**1. Introduction**

Buckwheat is a pseudocereal belonging to the *Polygonaceae*family that grows rapidly and is tolerant to cold [1]. Buckwheat is found almost everywhere but is mainly grown in the northern hemisphere [2]. Common buckwheat (*Fagopyrum esculentum* Möench) and tartary buckwheat (*F. tataricum* Gaertner) are the most consumed buckwheat species [1]. Among the edible parts of common buckwheat, the sprouts have attracted considerable attention in recent years [3] as they are considered to be a popular health food and are widely consumed because of their bioactive compounds [4,5]. The sprout-germination process induces the hydrolysis of triglycerides in the seeds and produces the energy required for various biochemical reactions through the tricarboxylic acid cycle [6], and the content of bioactive compounds in the seeds increases through chemical reactions, for example, the flavone glycoside content is known to increase during the germination of common buckwheat [7,8]. Common buckwheat

sprout (CS) has been studied for a variety of pro-health benefits, such as its antioxidant capacity [7,9] and anti-inflammatory effects [10,11].

CS has been reported to have more abundant flavone *C*-glucosides than those of the tartary species [12]. The flavone *C*-glucosides in CS are present as orientin and isoorientin, from luteolin as the parent, as well as vitexin and isovitexin, from apigenin as the parent (Figure S1). In addition, rutin is a representative flavonoid present in common buckwheat and CS [12]. A recent report revealed that quercetin-3-*O*-robinobioside (Q3R), which is an isomer of rutin, exists in CS [8]. Q3R is known to be present in cotyledon and immature common buckwheat, rooibos, mature saskatoon fruit, and jujube fruit [8,13–15]. To summarize, the six main flavonoids found in CS exist as three sets of isomer pairs, with differences due to the position and form of the sugar.

Flavonoids and their glycosides can be used as quality control markers for many phytomedicines and medicinal plants [16]. Various analytical techniques have been developed for the separation and detection of flavonoid glycosides, in which the most-widely employed method is reversed-phase high-performance liquid chromatography (HPLC), coupled with photodiode array (PDA) detection and/or mass spectrometry (MS) [14,16,17]. Qualitative analysis of flavonoids is possible using MS even when baseline separation has not been secured. However, structural isomers cannot be distinguished only on the basis of MS/MS information because they have the same molecular weight and similar fragment patterns [16,18,19]. Therefore, flavonoid glycosides need to be separated in order to enable their accurate quantitative analysis in food. Previous studies reported the effects of column temperature, composition of mobile phase, and flow rate on the separation of a flavonoid isomer for qualitative analysis [20,21]. Even with a column temperature of 40 ◦C and the addition of acid to solvents, orientin and Q3R in CS were not separatable with isoorientin and rutin, respectively [7,9]. Q3R and rutin in mature saskatoon and jujube fruits were not completely separated and were not suitable for quantification [13,22]. Therefore, a suitable HPLC method for quantitatively analyzing flavonoid isomers present in CS is necessary.

In this study, we introduce an HPLC analysis method that simultaneously quantifies two types of flavone-*C*-glycoside isomer and flavonol-*O*-glycoside isomer found in common buckwheat sprout extract (CSE). The HPLC conditions of the developed analytical method, including mobile phase, column temperature, and flow rate, were optimized. The developed method was validated by determining the range, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) for each compound.

### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Orientin, isoorientin, vitexin, and isovitexin (all with ≥99% purity) were purchased from Extrasynthese (Genay, France). Rutin hydrate (≥94%), HPLC-grade formic acid (≥98%), and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich Co., LLC (St. Louis, MO, USA). HPLC-grade water, methanol, and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
