Method for the Determination of 2'-Fucosyllactose (2'-FL), 3-Fucosyllactose (3-FL), 6'-Sialyllactose (6'-SL), 3'-Sialyllactose (3'-SL), Lacto-N-Tetraose (LNT), and Lacto-N-neoTetraose (LNnT) by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD): First Action 2022.04.

BACKGROUND: Method for simultaneous determination of six human milk oligosaccharides (HMOs) is described. The HMOs include 2'-fucosyllactose (2'-FL, CAS number 41263-94-9), 3-fucosyllactose (3-FL, CAS number 41312-47-4), 6'-sialyllactose (6'-SL, CAS number 35890-39-2), 3'-sialyllactose (3'-SL, CAS number 35890-38-1), lacto-N-tetraose (LNT, CAS number 14116-68-8), and lacto-N-neotetraose (LNnT, CAS number 13007-32-4). The method was designed to comply with the respective Standard Method Performance Requirements (SMPR, see also Table 1) respectively.

OBJECTIVE: Method is valid for six HMOs in infant formula and adult nutritional matrices, including samples with intact protein, protein hydrolysates, elemental formulations free of intact protein, and rice flour over the ranges defined in the SMPR (see also Table 2). Method not valid for determination of difucosyllactose (DFL/DiFL).

METHOD: For most samples, reconstitution with water followed by filtration. For products containing interferences (fructans and maltodextrins), hydrolysis with enzymes is used. After preparation, samples are analyzed using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The method provides for separation of six HMOs and other carbohydrates commonly found in infant formula and adult nutritional products (e.g., lactose, sucrose, and GOS).

RESULTS: This study includes data from multiple matrices evaluated by multiple labs globally. RSDr ranged from 0.0068-4.8% RSDr and spike recovery results ranged from 89.4-109%. Calibration fit optimal with a quadratic curve; alternately linear fit showed no statistically significant impact to data (when correlation passes).

CONCLUSION: This method was reviewed by the AOAC SPIFAN Expert Review Panel (ERP) and determined to meet the SMPRs for the six noted HMOs.

HIGHLIGHTS: The method was granted First Action Official MethodsSM status.