Determination of urinary free noradrenaline by reversed-phase high-performance liquid chromatography with on-line extraction and fluorescence derivatization.

A fully automated reversed-phase high-performance liquid chromatographic method with fluorimetric detection for the determination of urinary free noradrenaline was developed. Urine samples, diluted with buffer were injected into a boric acid gel column (12 microns, TSK: 10 x 4.6 mm I.D.) without prior extraction. Urinary noradrenaline was simultaneously extracted and derivatized with an alkaline mobile phase of pH 11, containing o-phthalaldehyde and 2-mercaptoethanol, in a boric acid gel column. After switching columns, the fluorescent derivatized catecholamines were separated with an ODS-4PW column (TSK) and a mobile phase of pH 2 and the fluorescence was monitored with excitation at 340 and emission at 440 nm. The retention times of noradrenaline and dopamine were 11.0 and 14.2 min, respectively. The detection limits for noradrenaline and dopamine were 0.2 and 20 ng, respectively. This method has the advantages of not requiring preliminary extraction of urinary catecholamines, high sensitivity and stability of o-phthalaldehyde-derivatized catecholamines.