Analysis of Six HumanMilk Oligosaccharides (HMO) in Infant Formula and Adult Nutritionals by 2AB Labeling and Quantification with HILIC-FLD: First Action 2022.02.

This article presents the development and single-laboratory validation of AOAC Official MethodSM 2022.02 for quantifying six human milk oligosaccharides (HMOs)—2’-fucosyllactose (2’ FL), 3-fucosyllactose (3FL), 3’-sialyllactose (3’ SL), 6’-sialyllactose (6’ SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT)—in infant formula and adult nutritional products. The method employs 2-aminobenzamide (2AB) fluorescent labeling combined with hydrophilic interaction liquid chromatography and fluorescence detection (HILIC–FLD), providing a non-toxic derivatization and automated removal of excess reagent. Validation demonstrated that the method meets AOAC Standard Method Performance Requirements (SMPRs) for accuracy, precision, linearity, and specificity across various product matrices, with the exception of LNT, which required an elevated limit of quantification due to matrix interferences in some formulas. The method’s scope excludes certain matrices containing casein hydrolysates, galactooligosaccharides, or goat/sheep milk-derived Neu5Gc forms for specific analytes. This method was approved as a First Action Official MethodSM by the AOAC Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN).