High-performance liquid chromatographic analysis of biogenic amines in cells and in culture media using on-line dialysis and trace enrichment.

A highly sensitive method is presented for the automatic quantitative detection of DOPA metabolites in low concentrations in cells derived from the neural crest using reversed-phase HPLC in combination with fluorescence and electrochemical detection. The HPLC system was combined with on-line dialysis and on-line trace enrichment for the detection of small quantities of DOPA metabolites in culture media. Parameters like detector settings, pH, dialysis time and flow-rates are evaluated and optimized. Static-continuous dialysis can be performed at a low flow-rate concomitant with a high dialysis efficiency (up to >65%) depending on the type of DOPA metabolite. Counterflow dialysis can be used to analyse, with low efficiencies (17-29%), samples consisting of large volumes. Samples containing up to at least 7% (w/v) protein can be analysed in the low flow-rate static-continuous mode. In this last mode of dialysis, limits of detection for dopamine, norepinephrine, epinephrine and n-methyldopamine in DMEM/HAMF12 medium samples are 100 fmol or even lower. Serotonin is detectable at 10 fmol at a signal/noise ratio of 3. Biogenic amines were detectable at a concentration of 10 fmol/microl in a volume of 100 microl medium with an intra- and inter-assay imprecision <6.4%. This method is applied to study the differentiation level of tumour cells in culture and slices of a tumour derived from the neural crest. With this system, we also detected the excretion of DOPA metabolites from PC-12 cells after treatment with prenylamine.