Validation of UPLC method for determination of rubrofusarin-6-O-gentiobioside from Cassia obtusifolia L.

The seed of Cassia obtusifolia L. contains various kinds of anthraquinone, anthraquinone glycosides and naphthalene derivatives and is widely used in Asia to treat eye inflammation. Also, the extract of Cassia obtusifolia L. was known to reduce the memory impairments and neuronal damage caused by 2VO in a mouse transient global ischemia model. Rubrofusarin-6-O-gentiobioside, one of a major component of Cassia obtusifolia L., has been known to be an important constituents of this herbal drug with a lot of biological acitivities including decrease of the expression of TGF-β1 and fibronectin and NF-κB DNA binding activity and radical scavenging effect. However, there was little analytical method for determination of rubrofusarin-6-O-gentiobioside in Cassia obtusifolia. In this study, a rapid and simple UPLC method with UV detection was developed and validated to determine rubrofusarin-6-O-gentiobioside in Cassia obtusifolia. The method was established on a reversed phase C18, Brownlee column (2.1 mm x 75 mm, 2.7µm) and detection wavelength was 281nm. The mobile phase was acetonitrile (0.1% trifluoroacetic acid) and water (0.1% trifluoroacetic acid) with an isocratic elution and sample analysis time was 8.0 min. UPLC method is validated in terms of linearity range, limit of quantification, selectivity, accuracy and precision. The retention time of rubrofusarin-6-O-gentiobioside was 2.1 min. Calibration curve showed good linearity (R²= 0.999) over the range of 5.0 – 500.0 µg/mL. The relative standard deviation for intra- and inter-day precision was found to be below 3%. The recovery rate ranged from 105.0% to 114.0% and limit of quantification was 7.0 µg/mL. This method can be applied to determine rubrofusarin-6-O-gentiobioside rapidly and be utilized to control the quality of Cassia obtusifolia L..