AOAC Guidance on FA Immunoassay Validation (August 2023)

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intermediate precision. Data used must meet other method performance

criteria (e.g., recovery of 50-150%).

5.2.4.2.5. Recovery Assessment

5.2.4.2.5.1. Data collected for the purposes of precision evaluation may also be used for

the recovery assessment.

5.2.4.2.5.2. If conducted separately from the precision assessment, evaluate each incurred matrix with six independent analyses (test portions) per concentration level at a minimum of three non-blank concentration levels

covering the analytical range.

5.2.4.3. Data Analysis and Reporting

5.2.4.3.1. Precision

5.2.4.3.1.1. Nested Designs: Repeatability and Intermediate Precision

5.2.4.3.1.1.1. Data generated from nested designs, such as those as described above, should be analyzed by an ANOVA capable of providing estimates of

intermediate precision and repeatability.

5.2.4.3.1.1.2. Annex C contains full instructions, R code, and example datasets for the

study designs described in this guidance.

5.2.4.3.1.2. Repeatability Only

5.2.4.3.1.2.1. In a situation where a study design for estimating repeatability alone is selected, the mean, standard deviation, and relative standard deviation should be calculated for each test material (i.e., each matrix- concentration combination). Formulas for standard deviation and relative standard deviation, as defined in Appendix F (3), are as follows:

Repeatability standard Deviation (s r ): s r = [ Σ (x r – x̅ )

2 /n] 0.5

Repeatability relative standard deviation (RSD r ): RSD = s r × 100/ x̅

5.2.4.3.1.2.2. The study report must include the standard deviation and RSD values for each test material, and all repeatability estimates must meet requirements set forth in the relevant SMPR or established by the ERP. In the absence of an SMPR and ERP, acceptable RSD r values for food allergen

immunoassays are generally ≤20%.

5.2.4.3.2. Sensitivity: LOD, LOQ

5.2.4.3.2.1. LOD will be estimated using a hypothesis test approach, with α = β = 0.05. The relationship between observed concentration and intermediate precision standard deviation must be taken into account in the estimation of LOD (also referred to as a precision profile estimation method for LOD). Full instructions

for the calculations to estimate LOD are in Annex C.

5.2.4.3.2.2. LOQ estimation will be based on the relationship between concentration and

intermediate precision standard deviation. Full instructions for the

calculations to estimate LOQ are in Annex C.

5.2.4.3.3. Recovery