SPIFAN Nutrients ERP Book_9-29-15

Fluor-02 SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Accuracy - a. Analyze the NIST SRM 1849a over six days using multiple instruments and compare results to the reported NIST-certified value.

b. Determine spike recovery from unfortified products (i.e., placebos). Spike each selected sample at 50 and 100% of the amounts found in the unfortified products and analyze in duplicate on each of three days. Use the overall mean of the unspiked unfortified samples for calculating recoveries. Reference sample -NIST Standard Reference Material 1849a Infant/Adult Nutritional Formula, or equivalent. SRM 1849a is a milk-based, hybrid infant/adult nutritional powder. One unit of SRM 1849a contains 10 packets each containing approximately 10 g of material.

Sample calculation (Conversion of mg/L to µg/100 g):

For the conversion of mg/L concentration (obtained from Chromatogram) to µg/100 g, mg/L concentration obtained was divided by 73.5 and multiplied by 100*1000 to get µg/100g.

Explanation of factor 73.5:

1.47 g of sample in 20 mL is equivalent to 73.5 g in 1 L and the fluoride concentration obtained from chromatogram is in mg/L, thus mg of fluoride in 73.5 g of sample.

Validation Results

Separation and Detection

Ultra centrifugal filter devices were used to filter the sample and remove the fats and proteins from the sample. Following the filtration, fluoride was separated from some common anions using an IonPac AS15 column and detected by suppressed conductivity. Figure 1 shows the Chromatogram of a 2.5 µL injection of the SRM 1849a sample. The retention time for fluoride is 8.05 min (peak shown in the zoomed chromatogram). Fluoride concentration was found to be almost equal in both placebo and fortified Child formula and IF milk based RTF whereas for AN High fat and AN high protein, placebos show higher concentrations of fluoride compared to that of the fortified samples. The chromatogram of Infant elemental formula did not show presence of fluoride in either placebo or fortified sample. To determine linearity, fluoride calibration standards were injected in triplicate over seven concentration levels covering the range of 20 –1000 µg/L. To establish stability of the analytical curve, three independent experiments were conducted using independently prepared standards. The calibration results show that the detection is linear over the concentration range, with a coefficient of determination > 0.9995. Calibration errors at each level of the calibration curve were > 5%. Check standards at the lowest point and midpoint of the analytical range were Linearity