Porous graphitic carbon chromatography-tandem mass spectrometry for the study of isoprostanes in human cerebrospinal fluid

摘要

F2-isoprostanes are produced by the non-enzymatic peroxidation of arachidonic acid in membrane phospholipids. This paper describes a new method for the determination of all four classes of F2-isoprostanes in human cerebrospinal fluid (CSF) involving separation on a I mm x 150 turn porous graphitic carbon (PGC) column and detection by triple quadrupole mass spectrometry in negative-ion electrospray mode. The sample pre-treatment consisted of an ultrafiltration step, following which 300 mu l of CSF sample could be injected directly onto a 14 1 mm x 10 mm PGC guard column functioning as a trap for the analytes. The loading solvent was Milli-Q water at 125 mu l/min. After 3 min, the sample was switched into the separation column. The F2-isoprostanes were separated in 20 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5 and a flow of 50 mu l/min The limit of detection (calculated as 3S/N) was approximately 40 pM (14 pg/ml). The assay was linear within the examined range (18-450 pg/ml), using CSF spiked with iPF2 alpha-III standard (r(2) > 0.995). Repeatability data were calculated for CSF spiked to 90 pg/ml and the relative standard deviation (RSD) obtained was 3% (n = 6). @ 2005 Elsevier B.V. All rights reserved.