CN109612819A - 基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术及其应用 - Google Patents
基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术及其应用 Download PDFInfo
- Publication number
- CN109612819A CN109612819A CN201811438956.4A CN201811438956A CN109612819A CN 109612819 A CN109612819 A CN 109612819A CN 201811438956 A CN201811438956 A CN 201811438956A CN 109612819 A CN109612819 A CN 109612819A
- Authority
- CN
- China
- Prior art keywords
- extraction
- sample
- enzymatic hydrolysis
- solidification
- pretreatment technology
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000005516 engineering process Methods 0.000 title claims abstract description 24
- 238000004853 microextraction Methods 0.000 title claims abstract description 17
- 238000007667 floating Methods 0.000 title claims abstract description 16
- 230000007071 enzymatic hydrolysis Effects 0.000 title claims abstract description 14
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 title claims abstract description 14
- 238000007711 solidification Methods 0.000 title claims abstract description 12
- 230000008023 solidification Effects 0.000 title claims abstract description 12
- 229960001117 clenbuterol Drugs 0.000 claims abstract description 23
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229940074095 ractopamine Drugs 0.000 claims abstract description 23
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 claims abstract description 23
- 235000013372 meat Nutrition 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000007787 solid Substances 0.000 claims abstract description 9
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 235000001968 nicotinic acid Nutrition 0.000 claims abstract description 6
- 229960003512 nicotinic acid Drugs 0.000 claims abstract description 6
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000000605 extraction Methods 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000011159 matrix material Substances 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 239000008351 acetate buffer Substances 0.000 claims description 7
- XSWSEQPWKOWORN-UHFFFAOYSA-N dodecan-2-ol Chemical compound CCCCCCCCCCC(C)O XSWSEQPWKOWORN-UHFFFAOYSA-N 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 238000002604 ultrasonography Methods 0.000 claims description 7
- 238000005251 capillar electrophoresis Methods 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 5
- 230000000996 additive effect Effects 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 108010060309 Glucuronidase Proteins 0.000 claims description 4
- 102000053187 Glucuronidase Human genes 0.000 claims description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 235000011007 phosphoric acid Nutrition 0.000 claims description 4
- 230000006920 protein precipitation Effects 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- IXADHCVQNVXURI-UHFFFAOYSA-N 1,1-dichlorodecane Chemical compound CCCCCCCCCC(Cl)Cl IXADHCVQNVXURI-UHFFFAOYSA-N 0.000 claims description 3
- HNTGIJLWHDPAFN-UHFFFAOYSA-N 1-bromohexadecane Chemical compound CCCCCCCCCCCCCCCCBr HNTGIJLWHDPAFN-UHFFFAOYSA-N 0.000 claims description 3
- 239000007832 Na2SO4 Substances 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 125000003118 aryl group Chemical group 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 25
- 238000001514 detection method Methods 0.000 abstract description 14
- 229960001399 clenbuterol hydrochloride Drugs 0.000 abstract description 12
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 abstract description 12
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000002203 pretreatment Methods 0.000 abstract description 7
- 238000012545 processing Methods 0.000 abstract description 5
- 239000012535 impurity Substances 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 239000002270 dispersing agent Substances 0.000 abstract description 3
- 239000000839 emulsion Substances 0.000 abstract description 3
- 230000014759 maintenance of location Effects 0.000 abstract description 3
- 230000003247 decreasing effect Effects 0.000 abstract description 2
- 239000006185 dispersion Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 235000013622 meat product Nutrition 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 102000009133 Arylsulfatases Human genes 0.000 description 3
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 235000015277 pork Nutrition 0.000 description 3
- 229960002052 salbutamol Drugs 0.000 description 3
- 108060007951 sulfatase Proteins 0.000 description 3
- XWTYSIMOBUGWOL-UHFFFAOYSA-N (+-)-Terbutaline Chemical compound CC(C)(C)NCC(O)C1=CC(O)=CC(O)=C1 XWTYSIMOBUGWOL-UHFFFAOYSA-N 0.000 description 2
- YREYLAVBNPACJM-UHFFFAOYSA-N 2-(tert-butylamino)-1-(2-chlorophenyl)ethanol Chemical compound CC(C)(C)NCC(O)C1=CC=CC=C1Cl YREYLAVBNPACJM-UHFFFAOYSA-N 0.000 description 2
- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- JSJCTEKTBOKRST-UHFFFAOYSA-N mabuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(C(F)(F)F)=C1 JSJCTEKTBOKRST-UHFFFAOYSA-N 0.000 description 2
- 229950004407 mabuterol Drugs 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 229960000195 terbutaline Drugs 0.000 description 2
- 229960000859 tulobuterol Drugs 0.000 description 2
- HNTZKNJGAFJMHQ-UHFFFAOYSA-N 2-methylpyridine-3-carboxylic acid Chemical compound CC1=NC=CC=C1C(O)=O HNTZKNJGAFJMHQ-UHFFFAOYSA-N 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- -1 Dodecane alcohol compound Chemical class 0.000 description 1
- 241001412225 Firmiana simplex Species 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- FRYDSOYOHWGSMD-UHFFFAOYSA-N [C].O Chemical compound [C].O FRYDSOYOHWGSMD-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 229940031098 ethanolamine Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 235000020997 lean meat Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
- G01N2001/4061—Solvent extraction
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术及其应用。经过酶解处理后的样品，杂质含量明显下降，再利用分散剂对萃取剂的分散作用、萃取剂在水中的乳化现象(浊相)以及萃取剂，本身所具有的在室温下溶解和低温条件下固化析出漂浮的特点，从而达到对烟酸克伦特罗和莱克多巴胺的分离和富集，使其检出灵敏度明显升高。该方法可应用于动物饲料和肉类等固体基质中的主要“瘦肉精”种类如CLB和RCT的残留分析前处理，与以往的分散液液萃取技术相比，具有低检出限(分别为CLB为0.19μg kg‑1，RCT为0.28μg kg‑1)、高灵敏度和高重现性等优点，特别是提取剂的用量仅在微升级，使前处理成本急剧下降、污染大大降低，故有较强的应用价值。
Description
技术领域
本发明属于药物检测领域，具体是指应用于饲料和肉类产品中“瘦肉精”检测领域，特别是指酶解辅助和固化漂浮液滴微萃取联用(EA-SFODM)的前处理技术与毛细管电泳(紫外检测器)CEC-UVD结合检测瘦肉精的方法。
背景技术
近年来，食品安全形势不容乐观，食品污染依然是危害公众健康的重要因素。“瘦肉精”是一类能够促进瘦肉生长，抑制脂肪生成、加速脂肪转化的药物，主要包括烟酸克伦特罗(clenbuterol，CLB)、甲基烟酸克伦特罗(methylclenbuterol)、莱克多巴胺(rectopamine，RCT)、沙丁胺醇(salbutamol)、马布特罗(mabuterol)、特布它林(terbutaline)和妥布特罗(tulobuterol)等。在中国，“瘦肉精”种类中应用最多的为CLB和RCT，该药物的半衰期长，在体内代谢慢，极易蓄积残留于动物体内，在肝脏中的平均浓度高达45mg/kg，人食用后易出现恶心、头晕、肌肉震颤、心悸等中毒病症，严重影响消费者的身体健康。
近年来，酶的催化和水解作用在前处理分析领域得到了越来越多的应用。酶是理想的催化剂可应用于多种样品基质中降解净化杂质，提高分析物的回收率，它能分解植物组织中复杂的多糖为简单的小分子如半乳糖醛酸等，其活性增效作用强化了复杂碳水化合物的水解。
固化漂浮有机液滴微萃取技术(Solidified Floating Organic DropletMicroextraction,SFODM)是近年来发展起来的一种新型绿色前处理技术，由三元溶剂系统组成，利用分散剂对萃取剂的分散作用、萃取剂在水中的乳化现象(浊相)以及萃取剂(多为十二烷醇类化合物)本身所具有的在室温(25℃)下溶解和低温条件下固化析出漂浮的特点，从而达到对目标分析物的分离和富集。该技术具有萃取剂用量仅微升级、操作相对简便、快捷和高富集倍数等优点，但其缺点是单独使用仅能应用于基质相对干净的水样分析。
而在肉产品和动物饲料产品中，由于含有大量的脂肪、蛋白质和淀粉纤维类化合物，基质干扰非常严重，故实现SFODM技术在该类样品中的应用，样品中大分子物质的净化技术至关重要。如何将酶的催化和水解作用应用于复杂样品基质的降解净化杂质的作用是本技术的关键。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足，而提供一种基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术。
本发明所采取的技术方案如下：一种酶解辅助前处理，减少基质干扰。
优选地，所述的待检样品为饲料样品或肉类样品。
优选地，所述的酶为β-葡萄糖醛酸苷和芳基硫酸酯酶组合物。
其中，水、乙酸缓冲液和酶的体积比为10:1:50-100
一种基于以上酶解辅助和固化漂浮液滴微萃取联用的前处理技术方法，包括以下步骤：
(1)酶辅助提取，包括以下步骤：
(1-1)将待检固体基质样品粉碎，置于容器中，加入水、乙酸缓冲液和酶，然后进行超声振摇和离心，取上清液；本步骤(1-1)所述的水、乙酸缓冲液和酶的体积比为10:1:50-100，且每克待检样品对应50-100ml酶，所述的酶为β-葡萄糖醛酸苷和芳基硫酸酯酶组合物，所述的待检样品为饲料样品或肉类样品；
(1-2)将上清液中添加1-5mL,20-80g/L NaCl和20-80μL正磷酸，并调节pH 2.0-8.0，得到混合溶液，将混合溶液在30-70℃水浴锅中加热2-10min，然后超声振摇1-5min；
(1-3)混合溶液在6000rpm离心1-5min，将沉淀蛋白和溶解性碳水化合物，取本步骤(1-3)的上清液，并将pH调至5-12.0；
(2)以20-60μL熔点13-15℃的1-十一烷醇、熔点22-24℃的1-十二烷醇、熔点17-18℃的2-十二烷醇、熔点17-18℃的溴代十六烷和熔点14-16℃的1,10-二氯癸烷中的一种或多种组合做萃取剂，对步骤(1-3)的上清液进行萃取，添加四种常规盐(NH4)2SO4、Na2SO4、NaCl、Mg2SO4，添加量为0.1-6.0g，置于15-65℃下萃取，样品pH 9-12，氮吹时间为0.5-5.0min，用缓冲液溶解后进毛细管电泳仪检测。
其中，所述样品可以为动物饲料，将样品冷冻干燥，粉碎保存。
其中，所述样品可以为肉类，将样品冷冻干燥，粉碎保存。
优选地，毛细管电泳(紫外检测器)CEC-UVD的分析条件为：UVD检测波长210nm，毛细管柱(75μm,I.D.×55cm)，运行电压20kv。
基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术萃取动物饲料中的
烟酸克伦特罗(CLB)或/和莱克多巴胺(RCT)的应用。
基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术萃取肉类中的烟酸
克伦特罗(CLB)或/和莱克多巴胺(RCT)的应用。
本发明的优点如下：
(1)首次将酶反应体系尝试应用于动物饲料和肉产品中“瘦肉精”的分析前处理，去除大分子基质干扰，使样品获得初步净化，便于和各种其它绿色前处理技术相联用，使前处理程序更加绿色和环保。
(2)将酶反应体系和微萃取体系有机地联用在一起，克服了过去SFODM程序仅能应用于水样分析的缺陷，二者的结合可扩展到肉食品和饲料等复杂固体基质样品，克服了微萃取技术在基质上的应用局限性。
(3)经过酶解处理后的样品，杂质含量明显下降，并且使CLB和RCT的检出灵敏度明显升高。该方法可应用于猪饲料和肉类样品等固体基质中的主要“瘦肉精”种类如CLB和RCT的残留分析前处理，与以往的分散液液萃取技术相比，具有低检出限(分别为CLB为0.19μgkg-1，RCT为0.28μg kg-1)、高灵敏度和高重现性等优点，特别是提取剂的用量仅在微升级，使前处理成本急剧下降、污染大大降低，非常符合目前前处理技术“绿色环保化”的发展趋势，故有较强的应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，根据这些附图获得其他的附图仍属于本发明的范畴。
图1是本方法的EA-SFODM流程图；
图2是本方法中溶液pH对CLB和RCT分离度的影响关系图；
图3是本方法对提取溶剂的种类和体积优化关系图；
图4是本方法对盐的种类和使用量的优化关系图；
图5是本方法对不同温度对CLB和RCT的平均ARs影响关系图；
图6是本方法中pH对CLB和RCT的ARs影响关系图；
图7是本方法中氮吹时间对ARs的影响关系图；
图8是本方法中2-十二烷醇加入后的分离和乳化现象图；
图9是本方法中2-十二烷醇和不同分散剂加入后的分离现象图；
图10是本方法中EA-SFODM技术检测WB1样品的电泳色谱；
图11是本方法中最适条件下以EA-SFODM方法测得实际样品色谱图；
图12是本方法中EA-SFODM方法的实际分离效果示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
如图1所示，
S101：酶辅助提取过程：
酶将待检固体基质样品粉碎，置于容器中，加入水、乙酸缓冲液和酶，然后进行超声振摇和离心，取上清液,所述的水、乙酸缓冲液和酶的体积比为10:1:50-100，且每克待检样品对应50-100ml酶，所述的酶为β-葡萄糖醛酸苷和芳基硫酸酯酶组合
S102：将上清液中添加1-5mL,20-80g/L NaCl和20-80μL正磷酸，并调节pH 2.0-8.0,得到混合溶液，将混合溶液在30-70℃水浴锅中加热2-10min，然后超声振摇1-5min
S103：混合溶液在6000rpm离心1-5min，将沉淀蛋白和溶解性碳水化合物，取本步骤(1-3)的上清液，并将pH调至5.0-12.0
S104：以20-60μL熔点13-15℃的1-十一烷醇、熔点22-24℃的1-十二烷醇、熔点17-18℃的2-十二烷醇、熔点17-18℃的溴代十六烷和熔点14-16℃的1,10-二氯癸烷中的一种或多种组合做萃取剂，对步骤(1-3)的上清液进行萃取，添加四种常规盐(NH4)2SO4、Na2SO4、NaCl、Mg2SO4，添加量为0.1-6.0g，置于15-65℃下萃取，样品pH 9-12，氮吹时间为0.5-5.0min，用缓冲液溶解后进毛细管电泳仪检测。
所述毛细管电泳(紫外检测器)CEC-UVD的分析条件为：UVD检测波长210nm，毛细管柱(75μm,I.D.×55cm)，运行电压20kv。
下面结合具体实施例对本发明的应用原理作进一步的描述。
实施例1
本实施例提供一种待检固体基质中瘦肉精成分的检测方法，采用CEC-UVD进行检测，所用的待检样品溶液通过权利要求1所述的前处理技术获得，该前处理技术包括有：
1、酶辅助提取过程，其特征在于以下步骤：
于离心管中的2g粉碎饲料样品添加10mL超纯水，1mL的HAC缓冲液(1M)和50mLβ-葡萄糖醛酸苷和芳基硫酸酯酶，均匀混合后，混合物在40℃培养1h。水解后加入丙酮，并进行超声振摇1min，6000rpm离心2min。上清液添加3mL 50g/L NaCl和60μL正磷酸(pH 2.0)，混合物在70℃水浴锅中加热10min，然后超声振摇2min。溶液在6000rpm离心2min以沉淀蛋白和溶解性碳水化合物。将上清液pH调至12.0后用于EA-SFODM程序中。
2、酶解辅助和固化漂浮液滴微萃取联用(EA-SFODM)过程，其特征在于以下步骤：
在最优萃取条件下，40μL 2-十二烷醇做提取溶剂，样品pH为12.0，硫酸铵添加量为4.0g，氮吹时间为1.0min，萃取温度为25℃。
本实施例的各参数参见附图1-9的实验数据最优获得，其为最适参数。
本实施例所述的CEC-UVD检查，基本仪器操作条件为：UVD检测波长210nm，毛细管柱(75μm,I.D.×55cm)，运行电压20kv。
实验例2：饲料中“瘦肉精”类成分的检测。
所开发的方法被应用于检测6种实际样品(WB1、WB2、PB1、PB2、YB1和YB2)中的CLB和RCT。结果表明：除YB2样品中的RCT被检测浓度为18.5μg/kg外，其它样品中均未检出CLB和RCT。YB2样品中RCT被检出说明部分动物饲料中确实存在添加的“瘦肉精”。近年来，我国已禁止了CLB的使用，已报道了多例养猪场或饲料加工厂添加RCT以取代CLB，沙丁胺醇和苯乙醇胺等“瘦肉精”种类。在20和50μg/kg添加水平下，对“瘦肉精”的提取回收率范围为74.1-92.6％。
实验例3：猪肉中“瘦肉精”类成分的检测。
样品前处理：4批猪肉分别购置于温州当地农贸市场(梧田、桥儿头、茶山和黄龙，为了增加样品的丰富性与代表性，我们分别在2017年5月15日，25日和30日在同一地点购买三个不同批次的样品，样品购买之后匀浆置于-20℃冰箱储存，保质期为一周。检测之前，将样品取出自然升至室温，加入适量浓度标准品。为保证标准品可以充分溶于肉样，加标后的肉样放置于黑暗干燥环境至少30min，每个样品做3组平行样。在实验的最优萃取条件下(40μL 2-十二烷醇做提取溶剂，样品pH为12.0，硫酸铵添加量为4.0g，氮吹时间为1.0min，萃取温度为25℃)。CLB和RCT的相关性系数R2在0.9983到0.9990之间；猪肉检测的最低检出限分别是CLB为0.19μg kg-1，RCT为0.28μg kg-1。最低定量限分别为CLB 0.63μg kg-1，RCT为0.93μg kg-1。
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。
Claims (4)
1.一种基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术，其特征在于包括以下步骤：
(1)酶辅助提取，包括以下步骤：
(1-1)将待检固体基质样品粉碎，置于容器中，加入水、乙酸缓冲液和酶，然后进行超声振摇和离心，取上清液；本步骤(1-1)所述的水、乙酸缓冲液和酶的体积比为10:1:50-100，且每克待检样品对应50-100ml酶，所述的酶为β-葡萄糖醛酸苷和芳基硫酸酯酶组合物，所述的待检样品为饲料样品或肉类样品；
(1-2)将上清液中添加1-5mL,20-80g/L NaCl和20-80μL正磷酸，并调节pH 2.0-8.0，得到混合溶液，将混合溶液在30-70℃水浴锅中加热2-10min，然后超声振摇1-5min；
(1-3)混合溶液在3000-6000rpm离心1-5min，将沉淀蛋白和溶解性碳水化合物，取本步骤(1-3)的上清液，并将pH调至5.0-12.0；
(2)以20-60μL熔点13-15℃的1-十一烷醇、熔点22-24℃的1-十二烷醇、熔点17-18℃的2-十二烷醇、熔点17-18℃的溴代十六烷和熔点14-16℃的1,10-二氯癸烷中的一种或多种组合做萃取剂，对步骤(1-3)的上清液进行萃取，添加四种常规盐(NH4)2SO4、Na2SO4、NaCl、Mg2SO4中的一种或多种组合，添加量为0.1-6.0g，置于15-65℃下萃取，样品pH 9-12，氮吹时间为0.5-5.0min，用缓冲液溶解后进毛细管电泳仪检测。
2.根据权利要求1基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术，其特征在于：所述待检固体基质样品为动物饲料；
a)根据权利要求1基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术，其特征在于：所述待检固体基质样品为肉类样品。
3.一种基于权利要求1或2所述酶解辅助和固化漂浮液滴微萃取联用的前处理技术萃取动物饲料中的烟酸克伦特罗或/和莱克多巴胺应用。
4.一种基于权利要求1或2所述酶解辅助和固化漂浮液滴微萃取联用的前处理技术萃取肉类中的烟酸克伦特罗或/和莱克多巴胺的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811438956.4A CN109612819A (zh) | 2018-11-28 | 2018-11-28 | 基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811438956.4A CN109612819A (zh) | 2018-11-28 | 2018-11-28 | 基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109612819A true CN109612819A (zh) | 2019-04-12 |
Family
ID=66006281
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811438956.4A Pending CN109612819A (zh) | 2018-11-28 | 2018-11-28 | 基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109612819A (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1487294A (zh) * | 2002-09-30 | 2004-04-07 | 华中科技大学同济医学院 | 一种快速检测生物样品中β2—受体兴奋剂的方法及其应用 |
| CN103512984A (zh) * | 2013-10-21 | 2014-01-15 | 中国动物卫生与流行病学中心 | 一种检测不同种类“瘦肉精”残留的样品前处理方法 |
| CN106770826A (zh) * | 2016-12-22 | 2017-05-31 | 温州医科大学 | 一种检测三氯生和甲基三氯生的微萃取方法 |
| CN107505417A (zh) * | 2017-01-16 | 2017-12-22 | 广东增源检测技术有限公司 | 生鲜肉及肉制品中瘦肉精的检测方法 |
-
2018
- 2018-11-28 CN CN201811438956.4A patent/CN109612819A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1487294A (zh) * | 2002-09-30 | 2004-04-07 | 华中科技大学同济医学院 | 一种快速检测生物样品中β2—受体兴奋剂的方法及其应用 |
| CN103512984A (zh) * | 2013-10-21 | 2014-01-15 | 中国动物卫生与流行病学中心 | 一种检测不同种类“瘦肉精”残留的样品前处理方法 |
| CN106770826A (zh) * | 2016-12-22 | 2017-05-31 | 温州医科大学 | 一种检测三氯生和甲基三氯生的微萃取方法 |
| CN107505417A (zh) * | 2017-01-16 | 2017-12-22 | 广东增源检测技术有限公司 | 生鲜肉及肉制品中瘦肉精的检测方法 |
Non-Patent Citations (1)
| Title |
|---|
| HUILI WANG 等: "An enzyme-assisted and nitrogen-blowing salt-induced solidified floating organic droplet microextraction fordetermination of clenbuterol and ractopamine in swine feedvia capillary electrophoresis", 《NIMAL FEED SCIENCE AND TECHNOLOGY》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fussell et al. | Determination of volatile fatty acids (C 2—C 5) and lactic acid in silage by gas chromatography | |
| Lavilla et al. | Ultrasound-assisted emulsification microextraction with simultaneous derivatization coupled to fibre optics-based cuvetteless UV–vis micro-spectrophotometry for formaldehyde determination in cosmetic samples | |
| Saaid et al. | In situ derivatization hollow fibre liquid-phase microextraction for the determination of biogenic amines in food samples | |
| Nadeem et al. | Determination of histamine in fish without derivatization by indirect reverse phase-HPLC method | |
| Yin et al. | Purification and determination of stachyose in Chinese artichoke (Stachys Sieboldii Miq.) by high-performance liquid chromatography with evaporative light scattering detection | |
| Moret et al. | Improvement of extraction procedure for biogenic amines in foods and their high-performance liquid chromatographic determination | |
| CN107677757B (zh) | 同时测定食品中香兰素、甲基香兰素、乙基香兰素的方法 | |
| Mafra et al. | Evaluation of biogenic amines in some Portuguese quality wines by HPLC fluorescence detection of OPA derivatives | |
| Pragst et al. | Are there possibilities for the detection of chronically elevated alcohol consumption by hair analysis? A report about the state of investigation | |
| CN103063782B (zh) | 水样品中PAEs类环境激素及代谢产物同时检测的方法 | |
| Ryu et al. | Validation of analytical methods for ethyl carbamate in nine food matrices | |
| Zhou et al. | Comparison of amberlite XAD-2/freon 11 extraction with liquid/liquid extraction for the determination of wine flavor components | |
| Huang et al. | Simultaneous determination of eight biogenic amines in the traditional Chinese condiment Pixian Douban using UHPLC–MS/MS | |
| Stolker et al. | Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment | |
| Takatsuki et al. | Liquid chromatographic determination of free and added niacin and niacinamide in beef and pork | |
| Csutoras et al. | Medium chain fatty acids and fatty acid esters as potential markers of alcoholic fermentation of white wines | |
| Conte et al. | Microwave Distillation− Solid Phase Adsorbent Trapping Device for the Determination of Off-Flavors, Geosmin and Methylisoborneol, in Catfish Tissue below Their Rejection Levels | |
| Lambropoulou et al. | Sensitive trace enrichment of environmental andiandrogen vinclozolin from natural waters and sediment samples using hollow-fiber liquid-phase microextraction | |
| Carneiro et al. | Doehlert matrix for the optimization of ultrasound dispersive liquid–liquid microextraction of melatonin in Argentine and Brazilian wine samples | |
| CN109612819A (zh) | 基于酶解辅助和固化漂浮液滴微萃取联用的前处理技术及其应用 | |
| Nezhadali et al. | Selective extraction of cholesterol from dairy samples Using a polypyrrole molecularly imprinted polymer and determination by gas chromatography | |
| Gao et al. | Determination of trace biogenic amines with 1, 3, 5, 7‐tetramethyl‐8‐(N‐hydroxysuccinimidyl butyric ester)‐difluoroboradiaza‐s‐indacene derivatization using high‐performance liquid chromatography and fluorescence detection | |
| Hayes et al. | Compositional relationships between organic matter in a grassland soil and its drainage waters | |
| Basler et al. | Microbial carbon recycling: an underestimated process controlling soil carbon dynamics–Part 2: AC 3-C 4 vegetation change field labelling experiment | |
| Polcaro et al. | Stereoselective analysis of acid herbicides in natural waters by capillary electrophoresis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190412 |
|
| RJ01 | Rejection of invention patent application after publication |