The method that the chromatograph joint used mensuration structured lipid of Solid-Phase Extraction and gas phase α, β position fatty acid and glyceride form
Technical field
The present invention relates to a kind of method of detection architecture fat, be specifically related to the method that the chromatograph joint used mensuration structured lipid of a kind of Solid-Phase Extraction and gas phase α, β position fatty acid and glyceride form.
Background technology
Lipid is the chief component of human diet, in human body three macro-energy nutrients, occupies an important position.Along with improving constantly of people's living standard, the mankind have proposed new requirement to the nutrition of lipid in meals.Eat oil, eat nutrient health oil, become the new demand that China consumer pays close attention to self health, guarantees normal nutrition intake.
Research shows, in grease, the content of TAGs is up to more than 95%.In TAGs, the composition of fatty acid, carbochain length, double key number order and position are that the Absorption And Metabolism in human body and nutritive value thereof have material impact to glyceride for the position distribution of fatty acid on the key factor that affects grease physicochemical property and nutritive value, especially glycerol backbone.In glyceride, the position of fatty acid can be divided into two large classes, i.e. α (Sn-1,3) position and β (Sn-2) position.In the intestines and stomach of human body, pancreatic lipase is the main enzyme of digestion lipid, has higher α position selectivity.Acyl group is on α position, and fatty acid is absorbed with the form of dissociating, and acyl group is when β position, is that the form with monoglyceride is absorbed, and the utilization that is more easily absorbed by the body of β position monoglyceride.This " the fatty acid acidylate position effect " of glyceride has very important meaning for the research and development of guiding function oil and fat product.
Structured lipid (StructuredLipid, SLs), by changing fatty acid on triacylglycerol skeleton, form and position distribution, the fatty acid with special dietary or physiological function is attached to ad-hoc location, thereby gives full play to physics and the trophic function character of various fatty acid.Structured lipid is as a class new type functional grease, and its exploitation is more and more wider with level of application.In the process of synthetic SLs, be often accompanied by the generation of TAG, DAG, MAG, FFA.In product, the separation of TAG, DAG, MAG, FFA is the prerequisite of optimizing reaction conditions, identification reaction product.In addition, set up a kind of high-throughout method of effectively measuring fast α in SLs, β position fatty acid composition, in SLs research work, have important realistic meaning.
All the time, the method for separated TAG, DAG, MAG, FFA is mainly TLC method and normal phase high performance liquid chromatography (NP-HPLC).The operation such as the making sheet of TLC method, balance, separation, extraction more complicated, sample is exposed in air after point sample, has increased the oxidation probability of fatty acid, especially larger for some long carbochain polyunsaturated fatty acid (LCPUFAs) impacts.In addition, because TLC method is difficult to quantitatively, and duration for existing, be subject to the shortcomings such as the impact of operator's operant level is larger, therefore, can only be as a kind of supplementary means of qualitative analysis, thus limited its further application.And NP-HPLC instrument is expensive, and need to first record the retention time of respective standard product, when carrying out sample analysis, need to collect respectively each component according to the retention time of each component, through GC, carry out fatyy acids, therefore have the shortcomings such as organic solvent consumption is large, time-consuming.With respect to traditional TLC method and HPLC method, solid phase extraction techniques have organic solvent consumption less, enrichment times is high, can be effectively to the feature such as TAG, DAG, the quick effectively separation of MAG, FFA in SLs synthetic product, in conjunction with GC method, can realize the composition analysis of measuring structured lipid α, β position fatty acid and glyceride.The present invention is optimized the elution requirement of solid phase extraction column separated TAG, DAG, MAG and FFA, set up the effectively separated fast of the TAG, the DAG that adopt commercial solid phase extraction column to complete to produce in SLs building-up process, MAG, FFA, in conjunction with GC method, realized the composition analysis of α, β position fatty acid and glyceride in SLs, the method is easy and simple to handle, effectively, be convenient to batch processing sample fast.
Summary of the invention
The method that the object of the present invention is to provide the chromatograph joint used mensuration structured lipid of a kind of Solid-Phase Extraction and gas phase α, β position fatty acid and glyceride to form, that the method has advantages of is easy and simple to handle, sample demand is low, need that solvent-oil ratio is few, qualitative and quantitative analysis result is accurate, detectability is low, analysis time is fast.
To achieve these goals, technical scheme of the present invention is: the chromatograph joint used mensuration structured lipid of a kind of Solid-Phase Extraction and gas phase α, the method that β position fatty acid and glyceride form, first utilize pure arachidonic acid-triglyceride (ARA-TAG), DHA-diglyceride (DHA-DAG), leukotrienes-monoglyceride (Ln-MAG), oleic acid-free fatty acid (O-FFA) standard items, by optimizing the eluent elution requirement of opposed polarity proportioning, TAG realize potpourri on same SPE pillar in, DAG, MAG, effective separation of FFA, it is characterized in that it comprises the steps:
1) separation of hybrid standard sample and fatty acid compositional analysis
(1) sample preparation: take respectively arachidonic acid-triglyceride, DHA-diglyceride, leukotrienes-monoglyceride, oleic acid-free fatty acid standard items, by arachidonic acid-triglyceride: DHA-diglyceride: leukotrienes-monoglyceride: the mass ratio of oleic acid-free fatty acid is 5-50mg:5-50mg:5-50mg:5-50mg, mix, with n-hexane dissolution, mass concentration is controlled between 5mg/mL~50mg/mL, obtain sample, and be placed in-20 ℃ of Refrigerator stores;
(2) pillar activation: solid phase extraction column is placed on solid-phase extraction device, and with the normal hexane of the packing volume in 5-10 times of solid phase extraction column, pillar being carried out to balance activation (is conventional method; Normal hexane is 5-10 times of packing volume); In solid phase extraction column, filler is florisil silica;
(3) loading: the sample of preparation in step (1) is transferred in solid phase extraction column, and sample loading volume is 1-2 times of the packing volume in solid phase extraction column, coutroi velocity <5mL/min;
(4) wash-out: with the eluent normal hexane of opposed polarity proportioning: absolute ether=85:15,70:30,25:75 (v/v) (the i.e. proportioning of three kinds of normal hexanes and absolute ether, first three groups eluent is three groups of different normal hexanes of proportioning and the potpourri of absolute ether), absolute ether: glacial acetic acid=95:5 (v/v) (the 4th group of eluent is the potpourri of absolute ether and glacial acetic acid) is drip washing pillar respectively, collect respectively eluent, the dry eluent of Nitrogen evaporator blowing down, obtains eluate;
(5) TLC detects: in the eluate of collecting to step (4), add normal hexane, mass concentration is controlled between 5mg/mL~50mg/mL, and vortex mixes, and obtains elution samples; Get elution samples point sample in silica gel plate (10cm * 20cm), be placed in chromatography cylinder and carry out thin-layer chromatography (carrying out according to a conventional method chromatography), developing agent used is normal hexane: absolute ether: glacial acetic acid=50:50:1 (v/v/v) (developing agent is the potpourri of normal hexane, absolute ether and glacial acetic acid), naturally after drying, being placed in iodine steam develops the color to spot and manifests completely, whether observation arachidonic acid-triglyceride, DHA-diglyceride, leukotrienes-monoglyceride, oleic acid-free fatty acid realize separation, and determine the type of each eluate;
(6) GC detects: the eluate that step (4) is collected carries out esterification derivatization treatment (carrying out according to a conventional method), according to the kind of the result judgement eluate of step (5), for the esterification reaction of organic acid of fatty acid, adopts dense H
2sO
4/ methyl alcohol heating is carried out, and for the esterification reaction of organic acid employing KOH/ methyl alcohol method of glyceride, carries out (carrying out according to a conventional method); Get step (5) elution samples 50 μ L, be dissolved in 2mL sherwood oil, vibration 1-2s, adds the dense H of 1mL5% (v/v)
2sO
4/ methanol solution (dense H
2sO
4concentration is 98%) or 1mL0.4mol/L (volumetric molar concentration) KOH/ methanol solution, vortex 5min, then adds the sodium-chloride water solution of 2mL0.9% (w/v), slightly vibrates 5-10 second, the centrifugal 5min of 5000rpm, finally gets supernatant and carries out GC analysis; GC analytical instrument is: Agilent7890A type gas chromatograph, fid detector; Chromatographic column: HP-FFAP (30m * 0.25mm * 0.5 μ m); Sample size 1 μ L; Split ratio 30:1; Temperature programme: 210 ℃ of initial temperature, keep 8min, 20 ℃/min is warming up to 240 ℃, and keeps 7min, obtains GC chromatogram, and the percentage composition of each fatty acid is calculated in data analysis;
(7) according to fatty acid in step (6), form (percentage composition of each fatty acid), the type of each eluate in integrating step (5), the fatty acid that obtains triglyceride, diglyceride, monoglyceride and free fatty acid forms;
2) analysis that structured lipid α, β position fatty acid and glyceride form
(1) analysis forming for structured lipid α, β position fatty acid, takes 300mg structured lipid in 30mL glass test tube, adds 20mg porcine pancreatic lipase, 2mLTris-HCl damping fluid (pH=8,2mol/L), after shaking up, add 0.5mL sodium taurocholate solution (1g/L), 0.2mLCaCl
2solution (220g/L) after vortex mixes, after concussion reaction 5min, adds 1mL absolute ether in 40 ℃ of water-baths, 1mLHCl solution (6mol/L), and after concussion evenly, the centrifugal 2min of 5000r/min, gets upper strata, and nitrogen blows to obtain sample;
(2) by the sample in step (1) by step 1) in (2)-(6) carry out, according to porcine pancreatic lipase to the acid-hydrolyzed selectivity of alpha position fat, obtain the composition of monoglyceride and free fatty acid, determine that the fatty acid of structured lipid α, β position forms;
(3) analysis forming for structured lipid glyceride, its sample is by step 1) in (2)-(7) carry out, determine the composition of glyceride.
The invention has the beneficial effects as follows:
1) adopt commercial florisil silica solid phase extraction column to complete the effectively separated fast of TAG, DAG, MAG, FFA in SLs product, in conjunction with vapor-phase chromatography implementation structure fat α, β position fatty acid compositional analysis;
2) the present invention only needs by easy steps such as sample preparation, activation, loading, wash-outs, just can complete TAG, DAG in SLs product, MAG, FFA and in same SPE pillar, realize separated;
3) solid phase extraction techniques that the present invention adopts, with respect to traditional TLC method, more quick and precisely; With respect to HPLC method, organic solvent consumption is few, without expensive instrument, processes, applicable more economically.
4) can implementation structure fat α, TAG, DAG, MAG, FFA form with the high flux of content and detect in the fatty acid the Nomenclature Composition and Structure of Complexes fat prod of β position, are applicable to the analysis of various structured lipid products.
Accompanying drawing explanation
The TLC analysis chart of the standard items potpourri of Fig. 1 after florisil silica solid phase extraction column separated and collected;
In Fig. 1:
1-4 is the sample collection liquid of the eluent drip washing of normal hexane: absolute ether=85:15 (v/v);
5-8 is the sample collection liquid of the eluent drip washing of normal hexane: absolute ether=70:30 (v/v);
9-12 is the sample collection liquid of the eluent drip washing of normal hexane: absolute ether=25:75 (v/v);
13-16 is the sample collection liquid of the eluent drip washing of absolute ether: glacial acetic acid=95:5 (v/v).
Fig. 2 normal hexane: absolute ether=85:15 (v/v) eluent wash-out, the fatty acid methyl ester chromatogram of the sample of collecting after the separation of florisil silica solid phase extraction column.
Fig. 3 normal hexane: absolute ether=70:30 (v/v) eluent wash-out, the fatty acid methyl ester chromatogram of the sample of collecting after the separation of florisil silica solid phase extraction column.
Fig. 4 normal hexane: absolute ether=25:75 (v/v) eluent wash-out, the fatty acid methyl ester chromatogram of the sample of collecting after the separation of florisil silica solid phase extraction column.
Fig. 5 absolute ether: glacial acetic acid=95:5 (v/v) eluent wash-out, the fatty acid methyl ester chromatogram of the sample of collecting after the separation of florisil silica solid phase extraction column.
In Fig. 6 embodiment 1, after Hydrolysis of Lard, use normal hexane: absolute ether=25:75 (v/v) eluent wash-out, the fatty acid methyl ester chromatogram of the sample of collecting after the separation of florisil silica solid phase extraction column.
The synthetic structured lipid TLC analysis chart of collecting after the separation of florisil silica solid phase extraction column in Fig. 7 embodiment 2;
In Fig. 7:
1-4 is the sample collection liquid of the eluent drip washing of normal hexane: absolute ether=85:15 (v/v);
5-12 is the sample collection liquid of the eluent drip washing of normal hexane: absolute ether=70:30 (v/v);
13-16 is the sample collection liquid of the eluent drip washing of normal hexane: absolute ether=25:75 (v/v);
17-19 is the sample collection liquid of the eluent drip washing of absolute ether: glacial acetic acid=95:5 (v/v).
High erucic acid rapeseed oil fatty acid methyl ester chromatogram in Fig. 8 embodiment 3.
In Fig. 9 embodiment 3, after high erucic acid rapeseed oil hydrolysis, use normal hexane: absolute ether=25:75 (v/v) eluent wash-out, the fatty acid methyl ester chromatogram of the sample of collecting after the separation of florisil silica solid phase extraction column.
Embodiment
In order to understand better the present invention, below in conjunction with embodiment, further illustrate content of the present invention, but content of the present invention is not only confined to the following examples.
The method that the chromatograph joint used mensuration structured lipid of a kind of Solid-Phase Extraction and gas phase α, β position fatty acid and glyceride form, first utilize pure arachidonic acid-triglyceride (ARA-TAG), DHA-diglyceride (DHA-DAG), leukotrienes-monoglyceride (Ln-MAG), oleic acid-free fatty acid (O-FFA) standard items, by optimizing the eluent elution requirement of opposed polarity proportioning, effective separation of TAG, DAG, MAG, FFA realize potpourri on same SPE pillar in, it comprises the steps:
1) separation of hybrid standard sample and fatty acid compositional analysis
(1) sample preparation: take respectively arachidonic acid-triglyceride, DHA-diglyceride, leukotrienes-monoglyceride, oleic acid-free fatty acid standard items, by arachidonic acid-triglyceride: DHA-diglyceride: leukotrienes-monoglyceride: the mass ratio of oleic acid-free fatty acid is 5-50mg:5-50mg:5-50mg:5-50mg, mix, with n-hexane dissolution, mass concentration is controlled between 5mg/mL~50mg/mL, obtain sample, and be placed in-20 ℃ of Refrigerator stores;
(2) pillar activation: solid phase extraction column is placed on solid-phase extraction device, with the normal hexane of the packing volume in 5-10 times of solid phase extraction column, pillar is carried out to balance activation (conventional method; Normal hexane is 5-10 times of packing volume); In solid phase extraction column, filler is florisil silica;
(3) loading: the sample of preparation in step (1) is transferred in solid phase extraction column, and sample loading volume is 1-2 times of the packing volume in solid phase extraction column, coutroi velocity <5mL/min;
(4) wash-out: with the eluent normal hexane of opposed polarity proportioning: absolute ether=85:15,70:30,25:75 (v/v) (the i.e. proportioning of three kinds of normal hexanes and absolute ether, first three groups eluent is three groups of different normal hexanes of proportioning and the potpourri of absolute ether), absolute ether: glacial acetic acid=95:5 (v/v) (the 4th group of eluent is the potpourri of absolute ether and glacial acetic acid) is drip washing pillar respectively, collect respectively eluent, the dry eluent of Nitrogen evaporator blowing down, obtains eluate;
(5) TLC detects: in the eluate of collecting to step (4), add normal hexane, mass concentration is controlled between 5mg/mL~50mg/mL, and vortex mixes, and obtains elution samples; Get elution samples point sample in silica gel plate (10cm * 20cm), be placed in chromatography cylinder and carry out thin-layer chromatography (carrying out according to a conventional method chromatography), developing agent used is normal hexane: absolute ether: glacial acetic acid=50:50:1 (v/v/v) (developing agent is the potpourri of normal hexane, absolute ether and glacial acetic acid), naturally after drying, being placed in iodine steam develops the color to spot and manifests completely, whether observation arachidonic acid-triglyceride, DHA-diglyceride, leukotrienes-monoglyceride, oleic acid-free fatty acid realize separation, and determine the type (seeing Fig. 1) of each eluate.
(6) GC detects: the eluate that step (4) is collected carries out esterification derivatization treatment (carrying out according to a conventional method), according to the kind of the result judgement eluate of step (5), for the esterification reaction of organic acid of fatty acid, adopts dense H
2sO
4/ methyl alcohol heating is carried out, and for the esterification reaction of organic acid employing KOH/ methyl alcohol method of glyceride, carries out (carrying out according to a conventional method); Get step (5) elution samples 50 μ L, be dissolved in 2mL sherwood oil, vibration 1-2s, adds the dense H of 1mL5% (v/v)
2sO
4/ methanol solution (dense H
2sO
4concentration is 98%) or 1mL0.4mol/L (volumetric molar concentration) KOH/ methanol solution, vortex 5min, then adds the sodium-chloride water solution of 2mL0.9% (w/v), slightly vibrates 5-10 second, the centrifugal 5min of 5000rpm, finally gets supernatant and carries out GC analysis; GC analytical instrument is: Agilent7890A type gas chromatograph, fid detector; Chromatographic column: HP-FFAP (30m * 0.25mm * 0.5 μ m); Sample size 1 μ L; Split ratio 30:1; Temperature programme: 210 ℃ of initial temperature, keep 8min, 20 ℃/min is warming up to 240 ℃, and keeps 7min, obtains GC chromatogram (seeing Fig. 2-5), and the percentage composition of each fatty acid is calculated in data analysis;
(7) according to fatty acid in step (6), form, the type of each eluate in integrating step (5), the fatty acid that obtains triglyceride, diglyceride, monoglyceride and free fatty acid forms;
2) analysis that structured lipid α, β position fatty acid and glyceride form: see embodiment 1-3.
Embodiment 1: a kind of method of α, β position fatty acid in fast detecting lard, it comprises the steps:
1) sample preparation: take 300mg lard in 30mL glass test tube, add 20mg porcine pancreatic lipase, 2mLTris-HCl damping fluid (pH=8,2mol/L), after shaking up, adds 0.5mL sodium taurocholate solution (1g/L), 0.2mLCaCl
2solution (220g/L) after vortex mixes, after concussion reaction 5min, adds 1mL absolute ether in 40 ℃ of water-baths, 1mLHCl solution (6mol/L), and after concussion evenly, the centrifugal 2min of 5000r/min, gets upper strata, and nitrogen blows to obtain sample;
2) pillar activation: get solid phase extraction column (in post, filler is 2g florisil silica), be placed on solid-phase extraction device, add 20mL normal hexane pillar is carried out to balance activation;
3) gained sample loading: by step 1), adds 8mL n-hexane dissolution, and vortex mixes.Get 2mL sample solution and transfer in solid phase extraction column, coutroi velocity <5mL/min;
4) wash-out: eluent and 12mL absolute ether: glacial acetic acid=95:5 (v/v) drip washing pillar of using respectively 12mL normal hexane: absolute ether=85:15,70:30,25:75 (v/v), collect respectively leacheate, every 3mL mono-pipe, each eluent is collected respectively 4 pipes, under Nitrogen evaporator, dry up, obtain eluate;
5) in the eluate of TLC detection: to step 4) collecting, add normal hexane, mass concentration is controlled between 5mg/mL~50mg/mL, and vortex mixes, and obtains elution samples; Get elution samples point sample in silica gel plate (10cm * 20cm), be placed in chromatography cylinder and carry out thin-layer chromatography, developing agent used is normal hexane: absolute ether: glacial acetic acid=50:50:1 (v/v/v), naturally after drying, being placed in iodine steam develops the color to spot and manifests completely, it is separated whether observation triglyceride, diglyceride, monoglyceride and free fatty acid are realized, and determine the type of each eluate, and the eluate of same type merges;
6) GC detects: by step 4) eluate collected carries out esterification derivatization treatment, according to step 5) the kind of result judgement eluate, for the esterification reaction of organic acid of fatty acid, adopt dense H
2sO
4/ methyl alcohol heating is carried out, and for the esterification reaction of organic acid employing KOH/ methyl alcohol method of glyceride, carries out; Get step 4) elution samples 50 μ L, be dissolved in 2mL sherwood oil, vibration 1-2s, adds the dense H of 1mL5% (v/v)
2sO
4/ methanol solution (dense H
2sO
4concentration is 98%) or 1mL0.4mol/L (volumetric molar concentration) KOH/ methanol solution, vortex 5min, then adds the sodium-chloride water solution of 2mL0.9% (w/v), slightly vibrates 5-10 second, the centrifugal 5min of 5000rpm, finally gets supernatant and carries out GC analysis; GC analytical instrument is: Agilent7890A type gas chromatograph, fid detector; Chromatographic column: HP-FFAP (30m * 0.25mm * 0.5 μ m); Sample size 1 μ L; Split ratio 30:1; Temperature programme: 210 ℃ of initial temperature, keep 8min, 20 ℃/min is warming up to 240 ℃, and keeps 7min, obtains GC chromatogram (seeing Fig. 6), and the percentage composition of each fatty acid is calculated in data analysis;
7) according to step 6) in fatty acid form, integrating step 5) in the type of each eluate, the fatty acid that obtains monoglyceride and free fatty acid forms, i.e. the composition of α, β position fatty acid in lard.
Embodiment 2: a kind of utilize the quick separated enzyme process of solid phase extraction column synthetic 1,3-arachidonic acid-diglyceride (1, the method that 3-ARA-DAG) in structured lipid, glyceride forms, it comprises the steps:
1) sample preparation: take 1.00g arachidonic acid (ARA) and 0.15g glycerine, add 0.12g immobilized lipase LipozymRMIM (account for substrate quality 10%), react 3h under 50 ℃ of water bath with thermostatic control magnetic agitation, obtain SLs reaction mixture;
2) pillar activation: get solid phase extraction column (in post, filler is 2g florisil silica), be placed on solid-phase extraction device, add 10mL normal hexane pillar is carried out to balance activation;
3) loading: take 50mg step 1) the SLs reaction product of synthesized, in test tube, adds 5mL normal hexane, and vortex mixes.Sample after mixing is transferred in solid phase extraction column to coutroi velocity <5mL/min;
4) wash-out: use respectively 12mL normal hexane: absolute ether=85:15,70:30,25:75 (v/v) and 12mL absolute ether: glacial acetic acid=95:5 (v/v) eluent drip washing pillar, collect respectively leacheate, every 3mL collects a pipe, every kind of eluent is collected 4 pipes, under Nitrogen evaporator, dry up, obtain eluate;
5) in the eluate of TLC detection: to step 4) collecting, add 10mL normal hexane, vortex mixes.Point sample, in silica gel plate (10cm * 20cm), is placed in chromatography cylinder and carries out thin-layer chromatography, and developing agent used is normal hexane: absolute ether: glacial acetic acid=50:50:1 (v/v/v), after naturally drying, is placed in iodine steam and develops the color to spot and manifest completely; According to the colour developing point of triglyceride, diglyceride, monoglyceride and free fatty acid, determine the type of eluate, the eluate of same type merges, and obtains the different component (seeing Fig. 7) of SLs reactant.
Embodiment 3: a kind of method of α, β position fatty acid in fast detecting high erucic acid rapeseed oil, it comprises the steps:
1) sample preparation: accurately take 50mg rapeseed oil in 30mL glass test tube, add 20mg porcine pancreatic lipase, 2mLTris-HCl damping fluid (pH=8,2mol/L), after shaking up, add 0.5mL sodium taurocholate solution (1g/L), 0.2mLCaCl2 solution (220g/L), after vortex mixes, in 40 ℃ of water-baths, concussion is reacted after 5min, add 1mL absolute ether, 1mLHCl solution (6mol/L), after concussion evenly, the centrifugal 2min of 5000r/min, get upper strata, nitrogen blows to obtain sample;
2) pillar activation: get solid phase extraction column (in post, filler is 2g florisil silica), be placed on solid-phase extraction device, add 20mL normal hexane pillar is carried out to balance activation;
3) gained sample loading: by step 1), adds 4mL n-hexane dissolution, and vortex mixes.Get 2mL sample solution and transfer in solid phase extraction column, coutroi velocity <5mL/min;
4) wash-out: use respectively 12mL normal hexane: absolute ether=85:15,70:30,25:75 (v/v) and 12mL absolute ether: glacial acetic acid=95:5 (v/v) eluent drip washing pillar, collect respectively leacheate, every 3mL mono-pipe, every kind of eluent is collected 4 pipes, and leacheate is dried up under Nitrogen evaporator, obtain eluate;
5) in the eluate of GC detection: to step 4) collecting, add 1mL normal hexane, vortex mixes, and obtains elution samples; And respectively former rapeseed oil sample and elution samples being carried out to esterification derivatization treatment, esterification reaction of organic acid adopts KOH/ methyl alcohol method to carry out; In elution samples, add 1mL0.4mol/L (volumetric molar concentration) KOH/ methanol solution, vortex 10min, then adds sodium-chloride water solution and the 1mL normal hexane of 2mL0.9% (w/v), slightly vibrates 5-10 second, the centrifugal 5min of 5000rpm, finally gets supernatant and carries out GC analysis; GC analytical instrument is: Agilent7890A type gas chromatograph, fid detector; Chromatographic column: HP-FFAP (30m * 0.25mm * 0.5 μ m); Sample size 1 μ L; Split ratio 30:1; Temperature programme: 210 ℃ of initial temperature, keep 6min, 20 ℃/min is warming up to 240 ℃, and keeps 4min, obtains GC chromatogram (Fig. 8,9), through data analysis, calculates the percentage composition of each fatty acid;
7) integrating step 6) fatty acid form, determine the composition of α in high erucic acid rapeseed oil, β position fatty acid, according to porcine pancreatic lipase, to the acid-hydrolyzed selectivity of alpha position fat, obtain the α position that erucic acid and peanut monoenoic acid in high erucic acid rapeseed oil are mainly distributed in glyceride.