About 180 mg of liver tissue of three mice was mixed with deionized water at a ratio of 100 mg/mL. The mixture was then homogenized for 2 min and stored at −80 °C till use. To extract metabolites from liver, a 200 μL of homogenized liver sample was mixed with 1.6 mL methanol and vortex for 1 min, followed by centrifugation at 4 °C for 10 min at 15000 rpm. 1.4 mL of the top solution was aspirated into a plastic tube and dried by N2 flow. After dissolving the dried sample with 200 μL methanol, a stock solution was prepared by diluting the sample 10 times. Thirty aliquots of the stock solution were then prepared with a volume of 50 μL per aliquot.
A mixture of 15 acid standards was prepared at a concentration of 10 μg/mL per acid. The acids included L-Proline, L-Cystine, L-Histidine, L-Phenylalanine, L-Tyrosine, L-Lysine, L-Glutamic acid, L-Aspartic acid, L-Leucine, nonadecanoic acid, hepadecanoic acid, heptanoic acid, nonanoic acid, pentadecanoic acid, and undecanoic acid. 20 μL of the acid mixture was added to each of the first 10 aliquots of the stock solution, while 24 μL and 100 μL of the acid mixture were added to each of the second 10 aliquots and the third 10 aliquots, respectively. Methanol was then added to each of the 30 aliquots to make the total volume of each aliquot to 200 μL. This resulted in three sample groups with spiked-in acid standards. The acid concentration in each of the spiked-in sample groups is 1.0 μg/mL, 1.2 μg/mL and 5.0 μg/mL, respectively.