高效液相色谱-电喷雾串联四级杆质谱法检测小鼠组织中花生四烯酸及其代谢产物  

Determination of Arachidonic Acid and Its Endogenous Eicosanoid Metabolites in Mice Tissues Using Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry

作　　者：谷艳[1,2] 石先哲[1] 赵素敏[1] 顾群[1] 路鑫[1] 许国旺[1] 

机构地区：[1]中国科学院大连化学物理研究所,中国科学院分离分析化学重点实验室,大连116023 [2]沈阳药科大学药学院,沈阳110016

出　　处：《分析化学》2010年第8期1089-1094,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.20835006;20675082;20805045)资助

摘　　要：采用反相高效液相色谱梯度洗脱,电喷雾串联质谱多反应检测模式,同时分离测定小鼠组织中花生四烯酸类代谢产物。组织样品经低温超声破碎细胞,采用乙酸乙酯进行两次萃取,LC-MS/MS检测。实验用3个内标,标准溶液稀释法建立了19种花生四烯酸类物质的标准曲线。方法学确证表明:各被测物质的线性相关系数均大于0.99;定量限(LOQ)为3～250μg/L;相对回收率在70.7%～123.0%之间;方法重复性(RSD)均小于20%。运用本法在小鼠肺、肝、脾组织中检测到了几种花生四烯酸类代谢产物,并对肺组织里的8种代谢产物进行了含量测定,结果为11-羟基二十碳四烯酸(58.3±11.6)ng/g,15-羟基二十碳四烯酸(52.6±15.5)ng/g,前列腺素E2(210±102)ng/g,前列腺素D2(20.3±13.9)ng/g,前列腺素J2(39.2±15.9)ng/g,血栓素B2(58.3±28.4)ng/g,前列腺素F2α(3.8±6.0)ng/g,花生四烯酸(1980±544)ng/g。本方法灵敏度高、重复性良好,为研究生物组织样品中花生四烯酸类物质的代谢状况提供了分析基础。A reversed phase liquid chromatography/tandem mass spectrometry method was developed to simultaneously analyze arachidonic acid（AA） and its metabolites such as prostaglandins（PGs）,thromboxane（TXB2）,epoxyeicosatrienoic acids（EETs）,dihydroxyeicosatrienoic acids（DHETs） and hydroxyeicosatetraenoic acids（HETEs） through the corresponding cyclooxygenase,cytochrome P450,and lipoxygenase pathways in mice tissues. Ethyl acetate extraction was employed for sample preparation after sonication in an ice bath,and the organic phase was dried under vacuum and dissolved in methanol for the LC-MS/MS analysis. 22 standard compounds were separated within 50 min using a gradient elution of acetonitrile and water with 0.05% formic acid（V/V） on a C18 column（250 mm×4.6 mm,5 μm）. PGB1 and deuterium labeled 15-HETE-d8 and AA-d8 were used as internal standards. Mass spectrometric detection was performed using negative electrospray ionization and multiple reaction monitoring. The method was validated and shown to be accurate（recovery values 70.7%-123.0%）,precise（RSD 20%） and sensitive（LOQ 3-250 μg/L）. The method was applied to separate arachidonic acid（AA） and its metabolites in lung,liver and spleen tissues in mice and quantify eicosanoid levels in lung tissue. The results were 11-HETE（58.3±11.6） ng/g,15-HETE（52.6±15.5） ng/g,PGE2（210±102） ng/g,PGD2（20.3±13.9） ng/g,PGJ2（39.2±15.9） ng/g,TXB2（58.3±28.4） ng/g,PGF2α（3.8±6.0） ng/g and AA（1980±544） ng/g,respectively.

关 键 词：高效液相色谱-串联质谱 花生四烯酸 组织 

分 类 号：S865.13[农业科学—野生动物驯养]