Quantitative and Pattern Recognition Analyses of Five Marker Compounds in Raphani Semen using High-Performance Liquid Chromatography

A rapid and simple high‐performance liquid chromatography (HPLC)‐photodiode array (PDA) analytical method was developed for the quantitative analysis of Raphani Semen (RS). This method was successfully used to determine the five main phenolic compounds found in RS specimens from different production regions. The compounds included sinapine thiocyanate (1), β‐d‐fructofuranosyl‐α‐d‐(6‐O‐sinapoyl)‐glucopyranoside (2), isorhamnetin 3,4′‐di‐O‐β‐d‐glucoside (3), β‐d‐(3‐O‐sinapoyl)‐fructofuranosyl‐α‐d‐(6‐O‐sinapoyl)‐glucopyranoside (4), and β‐d‐(3,4‐O‐disinapoyl)‐fructofuranosyl‐α‐d‐(6‐O‐sinapoyl)‐glucopyranoside (5). The marker compounds were separated using an Agilent Eclipse XDB‐C18 column (5.0 µm, 150 × 4.6 mm i.d.) by gradient elution with acetonitrile/water/0.1% trifluoroacetic acid (TFA) as the mobile phase (flow rate, 1.0 mL/min). This method was fully validated with respect to linearity, precision, accuracy, stability, and robustness. The HPLC analytical method was validated to conduct a pattern recognition analysis by repeatedly analyzing 56 seed samples including 55 RS (C01–C49 and K50–K55) and 1 Brassicae Semen samples. In addition, a content standard for RS was proposed. Compounds 1 and 4 were revealed as major components in the HPLC chromatogram, and their contents ranged from 0.06 to 0.20 and 0.02 to 0.35 mg/g, respectively. These results demonstrate the successful development of an analytical method suitable for evaluating the quality and distinguishing the origin of RS. In addition, we briefly describe the crucial liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analytical conditions for the precise simultaneous quantification of the marker compounds.