Simultaneous Determination of Eight Anthraquinones in Semen Cassiae by HPLC‐DAD

Abstract

Semen Cassiae (SC), a traditional Chinese herbal medicine for the treatment of various diseases, is known to contain active anthraquinone ingredients. However, since the content of some anthraquinones is too low, previous analytical methods only allow the quantitation of a few anthraquinones or a hydrolysis step has to be included in the sample preparation. A rapid and accurate method to examine the content of as many anthraquinones as possible in SC would be desirable. To develop a rapid, sensitive and accurate high-performance liquid chromatography-diode array detection (HPLC- DAD) method to simultaneously quantify eight major anthraquinones (obtusifolin-2-glucoside, aurantio-obtusin, aloe- emodin, rhein, obtusifolin, emodin, chrysophanol and physcion) in SC. The separation of anthraquinones was achieved on a C₁₈-column with a gradient elution using acetonitrile and 0.1% phosphoric acid. The detection wavelength was 278 nm and the analysis finished within 25 min. The limits of detection of these compounds ranged from 0.07 to 0.15 µg/mL while the limits of quantitation ranged from 0.24 to 0.51 µg/mL. All calibration curves showed good linearities (r² > 0.999) within the test ranges. This validated method was successfully used to analyse 22 batches of SC samples collected from various geographical locations. The method was validated to be simple, rapid, accurate and reliable to simultaneously determine eight major anthraquinones in SC. Meanwhile, a more specific anthraquinone, obtusifolin, was proposed to serve as a marker for SC, replacing chrysophanol.