Mono tip c18 cartridges

After weighing the liver tissues, they were homogenized in 4 volumes of cold water. A total of 50 μL of liver homogenate were treated with 18 volumes of n-hexane/isopropanol solution (3:2, v/v) [22 (link)], and lipid extracts were prepared. These extracts were then treated with methanolic sodium hydroxide as described previously [23 (link)], to convert sulfatides to their corresponding lysosulfatides (LS; sulfatides without fatty acids). The resulting LS samples were purified through Mono-tip C18 cartridges (GL Sciences, Tokyo, Japan) and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) together with the internal standard N-acetylated LS-sphinganine (d18:0) [23 (link)]. MALDITOF MS analysis was performed on a TOF/TOF 5800 system (AB Sciex, Framingham, MA, USA) in negative ion reflector mode with two-point external calibration using N-acetylated LS-d18:0 ([M–H]⁻ 584.310) and LS-(4E)-sphingenine (d18:1) ([M–H]⁻ 540.284) peaks. The following molecular species of LS based on the differences in sphingoid base structure were detected: LS-sphingadienine (d18:2), LS-d18:1, LSd18:0, LS-phytosphingosine (t18:0), LS-(4E)-icosasphingenine (d20:1), LS-icosasphinganine (d20:0), and LS-4Dhydroxyicosasphinganine (t20:0). The total sulfatide level in a sample (pmol/mg wet liver weight) was calculated as the sum of these seven LS species.