A total of 14 mouse liver samples
were used to prepare a pooled sample. About 60 mg of liver tissue
from each mouse 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 until use. To extract metabolites from liver tissue, 100 μL
of each homogenized liver sample was mixed with 20 μL of butylated
hydroxytoluene (BHT) mixture (50 mg of BHT into 1 mL methanol) and
800 μL methanol. The mixture was vortexed for 1 min followed
by centrifugation at 4 °C for 10 min at 15 000 rpm. A
portion (700 μL) of the supernatant was aspirated into a plastic
tube and dried by N2 flow. After dissolving the dried sample
with 100 μL of methanol, a stock solution was prepared by diluting
the sample 10 times. Twenty microliter aliquots of each of 14 mouse
liver exacts were combined to make a pooled sample for this work.
A mixture of 16 compound standards was prepared at a concentration
of 100 μg/mL for each compound, including three fatty acid (heptadecanoic
acid, heneicosanoic acid, and nonadecanoic acid), five triglycerides
(trilauroyl-glycerol, trimyristin, tripalmitin, tricaprylin, and tricaprin),
and eight phospholipids (PC(16:0/16:0), PC(16:0/14:0), PC(12:0/12:0),
PC(6:0/6:0), LysoPC(16:0/0:0), LysoPC(10:0), PC(18:2(9Z,12Z)/18:2(9Z,12Z)),
and PC(24:1(15Z)/24:1(15Z)). To prepare the spiked-in samples 100
μL of the pooled sample was added to each of three sample vials,
followed by addition of 20, 50, and 80 μL of the standard mixture
to the first, second, and third vial, respectively. Dichloromethane/methanol
(v/v = 2:1) was then added to each of the three vials to make the
total volume of 200 μL. This resulted in three sample groups
with spiked-in compound standards. The concentration of compound standards
in each of the spiked-in sample groups was 10, 25, and 40 μg/mL,
respectively.
were used to prepare a pooled sample. About 60 mg of liver tissue
from each mouse 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 until use. To extract metabolites from liver tissue, 100 μL
of each homogenized liver sample was mixed with 20 μL of butylated
hydroxytoluene (BHT) mixture (50 mg of BHT into 1 mL methanol) and
800 μL methanol. The mixture was vortexed for 1 min followed
by centrifugation at 4 °C for 10 min at 15 000 rpm. A
portion (700 μL) of the supernatant was aspirated into a plastic
tube and dried by N2 flow. After dissolving the dried sample
with 100 μL of methanol, a stock solution was prepared by diluting
the sample 10 times. Twenty microliter aliquots of each of 14 mouse
liver exacts were combined to make a pooled sample for this work.
A mixture of 16 compound standards was prepared at a concentration
of 100 μg/mL for each compound, including three fatty acid (heptadecanoic
acid, heneicosanoic acid, and nonadecanoic acid), five triglycerides
(trilauroyl-glycerol, trimyristin, tripalmitin, tricaprylin, and tricaprin),
and eight phospholipids (PC(16:0/16:0), PC(16:0/14:0), PC(12:0/12:0),
PC(6:0/6:0), LysoPC(16:0/0:0), LysoPC(10:0), PC(18:2(9Z,12Z)/18:2(9Z,12Z)),
and PC(24:1(15Z)/24:1(15Z)). To prepare the spiked-in samples 100
μL of the pooled sample was added to each of three sample vials,
followed by addition of 20, 50, and 80 μL of the standard mixture
to the first, second, and third vial, respectively. Dichloromethane/methanol
(v/v = 2:1) was then added to each of the three vials to make the
total volume of 200 μL. This resulted in three sample groups
with spiked-in compound standards. The concentration of compound standards
in each of the spiked-in sample groups was 10, 25, and 40 μg/mL,
respectively.