AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

Limits of Detection (LOD) and Quantification (LOQ ) were estimated by spiking the HMOs at approximately 10% of the desired LoQ in to blank formula (4 different matrices) and water (Table 7). The spiked samples were analysed ten times each to establish a mean and standard deviation for each. The LoD and LoQ were then estimated following equations 3 and 4 [27]. The milk-based formula contained native levels of 3’SL and 6’SL which were too high to allow the LoD and LoQ estimation in those matrices. Therefore, the estimation of LoD and LoQ for 3’SL and 6’SL could only be estimated from the spikes in water and soy-based formula.

= + × 3 (3) = + × 10 (4)

Where, LoD = limit of detection LoQ = limit of quantification = the mean concentration of HMO determined in the 10 replicates SD HMO = the standard deviation of the mean HMO concentration determined in the 10 replicates

Table 7: Spike levels used for estimation of LoD and LoQ

HMO Spike (mg/100g) a

2’FL

3FL

DFL

3’SL

LNT

LNnT

6’SL

Cow Milk Formula with FOS Formula with Partially Hydrolysed Protein Formula with Extensively Hydrolysed Protein

0.328

0.271

0.100 0.000 b 0.113

0.335 0.000 b

0.328

0.271

0.100 0.000 b 0.113

0.335 0.000 b

0.326

0.270

0.100 0.000 b 0.112

0.333 0.000 b

Soy Based Formula

0.318 0.327

0.263 0.271

0.097 0.100

0.113 0.101

0.110 0.113

0.325 0.335

0.150 0.131

Water

a Concentrations expressed as concentration found in reconstituted powders (25 g + 200g water) or “ready-to feed” liquid. b The milk-based formula contain native concentrations of 3’SL and 6’SL, which are too high for estimating LoD and LoQ using low level spikes.

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