Validity of spot urine samples for estimating systemic fluoride exposure in children across diverse fluoridation modalities

Objective Reliable biomarkers are essential for assessing children's fluoride exposure, yet 24-h urinary fluoride excretion (24 h-UFE), the gold standard, is logistically challenging to collect. The validity of spot urine samples as a proxy across fluoridation modalities has not been evaluated. This study is the first to assess the predictive accuracy of spot urine fluoride concentration (U FC ), creatinine-adjusted (U F/CR ), and specific gravity-adjusted (U F/SG ) for estimating 24 h-UFE across multiple fluoride exposure modalities. Methods In this multi-modality observational study, 178 children aged 4–6 years residing in regions with different fluoride modalities were included: water fluoridation (UK, Brazil), salt fluoridation (Colombia), milk fluoridation (Chile), and non-fluoridated areas (UK, Chile). Each child provided one 24-h urine sample and four spot samples during a separate session (post-breakfast, post-lunch, before bedtime, first morning void). Linear regression models assessed the predictive validity of U FC , U F/CR , and U F/SG . Results Mean urinary fluoride ranged 0.48–1.38 mg/L (U FC ), 1.13–2.30 mg/g (U F/CR ), and 0.55–1.70 mg/L (U F/SG ). UFC showed the strongest association with 24 h-UFE (mean R 2 = 77%), particularly in water-fluoridated areas (up to 85%). U F/CR and U F/SG correlations were weaker (mean R 2 = 58% and 61%). Accuracy improved when multiple spot samples were used. Timing of peak fluoride excretion varied: post-breakfast (water), post-lunch (salt), first morning void (milk and non-fluoridated areas). Conclusion Spot U FC provides a practical alternative for population-level monitoring of fluoride exposure in children, although accuracy depends on sampling time and fluoridation modality. This multi-modality study demonstrates variability in fluoride excretion across sources and informs optimized sampling strategies for public health surveillance.