Determination of catecholamines in pheochromocytoma cell (PC-12) culture medium by microdialysis-microbore liquid chromatography

An in vitro microdialysis system was constructed for the measurement of cat echolamines in pheochromocytoma cell culture medium. The novel microdialysi s device is composed of a petri dish, a dialysis membrane and two transmiss ion tubes. The dialysis membrane is located in the space of a petri dish su ch that it is immersed in the culture medium. Catecholamines contained in t he culture medium diffused into a designed dialysis membrane with sufficien t recovery (about 60%). Dialysates were collected by a sampling loop and in troduced by an on-line injector to a microbore liquid chromatographic syste m for analysis of catecholamines. This assay yielded a detection Limit of 0 .2-0.5 pg/injection with acceptable intra- and inter-assay reproducibilitie s in 5 mu l of dialysates. To evaluate the on-line microdialysis system, PC -12 cells were cultured in a petri dish within an incubator. The baseline c oncentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyan ide. In conclusion, the present microassay provides for the sensitive, dire ct measurement of catecholamines in culture medium while minimizing pretrea tment procedures for sample preparation. (C) 2000 Elsevier Science B.V. All rights reserved.