AOAC Final Action Methods in 2019

degasser, and HPLC peak area integrator or chromatography data processor. ( b ) Analytical column .—YMC PACK C8; 3 μm particle, 3.0 × 50 mm (YMC America, Inc.; Product No. OC12S03-0503WT), or equivalent. ( c ) Analytical balance .—Readable to 0.0001 g. ( d ) Oven .—Constant temperature oven capable of maintaining 50 ± 1°C. ( e ) Syringe filter .—ACRODISC disposable filter assembly, 0.45 μ pore size (Gelman Scientific; Cat. No. 4217). ( f ) Volumetric flasks .—Glass, class A, assorted sizes. ( g ) Volumetric pipets .—Glass, class A, assorted sizes. C. Reagents ( a ) Methanol .—HPLC grade (Fisher; Part No. A456). ( b ) Sodium phosphate monobasic .—HPLC grade (Macron; Part No. 7892-04). ( c ) Phosphoric acid, 85% .—Mallinckrodt; Part No. 3563-46. ( d ) Trizma base, ≥99.9% .—Sigma; Part No. T1503. ( e ) Hydrochloric acid, 6 N .—GFS Chemical; Part No. 504. ( f ) Laboratory water .—ASTM Type 1. ( g ) L-Tryptophan reference standard (C 11 H 12 N 2 O 2 ) .—United States Pharmacopeia (USP; Cat. No. 1700501). ( h ) 5-Methyl-DL-tryptophan (C 12 H 14 N 2 O 2 ) .—Sigma; Cat. No. M-0534. ( i ) Protease from Streptomyces griseus Type XIV (enzyme) .— Sigma; Cat. No. P-5147. D. Preparation of Standards and Solutions ( a ) HCl, 1.0 N solution .—Aliquot 167 mL of 6 N HCl (standardized) to a 1000 mL volumetric flask containing approximately 700 mL laboratory water and bring to volume with laboratory water. Store at room temperature. ( b ) Trizma buffer, 0.1M, pH 8.5 .—Weigh 6.055 g Trizma base and transfer into a 400 mL beaker. Bring to a volume of approximately 300 mL with laboratory water. While stirring, add 1.0 N HCl drop- wise until the pH of the solution reaches 8.5. Quantitatively transfer

Table 2017.03B. Working standard solutions

Tryptophan stock solution, mL

Internal standard stock solution, mL

Standard Very low

0.2 1.0 5.0

0.5 0.5 0.5 0.5

Low

Medium

High

10.0

solution from beaker to a 500 mL volumetric flask and bring to volume with laboratory water. Store at room temperature. ( 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 solution. Transfer the appropriate weight of protease into a volumetric flask of chosen volume according to amount needed for the assay ( see below). Rinse the weighing paper and the sides of the flask and add about 3/4 of volume with 0.1 M Trizma buffer, pH 8.5. Swirl the solution briefly (to avoid foaming) and allow to stand for about 5 min until all the powder is dissolved. Bring to volume with 0.1 M Trizma buffer, pH 8.5. Make solution homogeneous by gently inverting the flask several times. Do not shake, as this will cause foaming to occur. For 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 h 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 μmol (181 μg) tyrosine per minute at pH 7.5 and 37°C. Sigma P5147 is approximately 4 Sigma units per milligram solid material. To add 10 units per sample preparation, 10/4 U/mg or 2.5 mg enzyme is needed per 0.5 mL for addition to samples or 50 mg/10 mL volume, 125 mg/25 mL, and 250 mg/50 mL. ( d )  5-Methyl-DL-tryptophan internal standard solution .— Accurately weigh 0.0400 ± 0.0020 g 5-methyl-DL-tryptophan on a microanalytical balance and transfer to a 50 mL volumetric flask. Add approximately 25 mL laboratory water and a small stir bar.

Figure 2017.03A. Typical 5:50 standard chromatogram.

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