Direct analysis of prostaglandin-E-2 and -D-2 produced in an inflammatory cell reaction and its application for activity screening and potency evaluation using turbulent flow chromatography liquid chromatography-high resolution mass spectrometry

Abstract

Direct analysis of prostaglandin-E-2 (PGE(2)) and -D-2 (PGD(2)) produced from a RAW264.7 cell-based reaction was performed by liquid chromatography high-resolution mass spectrometry (LC-HRMS), which was online coupled with turbulent flow chromatography (TFC). The capability of this method to accurately measure PG levels in cell reaction medium containing cytokines or proteins as a reaction byproduct was cross-validated by two conventional methods. Two methods, including an LC-HRMS method after liquid liquid extraction (LLE) of the sample and a commercial PGE(2) enzyme-linked immunosorbent assay (ELISA), showed PGE(2) and/or PGD(2) levels almost similar to those obtained by TFC LC-HRMS over the reaction time after LPS stimulation. After the cross-validation, significant analytical throughputs, allowing simultaneous screening and potency evaluation of 80 natural products including 60 phytochemicals and 20 natural product extracts for the inhibition of the PGD(2) produced in the cell-based inflammatory reaction, were achieved using the TFC LC-HRMS method developed. Among the 60 phytochemicals screened, licochalcone A and formononetin inhibited PGD(2) production the most with IC50 values of 126 and 151 nM, respectively. For a reference activity, indomethacin and diclofenac were used, measuring IC50 values of 0.64 and 0.21 nM, respectively. This method also found a butanol extract of Akebia quinata Decne (AQ) stem as a promising natural product for PGD(2) inhibition. Direct and accurate analysis of PGs in the inflammatory cell reaction using the TFC LC-HRMS method developed enables the high-throughput screening and potency evaluation of as many as 320 samples in less than 48 h without changing a TFC column. (C) 2016 Elsevier B.V. All rights reserved.