Quantification of Neurotransmitters in Mouse Brain Tissue by Using Liquid Chromatography Coupled Electrospray Tandem Mass Spectrometry

A simple and rapid liquid chromatography tandem mass spectrometry method has been developed for the determination of BH4, DA, 5-HT, NE, EP, Glu, and GABA in mouse brain using epsilon-acetamidocaproic acid and isotopically labeled neurotransmitters as internal standards. Proteins in the samples were precipitated by adding acetonitrile, and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 mm x 100 mm, i.d., 3 mu m) column by adding a mixture of 10 mM ammonium formate in acetonitrile/water (75 : 25, v/v, 300 mu l/min) for BH4 and DA. To assay 5-HT, NE, EP, Glu, and GABA; a Luna 3 mu C-18 (3.0 mm x 150 mm, i.d., 3 mu m) column was used by adding a mixture of 1% formic acid in acetonitrile/water (20 : 80, v/v, 350 mu l/min). The total chromatographic run time was 5.5 min. The method was validated for the analysis of samples. The calibration curve was linear between 10 and 2000 ng/g for BH4 (r(2) = 0.995), 10 and 5000 ng/g for DA (r(2) = 0.997), 20 and 10000 ng/g for 5-HT (r(2) = 0.994), NE (r(2) = 0.993), and EP (r(2) = 0.993), and 0.2 and 200 mu g/g for Glu (r(2) = 0.996) and GABA (r(2) = 0.999) in the mouse brain tissues. As stated above, LC-MS/MS results were obtained and established to be a useful tool for the quantitative analysis of BH4, DA, 5-HT, NE, EP, Glu, and GABA in the experimental rodent brain.