Assessment of spectral variability encountered during FTIR analysis of fresh urine

Vibrational techniques, particularly infrared absorption spectroscopy, are promising tools for routine diagnosis due to their high chemical specificity, enabling the detection of disease-associated molecular changes. Urine is a valuable biofluid for identifying diagnostic biomarkers, as its molecular composition reflects the body's pathophysiological state, particularly that of the urinary system. However, the high variability of urine complicates the identification of spectroscopic markers. This variability arises not only from biological variations but also from experimental parameters within the analytical workflow. The objective of this study was to assess the impact of specific experimental parameters on the infrared signal, particularly two key time intervals: T1, from urine emission to the onset of sample drying, and T2, from the end of drying to spectral acquisition. After assigning infrared bands to urine's major urine components (urea, creatinine and uric acid), we analysed the spectral variability associated with these experimental parameters using principal component analysis on samples from healthy volunteers. Our results showed that infrared spectral profiles were strongly influenced by urine pH. Spectral changes were greater during T1, when urine remains in liquid form, while T2 had a comparatively smaller impact. Our study led to some recommendations for urine spectral analysis to minimise variability.