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FLUORESCENT HPLC ASSAY FOR 20-HETE AND OTHER P-450 METABOLITES OF ARACHIDONIC ACID
FLUORESCENT HPLC ASSAY FOR 20-HETE AND OTHER P-450 METABOLITES OF ARACHIDONIC ACID
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机译:花生四烯酸20-Hote和其他P-450代谢物的荧光HPLC测定
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摘要
The present invention provides a fluorescent HPLC assay for detecting the presence and/or measuring the level of 20-hydroxyeicosatetraenoic acid (20-HETE) and other P-450 metabolites of arachidonic acid in a sample. P-450 metabolites of arachidonic acid are first extracted from the sample and then labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. The labeling reaction is catalyzed by N,N-diisopropylethylamine. Next, the labeled P-450 metabolites are separated on a 4.5x250-mm, 5 muM particle size C18 reverse-phase HPLC column using a mobile phase of methanol:water:acetic acid (82:18:0.1, v/v/v) and an isocratic elution at a rate of about 1.3 ml per minute. Fluorescence intensities of the column eluent are monitored by a fluorescence detector. Quantitation of P-450 metabolites in a sample can be made by using an internal standard.
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机译:本发明提供了一种荧光HPLC测定法,用于检测样品中花生四烯酸的20-羟基二十碳四烯酸(20-HETE)和其他P-450代谢物的存在和/或水平。首先从样品中提取花生四烯酸的P-450代谢物,然后用2-(2,3-萘二亚氨基)乙基三氟甲磺酸盐标记。标记反应由N,N-二异丙基乙胺催化。接下来,使用甲醇:水:乙酸(82:18:0.1,v / v / v)的流动相在4.5x250 mm,5μM粒径C18反相HPLC色谱柱上分离标记的P-450代谢物)和等度洗脱,流速约为每分钟1.3 ml。柱洗脱液的荧光强度由荧光检测器监测。样品中P-450代谢物的定量可以使用内标物进行。
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