TY - JOUR
T1 - [72] Measurement of 5-Hydroxyeicosatetraenoic Acid (5-HETE) in Biological Fluids by GCMS
AU - Ogletree, Martin L.
AU - Schlesinger, Kenneth
AU - Nettleman, Mary
AU - Hubbard, Walter C.
N1 - Funding Information:
The authors thank Drs. J. M. Boeynaems and G. M. Bokoch for their advice in developing this assay and Dr. D. Taber for the generous gift of octadeuterated arachidonic acid. This work was supported by Grants GM 15431, HL 19153, and HL 26198 from the National Institutes of Health.
PY - 1982/1/1
Y1 - 1982/1/1
N2 - This chapter presents the highly sensitive and selective assay for 5-hydroxyeicosatetraenoic acid (5-HETE) employing racemic octadeuterated 5-HETE for stable isotope dilution measurements with quantitation via combined gas-liquid chromatography-mass spectrometry (GC–MS). Measurement of 5-HETE is employed as an index of 5-1ipoxygenation of arachidonic acid. Octadeuterated arachidonic acid is prepared from 5,8,11,14-eicosatetraynoic acid. Two independent methods are employed for standardization of ds-5- HETE, because the tendency of 5-HETE to form δ-lactone. Ultraviolet spectrophotometry at, λMeOHmax = 235 nm and gas–liquid chromatography with tricosanoic acid as internal standard should give agreement within ±5%. In purification by reversed-phase high performance liquid chromatography (HPLC), solvent is evaporated from the sample extract under a stream of nitrogen, and the residue is dissolved in a small volume of methanol for purification by reversed-phase HPLC. 5-HETE is eluted from μBondapak C18 column with a solvent of methanol-water -glacial acetic acid delivered at a flow rate of 1-2 ml/min.
AB - This chapter presents the highly sensitive and selective assay for 5-hydroxyeicosatetraenoic acid (5-HETE) employing racemic octadeuterated 5-HETE for stable isotope dilution measurements with quantitation via combined gas-liquid chromatography-mass spectrometry (GC–MS). Measurement of 5-HETE is employed as an index of 5-1ipoxygenation of arachidonic acid. Octadeuterated arachidonic acid is prepared from 5,8,11,14-eicosatetraynoic acid. Two independent methods are employed for standardization of ds-5- HETE, because the tendency of 5-HETE to form δ-lactone. Ultraviolet spectrophotometry at, λMeOHmax = 235 nm and gas–liquid chromatography with tricosanoic acid as internal standard should give agreement within ±5%. In purification by reversed-phase high performance liquid chromatography (HPLC), solvent is evaporated from the sample extract under a stream of nitrogen, and the residue is dissolved in a small volume of methanol for purification by reversed-phase HPLC. 5-HETE is eluted from μBondapak C18 column with a solvent of methanol-water -glacial acetic acid delivered at a flow rate of 1-2 ml/min.
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U2 - 10.1016/0076-6879(82)86232-0
DO - 10.1016/0076-6879(82)86232-0
M3 - Article
C2 - 7132773
AN - SCOPUS:0020012706
SN - 0076-6879
VL - 86
SP - 607
EP - 612
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -