Protocols for the measurement of the F sub(2)-isoprostane, 15(S)-8-iso-prostaglandin F sub(2 alpha ), in biological samples by GC-MS or GC-MS/MS coupled with immunoaffinity column chromatography

Arachidonic acid, the origin of the eicosanoids family, occurs in biological samples as free acid and as ester in lipids. Free arachidonic acid is oxidized to numerous metabolites by means of enzymes including cyclooxygenase (COX). Arachidonic acid esterified to lipids is attacked by reactive oxygen species (ROS) to generate numerous oxidized arachidonic acid derivatives. Generally, it is assumed that ROS-derived arachidonic acid derivatives are distinct from those generated by enzymes such as COX. Therefore, ROS-generated eicosanoids are considered specific biomarkers of oxidative stress. However, there are serious doubts concerning a strict distinction between the enzyme-derived eicosanoids and the ROS-derived iso-eicosanoids. Prominent examples are prostaglandin F sub(2 alpha ) (PGF sub(2 alpha )) and 15(S)-8-iso-prostaglandin F sub(2 alpha ) (15(S)-8-iso-PGF sub(2 alpha )) which have been originally considered to exclusively derive from COX and ROS, respectively. There is convincing evidence that both COX and ROS can oxidize arachidonic acid to PGF sub(2 alpha ) and 15(S)-8-iso-PGF sub(2 alpha ). Thus, many results previously reported for 15(S)-8-iso-PGF sub(2 alpha ) as exclusive ROS-dependent reaction product, and consequently as a specific biomarker of oxidative stress, require a careful re-examination which should also consider the analytical methods used to measure 15(S)-8-iso-PGF sub(2 alpha ). This prominent but certainly not the only example underlines more than ever the importance of the analytical chemistry in basic and clinical research areas of oxidative stress. In the present work, we report analytical protocols for the reliable quantitative determination of 15(S)-8-iso-PGF sub(2 alpha ) in human biological samples including plasma and urine by mass spectrometry coupled to gas chromatography (GC-MS, GC-MS/MS) after specific isolation of endogenous 15(S)-8-iso-PGF sub(2 alpha ) and the externally added internal standard [3,3',4,4'- super(2)H sub(4)]-15(S)-8-iso-PGF sub(2 alpha ) by immunoaffinity column chromatography (IAC). 15(S)-8-iso-PGF sub(2 alpha ) esterified to plasma lipids is hydrolysed by KOH. 15(S)-8-iso-PGF sub(2 alpha ) and [3,3',4,4'- super(2)H sub(4)]-15(S)-8-iso-PGF sub(2 alpha ) are analyzed as pentafluorobenzyl ester trimethylsilyl ether derivatives in the electron-capture negative-ion chemical ionization mode.