Development of analytical methodology for analysis of oxylipins in brain tissue and plasma

Oxylipins are bioactive oxygenated products of long chain (18-22 carbons) polyunsaturated fatty acids. They play an important role in different physiological processes acting as local hormones, so they may be used as biomarkers of these processes. In addition, the pathways and exact biological functions of many members of this family are not clear and need further investigation. To enable such investigations, it is important to have reliable and accurate analytical methods for oxylipin measurements in biospecimens such as blood and tissue. The analysis of oxylipins in biological matrices is challenging due to their low concentrations and the existence of many oxylipin isomers. So, the first objective of this thesis was to develop a sensitive LC-HRMS method for the quantitative analysis of oxylipins, that provides separation of isomers and works in a scan mode in order to enable comprehensive oxylipin profiling and further investigation of unknown oxylipins in real samples. To achieve this goal, different LC stationary phases were assessed to obtain maximum separation of oxylipin isomers and a C-18 UHPLC column was determined as the best choice for this separation. Also, different mobile phase additives were assessed, and it was found that 0.02% (v/v) acetic acid in mobile phase gives maximum sensitivity by increasing ionization efficiency. After LC optimization, three pairs of oxylipins among 65-standard mixture were still unresolved chromatographically. MS/MS fragmentation of these oxylipins was developed to resolve these three pairs. Thus, the final LC-MS method allows for a measurement of 62 oxylipins and seven deuterated standards in 40 min and with LLOQ 0.1-0.8 ng/ml. Another challenge for oxylipin analysis in plasma and brain is the complexity of the biological matrix that can affect the sensitivity and accurate quantitation of the method due to possible matrix effects. Thus, the second objective of this thesis was to develop and optimize sample preparation methodology to decrease a possible matrix effect and achieve the best limits of detection in biological matrices. Solid-phase extraction (SPE) was chosen for method development. During development, the following parameters were optimized: SPE sorbent type, elution solvent composition, elution solvent volume and sample treatment before loading. Due to the low abundance of oxylipins in biological matrices sample preparation requires preconcentration step following the extraction. This step was found to be critical for method reproducibility and was systematically optimized to decrease possible losses of analytes during this step. The final developed and optimized sample preparation method in combination with LC-MS was then applied to plasma and brain tissue samples. The average recovery was 70-97% and the matrix effect was 27-105%. High inter-individual variabilities and a wide range (0.26-681 ng/ml) of oxylipin concentrations in human plasma samples were found. Some high concentrations of oxylipins such as 408±35 ng/ml for 9-HETE were reported at first time. To accommodate this wide linear dynamic range, two injections were required, one with dilution for the accurate measurement of high abundance oxylipins and one with pre-concentration factor to enable the measurement of low abundance oxylipins. In general, the developed method allowed to detect 38 oxylipins in pooled plasma and accurately quantitated 25 of them. In rat brain tissue samples, the concentration range of oxylipins was narrower than in plasma 0.14-13.1 pg/mg of wet tissue and in general, 43 oxylipins were detected, among which 41 were accurately quantitated. The main issue with post-mortem analysis of oxylipins in brain tissue is the possibility of post-mortem formation of oxylipins, which can result in a 50-500x increase in oxylipin concentrations. Also, in vitro methods for the analysis of oxylipins in brain tissue do not allow multiple measurements to be performed with the same experimental animal over a period of time. This poses a critical limitation during the investigation of biochemical pathways in response to particular stimulus. In vivo solid-phase microextraction (SPME) could help solve these problems. In vivo SPME was performed in moving awake rats (n=15) in collaboration with the Centre for Addiction and Mental Health (CAMH) and the resulting extracts were analysed by LC-MS. Twenty (20) oxylipins were identified using authentic standards. In addition, 32 unknown peaks corresponding to expected oxylipin m/z were detected. Among these, 18 were unique to in vivo SPME while the rest were also detected in post-mortem SPE samples. Six (6) out of 32 unknowns were subsequently identified as oxylipins. Further characterization and identification of other unknowns will be performed in future. To the best of our knowledge, this is the largest oxylipin panel ever detected in vivo from the brain tissue of living animals and provides an important new tool in neuroscience