Simultaneous HPLC–MS–MS quantification of 8-iso-PGF 2α and 8,12-iso-iPF 2α in CSF and brain tissue samples with on-line cleanup

Abstract

Quantitation of isoprostanes such as 8-iso-PGF 2α and 8,12-iso-iPF 2α-VI in biological fluids has been proposed as a reliable test of oxidant stress and inflammation in a variety of disorders. This paper presents a liquid chromatography method with tandem mass spectrometry detection for the simultaneous analysis of these two isoprostanes in human CSF and brain tissue samples. An API 5000 triple quadrupole instrument (AB Sciex, Foster City, CA, USA) with an APCI ion source was used in this study. Aliquots of CSF samples (0.25 mL) were treated with a methanol:zinc sulfate mixture followed by on-line cleanup on an extraction column (Validated-C 18) with 0.1% formic acid. The brain tissue samples were homogenized and lipids were extracted using Folch solution. Solid-phase extraction columns (C 18) were used for the purification of the brain isoprostane fraction. Chromatographic separation was achieved using an analytical column (Synergi C 18 HydroRP) with 0.1% formic acid in water and a mixture of methanol:acetonitrile under isocratic conditions. The mass spectrometer was operated in the MRM scan and negative ion mode. The quadrupoles were set to detect the molecular ions [M−H] − and high mass fragments of isoprostanes: m/ z 353 → 193 amu (8-iso-PGF 2α) and m/ z 353 → 115 amu (8,12-iso-iPF 2α-VI) and their deuterated internal standards: m/ z 357 → 197 amu (8-iso-PGF 2α-d 4) and m/ z 364 → 115 amu (8,12-iso-iPF 2α-VI-d 11). The lower limit of quantification was 2.5 pg/mL for 8-iso-PGF 2α and 5.0 pg/mL for 8,12-iso-PF 2α-VI for the CSF method and 10.0 pg/0.1 g of tissue and 30.0 pg/0.1 g of tissue for 8-iso-PGF 2α and 8,12-iso-iPF 2α-VI, respectively, for the brain tissue method. No ion suppression or enhancement of the detection of 8-isoPGF 2α, 8,12-isoPF 2α-VI or both internal standards was found.