AOAC Guidance on FA Immunoassay Validation (August 2023)

5.2.4. Matrix Study 396 The matrix study is intended to provide data on precision (repeatability and intermediate precision), 397 sensitivity, and recovery. 398 5.2.4.1. Test Materials 399 5.2.4.1.1. Incurred test materials are required for evaluation of precision, sensitivity, and 400 recovery. See Annex A for description of best practices for incurred test material 401 preparation. 402 5.2.4.1.2. Minimum Number of Test Materials 403 5.2.4.1.2.1. At least 4 concentrations per matrix, including a zero/blank, must be included 404 in the study. One concentration should less than or equal to two times the 405 stated LOQ for the method. Other concentrations should span the calibration 406 range, e.g., at the middle of the calibration curve and upper end of the 407 calibration curve. 408 5.2.4.1.2.2. The minimum number of matrices and levels to be evaluated is dependent on 409 the parameter being estimated, as shown in Table 5.

410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436

Table 5: Required Test Materials for Quantitative Study Designs

Parameter Repeatability

Number of Matrices and Concentrations All matrices, 4 concentrations (blank, low, medium, and high) for each matrix All matrices, at least 3 concentrations (blank, low, medium) for each matrix All matrices, at least 3 concentrations (blank, low, medium) for each matrix All matrices, three non-blank concentrations (i.e., low, medium, and high)

Intermediate Precision

Sensitivity: LOD/LOQ Estimation

Recovery

5.2.4.2. Study Designs

5.2.4.2.1. A single, statistically valid study may be designed and utilized to provide estimates of precision (repeatability and intermediate precision), sensitivity, recovery, and lot-to- lot variability. Alternatively, individual studies may be designed for each performance parameter. Designs 1b and 2b below will provide sufficient data for all parameters in the Matrix Study and the lot-to-lot assessment required in the Robustness Study (5.2.5), if conducted on a sufficient number of test materials. 5.2.4.2.2. For food allergen immunoassays, intermediate precision study designs at a minimum must include multiple test portions , at least two test kit lots, and day/operator as a single confounded factor. Study designs given below may be used, but other designs

may also be able to give satisfactory data.

5.2.4.2.3. Precision: Repeatability and Intermediate Precision

5.2.4.2.3.1. Intermediate precision and repeatability can be estimated from one of several nested study designs. Depending on the design selected, results may be able to estimate repeatability, intermediate precision, variance from test kit lots,