Acrylamide ERP Resubmission Review

Linearity range was determined using 7-point calibration curves, each point being prepared in 384 triplicate. Linearity was assessed using either d 3 -AA or 13 C 3 - d 3 -AA as internal standard (IS) 385 and two LC-MS/MS instruments (Sciex 5500 and 6500 + ). Calibration curves were built with 386 the X-axis representing the concentration ratio (AA/AA IS ) and the Y-axis representing the area 387 ratios (Area AA /Area IS ). The concentration of AA in calibrant was in the 5-100 ng/mL 388 concentration range, representing 0.025-0.5 ng of AA injected on column. 389 For method selectivity, a mix containing AA along with 3-aminopropanamide, N-acetyl-ß- 390 alanine and lactamide, all at 1 µg/mL in water, was injected to demonstrate the basal resolution 391 from AA with the pre-cited potential interferences. 392 The shelf-life of the AA stock standard solution at 1 mg/mL in water (part E.a) and the 393 calibration solutions (part E.g) were tested for a 6-month storage period at -20°C. 394 Two separate stock standard solutions were prepared at a 6-month interval. Both solutions were 395 diluted within the linearity range and analyzed in the same sequence of analysis. The average 396 of peak area measured in five replicates from the stored solution shall not deviate by more than 397 15 % from the averaged peak area of five freshly prepared replicate solutions. 398 Two independent calibration curves were prepared at a 6-month interval. Freshly prepared 399 calibrants were used to build the reference calibration curve. The concentration calculated at 400 each datapoint in stored calibration solutions shall not deviate by more than 15 % from true 401 concentration. 402 403 (a) Method blank.— For each series of analysis, a method blank should be included. This is 404 carried out by performing successively extraction and clean-up procedures (starting from F.d) 405 in the absence of any test portion. Ensure that the peak area of AA in the method blank is absent 406 or less than 1/3 compared to the peak area of AA measured in the lowest calibrant. 407 (b) Positive control.— QC samples (Certified Reference Material, Quality Control Material or 408 in-house reference samples) must be regularly included during routine analysis. For matrices 409 without available reference material, positive controls are assessed through recovery of 410 additions of known amounts of AA. 411 412 The principle of the proposed method is based on EN 16618:2015 standard [11] pending major 413 modifications (see Figure 1) . Indeed, the EN 16618:2015 standard reported validation for 414 matrices (n=7) belonging to the category of bakery ware, potato products and roasted coffee. 415 I. Internal Quality Control Plan Results and Discussion

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