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 catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2-0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 microl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration 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 cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.
Cheng
F C
FC
Department of Medical Research, Taichung Veterans General Hospital, Taiwan. c1035@vghtc.vghtc.gov.tw
Kuo
J S
JS
Huang
H M
HM
Yang
D Y
DY
Wu
T F
TF
Tsai
T H
TH
D000818:Animals; D002395:Catecholamines / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D004563:Electrochemistry; D017551:Microdialysis; D016716:PC12 Cells; D051381:Rats
D002395:Catecholamines; D003470:Culture Media