NADA GRAHOVAC , MILICA ALEKSI´C , LATO PEZO , ANA ÐUROVI´C , ZORICA STOJANOVI´C , JELENA JOCKOVI´C , SANDRA CVEJI´C
Sunflower (Helianthus annuus L.) is a cross-pollinated species whose reproductive success depends heavily on pollinator attraction, largely influenced by nectar composition. Nectar is predominantly composed of sugars (up to 70%), mainly sucrose, glucose, and fructose, with minor sugars such as mannose, arabinose, xylose, and sugar alcohols (e.g., mannitol, meso-inositol) present in smaller quantities that vary with environmental and biological factors. In this study, a robust high-performance liquid chromatography method with refractive index detection (HPLC-RID) was developed and optimized for the simultaneous quantification of eight sugars (D-ribose, xylose, arabinose, fructose, mannose, glucose, sucrose, and maltose) and two sugar alcohols (mannitol and meso-inositol) in wild sunflower nectar.
Method optimization was performed using a Box–Behnken design (BBD) integrated with response surface methodology (RSM) to evaluate the effects and interactions of three critical parameters—column temperature (20–23 °C), acetonitrile concentration (80–85%), and flow rate (0.7–1.0 mL/min). The optimized conditions (20 °C, 82.5% acetonitrile, and 0.766 mL/min flow rate) achieved complete baseline separation (Rs > 1) of all analytes, including previously challenging co-eluting pairs such as glucose/mannitol and glucose/mannose.
The method demonstrated excellent linearity over 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. Limits of detection (LOD) and quantification (LOQ) ranged from 4.04 to 19.46 mg/L and 13.46 to 194.61 mg/L, respectively. Among the analytes, glucose showed the highest sensitivity (LOD = 4.04 mg/L; LOQ = 13.46 mg/L), while mannose exhibited the lowest (LOD = 19.46 mg/L; LOQ = 194.61 mg/L).
This validated, cost-effective, and environmentally friendly HPLC-RID method provides a reliable analytical tool for comprehensive sugar and sugar alcohol profiling in sunflower nectar and can be readily adapted for other botanical and food matrices following appropriate sample preparation.
Standard sugars and sugar alcohols — D-ribose, xylose, arabinose, mannose, mannitol, sucrose, and maltose — were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany), while fructose, glucose, and meso-inositol were obtained from Merck KGaA (Steinheim, Germany).
Individual stock solutions (10,000 µg/mL) of each compound were prepared by dissolving 0.02 g of analyte in 2 mL of HPLC-grade distilled water (Milli-Q, Millipore). Mixed working solutions containing all ten sugars and sugar alcohols were subsequently prepared at concentrations ranging from 100 to 500 µg/mL by serial dilution with Milli-Q water. Each working solution contained equal concentrations of all analytes.
HPLC-grade acetonitrile used in the mobile phase was obtained from J.T. Baker (Deventer, The Netherlands), and ultrapure water (Milli-Q) was used throughout the analyses.
Nectar samples were collected from tubular florets of wild sunflower (Helianthus annuus L.) plants cultivated during the summer of 2024 in the experimental fields of the Institute of Field and Vegetable Crops, Novi Sad, Serbia (Rimski Šančevi location).
Sugars and sugar alcohols were analyzed using an LC-2050C 3D HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a RID-20A refractive index detector (Shimadzu, Japan). Chromatographic separation was performed on a Nucleosil 100-5 NH₂ column (250 mm × 4.6 mm i.d., 5 µm particle size; Macherey-Nagel GmbH & Co. KG, Düren, Germany).
Data acquisition, integration, and processing were carried out using LabSolutions software (version 5.117, Shimadzu Corporation, Kyoto, Japan).
Prior to analysis, nectar samples were centrifuged using a LACE16R refrigerated centrifuge (COLO Lab Experts, Novo Mesto, Slovenia) and filtered through CHROMAFIL® RC syringe filters (0.45 µm pore size, 25 mm diameter; Macherey-Nagel, Düren, Germany).
Optimization of chromatographic conditions was conducted using a Box–Behnken design (BBD) implemented in Design Expert software (trial version, Stat-Ease Inc., Minneapolis, MN, USA). The effects of three independent factors — column temperature (20–23 °C), acetonitrile concentration (80–85%), and flow rate (0.7–1.0 mL/min) — were examined, while resolution between critical sugar pairs served as the response variable.
Statistical analyses were performed using TIBCO Statistica 13.3.0 (Palo Alto, CA, USA; 2017) and Microsoft Excel (Microsoft Corp., Redmond, WA, USA). Three predictive models — Linear, Linear Model with Main Effects Two-Way, and Quadratic — were evaluated for their ability to predict two dependent variables: Rgm and Rmg .
Analysis of variance (ANOVA) was applied to determine the statistical significance of model parameters and their interactions. Model performance was further assessed using multiple statistical criteria, including Root Mean Square Error (RMSE), Mean Bias Error (MBE), Mean Percentage Error (MPE), Sum of Squared Errors (SSE), Average Absolute Relative Deviation (AARD), and the coefficient of determination (R²), to confirm the accuracy and reliability of the proposed models.