Abstract

In this work, a highly selective ultra-high-performance liquid chromatography method was developed and validated to routinely estimate the concentration of dopamine (DA) in rat brain, using Xevo triple quadrupole mass spectrometry (Xevo-TQD-MS/MS) as a detecting system. Xevo TQD helps to minimize the unpredictability during quantification and to simplify the analytical procedure via automatic identification of daughter ions (product ions) in both positive and negative MS scans. The analyte and the internal standard (IS) (adrenaline) were separated on a Waters ACQUITY ultra-performance liquid chromatography Ethylene Bridged Hybrid C18 column (50 × 2.1 mm; 1.7 μm) using a mobile phase, consisting of acetonitrile and formic acid (0.1% w/v) (30: 70; v/v), with isocratic elution at a constant flow rate of 0.3 ml minute−1. The method has efficient separation and a short analysis time. The precursor-to-product ion transition of m/z 154.084→91.097 for DA and m/z 184.104→107.089 for adrenaline (IS) were observed using a positive electrospray ionization interface. The linear calibration curve was found throughout the range of 0.577–800 ng ml−1 (r2; 0.9999 ± 0.0001) for DA in rat brain homogenate, with mean recovery ranging from 81.04% to 85.41%. The intra-batch and inter-batch accuracy in percentage relative standard deviation (%RSD) were found to be in the range of 0.43%–2.29%. The method was later on successfully utilized for in vivo quantification of DA in rat brains.

Key words: Dopamine; Neurotransmitter; Parkinson's disease; UHPLC-Xevo-TQD-MS/MS; Validation