AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

2017.03 (JAN 2019) Amino-02-(ERP Review for Pubs) Tryptophan by enz hyd_Abbott Labs_Method_10172018

FOR ERP USE ONLY DO NOT DISTRIBUTE

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Jonathan Draher and Norman White Abbott Nutrition, Division of Abbott Laboratories

g) Add 3.0 mL of 0.1M, pH 8.5 Trizma buffer and 200 µ L methanol to all tubes including enzyme blanks. Cap tubes and vortex lightly (about 2 seconds) to mix contents. Be sure powder samples are completely dissolved but do not over agitate solution to avoid foaming. h) Incubate samples for 16 to 24 hours in heating unit previously set to 50°C. Remove samples from incubation unit after a minimum of 16 hours. Samples may be prepared for overnight incubation if removed within 24 hours. i) Let samples cool to room temperature. Remove caps and add 12 mL of methanol to each tube using a bottle re-pipettor. j) Dilute each tube to the 50 mL mark with Laboratory Water. k) Cap tubes and mix thoroughly by inversion. l) Attach a 0.45 µ m filter to a 3 or 5 cc syringe and transfer several mL of a sample to the syringe. Filter sample into an autosampler vial, and cap. Repeat with fresh syringe and filter for each sample and blank. m) Samples are ready for analysis or may be frozen for future analysis within 72 hours. F. HPLC Analysis and Equilibration a) Set HPLC pump to 0.5 mL/min flow rate, and equilibrate the analytical column with mobile phase for 30 minutes, or until a stable baseline is achieved. b) Set column oven to 30°C. c) The instrument method should designate an excitation wavelength of 295 nm and emission wavelength of 345 nm. The Agilent G1321A fluorescence detector lamp may be designated to be on during analysis only, due to the near instant warm up of the lamp. d) Load standards, enzyme blanks, and samples onto the autosampler tray. e) Sequence should include a minimum of four initial injections for equilibration. The first of the four injections should be a high standard to check that the TRP peak response does not exceed approximately 50% of full scale. If significantly over 50%, lower the PMT gain by one unit. This will decrease the signal by about ½. Results are best with high standard between 20% and 50% of full scale. f) The remaining three precision injections should be the 5.0/50 standard injections to provide for check of system equilibration by calculating the

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