Title:
FLUORESCENT HPLC ASSAY FOR 20-HETE AND OTHER P-450 METABOLITES OF ARACHIDONIC ACID
Document Type and Number:
WIPO Patent Application WO2001077674
Kind Code:
A3
Abstract:
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 4.5 X 250-mm, 5 mu M 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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Inventors:
ROMAN RICHARD J (US)
MAIER KRISTOPHER G (US)
MAIER KRISTOPHER G (US)
Application Number:
PCT/US2001/011321
Publication Date:
June 13, 2002
Filing Date:
April 06, 2001
Export Citation:
Assignee:
MCW RES FOUND INC (US)
ROMAN RICHARD J (US)
MAIER KRISTOPHER G (US)
ROMAN RICHARD J (US)
MAIER KRISTOPHER G (US)
International Classes:
G01N21/64; B01J20/281; G01N30/04; G01N30/06; G01N30/26; G01N30/34; G01N30/74; G01N30/86; G01N30/88; G01N33/92; G01N30/02; G01N30/62; (IPC1-7): G01N33/88
Other References:
MAIER K G ET AL: "A rapid, sensitive fluorescent HPLC assay for HETEs and EETs.", FASEB JOURNAL, vol. 14, no. 4, 15 March 2000 (2000-03-15), Annual Meeting of Professional Research Scientists: Experimental Biology 2000;San Diego, California, USA; April 15-18, 2000, pages A140, XP002195385, ISSN: 0892-6638
MAIER KRISTOPHER G ET AL: "Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid.", AMERICAN JOURNAL OF PHYSIOLOGY, vol. 279, no. 2 Part 2, August 2000 (2000-08-01), pages H863 - H871, XP001053692, ISSN: 0002-9513
YASAKA, YUTA ET AL: "2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate as a highly reactive ultraviolet and fluorescent labelling agent for the liquid chromatographic determination of carboxylic acids", J. CHROMATOGR. (1990), 508(1), 133-40, XP002195337
KISS LADISLAU ET AL: "Simultaneous analysis of 4- and 5-series lipoxygenase and cytochrome P450 products from different biological sources by reversed-phase high-performance liquid chromatographic technique.", ANALYTICAL BIOCHEMISTRY, vol. 261, no. 1, 15 July 1998 (1998-07-15), pages 16 - 28, XP002195386, ISSN: 0003-2697
MAIER KRISTOPHER G ET AL: "Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid.", AMERICAN JOURNAL OF PHYSIOLOGY, vol. 279, no. 2 Part 2, August 2000 (2000-08-01), pages H863 - H871, XP001053692, ISSN: 0002-9513
YASAKA, YUTA ET AL: "2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate as a highly reactive ultraviolet and fluorescent labelling agent for the liquid chromatographic determination of carboxylic acids", J. CHROMATOGR. (1990), 508(1), 133-40, XP002195337
KISS LADISLAU ET AL: "Simultaneous analysis of 4- and 5-series lipoxygenase and cytochrome P450 products from different biological sources by reversed-phase high-performance liquid chromatographic technique.", ANALYTICAL BIOCHEMISTRY, vol. 261, no. 1, 15 July 1998 (1998-07-15), pages 16 - 28, XP002195386, ISSN: 0003-2697
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