2h4 15 f2t isop

Total NP and IsoP, formed from the oxidation of DHA and AA, respectively, were extracted from the PFC, CER and ST, and quantified using published methods [33 (link),34 (link)] with modifications. Due to the small size of HC, the amount of tissue from this region was insufficient for analysis. Briefly, lipids were extracted from homogenized brain samples using the Folch method. The lipid extract was then saponified to release esterified NP and IsoP. Neutral lipids were removed from the resulting mixture, using hexane, and samples were acidified to pH 3 to protonate NP and IsoP carboxylic acid groups. An internal standard, [²H₄] 15-F_2t-IsoP (Cayman Chemicals, Ann Arbor, MI, USA), was added prior to extraction with ethyl acetate. NP and IsoP were then derivatized to form pentafluorobenzyl (PFB) esters and subjected to HPLC (Agilent 1050, Santa Clara, CA, USA), using the method described by Walter et al. [33 (link)]. NP and IsoP fractions were collected, converted to trimethylsilyl ether derivatives, and quantified using GC/MS [33 (link)]. Selective ion monitoring was used for analysis at m/z 593 for NP, m/z 569 for IsoP and m/z 573 for the internal standard, [²H₄] 15-F_2t-IsoP. The inter-assay CV was 10%.

Brain samples were prepared and analyzed for esterified 8-isoprostanes (8-IsoP) and prostaglandins (PGF2α) using gas chromatography-negative ion chemical ionization MS as described previously [19 (link)]. Separations were carried out on a DB-wax column (0.25 mm inner diameter × 15 m length × 0.25-μm film thickness). The oven temperature was programmed from 150 to 300 ºC held for 11 min. Samples were injected with a 1µL injection volume. Analyte peaks were obtained from ion chromatograms. [²H₄]-15-F2t-IsoP was used as internal standard (Cayman Chemical, Ann Arbor, MI USA). The major ion generated in the negative ion chemical ionization (NICI) mass spectrum of the pentafluorobenzyl (PFB) ester, trimethylsilyl ether (TMS) derivative of F2-IsoPs, was the m/z 569 carboxylate anion [M-181 (M-CH₂C₆F₅)]. The major ion generated in the NICI mass spectrum of the PFB ester, TMS ether derivative of F4-NPs, was the corresponding m/z 593 carboxylate anion. The ion generated by the [²H₄]-15-F2t-IsoP internal standard was m/z 573.