Abstract

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 (UFC), creatinine-adjusted (UF/CR), and specific gravity-adjusted (UF/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 UFC, UF/CR, and UF/SG. Results: Mean urinary fluoride ranged 0.48-1.38 mg/L (UFC), 1.13-2.30 mg/g (UF/CR), and 0.55-1.70 mg/L (UF/ SG). UFC showed the strongest association with 24 h-UFE (mean R2 = 77%), particularly in water-fluoridated areas (up to 85%). UF/CR and UF/SG correlations were weaker (mean R2 = 58% and 61%). Accuracy improved when multiple spot samples were used. Timing of peak fluoride excretion varied: post-breakfast (water), postlunch (salt), first morning void (milk and non-fluoridated areas). Conclusion: Spot UFC 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.