Direct detection of tetrahydrobiopterin (BH4) and dopamine in rat brain using liquid chromatography coupled electrospray tandem mass spectrometry

Abstract

A simple and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of tetrahydrobiopterin (BH4) and dopamine in rat brain using epsilon-acetamidocaproic acid (AACA) as an internal standard. Proteins in the samples were precipitated with acetonitrile and then the supernatants were separated by a Sepax Polar–Imidazole (2.1×100mm, i.d., 3μm) column using a mixture of 10mM ammonium formate in acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 300μl/min. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) and positive ion mode from m/z 242.1→166.0 for BH4, m/z 154.1→90.0 for dopamine and m/z 174.1→114.0 for AACA (IS). The total chromatographic run time was for 5.5min. The method was validated for the analysis of samples: the limit of detection was 10ng/g. The calibration curve was linear between 10–2000ng/g for BH4 (r2=0.995) and 10–5000ng/g for dopamine (r2=0.997) in the rat brain. Thus, good correlated LC–ESI/MS/MS results were obtained and found to be a powerful tool for the quantitative analysis of BH4 and dopamine in the rat brain.