AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

Recovery was assessed by spiking eight blank matrices with the seven HMOs at four different concentrations covering the desired range for each HMO specified in the SMPRs (Table 8). Spiking was performed by adding concentrated solutions of the HMOs, or adding powdered HMOs for high concentration levels. If the matrices were powders, they were reconstituted prior to spiking. If RTF, the matrices were spiked directly. After spiking the samples were homogenized, aliquoted, and stored frozen until analysis. None of the matrices contained native levels of 2’FL, 3FL, DFL, LNT or LNnT, therefore the recovery could be calculated according to equation 5. Most of the matrices contained native levels of 3’SL and 6’SL therefore the recovery was calculated after subtracting the amount of the HMO measured in the non-spiked sample according to equation 6. = × 100 (5) = ( − ) × 100 (6) Where, Rec = the spike-recovery expressed in % C m = the concentration of HMO measured in the spiked sample in mg/100 g C S = the concentration of HMO spiked into the sample in mg/100 g C n = the native concentration of HMO in the non-spiked sample in mg/100 g

Table 8: Matrices used for spike-recovery experiments

Spike Level 1 (mg/100g) a

Spike Level 2 (mg/100g) a

Spike Level 3 (mg/100g) a

Spike Level 4 (mg/100g) a

Matrix

HMO

2’FL 3FL DFL LNT

5.93 4.49 1.66 2.24 5.56 3.66 2.23 5.17 3.72 1.54 1.96 5.35 1.04 1.77 4.87 4.22 1.59 2.01 6.08

20.2 30.0 10.5 20.2 22.2 22.3 24.4 20.5 31.0 10.3 21.3 23.4 26.1 24.6 20.7 31.2 10.6 20.6 23.1 17.7 21.2

101 84.5 52.6 147 74.8 74.6 80.2 102 83.3 50.3 148 75.6 75.0 78.1 103 82.4 50.9 147 73.9 73.9 78.4

504 566 98.7 287 103 139 142 499 576 99.3 290 103 144 142 511 585 103 294 103 144 148

Adult Nutritional RTF with FOS & GOS

LNnT 3’SL 6’SL

2’FL 3FL DFL LNT

Infant Formula Elemental Powder b

LNnT 3’SL 6’SL

2’FL 3FL DFL LNT

Infant Formula Powder with FOS & GOS [1] b,c

LNnT 3’SL 6’SL

0.0 0.0

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