AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

B

A

0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 1.000 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 1.000

6.0

4.0

y = 0.0045x R² = 0.9997

2.0

0.0

0

50

100

150

200

250

-2.0

Concentration (%)

Peak Area Ratio (3'SL/IS)

-4.0

Difference Berween Actual and Calculated

0

50

100

150

200

250

-6.0

3'SL Concentration (µg/mL)

3'SL Concentration (µg/mL)

Figure 10: Calibration fit for 3’SL. A: Calibration curve. B: Plot of relative residuals

B

A

8.0

6.0

y = 0.0041x R² = 0.9989

4.0

2.0

0.0

0

50

100

150

200

250

-2.0

Concentration (%)

Peak Area Ratio (6'SL/IS)

-4.0

-6.0

Difference Berween Actual and Calculated

0

50

100

150

200

250

-8.0

6'SL Concentration (µg/mL)

6'SL Concentration (µg/mL)

Figure 11: Calibration fit for 6’SL. A: Calibration curve. B: Plot of relative residuals

Limit of detection and limit of quantification (LOD/LOQ) The LoD and LoQ were estimated by spiking low levels of each HMO in the different matrices as well as water. The level spiked represented about 10% of the desired LoQ specified in the SMPRs. The spiked samples were analysed 10 times and the mean and SD were combined to estimate LoD and LoQ. Three of the matrices tested were based on cow milk, and thus contained inherent amounts of 3’SL and 6’SL which were too high to enable us to estimate an LoD and LoQ. Thus for these two HMOs, LoD and LoQ were estimated only in a soy-based formula and in water. Based on the data for the other HMO, it seems likely that the estimate in soy-based formula should provide a good estimate for other types of formula. In general the estimated LoQs for all the HMOs meet the demands of the SMPR (Table 9). The only exception to this is DFL, for which there are some interferences in the chromatograms. We have identified that the laminaritriose internal standard used during these experiments contained a minor impurity which co-eluted at the retention time of DFL. That impurity contributed to the increased LoQ for DFL, however there are also matrix-based interferences which further increase the LoQ. The worst estimate for LoQ of DFL was 1.3 mg/100g. While this is higher than the desired LoQ of 1.2 mg/100g it

22