Development and Validation of a RP-HPLC Method for the Simultaneous Quantification of Flavonoids Markers in Mexican Propolis

Abstract

Acacetin (1), 4′,7-dimethyl naringenin (2), and 4′,7-dimethyl apigenin (3)—three of the main markers active components of propolis—were chosen for the development and validation of a suitable HPLC method for quality control of the crude drug. A 2³ factorial design was employed to study the main interactions of independent variables, and the efficient separation was achieved using an XBridge C₁₈ column with an isocratic binary phase consisting of deionized water (TFA; 0.1 % v/v) (eluent A) in methanol (eluent B) (45:65, v/v) at a flow rate of 1.0 mL min⁻¹. The method allows the quantification of 1–3; for each compound, a linear response was evaluated within the range of 100–1,000 μg mL⁻¹ for 1, 25–200 μg mL⁻¹ for 2, and 8–60 μg mL⁻¹ for 3. All the calibration curves showed good linearity within the test ranges (r ² ≥ 0.999). Percentage recoveries of the selected flavonoids 1–3 ranged between 98.3 and 101.2 % (RSD ≤2.0 %). The intra- and interday precision RSDs were no more than 2.0 %, while the repeatability variation was no more than 2.0 %. Finally, the method was applied to establish some seasonal and geographical variations of the markers in propolis obtained from several regions of Mexico. Thus, 4′,7-dimethyl apigenin (3) was selected as a marker compound from Mexican propolis, and, along with 2, could be useful for quality control procedures focused in to establish some geographical variation of Mexican propolis.