Comprehensive structural characterization and purity assessment of 1-kestose: quantification of structurally related impurities via an advanced HPLC-CAD platform

The widespread use of fructooligosaccharides (FOS) as prebiotics has increased the need for accurate quantification methods to ensure food quality. Since certified reference materials (CRMs) are essential for establishing metrological traceability for FOS analysis, a purity CRM for 1-kestose—the main component of FOS—was developed. Due to its complex structural features, such as regioisomeric glycosidic linkages and anomeric configurations, 1-kestose was characterized using two-dimensional nuclear magnetic resonance spectroscopy (NMR). Purity was determined using quantitative NMR (qNMR) and mass balance (MB) methods, yielding a certified value of 991 mg/g±3 mg/g ( k = 2) with metrological traceability to the International System of Units for mass (kg) and amount of substance (mol). MB analysis revealed 9.3 mg/g of structurally related impurities, and liquid chromatography coupled with a charged aerosol detector (HPLC-CAD) identified sucrose (2.7 mg/g) and nystose (5.4 mg/g) as the dominant impurities. Furthermore, a novel analytical strategy was developed to quantify structurally related impurities using the calibration curve of the principal component (1-kestose) obtained by HPLC-CAD. Compared with the traditional external standard method, this approach exhibited a bias of less than 0.3 mg/g, highlighting its potential for determining the purity of other oligosaccharides in the FOS family and related compounds. • A certified reference material of 1-kestose was firstly developed. • Comprehensive characterization of 1-kestose was conducted by 2D NMR. • A purity of 991 mg/g was established by qNMR and mass balance. • Quantification of structurally related impurities by HPLC-CAD.