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: Explicit correlation and basis set superposition error: The structure and energy of carbon dioxide dimer

authors: McMahon, Jordan D.

subjects: chemistry

abstract: We have investigated the slipped parallel and t-shaped structures of carbon dioxide dimer [(CO₂)₂] using both conventional and explicitly correlated coupled cluster methods, inclusive and exclusive of counterpoise (CP) correction. We have determined the geometry of both structures with conventional coupled cluster singles doubles and perturbative triples theory [CCSD(T)] and explicitly correlated cluster singles doubles and perturbative triples theory [CCSD(T)-F12b] at the complete basis set (CBS) limits using custom optimization routines. Consistent with previous investigations, we find that the slipped parallel structure corresponds to the global minimum and is 1.09 kJ mol⁻¹ lower in energy. For a given cardinal number, the optimized geometries and interaction energies of (CO₂)₂ obtained with the explicitly correlated CCSD(T)-F12b method are closer to the CBS limit than the corresponding conventional CCSD(T) results. Furthermore, the magnitude of basis set superposition error (BSSE) in the CCSD(T)-F12b optimized geometries and interaction energies is appreciably smaller than the magnitude of BSSE in the conventional CCSD(T) results. We decompose the CCSD(T) and CCSD(T)-F12b interaction energies into the constituent HF or HF CABS, CCSD or CCSD-F12b, and (T) contributions. We find that the complementary auxiliary basis set (CABS) singles correction and the F12b approximation significantly reduce the magnitude of BSSE at the HF and CCSD levels of theory, respectively. For a given cardinal number, we find that non-CP corrected, unscaled triples CCSD(T)-F12b/VXZ-F12 interaction energies are in overall best agreement with the CBS limit.

text: Explicit correlation and basis set superposition error: The structure and energy of carbon dioxide dimer We have investigated the slipped parallel and t-shaped structures of carbon dioxide dimer [(CO₂)₂] using both conventional and explicitly correlated coupled cluster methods, inclusive and exclusive of counterpoise (CP) correction. We have determined the geometry of both structures with conventional coupled cluster singles doubles and perturbative triples theory [CCSD(T)] and explicitly correlated cluster singles doubles and perturbative triples theory [CCSD(T)-F12b] at the complete basis set (CBS) limits using custom optimization routines. Consistent with previous investigations, we find that the slipped parallel structure corresponds to the global minimum and is 1.09 kJ mol⁻¹ lower in energy. For a given cardinal number, the optimized geometries and interaction energies of (CO₂)₂ obtained with the explicitly correlated CCSD(T)-F12b method are closer to the CBS limit than the corresponding conventional CCSD(T) results. Furthermore, the magnitude of basis set superposition error (BSSE) in the CCSD(T)-F12b optimized geometries and interaction energies is appreciably smaller than the magnitude of BSSE in the conventional CCSD(T) results. We decompose the CCSD(T) and CCSD(T)-F12b interaction energies into the constituent HF or HF CABS, CCSD or CCSD-F12b, and (T) contributions. We find that the complementary auxiliary basis set (CABS) singles correction and the F12b approximation significantly reduce the magnitude of BSSE at the HF and CCSD levels of theory, respectively. For a given cardinal number, we find that non-CP corrected, unscaled triples CCSD(T)-F12b/VXZ-F12 interaction energies are in overall best agreement with the CBS limit.

year: 2011

title: Manaaki whenua. Manaaki tangata: Care for the land, care for the people: Leading critical school reform with mana whenua and whānau Māori

authors: Ford, Therese

subjects: Critical school reform

abstract: Education disparities between Māori and Pākehā students in Aotearoa, have their origins in colonial education policies and practices which considerably advantaged Pākehā students and compromised the ability of Māori students to engage with learning and succeed.  Despite the promises of equality inherent in the Treaty of Waitangi,  this historical foundation has, for over 150 years, perpetuated intergenerational cycles of education failure for Māori resulting in high levels of endemic social and economic disadvantage in many Māori communities.
This thesis examines the leadership praxis of two Pākehā principals, to understand how, with more respectful connections with Māori communities, they were able to address education disparities and facilitate success for Māori students as Māori. Culturally responsive research methodologies guided two, in-depth case studies which employed mixed methods.
Two collaborative stories, with the voices of mana whenua and whānau Māori, identified the importance of school leaders developing critical levels of consciousness, through authentic, bidirectional relationships with Māori communities.  These relationships enabled the principals to understand the authentic local, pre-colonial iwi histories of the land. This in turn fostered connections between the schools and local Māori communities that enabled them to collaboratively respond to the implications of colonisation.  
The thesis challenges the monocultural, monolingual, unidirectional policies and practices that characterise the current model of colonial education.  It presents instead a decolonising model of critical school reform and a framework for implementation and evaluation.  It contends that honouring the Treaty of Waitangi requires an equity focused response with power sharing relationships, critically led by principals alongside Māori communities.

text: Manaaki whenua. Manaaki tangata: Care for the land, care for the people: Leading critical school reform with mana whenua and whānau Māori Education disparities between Māori and Pākehā students in Aotearoa, have their origins in colonial education policies and practices which considerably advantaged Pākehā students and compromised the ability of Māori students to engage with learning and succeed.  Despite the promises of equality inherent in the Treaty of Waitangi,  this historical foundation has, for over 150 years, perpetuated intergenerational cycles of education failure for Māori resulting in high levels of endemic social and economic disadvantage in many Māori communities.
This thesis examines the leadership praxis of two Pākehā principals, to understand how, with more respectful connections with Māori communities, they were able to address education disparities and facilitate success for Māori students as Māori. Culturally responsive research methodologies guided two, in-depth case studies which employed mixed methods.
Two collaborative stories, with the voices of mana whenua and whānau Māori, identified the importance of school leaders developing critical levels of consciousness, through authentic, bidirectional relationships with Māori communities.  These relationships enabled the principals to understand the authentic local, pre-colonial iwi histories of the land. This in turn fostered connections between the schools and local Māori communities that enabled them to collaboratively respond to the implications of colonisation.  
The thesis challenges the monocultural, monolingual, unidirectional policies and practices that characterise the current model of colonial education.  It presents instead a decolonising model of critical school reform and a framework for implementation and evaluation.  It contends that honouring the Treaty of Waitangi requires an equity focused response with power sharing relationships, critically led by principals alongside Māori communities.

year: 2020