title: Optimization of a solid phase extraction procedure for prostaglandin E2, F2 alpha and their tissue metabolites.
abstract: The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.
authors.affiliation: Mothers and Babies Research Centre, University of Newcastle, Australia.
authors.forename: Toni N
authors.initials: TN
authors.lastname: Welsh
authors.forename: Sonya
authors.initials: S
authors.lastname: Hubbard
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Mitchell
authors.forename: Sam
authors.initials: S
authors.lastname: Mesiano
authors.forename: Pawel K
authors.initials: PK
authors.lastname: Zarzycki
authors.forename: Tamas
authors.initials: T
authors.lastname: Zakar
mesh_terms: D002855:Chromatography, Thin Layer; D015237:Dinoprost / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015232:Dinoprostone / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D015237:Dinoprost; D015232:Dinoprostone