Optimization of high-performance liquid chromatographic assay for catecholamines. Determination of optimal mobile phase composition and elimination of species-dependent differences in extraction recovery of 3,4-dihydroxybenzylamine.

This paper describes the application of a window diagram technique to optimize the four components of eluent (sodium acetate, sodium heptanesulfonate, acetonitrile and pH adjusted by monochloroacetic acid), for complete separation of five catecholamine compounds and the internal standard (3,4-dihydroxybenzylamine, DHBA). In addition, studies were performed to address the problem of the variable recovery of DHBA from dog plasma due to a time-dependent loss of DHBA. We found that this phenomenon can be prevented by pH adjustment prior to addition of DHBA, allowing development of an accurate high-performance liquid chromatographic assay for plasma catecholamines in dogs.