**Developing and Validating a Method for Separating Flavonoid Isomers in Common Buckwheat Sprouts Using HPLC-PDA**

**Davin Jang <sup>1</sup> , Young Sung Jung <sup>2</sup> , Mi-Seon Kim <sup>1</sup> , Seung Eel Oh <sup>3</sup> , Tae Gyu Nam 3,\* and Dae-Ok Kim 1,2,\***


Received: 10 September 2019; Accepted: 1 November 2019; Published: 4 November 2019 -

**Abstract:** Buckwheat sprouts that are synthesized during the germination process are rich in flavonoids, including orientin, vitexin, rutin, and their isomers (isoorientin, isovitexin, and quercetin-3-*O*-robinobioside, respectively). The purpose of this study was to optimize and validate an analytical method for separating flavonoid isomers in common buckwheat sprout extract (CSE). Factors, such as range, linearity, precision, accuracy, limit of detection, and limit of quantification, were evaluated for each standard using high-performance liquid chromatography (HPLC). On the basis of resolution and symmetry, a column temperature of 40 ◦C with 0.1% (*v*/*v*) acidic water and acetonitrile as mobile phases, at a flow rate of 1 mL min−<sup>1</sup> were determined to be the optimal analytical conditions. Calibration curves for orientin, isoorientin, vitexin, isovitexin, and rutin exhibited good linearity with correlation coefficients of 0.9999 over the 6.25–100.00 µg mL−<sup>1</sup> range. Recovery values of 96.67–103.60% confirmed that the method was accurate for all flavonoids. The relative standard deviations of intra-day repeatability and inter-day reproducibility confirmed method preciseness, with values of less than 5.21% and 5.40%, respectively. The developed method was used to analyze flavonoids in CSE, with isomers satisfactorily separated and simultaneously quantified. We demonstrated that the developed HPLC method can be used to monitor flavonoids in buckwheat sprouts.

**Keywords:** common buckwheat sprout; flavonoid isomer; quercetin-3-*O*-robinobioside; validation; chromatographic separation
