Validated High-Throughput HPLC Method for the Analysis of Flavonol Aglycones Myricetin, Quercetin, and Kaempferol in Rhus coriaria L. Using a Monolithic Column

A rapid and simple reversed-phase high-performance liquid chromatographic method using a monolithic column was developed and validated for the separation and quantification of myricetin, quercetin, and kaempferol in Rhus coriaria L. The method employed the isocratic mobile phase acetonitrile-10 mM potassium dihydrogen orthophosphate buffer adjusted to pH 3.0 using orthophosphoric acid (38 + 62, v/v) at a flow rate of 4.0 mL/min; a Chromolith Performance RP-18e (100 x 4.6 mm) monolithic column kept at 40 degrees C; and UV detection at 370 nm. Although the elution order was identical and the selectivity was equivalent, the comparison between monolithic and particulate columns showed that the monolithic column could reduce the separation time to < 1 min without sacrificing column efficiency and selectivity. The method was validated according to International Conference on Harmonization guidelines. The validation characteristics included accuracy, precision, linearity, range, specificity, LOQ, and robustness. The calibration curves were linear (r > 0.999) over the concentration range of 0.88-88.3 micro/mL for myricetin, 0.95-95 microg/mL for quercetin, and 1.43-143.3 microg/mL for kaempferol. The recoveries for all three compounds were above 89%. Myricetin was found to be the major flavonol in the examined plant extracts, followed by minor quantities of quercetin and kaempferol.