Quantification of 6k-PGF1α and PGE2 via LC-MS/MS

6k-PGF_1α and PGE₂ in CM of ascTAM, ascTU and CAF were quantified as described previously [27 (link)] with slight modifications. Samples (1 mL) were spiked with 100 µL internal standard (PGE₂-d4 and 6k-PGF_1α-d4, each 9.8 ng/mL) in methanol and extracted using solid reverse phase extraction columns (Bond Elut Plexa, Agilent, Santa Clara, CA, USA). After elution and lyophilization, samples were resuspended in water/acetonitrile (70:30) with 0.02% formic acid (solvent A). Analysis was performed by LC-MS/MS on an Agilent 1290 device coupled to a QTrap 5500 mass spectrometer (AB Sciex, Darmstadt, Germany). Samples were separated at a flow rate pf 0.3 mL/min on a Synergi reverse-phase C18 column (2.1 × 250 mm; Phenomenex, Aschaffenburg, Germnay) using the following gradient: 1 min (0% solvent B: acetonitrile/isopropyl alcohol, 50:50, v/v), 3 min (25% B), 11 min (45% B), 13 min (60% B), 18 min (75% B), 18.5 min (90% B), 20 min (90% B), 21 min (0% B), 26 min (0% B). 6k-PGF_1α and PGE₂ were detected in scheduled multiple reaction monitoring mode (transitions: PGE₂ 351 → 271, PGE₂-d4 355 → 275, 6k-PGF_1α 369 → 163, 6k-PGF_1α-d4 373 → 167). For quantification, a 11-point calibration curve was used (0.06–60 ng/mL). Data analysis was performed using Analyst 1.7.2 and MultiQuant 2.1.1 (AB Sciex, Darmstadt, Germany).