Establishing homogeneity in a candidate honey certified reference material for sugars and hydroxymethylfurfural: insights into a complex natural matrix

Abstract Honey is a complex natural matrix requiring stringent quality control of its sugar profile and hydroxymethylfurfural (HMF). The development of certified reference materials (CRMs) for honey is essential to ensure analytical accuracy and traceability, with homogeneity being a fundamental requirement that directly affects certification validity. This study evaluates the within- and between-bottle homogeneity of a candidate honey CRM (Acacia hamulosa) intended for simultaneous certification of fructose, glucose, sucrose, and HMF. Following homogenization and bottling, sugars and HMF were determined using HPLC-RID and HPLC-PDA, respectively. Within-bottle assessment (n = 7) showed satisfactory reproducibility for fructose (RSD 2.79%), glucose (2.82%), and HMF (0.84%), while sucrose exhibited higher variability (5.87%). Between-bottle evaluation (n = 7) confirmed homogeneity, with RSD values of 1.83% (fructose), 1.86% (glucose), 4.61% (sucrose), and 1.85% (HMF). The analysis of variance (ANOVA) indicated no significant between-bottle variation except for sucrose. Multivariate analysis, including hierarchical cluster analysis (HCA) and principal component analysis (PCA), supported these findings, showing that variability arises from natural sugar composition, while HMF remained uniformly distributed. This study provides new insights into the homogeneity behaviour of major and trace-level analytes in a complex natural matrix and demonstrates that the candidate material satisfies the ISO 17034 homogeneity requirements.