Résumé
Alzheimer's disease (AD) is a neurodegenerative disease with complex aetiology. Due to its high content of polyunsaturated fatty acids (PUFA), the brain is highly susceptible to free radical-mediated oxidative damage. As a result, isoprostanes (IsoP) and neuroprostanes (NeuroP) are commonly observed lipid peroxidation products in brain. However, due to their low abundance, these metabolites are difficult to measure using traditional analytical tools. The aim of this work is to develop a highly sensitive and robust multiple reaction monitoring based LC-MS/MS method for the quantification of 24 different non-enzymatic F-IsoP and F4-NeuroP and to apply this method to analyse post-mortem brain samples from patients with AD. We analysed brain tissues from ten patients with AD and matched control brain tissue samples (0.1 mg) received from Brains for Dementia, UK. Samples were homogenised in 100% methanol and spiked with internal standards (d4-4(RS)-4-F4t-NeuroP, d4-10-F4t-NeuroP and d4-10-epi-10-F4t-NeuroP). Metabolites were enriched using two-step solid phase extraction (SPE) with a polymeric SPE column (HLB PRiME, Waters) and further separation was achieved by LC-MS/MS. Our assay has a linear dynamic range (R2 > 0.93) between 0.04ng/ml-20ng/ml for the 24 F-IsoP and F4-NeuroP. High intra- and inter-day precision (CV < 11%) was observed from the QC samples. Overall, F-IsoP and F4-NeuroP were present at higher levels in AD patient brain tissue compared to healthy subjects. Adrenic acid-derived dihomo-isofurans and docosahexaenoic acid-derived F4‐NeuroP were significantly higher in AD patients compared to healthy subjects (P < 0.01). This data demonstrate the potential to analyse of different classes of F-IsoP and F4-NeuroP providing new opportunities to study lipid peroxidation in neurodegenerative diseases such as AD.