Determination of urinary vanillylmandelic acid by direct injection and coupled-column chromatography with electrochemical detection

An automated column-switching system for determination of vanillylmandelic acid in urine is described. The liquid chromatographic system was composed of two separation columns with different selectivity properties, an octadecyl column coated with tributyl phosphate as stationary liquid phase and a silica-based anion exchanger. Urine samples were injected directly onto the first column, where vanillylmandelic acid was separated from the main part of the sample matrix. The internal standard isovanillylmandelic acid was co-eluting with vanillylmandelic acid, and a fraction of the eluate containing both substances was switched to the second column, where separation was performed. To assess peak purity, detection was performed with dual working electrodes in parallel mode. A relative standard deviation of 3.5% was obtained for determination of human urine samples containing 3 microM vanillylmandelic acid, and less than 0.1 microM could be detected.