AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

Figure 2: Overlay of chromatograms of HMO standards (green dotted line) and a goat milk-based formula (blue solid line) showing the overlap of peaks for 6'SL and 3'NeuGcL at a column temperature of 60°C..

Other oligosaccharides which may be added to infant formula also have the potential to interfere with the analysis. Maltodextrins may be added to formula as an energy source, or as a carrier for other components (e.g. vitamins and minerals). Most of the peaks from maltodextrins do not interfere with the HMOs, however maltotetraose elutes close to LNT, and has potential to interfere if present in high amounts. Maltodextrins are easily and specifically removed by treatment with amyloglucosidase (AMG), so treatment with this enzyme has been routinely included in the sample preparation. Beta galactooligosaccharides (GOS) are a common component of many formula, but the oligosaccharide profile changes between suppliers due to the different processes used to produce them [26, 32]. We tested a number of different GOS ingredients to assess their impact and found that different GOS generated interferences with DFL, 3’SL or 6’SL (Figure 4). In order to overcome this problem an optional 2 nd sample preparation was introduced, in which a β -galactosidase was added along with the AMG to remove the GOS and the maltodextrins at the same time. However, the β -galactosidase also partially hydrolyses LNT and LNnT converting them both to lacto-N-triose (LNT-II). Therefore, it is necessary to determine LNT and LNnT following the procedure without β -galactosidase treatment, then 3’SL, 6’SL and DFL can be determined in the sample treated with β -galactosidase. GOS do not interfere with the analysis of 2’FL or 3FL and neither are impacted with the β -galactosidase treatment, and thus can be determined using either approach for sample preparation. Fructans (inulin or fructooligosaccharides) and polydextrose may also be added to formula as sources of non-digestible oligosaccharides. Since the fructans are predominantly non-reducing oligosaccharides or have a ketose at the reducing end, they are not labelled by the 2AB and thus do not interfere with the analysis. When a polydextrose ingredient was analysed, a series of signals eluted early in the chromatogram before 2’FL and did not co-elute with any of the signals of interest (Figure 3). However, an increased baseline noise was observed in the region of DFL (Figure 3), thus the presence of polydextrose may be an issue if DFL is being analysed close to the LoQ, but otherwise should not be a problem.

18