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Linearity
Three independent experiments were run with eight levels of tryptophan ranging from approximately
0.2 to 30 mg/L. An internal standard linear regression constructed from the standards was used to back
calculate the concentration of each working standard. The error at each level was determined and then
the average computed for each level over the three runs.
Analytical Range
The analytical range for the method will be determined using the calculated limit of quantitation to
establish the low end. The high working standard concentration will be used to calculate an upper limit
based on a reasonable sample weight for a high protein sample.
LOQ
The Tryptophan limit of quantitation (LOQ) was determined based on the ability to distinguish sample
signal from the blank variability. The quantified Tryptophan mg/L amounts from enzyme blank pairs
were calculated from each of 11 calibration curves. The within-run standard deviation was determined
for the blank paired data, which was considered to constitute the noise for this analysis. The LOQ was
determined as ten times the blank RSD in mg/L as injected and then extrapolated to RTF and powder
samples using the typical sample preparation weight and dilution.
Ruggedness
Parameters varied during the validation to establish method ruggedness include: Precision runs
prepared by two analysts, samples run on two HPLC systems, two analytical columns from different
stationary phase batches were used, new working standards and mobile phases were prepared for each
precision run, and two different stock standard solutions were used throughout the validation.
Specificity
All of the unfortified matrices were expected to contain tryptophan from the protein component and,
therefore, could not be used as placebo samples to evaluate method specificity. To address this lack, a
protein free infant base powder product, ProPhree, was tested by quantitative analysis in duplicate in a
single run as a true placebo sample. In addition, specificity for the internal standard, 5-methyl-DL-
tryptophan was evaluated by analysis of the ProPhree and all SPIFAN matrices without the internal
standard addition. These chromatograms were overlaid with a standard chromatogram to determine
any co-eluting background peaks. Any co-eluting peaks would be quantified as percent of the average
internal standard area from the standard injections. The method’s use of Tryptophan native
fluorescence as the means of detection significantly benefits method specificity by limiting matrix
response.
AMINO-02 (FEBRUARY 2017)
SLV - FINAL REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE
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