Validation Study of HPLC-UV Determination Method of Urolithins in Broth Media

Urolithins are microbial metabolites derived from ellagitannins that have attracted increasing interest for their potential health benefits. Among them, urolithin A is particularly recognised for its role in inducing mitophagy. However, urolithin production is influenced by the differences in gut microbiota composition and metabotypes resulting from interindividual variability. To decipher microbial pathways responsible for urolithin biosynthesis, an accurate and cost-effective screening method is essential for studying their biotransformation in biological and fermentation systems. This study presents the development and validation of a high-performance liquid chromatography method as an accessible alternative to mass-spectrometry, offering an efficient, low-cost, and high-throughput screening of urolithins in anaerobic basal broth. Validation was performed according to ICH guidelines, assessing linearity, sensitivity, precision, accuracy, specificity, and matrix effects. The optimised method demonstrated excellent linearity (all r = 1) across the range of 3.125–100 μg/ml, with low detection and quantification limits, making it suitable for trace-level analysis. Precision and recovery rates were high (%RSD <5; recovery ≥98 %), and matrix effects were negligible (all p > 0.05), confirming its reliability and reproducibility. Its minimal solvent usage, excellent green profile, and reduced operational costs make it an environmentally sustainable and efficient tool for high throughput urolithin screening in fermentation and microbiome studies. • Urolithins, a group of gut microbial metabolites derived from ellagitannins with potent therapeutic properties such as mitophagy. • Individual differences in urolithin production are attributed to distinct gut microbial known as metabotypes. • A robust HPLC-MWD method was developed for the routine detection of urolithins in broth culture to investigate microbial biotransformation pathways. • The method provides high sensitivity, accuracy, and reproducibility with advantages in cost-efficiency and operational simplicity.