CN112345672A - 一种血清中脂肪酸质的提取方法 - Google Patents
一种血清中脂肪酸质的提取方法 Download PDFInfo
- Publication number
- CN112345672A CN112345672A CN202011146555.9A CN202011146555A CN112345672A CN 112345672 A CN112345672 A CN 112345672A CN 202011146555 A CN202011146555 A CN 202011146555A CN 112345672 A CN112345672 A CN 112345672A
- Authority
- CN
- China
- Prior art keywords
- fatty acid
- extracting
- supernatant
- solution
- steps
- 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
- 210000002966 serum Anatomy 0.000 title claims abstract description 54
- 235000014113 dietary fatty acids Nutrition 0.000 title claims abstract description 43
- 229930195729 fatty acid Natural products 0.000 title claims abstract description 43
- 239000000194 fatty acid Substances 0.000 title claims abstract description 43
- 150000004665 fatty acids Chemical class 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000000126 substance Substances 0.000 title claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 39
- 239000006228 supernatant Substances 0.000 claims abstract description 35
- 238000010438 heat treatment Methods 0.000 claims abstract description 33
- 238000002156 mixing Methods 0.000 claims abstract description 26
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 238000001212 derivatisation Methods 0.000 claims abstract description 22
- 239000003377 acid catalyst Substances 0.000 claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 11
- 230000010355 oscillation Effects 0.000 claims abstract description 11
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- 238000000861 blow drying Methods 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- WOXFMYVTSLAQMO-UHFFFAOYSA-N 2-Pyridinemethanamine Chemical compound NCC1=CC=CC=N1 WOXFMYVTSLAQMO-UHFFFAOYSA-N 0.000 claims description 6
- WCYAALZQFZMMOM-UHFFFAOYSA-N methanol;sulfuric acid Chemical compound OC.OS(O)(=O)=O WCYAALZQFZMMOM-UHFFFAOYSA-N 0.000 claims description 5
- 238000002203 pretreatment Methods 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 235000021588 free fatty acids Nutrition 0.000 abstract description 6
- 210000004369 blood Anatomy 0.000 abstract description 4
- 239000008280 blood Substances 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 230000032050 esterification Effects 0.000 abstract description 3
- 238000005886 esterification reaction Methods 0.000 abstract description 3
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 abstract description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract description 2
- 235000019581 fat taste sensations Nutrition 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract 3
- 239000000523 sample Substances 0.000 description 21
- 125000004432 carbon atom Chemical group C* 0.000 description 7
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 7
- 150000004670 unsaturated fatty acids Chemical group 0.000 description 7
- 239000012488 sample solution Substances 0.000 description 6
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 3
- 150000004671 saturated fatty acids Chemical group 0.000 description 3
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- UEEJHVSXFDXPFK-UHFFFAOYSA-N N-dimethylaminoethanol Chemical compound CN(C)CCO UEEJHVSXFDXPFK-UHFFFAOYSA-N 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- 235000021314 Palmitic acid Nutrition 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 229960002887 deanol Drugs 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 125000005456 glyceride group Chemical group 0.000 description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- 235000021313 oleic acid Nutrition 0.000 description 2
- 239000003760 tallow Substances 0.000 description 2
- DRCWOKJLSQUJPZ-DZGCQCFKSA-N (4ar,9as)-n-ethyl-1,4,9,9a-tetrahydrofluoren-4a-amine Chemical compound C1C2=CC=CC=C2[C@]2(NCC)[C@H]1CC=CC2 DRCWOKJLSQUJPZ-DZGCQCFKSA-N 0.000 description 1
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 235000019737 Animal fat Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- -1 Fatty acid compounds Chemical class 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 101001008429 Homo sapiens Nucleobindin-2 Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 239000005639 Lauric acid Substances 0.000 description 1
- 102100027441 Nucleobindin-2 Human genes 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000007368 endocrine function Effects 0.000 description 1
- 235000004626 essential fatty acids Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000021281 monounsaturated fatty acids Nutrition 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000005830 nonesterified fatty acids Chemical class 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- XDEPVFFKOVDUNO-UHFFFAOYSA-N pentafluorobenzyl bromide Chemical compound FC1=C(F)C(F)=C(CBr)C(F)=C1F XDEPVFFKOVDUNO-UHFFFAOYSA-N 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000010499 rapseed oil Substances 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及脂肪酸检测技术领域,具体地说,涉及一种血清中脂肪酸质的提取方法主要包括包含以下步骤:取血清样本,并对血清样本进行预处理;血清样本预处理完成后制得提取液,在提取液中加入酸催化剂,水浴加热;加热完成后,加入正己烷和生理盐水,涡旋振荡混匀后离心取上层清液,氮气吹干后,复溶;复溶后液体加入衍生化试剂,在密封反应系统中加热后,振荡摇匀,离心去上层清液收集;取离心后上清液置于C18反相色谱柱中进行色谱分析。该发明有效的提高了现有技术在对脂肪酸提取检测的不足,将甲酯化和HPLC‑MS检测技术相结合,大大提高血液中游离脂肪酸的提取和检测精度,操作流程简单易操作,提高脂肪酸的检测提取效率。
Description
技术领域
本发明涉及儿脂肪酸检测技术领域,具体地说,涉及一种血清中脂肪酸质的提取方法。
背景技术
脂肪酸是由碳、氢、氧三种元素组成的一类化合物,是中性脂肪、磷脂和糖脂的主要成分。
脂肪酸可分成两类:一类是分子内不带碳碳双键的饱和脂肪酸,如硬脂酸、软脂酸等;另一类是分子内带有一个或几个碳碳双键的不饱和脂肪酸,最常见的有油酸,油酸的碳链中只有一个碳碳双键,所以又叫单不饱和脂肪酸。一般脂肪酸化合物的碳链都较短,其长度一般在18-36个碳原子,最少的就是12个碳原子,如月桂酸。不管饱和的或不饱和的,生物体内脂肪酸的碳原子数大多是偶数,极少含有奇数碳原子,尤其是在高等动植物体内主要存在12碳以上的高级脂肪酸,一般在14-24个碳,以16和18碳脂肪酸最为常见。奇数碳原子脂肪酸仅在一些植物、反刍动物、海洋生物、石油酵母等体内部分存在。
含有多量饱和脂肪酸的甘油i酯在常温时往往是固体,例如牛油、羊油等,大多属动物脂肪。含有较多不饱和脂肪酸的甘油三酯在常温时往往是液体,例如玉米油、菜油等。植物和鱼类的油大多是不饱和脂肪酸的甘油酯。动物体内不能合成带有2-4个双键的不饱和脂肪酸,必须从食物中取得,因而这些脂肪酸就叫必需脂肪酸,也有人叫它维生素F。虽然已认为它们能降低血液中的胆固醇,但还没有证据能证明人会因为食物中缺乏这些脂肪酸而引起疾病。微生物中也含有不饱和脂肪酸,蓝细菌最独特之处是含有两个或多个双键组成的不饱和脂肪酸,而细菌通常只含有饱和脂肪酸和一个双键的不饱和脂肪酸。
游离脂肪酸又称非酯化脂肪酸(nonestesterified fatty acid NEFA),血清中含量很少,如用小量血清标本测定必须采用灵敏的方法,并要避免脂肪水解产生的脂肪酸的干扰。NEFA是由油酸,软脂酸,亚油酸等组成,大部分游离脂肪酸与白蛋白结合,存在于血液中。血清中游离脂肪酸的浓度与脂类代谢、糖代谢、内分泌功能有关,游离脂肪酸的浓度会因为糖尿病、重症肝障碍、甲状腺功能亢进等疾病而上升。
在人体血液中脂肪酸已经是反映机体状态的重要指标,现如今对血清中脂肪酸含量检测技术在实际使用中存在较大不足,由于检测过程中未对脂肪酸做甲酯化处理,使得最终脂肪酸检测结果精度较差,同时采用传统的气相色谱-质谱联用方法由于检测过程温度较高对于部分脂肪酸检测结果较差。
发明内容
本发明的目的在于提供一种血清中脂肪酸质的提取方法,以解决上述背景技术中提出的问题。
一种血清中脂肪酸质的提取方法,包含以下步骤:
步骤一:取血清样本,并对血清样本进行预处理;
步骤二:血清样本预处理完成后制得提取液,在提取液中加入酸催化剂,水浴加热;
步骤三:加热完成后,加入正己烷和生理盐水,涡旋振荡混匀后离心取上层清液,氮气吹干后,复溶;
步骤四:复溶后液体加入衍生化试剂,在密封反应系统中加热后,振荡摇匀,离心去上层清液收集;
步骤五:取离心后上清液置于C18反相色谱柱中进行色谱分析。
优选的,所述步骤一血清样本预处理方法具体为在血清样本中加入提取剂后在4℃下以3500r/min速度离心10-15min后取上层清液留样制得提取液。
优选的,所述提取剂为氯仿与甲醇以1∶3比例混合的混合溶液,且所述提取剂与血清样本的混合比为1-1.5∶1。
优选的,所述步骤二中酸催化剂具体为硫酸-甲醇(H2SO4/MeOH)溶液,所述酸催化剂与提取液的混合比为1∶6-10。
优选的,所述步骤二水浴加热温度为60-90℃,加热时间1-2h。
优选的,所述步骤三加热完成后液体与正己烷和生理盐水的调配比为1∶1∶2,所述涡旋振荡时间为30-60s,并在4℃下以3000r/min离心10-15min。
优选的,所述步骤三中氮气流速为2-4ml/min,吹干时间为3-5min,且吹干后复溶液为正己烷,所述复溶液与提取液剂量相同。
优选的,所述步骤四中衍生化试剂具体为氨甲基吡啶衍生化试剂,所述衍生化试剂与复溶液的混合比为0.1-0.3∶1。
优选的,所述步骤四中密封反应系统温度为80-90℃反应时间为10-15min,反应后振荡摇匀以5000-6000r/min速度离心5-10min后取上清液。
优选的,所述步骤五中检测流动相为甲醇/乙腈/水以60∶30∶10比例混合的混合液,柱温为20-25℃,流速在100-120ml/min之间。
与现有技术相比,本发明的有益效果:通过利用酸催化剂结合将脂肪酸先进行甲酯化,并且衍生化试剂采用二甲氨基乙醇,氨甲基吡啶衍经过试验显示相较于以往报道的二甲氨基乙醇、五氟苯甲基溴等试剂具有更好的质子亲和效率,结合HPLC-MS检测灵敏度显著提高,且该试剂绿色环保实用价值高,并且通过使用HPLC-MS进行检测避免了传统气相色谱-质谱联用方法中反应过程温度过高的问题。
具体实施方式
本发明公开了一种血清中脂肪酸质的提取方法,以下通过具体实施例对本发明作进一步详述。
实施例1
一种血清中脂肪酸质的提取方法,包含以下步骤:
步骤一:取脂肪酸样本溶液,并对脂肪酸样本溶液进行预处理;
步骤二:脂肪酸样本溶液预处理完成后制得提取液,在提取液中加入酸催化剂,水浴加热;
步骤三:加热完成后,加入正己烷和生理盐水,涡旋振荡混匀后离心取上层清液,氮气吹干后,复溶;
步骤四:复溶后液体加入衍生化试剂,在密封反应系统中加热后,振荡摇匀,离心去上层清液收集;
步骤五:取离心后上清液置于C18反相色谱柱中进行色谱分析。
进一步的,步骤一脂肪酸样本溶液预处理方法具体为在脂肪酸样本溶液中加入提取剂后在4℃下以3500r/min速度离心10min后取上层清液留样制得提取液,提取剂为氯仿与甲醇以1∶3比例混合的混合溶液,且提取剂与血清样本的混合比为1∶1。
进一步的,步骤二中酸催化剂具体为硫酸-甲醇(H2SO4/MeOH)溶液,酸催化剂与提取液的混合比为1∶6,步骤二水浴加热温度为60℃,加热时间1h。
进一步的,步骤三加热完成后液体与正己烷和生理盐水的调配比为1∶1∶2,涡旋振荡时间为30s,并在4℃下以3000r/min离心10min,步骤三中氮气流速为2ml/min,吹干时间为3min,且吹干后复溶液为正己烷,复溶液与提取液剂量相同。
进一步的,步骤四中衍生化试剂具体为氨甲基吡啶衍生化试剂,衍生化试剂与复溶液的混合比为0.1∶1,步骤四中密封反应系统温度为80℃反应时间为10min,反应后振荡摇匀以5000r/min速度离心5min后取上清液。
进一步的,步骤五中检测流动相为甲醇/乙腈/水以60∶30∶10比例混合的混合液,柱温为20℃,流速在100ml/min之间。
实施例2
一种血清中脂肪酸质的提取方法,包含以下步骤:
步骤一:取血清样本,并对血清样本进行预处理;
步骤二:血清样本预处理完成后制得提取液,在提取液中加入酸催化剂,水浴加热;
步骤三:加热完成后,加入正己烷和生理盐水,涡旋振荡混匀后离心取上层清液,氮气吹干后,复溶;
步骤四:复溶后液体加入衍生化试剂,在密封反应系统中加热后,振荡摇匀,离心去上层清液收集;
步骤五:取离心后上清液置于C18反相色谱柱中进行色谱分析。
进一步的,步骤一血清样本预处理方法具体为在血清样本中加入提取剂后在4℃下以3500r/min速度离心10min后取上层清液留样制得提取液,提取剂为氯仿与甲醇以1∶3比例混合的混合溶液,且提取剂与血清样本的混合比为1∶1。
进一步的,步骤二中酸催化剂具体为硫酸-甲醇(H2SO4/MeOH)溶液,酸催化剂与提取液的混合比为1∶6,步骤二水浴加热温度为60℃,加热时间1h。
进一步的,步骤三加热完成后液体与正己烷和生理盐水的调配比为1∶1∶2,涡旋振荡时间为30s,并在4℃下以3000r/min离心10min,步骤三中氮气流速为2ml/min,吹干时间为3min,且吹干后复溶液为正己烷,复溶液与提取液剂量相同。
进一步的,步骤四中衍生化试剂具体为氨甲基吡啶衍生化试剂,衍生化试剂与复溶液的混合比为0.1∶1,步骤四中密封反应系统温度为80℃反应时间为10min,反应后振荡摇匀以5000r/min速度离心5min后取上清液。
进一步的,步骤五中检测流动相为甲醇/乙腈/水以60∶30∶10比例混合的混合液,柱温为20℃,流速在100ml/min之间。
实施例3
一种血清中脂肪酸质的提取方法,包含以下步骤:
步骤一:取血清样本,并对血清样本进行预处理;
步骤二:血清样本预处理完成后制得提取液,在提取液中加入酸催化剂,水浴加热;
步骤三:加热完成后,加入正己烷和生理盐水,涡旋振荡混匀后离心取上层清液,氮气吹干后,复溶;
步骤四:复溶后液体加入衍生化试剂,在密封反应系统中加热后,振荡摇匀,离心去上层清液收集;
步骤五:取离心后上清液置于C18反相色谱柱中进行色谱分析。
进一步的,步骤一血清样本预处理方法具体为在血清样本中加入提取剂后在4℃下以3500r/min速度离心15min后取上层清液留样制得提取液,提取剂为氯仿与甲醇以1∶3比例混合的混合溶液,且提取剂与血清样本的混合比为1.5∶1。
进一步的,步骤二中酸催化剂具体为硫酸-甲醇(H2SO4/MeOH)溶液,酸催化剂与提取液的混合比为1∶10,步骤二水浴加热温度为90℃,加热时间2h。
进一步的,步骤三加热完成后液体与正己烷和生理盐水的调配比为1∶1∶2,涡旋振荡时间为60s,并在4℃下以3000r/min离心15min步骤三中氮气流速为4ml/min,吹干时间为5min,且吹干后复溶液为正己烷,复溶液与提取液剂量相同。
进一步的,步骤四中衍生化试剂具体为氨甲基吡啶衍生化试剂,衍生化试剂与复溶液的混合比为0.3∶1,步骤四中密封反应系统温度为90℃反应时间为15min,反应后振荡摇匀以6000r/min速度离心10min后取上清液。
进一步的,步骤五中检测流动相为甲醇/乙腈/水以60∶30∶10比例混合的混合液,柱温为25℃,流速在120ml/min之间。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的仅为本发明的优选例,并不用来限制本发明,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (10)
1.一种血清中脂肪酸质的提取方法,其特征在于:包含以下步骤:
步骤一:取血清样本,并对血清样本进行预处理;
步骤二:血清样本预处理完成后制得提取液,在提取液中加入酸催化剂,水浴加热;
步骤三:加热完成后,加入正己烷和生理盐水,涡旋振荡混匀后离心取上层清液,氮气吹干后,复溶;
步骤四:复溶后液体加入衍生化试剂,在密封反应系统中加热后,振荡摇匀,离心去上层清液收集;
步骤五:取离心后上清液置于C18反相色谱柱中进行色谱分析。
2.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤一血清样本预处理方法具体为在血清样本中加入提取剂后在4℃下以3500r/min速度离心10-15min后取上层清液留样制得提取液。
3.根据权利要求2所述的一种血清中脂肪酸质的提取方法,其特征在于:所述提取剂为氯仿与甲醇以1∶3比例混合的混合溶液,且所述提取剂与血清样本的混合比为1-1.5∶1。
4.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤二中酸催化剂具体为硫酸-甲醇(H2SO4/MeOH)溶液,所述酸催化剂与提取液的混合比为1∶6-10。
5.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤二水浴加热温度为60-90℃,加热时间1-2h。
6.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤三加热完成后液体与正己烷和生理盐水的调配比为1∶1∶2,所述涡旋振荡时间为30-60s,并在4℃下以3000r/min离心10-15min。
7.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤三中氮气流速为2-4ml/min,吹干时间为3-5min,且吹干后复溶液为正己烷,所述复溶液与提取液剂量相同。
8.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤四中衍生化试剂具体为氨甲基吡啶衍生化试剂,所述衍生化试剂与复溶液的混合比为0.1-0.3∶1。
9.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤四中密封反应系统温度为80-90℃反应时间为10-15min,反应后振荡摇匀以5000-6000r/min速度离心5-10min后取上清液。
10.根据权利要求1所述的一种血清中脂肪酸质的提取方法,其特征在于:所述步骤五中检测流动相为甲醇/乙腈/水以60∶30∶10比例混合的混合液,柱温为20-25℃,流速在100-120ml/min之间。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011146555.9A CN112345672A (zh) | 2020-10-22 | 2020-10-22 | 一种血清中脂肪酸质的提取方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011146555.9A CN112345672A (zh) | 2020-10-22 | 2020-10-22 | 一种血清中脂肪酸质的提取方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112345672A true CN112345672A (zh) | 2021-02-09 |
Family
ID=74359988
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011146555.9A Pending CN112345672A (zh) | 2020-10-22 | 2020-10-22 | 一种血清中脂肪酸质的提取方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112345672A (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102565270A (zh) * | 2012-01-05 | 2012-07-11 | 武汉工业学院 | 3-氯-1,2-丙二醇及其脂肪酸酯含量的检测方法 |
CN105445407A (zh) * | 2015-12-29 | 2016-03-30 | 成都普思生物科技股份有限公司 | 一种山桐子中脂肪酸和维生素e的检测方法 |
KR20180100915A (ko) * | 2017-03-03 | 2018-09-12 | 주식회사 에스씨엘헬스케어 | 민감도 및 특이성이 향상된 지방산 분석 방법 |
WO2020097593A1 (en) * | 2018-11-09 | 2020-05-14 | Nx Prenatal Inc. | Tandem-paired column chemistry for high-throughput proteomic exosome analysis |
-
2020
- 2020-10-22 CN CN202011146555.9A patent/CN112345672A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102565270A (zh) * | 2012-01-05 | 2012-07-11 | 武汉工业学院 | 3-氯-1,2-丙二醇及其脂肪酸酯含量的检测方法 |
CN105445407A (zh) * | 2015-12-29 | 2016-03-30 | 成都普思生物科技股份有限公司 | 一种山桐子中脂肪酸和维生素e的检测方法 |
KR20180100915A (ko) * | 2017-03-03 | 2018-09-12 | 주식회사 에스씨엘헬스케어 | 민감도 및 특이성이 향상된 지방산 분석 방법 |
WO2020097593A1 (en) * | 2018-11-09 | 2020-05-14 | Nx Prenatal Inc. | Tandem-paired column chemistry for high-throughput proteomic exosome analysis |
Non-Patent Citations (6)
Title |
---|
JOHN WILLIAMS等: "Fit-for-purpose biomarker LC-MS/MS qualification for the quantitation of very long chain fatty acids in human cerebrospinal fluid", 《BIOANALYSIS》 * |
MARTA I. AVELDANO等: "Quantitative release of fatty acids from lipids by a simple hydrolysis procedure", 《JOURNAL OF LIPID RESEARCH》 * |
PATRICK AUBOURG等: "Capillary gas-liquid chromatographic-mass spectrometric measurement of very long chain (C22 to C26) fatty acids in microliter samples of plasma", 《JOURNAL OF LIPID RESEARCH》 * |
XINGNAN LI等: "Improved LC-MS Method for the Determination of Fatty Acids in Red Blood Cells by LC-Orbitrap MS", 《ANALYTICAL CHEMISTRY》 * |
杨秀娟等: "基于超高效液相色谱-四极杆飞行时间质谱联用技术的血瘀模型大鼠血浆代谢组学分析", 《色谱》 * |
高永平等: "柱前衍生结合高效液相色谱-荧光检测法测定人体血浆中脂肪酸", 《分析科学学报》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Namal Senanayake et al. | Enzymatic incorporation of docosahexaenoic acid into borage oil | |
CN104651422B (zh) | 一种从深海鱼中提取甘油三酯型dha和epa的方法 | |
CN111088296B (zh) | 一种富集油脂中n-3多不饱和脂肪酸甘油酯的方法 | |
Irimescu et al. | Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols | |
Chen et al. | Synthesis of the structured lipid 1, 3-dioleoyl-2-palmitoylglycerol from palm oil | |
CN105087694B (zh) | 一种利用填充床反应器制备中长链甘油三酯的方法 | |
CN104186705A (zh) | 基于酶促酸解棕榈酸甘油三酯合成结构脂质的方法 | |
CN112280810A (zh) | 一种富含多不饱和脂肪酸的中长链甘油三酯的制备方法 | |
Antonio et al. | Biocatalytic ethanolysis of waste chicken fat for biodiesel production | |
Tavakoli et al. | Squid oil and fat production from squid wastes using subcritical water hydrolysis: free fatty acids and transesterification | |
Kurashige et al. | Enzymatic modification of canola/palm oil mixtures: Effects on the fluidity of the mixture | |
Prado et al. | Enzymatic hydrolysis of conjugated linoleic acid-enriched anhydrous milk fat in supercritical carbon dioxide | |
Huang et al. | Structure‐guided preparation of fuctional oil rich in 1, 3‐diacylglycerols and linoleic acid from Camellia oil by combi‐lipase | |
CN112266939B (zh) | 一种酶法富集油脂中多不饱和脂肪酸甘油酯的方法 | |
CN112592939B (zh) | 一种酶法富集n-3多不饱和脂肪酸的方法 | |
Kanjilal et al. | Synthesis and estimation of calorific value of a structured lipid-potential reduced calorie fat | |
CN112322669B (zh) | 一种提高n-3多不饱和脂肪酸甘油酯富集效率的方法 | |
Pedersen et al. | Studies of the Fatty Acid Specificity of the Lipase from Rhizomucor miehei Toward 20: 1n-9, 20: 5n-3, 22: 1n-9 and 22: 6n-3 | |
CN112345672A (zh) | 一种血清中脂肪酸质的提取方法 | |
Xu et al. | An Effective strategy for the production of lauric acid–enriched monoacylglycerol via enzymatic glycerolysis from black soldier fly (Hermetia illucens) larvae (BSFL) oil | |
Hoq et al. | Role of oleic acid solubilized in buffer—glycerol solution on adsorbed lipase during continuous hydrolysis of olive oil in a microporous hydrophobic membrane bioreactor | |
Sönnichsen et al. | A rapid and quantitative method for total fatty acid analysis of fungi and other biological samples | |
CN115651938A (zh) | 采用两步酶法生产高epa乙酯和高dha甘油酯的方法 | |
Shen et al. | Comparative analysis of DHA positional distribution and triacylglycerol molecular species in algal oil (Schizochytrium sp.) from different oil processing | |
Otero et al. | Enzymatic interesterification between pine seed oil and a hydrogenated fat to prepare semi-solid fats rich in pinolenic acid and other polyunsaturated fatty acids |
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: 20210209 |
|
RJ01 | Rejection of invention patent application after publication |