An improved LC–MS/MS method for the quantification of prostaglandins E2 and D2 production in biological fluids

We report an improved liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay that accurately measures prostaglandins D2 (PGD2) and E2 (PGE2) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/ml (0.20 pg, 0.55 fmol on-column), and the interday and intraday coefficients of variation were less than 5%. Both d4-PGE2 and d4-PGD2 were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8 h to measure PGD2 accurately, whereas preparation time did not affect PGE2 measurement due to its greater stability in biological samples. As an application of the method, PGD2 and PGE2 were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE2 but no PGD2, whereas the murine macrophage cell line RAW 264.7 produced PGD2 and only trace amounts of PGE2. This direct comparison showed that COX-2 gene expression can lead to differential production of PGD2 and PGE2 by epithelial cells and macrophages. Because PGE2 is antiasthmatic and PGD2 is proasthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macrophages in the lung contributes to the pathogenesis of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, and lung cancer.