AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

Repeatability (r) and intermediate reproducibility (iR) were assessed by analyzing the eight spiked samples, and seven commercial or pilot plant samples in duplicate on at least 6 different days by four different operators on three different instruments. The in-house statistical package Q-Stat, was used to calculate the robust SD rob (r) and SD rob (iR) using equations 8 and 9

( ) = 1.0484 × {| 1 − 2 |} =1 ,.. (8) ( ) = � 2 ( ) + 1 2 × 2 ( ) (9)

Where: n is the number of (single or duplicate) determinations

x i is the individual result within the set of single determinations with i going from 1 to n ; x i1 and x i2 are the two results within the set of duplicate determination with i going from 1 to n ; SD rob (b) is the robust standard deviation between the means of duplicates SD rob (r) is the robust standard deviation of repeatability SD rob (iR) is the robust standard deviation of intermediate reproducibility

RESULTS Method Development

We previously developed a method for the determination of oligosaccharides in human milk [15], and a method for the determination of 2’FL and LNnT in infant formula [20]. In both methods, the HMO were derivatized with 2AB, as described previously for N-glycan analysis [25]. The label is required for detection of the oligosaccharides which do not contain a chromophore or fluorophore and has been demonstrated to be incorporated in a quantitative manner [25]. It also introduces a certain amount of selectivity since the oligosaccharides containing an aldose at the reducing end are derivatized, while those with a ketose at the reducing end and non-reducing oligosaccharides are not derivatized. The method developed here follows the same principle as the previous methods, however the conditions for 2AB labelling have been modified, replacing the reducing agent sodium cyanoborohydride with picoline borane [28, 29]. Picoline borane has several advantages over sodium borohydride; it is less toxic [29], the reaction can be carried out in aqueous conditions [28], and the reaction time is shorter (1 hour instead of 2). After labelling with 2AB, the excess reagents used for the reaction are normally removed prior to chromatographic analysis. In our previous method, the excess reagents were removed by loading the sample on the guard column and washing away the excess reagents before switching the guard in-line with the analytical column for the analysis [15, 20, 30]. However, when running with UHPLC and using small injection volumes, it is possible to avoid this step, thus simplifying the method and the equipment requirements. Although everything is injected on the column, it is possible to achieve good separation of the excess reagents from the analytes, and we have not noticed a significant deterioration of the column performance or lifespan.

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