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 hours at room temperature, but blanks and samples must be prepared with the same enzyme solution at the same time. (Note: One Sigma Unit is defined to hydrolyze casein to produce 1.0 micromole (181 µ g) of Tyrosine per minute at pH 7.5 and 37 deg. Sigma P5147 is approximately 4 Sigma units per mg solid material. To add Ten Units per sample preparation: 10/4 U/mg or 2.5 mg enzyme needed per 0.5mL addition to samples, or 50 mg/10 mL volume, 125 mg/25mL, and 250 mg/50mL. d) 5-Methyl-DL-tryptophan, Internal Standard Solution. Accurately weigh 0.0400 ± 0.0020 grams of 5-methyl-DL-tryptophan on a Micro- Analytical balance, record the weight, and transfer to a 50 mL volumetric flask. Add about 25 mL of Laboratory Water, and a small stir bar. Due to the slow rate of dissolution, it is most convenient to place the flask in a refrigerator to stir overnight or until the powder dissolves. Once dissolved, rinse and remove the stir bar and bring to a final volume of 50 mL with water. Storage: Freeze solution in vials. Expiration date: 6 months. e) Tryptophan Standard Solution. Weigh 0.0100 ± 0.0010 grams of tryptophan and transfer to a 100 mL volumetric flask. Record weight. Add close to 50 mL of Laboratory Water and dissolve the tryptophan powder by sonicating the solution (about 3 minutes). Let solution cool to room temperature and bring to a final volume (100 mL) using Laboratory Water. Storage: freeze solution in vials. Expiration date: 6 months. f) Working Standard Solutions: Combine the appropriate aliquot of tryptophan and internal standard stock solutions together in 50 mL volumetric flasks (one for each level) following the table below: Standard TRP Stock Sol. Int. Std. Stock Very Low 0.2 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

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