Synergi reverse phase c18 column

Ascites samples (1 ml) were spiked with 100 μl deuterated internal standard and extracted using solid reverse phase extraction columns (Strata-X 33, Phenomenex). Fatty acids derivatives were eluted into 1.0 ml of methanol, lyophilized and resuspended in 100 ml of water/acetonitrile/formic acid (70:30:0.02, v/v/v; solvent A) and analyzed by LC-MS/MS on an Agilent 1290 separation system. Samples were separated on a Synergi reverse-phase C18 column (2.1×250 mm; Phenomenex) using a gradient as follows: flow rate =0.3 μl/min, 1 min (acetonitrile/isopropyl alcohol, 50:50, v/v; solvent B), 3 min (25% solvent B), 11 min (45% solvent B), 13 min (60% solvent B), 18 min (75% solvent B), 18.5 min (90% solvent B), 20 min (90% solvent B), 21 min (0% solvent). The separation system was coupled to an electrospray interface of a QTrap 5500 mass spectrometer (AB Sciex). Compounds were detected in scheduled multiple reaction monitoring mode. For quantification a 12-point calibration curve for each analyte was used. Data analysis was performed using Analyst (v1.6.1) and MultiQuant (v2.1.1) (AB Sciex).

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).

Free Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL), the most studied markers of AGE accumulation in tissues, were assessed on total liver extracts (n = 7/10 per group) after a hydrolyzing step with 0.6 M trichloroacetic acid (C2HCl3O2) and 6 M hydrochloric acid (HCl) for 12 h at 60 °C, by means of the liquid chromatography mass spectrometry (LC-MS) as previously described [33 (link)]. The chromatographic separation was performed in an UltiMate™ 3000 HPLC system (Dionex, Milan, Italy) provided by a high-resolution LTQ Orbitrap mass spectrometer (Thermo Scientific, Rodano, Italy) with an atmospheric pressure interface and an electrospray ionization (ESI) source. Liver extracts were processed through a Phenomenex Synergi reverse-phase C18 column (dimensions: 150 × 2.1 mm, particle size: 3 μm) at a flow rate of 200 μL/min. The composition of the gradient mobile phase was as follows: 95/5 to 40/60 in 25 min, 5 mM heptafluorobutanoic acid/acetonitrile. The monitored protonated molecular ions were 205.1188 m/z for CML and 379.2094 m/z for CEL. Calibration data made with CML and CEL were used for quantitative determination of the sample’s analytes.