Impact of sample mass on the reliability of toenail fluoride for exposure assessment.

OBJECTIVE: This study aimed to determine the minimum toenail mass needed for reliable fluoride analysis, and to compare fluoride extraction efficiency (%FE) using two protocols. METHODS: Toenail samples (n = 98 from 11 participants) were grouped into masses ranging from 1.0 to 5.0 mg (± 0.5 mg). Fluoride content was analyzed using a hexamethyldisiloxane(HMDS)-facilitated diffusion method and a fluoride-ion selective electrode. Pooled toenail samples (3.0 ± 0.5 mg) from four participants were analyzed using two protocols: protocol-A used 1 mL of HMDS-saturated 3N sulfuric acid/2 mL DI water; and protocol-B used 3 mL/3 mL. Samples were re-analyzed twice to calculate %FE. Minimum mass-threshold was determined by intraclass correlation coefficients (ICC) with 95%-confidence intervals (CI) from linear mixed-effects models and change-point analysis, accounting for intra-participant variability. F-tests compared the variance in %FE between protocols. RESULTS: Fluoride content ranged from 0.82 to 9.22 µg/g. Excellent reliability (ICC = 0.90, 95%CI:[0.71, 0.95]) was observed in samples ≥ 2.5 mg. A change-point was identified at 2.20 mg(95%CI:[1.70, 2.70 mg]). A less variable %FE was observed with protocol-A (43-58%) compared to protocol-B (38-80%) (F-ratio = 0.23, p = 0.02). CONCLUSION: Toenail sample mass affects the reliability of fluoride analysis. The use of a minimum toenail mass of about 2.2-2.5 mg increases the reliability of fluoride analysis at the participant level. Variability in %FE should be considered in future efforts to standardize analytical protocols. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13530-026-00321-y.