AOAC SPIFAN Nutrients ERP Reviewer Forms (December 7, 2022)

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method_HMO-05

Title: 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) Author: Philip Haselberger, Fang Tian, Renée Erney, Shuang Liu, Sho Wang, Qi Lin, Ding Yi Reviewer Name: Reviewer 1 Summary of Method: Powdered samples (25 g + 200g water) or liquid concentrates (5 g + 5 g) are reconstituted in water as outlined in the SMPRs. Then an aliquot (typically 1.5 g, but it may be adapted according to needs) is taken and transferred to a 25 mL volumetric flask. If samples contain fructans or maltodextrins, enzymes (fructanse and amyloglucosidase) are added and the volume made up to 25 mL with acetate buffer (pH 4.5). The flask is then incubated at 40°C for 45 min. If samples do not contain fructans or maltodextrins, the flask is made up to the volume with water and no enzymes are added. The prepared solutions are passed through a syringe filter into an autosampler vial and analysed by HPAEC PAD using a Carbopac PA1 column set and a gradient of sodium hydroxide and sodium acetate. Quantification is done against an external calibration of suitably controlled analytical standards, using preferably a quadratic calibration model (although the method states a linaer model may be applied).

Method Scope/Applicability: Quantification of 6 HMOs (2’-FL, 3-FL, 6’-SL, 3’-SL, LNT, and LNnT) in infant formula and adult nutritionals

General comments about the method: This is a fairly simple “dilute and shoot” method using HPAEC-PAD as the tool to make the measurements. HPAEC-PAD is a well-established technique for the determination of oligosaccharides and this approach should be straight-forward to implement in any lab with knowledge of such a system. The method also includes sufficient warnings about the quality of calibration standards and how to control them, which is a key aspect for HMO analysis.

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