Improving the detectability of low-abundance p-cresol in biological matrices by chemical derivatization and LC-MS/MS determination.

Gut microbiota produces a wide range of microbial metabolites with potential neuroactive properties. Among these, p-cresol, a by-product of tyrosine breakdown, has gained significant attention in various neuropsychiatric disorders, including autism spectrum disorder. However, current methods fail to detect p-cresol at trace levels in both the systemic circulation and brain, limiting the study of its role in neuropsychiatric disorders. There, the precise and accurate determination of p-cresol at low picogram levels is an unmet analytical need. To address this gap, we developed a highly-sensitive, validated method for quantifying p-cresol at low picogram levels in urine, plasma, and brain using chemical derivatization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that derivatization with 1,2-dimethylimidazole-5-sulfonyl chloride (5-DMIS-Cl or 5-DMISC) increased up to 40-fold the sensitivity compared to traditional dansyl derivatization. Therefore, a method based on 5-DMISC derivatization and sum of transitions was selected for validation. The method was accurate (recoveries 91-100 %) and precise (RSD <15 %) in all tested matrices, enabling detection down to100 pg/mL for urine, 20 pg/mL for plasma, and 0.04 pg/mg for brain tissue. The method was applied to plasma and brain samples from control and p-cresol-treated mice, revealing significant increases in p-cresol levels in treated animals. For the first time, we successfully quantified p-cresol levels in the brain, demonstrating its ability to cross the blood-brain barrier. In summary, this validated method offers a powerful tool for exploring the role of p-cresol -and potentially other phenolic compounds-in the microbiota-gut-brain axis and neuropsychiatric disorders.