Influence of pre-analytical factors and biological variability on the urinary volatilome

Volatile organic compounds (VOCs) in urine have shown increasing promise as non-invasive biomarkers for the early detection and monitoring of diseases. However, their volatile nature may make them highly susceptible to pre-analytical factors, presenting significant challenges for consistent biomarker discovery and reproducibility within metabolomic workflows. This study aimed to assess the impact of four critical pre-analytical variables, including fasting state, centrifugation procedures, room temperature (RT) stability, and freeze-thaw (FT) cycles, as well as intra- and inter-individual variability on the profiles of urinary VOC in general and volatile carbonyl compounds (VCC), in particular. Urine samples were analysed using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS). Our findings demonstrate that, although multivariate analysis did not discriminate between fasting and non-fasting states, univariate analysis revealed that the levels of distinct VOCs and VCCs significantly differed between both states. The urinary VOC or VCC profiles were very similar under both standard centrifugation and sequential mild pre-centrifugation followed by ultracentrifugation. These findings suggest that supernatants obtained from assays involving ultracentrifugation are suitable for volatilome analysis. RT stability studies demonstrated that VOC profiles remain stable for up to 21 h at RT, whereas VCC profiles showed alterations after 14 h. Compound-specific variations were also observed following repeated FT cycles, with several VOCs disclosing significant changes after two cycles, while VCC profiles remained stable, with no significant alterations detected. Both intra- and inter-individual variability were high, as evidenced by relative standard deviations exceeding 30% for most compounds and intraclass correlation coefficients below 0.4, indicating limited temporal reliability over a 2.5-month period with eight timepoint collections. These findings underscore the essential role of pre-analytical standardization in urinary volatile analysis and support the implementation of rigorous protocols to enhance data reproducibility and biomarker discovery in metabolomics.