AOAC SPIFAN Nutrients ERP Method Reviews (September 7, 2019)

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Method Optimization: Authors have demonstrated that the inline dialysis system is not required for method performance, but is used to enhance column life by removing high molecular weight substances that are co-extracted with the fluoride. Authors have done many spike tests to exclude potential interferences from various other anions (chloride, acetate, lactate, formate) as well as disaccharides (lactose, lactulose) and mixtures of sugars, organic acids, and inorganic ions without affecting recovery or observed coelutions. Authors have provided supporting references for use of horizontal baseline and peak height approach for integration. Analytical Range: 10 to 3000 mcg/100 g, broken into two ranges for higher accuracy (10-200 mcg/100 g for low, 200- 3000 mcg/100 g for high) SMPR = 30-200 mcg/100 g LOQ: 3.6 ± 0.8 mcg/100 g calculated over matrix (SRM 1849a and SPIFAN Child Formula Powder) signal- to-noise ratio. Well below SMPR SMPR = 30 mcg/100 g

• Performance Characteristics:

Accuracy/Recovery:

Spike recoveries in powders was 80-105%; RTF was 103-108%. Lowest recovery found for spike of SRM 1849a (all spike levels: 40, 80, 160 mcg/100 g) Spike recoveries in SPIFAN kit samples at 50 and 100 % native levels was 91-120%. Lowest for FOS/GOS powder (both levels 91%) and highest for high protein RTF (117-120%). SMPR (30-100 mcg/100g) = 80-120%, (>100 mcg/100g) = 90-110%

Precision (RSD r ):

Repeatability determined on spiked samples of SRM 1849a (3.8%), ready-to-feed liquid (2%), and adult ready-to-feed (0.8%) SMPR (30-100 mcg/100g) = 8%, (>100 mcg/100g) = 5%

Reproducibility (RSD R ):

Intermediate precision determined on all samples in duplicate on 6 days by 2 analysts with 2 different columns. 5-12 % RSDR observed. Highest for powder sample C1.1 Child Formula Powder (9.4%) SMPR (30-100 mcg/100g) = 15%, (>100 mcg/100g) = 10%

• System suitability:

3 blanks are used to equilibrate the system with column gradient then again between calibrants and check standards at beginning of run to check for carry over. Should be no carryover. Unclear

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