Determination of free and total catecholamines in human urine by HPLC with fluorescence detection.
A simple and highly sensitive method for the determination of free and total (free + conjugated) catecholamines (norepinephrine, epinephrine and dopamine) in human urine is described which employs HPLC with fluorescence detection. Conjugated catecholamines (sulfate form) are hydrolyzed by a sulfatase-mediated reaction to the corresponding free amines. After cation exchange chromatography on a Toyopak IC-SP S cartridge, catecholamines and isoproterenol (internal standard) in urine samples were converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. These compounds were separated within 8 min on a reversed phase column with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris-hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca 2 fmol per 100 microL injection volume.
D000328:Adult; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004298:Dopamine / Q000097:blood / Q000652:urine*; D004837:Epinephrine / Q000097:blood / Q000652:urine*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009638:Norepinephrine / Q000097:blood / Q000652:urine*; D012016:Reference Values; D013050:Spectrometry, Fluorescence
D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine