Investigating C₂₁ Steroidal Glycoside By Mass Spectrometry Technique

By virtue of its sensitivity, speed and accuracy, mass spectrometry is a powerful analytical technique that is ideally suited for screening active compounds in complicated extracts of natural product. The capability of MS to perform on line compositional and structural analyses through the use of hyphenated techniques such as LC/MS, MS/MS provides rich structural information at a rapid rate, which is unsurpassed by other techniques. In this thesis, we successfully developed electrospray ionization tandem mass spectrometry coupled with high-performance liquid chromatography to screen for C₂₁ steroidal glycosides with immunological activities form Stephanotis mucronata and Cynanchum chekiangense. In order to differentiate isomers in extracts, we also investigated energy-resolved mass spectrometry and the fragmentation mechanism of their complex combined with metal ions.Part 1: High-performance liquid chromatography/tandem mass spectrometry was developed for screening of immunological active C₂₁ steroidal glycosides from the stems of Stephanotis mucronata. In the mass spectra of C₂₁ steroidal glycosides, predominant [M+Na]⁺ ions were observed and used to determine the molecular mass, while fragmentation reaction 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 fraction, which provided accurate elemental compositions of fragment ions. Based on fragmentation patterns and comparison with standards, ten C₂₁ steroidal glycosides were identified and two of them are new compounds. This method combined with extracted ion current (EIC) spectra, has been also successfully applied to identify C21 steroidal glycosides in roots of Stephanotis mucronata. Total of 22 steroidal glycosides containing ten new ones were identified.Part 2: In order to differentiate the isomers of steroidal glycosides, the energy resolved mass spectrometry was investigated. Combined to HPLC/ESI-MSⁿ technique, eight C₂₁ steroidal glycosides in the roots of Cynanchum chekiangense were identified, and four of them are novel compounds.Part 3: The fragmentation patterns of compound induced by metal ion can be significantly altered, allowing new opportunities for structural determination. We have systemically investigated fragmentation patterns of C₂₁ steroidal glycosides induced by alkaline metal ions(Li⁺, Na⁺, K⁺, Cs⁺), alkaline-earth metal ion (Ca²⁺) and transition metal ions (Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺). And based on fragmentation patterns of C₂₁ steroidal glycoside-transition metal complex, the non-reducing end glycoside stereoisomers were differentiated.In addition, the principle of LC/MS technique and its extensive application in natural products analysis were reviewed in detail.