AOAC SPIFAN Nutrients ERP (December 7, 2022)

For ERP/Working Group Use Only DO NOT DISTRIBUTE Fluor-04 (SLV) Resubmission FOR ERP USE ONLY DO NOT DISTRIBUTE

Executive Summary A single laboratory validation (SLV) of “Fluoride determination in milk, soy, and water-based products using ion-selective electrode and direct measurement technique” has been completed. Five SPIFAN powder samples were analyzed in duplicate over six days. Results were calculated based on one of two least squares (log fit) standard curves (0.02 – 0.1 µg / 100 g or 0.1 -2.0 µg / 100 g). The results for three of the five samples were above the stated limit of detection of the ion-selective electrodes, which was far below the minimum analytical value required in the SMPR. The precision for these three products (0.7% to 4.1% RSD r ) also met the SMPR requirements. All five samples were spiked at three levels with NIST-traceable standard solutions, with recoveries that met the SMPR requirements. This method is applicable for all infant and adult nutritional products. Method Summary Fluoride is determined quantitatively using ion-selective electrodes. The fluoride ion-selective electrode is a solid-state electrode in which a single lanthanum fluoride crystal is sealed into a plastic electrode body. One face of the crystal is in contact with the internal reference solution and the other face is in contact with the sample. The crystal allows fluoride ions to pass through; this movement of ions effects an electrical potential, which is proportional to the activity of fluoride ions in the sample. The fluoride concentration of the sample is measured by comparing its potential to the potential of standards of known fluoride concentration. Samples and standards are treated with concentrated hydrochloric acid to dissolve and release any bound fluoride from protein and/or minerals in sample. Concentrated sodium citrate buffer is added to complex interfering ions and adjust the pH and ionic strength. The fluoride concentration is determined using a direct calibration measurement technique. A complete copy of this method can be found in Appendix A. Experimental The SLV was completed per AOAC Appendix L “AOAC Recommended Guidelines for Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Single-Laboratory Validation”. All SPIFAN matrices were prepared and analyzed in duplicate over six days by one analyst using two different electrode configurations. All products were reconstituted daily by mixing 25 g powder into 200 g lab water per the SMPR. Two samples from each reconstitution were weighed into disposable plastic cups. Concentrated hydrochloric acid was added and the samples were stirred for 10 minutes to hydrate and dissolve any suspended inorganic material and release any bound fluoride. Sodium citrate buffer was added to adjust the pH to > 5, which avoids any hydroxide interferences or the formation of hydrogen complexes of fluoride. The Single Laboratory Validation

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