Analysis of naphthoquinone derivatives in the Asian medicinal plant Eleutherine americana by RP-HPLC and LC–MS

The first analytical procedure for the determination of a new naphthopyrone, eleutherinoside A, together with the known bioactive compounds eleuthoside B, isoeleutherin, eleutherin and eleutherol in Eleutherine americana was established. Optimum HPLC separation of these naphthoquinone derivatives was possible on RP-12 column material, using water and acetonitrile as mobile phase. Flow-rate, detection wavelength and temperature were adjusted to 1.0 mL/min, 254 nm and 40 °C, respectively. Validation results indicated that the HPLC method is well suited for the determination of naphthoquinone derivatives in the bulbs of E. americana with a good linearity (r2 > 0.9996), precision (intra-day R.S.D. <4.70%, inter-day R.S.D. <5.68%) and recovery rates from 96.26 to 103.48%. Limit of detection (LOD) was found to be below 0.84 μg/mL for all five compounds. LC–MS analyses performed in positive and negative electrospray ionization mode assured peak purity and identity. The analysis of different E. americana samples from Thailand revealed that eleutherol (0.10–0.20%) was dominant in all specimens, followed by isoeleutherin and eleutherin. The new natural product 2,5-dimethyl-10-hydroxynaphthopyrone 8-O-β-d-glucopyranoside occurred in percentages of less than 0.05%.