6. AOACSPIFANMethods-2018Awards

273

D RAHER & W HITE : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . nnn , 2017 3

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) of 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 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 mL methanol 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.

Table 2017.03C. Tryptophan SLV data: linearity

R 2

Experiment

Relative calibration error range (low to high), %

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

(e) Syringe filter. — ACRODISC disposable filter assembly, 0.45 µm 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 (HCl), 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

). — (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. — 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

Table 2017.03D. Working Standard Preparation

Standard Trp stock solution, mL Internal standard stock solution, mL

Very low

0.2 1.0 5.0

0.5 0.5 0.5 0.5

Low

Medium

High

10.0