Eicompak sc 30ds

HPLC was used to quantify dopamine and serotonin levels in fly head extracts. Three- to 5-day-old male flies entrained in a 12:12 LD cycle were frozen by immersion in liquid nitrogen at ZT6 and ZT18 after at least 24-hour recovery from CO₂. Samples were acquired without disturbing the LD cycle. Fly heads were detached from their bodies by gentle vortexing. Per sample, three heads were transferred to an Eppendorf tube containing 100 μl of ice-chilled 0.1 M perchloric acid (pH 3.0) and homogenized using a hand tissue grinder. Homogenates were kept on ice for 10 min to denaturalize the proteins, followed by centrifugation at 14,000 rpm for 15 min at 4°C. The supernatant was transferred to an Ultrafree-MC filter with a pore size of 0.45 μm (Merck Millipore), filtered by centrifugation for 1 hour at 14,000 rpm at 4°C, and stored at −80°C until HPLC analysis. Ten microliters of the filtrate was analyzed using HPLC with electrochemical detection (EICOMPAK SC-30DS, EICOM) using a mobile phase consisting of 80% citrate-acetate buffer (0.1 M) (pH 3.5), methanol (20%) with sodium octane sulfonate (220 mg/liter) and EDTA-2Na (5 mg/liter), pumped at a rate of 360 μl/min through a 3.0 mm by 100 mm column (EICOMPAK SC-30DS, EICOM), as described by the manufacturer.