Fluorescent hplc assay for 20-hete and other p-450 metabolites of arachidonic acid

A method for analyzing P-450 metabolites of arachidonic acid in a biological sample, comprising the steps of:

• adding an internal standard into the sample;
• extracting P-450 metabolites of arachidonic acid and the added internal standard from the sample;
• labeling P-450 metabolites of arachidonic acid and the internal standard with a fluorescent material;
• loading P-450 metabolites of arachidonic acid and the internal standard, both of which are labeled with the fluorescent material, onto a 4.5×250-mm, 5 μM particle size C18 reverse-phase HPLC column;
• passing a mobile phase of methanol:water:acetic acid with a volume ratio of about 82:18:0.1 through the column isocratically at a rate of about 1.3 ml per minute;
• monitoring fluorescence intensity of eluent;
• and
• quantitating P-450 metabolites of arachidonic acid by calculating the peak ratio of a metabolite to the internal standard.

A method for clinically diagnosing of a disease associated with abnormalities in arachidonic acid metabolism, comprising the step of:

• analyzing P-450 metabolites of arachidonic acid from a sample of a human being comprising the steps of:
• extracting P-450 metabolites of arachidonic acid from the sample;
• labeling P-450 metabolites of arachidonic acid with a fluorescent material;
• loading P-450 metabolites of arachidonic acid labeled with the fluorescent material onto a 4.5×250-mm, 5 μM particle size C18 reverse-phase HPLC column;
• passing a mobile phase of methanol:water:acetic acid with a volume ratio of about 82:18:0.1 through the column isocratically at a rate of about 1.3 ml per minute;
• and
• monitoring fluorescence intensity of eluent.

A method for clinically diagnosing of a disease associated with abnormalities in arachidonic acid metabolism, comprising the step of:

• analyzing P-450 metabolites of arachidonic acid from a sample of a human being comprising the steps of:
• adding an internal standard into the sample;
• extracting P-450 metabolites of arachidonic acid and the added internal standard from the sample;
• labeling P-450 metabolites of arachidonic acid and the internal standard with a fluorescent material;
• loading P-450 metabolites of arachidonic acid and the internal standard, both of which are labeled with the fluorescent material, onto a 4.5×250-mm, 5 μM particle size C18 reverse-phase HPLC column;
• passing a mobile phase of methanol:water:acetic acid with a volume ratio of about 82:18:0.1 through the column isocratically at a rate of about 1.3 ml per minute;
• monitoring fluorescence intensity of eluent;
• and
• quantitating P-450 metabolites of arachidonic acid by calculating the peak ratio of a metabolite to the internal standard.