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

Abstract 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.