We report a simple, reliable, and validated method for the rapid screening and quantification of nine urolithin (UL) metabolites in human urine. Ultra-performance liquid chromatograph coupled with a tandem mass spectrometer (UPLC-MS/MS) was utilized for UL analysis following a simple sample preparation. Optimization of chromatographic and mass spectrometric conditions was performed to maximize the sensitivity and selectivity of the targeted analytes. A validation of the methodology was conducted to account for matrix interferences, linearity, method detection limits (MDLs), UL chemical stability, precision and accuracy of the ULs of interest. MDLs were achieved for the selected ULs ranging from 9.2-18.2 ng∙mL-1. Excellent linear coefficients of determination were obtained for the range of calibration standards of 5.0-5,000 ng∙mL-1, with R2 values between 0.9991 and 0.9998. The surrogate compound, 6,7-dihydroxycoumarin, was used to monitor the extraction efficiency and chrysin as the quantitative internal standard. The recoveries of the analytes were 88-99% with surrogate recoveries greater than 82%. This analytical method was developed and validated for processing samples associated with a human study, where it is hypothesized that walnut supplementation improves colonic health and lowers colorectal cancer risk, in part through enhancing UL formation.

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