DIETARY SUPPLEMENTS Validated High-Throughput HPLC Method for the Analysis of Flavonol Aglycones Myricetin, Quercetin, and Kaempferol in Rhus coriaria L. Using a Monolithic Column MORTEZA MEHRDAD and MAHNOOSH ZEBARDAST Shahid Beheshti University, Environmental Science Research Institute, Department of Environmental Pollutants, Evin 1983963113, Tehran, Iran GHAZALEH ABEDI Iran Polymer & Petrochemical Institute, Novel Drug Delivery Systems Department, PO Box 14185-458, Tehran, Iran MITRA NOURI KOUPAEI and HODA RASOULI Shahid Beheshti University, Faculty of Science, Department of Chemistry, Evin 1983963113, Tehran, Iran MOHAMMAD TALEBI 1 Kimiafaam Pharmaceutical Co., Farmanieh St 1953853351, Tehran, Iran 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 ´ 4.6 mm) monolithic column kept at 40°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 mg/mL for myricetin, 0.95–95 mg/mL for quercetin, and 1.43–143.3 mg/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. F lavonoids are secondary metabolites found ubiquitously in the plant kingdom. They are phenolic compounds that occur primarily as glycosides (1) and are subdivided into six classes—flavones, flavanones, isoflavones, flavonols, flavanols, and anthocyanins—that vary in their structural characteristics around the heterocyclic oxygen ring (2). Although differences in flavonoid glycoside composition occur from one plant to another, the flavonoid glycosides derive from only a few aglycones (without sugar moieties), and they are probably absorbed by human organisms in these free forms (3, 4). Epidemiological studies associate a direct relation between flavonoid intake and the reduction of risk of coronary diseases and some types of cancer (5, 6). Their wide biological, pharmacological, and medicinal properties have led to a growing interest in their isolation and separation from natural products (7, 8). Sumac is the common name for a genus Rhus that contains over 250 individual species of flowering plants in the family Anacardiaceae (9). Among these species R. coriaria L. (tanner’s sumac), which grows wild in the region from the Canary Islands through the Mediterranean region to Iran and Afghanistan, is commonly used as a spice by grinding the dried fruits with salt, and is also widely used as a medicinal herb in the Mediterranean and Middle East, particularly for wound healing (10). The known biological activity of extracts from sumac species has been critically reviewed (9). The flavonols myricetin (My), quercetin (Qu), and kaempferol (Km; Figure 1) are the main flavonoids found in R. coriaria L. (11, 12). Flavonols are of interest because they have a wide range of pharmaceutical properties, including antioxidative, anticarcinogenic, and antimutagenic (13, 14). Epidemiological studies have suggested that flavonol-rich diets are associated with reduced risk of cardiovascular diseases (15) and higher life expectancy (16). HPLC has become the most convenient analytical tool for separation and identification of flavonoids using various detection systems (17–26). These chromatographic procedures, which have been developed for the simultaneous measurement of different flavonoid aglycone families, MEHRDAD ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 4, 2009 1035 Received October 4, 2008. Accepted by AP November 3, 2008. 1 Corresponding author’s e-mail: talebi_md@yahoo.com Downloaded from https://academic.oup.com/jaoac/article/92/4/1035/5655935 by guest on 01 March 2023