BACKGROUND: Metabolomics and lipidomics analysis of various biological samples offer insights into potential mechanisms of health and disease development. Tissue samples, compared to other biological samples, are less elucidated due to challenges in sample collection and lack of standardized sample preparation protocols for reproducible tissue homogenization and broad-range metabolite extraction. RESULTS: Pork tissue samples were homogenized with six different solvent mixtures with increasing lipophilicity, followed by metabolites extraction using methanol for polar and methyl-tert-butyl ether (MTBE) in methanol (MeOH) for highly lipophilic compounds. Metabolite profiles of supernatant and homogenate extraction for three extract volumes were compared. Solvent dependent pipette tip blockage was addressed by introduction of a prewetting correction factor for non-polar homogenization solutions and low volume tissue homogenate pipetting. Upset plots were applied for multi-dimensional metabolite extraction efficiency evaluation for 24 different sample preparation conditions. The best-performing homogenization solution was PBS
MeOH (1:1
v/v), combined with a two-step polar metabolite and lipid extraction using MeOH and 75 % MTBE in MeOH employing the tissue homogenate. The optimized experimental conditions were applied on mouse pancreas tissues, providing evidence of varying metabolic pathway activities across different anatomical regions of an organ. SIGNIFICANCE: This study introduces a comprehensive tissue sample preparation and metabolite quantification workflow, covering highly polar to highly lipophilic metabolites using targeted high performance liquid chromatography electrospray ionization triple quadrupole-linear ion trap mass spectrometer (HPLC-ESI-QTRAP-MS/MS) for absolute quantitation of amino acids, organic acids and keto-acids, acyl-carnitines, and phospho-choline lipids.