AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

J. Calibration & Calculations

It is recommended to use bracketed calibration, injecting 6 standards followed by a maximum of 30 samples then 6 standards, etc. Use the instrument software to plot a six point calibration curve of against the concentration of HMO in µg/mL, forcing the line through the origin. For samples that do not contain GOS, the concentration of all HMOs can be determined from the results of preparation A For samples containing GOS: Concentrations of 2’FL, 3FL, LNT and LNnT should be determined from the results of preparation A. DFL, 3’SL and 6’SL should be determined from the results of preparation B For samples based on sheep milk or goat milk, or for elemental formula, follow the above guidelines, but 6’SL should be determined after running the analysis an additional time with the column temperature at 75°C The concentration (C D ) of the diluted sample is read from the calibration curve. The concentration of HMO in the ready to feed sample (or in reconstituted powders) is calculated according to equation 1. = × × 0.1 (1) Where, C RTF = Concentration of HMO in ready to feed sample or reconstituted sample in mg/100g C D = Concentration of HMO in the diluted sample read from the calibration curve in µg/mL V = volume to which the sample was diluted in mL m s = mass of sample diluted to volume V in g 0.1 = factor to convert µg/g to mg/100g For powdered products, the concentration of HMO in the powders can be calculated using equation 2 = × ( + ) (2) Where, C p = Concentration of HMO in powder samples in mg/100g C RTF = Concentration of HMO in the reconstituted sample in mg/100g (as calculated above) Validation design Table 5 summarizes the seven standard method performance requirements (SMPRs) for the seven HMOs. The validation experiments carried out were designed to test the method against those SMPRs. Calibration Fit was assessed by injecting calibration solutions of each HMO at 6 concentrations, adapted according to the HMO (Table 6), each level was prepared in triplicate. The ratio of the peak area of the HMO / the peak area of the internal standard was plotted against the concentration of the m p = mass of powder used to prepare reconstituted sample in g m w = mass of water used to prepare reconstituted sample in g

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