Objective: To clarify the chemical pattern of major tetracyclic triterpenoids and establish an HPLC method for determination of cucurbitacin B and D in Pedicellus Melo. Method: UPLC-ESI-Orbitrap-MS/MS was used to analyze the major tetracyclic triterpenoids on Agilent Eclipse XDB-C18 (4.6 mm×150 mm

5 μm)

with 0.1% formic acid solution-acetonitrile as the mobile phase for gradient elution at a flow rate of 0.5 mL·min-1. RP-HPLC was performed for the determination of cucurbitacin B and D in Pedicellus Melo on Inert Sustain C18 (4.6 mm×250 mm

5 μm)

with 0.1% glacial acetic acid solution-acetonitrile as the mobile phase for gradient elution at a flow rate of 1.0 mL·min-1. Result: In the qualitative analysis of chemical pattern

8 major tetracyclic triterpenoids were found and firstly identified in Pedicellus Melo

including cucurbitacin Ⅱa

isocucurbitacin Ⅱa

cucurbitacin I

cucurbitacin D

cucurbitacin B

and three isomers of deacetylatecucurbitacin B. In quantitative analysis

the separation degree of cucurbitacin B

D and related substances met the requirement

and the calibration curve showed good linearity for cucurbitacin B and D in the concentration range of 49.5-297.0 mg·L-1 (r=0.999 99) and 10.3-61.8 mg·L-1 (r=0.999 9). The average recoveries of cucurbitacin B and D were 99.18% (RSD 1.0%) and 99.13% (RSD 1.1%) respectively. Conclusion: The major tetracyclic triterpenoids were reported for the first time in Pedicellus Melo

and the rapid and reproducible HPLC method was established

which was suitable for the determination of cucurbitacin B and D in Pedicellus Melo.