Direct analysis of prostaglandin-E2 and -D2 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 IC 50 values of 126 and 151 nM, respectively. For a reference activity, indomethacin and diclofenac were used, measuring IC 50 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.