6. AOACSPIFANMethods-2018Awards

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Laboratory Water. While stirring, add 1.0N HCL drop wise until the pH of the solution reaches 8.5. Quantitatively transfer solution from beaker to a 500 mL volumetric flask and bring to volume with water. Storage: Room temperature. Expiration date: 6 months.

c) Protease Solution.

Weigh an amount of Protease enzyme material to be diluted to the desired volume such that 10 Sigma Units is delivered in 500 µL of solution. Transfer the appropriate weight of Protease into a volumetric flask of chosen volume according to amount needed for the assay (see below). Rinsing the weighing paper and the sides of the flask, add about 3/4 of volume with 0.1M Trizma buffer, pH 8.5. Swirl the solution briefly (avoid foaming), and allow it to stand for about 5 minutes until all the powder dissolves. Bring to volume mark with 0.1M Trizma buffer, pH 8.5. Make solution homogeneous by GENTLY inverting flask several times. DO NOT SHAKE, as this will cause foaming to occur. Alternate preparations: Prepare to 50, 25, or 10 mL volume as is adequate for each run at 0.5 mL per sample preparation. The Protease solution is stable for 6 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 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). Accurately weigh 0.0400 ± 0.0020 grams of 5-methyl-DL-tryptophan on a Micro-Analytical balance and transfer to a 50 mL volumetric flask. Add approximately 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. d) 5-Methyl-DL-tryptophan, Internal Standard Solution.

e) Tryptophan Standard Solution.

Weigh 0.0100 ± 0.0010 grams of tryptophan and transfer to a 100 mL volumetric flask. 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: