title: Refinement of LC-MS Methodology for the Analysis of Saponins

authors: Ball, Dylan Tane Charles

subjects: saponins

abstract: 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.

text: Refinement of LC-MS Methodology for the Analysis of Saponins 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.

year: 2010

title: Coastal evolution of the Rangitāiki Plains, Bay of Plenty, Aotearoa-New Zealand

authors: Rackham, Bailey

subjects: Rangitaiki Plains

abstract: The Rangitāiki Plains is a tectonically active alluvial plain in the Eastern Bay of Plenty, New Zealand. Throughout the mid-late Holocene (8 ka to present), the Rangitāiki Plains have undergone substantial geomorphological change, with the coastline prograding approximately 10 km over the past 6.5 ka, leaving relic shoreline features known as beach ridges. Previous studies of the Rangitāiki Plains have utilised tephrochronology and radiocarbon dating to determine the ages of several palaeoshorelines. These dates provided minimum ages for beach ridges and resulted in aging constraints; furthermore, limited textural and geochemical characterisation of beach ridges has hampered understanding of sediment provenance and reworking changes. Addressing these research gaps is important as this knowledge is especially relevant in coastal management, modern climate change, and increasing anthropogenic impacts on the coastline.  

Optically Stimulated Luminescence (OSL) dating on the Rangitāiki Plains produced older beach ridge ages than those derived via tephrochronology. OSL dating also showed that beach ridge ages coincided with volcanic eruption events. The OSL progradation rates extend to much older time frames than those based on tephrochronology, starting at 7.6 ka rather than 5.5 ka. New data concludes a prominent progradation peak up to 43.03 m yr⁻¹ between 5.202±0.368 ka and 5.187±0.447 ka, around the time of the Whakatāne eruption. By 4 ka, progradation had slowed to only 1.14 m yr⁻¹, suggesting that longshore drift occurred earlier than previously calculated (2 ka), or that progradation slowed before longshore drift commenced. XRF and XRD analysis of sediments from Rangitāiki Plains confirms that beach ridge sediments comprise of intermediate igneous sediments that have been tephra fingerprinted to the Taupō Volcanic Zone.

text: Coastal evolution of the Rangitāiki Plains, Bay of Plenty, Aotearoa-New Zealand The Rangitāiki Plains is a tectonically active alluvial plain in the Eastern Bay of Plenty, New Zealand. Throughout the mid-late Holocene (8 ka to present), the Rangitāiki Plains have undergone substantial geomorphological change, with the coastline prograding approximately 10 km over the past 6.5 ka, leaving relic shoreline features known as beach ridges. Previous studies of the Rangitāiki Plains have utilised tephrochronology and radiocarbon dating to determine the ages of several palaeoshorelines. These dates provided minimum ages for beach ridges and resulted in aging constraints; furthermore, limited textural and geochemical characterisation of beach ridges has hampered understanding of sediment provenance and reworking changes. Addressing these research gaps is important as this knowledge is especially relevant in coastal management, modern climate change, and increasing anthropogenic impacts on the coastline.  

Optically Stimulated Luminescence (OSL) dating on the Rangitāiki Plains produced older beach ridge ages than those derived via tephrochronology. OSL dating also showed that beach ridge ages coincided with volcanic eruption events. The OSL progradation rates extend to much older time frames than those based on tephrochronology, starting at 7.6 ka rather than 5.5 ka. New data concludes a prominent progradation peak up to 43.03 m yr⁻¹ between 5.202±0.368 ka and 5.187±0.447 ka, around the time of the Whakatāne eruption. By 4 ka, progradation had slowed to only 1.14 m yr⁻¹, suggesting that longshore drift occurred earlier than previously calculated (2 ka), or that progradation slowed before longshore drift commenced. XRF and XRD analysis of sediments from Rangitāiki Plains confirms that beach ridge sediments comprise of intermediate igneous sediments that have been tephra fingerprinted to the Taupō Volcanic Zone.

year: 2025

title: On equality and colourblindness

authors: Robertson, Neville

subjects: Maori

abstract: "I have a dream that my four children will one day live in a nation where they will not be judged by the color of their skin but by the content of their character." So said Martin Luther King, Jr, just over 40 years ago, in a speech that galvanised the US civil rights movement. King spoke of the United States but his dream is equally relevant in New Zealand. Has it been achieved? Some people would argue that not only has it been achieved but that the tables have been turned and now white folk are the ones who are oppressed. What is needed, they argue, is a level playing field. But how level is the playing field?

text: On equality and colourblindness "I have a dream that my four children will one day live in a nation where they will not be judged by the color of their skin but by the content of their character." So said Martin Luther King, Jr, just over 40 years ago, in a speech that galvanised the US civil rights movement. King spoke of the United States but his dream is equally relevant in New Zealand. Has it been achieved? Some people would argue that not only has it been achieved but that the tables have been turned and now white folk are the ones who are oppressed. What is needed, they argue, is a level playing field. But how level is the playing field?

year: 2004