Quantification of Δ12-PGJ3 by HPLC-MS

The estimation of Δ¹²-PGJ₃ was carried out as reported previously (25 (link)). Briefly, Δ¹²-PGJ₃ was quantified using a HPLC system consisting of LC-20AD UFLC pumps with a SIL-20AC autosampler (Shimadzu Corporation, Columbia, MD), a Luna™ phenyl-hexyl analytical column (2×150mm, 3 µm) (Phenomenex, Torrance, CA) developed with a 30 min isocratic elution with methanol/ water (70:30 v/v) containing 0.1 % acetic acid at a flow rate of 150µL/min and injection volume of 50µL. The negative ion electrospray tandem mass spectrometric analysis was carried out using API 2000 triple quadruple mass spectrometer (AB Sciex, Foster City, CA) at unit resolution with multiple reaction-monitoring mode (MRM). The source temperature was maintained at 450°C, electrospray voltage was −4500V and the declustering potential was set at −16V. Nitrogen was used as collision gas at −20eV and the dwell time was 150ms/ion. During MRM, Δ¹²-PGJ₃ was measured by recording the signal for the transition of the deprotonated molecule of m/z 331 to the most abundant fragment ion with m/z 269. Data were acquired and analyzed using Analyst software program version 1.5 (AB Sciex, Foster City, CA).