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 HClO 4 . The standard curve of cAMP was linear from 5.0 ng mL −1 to 992.0 ng mL −1 , with lower limits of detection and quantification of 0.5 ng mL −1 and 5.0 ng mL −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 (EC 50 ) of 40.6 ng mL −1 . The developed method indicated higher precision and selectivity than the commercial enzyme linked immunosorbent assay kits, which showed EC 50 of 66.6 ng mL −1 .