Pig urine for metabolomics LC-MS analysis (C18, HSS T3) (2017) Chemicals Acetonitril (ACN) (VWR) Formic acid (FA) Glycocholic acid (Glycine-113C) p-chlorophenylalanine Solutions H2O/ACN/ formic acid (95/5/0.1) (950 ml MILLIQ-water added 50 ml acetonitril and 1 ml FA.) ACN/IS: Internal standard solution (concentration: 0.1 mg / mL; final concentration: 0.01 mg / mL). Calculation, see bottom of document. Materials 2 ml Eppendorf tubes Pipettes and pipette tips Centrifuge Intern standards (IS) Glycocholic acid (Glycine-113C) solution in methanol (final concentration of 1 mg/ml) p-chlorophenylalanine solution in 2 % formic acid (final concentration 1 mg/ml) Sample preparation 180 μl urine is mixed with 20 μl ACN/IS Blank sample: H2O treated as a sample QC sample: 20 μl pool of each urine sample is treated as a normal sample Blind: 0.1% FA Protein precipitation in the fridge (20 min, 4°C). Centrifugation (10 min, 13,200 rpm, 4°C). The supernatant is transferred to vials with micro inserts (150 μl). Analysis on UHPLC-MS with C18 column (HSS T3) according to LC-MS procedure.
Pig urine for metabolomics LC-MS analysis (C18, HSS T3) (2017) Chemicals Acetonitril (ACN) (VWR) Formic acid (FA) Glycocholic acid (Glycine-113C) p-chlorophenylalanine Solutions H2O/ACN/ formic acid (95/5/0.1) (950 ml MILLIQ-water added 50 ml acetonitril and 1 ml FA.) ACN/IS: Internal standard solution (concentration: 0.1 mg / mL; final concentration: 0.01 mg / mL). Calculation, see bottom of document. Materials 2 ml Eppendorf tubes Pipettes and pipette tips Centrifuge Intern standards (IS) Glycocholic acid (Glycine-113C) solution in methanol (final concentration of 1 mg/ml) p-chlorophenylalanine solution in 2 % formic acid (final concentration 1 mg/ml) Sample preparation 180 μl urine is mixed with 20 μl ACN/IS Blank sample: H2O treated as a sample QC sample: 20 μl pool of each urine sample is treated as a normal sample Blind: 0.1% FA Protein precipitation in the fridge (20 min, 4°C). Centrifugation (10 min, 13,200 rpm, 4°C). The supernatant is transferred to vials with micro inserts (150 μl). Analysis on UHPLC-MS with C18 column (HSS T3) according to LC-MS procedure.
Protocol_for_untargeted_metabolomics_Urin_2017_ENG.docx