AOAC SN2-02 Reviewer Forms (July 25, 2023)

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• Monoacylglycerols, diacylglycerols, phospholipids, polar lipids, and free fatty acids prior to enzymatic digestion of triacylglycerols. Cons/Weaknesses Supporting Data General Comment: Precision determined using two different fat extractions using either Heptane-Isopropanol extraction in method, and also the Rose Gottlieb extraction. Precision tables report results as fatty acid in Sn2 position as weight percentage and mole percentage of the fat not in the units of the actual infant formula products which is grams fatty acid in Sn2 position per 100g of reconstituted product. Method Optimization: No optimization given. No data demonstrating the specificity and efficacy of the CALB enzyme which is critical component to this analysis. Should this data be included as part of SPIFAN review? This is based on AOCS standard, do we presume that this was part of their standard development? If this was a new method we would need to see this data. Performance Characteristics: Analytical Range: Annex D contains information about assessment of range based on grams fat at sn2 position per 100g of fat, but these are not converted to limits based on either powder or as SMPR requires, reconstituted powder. LOQ: 0.003g/100g meets the requirements in given in SMPR 2022.004 of less than or equal to 0.003g/100g Accuracy/Recovery: Accuracy was assessed in two ways. The recovery of model triacylglycerides with known fatty acid composition in each of the Sn1, Sn2, and Sn3 positions. This recovery demonstrated accuracy of the enzymatic transesterification technique. Recovery ranged from 90–98%. Recovery in an adult nutritional product was also assessed by comparison with an anhydrous fat milk sample demonstrated the recovery through the fat extraction. The recovery measured for palmitate, stearate and oleate was 98%, 97%, and 98% respectively. Precision (RSD r ): Repeatability values appear to be within SMPR limits, however as the concentration ranges aren’t specified in the same units as the SMPR it is difficult to be sure. Reproducibility (RSD R ): Not required for SLV. System suitability: No system suitability criteria are given in SLV report.

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