Development and validation of high performance liquid chromatography method for the measurements of biogenic amines

Many important biogenic amines (dopamine, noradrenaline and serotonin) are produced from amino acids by enzyme-catalysed processes and play a prominent role in neuronal functions and therefore, they serve as pharmacological target for the treatment of neurological disorders, such as Alzheimer’s disease or Parkinson’s disease. The aim of the current study was to optimize a high-performance liquid chromatography method that allows selective separation of eight biogenic amines and some of their metabolites (levodopa, 3,4-dihydroxyphenylacetic acid, noradrenaline, 5-hydroxyindoleacetic acid, homovanillic acid, dopamine, serotonin and 3-methoxythyramine) using 3 internal standards with electrochemical detection. During the development of our method, we optimized the amount of ion pairing component, pH and the amount of organic phase. Several selective methods were tested, but the most effective one was used for validation process for mouse and rat brain regions, including the striatum, cortex and hippocampus. During validation, the limit of detection, the limit of quantification, recovery, intraday and interday precisions were determined for the eight analytes. The ranges of recovery were between 87 and 120%, the intraday and interday precision were < 10% in all cases. The limit of detection and quantification ranged around 2 and 10 ng/ml, respectively. The developed and optimized method ensures the measurement of the aforementioned biogenic amines from mouse and rat brain regions