99 / 164
Analytical Range
The analytical range was determined to be 0.18 – 300 mg/100g. The LOQ was calculated to be 0.18
mg/100g. The upper limit was calculated by multiplying the concentration of the High Working Standard
(30 mg/L) by the typical sample dilution of 50mL and dividing this by the lowest sample weight used for
an RTF sample (0.5g) and multiplying that result by 100g to give units of mg/100g. In practice, for
extremely high concentration samples, the sample preparation weight or sample dilution can be altered
to extend the effective range. Very high tryptophan levels would not be encountered in an intact protein
sample, but could occur in a highly fortified nutritional product. For these reasons the analytical range of
0.18 – 300 mg/100g was acceptable for all 14 SPIFAN matrices and would likely be acceptable for the
vast majority of samples tested by this method.
LOQ
The limit of quantitation requirement of < 0.4mg/100g reconstituted final product was met. The within-
run standard deviation of the enzyme blank was calculated as 0.007mg/L as injected. The LOQ
concentration as injected is 0.07 mg/L (10 times the within-run SD) or 0.0035 mg per 50mL sample
preparation volume. Given the reconstituted powder sample addition of 2.0g and RTF formula addition
of 2.0g, the calculated powder and RTF sample LOQ is 0.18 mg/100g.
Ruggedness
The method performance achieved with the variables enumerated above is demonstration of the
method ruggedness under typical long term variables.
Specificity
Typical standard, SRM 1849a, and enzyme blank chromatograms are provided below. Chromatograms of
the protein free placebo powder sample prepared with and without internal standard addition are
presented under the Specificity-Placebo heading, along with a typical enzyme blank. No internal
standard interference is present in the placebo and the tryptophan peak area was found to be only
slightly below the average blank TRP area (data not shown). A quantitative, duplicate analysis of the
placebo sample gave a mean result of -0.40 mg/100g powder, equal to -0.040 mg/100g in the 25/225
reconstituted sample. This result is less than 1/3rd the method LOQ as determined above and below the
method LOD, representing no quantifiable tryptophan in the placebo powder. Chromatograms for all of
the SPIFAN matrices prepared without internal standard for chromatographic specificity showed no
internal standard baseline interference for any of these matrices.
AMINO-02 (FEBRUARY 2017)
SLV - FINAL REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE
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