CN103245752B - A kind of method detecting fatty acid chloropropanol ester pH-titration in food - Google Patents
A kind of method detecting fatty acid chloropropanol ester pH-titration in food Download PDFInfo
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Abstract
The present invention relates to a kind of method detecting grease in food, especially one utilizes gas chromatograph-mass spectrometer (GCMS) to detect fatty acid 3-chloro-1 in food, 2-propylene glycol monoester, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, the method of fatty acid 2-chloro-1,3-PD diester four class ester content.The present invention adopts amino solid-phase extraction column, utilize n-hexane-ethyl acetate mixed solvent wash-out, and adopt the GC-MS method of basic hydrolysis-boronic acid derivative to measure, comprise sample preparation, extract and separate, basic hydrolysis process, derivatization treatment, gas chromatograph-mass spectrometer (GCMS) analysis, the preparation of typical curve, quantivative approach seven steps, need solvent kind and consumption few, simple to operate, highly sensitive, separating power is strong, the qualitative analysis is accurate, detectability is low, analysis time is fast.
Description
Technical field
The present invention relates to a kind of method detecting grease in food, especially one utilizes gas chromatograph-mass spectrometer (GCMS) to detect fatty acid 3-chloro-1 in food, 2-propylene glycol monoester, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, the method of fatty acid 2-chloro-1,3-PD diester four class ester content.
Background technology
Propylene chlorohydrin is the food processing process pollutant known, especially 3-chloro-1,2-propylene glycol (being called for short 3-MCPD), because it has the effect such as Toxicity of Kidney, genotoxicity, immunosupress and potential carcinogenicity by extensive concern, and fatty acid chloropropanol ester (chlorine propyl ester) is propylene chlorohydrin and fatty acid esterification and the class material formed, it is emerging food-safety problem in the world in recent years, especially in grease based food, the pollution of fatty acid 3-chlorine-1,2-propylene glycol ester (being called for short 3-MCPD ester) is the most serious.The toxicity of current 3-MCPD ester is still not fully aware of, mainly multiple toxicity is had because of the hydrolysate of chlorine propyl ester to the worry of its health risk, therefore, chlorine third ester hydrolysis adds the exposure level of 3-MCPD, and intake may be tentative every day 5 ~ 100 times of maximal tolerance dose of 3-MCPD.3-MCPD ester has 3-MCPD monoesters and dibasic acid esters two kinds of isomeride, and structural difference may cause the difference of toxicological action.Manliu etc. test with rat, find that 3-MCPD diester can cause rat spermatozoa to reduce (namely having genotoxicity), and 3-MCPD monoesters is not only same with 3-MCPD diester has genotoxicity, also can make Rat renal tubular necrosis (i.e. Toxicity of Kidney).Find in the lipase hydrolysis test of simulation gastrointestinal model, 3-MCPD monoesters is hydrolyzed into the speed of speed considerably beyond 3-MCPD diester of 3-MCPD.Given this, be necessary to set up to 3-MCPD mono-/dibasic acid esters carries out the method for separation and detection respectively, the ratio of 3-MCPD monoesters, dibasic acid esters in acquisition food, thus contributes to assessing the health risk of 3-MCPD ester more accurately.
More with Hydrolyze method indirect determination fatty acid chloropropanol ester total quantifier elimination report in the world at present, but the research detected respectively fatty acid chloride propyl alcohol list/dibasic acid esters content in food is very few, the people such as Seefelder utilize aminopropyl (aminopropyl) solid phase extraction, normal hexane in varing proportions, ether, the mixed liquor wash-out of methylene chloride and ethyl acetate, 3-MCPD monoesters is separated with dibasic acid esters, respective content is detected respectively again in conjunction with GC-MS, because solvent for use kind is many, solvent ratios is complicated, operate very loaded down with trivial details (Janis C, Owen W P, Ray H L, et al. Jr. J. Agric. Food Chem. 1984, 32 (3), 474-476.), the people such as Zelinkov á utilize silica gel solid phase extraction, adopt the sherwood oil of different proportion and ether mixed solvent carry out wash-out be separated MCPD mono-/dibasic acid esters, then analyze in conjunction with GC-MS, but its shortcoming is that required eluting solvent ratio is many, and consumption solvent volume reaches 850 ml, complex operation, sensitivity is low, and to serious (Zuzana Z, the Doleqal M of instrumental pollution, Jan V. Eur Food Res Technol. 2009,228:571 – 578.).
For overcoming above-mentioned shortcoming, the present invention adopts amino solid-phase extraction column, utilize n-hexane-ethyl acetate mixed solvent wash-out, and adopt the GC-MS method of basic hydrolysis-boronic acid derivative to measure fatty acid 3-chlorine-1,2-propylene glycol monoesters, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, fatty acid 2-chloro-1,3-PD two ester content.
Summary of the invention
The present invention adopts gas chromatograph-mass spectrometer (GCMS) to detect food fatty acids 3-chlorine-1,2-propylene glycol monoesters, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, fatty acid 2-chloro-1,3-PD diester four class monoesters or dibasic acid esters, comprise the following steps:
(1) process of sample
Take foodstuff samples to be measured in right amount in plastic centrifuge tube, add mixing inner mark solution, ultrasonicly to mix, wherein fatty acid 3-chlorine-1,2-propylene glycol monoesters, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, interior mark corresponding to fatty acid 2-chloro-1,3-PD diester is respectively d
5-palmitic acid 3-chlorine-1,2-propylene glycol monoesters, d
5-palmitic acid 3-chlorine-1,2-propylene glycol diester, d
5the chloro-1,3-PD monoesters of-stearic acid 3-, d
5the chloro-1,3-PD diester of-stearic acid 3-;
(2) extract and separate
Upper prop: by the sample that mixes and inner mark solution, to proceed to after normal hexane activation in solid-phase extraction column, and twice with n-hexane centrifuge tube, cleansing solution is proceeded in solid-phase extraction column in the lump, the filler of solid-phase extraction column is one in amino filler, zeyssatite or potpourri, the weight of filler is between 0.5g to 5.0 g, and wherein the granularity of amino filler is between 50 order to 400 orders;
Wash-out: be that fatty acid chloride propyl alcohol dibasic acid esters elutes by eluting solvent with normal hexane, eluent is collected with glass test tube, and then with normal hexane-ethyl acetate mixed solvent wash-out fatty acid chloride propyl alcohol monoesters, this eluent is collected with another glass test tube, wherein in normal hexane-ethyl acetate mixture, any volume ratio of normal hexane and ethyl acetate can be one in 10:0,9:1,8:2,7:3; Any flow velocity adopted during wash-out can be in 0.5 ml/min, 1.0 ml/min, 1.5 ml/min;
(3) basic hydrolysis process
Respectively step (two) gained eluent is placed in nitrogen flow down slowly be concentrated near dry, add methyl tert-butyl ether-ethyl acetate solution dissolved residue, add sulfuric acid propanol solution again, then add after hydrolysing agent is hydrolyzed 1 min, add glacial acetic acid solution rapidly, abundant vortex mixing, then normal hexane degreasing is added, leave standstill after abundant vortex, after obvious layering, remove normal hexane layer, then use n-hexane extraction 1 time, discard normal hexane phase, aqueous phase is for derivatization, and wherein hydrolysing agent is sodium methoxide-methanol solution or sodium hydroxide solution;
(4) derivatization treatment
Derivatization reagent is added in above-mentioned scavenging solution, at 80 DEG C, 20 min are derived after vortex mixing, be cooled to after room temperature until it, add the abundant vortex of ethyl acetate, after stratification, ethyl acetate layer is transferred in another test tube, re-extract 2 times again, merge 3 extracts, flow down in nitrogen and slowly dry up, after residue dissolves with isooctane, with centrifugation 5 min of 12000 turns/min, get supernatant for the analysis of GC-MS sample introduction, wherein derivatization reagent is phenylboric acid or seven fluorine bytyry imidazole solution;
(5) gas chromatograph-mass spectrometer (GCMS) analysis
Step (three), (four) and step (five) gained supernatant are detected by gas chromatograph-mass spectrometer (GCMS) respectively, needed for gas chromatograph-mass spectrometer (GCMS), condition is as follows:
Chromatographic column: DB-5MS capillary column; Injector temperature: 280 DEG C; Post begins warm: 80 DEG C keep 1 min, then are warming up to 190 DEG C with the speed program of 10 DEG C/min, finally rise to 300 DEG C with the speed of 40 DEG C/min, keep 5 min; Carrier gas: high-pure helium, flow velocity 3.0 mL/min; Input mode: Splitless injecting samples; Sampling volume: 1 μ L;
Ion gun: electron impact ion source (EI); Electron-multiplier gain :+250V; Filament current: 100 μ A; Trap temperature: 230 DEG C; Transmission line temperature: 250 DEG C; Discrimination box temperature: 50 DEG C; Solvent delay: 3 min; Mass spectrum acquisition time: 3-29.75 min; Quality of scanning number scope: 90-300; Sweep speed: 0.65 s/ time; The quota ion of fatty acid 3-chlorine-1,2-propylene glycol and fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z147 and m/z196, interior mark d
5-3-chlorine-1,2-propylene glycol and d
5the quota ion of-fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z150 and m/z201;
(6) preparation of typical curve
Accurately take appropriate fatty acid 3-chloro-1 respectively, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, fatty acid 2-chloro-1,3-PD diester standard items, in 10ml volumetric flask, are settled to scale with after acetic acid ethyl dissolution, abundant mixing obtains standard reserving solution, places and preserve in-24 DEG C of refrigerators; Get the standard working solution that appropriate storing solution normal hexane is diluted to 25 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500ng/ml more respectively, respectively add amount identical with sample inner mark solution, then carry out Treatment Analysis according to step (three), (four) and (five);
(7) quantivative approach
With standard items fatty acid 3-chlorine-1,2-propylene glycol monoesters, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, the chloro-1,3-PD diester of fatty acid 2-marks peak area ratio and sample introduction Mass Calculation equation of linear regression, by this equation to the fatty acid 3-chloro-1 in sample with interior separately, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, the chloro-1,3-PD diester of fatty acid 2-carries out quantitatively.
Method provided by the present invention need solvent kind and consumption few, simple to operate, highly sensitive, separating power is strong, the qualitative analysis is accurate, detectability is low, analysis time is fast.
Embodiment
Experimental apparatus and material:
3800 type gas chromatographs and Saturn 2200 type gas chromatography-GC-MS (Varian company of the U.S.); G-560 E type eddy mixer (Scientific Industries company of the U.S.); KQ-500B type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); DHG-9123A type electric heating constant-temperature blowing drying box (the upper grand testing equipment company limited of Nereid); N-EVAPTM 111 type Nitrogen evaporator (Organomation Associates company of the U.S.), Microfuge type supercentrifuge (Beckman company of the U.S.); CP 225D electronic balance (accurately to 0.01mg, German Sartorious company, for weighing standard items); YP202 electronic balance (Shanghai goods company, for sample weighing), 6 ml polypropylene solid-phase extraction column column casings (Agela company, joins sieve plate).Glassware uses toast 3 h at 220 DEG C after.1.5ml plastic centrifuge tube, disposable syringe (1ml, ShangHai ShuangGe Industry Co., Ltd) and plastic dropper (1ml and 5ml, Xin Kang Medical Devices Co., Ltd.), 10ml glass test tube.
Peanut wet goods edible vegetable oil and common brand baby milk powder (comprising for one, two, three stages) are all purchased from local supermarket, Foochow, and in milk powder, fat-extraction is by first method in the standard GB/T/T5413.3-2010 mensuration of fat " in infant food and the dairy products " method.Methyl tert-butyl ether, sodium methoxide, sodium chloride, n-propanol, the concentrated sulphuric acid, ethyl acetate (heavily steaming) and glacial acetic acid are analyzes pure (Shanghai Chemical Reagent Co., Ltd., Sinopharm Group).Normal hexane is the residual level of agriculture (Fisher company), and methyl alcohol, acetone are chromatographically pure ((Fisher company), phenylboric acid (10 g/ bottles, Organics company).Water is pure water.Solid phase extraction column stuffing CNWBOND NH
2amino (230 ~ 400 orders, Shanghai ANPEL Scientific Instrument Co., Ltd.) methyl tert-butyl ether/ethyl acetate mixtures (V/V=8:2); Sulfuric acid/propyl alcohol (V/V=0.5:100); Sodium methoxide/methyl alcohol (0.5 mol/L, namely 2.16g sodium methoxide is dissolved in 40ml methyl alcohol); Glacial acetic acid solution (1.0 mL glacial acetic acids are dissolved in 30 mL 20% sodium chloride solutions); (2.5 g phenylboric acids are dissolved in 20 mL acetone/water (V/V=19:1) PBA solution.
Palmitic acid 3-MCPD monoesters (purity 98%, CAS 30557-04-1), palmitic acid 3-MCPD diester (purity 98%, CAS 51930-97-3), stearic acid 2-MCPD diester (purity 98%, CAS 267-56-4) and deuterated isotope palmitic acid d
5-3-MCPD monoesters (purity 99%), palmitic acid d
5-3-MCPD diester (purity 99%, CAS 1185057-55-9) and stearic acid d
5-2-MCPD diester (purity 99%) and other MCPD ester standard items are all purchased from TRC company (Canada).
Embodiment 1:
(1) process of sample
Accurately take appropriate d respectively
5-palmitic acid 3-chlorine-1,2-propylene glycol monoesters, d
5-palmitic acid 3-chlorine-1,2-propylene glycol diester, d
5the chloro-1,3-PD monoesters of-stearic acid 2-, d
5the chloro-1,3-PD diester of-stearic acid 2-, in 10ml volumetric flask, is settled to scale with after acetic acid ethyl dissolution, and fully mixing obtains standard reserving solution, places and preserve in-24 DEG C of refrigerators; Get the mixing inner mark solution that appropriate storing solution normal hexane is diluted to 2.0 μ g/ml more respectively.Take 0.1 g edible oil sample in 1.5ml plastic centrifuge tube, add 25 μ l mixing inner mark solutions, ultrasonic mixing 5 min at 45 DEG C.
(2) extract and separate
Upper prop: by the sample that mixes and inner mark solution, (amino filler weight is 0.5g to proceed to the amino solid-phase extraction column of 6.0 ml after the activation of 4.0ml normal hexane, granularity is 230 ~ 400 orders) in, and twice with 100 μ l n-hexane centrifuge tubes, cleansing solution is proceeded in the lump in amino solid-phase extraction column;
Wash-out: with 6.0ml normal hexane for fatty acid chloride propyl alcohol dibasic acid esters elutes by eluting solvent, eluent is collected with 10 ml glass test tubees, and then be eluting solvent wash-out fatty acid chloride propyl alcohol monoesters monoesters with 6.0ml normal hexane and ethyl acetate with normal hexane-ethyl acetate solution that volume ratio 7:3 mixes, collect this eluent with another glass test tube; During wash-out, flow velocity is 1.0 ml/min.
(3) basic hydrolysis process
Respectively step (two) gained eluent is placed in nitrogen flow down slowly be concentrated near dry, adding 0.5ml by methyl tert-butyl ether and ethyl acetate volume ratio is methyl tert-butyl ether-ethyl acetate solution dissolved residue that 4:1 mixes, add again and be dissolved in the formulated sulfuric acid propanol solution of 100ml propyl alcohol with 98% concentrated sulphuric acid of 0.5ml, then adding 0.5 mL concentration is after the sodium methoxide-methanol solution of 0.5mol/L is hydrolyzed 1 min, add rapidly 3.0ml 1ml glacial acetic acid and be dissolved in glacial acetic acid solution formulated in 29ml saturated sodium-chloride, abundant vortex mixing is to neutralize excessive sodium methoxide, then 3.0 ml normal hexane degreasings are added, after abundant vortex, leave standstill, normal hexane layer is removed after obvious layering, use 3.0 ml n-hexane extraction 1 time again, discard normal hexane phase, aqueous phase is for derivatization.
(4) derivatization treatment
Add after 2.5g phenylboric acid is first dissolved in 20 mL acetone by 0.25 ml in above-mentioned scavenging solution, again gained solution is mixed prepared phenylboric acid solution with water according to volume ratio 19:1, at 80 DEG C, 20 min are derived after vortex mixing, be cooled to after room temperature until it, add the abundant vortex of 2.0 ml ethyl acetate, after stratification, ethyl acetate layer is transferred in another test tube, re-extract 2 times again, merge 3 extracts, flow down in nitrogen and slowly dry up, after residue dissolves with 0.5 ml isooctane, with centrifugation 5 min of 12000 turns/min, get supernatant for the analysis of GC-MS sample introduction.
(5) gas chromatograph-mass spectrometer (GCMS) analysis
Step (three), (four) and step (five) gained supernatant are detected by gas chromatograph-mass spectrometer (GCMS) respectively, needed for gas chromatograph-mass spectrometer (GCMS), condition is as follows:
Chromatographic column: DB-5MS capillary column; Injector temperature: 280 DEG C; Post begins warm: 80 DEG C keep 1 min, then are warming up to 190 DEG C with the speed program of 10 DEG C/min, finally rise to 300 DEG C with the speed of 40 DEG C/min, keep 5 min; Carrier gas: high-pure helium, flow velocity 3.0 mL/min; Input mode: Splitless injecting samples; Sampling volume: 1 μ L;
Ion gun: electron impact ion source (EI); Electron-multiplier gain :+250V; Filament current: 100 μ A; Trap temperature: 230 DEG C; Transmission line temperature: 250 DEG C; Discrimination box temperature: 50 DEG C; Solvent delay: 3 min; Mass spectrum acquisition time: 3-29.75 min; Quality of scanning number scope: 90-300; Sweep speed: 0.65 s/ time; The quota ion of fatty acid 3-chlorine-1,2-propylene glycol and fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z147,196, interior mark d
5-3-chlorine-1,2-propylene glycol and d
5the quota ion of-fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z150,201.
(6) preparation of typical curve
Accurately take appropriate fatty acid 3-chloro-1 respectively, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, fatty acid 2-chloro-1,3-PD diester standard items, in 10ml volumetric flask, are settled to scale with after acetic acid ethyl dissolution, abundant mixing obtains standard reserving solution, places and preserve in-24 DEG C of refrigerators; Get the standard working solution that appropriate storing solution normal hexane is diluted to 25 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500ng/ml more respectively, respectively add amount identical with sample inner mark solution, then carry out Treatment Analysis according to step (three), (four) and (five).
(7) quantivative approach
With standard items fatty acid 3-chlorine-1,2-propylene glycol monoesters, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, the chloro-1,3-PD diester of fatty acid 2-marks peak area ratio and sample introduction Mass Calculation equation of linear regression, by this equation to the fatty acid 3-chloro-1 in sample with interior separately, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, the chloro-1,3-PD diester of fatty acid 2-carries out quantitatively.Pass through interpretation of result, each fatty acid chloride propyl alcohol list/dibasic acid esters and the interior target recovery thereof are all between 92.2 ~ 102%, fatty acid 3-chloro-1,2-propylene glycol monoester, fatty acid 3-chloro-1, the detection limit of 2-propylene glycol diesters, fatty acid 2-chlorine-1,2-propylene glycol monoesters, fatty acid 2-chlorine-1,2-propylene glycol diester is respectively 83 μ g/kg, 125 μ g/kg, 92 μ g/kg, 100 μ g/kg, quantitative limit is respectively 278 μ g/kg, 417 μ g/kg, 243 μ g/kg, 333 μ g/kg; Fatty acid 3-chloro-1,2-propylene glycol monoester, fatty acid 3-chloro-1,2-propylene glycol diesters, fatty acid 2-chloro-1, the regression equation of 2-propylene glycol monoester, fatty acid 2-chlorine-1,2-propylene glycol diester is respectively: Y=0.0246X+0.0169, Y=0.0274X-0.2664, Y=0.0174X-0.0664, Y=0.0133X-0.0290, good in 25 ~ 500ng scope linear relationship, coefficient R is all more than 0.9994.Record fatty acid 3-chlorine-1,2-propylene glycol monoester content between 83 μ g/kg ~ 5.9 mg/kg, fatty acid 3-chlorine-1,2-propylene glycol two ester content is between 125 μ g/kg ~ 1.2 mg/kg; The content of fatty acid 2-chlorine-1,2-propylene glycol monoesters between 92 μ g/kg ~ 2.5 mg/kg, the content of fatty acid 2-chlorine-1,2-propylene glycol diester is between 100 μ g/kg ~ 0.7 mg/kg.
Embodiment 2:
(1) process of sample
Accurately take appropriate d respectively
5-palmitic acid 3-chlorine-1,2-propylene glycol monoesters, d
5-palmitic acid 3-chlorine-1,2-propylene glycol diester, d
5the chloro-1,3-PD monoesters of-stearic acid 2-, d
5the chloro-1,3-PD diester of-stearic acid 2-, in 10ml volumetric flask, is settled to scale with after acetic acid ethyl dissolution, and fully mixing obtains standard reserving solution, places and preserve in-24 DEG C of refrigerators; Get the mixing inner mark solution that appropriate storing solution normal hexane is diluted to 2.0 μ g/ml more respectively.Take the milk powder 1g after mixing, according to first method in the standard GB/T/T5413.3-2010 mensuration of fat " in infant food and the dairy products " method, fat-extraction is carried out to it, then the fat-extraction thing of 0.1g milk powder is got in 1.5ml plastic centrifuge tube, add 25 μ l mixing inner mark solutions, ultrasonic mixing 5 min at 45 DEG C;
(2) extract and separate
Upper prop: by the sample that mixes and inner mark solution, (amino filler weight is 0.5g to proceed to the amino solid-phase extraction column of 6.0 ml after the activation of 4.0ml normal hexane, granularity is 230 ~ 400 orders) in, and twice with 100 μ l n-hexane centrifuge tubes, cleansing solution is proceeded in the lump in amino solid-phase extraction column;
Wash-out: with 6.0ml normal hexane for fatty acid chloride propyl alcohol dibasic acid esters elutes by eluting solvent, eluent is collected with 10 ml glass test tubees, and then be eluting solvent wash-out fatty acid chloride propyl alcohol monoesters monoesters with 6.0ml normal hexane and ethyl acetate with normal hexane-ethyl acetate solution that volume ratio 7:3 mixes, collect this eluent with another glass test tube; During wash-out, flow velocity is 1.0 ml/min.
(3) basic hydrolysis process
Respectively step (two) gained eluent is placed in nitrogen flow down slowly be concentrated near dry, adding 0.5ml by methyl tert-butyl ether and ethyl acetate volume ratio is methyl tert-butyl ether-ethyl acetate solution dissolved residue that 4:1 mixes, add again and be dissolved in the formulated sulfuric acid propanol solution of 100ml propyl alcohol with 98% concentrated sulphuric acid of 0.5ml, then adding 0.5 mL concentration is after the sodium methoxide-methanol solution of 0.5mol/L is hydrolyzed 1 min, add rapidly 3.0ml 1ml glacial acetic acid and be dissolved in glacial acetic acid solution formulated in 29ml saturated sodium-chloride, abundant vortex mixing is to neutralize excessive sodium methoxide, then 3.0 ml normal hexane degreasings are added, after abundant vortex, leave standstill, normal hexane layer is removed after obvious layering, use 3.0 ml n-hexane extraction 1 time again, discard normal hexane phase, aqueous phase is for derivatization.
(4) derivatization treatment
Add after 2.5g phenylboric acid is first dissolved in 20 mL acetone by 0.25 ml in above-mentioned scavenging solution, again gained solution is mixed prepared phenylboric acid solution with water according to volume ratio 19:1, at 80 DEG C, 20 min are derived after vortex mixing, be cooled to after room temperature until it, add the abundant vortex of 2.0 ml ethyl acetate, after stratification, ethyl acetate layer is transferred in another test tube, re-extract 2 times again, merge 3 extracts, flow down in nitrogen and slowly dry up, after residue dissolves with 0.5 ml isooctane, with centrifugation 5 min of 12000 turns/min, get supernatant for the analysis of GC-MS sample introduction.
(5) gas chromatograph-mass spectrometer (GCMS) analysis
Step (three), (four) and step (five) gained supernatant are detected by gas chromatograph-mass spectrometer (GCMS) respectively, needed for gas chromatograph-mass spectrometer (GCMS), condition is as follows:
Chromatographic column: DB-5MS capillary column; Injector temperature: 280 DEG C; Post begins warm: 80 DEG C keep 1 min, then are warming up to 190 DEG C with the speed program of 10 DEG C/min, finally rise to 300 DEG C with the speed of 40 DEG C/min, keep 5 min; Carrier gas: high-pure helium, flow velocity 3.0 mL/min; Input mode: Splitless injecting samples; Sampling volume: 1 μ L;
Ion gun: electron impact ion source (EI); Electron-multiplier gain :+250V; Filament current: 100 μ A; Trap temperature: 230 DEG C; Transmission line temperature: 250 DEG C; Discrimination box temperature: 50 DEG C; Solvent delay: 3 min; Mass spectrum acquisition time: 3-29.75 min; Quality of scanning number scope: 90-300; Sweep speed: 0.65 s/ time; The quota ion of fatty acid 3-chlorine-1,2-propylene glycol and fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z147,196, interior mark d
5-3-chlorine-1,2-propylene glycol and d
5the quota ion of-fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z150,201.
(6) preparation of typical curve
Accurately take appropriate fatty acid 3-chloro-1 respectively, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, fatty acid 2-chloro-1,3-PD diester standard items, in 10ml volumetric flask, are settled to scale with after acetic acid ethyl dissolution, abundant mixing obtains standard reserving solution, places and preserve in-24 DEG C of refrigerators; Get the standard working solution that appropriate storing solution normal hexane is diluted to 25 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500ng/ml more respectively, respectively add amount identical with sample inner mark solution, then carry out Treatment Analysis according to step (three), (four) and (five);
(7) quantivative approach
With standard items fatty acid 3-chlorine-1,2-propylene glycol monoesters, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, the chloro-1,3-PD diester of fatty acid 2-marks peak area ratio and sample introduction Mass Calculation equation of linear regression, by this equation to the fatty acid 3-chloro-1 in sample with interior separately, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, the chloro-1,3-PD diester of fatty acid 2-carries out quantitatively.Pass through interpretation of result, each fatty acid chloride propyl alcohol list/dibasic acid esters and the interior target recovery thereof are all between 92.2 ~ 102%, fatty acid 3-chloro-1,2-propylene glycol monoester, fatty acid 3-chloro-1, the detection limit of 2-propylene glycol diesters, fatty acid 2-chlorine-1,2-propylene glycol monoesters, fatty acid 2-chlorine-1,2-propylene glycol diester is respectively 95 μ g/kg, 142 μ g/kg, 84 μ g/kg, 122 μ g/kg, quantitative limit is respectively 316 μ g/kg, 473 μ g/kg, 298 μ g/kg, 406 μ g/kg.Fatty acid 3-chloro-1,2-propylene glycol monoester, fatty acid 3-chloro-1,2-propylene glycol diesters, fatty acid 2-chloro-1, the regression equation of 2-propylene glycol monoester, fatty acid 2-chlorine-1,2-propylene glycol diester is respectively: Y=0.0246X+0.0169, Y=0.0274X-0.2664, Y=0.0174X-0.0664, Y=0.0133X-0.0290, good in 25 ~ 500ng scope linear relationship, coefficient R is all more than 0.9994.Record fatty acid 3-chlorine-1,2-propylene glycol monoester content between 95 μ g/kg ~ 1.3 mg/kg, the content of fatty acid 3-chlorine-1,2-propylene glycol diester is between 142 μ g/kg ~ 0.6mg/kg; Fatty acid 2-chlorine-1,2-propylene glycol monoesters, fatty acid 2-chlorine-1,2-propylene glycol diester all do not detect.
Claims (9)
1. detect a method for fatty acid chloride propyl alcohol pH-titration in food, it is characterized in that: comprise the following steps:
(1) process of sample
Take foodstuff samples to be measured in right amount in plastic centrifuge tube, add mixing inner mark solution, ultrasonicly to mix;
(2) extract and separate
Upper prop: by the sample that mixes and inner mark solution, proceed in solid-phase extraction column, and twice with n-hexane centrifuge tube, proceeded in the lump in solid-phase extraction column by cleansing solution, the filler of described solid-phase extraction column is amino filler or amino and diatomaceous potpourri filler;
Wash-out: be that fatty acid chloride propyl alcohol dibasic acid esters elutes by eluting solvent with normal hexane, collect eluent with glass test tube, and then with normal hexane-ethyl acetate mixed solvent wash-out fatty acid chloride propyl alcohol monoesters, collect this eluent with another glass test tube;
(3) basic hydrolysis process
Respectively step (two) gained eluent is placed in nitrogen flow down slowly be concentrated near dry, add methyl tert-butyl ether-ethyl acetate solution dissolved residue, then add sulfuric acid propanol solution, then add after hydrolysing agent is hydrolyzed 1 min, add glacial acetic acid solution rapidly, abundant vortex mixing, then adds normal hexane degreasing, leaves standstill after abundant vortex, normal hexane layer is removed after obvious layering, use n-hexane extraction again 1 time, discard normal hexane phase, aqueous phase is for derivatization;
(4) derivatization treatment
Derivatization reagent is added in the aqueous phase of step (three) gained, at 80 DEG C, derive 20 min after vortex mixing, be cooled to after room temperature until it, add the abundant vortex of ethyl acetate, after stratification, ethyl acetate layer is transferred in another test tube, then re-extract 2 times, merge 3 extracts, flow down in nitrogen and slowly dry up, after residue dissolves with isooctane, with centrifugation 5 min of 12000 turns/min, get supernatant for the analysis of GC-MS sample introduction;
(5) gas chromatograph-mass spectrometer (GCMS) analysis
Detected by gas chromatograph-mass spectrometer (GCMS) by above-mentioned steps gained supernatant respectively, needed for gas chromatograph-mass spectrometer (GCMS), condition is as follows:
Chromatographic column: DB-5MS capillary column; Injector temperature: 280 DEG C; Post begins warm: 80 DEG C keep 1 min, then are warming up to 190 DEG C with the speed program of 10 DEG C/min, finally rise to 300 DEG C with the speed of 40 DEG C/min, keep 5 min; Carrier gas: high-pure helium, flow velocity 3.0 mL/min; Input mode: Splitless injecting samples; Sampling volume: 1 μ L;
Ion gun: electron impact ion source (EI); Electron-multiplier gain :+250V; Filament current: 100 μ A; Trap temperature: 230 DEG C; Transmission line temperature: 250 DEG C; Discrimination box temperature: 50 DEG C; Solvent delay: 3 min; Mass spectrum acquisition time: 3-29.75 min; Quality of scanning number scope: 90-300; Sweep speed: 0.65 s/ time; The quota ion of fatty acid 3-chlorine-1,2-propylene glycol and fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z147 and m/z196, and the quota ion of interior mark d5-3-chlorine-1,2-propylene glycol and d5-fatty acid 2-chlorine-1,2-propylene glycol is respectively m/z150 and m/z201;
(6) preparation of typical curve
Accurately take appropriate fatty acid 3-chloro-1 respectively, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, fatty acid 2-chloro-1,3-PD diester standard items, in 10ml volumetric flask, are settled to scale with after acetic acid ethyl dissolution, abundant mixing obtains standard reserving solution, places and preserve in-24 DEG C of refrigerators; Get the standard working solution that appropriate storing solution normal hexane is diluted to 25 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500ng/ml more respectively, respectively add amount identical with sample inner mark solution, then carry out Treatment Analysis according to step (three), (four) and (five);
(7) quantivative approach
With standard items fatty acid 3-chlorine-1,2-propylene glycol monoesters, fatty acid 3-chloro-1,2-propylene glycol diesters, the chloro-1,3-PD monoesters of fatty acid 2-, the chloro-1,3-PD diester of fatty acid 2-marks peak area ratio and sample introduction Mass Calculation equation of linear regression, by this equation to the fatty acid 3-chloro-1 in sample with interior separately, 2-propylene glycol monoester, fatty acid 3-chlorine-1,2-propylene glycol diester, fatty acid 2-chloro-1, ammediol monoesters, the chloro-1,3-PD diester of fatty acid 2-carries out quantitatively.
2. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as claimed in claim 1, it is characterized in that: fatty acid 3-chloro-1,2-propylene glycol monoester, fatty acid 3-chloro-1,2-propylene glycol diesters, fatty acid 2-chloro-1, ammediol monoesters, fatty acid 2-chloro-1, interior mark corresponding to ammediol diester is respectively d5-palmitic acid 3-chlorine-1,2-propylene glycol monoesters, d5-palmitic acid 3-chlorine-1,2-propylene glycol diester, d5-stearic acid 2-chloro-1, the chloro-1,3-PD diester of ammediol monoesters, d5-stearic acid 2-.
3. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as claimed in claim 1, is characterized in that: solid-phase extraction column needs to activate through normal hexane before upper prop.
4. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as described in claim 1 or 3, is characterized in that: the weight of the filler of solid-phase extraction column is between 0.5g to 5.0 g.
5. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as claimed in claim 1, is characterized in that: the granularity of amino filler is between 50 order to 400 orders.
6. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as claimed in claim 1, is characterized in that: in eluting solvent normal hexane-ethyl acetate mixture, the volume ratio of normal hexane and ethyl acetate is in 9:1,8:2,7:3.
7. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as claimed in claim 1, it is characterized in that: the flow velocity of employing when eluting solvent normal hexane and normal hexane-ethyl acetate mixture carry out wash-out is in 0.5 ml/min, 1.0 ml/min, 1.5 ml/min.
8. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as claimed in claim 1, is characterized in that: hydrolysing agent is sodium methoxide-methanol solution or sodium hydroxide solution.
9. a kind of method detecting fatty acid chloride propyl alcohol pH-titration in food as claimed in claim 1, is characterized in that: derivatization reagent is phenylboric acid or seven fluorine bytyry imidazole solution.
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| CN111103277A (en) * | 2020-01-21 | 2020-05-05 | 滁州学院 | Chloropropanol sensitive gas sensor, preparation method thereof, detection device containing sensor and detection method |
| CN113640396B (en) * | 2020-05-11 | 2023-06-20 | 华奇（中国）化工有限公司 | Identification method and application of ester compounds in fatty acid derivatives |
| CN112255350A (en) * | 2020-10-14 | 2021-01-22 | 青岛天祥食品集团有限公司 | Method for rapidly detecting 3-MCPD and 3-MCPPDE in edible vegetable oil |
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