HPLC analysis for brain serotonin (5-HT) concentration

The levels of 5-HT neurotransmitter in the resident male mice brain were determined using high performance liquid chromatography (HPLC). Mice were euthanized with diethylether (not more than 5 ml via the respiratory route by exposing them to ether for approximately 2 min in a transparent jar) and brain carefully removed from the skull of mouse according to the guidelines set by American Veterinary Medical Association, (2013). Brain samples from both hemispheres were dissected on ice to isolate the prefrontal cortex (PFC) from a thick section in a cryostat. Both PFC and the remaining part of the brain were kept in tissue containers at -80°C until use. Frozen brain samples of mice were weighed in 15 ml falcon tube using electronic balance. The brain tissues were homogenized on ice in 700 µl of ice-cold 0.1% formic acid solution in methanol (1:1, v/v) for 60 s at 60 Hz 30 pulses using a micro-ultrasonic cell disrupter at 30 amplitudes. Brain homogenates were kept on ice for 15 min to aid the precipitation of proteins and cell debris. The homogenates were centrifuged at 14,000 rpm for 20 min at 4°C and the supernatant was collected into a labelled 1.5 ml reaction tube to measure 5-HT in the next section.

HPLC analysis of 5-HT in the supernatant from the brain homogenate was performed on a Shimadzu Prominence reverse phase HPLC consisting of a binary solvent delivery system (LC—20AB), a degasser (DGU-20A3), an auto-sampler (SIL—20ACHT), a column temperature controller (CTO—10AS VP) and a photo diode array detector (SPD—M20A). HPLC stationary phase used was a TSK gel C18 column (diameter 3 μm, length × width, 150 mm × 4.6 mm) while the mobile phase comprised 0.05% formic acid: methanol (90%:10%, v/v) at a flow rate of 0.70 ml/min. The injection volume was 20 µl and the runtime was set at 15 min. The column was heated to 30°C and 5-HT was detected at an emission wavelength of 280 nm. Peaks were identified by comparing their retention time in the sample (tissue extracts) solution with that of standard solution. LC workstation (Shimadzu) software was used to control the HPLC components and to process data.