Amino-01 (July 2018)

Single Laboratory Validation Report for Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals

- Taurine (e.g. Sigma Aldrich 86329) - Norvaline (e.g. Sigma Aldrich N7627) - Sodium hydroxide pellets, reagent grade (e.g. Merck 106498) - Sodium hydroxide solution, 1 M (e.g. Merck 109137) - Optional: sodium hydroxide solution, 6 M (e.g. Merck 137032) - Hydrochloric acid fuming 37 % (12 M) GR for analysis (e.g. Merck 100317)

- Hydrochloric acid, 1 M (e.g. Merck 109057) - Hydrochloric acid, 0.1 M (e.g. Merck 109060) - Laboratory Water Grade Type 1

Reagents

3.2.1 Solutions to prepare - Sodium hydroxide (NaOH) solutions at 6 M, 0.2 M, and 0.05 M - Hydrochloric acid (HCl) solution at 0.2 M

- 1% DDP in 0.2 M NaOH - 0.1% phenol in 12 M HCl

3.2.2 AccQ•Tag™ Ultra Derivatization kit Prepare the reagents included in the kit following the manufacturer’s instructions.

3.2.2.1 AccQ•Tag™ Ultra Borate buffer (reagent 1) Ready-to-use solution. Alternative reagent: 5 % (W/V) sodium tetraborate in water.

3.2.2.2 AccQ•Tag™ Ultra reagent (vial 2A and 2B) Reconstitute AccQ•Tag™ Ultra reagent (vial 2A) according to the manufacturer’s instructions: a. Preheat a heating block to 55 °C. b. Tap vial 2A lightly before opening to ensure all AccQ•Tag™ Ultra reagent powder is at the bottom of the vial. c. Rinse a clean micropipette by drawing and discarding 1 mL of AccQ•Tag™ Ultra reagent diluent from vial 2B (ready-to-use solution). Repeat 2 times. d. Draw 1.0 mL from vial 2B and transfer it to the AccQ•Tag™ Ultra reagent powder in vial 2A. Cap the vial tightly. e. Vortex mix for approximately 10 seconds. f. Heat vial 2A on top of the preheated heating block until the AccQ•Tag™ Ultra reagent powder is dissolved. Do not heat the reagent for longer than 10 minutes. Once reconstituted, the AccQ•Tag™ Ultra reagent is approximately 10 mM. Store reconstituted AccQ•Tag™ Ultra reagent in a desiccator at room temperature for up to 1 week. Caution: AccQ•Tag™ Ultra reagent reacts with atmospheric moisture. Seal the container tightly when not in use. Do not refrigerate. Do not use discolored reagent, especially if it is yellow or green.

Jaudzems, Guthrie, Lahrichi, and Fuerer – January 2018

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