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

Background:

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.

Aims:

The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT.

Methods:

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.

Results:

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.

Conclusions:

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.