Determination of Polyphenols in Honey Using Liquid–Liquid Extraction and HPLC-PDA with MS Confirmation

Polyphenols are present in honey due to the collection of nectar and pollen. These bioactive compounds may be used for assessing honey’s composition, antioxidant capacity and overall quality. This study presents the development and validation of an optimized liquid chromatography coupled with photodiode array detection with mass spectrometry confirmation (HPLC-PDA-MS) method for the determination of eight phenolic acids (chlorogenic acid, rosmarinic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, vanillic acid, and gallic acid) and four flavones (apigenin, rutin, luteolin, and quercetin) in diverse honey matrices. Method optimization focuses on improving chromatographic resolution, ionization efficiency and sensitivity through fine-tuning of mobile phase composition, gradient profile, and MS parameters. Validation followed international guidelines, evaluating linearity, accuracy, precision, limits of detection (LOD), limits of quantification (LOQ), recovery, and matrix effects. The method demonstrated excellent linearity (R2 ≥ 0.998), precision below 5%, and recoveries ranging from 85% to 110% across all the tested polyphenols. Repeatability test, assessed by injecting mixed standard solution six times in a row, resulted in RSD values below 3%. The LOD and LOQ values were in the low ng/g range, confirming the method’s high sensitivity. The optimized method was tested on four honey samples with different botanical origins. For sample preparation, a liquid–liquid extraction (LLE) protocol was followed, and the obtained chromatograms showed differences in phenolic composition. Overall, the validated HPLC-PDA-MS method provides a robust, sensitive, and reproducible analytical tool for the reliable quantification of polyphenols in honey. Its applicability across multiple honey types supports its use in routine quality control, authenticity verification, and research on honey’s bioactive profile.