Determination of catecholamines and related compounds in mouse urine using column-switching HPLC

Abstract

We have developed an analytical method for the determination of catecholamines and related compounds in mouse urine by column-switching HPLC. Selective extraction of the catechol compounds was performed using a precolumn modified with phenylboronic acid, which has a pH dependent affinity for the catechol structures. The pretreatment buffer, which facilitated binding of the catechols to the precolumn, was optimized to ensure high analyte recoveries and good peak shapes. We found that using the same acetonitrile content in the pretreatment buffer and hydrophilic interaction liquid chromatography mobile phase was necessary to improve peak shapes. Eight catechol compounds were selectively extracted and separated using 100 mmol L-1 ammonium formate/acetonitrile (20/80 v/v, pH 8.0) for the extraction step, and 20 mmol L-1 ammonium formate (pH 2.5)/acetonitrile (20/80 v/v) for elution and separation. Native fluorescence of the separated catechol compounds was monitored, and the limits of detection, corresponding to a signal to noise ratio of 3, were 9-58 nmol L-1. Five catechol compounds (dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxymandelic acid) were successfully quantified in mouse urine. Intra- and inter-day precisions were less than 10%, and performance was superior to that afforded by manual sample pretreatment.