A simple, efficient and rapid HPLC-UV method for the detection of 5-HT in RIN-14B cell extract and cell culture medium.

ABSTRACT

5-Hydroxytryptamine (also known as 5-HT, serotonin) is one of the monoamine neurotransmitters which is distributed widely in plasma and brain of mammals and plays important roles in physiological manipulations. In the present method, we describe the development of a simple, efficient and rapid high performance liquid chromatographic method coupled with ultraviolet (HPLC-UV) detector for the qualitative and quantitative analysis of 5-HT in both cell extract and cell culture medium (RIN-14B). The experiments use repeated freeze-thaw cycles followed by centrifugation and direct injection of the supernatant into the chromatography. An analytical C18 column (Agilent Zorbax Extend, 4.6 × 250 mm, 5 µm.) was taken for chromatographic separation; the mobile phase was 0.05 mol/L potassium dihydrogen phosphate (KH2PO4)/acetonitrile (9010 v/v). Isocratic elution is established at the flow rate of 1.0 mL/min. The time required for this chromatographic run is 8 min. Over the concentration range of 0.1-10 µg/mL, the calibration curve is linear in this method. Other unique characteristics and advantages include high accuracy (92.02-103.28%) and high precision (intra- and inter-day coefficients of variation ≤ 4.69%). This method is applicable for the investigation of drug/condition-response relationships in the function of synthesis and secretion of 5-HT in cultured RIN-14B cells in various in vitro studies.