12 13 dihome d4

Total lipid fractions were extracted from purified beef tallow samples by single-step deproteinization using methanol. The oxidized fatty acid fraction was purified from the lipid fractions by solid-phase extraction with Oasis HLB columns (Waters Corporation, MA, USA). Hydroxy fatty acids were separated using a high-performance liquid chromatography system (Nexera LC-30AD, Shimadzu Corporation, Kyoto, Japan) equipped with an XBridge C18 column (particle size 3.5 µm, length 150 mm, inner diameter 1.0 mm; Waters) and analyzed on a triple quadrupole mass spectrometer (LC-MS-8040; Shimadzu, Kyoto, Japan).
Mass spectrometric analysis of hydroxy fatty acids was performed in negative ion mode with an injection volume of 15 µL containing 0.5 mg of the oxidized fatty acid fraction and 1500 pg of the internal standard (12,13-diHOME-d4, 13S-HODE-d4, 13-KODE-d3, 12,13-EpOME-d4; Cayman chemicals, Ann Arbor, Michigan USA). The quantification of hydroxy fatty acids was identified and quantified by multiple-reaction monitoring as reported for the determination of other lipid metabolites [47 (link)]. For quantitation, calibration curves were prepared for each compound, and recoveries were monitored using deuterated internal standards. Data analysis was performed using LabSolutions software (Shimadzu, Kyoto, Japan).

Prostanoids and hydroxy fatty acids were extracted from whole lung tissue as described^{41 (link),42 (link)}. Briefly, lung tissue was homogenised in methanol, then diluted in water to a final concentration of 15 % (v/v) methanol/water. Internal standards were added (20 ng each of PGB₂-d4, 12-HETE-d8, 8,9DHET-d11, 8(9)EET-d11 and 12(13)DiHOME-d4; Cayman Chemical, Ann Arbor, USA). Samples were semi-purified using solid-phase extraction (500 mg C18-E cartridges; Phenomenex, Macclesfield, UK). Analytes were eluted in methyl formate, dried under nitrogen and reconstituted in ethanol, then stored at −20 °C until analysis. Analyte separation was performed using ultraperformance liquid chromatography (Acquity; Waters, Wilmslow, UK) and a C18 column (Acquity UPLC BEH; 1.7 μm; 2.1 × 50 mm; Waters, Wilmslow, UK), before multiple reaction monitoring on a triple quadrupole mass spectrometer with electrospray ionisation (Xevo TQ-S; Waters, Wilmslow, UK). Quantitation was performed using calibration lines constructed with commercially available standards (Cayman Chemicals, Ann Arbor, UK).