Simultaneous Determination of Six Components in the Traditional Herbal Medicine 'Oryeongsan' by HPLC-DAD and LC-MS/MS

A simple high performance liquid chromatography - diode array detector (HPLC-DAD) method has been developed and validated for simultaneous determination of the six components (cinnamic acid, cinnamaldehyde, eugenol, atractylenolide I, atractylenolide III, and ergosterol) in Oryeongsan. In addition, identification of six marker compounds was conducted by a LC-MS/MS analysis. The six compounds in Oryeongsan were separated on Shishedo $C_{18}$ column (5 ${\mu}m$, $4.6{\times}250$ mm) at a column temperature of $30^{\circ}C$. The mobile phase was a mixture of 0.1% trifluoroacetic acid (TFA) water and acetonitrile employing gradient elution at a flow rate of 1.0 mL/min. The detection wavelength was set at 205, 250, 280, and 330 nm. The developed method had good linearity ($R^2$ > 0.9997) and the limit of detection (LOD) and limit of quantification (LOQ) were observed within the ranges 0.01~0.15 and 0.05~0.45 ${\mu}g/mL$, respectively. The relative standard deviation (RSD) values of intra- and inter-day testing were indicated that less than 3% and 90.31~103.31% of accuracy. The results of recovery test were 90.56~106.72% and RSD range was measured from 0.84 to 2.95%. In conclusion, this HPLC-DAD method has been successfully applied to the simultaneous determination of six compounds in Oryeongsan samples.