Simultaneous determination of five marker constituents in traditional Chinese medicinal preparation Le-Mai granule by high performance liquid chromatography

An HPLC method for the simultaneous determination of five marker constituents was established for the quality control of traditional Chinese medicinal preparation Le-Mai granule. The marker constituents were danshensu, protocatechuic acid and protocatechualdehyde from Salviae miltiorrhizae bunge; paeoniflorin from Radix paeoniae rubra and ferulic acid from Rhizoma chuanxiong. Extracted samples were successfully separated on a Diamonsil C18 column (150 mm × 4.6 mm i.d., 5 μm) at 25 °C. The mobile phase was a mixture of methanol and 1.0% acetic acid employing gradient elution at a flow rate of 1.0 mL/min. Detection was accomplished with a diode-array detector and chromatograms were recorded at 230, 262, 280 and 322 nm. The compounds were identified by comparing their retention times and UV spectra in the 200-400 nm range with authentic standards. Regression equations revealed good linear relationship (correlation coefficients: 0.9993-0.9999) between the peak areas of the constituents and their concentrations. The average recoveries (n = 3) were between 96.2 and 102.5%. The proposed method has been successfully applied to the simultaneous determination of the five marker constituents in three lots of Le-Mai granule.