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Determination of free and total catecholamines in human urine by HPLC with fluorescence detection
Author(s) -
Nohta Hitoshi,
Mitsui Akane,
Umegae Yoshihiko,
Ohkura Yosuke
Publication year - 1987
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130020104
Subject(s) - chemistry , chromatography , detection limit , high performance liquid chromatography , elution , fluorescence , urine , hydrochloric acid , hydrolysis , organic chemistry , biochemistry , physics , quantum mechanics
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‐SPS 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 μL injection volume.