Hplc apparatus lc 10advp

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.

Skin biopsies were homogenized in chloroform/methanol/Tris–HCl 50 mM pH 7.4 (2 : 1 : 1, v/v) containing 10 pmol of [2H]₈-AEA, [2H]₄-PEA and [2H]₄-OEA, and 50 pmol of [2H]₅-2-AG as internal deuterated standards (purchased from Cayman Chemicals, Ann Arbor, MI). The lipid-containing organic phase was dried down, weighed and prepurified by open-bed chromatography on silica gel. Fractions obtained by eluting the column with 9 : 1 (by vol) chloroform/methanol were analysed by liquid chromatography– atmospheric pressure chemical ionization–mass spectrometry (LC– APCI–MS) by using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2010) quadrupole MS via a Shimadzu APCI interface. LC–APCI–MS analyses were carried out in the selected ion monitoring mode, using m/z values of 356 and 348 (molecular ions +1 for deuterated and undeuterated AEA), 304 and 300 (molecular ions +1 for deuterated and undeuterated PEA), 330 and 326 (molecular ions +1 for deuterated and undeuterated OEA), and 384.35 and 379.35 (molecular ions +1 for deuterated and undeuterated 2-AG). AEA, OEA, PEA and 2-AG concentrations were calculated by isotope dilution and are expressed as pmol per g of wet tissue weight. The concentrations of 2-AG were obtained by adding up to the amounts of the 2-isomer also those of the 1(3)-isomer, which mostly originates from the isomerization of the former during work-up.

Murine or human skeletal muscle cells were collected and sonicated in a solution containing 50 mmol/L chloroform/methanol/cell media (2:1:1, vol/vol). Muscle tissues were first dounce-homogenized in a solution containing 50 mmol/L chloroform/methanol/Tris·HCl, pH 7.5 (2:1:1, vol/vol) and then sonicated for 8 min. After sonication, internal standards [[2H]8 anandamide (AEA) 10 pmol; [2H]5 2-arachidonoylglycerol (2-AG)] were added to the solutions. The organic phase containing lipids was dried down, weighed, and purified by open-bed chromatography on silica gel. Fractions were obtained by eluting the column with 99:1, 90:10, and 50:50 (vol/vol) chloroform/methanol. The 90:10 fraction was used for AEA and 2-AG quantification by liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry by using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS2020) quadrupole mass spectrometry via a Shimadzu atmospheric pressure chemical ionization interface as previously described [49 (link)]. The amount of endocannabinoids in both cells and tissues, quantified by isotope dilution with the above-mentioned deuterated standards, is reported as pmol/mg of the total amount of lipids extract.