Sensitive determination of monoamine neurotransmitters, their main metabolites and precursor amino acids in different mouse brain components by liquid chromatography-electrospray tandem mass spectrometry after selective sample clean-up.

Abstract

For the assessment of diets and supplements formulated for the treatment of phenylketonuria, a highly sensitive and selective method was developed and validated for the quantification of dopamine (DA), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), phenylalanine, tyrosine and tryptophan in mouse cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform cortex and bulbus olfactorius. Samples were extracted by deproteinization with acetonitrile, and the extracts were cleaned up by strong anion exchange and weak cation exchange applied sequentially. The substances were detected by rapid liquid chromatography tandem mass spectrometry. Matrix components were largely removed by the clean-up, resulting in low matrix effects. The lower limits of quantification for an extracted tissue mass of 100 mg were 0.3, 0.3, 0.2 and 2 ng/g for DA, 5-HT, 5-HIAA and DOPAC, respectively. The mean true extraction recoveries were 80-102%. The relative intra-laboratory reproducibility standard deviations were generally <11% at concentrations of 20-1000 ng/g for DA, 5-HT, 5-HIAA and DOPAC and 7% at concentrations of 5-50 μg/g for the amino acids. This method was successfully used in a phenylketonuria mice study including nearly 300 brain tissue samples and for small sample masses (for example, 2 mg of bulbus olfactorius).