Amino-01 (July 2018)

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

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PERFORMANCE CHARACTERISTICS

Method Performance Requirements (SMPR) According to their concentration in reconstituted powders or ready-to-feed liquids, analytes are categorized into three groups: 0.5-5.0, 5-150, and 150-2500 mg/100 g reconstituted product. Table 2 from AOAC SMPR 2014.013 (below) indicates the different requirements for each group. Table 2. Method performance requirements Parameter Minimum acceptable criteria Analytical range 0.4–2500b .4–2500b Limit of quantitation (LOQ) ≤0.4b 0.5-5 ±12 Recovery 5-150 ±10 150-2500 ±7 0.5-5 ≤7 Repeatability (RSD r ) 5-150 ≤5 150-2500 ≤3 0.5-5 ≤11 Reproducibility (RSD R ) 5-150 ≤8 150-2500 ≤5

a Concentrations apply to: (a) "ready-to-feed” liquids “as is”; (b) reconstituted powders (25 g into 200 g water); and (c) liquid concentrates diluted 1:1 by weight using water. b mg/100 g reconstituted final product. The results from the following compounds may fall slightly outside of the performance parameters due to the diverse nature of amino acids regarding the degradation, conversion, and/or hydrolysis: Threonine, Serine, Tyrosine, Methionine, Cystine/Cysteine, Valine, and Isoleucine.

Note: The requirements listed here correspond to the updated version proposed by the amino acid working group. This proposal will be reviewed by the stakeholder panel during the August 2018 annual meeting.

Linearity For each analyte, three standard curves with six to ten calibration levels between 0.25 and 50 pmol/µL were injected (1 µL). The concentration of the internal standard norvaline (Nva) was 10 pmol/µL. The calibration range (0.25 to 50 pmol/μL) corresponds to concentrations between 2.5 and 500 mg/100 g reconstituted final product (see section 4.3 for conversion details). Note: concentrations lower than 2.5 mg / 100 g reconstituted final product (as listed in the SMPR) were not tested. However, all samples tested were above this value (lowest value was 3.5 for taurine in sample 9). Sample dilution can be done if values fall above standard curves.

Concentration range

Slope

Intercept

Coefficient of determination R 2

Standard deviation of residuals

Analyte

Unit

Central value 0.982 0.948 0.901 1.834

Slope=0? (Y/N)

Central value -0.043 -0.194 0.190 0.049

Intercept=0? (Y/N)

Min

Max

Ala Arg Asp

pmol/µL pmol/µL pmol/µL pmol/µL

0.25 0.50 0.25 0.25

50 50 50

N N N N

Y Y Y Y

0.999 0.999 0.998 1.000

0.533 0.569 0.637 0.093

Cys2

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Jaudzems, Guthrie, Lahrichi, and Fuerer – January 2018

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