AOAC Final Action Methods in 2019

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 μ 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 then 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 experiment. Weigh liquid samples and reconstituted powders directly into tared 50 mL centrifuge tubes using a disposable transfer pipet. For nonreconstituted powder samples, weigh into a tared tube with a spatula. Add 0.5 mL (500 μL) protease enzyme solution to all samples. Prepare no fewer 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 enzyme blanks. Cap tubes and mix with 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 samples for 16–24 h in heating unit previously set to 50°C. ( b ) Remove samples from incubation unit after a minimum of 16 h. Allow samples to cool to room temperature. Remove caps and add 12 mL methanol to each tube. Dilute each tube to 50 mL volume with laboratory water. ( c ) Cap tubes and mix thoroughly by inversion. Attach a 0.45 μm filter to a 3 cc syringe and transfer several milliliters of each sample to the syringe. Filter the samples into an autosampler vial, cap, and analyze samples. L. Instrumental Analysis and System Suitability ( a ) Set HPLC pump to a 0.5 mL/min flow rate and equilibrate the analytical column with mobile phase for 30 min. Set column oven to 30°C. The instrument method designates an excitation wavelength of 295 nm and emission wavelength of 345 nm. The Agilent G1321A fluorescence detector lamp is designated to be on during analysis only because of the near-instant warm up of the lamp. ( b ) Load standards, enzyme blanks, and samples onto the autosampler tray. The sequence includes a minimum of four initial

injections for equilibration. The first of the four injections is a high standard to check that the tryptophan peak response does not exceed approximately 50% of full scale. If significantly over 50%, lower the PMT gain by 1 unit. This will decrease the signal by about one-half. Results are best with high standard between 20 and 50% of full scale. The remaining three precision injections are 5.0:50 change to middle (MID) standard injections (Figure 2017.03A ) and provide a check of system equilibration by calculating the percent difference in the last two middle standard injection tryptophan peak areas. The system is considered equilibrated if the areas agree within approximately 1%. If not equilibrated, an additional middle standard injection is added, and the system is rechecked for area agreement. This is a pre-run suitability check only. ( c ) Once the system is equilibrated, the sequence is ordered with the four standard levels (high to low is recommended), blanks (Figure 2017.03B ), samples, and bracketing standard injections. Repeat the calibration after approximately 20 sample injections. Following the sample analysis, rinse the analytical column with a solution of approximately 25% methanol in water. For longer storage, the column is best left in 60–80% organic solvent, preferably acetonitrile. ( d ) System suitability requirements .—( 1 ) Resolution .—In the laboratory control sample (Figure 2017.03C ), the resolution of the tryptophan peak from the small peak following shall be >1.25. ( 2 ) USP tailing .—The USP tailing factor may be calculatedwithin the chromatography software and should be <1.59 for tryptophan and <1.26 for 5-methyl-tryptophan. Excessive tailing indicates the need for a new column to restore expected performance. ( 3 ) Residual error of linear regression .—The residual error for any one point of the 4-point linear regression is not to exceed 5%. In the event of excessive error, repeat the analysis with fresh standard solutions. References: (1) Narahashi, Y., & Yanagita, M. (1967) J. Biochem . 62 , 633–641

(2) Yamaskov, I.A., Tichonova, T.V., & Davankov, V.A. (1986) Enzyme Microb. Tech . 8 , 241–244 AOAC SMPR2014.013 J. AOAC Int . 98 , 1073(2015) DOI: 10.5740/jaoac.int.SMPR2014.013 J. AOAC Int . 101 , 824(2018) DOI: 10.5740/jaoacint.17-0257 (First Action) J. AOAC Int . 102 , 1567(2019) DOI: 10.5740/jaoacint.18-0399 (Final Action)

Posted: July 19, 2017, February 2018, October 2018, August 2019

© 2019 AOAC INTERNATIONAL