Targeted metabolomics for the analysis of p-Cresol in mouse brain by HPLC-ESI-MS/MS: a new tool for drug discovery

p-Cresol is gaining increasing attention due to its potential health impact. The primary source of p-cresol in humans is the intestinal microflora in the ileum, primarily Clostridium difficile and Pseudomonas stutzeri, which can convert the aromatic amino acid tyrosine into 4-hydroxyphenylacetic acid, which is then decarboxylated to p-cresol. Once produced, the compound is converted into p-cresyl-glucuronide and p-cresyl-sulfate; the latter, along with the parent compound itself, is then eliminated through urine via organic anion transporters (OAT)5, which are also expressed in the brain, where they are responsible for the efflux across the blood-brain barrier (BBB). p-Cresol and its metabolites, which fall into the class of uremic toxins, are implicated in numerous pathologies, producing central nervous system (CNS), immune system, and cardiovascular complications in patients suffering from chronic renal failure (CKD). Elevated levels of urinary p-cresol and its conjugated derivative p-cresyl-sulfate have been found in autistic children with altered intestinal microbiota, suggesting that this molecule may contribute to worsening autism severity and gut dysfunction1,2. Furthermore, p-cresol has been demonstrated to interfere with dopamine metabolism, indicating its involvement in the pathophysiology of post-traumatic stress disorder (PTSD) and Parkinson's disease (PD), where this neurotransmitter plays a key role3. However, the evidence on the presence and concentration of p-cresol in the CNS is virtually unknown, highlighting the need to develop an analytical method capable of precisely and accurately quantifying this compound at a very low concentration. To address this issue, a new HPLC-MS/MS method was optimized and validated for the first time for targeted metabolomics of this phenolic compound in brain areas, allowing its distribution within the CNS to be defined in detail. Given the high volatility of the analyte, the sample preparation was based on the derivatization of p-cresol with dansyl chloride4. The analysis of the target analyte in brain homogenates was carried out by optimizing a reversed-phase HPLC method coupled with electrospray ionization-mass spectrometry (ESI-MS/MS) detection, using a triple quadrupole in multiple reaction monitoring (MRM) mode. A Discovery® HS-F5 column (150 mm × 2.1 mm, 3 μm) was selected for the HPLC analysis, with a mobile phase composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The analytical method optimized in this study was validated to show compliance with international requirements. Furthermore, it was applied to quantify p-cresol concentration in brain tissues from adult male and female C57/BL6 mice, determining its distribution in seven different brain areas (thalamus, hypothalamus, hippocampus, cortex, frontal cortex, cerebellum, and striatum). In addition, the levels of p-cresol were correlated with those of noradrenaline, dopamine, and its metabolites in the cortex to explore potential interactions between these compounds. Finally, given the role of OATs in the transport of the uremic toxins and their involvement in BBB permeability, the mode of interaction of p-cresol and its metabolites with OAT-1 was further explored through molecular docking. By establishing a reliable method for detecting and quantifying p-cresol in various brain regions, this research provides a foundation for further investigations of its role in neurodevelopmental and neurodegenerative diseases. Moreover, future work will address whether targeting these transporters may decrease central accumulation of p-cresol, thus providing new strategies for the therapeutic treatment of complex pathologies, such as autism, PTSD, and PD.