AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

Calibration fit A linear model forced through zero was used to fit the calibration data for all HMOs (Figure 5 - Figure 11), and the model appears to be a good fit. The relative residuals were calculated and plotted against the analyte concentration (Figure 5 - Figure 11), for all but two points the predicted concentration is within 5 % of the actual concentration. The two points beyond that limit were both for 6’SL, one point at 3.73 µg/ml (-7.3 %), and one point at 74.7 µg/mL (+5.9 %). For 2’FL and 3FL the plots of the relative residuals (Figure 5 and Figure 6) are not randomly distributed, there is a tendency for underestimation at low concentrations. However, the magnitude of the deviation is small enough that it does not have a significant impact on the analytical results.

B

A

Figure 5: Calibration fit for 2'FL. A: Calibration curve. B: Plot of relative residuals

A

B

6.0

4.000

3.500

y = 0.0055x R² = 0.9998

4.0

3.000

2.0

2.500

2.000

0.0

0

100 200 300 400 500 600 700

1.500

-2.0

1.000

Concentration (%)

Peak Area Ratio (3FL/IS)

0.500

-4.0

0.000

Difference Berween Actual and Calculated

0

100 200 300 400 500 600 700

-6.0

3FL Concentration (µg/mL)

3FL Concentration (µg/mL)

Figure 6: Calibration fit for 3FL. A: Calibration curve. B: Plot of relative residuals

20