AOAC OMA# 2017.03 (Final Action)-OMB

1246  D raher & W hite : J ournal of AOAC I nternational V ol . 101, N o . 4, 2018

the powder dissolves (due to slow dissolution, this solution should be prepared 1 day before analysis is intended). Once dissolved, rinse and remove the stir bar and dilute the solution to a final volume of 50 mL with water. Store the solution frozen for future use. ( e ) Trp standard solution .—Weigh 0.0100 ± 0.0010 g Trp and transfer it into a 100 mL volumetric flask. Add approximately 50 mL laboratory water and dissolve the Trp powder by sonicating the solution (approximately 3 min). Let the solution cool to room temperature and dilute to a final volume of 100 mL using laboratory water. Store the solution frozen for future use. ( f ) Working standard solutions .—Combine the appropriate aliquot of Trp and internal standard stock solutions together in 50 mL volumetric flasks (one for each level), following the parameters listed in Table 2017.03D . Add 12.0 mL methanol to each of the flasks containing the standard–internal standard mixtures and dilute to volume with laboratory water. Transfer diluted standards to the autosampler vials. ( g ) Mobile phase (methanol–0.05 M buffer; 18 + 82, pH 2.3) .—Dissolve 6.9 ± 0.1 g sodium phosphate monobasic and quantitatively transfer to a beaker containing approximately 900 mL laboratory water. Add 2.0 mL concentrated phosphoric acid (85%) with a class A volumetric pipet while stirring. This adjusts the pH to 2.3. Transfer to a 1 L volumetric flask and dilute to volume with laboratory water. Filter the buffer solution through a 0.45 μm filter and transfer approximately 500 mL filtered buffer into a 1 L volumetric flask. Add 180 mLmethanol and swirl to mix. Allow to degas and subsequently dilute to volume with filtered buffer. Mix flask contents thoroughly and transfer to a 1 L HPLC system reagent reservoir. ( h ) Column rinse solution .—Prepare a maintenance solution of approximately 25% methanol in water to use as a system rinse following each analysis run. E. Sample Preparation ( a ) Set the heating unit to 50°C prior to the experiment. Weigh liquid samples and reconstituted powders directly into tarred 50 mL centrifuge tubes using a disposable transfer pipet. For nonreconstituted powder samples, weigh into a tarred tube with a spatula. Add 500 μL protease enzyme solution to all samples. Prepare no less than two blank enzyme solutions per run. Blank solutions contain enzyme, internal standard, and buffer only. Add 0.5 mL 5-methyl-DL-tryptophan internal standard to all tubes, including the enzyme blanks. Add 3.0 mL of 0.1 M pH 8.5 Trizma buffer and 200 μL methanol to all tubes, including the enzyme blanks. Cap the tubes and mix on a vortex mixer lightly to mix contents. It is important to be sure that powder samples are completely dissolved, but do not overagitate the solution to avoid foaming. Incubate the samples for 16–24 h in a heating unit previously set to 50°C.

Table 2017.03C. Tryptophan SLV data: Linearity

Relative calibration error range (low to high), %

R 2

Experiment

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9

0.99999 0.99994 0.99993 0.99996 0.99998 0.99996 0.99998 0.99997 1.00000 0.99999

–0.587 to 0.931 –4.569 to 0.519 –0.847 to 4.748 –1.165 to 0.956 –0.961 to 1.224 –0.658 to 3.395 –0.949 to 4.261 –0.476 to 3.158 –3.670 to 0.187 –0.947 to 0.850

Day 10

( 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 .—Add 167 mL of 6 N HCl (standardized) to a 1000 mL volumetric flask containing approximately 700 mL laboratory water and dilute to volume with laboratory water. Store at room temperature. ( b ) Trizma buffer, 0.1 M, pH 8.5 .—Weigh 6.055 g Trizma base and transfer into a 400 mL beaker. Dilute 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 the solution from the beaker to a 500 mL volumetric flask and dilute 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 the amount needed for the assay ( see below). Rinse the weighing paper and the sides of the flask and add about three-fourths of the volume with 0.1 M Trizma buffer, pH 8.5. Swirl the solution briefly (to avoid foaming) and allow it to stand for about 5 min until all the powder has dissolved. Dilute to volume with 0.1 M Trizma buffer, pH 8.5. Make the solution homogeneous by gently inverting the flask several times. Note : Do not shake, as this will cause foaming to occur. Alternate preparations .—Prepare a 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 of solid material. To add 10 units per sample preparation, 10/4 U/mg or 2.5 mg enzyme are needed per 0.5 mL addition to samples or volumes of 50 mg/10 mL, 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. Place the flask in a refrigerator to stir overnight or until

Table 2017.03D. Working standard preparation

Trp 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