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

e) Typical PMT setting: 7 for Agilent G1321A (see F.3.a) f) Run time: 8.5 minutes, or as needed to fully collect the 5-methyl-DL-tryptophan peak.

D. REAGENTS 1. Standards

L-Tryptophan USP Standard Cat #1700501, or equivalent. 2. Chemicals a) Methanol, HPLC grade b) Sodium Phosphate Monobasic, HPLC grade. c) Phosphoric Acid, (85%) d) Trizma Base e) Hydrochloric Acid, 6N.

f) Laboratory Water – minimum of 18 mega ohm conductivity g) 5-Methyl-DL-tryptophan Sigma Cat # M-0534, or equivalent. h) Protease from Streptomyces griseus : Sigma Cat # P-5147, or equivalent. 3. Solutions a) Hydrochloric Acid, 1.0N Solution. b) Trizma Buffer, 0.1M, pH 8.5. c) Protease Solution - Weigh 0.125 ± 0.010 grams to 25 mL with Trizma buffer. Scale volume as needed with proportional weight. Expiration: 6 hours. d) 5-Methyl-DL-tryptophan, Internal Standard Solution - 0.0400 ± 0.0020 grams to 50 mL with water. Initially add ~25 mL water and stir with small stir bar. Due to slow dissolution, may stir overnight in a refrigerator. Store frozen. Expiration: 6 months e) Tryptophan Stock Standard Solution - (0.0100 ± 0.0010 grams per 100 mL) Store frozen. Expiration: 6 months. h) Working Standard Solutions: Combine the appropriate aliquot of tryptophan and internal standard stock solutions in separate 50 mL volumetric flasks for four working levels. Standard TRP Stock Sol. Int. Std. Stock VERY LOW 0.20 mL 0.5 mL LOW 1.0 mL 0.5 mL MID 5.0 mL 0.5 mL HIGH 10.0 mL 0.5 mL