Simultaneous quantification of neuroactive dopamine serotonin and kynurenine pathway metabolites in gender-specific youth urine by ultra performance liquid chromatography tandem high resolution mass spectrometry

• This is a high throughput and sensitive analytical method with derivatization-SPE-UPLC-ESI-HRMS/MS. The analytes cover three metabolic pathways with targeted metabolomics, to study the gender-based difference of metabolism.
• Neurotransmitters are highly polar analytes, and they can hardly separate on the routine RP columns. In this study, we originally developed a sample preparation step with derivatization process before SPE procedure to reduce the analytes’ polarities. So they were better retained on the common RP C18 SPE cartridges for better purification and the C18 analytical columns for better separation.
• Significant differences in metabolite profile and their turnover in human urine from 100 healthy young volunteers between genders (Female:Male = 1:1) were found.
• Concentrations of metabolites in serotonin route were significantly higher in females, and concentrations of metabolites in kynurenine pathway were significantly higher in males. These findings will offer better understanding of the nature and roles of the network of such neurotransmitters, which are pre-clinically and clinically applied as a biochemical index for certain mental diseases and the monitoring of medical therapy.

Neuroactive metabolites in dopamine, serotonin and kynurenine metabolic pathways play key roles in several physiological processes and their imbalances have been implicated in the pathophysiology of a wide range of disorders. The association of these metabolites’ alterations with various pathologies has raised interest in analytical methods for accurate quantification in biological fluids. However, simultaneous measurement of various neuroactive metabolites represents great challenges due to their trace level, high polarity and instability. In this study, an analytical method was developed and validated for accurately quantifying 12 neuroactive metabolites covering three metabolic pathways in youth urine by ultra performance liquid chromatography coupled to electrospray tandem high resolution mass spectrometry (UPLC-ESI-HRMS/MS). The strategy of dansyl chloride derivatization followed by solid phase extraction on C18 cartridges were employed to reduce matrix interference and improve the extraction efficiency. The reverse phase chromatographic separation was achieved with a gradient elution program in 20 min. The high resolution mass spectrometer (Q Exactive) was employed, with confirmation and quantification by Target-MS/MS scan mode. Youth urine samples collected from 100 healthy volunteers (Female:Male = 1:1) were analyzed to explore the differences in metabolite profile and their turnover between genders. The results demonstrated that the UPLC-ESI-HRMS/MS method is sensitive and robust, suitable for monitoring a large panel of metabolites and for discovering new biomarkers in the medical fields.

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