An automated direct-injection HPLC-method for the electrochemical/fluorimetric quantitation of monoamines and related compounds optimized for the screening of large numbers of animals.

An automated, highly stable HPLC system is described allowing the quantitation of monoamines and related compounds with virtually online data processing and statistical evaluation of the mean values of various differently treated groups of animals. The system is highly suited to the screening of drugs involving large numbers of samples. To ensure long-term, uninterrupted performance, two robust chromatographic systems were developed optimized to separate neighboring peaks as widely as possible. This was achieved by using mobile phases of relatively low pH to retard acidic compounds, and optimal concentrations of the ion-pairing reagent to manipulate the retention times of amines on the RP-18 column, resulting also in clearcut separations from the solvent/tissue peak. Direct injection of deproteinized tissue samples, requiring no clean up procedures is used. One faster system allows measurement of the main biogenic amines, metabolites and TRP, whereas another system allows the simultaneous quantitation of DOPA, 5HTP, NA, MOPEG, NMN, A, MN, VMA, DA, 3MT, DOPAC, HVA, 5HT, 5HIAA and TYR. By pretreatment of animals with a decarboxylase inhibitor the latter system offers the possibility of detecting drug effects in the same animal on either dopamine and serotonin turnover or noradrenaline and serotonin turnover, depending on the brain area chosen. The system described has been running for over a year without major disturbance and with minimal technician attendance.