Porous graphitic carbon chromatography-tandem mass spectrometry for the study of isoprostanes in human cerebrospinal fluid.
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 1 mm x 150 mm 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 microl of CSF sample could be injected directly onto a 1 mm x 10 mm PGC guard column functioning as a trap for the analytes. The loading solvent was Milli-Q water at 125 microl/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 microl/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 iPF2alpha-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).
Bohnstedt
Kristina Claeson
KC
Department of Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden.
Karlberg
Bo
B
Basun
Hans
H
Schmidt
Staffan
S
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D028441:F2-Isoprostanes / Q000134:cerebrospinal fluid*; D006108:Graphite; D006801:Humans; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D028441:F2-Isoprostanes; D006108:Graphite