Derivatization of F2-isoprostanes with 1-pyrenyldiazomethane and their subsequent determination by fluorescence high-performance liquid chromatography

Abstract

F(2)-isoprostanes are produced during free radical oxidation of cell phospholipids and are considered reliable biomarkers of oxidative stress. Currently, mass spectroscopy is the method of choice to detect F(2)-isoprostanes. However, due to numerous isomeric forms, analysis of F(2)-isoprostanes using MS detectors requires preliminary chromatographic separation. The current study was undertaken to develop a method of HPLC separation and quantification of different isomers of prostaglandin F(2alpha), including 8-iso-PGF(2alpha), following derivatization with 1-pyrenyldiazomethane (PDAM) to a highly fluorescent 1-pyrenylmethyl ester. HPLC separation and quantification of 1-pyrenylmethyl esters of PGF(2alpha) isomers at picogram level are complicated by numerous interfering products of the degradation of 1-pyrenyldiazomethane. A procedure of derivatization and purification was developed to reduce these interfering contaminants. The procedure of derivatization includes sorption of PGF(2alpha) isomers from solution in a hexane:ethyl acetate mixture (10:1) on a cellulose support prepared in the form of small (4 x 4mm) filter paper squares. Bound PGF(2alpha) isomers are derivatized by 1-pyrenyldiazomethane dissolved in the same hexane:ethyl acetate mixture. During subsequent washing of the cellulose squares by the hexane:ethyl acetate mixture (10:1), fluorescent derivatives of PGF(2alpha) remain bound to cellulose while a significant portion of the contaminants are washed out. The 1-pyrenylmethyl esters of PGF(2alpha) can be quantitatively extracted from cellulose by an ethyl acetate:methanol (1:1) mixture. The next step in eliminating interference is a solid-phase extraction on silica cartridges using ethyl acetate for application of the sample and an ethyl acetate:methanol (1:1) mixture for elution. Final purification is achieved by normal-phase HPLC with wet ethyl acetate as the mobile phase. This chromatographic method displays remarkable resolution in the separation of different PGF(2alpha) isomers and can be used not only for sample purification but also for pre-MS separation. The purified 1-pyrenylmethyl esters of PGF(2alpha) were quantitatively analyzed by reverse-phase HPLC with fluorescent detection, with a detection limit of 5-10 pg.