Quantification of serotonin (5-hydroxytryptamine, 5-HT) and its metabolite, 5- hydroxyindoleacetic acid (5-HIAA) by high pressure liquid chromatography and electrochemical detection (HPLC-ECD)

Mice were immediately sacrificed after captopril administration for 21 days followed by series of behavioral test. Brain tissue blocks were rapidly dissected in ice and homogenized with ice-cold 0.4M perchloric acid, then incubated on ice for 1 h. After centrifugation at 21,130 g for 30 min at 4°C, the supernatant was filtered using an appropriate column (#Sc1000-1Kt, SigmaPrep spin column). Filtered supernatants were directly injected onto the Nova-Pak C18 reversed-phase column. The mobile phase consisted of 0.1M sodium phosphate monobasic, 0.1mM EDTA, 1mM sodium octyl sulfate, 0.003% trimethylamine, and 10% methanol at pH 3.7. The external standard solutions for each analyte were 5-hydroxytryptamine (5-HT; sc-298707, Santa Cruz) and 5-hydroxyindole-3-acetic acid (5-HIAA; #H8876, Sigma-Aldrich) solutions in HPLC grade water with 0.4M perchloric acid. The flow rate was kept constant at 1 ml/min. Chromatographic peak analysis was accompanied by identification of unknown peaks in a sample matched according to retention times.