Abstract: To meet the traceability requirements for detecting fructooligosaccharide in foods, a purity certified reference material of 1F-β-fructofuranosyl nystose was developed. The candidate certified reference material of 1F-β-fructofuranosyl nystose was structurally characterized using triple quadrupole mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. One- and two-dimensional NMR spectroscopy including 1H and 13C, 1H-1H correlation spectroscopy (COSY) and heteronuclear single-quantum coherence (HSQC) were employed to systematically elucidate the glycosyl composition, stereochemical configurations, and glycosidic linkages of 1F-β-fructofuranosyl nystose. Its structure was confirmed as α-D-glucopyranosyl-(1→2)-β-D-Fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl. Quantitative analysis of the certified reference material was conducted by the mass balance (MB) method and quantitative nuclear magnetic resonance (qNMR) method. In the MB analysis, structurally related impurities were analyzed using high performance liquid chromatography coupled with a charged aerosol detector (HPLC-CAD). Under optimized chromatographic conditions, baseline separation between fructosyl-oligosaccharide isomers and 1F-β-fructofuranosyl nystose was achieved. Moisture content was measured using Karl Fischer titration. Volatile components were analyzed by headspace gas chromatography with a flame ionization detector (GC-FID), and non-volatile components by inductively coupled plasma-mass spectrometry (ICP-MS). The major component purity was calculated by deducting the sum of all quantified impurities. For the qNMR method, deuterium oxide was used as the solvent, with potassium hydrogen phthalate as the internal standard, and the characteristic proton signals of 1F-β-fructofuranosyl nystose were selected for integration to calculate the purity. The final certified purity value was obtained as the average of the results from these two methods. The homogeneity and stability of the reference material were evaluated using HPLC-CAD. Uncertainties arising from value assignment, homogeneity, and stability were systematically assessed. Finally, the certified purity value of 1F-β-fructofuranosyl nystose was determined to be 92.52% with a relative expanded uncertainty of 0.50% (k = 2). This certified reference material can serve as a reliable basis for metrological traceability in fructooligosaccharide determination.

Key words: 1F-β-fructofuranosyl nystose; certified reference material; charged aerosol detector; nuclear magnetic resonance spectroscopy; fructooligosaccharide; prebiotic