Novel orthogonal liquid chromatography methods to dose neurotransmitters involved in Parkinson's disease

• Orthogonal RP-IPC and HILIC methods allow to quantify GABA, DA and its metabolites.
• The two methods were directly applied to mouse neuronal tissue homogenates.
• Orthogonal approaches compensate inherent limits of all chromatographic methods.

Parkinson's disease is a multifactorial neurodegenerative disorder, characterized by a reduction of dopamine (DA) levels. The molecular pathways involved in the pathogenesis of disease have not yet been fully disclosed. Therefore, developing new diagnostic methods and tools to evaluate the depletion of DA and of some of its metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine) is of outstanding importance for biochemical evaluations.Moreover, neurons responsible for DA release also produce the neurotransmitter gamma-aminobutyric acid (GABA), thus, quantitative measurements of GABA levels can have a relevant impact for a further understanding of the biochemical processes involved in the neurodegenerative event.In the present study, two HPLC methods based on the reversed-phase ion-pairing chromatography (RP-IPC) and the hydrophilic liquid interaction chromatography (HILIC) concepts were developed to allow the quantification of DA and its metabolites as well as GABA levels in mouse striatal and cortical tissue homogenates.The two fairly orthogonal HPLC methods were directly applied to the biological samples, without preliminary derivatization of the compounds of interest. A high level of selectivity was obtained for DA metabolites and GABA by running the gradient RP-IPC method with a volatile ion-pairing reagent, which makes it suitable for the quantitative assay of four out of five compounds. Matrix deriving interferences unabled the base-line separation of DA which was instead successfully achieved with the HILIC-based method.To avail of HPLC methods providing distinct selectivity profiles, makes possible the correct species quantification and allows to compensate the intrinsic limits characterizing all chromatographic methods.

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