-65min under a 254nm low-pressure mercury lamp to obtain a fatty acid P-B reaction product;
3) performing N, N-diethylethylenediamine derivatization reaction on the reaction product of the fatty acid P-B in the step 2):
adding the reaction product of the fatty acid P-B obtained in the step 2) into a DEEA (diethyl ether-ethyl ether) reactant, uniformly mixing, performing ultrasonic treatment at 35-45 ℃ for 4-6min to perform derivatization reaction, and drying by using nitrogen after the derivatization reaction; in the step, after redissolving a product dried by nitrogen by using a redissolving solution 1, adding a salt scavenger, whirling, discarding an upper-layer solvent, drying by using nitrogen, redissolving by using a redissolving solution 2, and filtering by using an organic phase filter membrane;
4) fatty acid characterization method: performing mass spectrometry on the product obtained in the step 3) to obtain a spectrogram and spectrum peak data, and determining the quasi-molecular ion peak corresponding to each fatty acid derivative; each double bond in the fatty acid-derived product will generate two characteristic fragment ions, namely a pair of diagnostic ions, and the molecular formulas of 1 pair of diagnostic ions are respectively in the following rules, C(x-n)H(2(x-n-a)+4)O2N+, C(x-n+3)H(2(x-n-a)+10)ON+(X is the total number of carbons, n is the position of the first double bond counted from the methyl end, a is the number of the double bond counted from the methyl end, e.g., a ═ 1 indicates the 1 st double bond, a ═ 2 indicates the 2 nd double bond). The position of the double bond can be judged according to the diagnosis ion, and simultaneously, the unsaturated fatty acid with the double bond position isomer can be detectedThe positions of the contained double bonds are different, the derivative products respectively corresponding to the double bonds respectively generate diagnostic ions with different m/z in mass spectrum collision induced dissociation, and the positions of the double bonds contained in the unsaturated fatty acid with the double bond position isomer are determined, so that the qualitative analysis of the free fatty acid in the sample to be detected is realized.
Compared with the prior art, the unsaturated fatty acid determination kit has the beneficial effects that: the kit provided by the invention is convenient to use, can realize accurate qualitative of unsaturated fatty acid under the condition of no standard substance, and can realize accurate structural analysis of unsaturated fatty acid with high sensitivity.
Drawings
FIG. 1 is a schematic flow chart of the use process of the kit of the present invention.
In fig. 2: a is mass spectrum 73Da neutral loss positive ion mode (+ NLS73Da) mass spectrum of fatty acid derivative products in echium oil, B is secondary mass spectrum of C18:1, C is secondary mass spectrum of C18:2, D is secondary mass spectrum of C18:3, and E is secondary mass spectrum of C18: 4.
In fig. 3: a is a mass spectrum 73Da neutral loss positive ion mode (+ NLS73Da) mass spectrum of a fatty acid derivative product in the ARA bacterial oil, B is a secondary mass spectrum of C18:1, C is a secondary mass spectrum of C18:2, D is a secondary mass spectrum of C18:3, and E is a secondary mass spectrum of C20: 4.
In fig. 4: a is a mass spectrum 73Da neutral loss positive ion mode (+ NLS73Da) mass spectrum of a fatty acid derivative product in DHA algae oil, B is a secondary mass spectrum of C18:1, C is a secondary mass spectrum of C18:2, D is a secondary mass spectrum of C18:3, and E is a secondary mass spectrum of C22: 6.
Abbreviations for fatty acids used in the present invention:
c16:1n7, palmitoleic acid; c17:1, heptadecenoic acid; c18:3n3, alpha-linolenic acid; c18:3n6, gamma-linolenic acid; c18:2n6, linoleic acid; c18:1n9, oleic acid; c18:1n7, vaccenic acid; c20:4n6, ARA; c20:3n6, eicosatrienoic acid; c20:2n6, eicosadienoic acid; c20:1n9, eicosenoic acid; c22:1n9, erucic acid; c22:6n-3, DHA.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with specific examples, but should not be construed as limiting the present invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and substance of the invention.
In the following examples, reagents such as n-hexane, acetone, ethanol, chloroform, formic acid and the like are all chromatographically pure reagents; the potassium hydroxide solution is prepared by taking ethanol/water (95:5, v/v) as a solvent, and the hydrochloric acid is prepared by taking ultrapure water as a solvent; the DEEA solution, the CMPI solution and the TEA solution are prepared by using chromatographic grade acetonitrile as a solvent.
In the examples described below, the P-B reaction was carried out by transferring it to a quartz cuvette.
In the following examples, the mass spectrometry conditions were 4000Q-Trap mass spectrometer detectors, Applied Biosystems, USA. Electrospray Ionization source (Electrospray Ionization, ESI): a positive ion mode; mass spectrometry scan mode: 73Da neutral loss scan (73Da neutral loss scan) -Enhanced ion scan (Enhanced product ion scan, EPI); ion Source Gas 1(Ion Source Gas 1, GS 1): 20 Psi; ion Source Gas 2(Ion Source Gas 1, GS 1): 20 Psi; collision Energy (CE): 35eV (neutral loss), 48eV (second order); declustering Potential (DP): 40 eV; inlet voltage (EP): 5V, and (5); collision Cell extension Potential (CXP): 10V; the mass range is as follows: 200-650m/z
Example 1
A kit for accurately identifying a mass spectrum structure of unsaturated fatty acids in grease comprises an alkaline hydrolysis liquid, a hydrolysis reaction quenching agent, an extracting solution, a PB reaction liquid, a DEEA (dehydroepiandrosterone) reaction agent, a salt cleaning agent and a complex solution, wherein the specific components are as follows:
alkali hydrolysate: 0.35mol/L potassium hydroxide solution, 200 mu L;
② a quenching agent for hydrolysis reaction: 0.5mol/L hydrochloric acid, 500 mu L;
③ extracting solution: n-hexane, 9 mL;
fourthly patent OA-Buchi (P-B) reaction solution: acetone/water/ethanol (570/380/50, v/v/v), 1 mL;
fifthly, DEEA reactant: n, N-diethyl ethylenediamine (N, N-diethyl-1, 2-ethylenediamine, DEEA) solution: 20 mu mol/mL, 50 mu L; 2-chloro-1-methylpyridinium iodide (2-chloro-1-methylpyridinium iodide, CMPI) solution: 20 mu mol/mL, 50 mu L; triethylamine (triethylamine, TEA) solution: 20 mu mol/mL, 30 mu L;
sixthly, salt cleaning agent: formic acid/water (90/10, v/v/v), 5 mL;
and seventh, recovering the solution 1: chloroform, 1 mL;
and (2): acetonitrile, 1 mL.
The kit provided by the embodiment is used for qualitative analysis of fatty acid in echium oil, and comprises the following specific operation steps:
1) weighing 3mg of echium oil (sample to be detected), diluting with 3mL of chloroform/methanol (1:1, v/v), taking 10 mu L of diluent, and drying by nitrogen; then adding 200 mu L of alkaline hydrolysis liquid 0.35M KOH (dissolved in ethanol/water (95:5, v/v)), carrying out ultrasonic treatment for 2 minutes, carrying out water bath at 80 ℃ for 2 hours, adjusting the pH of the solution to 3 by using 0.5MHCl aqueous solution of hydrolysis quenching agent, then extracting fatty acid by using 3mL of extracting solution n-hexane, repeatedly extracting for 3 times, combining n-hexane layers, and drying by nitrogen;
2) photochemical derivatization of unsaturated fatty acids: redissolving the fatty acid extract obtained in the step 1) and dried by nitrogen by using 1mL of acetone/water/ethanol (570/380/50, v/v/v) solution of P-B reaction solution, and reacting for 60 min under a 254nm low-pressure mercury lamp;
3) derivatization of the fatty acid P-B reaction product with N, N-diethylethylenediamine: after the fatty acids had reacted via PB, DEEA reagent 50. mu.L of 20. mu. mol/mL 2-chloro-1-methylpyridinium iodide (CMPI) and 30. mu.L of 20. mu. mol/mL Triethylamine (TEA) were added, vortexed for 60 s; then adding 50 mu L of 20 mu mol/mL derivatization reagent N, N-diethyl ethylenediamine (N, N-diethyl-1, 2-ethylenediamine, DEEA), performing derivatization reaction at 40 ℃ by ultrasonic wave for 5min, and drying by using nitrogen after the derivatization reaction;
then, redissolving the product chromatogram by nitrogen blow-drying with redissolving solution 1(1mL of chloroform), adding 1mL of salt scavenger after vortexing for 30s, vortexing for 2min, discarding the upper layer solvent, repeating the operation for 5 times, blowing-drying by nitrogen, redissolving by 1mL of chromatographic pure acetonitrile, and filtering by a 2-micron organic phase filter membrane;
4) fatty acid characterization method: injecting the product obtained in the step 3) by a syringe pump at a flow rate of 20 μ L/min, performing mass spectrometry, determining the excimer ion peak corresponding to each fatty acid derivative according to the spectrogram and spectral peak data of free fatty acid derivatives in Echium oil shown in FIG. 2, and determining the type of fatty acid, such as C16:1, C18:1, C18:2, C18:3, C18:4, C20:2 (FIG. 2A); meanwhile, the position of the double bond of the unsaturated fatty acid can be judged according to fragment ions. For example, the C18:1 derivative product can generate 1 pair of diagnostic ions (m/z 198.1,224.1), thereby judging the position of the double bond to be C18:1n9 (FIG. 2B); the derived product of C18:2 produced two pairs of diagnostic ions (m/z 198.1,224.1), (m/z 238.1,264.1), so that the position of the double bond could be judged as C18:2n6 FIG. 2C); the derived product of C18:3 produced six pairs of diagnostic ions (m/z 156.1,182.1), (m/z 196.1,222.1) (m/z 236.1,272.1), (m/z 198.1,224.1), (m/z 238.1,264.1), (m/z 278.1,304.1) so that the positions of the double bonds could be judged as C18:3n6 and C18:3n3 (FIG. 2D); the derived product of C18:4 produced four pairs of diagnostic ions (m/z 156.1,182.1), (m/z 196.1,222.1) (m/z 236.1,272.1), (m/z276.1,302.1), and the position of the double bond of C18:4 was judged as C18:4n3 (FIG. 2E). Therefore, the kit can realize accurate qualitative analysis of unsaturated fatty acid in a sample to be detected under the condition of no standard substance.
Example 2
4) Example 2 differs from example 1 in that: replacing Echium oil with ARA bacterial oil 3mg to obtain sample to be tested. The spectrum and peak data of free fatty acid derivatives in ARA bacterial oil are shown in FIG. 3, so that the types of fatty acids can be judged, such as C16:1, C18:1, C18:2, C18:3, C20:1, C20:2, C20:3 and C20:4 (FIG. 3A); meanwhile, for the position of the double bond of unsaturated fatty acid, for example, the derivative product of C18:1 can generate 2 pairs of diagnostic ions (m/z 198.1,224.1) (m/z 226.1,252.1), thereby judging the position of the double bond to be C18:1n9, C18:1n7 (FIG. 3B); the derived product of C18:2 produced two pairs of diagnostic ions (m/z 198.1,224.1), (m/z 238.1,264.1), so that the position of the double bond could be judged as C18:2n6 FIG. 3C); the derived product of C18:3 produced six pairs of diagnostic ions (m/z 156.1,182.1), (m/z 196.1,222.1) (m/z 236.1,272.1), (m/z 198.1,224.1), (m/z 238.1,264.1), (m/z 278.1,304.1) so that the positions of the double bonds could be judged as C18:3n6 and C18:3n3 (FIG. 3D); the derived product of C20:4 produced four pairs of diagnostic ions (m/z 156.1,182.1), (m/z 196.1,222.1) (m/z 236.1,272.1), (m/z276.1,302.1), and the position of the double bond of C18:4 was judged to be C20:4n6 (FIG. 3E). Therefore, the kit can realize accurate qualitative analysis of the free unsaturated fatty acid in the sample to be detected under the condition of no standard substance.
Example 3
Example 3 differs from example 1 in that: replacing Echium oil with DHA algae oil 3mg to obtain the sample to be tested. The spectrum and the peak data of the free fatty acid derivatives in the DHA algal oil are shown in FIG. 4, so that the types of the fatty acids can be judged, such as C16:1, C18:1, C18:2, C20:2, C22:1, C22:5C22:6 (FIG. 4A); meanwhile, for the position of the double bond of unsaturated fatty acid, for example, the derivative product of C18:1 can generate 2 pairs of diagnostic ions (m/z 198.1,224.1), thereby judging the position of the double bond to be C18:1n9 (FIG. 4B); the derived product of C18:2 produced two pairs of diagnostic ions (m/z 198.1,224.1), (m/z 238.1,264.1), so that the position of the double bond could be judged as C18:2n6 (FIG. 4C); the derived product of C18:3 produced three pairs of diagnostic ions (m/z 156.1,182.1), (m/z 196.1,222.1) (m/z 236.1,272.1), and the position of the double bond was judged to be C18:3n6 (FIG. 4D); the derived product of C22:6 produced six pairs of diagnostic ions (m/z 128.1,154.1) (m/z 168.1,194.1), (m/z 208.1,234.1) (m/z 248.0,274.0), (m/z 288.0,314.1), (m/z 328.0,354.1) and judged the position of the double bond of C18:4 as C20:4n6 (FIG. 4E). Therefore, the kit can realize accurate qualitative analysis of the free unsaturated fatty acid in the sample to be detected under the condition of no standard substance.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and changes can be made without departing from the inventive concept of the present invention, and these modifications and changes are within the protection scope of the present invention.
Claims (4)
1. An application method of a kit for accurately identifying the structure of unsaturated fatty acid in grease is characterized by comprising the following steps:
1) weighing 1-6 mg of a sample to be tested of grease, mixing and diluting the sample with chloroform/methanol, and blowing the mixture with nitrogen for drying; then adding alkaline hydrolysis liquid for ultrasonic dispersion, carrying out water bath at the temperature of 75-85 ℃ for 1.5-2.5 hours, adding a hydrolysis quenching agent for quenching reaction, extracting fatty acid by using extracting solution n-hexane, and drying by nitrogen;
2) photochemical derivatization of unsaturated fatty acids
Re-dissolving the fatty acid extract obtained in the step 1) by nitrogen drying with a P-B reaction solution, and reacting for 55-65min under a 254nm low-pressure mercury lamp to obtain a fatty acid P-B reaction product;
3) step 2) N, N-diethylethylenediamine derivatization reaction of a fatty acid P-B reaction product:
adding the reaction product of the fatty acid P-B obtained in the step 2) into a DEEA (diethyl ether-ethyl ether) reactant, uniformly mixing, performing ultrasonic treatment at 35-45 ℃ for 4-6min to perform derivatization reaction, and drying by using nitrogen after the derivatization reaction; in the step, after redissolving a product dried by nitrogen by using a chromatographic grade complex solution 1, adding a salt scavenger, whirling, discarding an upper layer solvent, drying by nitrogen again, redissolving by using a chromatographic grade complex solution 2, and filtering by using an organic phase filter membrane;
4) fatty acid characterization method: performing mass spectrometry on the product obtained in the step 3) to obtain a spectrogram and spectrum peak data, and determining the quasi-molecular ion peak corresponding to each fatty acid derivative; each double bond in the fatty acid derived product will generate two characteristic fragment ions, namely 1 pair of diagnostic ions, and the molecular formulas of 1 pair of diagnostic ions have the following respective laws, C(x-n)H(2(x-n-a)+4)O2N+, C(x-n+3)H(2(x-n-a)+10)ON+Wherein X is the total number of carbons, n is the position of the first double bond counted from the methyl terminus, and a is the number of the double bond counted from the methyl terminus; the position of the double bond can be judged according to the diagnosis ion, and the position of the double bond contained in the unsaturated fatty acid with the double bond position isomer is different,the corresponding derivative products respectively generate diagnostic ions with different m/z in mass spectrum collision induced dissociation, and the position of a double bond contained in unsaturated fatty acid with double bond position isomers is determined, so that qualitative analysis is realized on free fatty acid in a sample to be tested for grease;
the kit comprises an alkaline hydrolysis liquid, a hydrolysis reaction quenching agent, an extracting solution, a P-B reaction liquid, a DEEA reagent, a salt cleaning agent and a complex solution, and the specific components are as follows:
alkali hydrolysate: 0.3-0.4mol/L potassium hydroxide solution, 100-;
hydrolysis reaction quenching agent: 0.4-0.6mol/L hydrochloric acid, 400-;
extracting solution: n-hexane, 5-15 mL;
P-B reaction solution: the volume ratio of the acetone/water/ethanol mixed solution is (550-: (350-400): (40-60), 0.5-1.5 mL;
DEEA reagent: n, N-diethylethylenediamine solution: 15-25 mu mol/mL, 40-60 mu L; 2-chloro-1-methylpyridinium iodide solution: 15-25 mu mol/mL, 40-60 mu L; triethylamine solution: 15-25 mu mol/mL, 20-40 mu L;
salt cleaning agent: 2-10mL of formic acid aqueous solution with volume fraction of 85-95%;
compound solution 1: 0.5-2mL of chloroform;
and (3) preparing a complex solution 2: acetonitrile, 0.5-2 mL.
2. The method of using the kit according to claim 1, wherein the potassium hydroxide solution is prepared using 90-95% by volume of an aqueous ethanol solution as a solvent.
3. The method for using the kit according to claim 1, wherein the three solutions of the DEEA reagent are prepared using chromatographic grade acetonitrile as a solvent.
4. The application method of the kit according to claim 1, wherein the amount of the sample to be tested for the oil is 3-10 mg, and the sample to be tested for the oil is diluted to 0.3-1mg/mL by using a chloroform/methanol mixed solution.
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