Amino-01 (July 2018)

Amino-01 (JULY 2018) 180124 AA SPIFAN SLV Appendix J

FOR ERP USE ONLY DO NOT DISTRIBUTE

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

SelectSPIFAN II samples subjected to nitrogen flush priorothydrolysis.

Sample 1 Child formulapwd 6 NIST1849a

7 InfantFormulaPowderPartially Hydrolyzed Milk Based 8 InfantFormulaPowderPartially Hydrolyzed Soy Based

11 AdultNutFormula low fat 16 InfantFormPWDsoy based NIST1869beta

Sample:

Original validation

Sparged with nitrogen trial 1

Sparged with nitrogen trial 2

1 Child formula pwd

resultnot sparged

% Gain or loss adding in nitrogen sparge prior to hydrolysis …ornegligible difference

%Recovery

%Recovery

%Recovery

avg.ofdups

avg.ofdups

avg.ofdups

%Recovery

%Recovery

AMINO ACIDS

Spike 1

Spike 1

Spike 2

Spike 1

Spike 2

(mg/100g)

(mg/100g)

(mg/100g)

0.384

0.393

5.1

98%

96%

94%

112%

111%

0.3975

His

-

0

-

100% 109%

-

-

0%

0%

0

Tau Ser Arg Gly Asp Glu Thr Ala Pro Lys Tyr Cys Met Val

0.746

0.71

-15.1

92% 94% 98% 98% 90% 97% 96% 96% 94% 92% 97% 97% 98% 98% 101% -

96% 94% 95% 97% 91% 91% 95% 94% 95% 95% 94% 94% 93% 94% 95% -

91% 98%

93% 96%

0.893

0.5625

0.589 0.328 1.357 3.167 0.669 0.563 1.315 1.208 0.689 0.124 0.968 0.883 1.476 0.735 0.35

-0.9 -1.7 -9.3 -8.1 -6.7 -4.0 -4.0 -9.6 -4.4

97%

0.5765

0.32

100% 113% 107% 100% 102% 101% 108% 100% 102%

102% 106% 104% 101% 100% 100% 93%

100% 105% 101%

0.334 1.411

1.311

3.0605 0.6685 0.5465

3.2405 0.7075 0.5765 1.3145

99%

100%

1.279 1.176

98% 98% 96% 97% 93%

1.265

0.6615

97% 90% 94%

0.6875

-

-

0.161 0.328 0.959

0.3435

4.8 4.4 4.0

89% 94% 94%

0.925 0.835 1.425

101% 102% 100% 101%

101% 101%

0.85

Ile

-2.4 -1.2

100% 100%

99%

1.4875

Leu Phe

0.6985

100%

0.724

% Gain or loss adding in nitrogen sparge prior to hydrolysis

Sample:

Original validation

Sparged with nitrogen trial 1

Sparged with nitrogen trial 2

6 NIST 1849a

resultnot sparged

…ornegligible difference

%Recovery

%Recovery

%Recovery

avg.ofdups

avg.ofdups

avg.ofdups

Recovery from NIST CoA

Recovery from NISTCoA

Recovery from NISTCoA

%Recovery

%Recovery

AMINO ACIDS

Spike 1

Spike 1

Spike 2

Spike 1

Spike 2

(mg/100g)

(mg/100g)

(mg/100g)

0.316 35.76 0.575 0.374 0.243 1.075 2.623 0.454 1.147 1.033 0.553 0.448 0.828 0.749 1.234 0.581 0.58 -

100.4

0.313

99.5 96.0 88.6

3.7 1.0

96% 98% 88% 93% 97% 98% 92% 97% 97% 97% 94% 92% 97% 96% 98% 98% -

95% 97% 91% 97% 97% 97% 94% 97% 98% 97% 96% 95% 95% 95% 97% 97% -

97% 99% 93% 96%

96% 98% 91% 97%

93% 97%

0.3085 37.1995 0.6505

98.0

His

97.7 79.9 93.5

35.146

101.6

Tau Ser Arg Gly Asp Glu Thr Ala Pro Lys Tyr Cys Met Val

0.638 0.408 0.245 1.133 2.741 0.595 0.473 1.176 1.105 0.575 0.131 0.457 0.847 0.764 1.268 0.592

-12.7

104%

90.3 97.3

102.0 101.7 105.9 105.8

0.9 1.0

95% 98%

0.389

100.8 100.5 101.3

101% 106% 103%

101% 107% 102% 102% 100% 106% 95%

0.2425 1.1425

100.6 106.8 106.3

-1.5 -3.2 -6.2

105% 103% 100% 100% 100% 101%

101% 101%

2.754 0.615 0.469

90.6 99.8 96.0

93.0

93%

96.1

104.0

0.1

102%

103.1

98.4

-1.8

99%

1.1645

97.4

102.3 108.4

109.4 112.7 101.9 104.6 111.4 115.8 100.6 102.1

0.8

105%

1.056

104.6 110.7 101.9 103.0 110.1 112.7

-1.3

96% 99% 95% 92% 90% 97% 97%

95%

95%

0.5645

-

7.3

103%

110%

0.131

102.5 108.9 113.5

-1.5

94%

94%

0.45

7.5 9.6 2.3 2.3

101% 101% 101% 100%

101% 102% 101% 100%

0.8365

0.744

Ile

97.9

1.2595

99.9

Leu Phe

100.2

0.593

102.2

% Gain or loss adding in nitrogen sparge prior to hydrolysis

Sample: 7 Infant Formula Powder Partially Hydrolyzed Milk Based

Original validation

Sparged with nitrogen trial 1

Sparged with nitrogen trial 2

resultnot sparged

%Recovery

%Recovery %Recovery

%Recovery %Recovery

avg.ofdups (mg/100g)

avg.ofdups (mg/100g)

AMINO ACIDS

…ornegligible difference

Spike 1

Spike 1

Spike 2

Spike 1

Spike 2

0.218

12.2

100% 97% 93% 92% 98% 99% 95% 98% 96% 97% 93% 97% 90% 94% 94% 96% 97% 105%

- - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - -

114%

109%

0.212

His

45.553

-

-

-

41.3015

Tau Ser Arg Gly Asp Glu Thr Ala Pro Lys Tyr Cys Met Val

0.45

-1.3

87% 97% 99%

97% 97% 99%

0.595 0.287

0.305

5.2 1.7

0.23

0.2255 1.4045

1.358 2.111 0.732 0.596 1.091 0.337 0.217 0.239 0.727 0.822 1.256 0.389 0.69

-0.2 2.9 3.0 1.7 3.2 2.0 4.6 4.8 3.9 3.9 4.1 4.5 -4.4

106% 102%

104% 101% 100%

2.157

95%

0.8165 0.6025 0.6915 1.1045

100%

99% 98% 99% 97% 95% 94% 97% 96% 99%

99% 99% 97% 89% 94% 99% 99%

0.337 0.302 0.238

0.7035

0.77

Ile

100% 101%

1.256

Leu Phe

101%

0.3945

Jaudzems, Guthrie, Lahrichi, and Fuerer – January 2018

page 2/6