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.