Validation of spectrophotometric methods for determination of flavonoids in propolis

The increasing use of propolis preparations now days requires adequate analytical procedures for quantitative determination of main active components (different polyphenols, including flavonoids and phenolic acids). Rapid spectrophotometric methods have been used for years and are useful methods for routine control of propolis samples. The aim of this work was to improve and validate two spectrophotometric methods for determination of different groups of flavonoids in propolis. Flavones and flavonols form stable complexes with AlCl3, while flavanones and flavanonols react with 2, 4-dinitrophenylhidrazine (2, 4-DNP). Both given methods were optimized using a mixture of flavonoids. Galangin was later used to make the calibration curve for the method with AlCl3 (absorbance was measured at 415 nm), while pinocembrine was used as a standard for 2, 4-dinitrophenylhidrazine method (λ = 495 nm). After the conditions were optimized, we have investigated the following validation characteristics: linearity, accuracy, repeatability, intermediate precision, stability, and selectivity. To prove that improved and validated methods are suitable for intended purpose, we have analyzed nine ethanolic propolis extracts from different regions of Croatia to establish the differences in their composition. Investigated validation parameters for both methods satisfied the acceptance criteria for natural drug preparations (correlation coefficient ł 0.999 ; recovery – AlCl3 method 99.17 – 101.25 % and 2, 4-DNP method 98.67 – 101.46% ; repeatability (RSD(n = 6)) 1.61% and 4.77% ; intermediate precision (RSD(n = 18)) 3.21% and 4.03%). Tested colorimetric methods showed to be appropriate for establishing the differences in the composition of propolis samples (e.g. composition of propolis sample from peninsula Pelješac: mass concentration g(flavones, flavonols) = 320.95 mg/ml / RSD = 3.84%, g(flavanones, flavanonols) = 546.49 mg/ml / RSD = 3.28% ; composition of propolis sample from central Croatia: g(flavones, flavonols) = 54.87 mg/ml / RSD = 1.93%, g(flavanones, flavanonols) = 254.06 mg/ml / RSD = 3.24%).