Single-Laboratory Validation for the Determination of Flavonoids in Hawthorn Leaves and Finished Products by LC-UV

Suitably validated analytical methods that can be used to quantify medicinally active phytochemicals in natural health products are required by regulators, manufacturers, and consumers. Hawthorn (Crataegus) is a botanical ingredient in natural health products used for the treatment of cardiovascular disorders. A method for the quantitation of vitexin-2 ''-O-rhamnoside, vitexin, isovitexin, rutin, and hyperoside in hawthorn leaf and flower raw materials and finished products was optimized and validated according to AOAC International guidelines. A two-level partial factorial study was used to guide the optimization of the sample preparation. The optimal conditions were found to be a 60-minute extraction using 50: 48: 2 methanol: water: acetic acid followed by a 25-minute separation using a reversedphased liquid chromatography column with ultraviolet absorbance detection. The single-laboratory validation study evaluated method selectivity, accuracy, repeatability, linearity, limit of quantitation, and limit of detection. Individual flavonoid content ranged from 0.05 mg/g to 17.5mg/g in solid dosage forms and raw materials. Repeatability ranged from 0.7 to 11.7% relative standard deviation corresponding to HorRat ranges from 0.2 to 1.6. Calibration curves for each flavonoid were linear within the analytical ranges with correlation coefficients greater than 99.9%. Herein is the first report of a validated method that is fit for the purpose of quantifying five major phytochemical marker compounds in both raw materials and finished products made from North American (Crataegus douglasii) and European (Crataegus monogyna and Crataegus laevigata) hawthorn species. The method includes optimized extraction of samples without a prolonged drying process and reduced liquid chromatography separation time.