Quantification of intracellular and extracellular prostanoids stimulated by A23187 by liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

Prostanoids are bioactive substances that contribute to various biological and pathological processes. To evaluate both extracellular and intracellular levels of prostanoids at the same time, we developed methods for quantification of extracellular and intracellular levels of prostanoids, including prostaglandin E 2 (PGE 2), PGD 2, PGF 2α, 6-keto PGF 1α, and TXB 2, in cultured cells using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and we validated the LC/MS/MS methods. A solid-phase extraction cartridge was used for extraction of prostanoids. The prostanoids were separated by a C 18 column with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). Calibration curves of extracellular measurement for the prostanoids were linear in the range from 0.1 to 100 ng/mL ( r 2 > 0.999), and those of intracellular measurement were linear in the range from 0.05 to 50 ng ( r 2 > 0.999). Validation assessment showed that both methods of extracellular and intracellular measurements were highly reliable with good accuracy and precision. We also applied the methods to human airway epithelial Calu-3 cells and human lung adenocarcinoma epithelial A549 cells.