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

Abstract

We report an improved LC-MS-MS assay that accurately measures prostaglandins D 2 (PGD 2 ) and E 2 (PGE 2 ) 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 inter-day and intra-day coefficients of variation were less than 5%. Both d 4 -PGE 2 and d 4 -PGD 2 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 PGD 2 accurately, whereas preparation time did not affect PGE 2 measurement due to its greater stability in biological samples. As an application of the method, PGD 2 and PGE 2 were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE 2 but no PGD 2 while the murine macrophage cell line RAW 264.7 produced PGD 2 and only trace amounts of PGE 2 . This direct comparison showed that COX-2 gene expression can lead to differential production of PGD 2 and PGE 2 by epithelial cells and macrophages. Since PGE 2 is anti-asthmatic and PGD 2 is pro-asthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macropahges in the lung contributes to the pathogenesis of COPD, bronchiectasis, asthma, and lung cancer.

LC/MS/MS method for analysis of E2 series prostaglandins and isoprostanes

“…The 351/189 extracted ion chromatogram had less peaks indicating higher specifi city of this mass transition as compared with 351/271. Although 351/271 mass transition was previously used for PGE 2 and PGD 2 quantifi cation ( 29,32,(34)(35)(36), the presented results indicate that 351/189 mass transition gains higher selectivity for the analysis. There were two unidentifi ed peaks eluted before and after 8-isoPGE 2 that were found in both 20:4n-6 standard and strate the appropriateness of the developed method for PGE 2 and entPGE 2 analysis.…”

“…However, because PGE 2 , PGD 2 , and 15-series isoPGE 2 have the same molecular weight and similar product ion spectrums, it is important to separate them in time before MS/MS detection. A number of HPLC conditions have been used to resolve PGE 2 /PGD 2 -like molecules ( 13,20,29,(31)(32)(33)(34)(35)(36). However, to the best of our knowledge, none of the reported conditions allowed for PGE 2 , PGD 2 , and major isoPGE 2 separation in a single run without derivatization.…”

“…Liquid chromatography with tandem mass spectrometer detection (LC/MS/MS) methods are considered to be the most specifi c and less laborious for isoPG measurement as compared with enzyme immunoassay and radioimmunoassay methods, high performance liquid chromatography (HPLC) with ultraviolet or fl uorescence detection, and gas chromatography with MS or fl ame ionization detection because of a better isomer separation and no derivatization or multi-step purifi cation requirements ( 29,30 ). However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE 2 /D 2 and isoPGE 2 such as PGE 2 , entPGE 2 , PGD 2 , 8-isoPGE 2 , and 11 ␤ -PGE 2 without derivatization and multiple HPLC separations ( 13,20,29,(31)(32)(33)(34)(35)(36).…”

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma

“…Consequently, we selected ACN precipitation, followed by SPE clean up, could not be analyzed in this mode [24,[36][37][38]. Only one publication measured AA in positive mode (using a Micromass Quatro Micro spectrometer from Waters) [39], but these results could not be reproduced using our instrument.…”

Repeated application of glucocorticoids exacerbate pruritus via inhibition of prostaglandin D₂ production of mast cells in a murine model of allergic contact dermatitis

“…Therefore, in the search for another factor that may be involved in our pruritus model, we focused on prostaglandin (PG) D 2 in the present study. PGD 2 , which is produced primarily by skin mast cells (Lewis et al, 1982;Peters et al, 1984) and macrophages (Cao et al, 2008), acts as an endogenous anti-pruritic agent (Arai et al, 2004;Honma et al, 2005) by suppressing histamine release from mast cells (Chan et al, 2000;Hashimoto et al, 2005).…”