The stems of the Chinese traditional medicine Stephanotis mucronata were screened for immunologically active pregnane glycosides using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry. In the mass spectra of pregnane glycosides, predominant [M+Na]+ ions were observed and used to determine the molecular masses, while fragmentation reactions of the [M+Na]+ ions were recorded to provide information on the primary sequences of oligosaccharide chains in terms of classes of monosaccharide. Fragment ions from the side-chain cleavage of aglycone portions can provide mass information about side-chain substitutions. To further confirm the fragment ion structures, Fourier transform ion cyclotron resonance tandem mass spectrometry (MSn) with low-energy collision-induced dissociation was performed using samples collected from HPLC fractions, which provided accurate elemental compositions of fragment ions. Based on fragmentation patterns and comparison with standards, ten pregnane glycosides were identified as stemucronatosides C, D, F, and G, mucronatosides A, B, and C, stephanoside E, and two glycosides that are identified in the S. mucronata extracts for the first time. The latter two pregnane glycosides are 12-O-cinnamoyldeacetylmetaplexigenin-3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside and 12-O-cinnamoyl-20-O-acetyl (20S)-pregn-6-ene-3β,5α,8β,12β,14β,17β,20-heptaol 3-O-β-D-thevetopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside. Copyright © 2005 John Wiley & Sons, Ltd.