Refinement of LC-MS Methodology for the Analysis of Saponins
Ball, D. T. C. (2010). Refinement of LC-MS Methodology for the Analysis of Saponins (Thesis, Master of Science (MSc)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/4275
Permanent Research Commons link: https://hdl.handle.net/10289/4275
Systematic evaluation of the factors affecting LC-MS detection of some saponins and sapogenins associated with hepatogenous photo-sensitisation diseases in livestock, as determined using a Thermo Finnigan LCQ Advantage mass spectrometer, showed that ion responses could be substantially improved by careful selection of analysis parameters. Eluent composition, capillary temperatures and flow rate were found to significantly affect the ionisation of the target analytes. Optimisation of ionisation conditions resulted in a more than 100 fold (2 orders of magnitude) improvement in the detection of the model sapogenins and sapogenin glycosides relative to conditions utilised in previous studies at the University of Waikato. Optimal ESI and APCI responses were achieved using formic acid buffered acetonitrile-water eluent mixtures which possessed 30-40% acetonitrile for sapogenin glycosides or 50-60% acetonitrile for sapogenins and capillary interface temperatures in the range 275-325 C. Lower eluent flow rates also results in increased detector responses. Optimised ESI responses were greater by ca 2-10 times when compared to optimised APCI responses.Analyses of full scan and MSn ESI and APCI mass spectral fragmentation data showed that the molecular weight of conjugated sapogenin glycosides could be readily determined from [M+H]+ ions observed in full scan spectra. Under MS2 conditions sapogenin glycosides [M+H]+ ions fragmented to afford ions attributable to the protonated parent sapogenin and a series of other ions that were found to present in the ESI and APCI mass spectra of parent sapogenins, including m/z 399, m/z 273 and m/z 255 ions. Detailed analysis of full scan, MS2 and MS3 ESI and APCI fragmentation and chromatographic data determined for sarsasapogenin, episarsasapogenin, smilagenin, 20,20,23-2H3-sarsasapogenin and 2,2,4,4-2H4-episarsasapogenin, tigogenin and diosgenin showed that, unlike previously reported EI-GC-MS data, neither LC-MS ion ratio data, or retention time data could not be used to distinguish isomeric 3α/β, 5 α/β, 25(R/S)-spirostanol sapogenins of the type known to be present in the rumen and other GI tracts samples recovered from livestock suffering hepatogenous photo-sensitisation diseases.Based on the results reported in this thesis it is apparent that a combination of EI-GC-MS and LC-ESI-MSn data will be required to identify conjugated sapogenins present in rumen and other GI tract samples recovered from livestock suffering from hepatogenous photo-sensitisation diseases since LC-MSn data alone is not capable of distinguishing isomeric forms of free and conjugated sapogenins. It has been hypothesised that genetic factors may influence the nature of conjugates in the rumen and their metabolism to alternate conjugated analogues. The availability of combination of EI-GC-MS and optimised LC-MS methods to probe the nature and distribution of free and conjugated sapogenins in rumen and GI tact samples may assist in the identification of susceptible and resistant animals.
The University of Waikato
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