Determination of intracellular dopamine by liquid chromatography-fluorescence detection with post-column derivatization using the Konig reaction

Dopamine is an important neurotransmitter, and the disruption of dopaminergic homeostasis causes various neurological diseases such as Parkinson's disease. Analysis of intracellular dopamine levels is important to understand the pathology of neurological diseases. We have developed a new method for the fluorometric detection of dopamine by adopting the Konig reaction, which is commonly used for the detection of cyanide, thiocyanate, and selenocyanate, and demonstrated that it can be applied to the determination of intracellular dopamine levels. The present method only requires a conventional LC system with isocratic elution and post-column derivatization and is simple to perform. The LOD, LOQ, and linearity range were 10.8 nM, 32.8 nM, and 0.05-10 mu M, respectively, with accuracies of 101.8-106.3 % and precisions within 5 %, which are sufficient for the quantification of intracellular dopamine. We also determined dopamine levels in PC12 cells and found that the levels increased and decreased when the cells were exposed to L-dopa and cyanide, respectively, possibly because of the conversion of L-dopa into dopamine and the depletion of intracellular dopamine by exposing cells to cyanide, respectively. These results suggest the applicability of the present method, and that this new use of the Konig reaction offers a reliable and useful means of quantifying intracellular dopamine.