Acrylamide ERP Resubmission Review

(o)

Disposable liners for SPE manifold .—(e.g., art. 57059, Sigma Aldrich).

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Equipment for automated SPE:

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(p) Automated Solid Phase Extraction system .—(e.g., Gilson GX-274 ASPEC™ equipped 172 with 3-mL cartridges mobile racks). 173 Note: Detailed parameters about automated SPE are provided in Table 3 . 174

D.

Chemicals and reagents

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(a) Acrylamide, analytical standard .—purity ≥ 99 % (e.g., art. 23701, Sigma Aldrich). 176 (b) Acrylamide, internal standard .—(e.g., 2,3,3-d 3 -Acrylamide, D enrichment ≥ 98 %, 177 purity ≥ 98 %, art. 636568) or 178 (c) Alternative acrylamide, internal standard .—(e.g., 13 C 3 -2,3,3-d 3 -Acrylamide, D 179 enrichment ≥ 98 %, 13 C enrichment ≥ 99 %, purity ≥ 98 %, art. 798924) both from 180 Sigma Aldrich. 181 (d) Isooctane .—for analysis EMSURE® ACS, Reag. Ph Eur (e.g., art 1.04727, Sigma 182 Aldrich). 183 (a) Methanol .—hypergrade for LC-MS LiChrosolv® (e.g., art. 106035, Sigma Aldrich). 184 (b) Methanol .—for analysis EMSURE® ACS, Reag. Ph Eur (e.g., art. 1.06009, Sigma 185 Aldrich). 186 (c) Water .—e.g., ultrapure water (e.g., Milli Q water) or LC-MS Grade (LiChrosolv®, art. 187 1.15333, Merck). 188 (d) Alpha-amylase (e.g., Fungamyl® 2500 SG, Novozymes). 189 E. Reagents and solution preparation 190 (a) Acrylamide stock standard solution 1000 µg/mL in water .—Weigh 10.00 mg ± 0.01 mg 191 of AA into a 10-mL volumetric flask. Dilute to volume with water. This solution is stable at 192 - 20 °C for at least 6 months. 193 (b) Acrylamide working standard solution 0.5 µg/mL in water .—Transfer 100 µL of the 194 stock solution (E.a) into a 200-mL volumetric flask, and complete to the mark with water. Store 195 at 4 °C for maximum 3 months. 196 (c) 2,3,3-d 3 -acrylamide (d 3 -AA) stock standard solution 200 µg/mL in water (Internal 197 Standard (IS)) .—Weigh 10.00 mg ± 0.01 mg of d 3 -AA into a 50-mL volumetric flask. Dilute 198 to volume with water. Store at - 20 °C for maximum 9 months. 199

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