Amino-01 (July 2018)

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

3.5.5 Amino acids calibration standards preparation The table below shows how to prepare 0.5 mL calibration standards at (0 to 25) pmol/µL (final concentration of amino acids after derivatization) and Nva at 10 pmol/µL (final concentration after derivatization). If quantification of taurine is not needed, replace the taurine solution by water. Amino acid concentrations (each, final, after derivatization) Solution 25 pmol/µL 10 pmol/µL 5 pmol/µL 1 pmol/µL 0.5 pmol/µL 0 pmol/µL Amino acid solution 50* µL 100** µL 50** µL 100*** µL 50*** µL 0 µL Taurine solution 50 a µL 100 b µL 50 b µL 100 c µL 50 c µL 0 µL 2.5 mM Nva in 0.1 M HCl 20 µL 20 µL 20 µL 20 µL 20 µL 20 µL 0.1 M HCl 380 µL 280 µL 380 µL 280 µL 380 µL 480 µL * 2.5 mM AA stock solution, ** 0.5 mM AA solution, *** 0.05 mM AA solution. a 2.5 mM Tau stock solution, b 0.5 mM Tau solution, c 0.05 mM Tau solution. The above amino acid solutions are stable for 1 week when stored at 4 °C (± 2 °C). 3.5.6 Derivatization (of samples, cystine standards, and amino acids standards) Derivatization converts free amino acids into highly stable derivatives. Standards and samples are derivatized following the manufacturer’s instruction as described below: a. Preheat a heating block to 55 °C. b. With a micropipette, add 70 μL of AccQ•Tag™ Ultra Borate buffer (reagent 1, see section 3.2.2.1) to a clean 12 x 32 mm glass screw neck total recovery vial. c. Add 10 μL of calibration standard (3.5.5), neutralized sample solution (3.5.4), or neutralized converted cystine standard (3.5.4) to the vial. d. Vortex mix briefly. e. Add 20 μL of reconstituted AccQ•Tag™ Ultra reagent (3.2.2.2) to the sample vial. f. Mix the solution immediately by pipetting up and down several times. Vortex mix immediately for several seconds and tap the vial to ensure no bubble is trapped. g. Let stand for 1 minute at room temperature. h. Heat the vial in a heating block for 10 minutes at 55 °C (± 1 °C). - Allow the chromatographic system to stabilise before injecting standards and samples. Make sure the system pressure and initial conditions are stable before performing injections (around 9000 psi). - Before starting a series of analyses, inject two blanks (water) to condition the column. - Inject 1 µL of each derivatized calibration standards, and then inject 1 µL of derivatized sample solutions. Perform single injections. Add a blank injection (water) at the end of each calibration series. - Perform UHPLC under the following conditions: Column temperature: 50 °C UV detector: 260 nm Injection volume: 1 μL Flow rate: 0.4 mL/min 3.5.7 UHPLC separation - Prime solvent lines for 5 min. - Prime wash / sample syringes for 4 cycles.

Jaudzems, Guthrie, Lahrichi, and Fuerer – January 2018

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