In this work, the separation of ginsenoside Rb-3 and Re from the crude extracts of caudexes and leaves of Panax notoginseng (CECLPN) was studied with reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic separation was achieved using a SynChropak C-18 column (at 25 degrees C). A satisfied separation of the compounds was obtained in less than 40 min with a flow rate of 1.0ml/min. Chromatography was performed using ternary mobile phase composed of methanol, water and phosphoric acid, 65:33:1.4 (v/v). UV detection was accomplished at 203 nm. Ginsenoside Rb3 and Re found in the CECLPN constitute up to 39.25% and 26.65% of the dry powder, respectively. Ginsenoside Rb3 and Re were prepared by semi-preparative HPLC/ELSD using gradient elution system of methanol-water = 70:30 -> 65:35 at a flow rate of 30 ml/min with a sample load of 300-400 mg. With this chromatographic condition, each individual ginsenoside Rb3 and Re in purity of more than 97% were gained. The products were confirmed by spectroscopic (UV, IR, ESI-MS/MS, NMR) and HPLC methods. The methods were validated for linearity, precision, recovery, limits of detection (LOD) and limits of quantification (LOQ). The results show that the proposed method appears to be an adequate method for quality control of CECLPN and a useful tool for pharmaceutical study of ginsenosides. (c) 2006 Elsevier B.V. All rights reserved.