CN105758973A - Solid support liquid-liquid extraction pretreatment method of urethane in wine - Google Patents
Solid support liquid-liquid extraction pretreatment method of urethane in wine Download PDFInfo
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- CN105758973A CN105758973A CN201410797553.4A CN201410797553A CN105758973A CN 105758973 A CN105758973 A CN 105758973A CN 201410797553 A CN201410797553 A CN 201410797553A CN 105758973 A CN105758973 A CN 105758973A
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Abstract
The purpose of the invention is to provide a solid support liquid-liquid extraction pretreatment method of urethane in wine. The method comprises the steps of extracting purification, concentration and water removal. The pretreatment method has the advantages of avoiding of the emulsification phenomenon of liquid-liquid extraction, enhancement of the extraction ability, improvement of the problem of use of a large amount of an extraction solvent in solid extraction, solvent saving, concentration process simplification and pretreatment efficiency increase.
Description
Technical field
The present invention relates to the efficient sample-pretreating method of a kind of Dietary Ethyl Carbamate in Wine, adopt the detection technique of gas chromatography-mass spectrometry analysis Dietary Ethyl Carbamate in Wine content simultaneously.
Background technology
Urethanes (EthylCarbamate, EC) is a kind of many sites carcinogen, may result in the diseases such as pulmonary carcinoma, lymphatic cancer, hepatocarcinoma, skin carcinoma, assert that it is 2A class carcinogen through international cancer research institution (IARC).The urethanes that research shows in wine has 2 main forming feature: 1, the cyanogen glycosides in Semen Vitis viniferae is degraded under the effect of enzyme and produced cyanide, is then oxidized to cyanate, then generates urethanes with ethanol synthesis；2, the arginine breakdown in Sucus Vitis viniferae is carbamide by yeast, and carbamide generates urethanes with ethanol synthesis.It follows that urethanes is inevitable association product in wine.
The harm of the mankind is caused by urethanes mainly through No alcoholic beverages, so America and Europe etc. are national in recent years has strictly formulated the limit standard of EC in wine, wherein the state such as Canada, Czech specifies that the EC content in table wine must not exceed 30 μ g/L, the U.S. must not exceed 15 μ g/L, and China not yet formulates the limit standard of EC in wine.
At present, detect the pre-treating method of EC in wine both at home and abroad and mainly have following methods: 1, liquid-liquid extraction.This method does not need special installation, the advantage with low cost, but liquid-liquid extraction is inefficient, generally requires and carries once again, thus operating procedure more, consuming time longer, extractant consumption is also bigger；2, Solid-Phase Extraction.The pre-treating method of current European Union, the U.S. and international wine Organisation recommendations is exactly solid phase extraction, it integrates extraction and purifies, but this method need to (dichloromethane itself be also 2A class carcinogen with 160mL dichloromethane eluent, too much contact is also unfavorable for the health of experimenter), so needing to rotate evaporation to blow two-step enrichment with nitrogen, length consuming time, treatment effeciency are low.3, solid-phase microextraction.This method does not need complicated pre-treatment, is full automatic process, but fiber used is expensive, and access times are limited, so this method is not suitable for the routine testing of manufacturing enterprise.
Summary of the invention
It is an object of the invention to overcome existing Sample Pretreatment Technique to process time length, consume the shortcoming and defect such as solvent is many, for the Sample Pretreatment Technique of detection a kind of simple and effective of offer of Dietary Ethyl Carbamate in Wine.For reaching above-mentioned purpose, technical scheme is as follows:
A kind of solid phase that urethanes in wine is carried out supports the pre-treating method of liquid-liquid extraction, and described method comprises the steps:
(1) extract and purify: weighing wine samples, described wine samples is loaded on extraction column, then stands, then use dichloromethane eluent；
(2) concentration: the eluent that step (1) obtains is carried out nitrogen and blows；
(3) dewater: in the concentrated solution that step (2) obtains, add desiccant, then stand, be filtered with filter；
In step (2), described eluting liquid nitrogen is blown to 1mL.
The present invention adopts the solid phase in conjunction with liquid-liquid extraction and Solid-Phase Extraction advantage to support liquid-liquid extraction method.After loading, sample liquid forms one layer of thin liquid film on extraction stuffing surface, and after adding eluant, eluant is wrapped in sample exterior, carries out the liquid-liquid extraction of microcosmic between two liquid films.Extraction stuffing is kieselguhr, its natural micropore rich surface hydroxyl, aqueous sample solution can be adsorbed, form liquid film, eluting organic solvent and the intermembranous formation microcosmic liquid-liquid extraction of sample liquid, both avoided emulsion, raising extracting power that liquid-liquid extraction oscillating operation brings, improved again the problem using a large amount of extractant in existing Solid-Phase Extraction, save solvent, simplify concentration process, improve pre-treatment efficiency.
Accompanying drawing explanation
Fig. 1 be 1000 μ g/L urethanes standard substance total ions chromatogram (on) and mass spectrum (under).
Detailed description of the invention
At some existing pre-treating methods, use ether as eluting solvent, but the healthy of experimenter may be had a negative impact by ether.Blow at nitrogen journey needs be blown to absolutely dry it addition, adopt ether to carry out the eluent that affords, then redissolve in the middle of ethanol.There is the problem that following two is likely to affect measurement accuracy in this process: the boiling point of (1) urethanes is relatively low, and nitrogen is blown to the dry volatilization being likely to result in a small amount of urethanes；(2) due to production technology, ethanol used itself is likely to just contain the urethanes of low content.
And in the present invention, have employed dichloromethane as eluting solvent, it is to avoid the use of ether.Further, the concentrated solution that nitrogen obtains after blowing can be made directly follow-up process step, it is not necessary to redissolves in ethanol after drying up.Visible, in existing pre-treating method, problem that may be present is all avoided in the present invention.Therefore, the pre-treating method of the present invention possesses simply efficiently, consumption of organic solvent is little, inorganic salt consumption is little, concentration step loss is little, poison many-sided advantages such as little.
In the step (1) of the present invention, it is preferred to use time of repose be 10-12min.Too short if left standing for the time, then wine samples filler in extraction column cannot be made fully to be combined, cause follow-up eluting poor effect；If left standing for overlong time, then the unnecessary time can be caused to waste.
In the step (1) of the present invention, relative to the wine samples of 2g, it is preferred that methylene chloride is 12-15mL.If methylene chloride is too small, then completely by the determinand eluting in sample, measurement result cannot be caused less than normal；If methylene chloride is too much, then cause unnecessary organic solvent to waste, and also make nitrogen blow time increase.
In the step (1) of the present invention, it is preferred that dichloromethane flow velocity is 1.5-2mL/min.If flow velocity is too fast, then completely by the determinand eluting in sample, measurement result cannot be caused less than normal；If flow velocity is excessively slow, then the unnecessary time can be caused to waste.
Embodiment
It follows that the present invention is illustrated in further detail by embodiment, but the present invention is not limited only to these embodiments.
Embodiment 1
1.1 instruments and reagent
7890A-5975C gas chromatograph-mass spectrometer (Agilent company), 12 negative pressure SPE devices (Beaune Ai Jieer Science and Technology Ltd.), BSA224S-CW electronic balance (Sai Duolisi group), TTL-DCII Nitrogen evaporator (with Tai Lian development in science and technology company limited), QL-902 vortex mixer (Haimen its woods Bel instrument manufacturing company limited).
Urethanes standard substance (CAS51-79-6, AccuStandard company).
Acetonitrile (chromatographically pure, Fisher company), dichloromethane (chromatographically pure, ClemanChemical company), anhydrous sodium sulfate (analytical pure, Beijing Chemical Plant), CleanertEC-SPE post (Beaune Ai Jieer Science and Technology Ltd.).
Wine samples is purchased from supermarket.
1.2 experimental techniques
1.2.1 the preparation of standard solution
Storing solution: accurately weigh urethanes standard substance 0.0100g, adds acetonitrile and dissolves and be diluted to 10mL, and concentration is 1000mg/L.Refrigerated storage (less than-18 DEG C).
Use liquid: accurately pipette urethanes storing solution, with the use liquid that dilution in acetonitrile is 20 μ g/mL.Refrigerated storage (less than-18 DEG C).
1.2.2 sample pre-treatments
(1) extract and purify: weighing 2g (being accurate to 0.01g) uniformly wine in 10mL centrifuge tube, after vortex mixing, wine sample is loaded on extraction column, after standing 10-12min, use 12-15mL dichloromethane eluent, coutroi velocity is about 1.5-2mL/min, and whole eluents are collected in test tube.
(2) concentration: eluent gentle nitrogen at 30 DEG C is blown to 1mL.
(3) dewater: in 1mL concentrated solution, add 0.7-0.8g anhydrous sodium sulfate, after vortex mixing, stand 3-5min.After 0.22-0.45 μm of nylon leaching film crossed by sample, treat that GC/MS analyzes.
1.2.3 instrument condition
Chromatographic column: VF-WAXms capillary column, 30m × 0.25mm × 0.25 μm, constant current mode, flow velocity 1.0mL/min；Injection port: pulse is shunt mode not, pulsating pressure 30psi, temperature 180 DEG C, sample size 2 μ L；Heating schedule: 60 DEG C keep 1min, rise to 140 DEG C with 5 DEG C/min,；Rear running temperature 220 DEG C, rear operation time 10min；Carrier gas: He, purity >=99.999%.Mass detector testing conditions: Salbutamol Selected Ion Monitoring pattern (SIM), quota ion is 62, and qualitative ion is 45,74,89；Electron impact ion source (EI): 70eV；Ion source temperature: 230 DEG C；Level Four bar temperature: 150 DEG C；Interface temperature: 280 DEG C；Solvent delay: 15min.
The range of linearity of 1.3 methods
Under the experimental technique of 1.2, the chromatographic peak profile of urethanes is sharp-pointed, symmetrical, and retention time is about 16.2min.With the acetonitrile standard solution by the standard solution stepwise dilution of 20 μ g/mL to be concentration be 1000,500,200,100,50,20,10,5,0 μ g/L, with the peak area of urethanes to concentration drawing standard curve, linear equation is y=137.1x-680.3, regression coefficient R2=0.998.1000 μ g/L urethanes standard substance chromatograms and mass spectrum are shown in Fig. 1.
The mark-on of 1.4 methods reclaims and Precision Experiment
Weigh 2g (being accurate to 0.01g) uniformly wine, add standard solution and make interpolation concentration respectively 0.02mg/kg, 0.05mg/kg, 0.10mg/kg of sample, it is analyzed by the experimental technique of 1.2, the average recovery rate after blank, relative standard deviation (RSD) is detained with quantitative limit (in quota ion 10 times of signal to noise ratios, i.e. LOQ=10 × S/N) in Table 1 under each pitch-based sphere.
Table 1 urethanes adds the average recovery rate of recovery experiment, relative standard deviation and quantitative limit
Claims (5)
1. the urethanes in wine carrying out solid phase and supports a pre-treating method for liquid-liquid extraction, described method comprises the steps:
(1) extract and purify: weighing wine samples, described wine samples is loaded on extraction column, then stands, then use dichloromethane eluent；
(2) concentration: the eluent that step (1) obtains is carried out nitrogen and blows；
(3) dewater: in the concentrated solution that step (2) obtains, add desiccant, then stand, be filtered with filter；
It is characterized in that, in step (2), described eluting liquid nitrogen is blown to 1mL.
2. pre-treating method as claimed in claim 1, it is characterised in that it is one or more that step (1) meets in following condition:
The amount of weighing of described wine samples is 2g；
The filler of described extraction column is kieselguhr, it is preferable that described extraction column is CleanertEC-SPE post；
The time of described standing is 10-12min；
The consumption of described dichloromethane is 12-15mL；And/or
During described eluting, the flow velocity of dichloromethane is 1.5-2mL/min.
3. pre-treating method as claimed in claim 1 or 2, it is characterised in that the described nitrogen in step (2) blows and carries out at 30 DEG C.
4. the pre-treating method as according to any one of claim 1-3, it is characterised in that it is one or more that step (3) meets in following condition:
Described desiccant is anhydrous sodium sulfate, it is preferable that the consumption of described anhydrous sodium sulfate is 0.7-0.8g；
The time of described standing is 3-5min；And/or
Described filter is nylon leaching film, it is preferable that the aperture of described nylon leaching film is 0.22-0.45 μm.
5. one kind adopts Gas chromatographyMass spectrometry that the urethanes in wine is carried out quantitative method, it is characterised in that described method adopts the pre-treating method according to any one of claim 1-4.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011136329A1 (en) * | 2010-04-28 | 2011-11-03 | 株式会社日立ハイテクノロジーズ | Adsorbent and method for producing same |
| CN102778519A (en) * | 2012-08-16 | 2012-11-14 | 江苏中烟工业有限责任公司 | Method for detecting ethyl carbamate content of side stream smoke of cigarettes through combination utilization of gas chromatography-mass spectrometry (GC-MS) |
| CN102788858A (en) * | 2012-08-16 | 2012-11-21 | 江苏中烟工业有限责任公司 | Method for LC-MS/MS (Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry) jointly detecting ethyl carbamate content in cigarette main stream smoke |
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2014
- 2014-12-18 CN CN201410797553.4A patent/CN105758973A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011136329A1 (en) * | 2010-04-28 | 2011-11-03 | 株式会社日立ハイテクノロジーズ | Adsorbent and method for producing same |
| CN102778519A (en) * | 2012-08-16 | 2012-11-14 | 江苏中烟工业有限责任公司 | Method for detecting ethyl carbamate content of side stream smoke of cigarettes through combination utilization of gas chromatography-mass spectrometry (GC-MS) |
| CN102788858A (en) * | 2012-08-16 | 2012-11-21 | 江苏中烟工业有限责任公司 | Method for LC-MS/MS (Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry) jointly detecting ethyl carbamate content in cigarette main stream smoke |
Non-Patent Citations (6)
| Title |
|---|
| ESHWAR JAGERDEO 等: "Analysis of Ethyl Carbamate in Wines Using Solid-Phase Extraction and Multidimensional Gas Chromatography/Mass Spectrometry", 《J. AGRIC. FOOD CHEM.》 * |
| QIANG XIA 等: "An Improved and Validated Sample Cleanup Method for Analysis of Ethyl Carbamate in Chinese Liquor", 《 JOURNAL OF FOOD SCIENCE》 * |
| 李华 等: "固相萃取结合GC/MS法测定葡萄酒中氨基甲酸乙酯", 《食品与生物技术学报》 * |
| 梁孟军 等: "不同酒类中氨基甲酸乙酯前处理方法的研究", 《职业与健康》 * |
| 梁新红: "中国葡萄酒中氨基甲酸乙酯的研究", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 石维妮 等: "发酵性食品中的氨基甲酸乙酯含量调研", 《中国酿造》 * |
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