Lcms 2020 quadrupole ms

Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) analyses of AEA, 2-AG, PEA and OEA levels were carried out as previously described (Bisogno et al., 1997 (link); Marsicano et al., 2002 (link)). Briefly, plantar paws were homogenized in a solution of chloroform/methanol/Tris-HCl 50 mM pH 7.4 (2:1:1 by vol.) containing 10 pmol of [²H]₈-AEA, and 5 pmol each of [²H]₅-2-AG, [²H]₄-PEA and [²H]₂-OEA as internal deuterated standards. The lipid-containing organic phase was pre-purified by open-bed chromatography on silica gel, and fractions obtained by eluting the column with a solution of chloroform/methanol (90:10 by vol.) were analyzed by LC-APCI-MS by using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2020) quadrupole MS via a Shimadzu APCI interface. LC-APCI-MS analyses of AEA, 2-AG, PEA and OEA were carried out in the selected ion monitoring (SIM) mode, using m/z values of molecular ions +1 for deuterated and undeuterated compounds, respectively, as follows: 356 and 348 (AEA), 384.35 and 379.35 (2-AG), 304 and 300 (PEA), 328 and 326 (OEA). AEA, 2-AG, PEA and OEA levels were calculated on the basis of their area ratio with the internal deuterated standard signal areas, and their amounts (pmol) were normalized per g or mg of plantar paw.

Plasma sample collection was performed as previously described (Petrosino et al., 2016 (link)). The levels of [¹³C]₄-PEA in rat plasma and tissues were measured using the protocol previously described for PEA (Bisogno et al., 1997 (link); Di Marzo et al., 2001 (link); Marsicano et al., 2002 (link); Petrosino et al., 2016 (link)), except that N-heptadecanoyl-ethanolamine was added as internal standard instead of [²H]₄-PEA. Briefly, plasma and tissues were homogenized in chloroform/methanol/50 mM Tris-HCl pH 7.4 (2:1:1 by vol.) containing 10 pmol of N-heptadecanoyl-ethanolamine. The lipid-containing organic phase was pre-purified by open-bed silica gel chromatography, and the fractions obtained by eluting with chloroform/methanol (90:10 by vol.) were analyzed by LC-APCI-MS using a Shimadzu high-performance liquid chromatography apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2020) quadrupole MS via a Shimadzu APCI interface. LC-APCI-MS analysis of PEA was carried out in the selected ion monitoring mode, using m/z values of 314 and 304 (molecular ions +1 for the standard and [¹³C]₄-PEA, respectively); retention times were 17 and 13 min, respectively. [¹³C]₄-PEA levels were calculated on the basis of their area ratios with the internal standard signal areas to give the amounts in pmol/ml of volume or pmol/g of tissue.