Quantification of Fluoride in Serum, Bone, and Teeth in a Murine Model Using Hexamethyldisiloxane-Facilitated...

Quantifying fluoride levels in biological tissues is essential for understanding fluoride exposure, metabolism, and toxicity. This protocol describes a sensitive and reproducible method for measuring fluoride ion mass in serum, bone, and teeth using hexamethyldisiloxane (HMDS)‑facilitated diffusion followed by ion‑selective electrode (ISE) analysis. Biological specimens, including serum, femoral bone, and mandibular incisors, are collected from mice exposed to controlled fluoride treatments. Bone and tooth specimens are ashed, milled, and hydrated, while serum is processed directly. Each specimen is transferred to a diffusion dish containing ultrapure water and sealed with a petrolatum‑lined lid bearing sodium hydroxide droplets that serve as fluoride traps. Injection of HMDS‑saturated sulfuric acid initiates fluoride release and diffusion, allowing liberated fluoride ions to be quantitatively captured in the traps. Following overnight diffusion, the fluoride trap droplets are combined into a single sample, acidified to the required pH range, and adjusted to a defined volume for ISE measurement. Calibration diffusion dishes prepared with known fluoride masses generate a standard curve for quantification, and quality control samples verify electrode stability. Representative results from adolescent and mature mice exposed to 0 or 125 ppm fluoride demonstrate the method's ability to detect age‑ and dose‑dependent differences in fluoride levels across serum, bone, and teeth. This protocol provides a robust, calibration‑based approach for quantifying fluoride in diverse biological matrices and is well‑suited for studies investigating fluoride exposure, tissue deposition, and toxicological outcomes in murine models. A key advantage of this method is its ability to accurately quantify fluoride in extremely small specimens, including the 3-5 mg of ash obtained from individual mouse incisors, because the diffusion process concentrates all released fluoride into a measurable sample volume of less than 100 µL.