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

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 × 100 mm, i.d., 3 μm) column using a mixture of 10mM ammonium formate in acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 300 μl/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 5.5 min. The method was validated for the analysis of samples: the limit of detection was 10 ng/g. The calibration curve was linear between 10-2000 ng/g for BH4 (r(2)=0.995) and 10-5000 ng/g for dopamine (r(2)=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.