Simultaneous quantitation of oleanolic and ursolic acids by RP-HPLC-DAD on active ingredient from Tabebuia hypoleuca leaves.

The genus Tabebuia is widely used in traditional medicine of tropical and subtropical countries. We recently reported the antitumor activity toward several human cell lines of the ethyl acetate extract of the leaves of Tabebuia hypoleuca, a species endemic to Cuba, together with the high content of triterpenic acids. This work describes the development and the validation of a reversed-phase high-performance liquid chromatography with diode array detection method for the simultaneous quantitation of the contents of oleanolic acid (OA) and ursolic acid (UA). The method was carried out on a C18 column, using methanol and water as mobile phase (ratio 95:5 v/v), at the flow rate of 0.4 mL/min, recorded at λ = 210 nm. The retention times were 16.371 ± 0.045 and 16.943 ± 0.052 min for OA and UA, respectively, with a resolution between peaks of 1.29. The average contents of OA and UA within three sets of the ethyl acetate extract, considered as an active pharmaceutical ingredient (API) were 102.93 and 244.28 mg/g, respectively. The linearity of the response was found to be 10-700 μg/mL for OA and its equation was y = 0.2946x - 2.0432 (r2 = 0.9924). The linearity range for UA was 5-1000 μg/mL and its equation was y = 0.3284x - 17.508 (r2 = 0.9865). The limit of detection and limit of quantification of these constituents were 6.50 and 19.81 μg/mL for OA, 0.75 and 1.98 μg/mL for UA. OA and UA may be used as markers for the quality assessment of formulations containing this API. The developed method is accurate, specific, precise, reproducible and may be applied as a simple and rapid quality-control procedure for medicinal plant extracts with similar triterpene patterns.