An efficient and label-free LC-MS/MS method for assessing drug's activity at dopamine and serotonin transporters using transporter-transfected HEK293T cells.

Abstract

Dopamine transporter (DAT) and serotonin transporter (SERT) are targets for many psychoactive substances. Functional assays including uptake inhibition and release assays often involve radiolabeled compounds like [3H]-dopamine and [3H]-serotonin to assess drug activity at transporters, which have high requirements on handling radioactive samples. The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT. A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established using transporter-transfected human embryonic kidney 293T (HEK293T) cells. This method was evaluated by testing the effects of amphetamine and cocaine in the assay procedure. The limits of detection of this method were 0.2 nM for both dopamine (DA) and serotonin (5-HT), with good linearities in the range of 0.5-160 nM. Amphetamine and cocaine's IC50 and EC50 on DAT and SERT determined by this method were consistent with previous reports. A rapid, reliable and label-free LC-MS/MS method for assessing drug activity was established, which affords an attractive alternative for those laboratories that do not have a radiation license or capabilities.