Review Team (February 2016)

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

F. Procedure

a) Acid Hydrolysis procedure: 1) Accurately weigh 0.5 g of finely ground sample into a digestion vessel. 2) Add a speck of phenol (Approx. 10 mg) and 5mL of 6M HCl. 3) Purge slowly with nitrogen on top of the vial solution to remove all the atmospheric oxygen. 4) Immediately seal with relse cap using crimper and ensure the sealing is tight. 5) Keep the sealed vials in a hot air oven, initially at room temperature. 6) Raise the hot air oven temperature to 110°C±5°C and hydrolyze the samples for 20 hours. 7) Cool the vials to room temperature and quantitatively transfer the contents into a 50 ml standard volumetric flask. Makeup the flask with laboratory water and mix contents. 8) Filter a portion of the sample solution through 0.22 μm PTFE filters into a 15 ml disposable centrifuge tube. 9) Dilute the filtered aliquots further, depending on the protein content present in the samples (See Table 5) 10) Take 100 μL of the final diluted sample into the bottom of a micro centrifuge tube and add 50 μL of 0.05mM Norvaline internal standard.

11) Dry the tubes under gentle stream of nitrogen at 40°C. 12) Proceed for derivatization with the dried sample tubes. Table 5: Protein % Sample weight (g) Makeup Vol. (ml)

Volume to be taken for drying (μL)

Further dilutions

< 5.0

0.50 0.50 0.50 0.50

50.0 50.0 50.0 50.0

1.0 ml to 10 ml 0.5 ml to 10 ml 0.75 ml to 25 ml 1.0 ml to 50 ml

100.0 100.0 100.0 100.0

5.0 to 20.0 20.0 to 40.0 40.0 to 60.0

b) Oxidative acid hydrolysis procedure: 1) Accurately weigh 0.5 g of finely ground sample into a digestion vessel. 2) Add 3 to 4 mL of cold performic acid solution and ensure the sample is completely dispersed. 3) Keep in a refrigerator at 4 ˚C for 16 hours. 4) Evaporate residual performic acid solution under nitrogen at 50˚C 5) Add 5 mL of 6M Hydrochloric acid and proceed as per the acid hydrolysis procedure (from step 3 to 12). c) Derivatization Procedure: 1) Add 20 μL of 20 mM Hydrochloric acid solution to the bottom of the dried sample tube and vortex for 2 minutes. 2) Add 60 μL of Borate buffer (Vial 1 from derivatization kit) and vortex for 30 seconds. 3) Add 20 μL of derivatizing agent and vortex for 30 seconds. 4) Let it stand for 1 minute at room temperature and heat at 55°C in a preheated water bath for 5 minutes. 5) Vortex the contents in the whole tube and centrifuge the tube at 2000 rpm for 2 minutes to bring down all the liquid to the bottom of the vial. 6) Transfer the derivatized sample solution into total recovery vials and inject 1μL into UHPLC- DAD. d) Alkaline hydrolysis procedure: 1) Accurately Weigh 0.5 g of finely ground sample into a digestion vessel. 2) Add 5 mL of 4N Sodium Hydroxide solution

3) Seal the vial with relse cap using crimper and ensure the sealing is tight. 4) Keep the sealed vials in a hot air oven, initially at room temperature.

5