Amino-01 (July 2018)

Single Laboratory Validation Report for Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals

Intermediate reproducibility Appendix E details the reproducibility requirements for each analyte/matrix pair according to the analyte concentrations listed in Appendix A. Intermediate reproducibility results (based on robust statistics) are detailed below for overview and are presented in their full-sized version in Appendix F.

SPIFAN II SLV kit product

Analyte 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 5.6% 7.6% 6.4% 3.7% 6.3% 12.0% 7.5% 4.3% 7.5% 9.3%

His

4.9% 7.8% 4.6% 11.0% 6.8% 4.7% 7.5% 7.6%

9.2%

Tau N/A N/A N/A N/A N/A

2.3% 4.2% 4.2% 10.2% 2.2%

N/A

6.3% 7.1% 3.7% 7.7% 6.6% 6.2% 3.9% 4.4%

Ser Arg

7.4% 10.6% 6.9% 5.7%

5.8%

9.0% 4.2% 5.3% 2.3% 2.4% 3.5% 7.4% 6.3% 3.0% 5.0% 7.9% 5.3%

8.8% 9.7% 6.6% 7.2%

3.7% 2.1% 2.0%

7.9%

7.6% 2.7% 6.4% 3.0%

8.1%

6.4% 5.1%

8.2%

4.3% 6.4% 8.0%

8.1%

2.0%

Gly 3.0% 3.9% 2.6% 5.5% 2.5% 1.0% 1.9% 3.1% 4.3% 5.0% 5.0% 7.2% 6.4% 3.7% 4.1% 9.4% Asp 2.0% 3.4% 1.7% 2.1% 4.2% 3.3% 4.2% 3.2% 4.3% 3.3% 1.1% 4.3% 2.3% 1.2% 6.8% 5.2% Glu 1.4% 3.7% 1.7% 2.0% 3.2% 1.2% 3.8% 2.3% 3.0% 2.9% 2.2% 4.4% 3.5% 3.2% 5.6% 5.1%

0.0% 6.2% 6.0% 3.9% 1.8% 3.8% 3.1% 1.5% 4.5%

Intermediate reproducibility data, color-coded with respect to reproducibility requirement values. From the table above (and Appendix F), 91% of the values are within the requirements and 7% outside. 2% could not be attributed (blank values for taurine). Valine and Isoleucine show high CViR.This is most likely because those amino acids are released slower than the others upon hydrolysis. Prolonging the hydrolysis step would however, negatively affect other amino acids such as Serine, Tyrosine, Threonine, Cysteine/Cystine and Methionine. Recovery Appendix G details the recovery requirements for each analyte/matrix pair according to the analyte concentrations listed in Appendix A. Spike recovery results (based on robust statistics) are detailed below for overview and are presented in their full-sized version in Appendix H. Thr 3.1% 2.1% 2.6% 3.9% 3.1% 2.7% 1.3% 1.6% 3.6% 1.9% 0.8% 4.8% 2.8% 0.9% 2.6% 5.4% 2.5% 4.9% 5.1% Ala 2.0% 1.3% 3.5% 3.4% 3.7% 1.3% 4.3% 3.4% 3.5% 2.7% 1.9% 5.3% 4.0% 2.3% 4.6% 4.5% 2.5% 4.2% 5.6% Pro 0.6% 2.8% 1.8% 2.4% 1.9% 1.1% 1.4% 2.1% 4.2% 3.2% 3.0% 4.0% 2.3% 2.7% 3.8% 5.5% 2.1% 4.6% 5.3% Lys 1.4% 3.2% 1.6% 2.8% 3.5% 3.0% 7.4% 7.6% 2.3% 3.9% 3.9% 3.4% 3.9% 4.9% 11.3% 8.9% 6.2% 5.1% 3.1% Tyr 4.3% 5.5% 2.5% 6.2% 3.8% 2.9% 5.2% 5.6% 5.8% 2.5% 6.1% 4.3% 3.7% 1.8% 7.1% 9.2% 4.4% 4.9% 7.4% xCys 4.2% 10.6% 5.1% 3.8% 5.1% 3.4% 3.9% 5.2% 6.5% 2.4% 3.4% 3.5% 10.5% 3.7% 8.1% 8.1% 5.2% 2.6% 6.1% Met 5.7% 3.4% 1.4% 9.1% 7.7% 2.3% 2.1% 4.1% 4.1% 1.9% 1.8% 1.4% 3.3% 2.5% 4.0% 6.3% 1.9% 6.8% 7.2% Val 10.0% 0.8% 9.4% 5.5% 13.3% 6.7% 9.4% 13.8% 13.9% 7.4% 4.3% 11.9% 2.5% 6.1% 12.8% 15.0% 6.5% 11.4% 12.2% Ile 10.2% 0.5% 9.6% 8.3% 15.7% 8.6% 10.8% 12.6% 14.5% 8.8% 7.9% 13.7% 3.5% 8.0% 13.7% 14.1% 6.2% 11.8% 12.1% Leu 1.8% 3.7% 2.4% 2.3% 1.8% 1.3% 2.7% 2.6% 5.2% 4.4% 3.4% 0.9% 3.1% 2.3% 6.2% 7.9% 1.8% 4.3% 7.5% Phe 5.5% 5.2% 4.0% 5.2% 3.3% 2.5% 8.3% 6.4% 5.5% 6.1% 5.2% 5.5% 5.8% 4.4% 9.4% 8.5% 3.3% 3.7% 5.8% His 98.4% 99.9% 98.6% 90.1% 100.1% 92.6% 99.6% 108.9% 94.8% 103.3% 95.1% 102.1% 96.0% 102.5% 97.9% 97.2% 97.7% 98.7% 97.5% 98.5% Tau 100.3% 101.0% 99.1% 94.0% 96.0% 97.0% 97.0% 97.9% 96.2% 100.0% 95.9% 109.6% 99.7% 99.2% 98.2% 97.9% 99.2% 100.1% 99.4% 98.8% Ser 109.5% 103.0% 96.6% 91.4% 79.8% 104.0% 93.2% 95.3% 97.5% 95.0% 93.2% 102.1% 93.0% 94.0% 96.8% 96.4% 91.8% 95.6% 99.1% 96.2% Arg 96.5% 98.3% 101.4% 90.8% 92.4% 94.8% 91.9% 93.1% 93.2% 91.6% 93.8% 104.4% 90.3% 92.6% 94.5% 97.1% 95.7% 99.5% 95.9% 95.2% Gly 100.2% 99.4% 100.4% 94.3% 98.4% 97.9% 97.5% 97.0% 97.1% 104.4% 97.5% 107.2% 106.2% 102.3% 97.0% 98.2% 101.2% 99.4% 98.6% 99.7% Asp 112.7% 110.5% 102.8% 101.1% 108.0% 105.2% 104.9% 108.5% 114.1% 107.4% 104.1% 112.1% 108.5% 108.9% 106.6% 103.6% 105.0% 102.3% 105.5% 106.9% Glu 106.9% 103.5% 105.5% 94.2% 103.6% 103.4% 98.6% 102.1% 110.4% 103.6% 99.6% 109.7% 103.3% 103.8% 101.1% 100.0% 101.3% 98.7% 98.4% 102.5% Thr 100.0% 99.2% 98.6% 92.0% 90.5% 99.6% 94.8% 95.7% 97.2% 97.2% 94.8% 104.6% 96.8% 97.2% 96.6% 96.7% 96.1% 97.2% 97.7% 97.0% Ala 102.2% 102.2% 101.7% 95.7% 99.9% 99.5% 98.0% 101.1% 104.2% 101.5% 98.2% 107.6% 102.1% 101.7% 99.9% 98.9% 100.1% 99.7% 99.3% 100.7% Pro 101.0% 100.5% 103.5% 93.7% 97.4% 100.2% 95.5% 99.1% 101.4% 99.1% 94.3% 106.3% 98.8% 99.5% 98.4% 97.5% 99.0% 99.3% 97.0% 99.0% Lys 107.7% 106.2% 106.6% 98.9% 100.5% 100.6% 97.3% 105.8% 112.9% 100.5% 97.1% 104.3% 102.5% 101.9% 102.5% 102.1% 104.3% 104.1% 99.6% 102.9% Tyr 99.9% 99.9% 98.4% 93.9% 92.8% 96.1% 92.6% 94.1% 95.7% 97.9% 93.5% 102.2% 95.7% 97.3% 96.8% 97.2% 96.8% 99.1% 96.9% 96.7% xCys 102.0% 102.6% 103.3% 94.0% 96.4% 99.4% 96.6% 99.8% 99.7% 101.3% 102.2% 100.6% 98.7% 100.4% 100.8% 99.9% 101.8% 96.1% 97.2% 99.6% Met 88.7% 90.1% 94.6% 90.2% 89.6% 95.4% 89.8% 88.2% 90.2% 95.8% 90.2% 102.5% 93.9% 94.9% 92.7% 93.6% 92.7% 93.6% 90.8% 92.5% Val 94.0% 101.2% 100.5% 85.4% 101.5% 91.6% 94.3% 98.4% 100.0% 98.3% 95.3% 107.9% 100.5% 99.3% 98.1% 97.3% 99.8% 102.8% 93.4% 97.9% Ile 94.2% 101.0% 99.7% 87.0% 101.3% 89.8% 93.8% 97.7% 98.7% 101.2% 96.1% 107.6% 99.9% 99.6% 97.6% 97.5% 99.5% 102.1% 94.8% 97.9% Leu 100.2% 101.5% 102.2% 91.4% 98.9% 96.8% 95.8% 98.8% 102.0% 99.6% 96.4% 107.9% 101.5% 100.1% 99.0% 98.3% 99.9% 101.6% 95.7% 99.3% Phe 99.9% 100.8% 100.3% 93.0% 98.4% 96.8% 96.8% 95.9% 96.4% 100.2% 96.4% 108.2% 102.1% 100.0% 98.8% 98.2% 99.4% 100.8% 97.5% 98.9% SPIFAN II SLV kit product Analyte 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 av

Recovery data, color-coded with respect to performance requirement values.

Jaudzems, Guthrie, Lahrichi, and Fuerer – January 2018

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