CN109828047A - The kit of the accurate Structural Identification of unsaturated fatty acid in a kind of grease - Google Patents
The kit of the accurate Structural Identification of unsaturated fatty acid in a kind of grease Download PDFInfo
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Abstract
The invention discloses a kind of kits of the accurate Structural Identification of unsaturated fatty acid in grease, have many advantages, such as specific height, are able to achieve accurate Structural Identification.The kit includes alkaline hydrolyzate, hydrolysis quencher, extracting solution, P-B reaction solution, and DEEA reactant, salt cleaning agent, redissolution liquid, what is utilized is the position of mass-spectrometric technique analysis fatty acid and its double bond, improves food inspection quality.The present invention has many advantages, such as high sensitivity, and Structural Identification is accurate, and is avoided that operating error caused by corresponding Self-made reagent, while can promote the simplicity of operation and the stability of structure.
Description
Technical field
The present invention relates to a kind of kit, the unsaturation of a kind of agricultural product such as grease or field of food is particularly related to
Determination of fatty acid kit.
Technical background
Edible oil and fat are the important sources of dietary lipids, are played an important role in a variety of biological functions of the mankind, it
Be energy source, it is sweet containing a large amount of essential fatty acids (such as linoleic acid plus linolenic acid) and liposoluble vitamin (A, D, E and K)
The main component (accounting for 95%-98%) of oily three esters (triacylglycerols, TAGs) edible oil, triglycerides be fatty acid most
Main existence form, cis/trans configuration of the carbon chain lengths of fatty acid, double key number and double bond etc. is to the physico of grease
It learns property and nutrition property has an important influence.
The position of double bond of unsaturated fatty acid and its function are closely related, and fatty acid radical is according to first double bond apart from methyl end
Position it is different, it is serial that n-3, n-6, n-7 and n-9 etc. can be divided into.N-3 group and n-6 group are wherein concerned with by researcher extensively.
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., n-3 system class mainly has α-flax
Sour (alfa-linolenic acid C18:3n-3, LNA), eicosapentaenoic acid (eicosapentaenoic acid C20:
5n-3, EPA) and the long-chains PUFA such as docosahexaenoic acid (docosahexaenoic acid C22:6n-3, DHA).Due to people
Class and other mammals itself cannot synthesize these fatty acid, it is necessary to be provided by food, so referred to as essential fatty acid is very
In more situations, this two race PUFAs functionally mutually coordinated restriction, the common vital movement for adjusting organism.But this two races rouge
Fat acid in many functions there is also difference, for example, n-6PUFAs excessively will lead to platelet aggregation and thrombosis, and n-
3PUFAs then has opposite effect.And compared with n-6PUFAs, n-3PUFAs can more make insulin keep higher sensibility,
To play stronger function of blood sugar reduction.More and more researches show that the accurate structure of fatty acid and its horizontal and sugar, rouge generation
Thank exception and the risk factor of other cardiovascular diseases, such as it is fat, hypertension is closely related.Since polyunsaturated fatty acid is in people
The important function of body health aspect, many new resources greases containing unsaturated fatty acid are constantly discovered and are excavated, including animal
Grease, vegetable fat and microbial oil.With the appearance of new product, the method that analyzes and identifies of fatty acid in grease is also mentioned
Higher requirement is gone out.
The Mass Spectrometric Identification method of traditional fatty acid position of double bond has gas-chromatography tandem mass spectrometry, is able to achieve every kind of fat
The differentiation of acid, but this method needs to carry out sample complicated derivative reaction, and can only accurately be reflected to 1 double bond
It is fixed.For the unsaturated fatty acid more than number of double bonds, often there is the phenomenon that co-elute in gas-chromatography, cannot achieve position of double bond
Accurate judgement.
Summary of the invention
The technical problem to be solved by the present invention is to provide insatiable hunger in a kind of grease in view of the above shortcomings of the prior art
With the kit of the accurate Structural Identification of fatty acid mass spectrum, the accurate qualitative of unsaturated fatty acid is realized under conditions of no mark product,
It is easy to use.
The present invention is to solve technical problem set forth above, used technical solution are as follows:
The kit of the accurate Structural Identification of unsaturated fatty acid in a kind of grease, including alkaline hydrolyzate, hydrolysis are quenched
Agent, extracting solution, P-B reaction solution, DEEA reactant, salt cleaning agent redissolve liquid, and concrete composition ingredient difference is as follows:
1. alkaline hydrolyzate: 0.3-0.4mol/L potassium hydroxide solution, 100-300 μ L；
2. hydrolysis quencher: 0.4-0.6mol/L hydrochloric acid, 400-600 μ L；
3. extracting solution: n-hexane, 5-15mL；
Paten 4. ò-B ü chi (P-B) reaction solution: acetone/water/ethyl alcohol mixed solution, three's volume ratio are (550-
600): (350-400): (40-60), 0.5-1.5mL；
5. DEEA reactant: N, N- diethyl ethylenediamine (N, N-diethyl-1,2-ethanediamine, DEEA) are molten
Liquid: 15-25 μm of ol/mL, 40-60 μ L；The chloro- 1- methyl pyridinium iodide (2-chloro-1-methylpyridinium of 2-
Iodide, CMPI) solution: 15-25 μm of ol/mL, 40-60 μ L；Triethylamine (triethylamine, TEA) solution: 15-25 μ
Mol/mL, 20-40 μ L；
6. salt cleaning agent: the aqueous formic acid of volume fraction 85-95%, 2-10mL；
7. redissolving liquid 1: chloroform, 0.5-2mL；
8. redissolving liquid 2: acetonitrile, 0.5-2mL.
According to the above scheme, for kit of the present invention in use, the dosage of grease sample to be tested is 1~6mg, grease is to be measured
Sample is diluted to 0.3-2mg/mL with chloroform/methanol mixed solution in advance.
According to the above scheme, the potassium hydroxide solution is solvent configuration, the hydrochloric acid with ethanol/water (95:5, v/v)
It is configured by solvent of ultrapure water.
According to the above scheme, the DEEA solution, CMPI solution, what TEA solution was configured using chromatographic grade acetonitrile as solvent
Solution.
Kit of the present invention is primarily adapted for use in the unsaturated fatty acid in vegetable oil, bacterium oil and algae oil etc., specifically
Application method is as follows:
1) 1~6mg grease is weighed, after chloroform/methanol mixed diluting, nitrogen drying；Then alkaline hydrolyzate ultrasound point is added
After dissipating, 75-85 DEG C water-bath 1.5-2.5 hour, hydrolysis quencher quenching reaction is added, is then extracted with extracting solution n-hexane fatty
Acid, nitrogen drying；
2) the photochemical derivatizationization reaction of unsaturated fatty acid
By the fatty acid extract of the drying of nitrogen obtained by step 1), redissolved with P-B reaction solution, it is anti-under 254nm low pressure mercury lamp
55-65min is answered, fatty acid P-B reaction product is obtained；
3) by the N of step 2) fatty acid P-B reaction product, N- diethyl ethylenediamine derivative reaction:
After the mixing of DEEA reactant is added in step 2) fatty acid P-B reaction product, 35-45 DEG C of ultrasound 4-6min spreads out
Biochemical reaction, with being dried with nitrogen after derivative reaction；After the product being dried with nitrogen in the step is redissolved with redissolution liquid 1, salt is added
Class scavenger is abandoned upper layer solvent after vortex, then is dried with nitrogen, and is redissolved with liquid 2 is redissolved, then by organic phase filter membrane filtering；
4) qualitative method of fatty acid: products therefrom is analyzed by mass spectrometry in step 3), obtains spectrogram and spectral peak data, really
Surely the quasi-molecular ion peak of each derivative of fatty acid is corresponded to；It is broken to generate two characteristics for each double bond in fatty acid derivative products
Piece ion, that is, be known as a pair of of diagnosis ion, and the molecular formula of 1 pair of diagnosis ion is respectively present following rule, C(x-n)H(2(x-n-a)+4)
O2N+,C(x-n+3)H(2(x-n-a)+10)ON+(X is total carbon number, and n is the position taken from methyl end first double bond of counting mode, and a is
The serial number from the methyl end counting mode double bond, such as a=1 is taken to refer to that the 1st double bond, a=2 refer to the 2nd double bond).According to examining
Dialysis can determine whether out the position of double bond, simultaneously for there are the unsaturated fatty acid of double-bond positional isomerization body, contained double bonds
Position it is different, corresponding derivative products generate the diagnosis ion of different m/z respectively in MS/MS collision induction dissociation,
Determine that there are the double bond containing positions of institute in the unsaturated fatty acid of double-bond positional isomerization body, thus to the free rouge in sample to be tested
Fat acid realizes qualitative analysis.
Compared with prior art, the beneficial effect of unsaturated fatty acid assay kit of the invention is mainly manifested in: this
It is easy to use to invent configured kit, accurate qualitative, the Gao Ling of unsaturated fatty acid can be realized under conditions of no mark product
The accurate structural analysis of unsaturated fatty acid is realized quickly.
Detailed description of the invention
Fig. 1 is the outline flowchart of kit use process of the present invention.
In Fig. 2: A is the mass spectrum 73Da neutral loss positive ion mode (+NLS73Da) of fatty acid derivative products in blue dragonfly oil
Mass spectrogram, second order ms figure of the B for C18:1, second order ms figure of the C for C18:2, second order ms figure of the D for C18:3, E C18:
4 second order ms figure.
In Fig. 3: A be ARA bacterium oil in fatty acid derivative products mass spectrum 73Da neutral loss positive ion mode (+
NLS73Da) mass spectrogram, B are the second order ms figure of C18:1, and C is the second order ms figure of C18:2, and D is the second order ms of C18:3
Figure, E are the second order ms figure of C20:4.
In Fig. 4: A be DHA algal oil in fatty acid derivative products mass spectrum 73Da neutral loss positive ion mode (+
NLS73Da) mass spectrogram, B are the second order ms figure of C18:1, and C is the second order ms figure of C18:2, and D is the second order ms of C18:3
Figure, E are the second order ms figure of C22:6.
The fatty acid abbreviation explanation used in the present invention:
C16:1n7, palmitoleic acid；C17:1,17 carbon monoenoic acids；C18:3n3, alpha-linolenic acid；C18:3n6, γ-flax
Acid；C18:2n6, linoleic acid；C18:1n9, oleic acid；C18:1n7, vaccenic acid；C20:4n6, ARA；C20:3n6,20 carbon triolefins
Acid；C20:2n6, eicosadienoic acid；C20:1n9,20 carbon monoenoic acids；C22:1n9, erucic acid；C22:6n-3, DHA.
Specific embodiment
It is right combined with specific embodiments below in order to make those skilled in the art more fully understand technical solution of the present invention
The present invention is described in further detail, but should not be construed as limiting the invention.Without departing substantially from spirit of that invention and essence
In the case where, modifications or substitutions made by the method for the present invention, step or condition are all belonged to the scope of the present invention.
In following embodiments, n-hexane, acetone, ethyl alcohol, chloroform, the reagents such as formic acid are chromatography pure reagent；The hydrogen
Potassium oxide solution is solvent configuration with ethanol/water (95:5, v/v), and the hydrochloric acid is configured by solvent of ultrapure water；Described
DEEA solution, CMPI solution, the solution that TEA solution is configured using chromatographic grade acetonitrile as solvent.
In following embodiments, P-B reaction, which need to be transferred in quartz colorimetric utensil, to be carried out.
In following embodiments, mass spectral analysis condition is the inspection of U.S. Applied Biosystems company 4000Q-Trap mass spectrum
Survey device.Electrospray ionisation source (Electrospray Ionization, ESI): positive ion mode；Scanning of the mass spectrum mode: in 73Da
Property lose scanning (73Da neutral loss scan)-enhancing ion scan (Enhanced product ion scan,
EPI)；Ion source gas 1 (Ion Source Gas 1, GS1): 20Psi；Ion source gas 2 (Ion Source Gas 1,
GS1): 20Psi；Impact energy (collision energy, CE): 35eV (neutral loss), 48eV (second level)；Remove cluster voltage
(declustering potential, DP): 40eV；Entrance potential (Entrance Potential, EP): 5V；Collision cell is defeated
Out voltage (Collision Cell Exit Potential, CXP): 10V；Mass range: 200-650m/z
Embodiment 1
The kit of the accurate Structural Identification of unsaturated fatty acid mass spectrum in a kind of grease, including alkaline hydrolyzate, hydrolysis
Quencher, extracting solution, PB reaction solution, DEEA reactant, salt cleaning agent redissolve liquid, and concrete composition ingredient difference is as follows:
1. alkaline hydrolyzate: 0.35mol/L potassium hydroxide solution, 200 μ L；
2. hydrolysis quencher: 0.5mol/L hydrochloric acid, 500 μ L；
3. extracting solution: n-hexane, 9mL；
Paten 4. ò-B ü chi (P-B) reaction solution: acetone/water/ethyl alcohol (570/380/50, v/v/v), 1mL；
5. DEEA reactant: N, N- diethyl ethylenediamine (N, N-diethyl-1,2-ethanediamine, DEEA) are molten
Liquid: 20 μm of ol/mL, 50 μ L；The chloro- 1- methyl pyridinium iodide of 2- (2-chloro-1-methylpyridinium iodide,
CMPI) solution: 20 μm of ol/mL, 50 μ L；Triethylamine (triethylamine, TEA) solution: 20 μm of ol/mL, 30 μ L；
6. salt cleaning agent: formic acid/water (90/10, v/v/v), 5mL；
7. redissolving liquid 1: chloroform, 1mL；
8. redissolving liquid 2: acetonitrile, 1mL.
Kit described in the present embodiment to fatty acid qualitative analysis in blue dragonfly oil, specific steps are as follows:
1) 3mg blue dragonfly oil grease (sample to be tested) is weighed, after 3mL chloroform/methanol (1:1, v/v) dilution, takes 10 μ L dilute
Release liquid, nitrogen drying；Then 200 μ L alkaline hydrolyzate 0.35M KOH (being dissolved in ethanol/water (95:5, v/v)), 2 points of ultrasound is added
Clock, 80 DEG C water-bath 2 hours, pH value of solution is adjusted to~3 quenching reactions with hydrolysis quencher 0.5MHCl aqueous solution, is then mentioned with 3mL
It takes liquid n-hexane to extract fatty acid, extracts 3 times repeatedly, n-hexane is laminated simultaneously, nitrogen drying；
2) the photochemical derivatizationization reaction of unsaturated fatty acid: the fatty acid extract of the drying of nitrogen obtained by step 1) is used
P-B reaction solution 1mL acetone/water/ethyl alcohol (570/380/50, v/v/v) solution redissolves, and reacts under 254nm low pressure mercury lamp
60min；
3) N of fatty acid P-B reaction product, N- diethyl ethylenediamine derivative reaction: fatty acid is added after PB reacts
The chloro- 1- methyl pyridinium iodide (2-chloro-1-methylpyridinium of 2- of 50 μ L20 μm ol/mL of DEEA reactant
Iodide, CMPI) and 30 μ L, 20 μm of ol/mL triethylamines (triethylamine, TEA), vortex 60s；Then 50 μ are added
Derivative reagent N, the N- diethyl ethylenediamine (N, N-diethyl-1,2-ethanediamine, DEEA) of L20 μm of ol/mL, 40 DEG C
Ultrasonic 5min performs the derivatization reaction, with being dried with nitrogen after derivative reaction；
Then, it is dried with nitrogen product chromatographic grade redissolution liquid 1 (1mL chloroform) to redissolve, after vortex 30s, salt scavenger is added
1mL, vortex 2min abandon upper layer solvent, repeat this operation 5 times, then be dried with nitrogen, and redissolved with the trifluoroacetic acid aqueous solution of 1mL, then
It is filtered by 2 μm of organic phase filter membranes；
4) qualitative method of fatty acid: products therefrom injection needle pumps sample introduction in step 3), and sample introduction flow velocity is 20 μ L/min, into
Row mass spectral analysis, the spectrogram and spectral peak data of the derivative of fatty acid that dissociates in blue dragonfly oil as shown in Figure 2, determines corresponding each fat
The quasi-molecular ion peak of acid derivative, it can be determined that go out the type of fatty acid, such as C16:1, C18:1, C18:2, C18:3, C18:
4, C20:2 (Fig. 2A)；Simultaneously for the position of double bond of unsaturated fatty acid, can be judged according to fragment ion.For example, C18:
1 derivative products can produce 1 pair of diagnosis ion (m/z 198.1,224.1), to judge that the position of double bond is C18:1n9
(Fig. 2 B)；Derivative products two pairs of diagnosis ions (m/z 198.1,224.1) of generation of C18:2, (m/z 238.1,264.1), from
And may determine that the position of double bond is C18:2n6 Fig. 2 C)；The derivative products of C18:3 generate six couples of diagnosis ion (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) is C18:3n6 and C18:3n3 (Fig. 2 D) so as to the position for judging double bond；C18:
4 derivative products four pairs of diagnosis ions (m/z156.1,182.1) of generation, (m/z 196.1,222.1) (m/z 236.1,
272.1), (m/z276.1,302.1) judges that the position of double bond of C18:4 is C18:4n3 (Fig. 2 E).Therefore, which can
Under the conditions of no mark product, realizes and accurate qualitative analysis is realized to the unsaturated fatty acid in sample to be tested.
Embodiment 2
4) embodiment 2 difference from example 1 is that: use ARA bacterium oil 3mg replacement blue dragonfly oil grease as to test sample
Product.The spectrogram of free derivative of fatty acid and spectral peak data are as shown in figure 3, so as to judge fatty acid in ARA bacterium oil
Type, such as C16:1, C18:1, C18:2, C18:3, C20:1, C20:2, C20:3, C20:4 (Fig. 3 A)；Simultaneously for unsaturated lipid
The position of double bond of fat acid, such as the derivative products of C18:1 can produce 2 couples of diagnosis ion (m/z 198.1,224.1) (m/
Z226.1,252.1), to judge that the position of double bond is C18:1n9, C18:1n7 (Fig. 3 B)；The derivative products of C18:2 generate
Two pairs of diagnosis ions (m/z 198.1,224.1), (m/z 238.1,264.1), so as to judge that the position of double bond is
C18:2n6 Fig. 3 C)；The derivative products of C18:3 generate six pairs of diagnosis 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 as to judge that the position of double bond is C18:3n6 and C18:3n3 (Fig. 3 D)；The derivative products of C20:4 generate four pairs of diagnosis
Ion (m/z 156.1,182.1), (m/z 196.1,222.1) (m/z 236.1,272.1), (m/z276.1,302.1) sentences
The position of double bond of disconnected C18:4 out is C20:4n6 (Fig. 3 E).Therefore, which can realize under the conditions of no mark product and treat test sample
Free unsaturated fatty acid in product realizes accurate qualitative analysis.
Embodiment 3
Embodiment 3 difference from example 1 is that: use DHA algal oil 3mg replacement blue dragonfly oil grease as to test sample
Product.The spectrogram of free derivative of fatty acid and spectral peak data are as shown in figure 4, so as to judge fatty acid in DHA algal oil
Type, such as C16:1, C18:1, C18:2, C20:2, C22:1, C22:5C22:6 (Fig. 4 A)；Simultaneously for unsaturated fatty acid
Position of double bond, such as the derivative products of C18:1 can produce 2 pairs of diagnosis ions (m/z 198.1,224.1), to judge double
The position of key is C18:1n9 (Fig. 4 B)；The derivative products of C18:2 generate two pairs of diagnosis ions (m/z 198.1,224.1), (m/
Z238.1,264.1), so as to judge that the position of double bond is C18:2n6 (Fig. 4 C)；The derivative products of C18:3 generate three pairs
It diagnoses ion (m/z 156.1,182.1), (m/z 196.1,222.1) (m/z 236.1,272.1), it is double so as to judge
The position of key is C18:3n6 (Fig. 4 D)；The derivative products of C22:6 generate six couples of diagnosis ion (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) position of double bond for judging C18:4 is C20:4n6 (Fig. 4 E).Therefore, which can realize under the conditions of no mark product
Accurate qualitative analysis is realized to the free unsaturated fatty acid in sample to be tested.
The above is only a preferred embodiment of the present invention, it is noted that come for those of ordinary skill in the art
It says, without departing from the concept of the premise of the invention, several modifications and variations can also be made, these belong to of the invention
Protection scope.
Claims (6)
1. the kit of the accurate Structural Identification of unsaturated fatty acid in a kind of grease, it is characterised in that including alkaline hydrolyzate, hydrolysis
It reacts quencher, extracting solution, P-B reaction solution, DEEA reactant, salt cleaning agent, redissolve liquid, concrete composition ingredient is respectively such as
Under:
1. alkaline hydrolyzate: 0.3-0.4mol/L potassium hydroxide solution；
2. hydrolysis quencher: 0.4-0.6mol/L hydrochloric acid；
3. extracting solution: n-hexane；
4. P-B reaction solution: acetone/water/ethyl alcohol mixed solution, three's volume ratio are (550-600): (350-400): (40-
60)；
5. DEEA reactant: N, N- diethyl ethylenediamine solution: 15-25 μm of ol/mL；The chloro- 1- methyl pyridinium iodide of 2- is molten
Liquid: 15-25 μm of ol/mL；Triethylamine solution: 15-25 μm of ol/mL；
6. salt cleaning agent: the aqueous formic acid of volume fraction 85-95%；
7. redissolving liquid 1: chloroform；
8. redissolving liquid 2: acetonitrile.
2. the kit of the accurate Structural Identification of unsaturated fatty acid in a kind of grease, it is characterised in that including alkaline hydrolyzate, hydrolysis
Quencher, extracting solution, P-B reaction solution, DEEA reactant are reacted, salt cleaning agent redissolves liquid, and concrete composition ingredient is respectively such as
Under:
1. alkaline hydrolyzate: 0.3-0.4mol/L potassium hydroxide solution, 100-300 μ L；
2. hydrolysis quencher: 0.4-0.6mol/L hydrochloric acid, 400-600 μ L；
3. extracting solution: n-hexane, 5-15mL；
4. P-B reaction solution: acetone/water/ethyl alcohol mixed solution, three's volume ratio are (550-600): (350-400): (40-
60), 0.5-1.5mL；
5. DEEA reactant: N, N- diethyl ethylenediamine solution: 15-25 μm of ol/mL, 40-60 μ L；The chloro- 1- picoline of 2-
Iodide solution: 15-25 μm of ol/mL, 40-60 μ L；Triethylamine solution: 15-25 μm of ol/mL, 20-40 μ L；
6. salt cleaning agent: the aqueous formic acid of volume fraction 85-95%, 2-10mL；
7. redissolving liquid 1: chloroform, 0.5-2mL；
8. redissolving liquid 2: acetonitrile, 0.5-2mL.
3. the kit of the accurate Structural Identification of unsaturated fatty acid, feature in a kind of grease according to claim 1 or 2
It is that the potassium hydroxide solution is configured by solvent of the ethanol water of volume fraction 90-95%.
4. the kit of the accurate Structural Identification of unsaturated fatty acid, feature in a kind of grease according to claim 1 or 2
It is in the DEEA reactant that three kinds of solution are configured by solvent of chromatographic grade acetonitrile.
5. the kit of the accurate Structural Identification of unsaturated fatty acid, feature exist in a kind of grease according to claim 2
In the kit in use, the dosage of grease sample to be tested is 3~10mg, grease sample to be tested is mixed with chloroform/methanol in advance
It closes solution and is diluted to 0.3-1mg/mL.
6. the application method of kit described in claim 2, it is characterised in that specifically used method is as follows:
1) 1~6mg grease is weighed, after chloroform/methanol mixed diluting, nitrogen drying；Then after alkaline hydrolyzate ultrasonic disperse being added,
75-85 DEG C water-bath 1.5-2.5 hours, be added hydrolysis quencher quenching reaction, then with extracting solution n-hexane extract fatty acid, nitrogen
Drying；
2) the photochemical derivatizationization reaction of unsaturated fatty acid
By the fatty acid extract of the drying of nitrogen obtained by step 1), is redissolved with P-B reaction solution, react 55- under 254nm low pressure mercury lamp
65min obtains fatty acid P-B reaction product；
3) by the N of step 2) fatty acid P-B reaction product, N- diethyl ethylenediamine derivative reaction:
After the mixing of DEEA reactant is added in step 2) fatty acid P-B reaction product, 35-45 DEG C of ultrasound 4-6min is performed the derivatization
Reaction, with being dried with nitrogen after derivative reaction；After the product being dried with nitrogen in step chromatographic grade redissolves the redissolution of liquid 1, it is added
Salt scavenger is abandoned upper layer solvent after vortex, then is dried with nitrogen, and redissolves liquid 2 with chromatographic grade and redissolve, and then filters by organic phase
Film filtering；
4) qualitative method of fatty acid: products therefrom is analyzed by mass spectrometry in step 3), obtains spectrogram and spectral peak data, determination pair
Answer the quasi-molecular ion peak of each derivative of fatty acid；In fatty acid derivative products each double bond can generate two characteristic fragments from
Son, that is, be known as a pair of of diagnosis ion, and the molecular formula of 1 pair of diagnosis ion is respectively present following rule, C(x-n)H(2(x-n-a)+4)O2N+,
C(x-n+3)H(2(x-n-a)+10)ON+, wherein X is total carbon number, and n is the position taken from methyl end first double bond of counting mode, and a is
Take the serial number from the methyl end counting mode double bond；The position that can determine whether out double bond according to diagnosis ion, simultaneously for presence
The unsaturated fatty acid of double-bond positional isomerization body, the double bond containing position of institute is different, and corresponding derivative products are in mass spectrum
The diagnosis ion of different m/z is generated in collision induced dissociation respectively, determines that there are the unsaturated fatty acids of double-bond positional isomerization body
The middle double bond containing position of institute, to realize qualitative analysis to the free fatty acid in sample to be tested.
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