*2.5. Method Validation*

Stock solutions of LOX, cis-LOX, and trans-Lox (40 mg/mL) were prepared in methanol to generate a calibration curve and linearity. The concentrations of LOX in plasma used to generate the calibration curve were 0.1, 0.2, 0.5, 1.0, 5.0, 10.0, 20.0, and 40.0 μg/mL. The concentrations of cis-LOX and trans-LOX used to generate their calibration curves were 0.2, 0.5, 1.0, 5.0, 10.0, 20.0, and 40.0 μg/mL. An amount of 10 μL of these samples was processed as mentioned above for LC-MS/MS analysis. Area peak ratios of analytes/IS versus concentration of samples were used to prepare the calibration curves. The calibration equation of LOX was *y* = 8 × 10−7*x* + 0.0002 (*R*<sup>2</sup> = 0.997). The calibration equations for cis-LOX and trans-LOX were *y* = 2 × 10−6*x* + 0.0002 (*R*<sup>2</sup> = 0.996) and *y* = 1 × 10−6*x* − 0.0002 (*R*<sup>2</sup> = 0.997), respectively.

To evaluate the accuracy and precision of LOX measurements, mouse plasma was spiked with a known concentration of LOX or QC samples at 0.2, 1.0, 10.0, 40.0 μg/mL (*n* = 5). Similarly, the accuracy and precision of cis-LOX and trans-LOX measurements were evaluated by spiking mouse plasma with a known concentration of either compound or QC samples at 0.5, 5.0, and 40.0 μg/mL (*n* = 5). Moreover, the accuracy and precision of intraday and interday were analyzed on the same day and five consecutive days at each concentration.
