Abstract
Objectives: Validate fluoride assay for small volumes of human milk using a fluoride ion-selective electrode (F-ISE; Orion™ 9609BNWP).
Methods: Fluoride measurement reliability was evaluated across decreasing volumes of human milk samples. Two separate pools of human milk (pool 1 and pool 2) were analysed in duplicate at six volumes (1.0, 0.5, 0.25, 0.125, 0.0625, and 0.0313 mL). All samples were diluted 1:1 with low-level total ionic strength adjustment buffer (TISAB) solution. Fluoride concentrations were measured using an F-ISE with a calibration curve of 0.005, 0.01, and 0.05 mg/L. Mean values across duplicates were utilized for statistical analysis. One-way ANOVA with Sidak adjustment compared each sample volume against the 1 mL reference volume to assess measurement reliability with decreasing volumes.
Results: Fluoride concentrations in the 1 mL reference volume were 0.012 and 0.006 mg/L in pools 1 and 2, respectively. Fluoride concentrations obtained from 0.5 mL of milk were not significantly different from the 1 mL reference volume in either milk pool (pool 1: -3.36% difference, p>0.99; pool 2: -5.63% difference, p >0.99). Fluoride concentrations obtained from 0.25 mL of pool 1 were not significantly different from the 1 mL reference (-10.08% difference, p >0.92); however, that obtained from 0.25 mL of pool 2 did differ significantly (-25% difference, p< 0.05). Measurements from volumes ≤0.125 mL differed in both pooled milk samples from the 1 mL reference volume (all p< 0.05).
Conclusions: Fluoride concentrations obtained using the F-ISE were reliable in human milk sample volumes ≥0.5 mL. Volumes below this threshold produced inconsistent results. These findings establish reliable fluoride quantification in human milk volumes ≥0.5 mL, supporting future research when sample availability is restricted.