CN106124604B - The mass spectrometric analysis method of free fatty in a kind of edible oil based on Derivative - Google Patents
The mass spectrometric analysis method of free fatty in a kind of edible oil based on Derivative Download PDFInfo
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Abstract
The mass spectrometric analysis method of free fatty, includes the following steps in a kind of edible oil based on Derivative：1) edible oil sample to be measured is diluted with solvent, carries out derivative reaction after internal standard is added, obtains the sample introduction solution of sample to be tested, be analyzed by mass spectrometry；2) qualitative analysis：According to the mass-to-charge ratio in gained spectrogram, the quasi-molecular ion peak of corresponding free-fat acid derivative is determined；3) quantitative analysis：Fatty acid standards are configured to a series of standard solution of concentration, derivative reaction is carried out after being separately added into internal standard, the sample introduction solution to get standard samples is analyzed by mass spectrometry, and draws standard curve, and quantitative analysis is carried out to edible oil sample to be measured.It is complicated that the present invention solves edible oil mesostroma, free fatty acid content is low, ionizing efficiency is low when being difficult to purify and be enriched with, and being detected under mass spectrum negative ion mode and the insufficient difficulty of detection sensitivity, realizes efficient, the highly sensitive qualitative and quantitative analysis of free fatty in edible oil.
Description
Technical field
The present invention relates to a kind of mass spectrometric analysis method of free fatty in edible oil based on Derivative, belong to point
Analyse detection field.
Technical background
The main component of edible oil is triglycerides (triacylglycerols, TAG account for 95%~98%), glycerine three
Ester is condensed by 1 glycerol molecule and 3 fatty acid molecules, aliphatic acid account for triglycerides molecular weight composition 95% with
On.Aliphatic acid (Fatty acid, FA) is the aliphatic carboxylic acid of length not equal (C4~C36) a series of, is simplest one kind
Lipid compounds are the ingredients for forming other lipids.Aliphatic acid plays an important role on maintaining health, fat in diet
The composition of fat acid and the close phase of incidence of the various diseases such as content and tumour, coronary heart disease, cardiovascular and cerebrovascular diseases and senile dementia
Guan Xing.Therefore, the composition of aliphatic acid and its proportioning become the most important index for weighing edible oil nutritive value.
Free fatty (Free fatty acid, FFA) is also known as non-esterified fatty acid (nonestesterified
Fatty acid, NEFA), be decomposing neutral fat at substance.Free fatty in edible oil is the hydrolysis of triglycerides
Product, due to both containing hydrophilic radical in its molecule or containing hydrophobic grouping, it is intended to concentrate on the surface of edible oil, reduce table
Face tension increases oxygen diffusion rate in edible oil and fat, and then accelerates the oxidation of edible oil.Also, free fatty does not have
Triglycerides is stablized, it is easier to which the oxidation and rancid for leading to oil influences the quality and function of oil.FFAs compositions can in raw oil
The oily degree of injury of quality and assessment for describing expressed oil.FFA ingredients can be used as oil product in different in moisture, temperature, oxygen-containing
Degradation parameter during amount, illumination storage and pan-fried stir-fry.
The method that tradition measures free fatty in edible oil mainly has titration measuring acid value and gas chromatography-mass spectrography
(GC-MS).Acid value of lipids is indicated with neutralizing the milligram number of the potassium hydroxide in 1 gram of grease needed for free fatty, is to weigh
One important indicator of oil quality.Potassium hydroxide titration is mostly used to the measurement of acid value of lipids both at home and abroad at present and is dissolved in ethyl alcohol
In oil to phenolphthalein terminal point, although easy to operate, be not easy to realize automated analysis, and this method needs a large amount of reagent, structure
At potential environmental pollution, in addition, what titration measuring acid value obtained is the fatty acid total amount that dissociates in edible oil, it cannot be to each trip
Qualitative analysis is carried out from aliphatic acid, the content of various free fatties can not be obtained；Gas chromatography (GC) is capable of providing food
With the qualitative and quantitative information of each free fatty in oil, but edible oil mesostroma is complicated, and free fatty acid content is low, it is strong to have
Polarity and weak volatility, therefore the free fatty in edible oil is difficult directly to be detached through GC, sample needs to first pass through complexity
Isolating and purifying process, (free fatty or use magnetic nanoparticle are enriched with the trip in edible oil in such as liquid-liquid extraction edible oil
From aliphatic acid etc.), then GC separation, qualitative will could be carried out after the free fatty being purified carries out esterification derivative again
With the free fatty in quantitative analysis edible oil, whole process is extremely complex, cumbersome, time-consuming, and needs to expend a large amount of organic
Solvent, in addition, disadvantages such as that there is also stability are poor for this method, and detection sensitivity is low.
Therefore, fatty acid analysis technology of dissociating in existing edible oil is in analysis throughput, sensitivity for analysis and accuracy
Aspect all comes with some shortcomings.
Invention content
The technical problem to be solved by the present invention is to provide a kind of based on derivatization in view of the above shortcomings of the prior art
The mass spectrometric analysis method of free fatty in the edible oil of technology solves edible oil mesostroma complexity, free fatty acid content
It is low, it is difficult to which that ionizing efficiency is low when purifying and enrichment and free fatty are detected under mass spectrum negative ion mode and detects
Under-sensitive is difficult, realizes efficient, the highly sensitive qualitative and quantitative analysis of free fatty in edible oil.
The present invention is to solve technical problem set forth above, and used technical solution is：
The mass spectrometric analysis method of free fatty, includes the following steps in a kind of edible oil based on Derivative：
1) edible oil sample to be measured is diluted with solvent, after internal standard is added, reaction is performed the derivatization, after reaction in edible oil
Free fatty be converted into positively charged free-fat acid derivative, obtain the sample introduction solution of sample to be tested；
2) the sample introduction solution of sample to be tested obtained by step 1) is analyzed by mass spectrometry, acquires the spectrum of free-fat acid derivative
Figure and spectral peak data；
3) qualitative analysis：According to the mass-to-charge ratio in spectrogram obtained by step 2), the standard of corresponding free-fat acid derivative is determined
Molecular ion peak, to carry out qualitative analysis to the free fatty in sample to be tested；
4) quantitative analysis：Fatty acid standards are diluted to a series of standard solution of concentration with solvent, are separately added into interior
It after mark, is reacted being performed the derivatization under the same conditions with step 1), free fatty is converted into positively charged trip after reaction
From derivative of fatty acid, the sample introduction solution that gets standard samples；
5) a, the sample introduction solution of standard sample obtained by step 4) is analyzed by mass spectrometry, acquisition free-fat acid derivative
Spectrogram and spectral peak data, using free fatty in standard solution and interior target concentration proportion as abscissa, free fatty derives
The quasi-molecular ion peak intensity rate of object and internal standard derivative is ordinate, draws standard curve；B, by spectrogram obtained by step 2)
In dissociate derivative of fatty acid and internal standard derivative quasi-molecular ion peak intensity rate combined standard curve, to edible oil to be measured
Free fatty in sample carries out quantitative analysis.
By said program, the edible oil sample to be measured includes rapeseed oil, sesame oil, corn oil, linseed oil, olive
Oil, perilla herb oil, lard, fish oil etc..
By said program, used derivative reagent is amino (- NH there are one one end contains2), the other end is containing there are one uncles
The compound of amido, general formula are expressed as：NH2-(CH2)n-NR2, wherein n value ranges are 2~4, and R group is methyl, ethyl
Deng.Preferably, derive effect when n=2 and Mass Spectrometer Method effect is ideal, such as derivative reagent selects N, N- dimethyl second
Diamines and N, N- diethyl ethylenediamines etc..It is anti-that amidation occurs for the carboxyl in the amino and free fatty in the derivative reagent
It answers, the tertiary amino group of the derivative reagent other end is introduced into free fatty, to introduce the positive charge of an easily ionizable
Group converts negatively charged free fatty to positively charged free-fat acid derivative.
By said program, the condition of the derivative reaction is：Internal standard is added after edible oil sample is diluted with solvent, urges
Then derivative reagent is added in agent vortex mixing, reaction temperature is 38~42 DEG C, and the reaction time is 2~10min.
By said program, in the derivative reaction, with deuterated hexadecanoic acid (FA16:It is 0-d4) internal standard, with the chloro- 1- of 2-
Methyl pyridinium iodide (2-chloro-1-methylpyridinium iodide, CMPI) and triethylamine
(triethylamine, TEA) is catalyst, with N, N- diethyl ethylenediamines (N, N-diethyl-1,2-ethanediamine,
DEEA) it is derivative reagent, solvent is using organic solvents such as acetonitriles.Preferably, in derivative reaction system, the concentration of aliphatic acid
For 0.5~200nmol/L, interior a concentration of 5~15nmol/L of target, a concentration of 0.5~0.7 μm of ol/mL of catalyst of triethylamine,
A concentration of 0.1~0.5 μm of ol/mL of the chloro- 1- methyl pyridinium iodides of catalyst 2-, derivative reagent N, N- diethyl ethylenediamine
A concentration of 0.6~1.2 μm of ol/mL.
By said program, the present invention performs the derivatization reaction to free fatty in edible oil, and free fatty is converted into
Positively charged free-fat acid derivative, when being analyzed by mass spectrometry in the positive-ion mode, free-fat acid derivative is in matter
Spectrum collision-induced cracking in will produce a fixed mass number neutral group (such as derivative reagent be N, N- diethyl ethylenediamines
When, neutral group is NH- (CH2CH3)2, mass number 73Da), therefore mass spectrum neutral loss scan pattern is used, scanning is neutral
Missing mass number 73Da (NLS73), is capable of the quasi-molecular ion peak for detecting each derivative of fatty acid of selectivity, and then realizes
The qualitative and quantitative analysis of free fatty in edible oil sample, as shown in figs. 10-11.
By said program, sample introduction solution is pretreated preferably through drying, redissolution, purifying etc. obtained by step 1) and step 4)
Journey is analyzed by mass spectrometry again.The preprocessing process is more than primary, wherein dry using dryings such as nitrogen, helium, argon gas；
It redissolves using organic solvents such as chloroforms；Purifying includes extracting and filtering etc., wherein extraction is mixed using what formic acid, water, chloroform formed
Close solution；Filtering uses aperture for 0.1~5 μm of organic phase filter membrane.
By said program, the condition of the mass spectral analysis is：Injection needle pumps sample introduction, and sample introduction flow velocity is 10~30 μ L/min；
Electron spray ionisation source (Electrospray Ionization, ESI)：Positive ion mode；Scanning of the mass spectrum pattern：73Da neutrality is lost
Lose scanning (73Da neutral loss scan)；Ion source gas 1 (Ion Source Gas 1, GS 1)：12kPa；Collision
Energy (collision energy, CE)：30~35eV；Remove cluster voltage (declustering potential, DP)：70~
80eV；Entrance potential (Entrance Potential, EP)：10V；Collision cell output voltage (Collision Cell Exit
Potential, CXP)：10V；Mass range：200~550m/z.
By said program, the condition of step 4) the derivedization reaction is identical as step 1)；Mass spectral analysis in step 4)
Condition it is identical as step 2)；Solvent in the step 4) is identical with step 1).
By said program, step 4) the Plays solution is the mixed solution of one or more of aliphatic acid, and the mark
The concentration range of aliphatic acid is in 0.5~200nmol/L in quasi- solution.
The present invention proposes a kind of mass spectrometric analysis method of free fatty in the edible oil based on Derivative.The party
Method is using one end containing there are one amino (- NH2), the other end contains the compound there are one tertiary amine groups, such as N, N- dimethyl-ethylenediamines
As derivative reagent, the derivative for free fatty in edible oil.In amino group and free fatty in derivative reagent
Carboxyl occur amidation process, the tertiary amino group of the derivative reagent other end is introduced into free fatty, to introduce
The positive charge group of one easily ionizable, and convert negatively charged free fatty to positively charged free fatty and derive
Object.
Compared with prior art, the beneficial effects of the invention are as follows：
1, edible oil mesostroma is complicated, and free fatty acid content is low, and traditional gas chromatography-mass spectrometry analysis method needs head
First the free fatty in edible oil is enriched with and is purified, methyl esters then is carried out to the free fatty after enriching and purifying again
Change and carry out gas chromatography-mass spectrometry analysis after deriving, operating procedure is extremely complex and detection sensitivity is low.And in the present invention, excellent
Under the derivatization conditions of change, amino group in derivative reagent specificity and the free fatty containing carboxylic group in edible oil
Amidation condensation occurs for class compound, there is no need to which the free fatty in edible oil sample is enriched with and is purified, directly
Reaction can be performed the derivatization after edible oil is diluted, the reaction time only needs 5 minutes or so, and it is multiple to solve edible oil mesostroma
Miscellaneous, free fatty acid content is low, and traditional analysis purifying and enriching step are complicated, the disadvantage of required time length.
2, amidation occurs for the carboxyl in the amino group and free fatty in derivative reagent of the present invention anti-
It answers, the tertiary amino group of the derivative reagent other end is introduced into free fatty, to introduce the positive charge of an easily ionizable
Group, and convert negatively charged free fatty to positively charged free-fat acid derivative, successfully realize
Derivatization product is detected under positive ion mode, improves ionizing efficiency, ionization is reduced and inhibits, and substantially increases electrospray ionization mass spectrum spirit
Sensitivity；Meanwhile solving that ionizing efficiency when free fatty is detected under mass spectrum negative ion mode is low and detection sensitivity
Insufficient difficulty realizes the highly sensitive detection and analysis of free fatty in edible oil.
3, after the present invention performs the derivatization reaction to free fatty in edible oil, mass spectrum point is carried out in the positive-ion mode
When analysis, the neutral group that free-fat acid derivative will produce a fixed mass number in MS/MS collision inducing lysis (such as spreads out
Raw reagent is N, and when N- diethyl ethylenediamines, neutral group is NH- (CH2CH3)2, mass number 73Da), therefore using in mass spectrum
Property lose scan pattern, scanning neutral loss mass number 73Da (NLS73), be capable of selectivity detects each derivative of fatty acid
Quasi-molecular ion peak.Compared to conventional method, free-fat in the edible oil of the present invention based on Derivative
Acid mass spectrometric analysis method in edible oil dissociate fatty acid compound have it is highly selective, can specificity in edible oil
Free acid kind compound carries out high-selectivity analysis, is particularly suitable for analyzing the free acid kind in complex matrices sample
Compound.
4, method of the invention is easy to operate, carries out derivative reaction after edible oil sample to be measured directly dilution, obtains
Reaction product is directly entered mass spectrum, is divided in the positive-ion mode using neutral loss scan without carrying out chromatography post separation
Analysis, method is simple and efficient, can realize efficient, highly selective, the highly sensitive qualitative, quantitative point of free fatty in edible oil
Analysis.
Description of the drawings
Fig. 1 is 16 kinds of fatty acid standards (capric acid (10 of mass spectral analysis:0), lauric acid (12:0), myristic acid (14:0)、
Pentadecanoic acid (15:0), palmitoleic acid (16:1), palmitic acid (16:0), leukotrienes (18:3), linoleic acid (18:2), oleic acid (18:
1), stearic acid (18:0), nonadecylic acid (19:0), eicosapentaenoic acid (EPA, 20:5), arachidonic acid (ARA, 20:4), two
Ten carbon enoic acids (20:1), arachidic acid (20:0), docosahexaenoic acid (DHA, 22:6)) derivative and internal standard (deuterated hexadecane
Acid, FA16:0-d4 (IS)) derivative carries out the mass spectrum of neutral loss scan (73Da) (+NLS73Da) in the positive-ion mode
Figure；
In Fig. 2：A is the mass spectrogram of the free-fat acid derivative in olive oil sample, and b is free fatty in olive oil
Contain spirogram；
In Fig. 3：A is the mass spectrogram of free derivative of fatty acid in linseed oil sample, and b is free-fat in linseed oil
Acid contains spirogram；
In Fig. 4：A is mass spectrogram of the purple perilla oil samples middle reaches from derivative of fatty acid, and b is free fatty in perilla herb oil
Containing spirogram；
In Fig. 5：A is mass spectrogram of the sesame oil samples middle reaches from derivative of fatty acid, and b is free fatty in sesame oil
Containing spirogram；
In Fig. 6：A is the mass spectrogram of the free-fat acid derivative in corn oil samples, and b is the free-fat in corn oil
Acid contains spirogram；
In Fig. 7：A is the mass spectrogram of the free-fat acid derivative in vegetable seed oil samples, and b is free fatty in rapeseed oil
Contain spirogram；
In Fig. 8：A is the mass spectrogram of the free-fat acid derivative in fish oil sample, and b is that free fatty contains in fish oil
Spirogram；
In Fig. 9：A is the mass spectrogram of the free fatty of lard, and b is the free fatty acid content figure of lard；
Figure 10 is free fatty derivative reaction schematic diagram；
Figure 11 is the mass spectral analysis schematic diagram of free-fat acid derivative.
Specific implementation mode
In order to make those skilled in the art more fully understand technical scheme of the present invention, with reference to specific embodiment pair
The present invention is described in further detail.
The aliphatic acid abbreviation explanation used in the present invention：10:0, capric acid；12:0, lauric acid；14:0, myristic acid；15:
0, pentadecanoic acid；16:1, palmitoleic acid；16:0, palmitic acid；18:3, leukotrienes；18:2, linoleic acid；18:1, oleic acid；18:0,
Stearic acid；19:0, nonadecylic acid；20:5, eicosapentaenoic acid；20:4, arachidonic acid；20:1, eicosenoic acid；20:0,
Arachidic acid；22:6, docosahexaenoic acid；IS, internal standard：Deuterated hexadecanoic acid (FA16:0-d4).
Mass spectral analysis condition is the 4000Q-Trap mass spectrums inspection of Applied Biosystems companies of the U.S. in following embodiments
Survey device.
In following embodiments, the establishment step of standard curve is as follows：
1) fatty acid standards are made into isopropanol to the stock solution of 1mg/mL respectively, are then diluted to respectively with acetonitrile
A concentration of 0.5nmol/L, 1nmol/L, 2nmol/L, 20nmol/L, 50nmol/L, 100nmol/L, 150nmol/L,
200nmol/L Fatty acid standards liquid；
2) each that 10 μ L1 μm ol/L internal standards d4-16 are added in the acetonitrile for taking 50 μ L Fatty acid standards liquid to 810 μ L respectively:0
After vortex 10s mixings, the chloro- 1- methyl pyridinium iodides (CMPI) of 2- and 30 μ L, 20 μ of 20 μ L20 μm ol/mL are added
Mol/mL triethylamines (TEA), vortex 60s；Then derivative reagent N, the N- diethyl ethylenediamine of 50 μ L20 μm ol/mL is added,
40 DEG C of ultrasound 5min, derivative reaction are completed；
3) solution after derivative reaction obtained by step 2) is dried up with nitrogen, then uses the chloroform of 1mL to redissolve, is vortexed
After 30s, it is 10 that volume ratio, which is added,:90:20 formic acid：Water：Chloroformic solution 1mL, vortex 2min abandon upper layer solvent；Repeat this behaviour
Make 2 times, then dried up with nitrogen, the trifluoroacetic acid aqueous solution of 1mL is used in combination to redissolve, then passes through 2 μm of organic phase filter membrane and filter, marked
The sample introduction solution of quasi- sample；
4) the sample introduction solution of standard sample obtained by step 3) is analyzed by mass spectrometry, acquires the spectrum of free-fat acid derivative
Figure and spectral peak data (see Fig. 1), using aliphatic acid in standard solution and interior target concentration proportion as abscissa x, derivative of fatty acid
Quasi-molecular ion peak intensity rate with internal standard derivative is ordinate y, draws standard curve (being shown in Table 1).
Fig. 1 is 15 kinds of fatty acid standards (10:0、12:0、14:0、15:0、16:1、16:0、18:3、18:2、18:1、
18:0、20:5、20:4、20:1、20:0、22:6) it is analyzed by mass spectrometry after derivative, carries out in 73Da in the positive-ion mode
Property lose (+NLS73Da) scanning mass spectrogram.
Table 1 is the standard curve of 16 kinds of free fatties, wherein using free fatty and internal standard concentration proportion as abscissa,
Free-fat acid derivative is ordinate with internal standard derivative peak intensity rate.
Table 1
5) method veracity and precision：Using corn oil as representative, basic, normal, high concentration is added in sample diluting liquid
Aliphatic acid hybrid standard product solution, 3 samples of each parallel preparation of concentration are accurate to characterize with the rate of recovery and relative standard deviation
Degree, the evaluation of withinday precision and day to day precision is to prepare 3 samples respectively within 3 working days, repeats sample introduction 3 times, meter
Calculate its relative standard deviation value.The calculation formula of free-fat acid recovering rate is the rate of recovery=(sample surveyed after addition standard items
Middle FFA contents-be not added with standard items survey actual sample in FFA contents)/actual interpolation amount, as can be seen from Table 2, sample to be tested
The rate of recovery 85.3~117.6%, method repeatability (RSD in tolerance interval<10%).
Table 2
Embodiment 1
The mass spectrometric analysis method of free fatty, includes the following steps in a kind of olive oil based on Derivative：
1) it weighs 2mg olive oil and is diluted to 0.2mg/ml with n-hexane, as sample to be tested；
It takes 60 μ L samples to be tested that 830 μ L dilution in acetonitrile are added, 10 μ L1 μm ol/L internal standards FA16 is then added:0-d4 is vortexed
After 10s mixings, the chloro- 1- methyl pyridinium iodides (CMPI) of 2- and 30 μ L, 20 μm of ol/mL tri- of 20 μ L20 μm ol/mL are added
Ethamine (TEA), vortex 60s；Then derivative reagent N, the N- diethyl ethylenediamine (DEEA) of 50 μ L20 μm ol/mL is added, 40
DEG C ultrasound 5min, derivative reaction are completed；
Solution after gained derivative reaction is dried up with nitrogen, and the chloroform of 1mL is then used to redissolve, and after vortex 30s, body is added
Product is than being 10:90:20 formic acid：Water：Chloroformic solution 1mL, vortex 2min abandon upper layer solvent；This operation 2 times is repeated, then uses nitrogen
Air-blowing is dry, and the trifluoroacetic acid aqueous solution of 1mL is used in combination to redissolve, and then passes through 2 μm of organic phase filter membrane and filters, obtains the sample introduction of sample to be tested
Solution；
2) the sample introduction solution of sample to be tested obtained by step 1) is analyzed by mass spectrometry, acquires the spectrum of free-fat acid derivative
Figure and spectral peak data, as shown in Figure 2；
Wherein, the condition of mass spectral analysis is：Injection needle pumps sample introduction, and sample introduction flow velocity is 20 μ L/min, is analyzed by mass spectrometry,
Mass spectral analysis condition is Applied Biosystems companies of U.S. 4000Q-Trap mass detectors.Electron spray ionisation source
(Electrospray Ionization, ESI)：Positive ion mode；Scanning of the mass spectrum pattern：73Da neutral loss scans (73Da
neutral lossscan)；Ion source gas 1 (Ion Source Gas 1, GS1)：12kPa；Impact energy (collision
Energy, CE)：32eV；Remove cluster voltage (declustering potential, DP)：75eV；Entrance potential (Entrance
Potential, EP)：5V；Collision cell output voltage (Collision Cell Exit Potential, CXP)：10V；Quality model
It encloses：210-550m/z
3) qualitative analysis：According to the mass-to-charge ratio in spectrogram obtained by step 2), the standard of corresponding free-fat acid derivative is determined
Molecular ion peak, to carry out qualitative analysis to the free fatty in sample to be tested；According to mass spectrogram 2a, olive can be obtained
It is respectively containing 8 kinds of free fatties in oil：14:0、16:0、18:3、18:2、18:1、18:0 and 20:0；
4) quantitative analysis：By the quasi-molecular ion of dissociate in spectrogram obtained by step 2) derivative of fatty acid and internal standard derivative
Peak intensity ratio 1.401 (14:0)、1.577(16:0)、0.012(18:3)、0.168(18:2)、1.622(18:1)、1.206
(18:And 0.2156 (20 0):0), combined standard curve (table 1) determines the free fatty in olive oil sample to be tested
Amount analysis, the content for obtaining free fatty is as shown in Figure 2 b, respectively：424.94μg/g(14:0)、536.70μg/g(16:
0)、37.63μg/g(18:3)、157.64μg/g(18:2)、1545.95μg/g(18:1)、562.14μg/g(18:0) and
11.67μg/g(20:0)。
Embodiment 2
The mass spectrometric analysis method of free fatty in a kind of linseed oil based on Derivative, not with embodiment 1
It is with place：Olive oil sample to be tested in embodiment 1 replaces with linseed oil sample to be tested.
Fig. 3 a and Fig. 3 b are that the mass spectrogram of free derivative of fatty acid and free fatty contain in linseed oil sample to be tested
Amount.According to mass spectrogram 3a, it is known that contain 8 kinds of free fatties in linseed oil sample to be tested, respectively 14:0、16:1、
16:0、18:3、18:2、18:1、18:0 and 20:1；Free derivative of fatty acid and internal standard in gained linseed oil sample to be tested
The quasi-molecular ion peak intensity rate of derivative is respectively：0.200(14:0)、0.006(16:0)、0.122(18:3)、0.116
(18:2)、0.199(18:1)、0.681(18:And 0.003 (20 0):0), combined standard curve (table 1), waits for linseed oil
Free fatty carries out quantitative analysis in sample, obtains content such as Fig. 3 b institutes of free fatty in linseed oil sample to be tested
Show, respectively：112.14μg/g(14:0)、169.42μg/g(16:1)、292.42μg/g(16:0)、593.25μg/g(18:
3)、174.50μg/g(18:2)、244.89μg/g(18:1)、504.76μg/g(18:And 62.53 μ g/g (20 0):1).
Embodiment 3
The mass spectrometric analysis method of free fatty, the difference with embodiment 1 in a kind of perilla herb oil based on Derivative
Place is：Olive oil sample to be tested in embodiment 1 replaces with perilla herb oil sample to be tested.
Fig. 4 a and Fig. 4 b are the mass spectrogram and free fatty acid content of free derivative of fatty acid in perilla herb oil sample to be tested.
According to mass spectrogram 4a, it is known that contain 7 kinds of free fatties in perilla herb oil sample to be tested, respectively 14:0、16:0、18:3、
18:2、18:1、18:0 and 20:0；The standard of free derivative of fatty acid and internal standard derivative in gained linseed oil sample to be tested
Molecular ion peak intensity rate is respectively：1.151(14:0)、1.356(16:0)、0.284(18:3)、0.129(18:2)、
0.106(18:1)、0.964(18:And 0.015 (20 0):0), combined standard curve (table 1), to perilla herb oil sample to be tested middle reaches
Quantitative analysis is carried out from aliphatic acid, the content for obtaining free fatty in perilla herb oil sample to be tested is as shown in Figure 4 b, respectively：
422.05μg/g(14:0)、457.73μg/g(16:0)、689.84μg/g(18:3)、120.83μg/g(18:2)、34.38μg/g
(18:1)、435.69μg/g(18:And 8.96 μ g/g (20 0):0).
Embodiment 4
The mass spectrometric analysis method of free fatty, the difference with embodiment 1 in a kind of sesame oil based on Derivative
Place is：Olive oil sample to be tested in embodiment 1 replaces with sesame oil sample to be tested.
Fig. 5 a and Fig. 5 b are the mass spectrogram and free fatty acid content of free derivative of fatty acid in sesame oil sample to be tested.
According to mass spectrogram 5a, it is known that contain 7 kinds of free fatties in sesame oil sample to be tested, respectively 14:0、16:0、18:3、
18:2、18:1、18:0 and 20:0.The standard point of free derivative of fatty acid and internal standard derivative in gained sesame oil sample to be tested
Daughter ion peak intensity ratio is respectively：1.327(14:0)、1.442(16:0)、0.013(18:3)、0.786(18:2)、1.435
(18:1)、1.358(18:And 0.049 (20 0):0), combined standard curve (table 1), to the fat that dissociates in sesame oil sample to be tested
Fat acid carries out quantitative analysis, and the content for obtaining free fatty in sesame oil sample to be tested is as shown in Figure 5 b, respectively：479.67
μg/g(14:0)、527.95μg/g(16:0)、34.20μg/g(18:3)、1382.37μg/g(18:2)、1229.63μg/g(18:
1)、641.49μg/g(18:And 64.05 μ g/g (20 0):0).
Embodiment 5
The mass spectrometric analysis method of free fatty, the difference with embodiment 1 in a kind of corn oil based on Derivative
Place is：Olive oil sample to be tested in embodiment 1 replaces with corn oil sample to be tested.
Fig. 6 a and Fig. 6 b are the mass spectrogram and free fatty acid content of free derivative of fatty acid in corn oil sample to be tested.
According to mass spectrogram 6a, it is known that contain 5 kinds of free fatties in corn oil sample to be tested, respectively 14:0、16:0、18:3、
18:2 and 18:0；The quasi-molecular ion peak intensity of free derivative of fatty acid and internal standard derivative in gained corn oil sample to be tested
Spending ratio is respectively：0.848(14:0)、0.622(16:0)、0.005(18:3)、0.059(18:And 0.292 (18 2):0),
Combined standard curve (table 1) carries out quantitative analysis to free fatty in corn oil sample to be tested, obtains corn oil sample to be tested
The content of middle free fatty is as shown in Figure 6 b, respectively：236.04μg/g(14:0)、146.06μg/g(16:0)、15.10μ
g/g(18:3)、65.93μg/g(18:And 124.89 μ g/g (18 2):0).
Embodiment 6
A kind of mass spectrometric analysis method of the vegetable seed oil samples middle reaches from aliphatic acid based on Derivative, with embodiment 1
The difference is that：Olive oil sample to be tested in embodiment 1 replaces with rapeseed oil sample to be tested.
Fig. 7 a and Fig. 7 b are the mass spectrogram and free fatty acid content of free derivative of fatty acid in rapeseed oil sample to be tested.
According to mass spectrogram 7a, it is known that contain 7 kinds of free fatties in rapeseed oil sample to be tested, respectively 14:0、16:0、18:3、
18:2、18:1、18:0 and 20:1；The standard point of free derivative of fatty acid and internal standard derivative in gained rapeseed oil sample to be tested
Daughter ion peak intensity ratio is respectively：1.046(14:0)、1.086(16:0)、0.046(18:3)、0.162(18:2)、0.528
(18:1)、1.061(18:And 0.018 (20 0):1), combined standard curve (table 1), to the fat that dissociates in rapeseed oil sample to be tested
Fat acid carries out quantitative analysis, and the content for obtaining free fatty in rapeseed oil sample to be tested is as shown in Figure 7b, respectively：354.80
μg/g(14:0)、335.75μg/g(16:0)、115.57μg/g(18:3)、118.20μg/g(18:2)、383.82μg/g(18:
1)、486.17μg/g(18:And 114.91 μ g/g (20 0):1).
Embodiment 7
The mass spectrometric analysis method of free fatty in a kind of fish oil based on Derivative, it is different from embodiment 1 it
Be in：Olive oil sample to be tested in embodiment 1 replaces with fish oil sample to be tested.
Fig. 8 a and Fig. 8 b are the mass spectrogram and free fatty acid content of free derivative of fatty acid in fish oil sample to be tested.Root
According to mass spectrogram 8a, it is known that containing 12 kinds of free fatties in fish oil sample to be tested, respectively：14:0、16:1、16:0、18:
3、18:2、18:1、18:0、20:5、20:4、20:1、20:0 and 22:6；The free fatty of gained fish oil sample to be tested derives
The quasi-molecular ion peak intensity rate of object and internal standard derivative is respectively：0.661(14:0)、0.0903(16:1)、1.247(16:
0)、0.029(18:3)、0.242(18:2)、0.269(18:1)、1.097(18:0)、0.124(20:5)、0.0182(20:4)、
0.0218(20:1)、0.0249(20:And 0.0218 (22 0):6), combined standard curve (table 1), in fish oil sample to be tested
Free fatty carries out quantitative analysis, and the content for obtaining free fatty in fish oil sample to be tested is as shown in Figure 8 b, respectively：
206.45μg/g(14:0)、28.58μg/g(16:1)、482.23μg/g(16:0)、88.58μg/g(18:3)、276.23μg/g
(18:2)、136.66μg/g(18:1)、593.43μg/g(18:0)、357.76μg/g(20:5)、116.12μg/g(20:4)、
156.38μg/g(20:1)、30.61μg/g(20:And 116.52 μ g/g (22 0):6).
Embodiment 8
The mass spectrometric analysis method of free fatty in a kind of lard based on Derivative, it is different from embodiment 1 it
Be in：Olive oil sample to be tested in embodiment 1 replaces with lard sample to be tested.
Fig. 9 a and Fig. 9 b are the mass spectrogram and free fatty acid content of free derivative of fatty acid in lard sample to be tested.Root
According to mass spectrogram 9a, it is known that contain 9 kinds of free fatties in lard sample to be tested, respectively 14:0、16:1、16:0、18:3、
18:2、18:1、18:0、20:1 and 20:0；The free-fat acid derivative of gained lard sample to be tested and internal standard derivative
Quasi-molecular ion peak intensity rate is respectively：0.319(14:0)、0.059(16:1)、0.957(16:0)、0.0209(18:3)、
0.457(18:2)、1.200(18:1)、0.822(18:0)、0.0366(20:And 0.0191 (20 1):0), combined standard curve
(table 1) carries out quantitative analysis to free fatty in lard sample to be tested, obtains containing for free fatty in lard sample to be tested
It measures as shown in figure 9b, respectively：54.25μg/g(14:0)、14.54μg/g(16:1)、282.89μg/g(16:0)、65.58μg/
g(18:3)、428.82μg/g(18:2)、1086.41μg/g(18:1)、384.36μg/g(18:0)、191.56μg/g(20:1)
And 16.06 μ g/g (20:0).
In conclusion the method for the present invention is easy to operate, derivative reaction is carried out after edible oil sample to be measured directly dilution,
Obtained reaction product is directly entered mass spectrum, uses neutral loss scan in the positive-ion mode without carrying out chromatography post separation
It is analyzed, method is simple and efficient, can realize the efficient, highly selective, highly sensitive qualitative of free fatty in edible oil
Quantitative analysis.
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 the present invention's
Protection domain.
Claims (9)
1. the mass spectrometric analysis method of free fatty in a kind of edible oil based on Derivative, it is characterised in that including as follows
Step：
1) edible oil sample to be measured is diluted with solvent, after internal standard is added, performs the derivatization reaction, the trip after reaction in edible oil
It is converted into positively charged free-fat acid derivative from aliphatic acid, obtains the sample introduction solution of sample to be tested；
2) the sample introduction solution of sample to be tested obtained by step 1) is analyzed by mass spectrometry, acquire free-fat acid derivative spectrogram and
Spectral peak data；
3) qualitative analysis：According to the mass-to-charge ratio in spectrogram obtained by step 2), the quasi-molecule of corresponding free-fat acid derivative is determined
Quasi-molecular ions, to carry out qualitative analysis to the free fatty in sample to be tested；
4) quantitative analysis：Fatty acid standards are diluted to a series of standard solution of concentration with solvent, after being separately added into internal standard,
It is reacted being performed the derivatization under the same conditions with step 1), free fatty is converted into positively charged free-fat after reaction
Acid derivative, the sample introduction solution to get standard samples；
5) a, the sample introduction solution of standard sample obtained by step 4) is analyzed by mass spectrometry, acquire the spectrogram of free-fat acid derivative
With spectral peak data, using free fatty in standard solution and interior target concentration proportion as abscissa, free-fat acid derivative with
The quasi-molecular ion peak intensity rate of internal standard derivative is ordinate, draws standard curve；B, by spectrogram middle reaches obtained by step 2)
Quasi-molecular ion peak intensity rate combined standard curve from derivative of fatty acid and internal standard derivative, to edible oil sample to be measured
In free fatty carry out quantitative analysis；
Wherein, derivative reagent is that one end contains there are one amino, and the other end contains the compound there are one tertiary amine groups, and general formula is expressed as：
NH2-(CH2)n-NR2, wherein n value ranges are 2~4, and R group is the alkyl that carbon number is less than 2.
2. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 1
Method, it is characterised in that the derivative reagent selects N, N- diethyl ethylenediamines.
3. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 1
Method, it is characterised in that the condition of the derivative reaction is：Internal standard is added after edible oil sample is diluted with solvent, catalyst mixes
It is even, derivative reagent is then added, reaction temperature is 38~42 DEG C, and the reaction time is 2~10min.
4. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 1
Method, it is characterised in that in the derivative reaction, using deuterated hexadecanoic acid as internal standard, with the chloro- 1- methyl pyridinium iodides of 2-
It is catalyst with triethylamine, with N, N- diethyl ethylenediamines are derivative reagent.
5. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 4
Method, it is characterised in that in derivative reaction system, a concentration of 0.5~200nmol/L of aliphatic acid, interior target a concentration of 5~
15nmol/L, a concentration of 0.5~0.7 μm of ol/mL of catalyst of triethylamine, the chloro- 1- methyl pyridinium iodides of catalyst 2-
A concentration of 0.1~0.5 μm of ol/mL, a concentration of 0.6~1.2 μm of ol/mL of derivative reagent N, N- diethyl ethylenediamine.
6. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 4
Method, it is characterised in that sample introduction solution obtained by step 1) and step 4) passes through drying, redissolves, is analyzed by mass spectrometry again after purification.
7. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 1
Method, it is characterised in that the condition of the mass spectral analysis is：Injection needle pumps sample introduction, and sample introduction flow velocity is 10~30 μ L/min；Electron spray
Ionization source：Positive ion mode；Scanning of the mass spectrum pattern：73Da neutral loss scans；Ion source gas 1：12kPa；Impact energy：30~
35eV；Remove cluster voltage：70~80eV；Entrance potential：5~10V；Collision cell output voltage：5~10V；Mass range：200~
550m/z。
8. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 1
Method, it is characterised in that step 4) the Plays solution is the mixed solution of one or more of aliphatic acid, and the standard solution
The concentration range of middle aliphatic acid is in 0.5~200nmol/L.
9. the mass spectral analysis side of free fatty in a kind of edible oil based on Derivative according to claim 1
Method, it is characterised in that the edible oil sample to be measured mainly include rapeseed oil, sesame oil, corn oil, linseed oil, olive oil,
Perilla herb oil, lard, fish oil.
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| CN108931603A (en) * | 2018-04-23 | 2018-12-04 | 国家烟草质量监督检验中心 | A method of based on carboxylic acid metabolite in stable isotope labeling mass spectrometric hyphenated technique detection blood plasma |
| CN109374723B (en) * | 2018-09-30 | 2021-07-27 | 中国农业科学院油料作物研究所 | Free fatty acid mass spectrum qualitative and quantitative analysis method based on double-derivative technology |
| CN109828047B (en) * | 2019-02-25 | 2021-08-31 | 中国农业科学院油料作物研究所 | Kit for identifying accurate structure of unsaturated fatty acid in grease |
| CN110441383A (en) * | 2019-07-05 | 2019-11-12 | 清华大学 | Method based on epoxidation and Mass Spectrometric Identification unsaturated lipids carbon-carbon double bond position |
| CN111579633B (en) * | 2020-05-28 | 2023-03-10 | 山东省分析测试中心 | Fatty acid MALDI mass spectrometry imaging method based on in-situ derivatization and application thereof |
| CN113156015A (en) * | 2021-04-25 | 2021-07-23 | 云南锡业锡化工材料有限责任公司 | Method for analyzing content of industrial coconut oil component by internal standard method without derivatization treatment |
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