96 / 164
Introduction
A single laboratory validation of the method described below has been completed and is being
submitted to AOAC SPIFAN for consideration as a dispute resolution method for total Tryptophan.
Method Summary
This isocratic reverse phase HPLC method allows for the determination of total tryptophan in infant,
pediatric, and adult nutritional products. Protein in a sample containing not over approximately 50 mg
of protein is hydrolyzed with 10 Sigma Units of Protease enzyme sourced from Streptomyces griseus
bacteria, selected for its non-specific protease activity, fast rate of hydrolysis, and low self-digestion.
After a 50 degrees Centigrade overnight (16 to 24 hour) digestion of sample and enzyme blank
preparations in pH 8.5 Trizma buffer, the sample preparation, with added 5-methyl-DL-tryptophan
internal standard, is diluted to approximately 50 mL with water and methanol at approximately 25% of
final volume. Following mixing and filtration, the sample is injected onto a C8 HPLC column with pH 2.3
Sodium phosphate/methanol mobile phase. The Tryptophan and 5-Methyl-DL-tryptophan are detected
by native fluorescence at an excitation wavelength of 295 nm and emission wavelength of 345 nm.
Tryptophan is quantified by internal standard calibration mode using reference standard to internal
standard amount and response ratios.
Single Laboratory Validation
Experimental
Precision
All fourteen fortified SPIFAN matrices were prepared and analyzed in duplicate on six days. New
powder reconstitutions were prepared on each day. Powder samples were blanketed with nitrogen and
frozen between uses as specified.
Accuracy
Overspike experiments were conducted on the following SPIFAN matrices: SRM 1849a, RTF sample
00729RF00, powder Lots 410057652Z, 00859RF00, K16NTAV, 41045651Z, 00795RF, and E10NWZC. For
each run two predetermined target amounts of the SPIFAN matrices were weighed into separate 50 mL
centrifuge tubes for both low and high overspikes. The samples were then spiked with a Sigma BSA
solution at about 50% and 100% of the previously determined total tryptophan levels, maintaining total
protein addition of sample plus BSA spike to approximately 50 mg maximum. The weight of the spike
addition was recorded and then mixed with the sample into the enzyme hydrolysis preparation after all
reagents were added. The BSA spiking solution was also prepared for analysis in triplicate with each
overspike run. The BSA spike solution weight and the mg/g Tryptophan content determined from five
triplicated analyses were used to calculate the theoretical spike. Sample preparations were hydrolyzed
and prepared for analysis according to the method procedure described. Accuracy was further assessed
by analysis of SRM 1849a and SRM 927e (BSA solution).
AMINO-02 (FEBRUARY 2017)
SLV - FINAL REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE
2