AOAC Guidance on FA Immunoassay Validation (November 2023)

5.2.4. Matrix Study 398 The matrix study is intended to provide data on precision (repeatability and intermediate precision), 399 LOD/LOQ, and recovery. 400 5.2.4.1. Test Materials 401 5.2.4.1.1. Incurred test materials are required for evaluation of precision, LOD/LOQ, and 402 recovery. See Annex A for description of best practices for incurred test material 403 preparation. 404 5.2.4.1.2. Minimum Number of Test Materials 405 5.2.4.1.2.1. At least 4 concentrations per matrix, including a zero/blank, must be included 406 in the study. One concentration should less than or equal to two times the 407 stated LOQ for the method. Other concentrations should span the calibration 408 range, e.g., at the middle of the calibration curve and upper end of the 409 calibration curve. 410 5.2.4.1.2.2. All claimed matrices must be evaluated, and specific levels to be included are 411 dependent on the parameter being estimated, as shown in Table 5.

412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437

Table 5: Required Test Materials for Quantitative Study Designs

Parameter Repeatability

Matrices and Concentrations

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

Intermediate Precision

LOD and 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), LOD/LOQ, 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. Additional information about the principles and requirements for nested designs 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 can be found in Annex C of this document.

may also be able to give satisfactory data.

5.2.4.2.3. Precision: Repeatability and Intermediate Precision