2017.03 Amino Acids (Tryptophan) - Amino-02

2017.03 (JULY 2018) Amino-02 TRP_AOAC_2017.03_MLT_Corrected 080218

FOR ERP USE ONLY DO NOT DISTRIBUTE

Add 12.0 mL of methanol to each of the four flasks containing the standard/internal standard mixtures, and bring to volume with water. Transfer diluted standards to vials for frozen storage. Expiration: 6 months. g) Mobile Phase: (18:82, Methanol/0.05 M Phosphate Buffer, pH 2.3) h) Column Rinse Solution: (25% methanol in water)

E. PROCEDURE

1. Sample Preparation

a) Set heating unit to 50°C prior to experiment. b) Using an analytical balance, tare a 50 mL centrifuge tube.

c) Weigh liquid samples and reconstituted powders directly into tarred centrifuge tubes using a disposable transfer pipette. For non-reconstituted powder samples weigh into a tarred tube with a spatula. Record weights. d) Add 0.5 mL (500 µ L) of protease enzyme solution to all samples. e) Prepare no fewer than two "blank" enzyme solutions per run. Blank solutions will contain enzyme, internal standard, and buffer only. Otherwise follow sample preparation. f) Add 0.5 mL of 5-methyl-DL-tryptophan internal standard to all tubes including the enzyme blanks. 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.