Plasma
For the metabolomics study, plasma samples were processed through a
simple protein precipitation technique using 150 µL of ice-cold
methanol, with plasma to methanol ratio was of 1:3. After the addition
of methanol, the mixture was then vortexed and centrifuged at 14,000 rpm
for 10 min at 4 °C. The resulting supernatant was transferred to a new
tube and diluted with water containing IS (reserpine; 4
µg.mL-1) at a ratio of 2:1. Finally, 5 µL of sample
was injected into the UHPLC-Orbitrap-MS system after slight vortexing
and spinning down. A quality control (QC) sample was made by gathering
identical volumes of plasma samples and then diluting them with water
containing IS (reserpine; 4 µg.mL-1) using the same
ratio mentioned above. QC samples were used to evaluate the
repeatability and robustness of the instrumental system and were
analyzed before starting the sequence for column conditioning and after
every ten samples in the analytical batch. Additionally, test mixtures
containing a few commercially available validated standards were run at
the beginning, middle and end of the analytical batch. The test mixture
contained the following compounds: caffeine (0.5
µg.mL-1) and acetaminophen (0.5
µg.mL-1) for positive mode and glycocholic acid (0.5
µg.mL-1) and hippuric acid (0.5
µg.mL-1) for negative mode.
For the lipidomics study, 25 µL of PC (16:0/18:1)-d31 (4
µg.mL-1; internal standard for positive mode), 25 µL
of arachidonic acid-d8 (4 µg.mL-1;
internal standard for negative mode), and 50 µL of 0.1 M NaCl were added
to 50 µL of the plasma samples. Lipid extraction was then performed by
the addition of 250 µL of ice-cold chloroform/methanol (1:2; v/v) to the
plasma mixture. The mixture was then vortexed for 1 min, kept at room
temperature for 1 h, and centrifuged at 14,000 rpm for 10 min at 4 °C.
The clear supernatant was transferred to a new tube and evaporated to
dryness under a nitrogen stream at 37 °C. Finally, the dried residue was
reconstituted using 60 µL of ice-cold chloroform/methanol (1:1; v/v)
before being injected into the instrument for analysis. A QC sample was
also prepared by gathering identical volumes from each sample after
reconstitution in order to assess the repeatability and robustness of
the instrument. All QC samples were run following a sequence similar to
that of the metabolomics analysis.