Development of a chiral SFC-MS/MS and reversed-phase LC-MS/MS platform for the quantitative metabolic profiling of octadecanoid oxylipins

Octadecanoids are broadly defined as oxylipins (i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem-MS detection for quantification of a broad panel of octadecanoids. The platform included 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 minutes, while the achiral LC method separated 67 species in 13.5 minutes. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods (R2>0.995) and LLOQ ranged from 0.03-6.00 ng/mL for SFC and 0.01-1.25 ng/mL for LC. The average accuracy in solvent and surrogate matrix ranged from 89-109% in SFC and from 106-220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentration) and 26% (at low concentration). Validation in surrogate matrix showed negligible matrix effects (<16% for all analytes) and average recoveries ranged from 71-83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little studied compounds.