Bioactivity assay of porcine relaxin based on cAMP accumulation in THP-1 cells quantified by LC–MS/MS

Abstract

This study describes a reliable bioactivity assay of porcine relaxin (pRLX) based on cyclic adenosine 3′,5′-monophosphate (cAMP) accumulation in the human monocyte cell-line quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). As a result, the LC–MS/MS was based on a positive selected reaction monitoring of cAMP with a stable internal standard, 8-Br-cAMP and a protein precipitation procedure by HClO4. The standard curve of cAMP was linear from 5.0ngmL−1 to 992.0ngmL−1, with lower limits of detection and quantification of 0.5ngmL−1 and 5.0ngmL−1, respectively. The satisfactory validation data including stability assay were obtained. When measured by the LC–MS/MS, the pRLX sample showed a time- and dose-dependent stimulation of cAMP with the concentration for 50% of the maximal effect (EC50) of 40.6ngmL−1. The developed method indicated higher precision and selectivity than the commercial enzyme linked immunosorbent assay kits, which showed EC50 of 66.6ngmL−1.