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.
Slingerland
R J
RJ
Academic Medical Center, University of Amsterdam, Department of Pediatrics, The Netherlands.
Van Kuilenburg
A B
AB
Bodlaender
J M
JM
Overmars
H
H
Voûte
P A
PA
Van Gennip
A H
AH
D000818:Animals; D015306:Biogenic Monoamines / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017077:Culture Media, Conditioned / Q000737:chemistry*; D003846:Deoxyepinephrine / Q000032:analysis; D003956:Dialysis; D004295:Dihydroxyphenylalanine / Q000032:analysis / Q000378:metabolism; D004298:Dopamine / Q000032:analysis; D004355:Drug Stability; D004837:Epinephrine / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D009638:Norepinephrine / Q000032:analysis; D016716:PC12 Cells / Q000378:metabolism*; D051381:Rats
D015306:Biogenic Monoamines; D017077:Culture Media, Conditioned; D004295:Dihydroxyphenylalanine; D003846:Deoxyepinephrine; D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine