AOAC SPIFAN Nutrients ERP (December 7, 2022)

HMO-02 (Analysis Paper) 10-2022 FOR ERP USE ONLY DO NOT DISTRIBUTE

HMO. A linear model forced through the origin was used to fit the data for calibration purposes. The relative residuals were calculated and plotted against the analyte concentration.

Table 5: Summary of Method Performance Requirements for HMO Analysis

Conc. Range 1 (mg/100g)

LoQ 1 (mg/100g)

Recovery (%) 85 - 110 90 - 110 85 - 110 90 - 110 85 - 110 90 - 110 85 - 110 90 - 110 85 - 110 90 - 110 85 - 110 90 - 110 85 - 110 90 - 110

RSD r (%)

RSD R (%) ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10 ≤ 10

SMPR

HMO

2020.003 2020.003 2020.004 2020.004 2021.004 2021.004 2021.006 2021.006 2021.007 2021.007 2021.003 2021.003 2021.005 2021.005

2’FL 2’FL

5.0 - 20 >20 - 500 5.0 - 20 >20 - 100 4.0 - 20 >20 - 600 1.5 - 20 >20 - 100 2.0 - 20 >20 - 300 1.5 - 20 >20 - 150 2.0 - 20 >20 - 150

≤ 4.0 ≤ 4.0 ≤ 4.0 ≤ 4.0 ≤ 3.2 ≤ 3.2 ≤ 1.2 ≤ 1.2 ≤ 1.6 ≤ 1.6 ≤ 1.2 ≤ 1.2 ≤ 1.6 ≤ 1.6

≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5 ≤ 5

LNnT LNnT

3FL 3FL DFL DFL LNT LNT 3’SL 3’SL 6’SL 6’SL

≤ 10

≤ 10 1. Concentrations apply to i) “ready to feed” liquids “as-is”, ii) reconstituted powders (25 g + 200g water), iii) liquid concentrates diluted 1:1 by weight

Table 6: Concentration of HMO Solutions used to Assess Calibration Fit

Level 1 (µg/mL)

Level 2 (µg/mL)

Level 3 (µg/mL)

Level 4 (µg/mL)

Level 5 (µg/mL)

Level 6 (µg/mL)

HMO

2’FL 3FL DFL LNT

10.3 10.5 4.33 6.90 8.98 3.24 3.73

113 116 30.3 60.4 44.9 30.4 35.0

227 232 60.6 121 89.8 64.8 74.7

350 358 95.3 190 135 105 121

494 505 130 259 180 146 168

618 632 173 345 224 194 224

LNnT 3’SL 6’SL

12