Liquid chromatography–coupled tandem mass spectroscopy
(LC–MS/MS)
Quantification of metabolites Quinolinic acid (QUIN), and
neurotransmitters glutamate, GABA was performed by LC-MS/MS.
Sample preparation : Within the supernatant, which was used for
HPLC, 100 µl extraction solvent containing 0.1 M formic acid was added
and centrifuged at 2000 rpm for 5 min at 0°C. The supernatant was again
collected and immediately used for LC-MS/MS. One hundred sixty
microliter of each sample was loaded into a 96 well-plate and subjected
for analysis using LC–MS/MS (Roy et al., 2014).
LC-MS/MS system consisted of an ultra-HPLC (Agilent 1290 II) coupled to
an Applied Bio systems API 4000 QTRAP triple quadrupole mass
spectrometer (ABSciex). HPLC separation was achieved with a Waters
Acquity UPLC BEH C18 column (50 mm length, 2.1 mm internal diameter, 130
Å pose size and 1.7 µm particle size). The column temperature was
maintained at 25°C. The mobile phase, pumped at 0.2 mL/min, was
consisted of methanol (eluent A) and 0.05% (v/v) formic acid in water
with 1mM of heptafluorobutyric acid (eluent B). The chromatographic
gradient run was optimized according to the separation of analyte peaks
and their shape. The total chromatographic run time was 8 minutes for a
single run. The gradient run was started with 5% of eluent A and was
maintained for 1 minute at a flow rate of 0.2 ml/min. After that it was
shifted to 100% eluent A with a flow rate of 0.4 ml/min in next 1.5
min. This condition was maintained for the next 3 min and after that
first line condition was achieved in next 0.5 min and was maintained for
next 2 min. The mass spectrometer was operated in positive ionisation
utilising an electro spray ionisation (ESI) source. GABA, Glu, QUIN
standards (Sigma-Aldrich, MO, USA; 100ng/ml in water) were infused into
mass spectrometer using a pump with microsyringe (Hamilton, Switzerland)
at the rate of 5µl/min to tune the MS/MS system at different transitions
of those compounds. Compound and instrument dependent parameters were
optimized to get the maximum ion intensity using the inbuilt algorithm
of Analyst 4.1 software. The multiple reaction monitoring (MRM)
transitions of 104/87, 104/58.1 were used for GABA, 148/130, 148/84.2
for glutamate, and 121.83/77.8, 121.83/93.9 were used for QUIN. Nitrogen
was used for the collision in MRM.