Box–Behnken-Assisted Optimization of High-Performance Liquid Chromatography Method for Enhanced Sugar Determination in Wild Sunflower Nectar

Sunflower (Helianthus annuus L.) is a cross-pollinated species that relies on pollinators, attracted by itsnectar composition. Nectar consists primarily of sugars (up to 70%), with sucrose, glucose, and fructose being dominant, while minor components such as mannose, arabinose, xylose, and sugar alcohols (e.g., mannitol and inositol) occur in lower concentrations and vary with biotic and abiotic factors. This study developed a robust high-performance liquid chromatography method with refractive index detection (HPLC-RID) for the simultaneous quantification of eight sugars (D-ribose, xylose, arabinose, fructose, mannose, glucose, sucrose, and maltose) and two sugar alcohols (mannitol, meso-inositol) in wild sunflower nectar. A Box–Behnken design (BBD), coupled with response surface methodology (RSM), was used to systematically optimize column temperature (20–23 °C), acetonitrile concentration (80–85%), and flow rate (0.7–1 mL/min), while achieving baseline separation of critical sugar pairs, including the previously co-eluting glucose/mannitol and glucose/mannose. Satisfactory resolution (Rs > 1 for all analytes) was achieved under optimized separation conditions comprising a column temperature of 20 °C, 82.5% acetonitrile, and a flow rate of 0.766 mL/min. The RSM efficiently evaluated factor interactions to maximize chromatographic performance, resulting in an optimized protocol that provides a cost-effective and environmentally friendly alternative to conventional sugar analysis methods. Method validation confirmed satisfactory linearity across relevant concentration ranges (50–500 mg/L for most sugars; 50–5500 mg/L for fructose and glucose), with correlation coefficients (R) between 0.985 and 0.999. The limits of detection (LOD) and quantification (LOQ) for the analyzed sugars and sugar alcohols ranged from 4.04 to 19.46 mg/L and from 13.46 to 194.61 mg/L, respectively. Glucose exhibited the highest sensitivity showing LOD of 4.04 and LOQ of 13.46 mg/L, whereas mannose was identified as the least sensitive analyte, with LOD of 19.46 mg/L and LOQ of 194.61 mg/L. The described method represents a reliable tool for sugar and sugar alcohol analysis in sunflower nectar and can be extended to other plant and food matrices with suitable sample preparation.