AOAC SPIFAN ERP Reviewer Forms (March 16, 2017)

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

The method does not include: • Example calculations

o Height or area? Calculations are based on sample area (MLT Step 3.3.12, page 8). o How the standard curve data is treated to create a calibration? Injection sequence is specified (MLT Step 3.3.11, page 8). o Sample calculation. Sample calculation added (MLT Step 3.3.12, page 8) • System suitability o What sequence of injections should be used for calibrants and unknowns; o Performance criteria for standard curve (r) and read-backs. o These points are addressed in MLT Step 3.3.11, page 8. The Study Director noted that a clarification was made to the original method, to one step of the sample preparation, concerning elution from the immunoaffinity column: • Bio-02 (3/2016), page 3 Step 13: Introduce a reacti-vial and elute the analyte under gravity with 2mL methanol. Elute further with additional 1mL of methanol. Back flush at least 3 times when eluting. • 2016.02-MLT (3/2017), page 7 step 12, italics added: Introduce a reacti-vial and elute the analyte under gravity with 2mL methanol. Elute further with additional 1mL of methanol. Back flush at least 3 times when eluting and this can be achieved by gentle up and down motion of the syringe plunger to maximize the elution . Pros/Strengths: • The sample preparation is straightforward and uses conventional, widely practiced techniques • The quantification uses conventional HPLC w UV detection. • The validation report documented similar analytical results for the majority of samples from a second brand of immunoaffinity column; most samples produced results within ± 5%. The milk based IF powder result was 10% lower - 3 mcg/100g powder, or c 0.4 mcg/100mL (4 mcg/L) as fed; although the absolute difference is small, further investigation is warranted because of the commonness of this matrix. (Table 5) Cons/Weaknesses • Text needs to be added to describe system suitability and calculations. Done, see above. • The values in the SLV are not consistently reported in the SMPR units (mcg/100g reconstituted) The MLT did not use the units of the SMPR either. Method Safety Concerns: None

Supporting Data • General Comment:

The validation report stated that while a reagent blank and a matrix blank prepared from infant elemental powder were devoid of interferences, the placebo (non-fortified) products child formula powder, adult nutritional RTF high fat, and infant formula RTF milk based showed a response near the retention time of

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