A strategy is described for method evaluation to manage the influence of endogenous compounds to induce bias in pharmaceutical quantification from blood samples of different animal species resulting from ionisation matrix effects. The approach introduces the possibility of simultaneous cross animal species calibration for ethical reasons using a simple indicative test, which also rapidly demonstrates the utility of a test assay in other matrices. A quantitative Turboflow LC-MS/MS assay was evaluated using samples prepared in species matched controlled matrices with deuterated internal standardisation. The method was subsequently tested to assess the analysis of samples from multiple animal species using calibration samples from a single matrix origin. The object of this was to enable the substitution of analytical control samples in rodent plasma, reducing the plasma volume required, therefore the number of rodents used indirectly to support development studies. The method was unsuccessful due to concentration bias in identically prepared test samples. The bias origin was investigated using a fixed ratio solution of the analyte and its deuterated analogue with matrix test aliquots. The investigation identified non equivalent relative ionisation efficiency between compounds. The ratio method is proposed as an evaluation strategy for matrix effects causing non-equivalent response ratios. The origin of this deviation was identified using a post column standard infusion test highlighting a region of ionisation suppression co-eluting with the analyte. Full scan acquisition analysis revealed co-elution of endogenous glycerophospholipids. The relative expression of these compounds between species was investigated, employing a precursor ion scanning method of a common product ion indicative of phosphotidyl choline compounds; the human diversity was also investigated. Distribution was found to differ between animal species, which was further tested by construction of a model using partial least squares regression which correctly identified the species origin of all non-primate species tested. The mechanism of differentiation between compound and deuterated analogue by endogenous phosphotidyl choline was proposed as micellar phase equilibrium following elution from the HPLC column. A new Turboflow LC-MS/MS assay was optimised with attention to the resolution of glycerophospholipid interferences and retested for applicability between species. It was simultaneously applied to the analysis of small volume whole blood samples via dried blood spots on filter paper. Precision and bias were acceptable for the analysis of rat and mouse samples using human control plasma. Analysis of incurred ex-vivo samples from rats was successful in demonstrating equivalence between calibration sources.

Glycosyltransferases (GTs) are essential for the biosynthesis and diversification of many therapeutically important natural products. Of these, UDP-sugar: sterol glucosyltransferases (UGTs) (2.4.1.173) catalyse the synthesis of therapeutically important steryl glycosides (SGs). Guided by the sequence similarity with a previously characterised N-terminally truncated UGT from Saccharomyces cerevisiae (UGT51), this study reports the cloning of the gene fragment encoding the C-terminal catalytic domains from related yeasts and the expression and characterisation of their encoded products produced. N-terminally histidine tagged proteins were purified for in vitro assays against a panel of sterol and steroidal acceptors. Liquid chromatography-mass spectrometry (LC-MS) and kinetic analysis led to the successful characterisation of two novel UGTs from Pichia angusta and Kluyveromyces lactis. In addition, testosterone was shown to be utilized by all UGTs, including the previously characterised S. cerevisiae UGT51. Random mutagenesis of UGTs and homology modelling of the S. cerevisiae UGT revealed structural similarities with family 1 bacterial glycopeptide GTs. Given the structural and mechanistic similarities among GT family 1 UGTs, this approach may provide a template for genetic manipulation of novel UGTs from other members of the GT superfamily with a better understanding of catalytic domains and for broadening their scope in drug development. It may also aid the development of a generic process in the synthesis of SGs.

No description available.

A range of novel substrates for the detection of bacterial hydrolyases have been examined in both liquid and solid media. The potential inhibitory effect of these substrates on both Gram-positive and Gram-negative organisms has been evaluated. In addition a range of alternative substrates possessing an appropriate chemical configuration have also been evaluated as substrates in this thesis. It has been demonstrated that substrates based on 4-aminophenol, particularly the dichloro derivative produce intensely coloured reaction products upon hydrolysis and subsequent coupling with 1-naphthol. A derivative of 1-naphthol, 3,5-dihydroxy-2-naphthoic acid, was shown to react favourably with a number of released core compounds, producing a wide variety of coloured complexes. In comparison with o-nitropheny1-13-D-galactoside (ONPG), the novel substrates for the detection of P-galactosidase activity showed wide differences in K. and V. values. Overall these substrates performed well in liquid media, especially the dichloro derivatives for detection of a range of bacterial hydrolyases. The substrates have also been evaluated in solid media and have been shown to produce intensely coloured reaction products. The fact that these substrates offer themselves to the production of novel dual substrate systems indicates their potential diagnostic applications. Use of L-alanyl-DEPPD in combination with 1-naphthyl-J3-D-galactoside has been shown to be of diagnostic potential for the detection of 13-galactosidase activity in Gram-negative organisms. In addition, a combination of substrates, L-prolyl-4-amino-2,6-dichlorophenol and 1-naphthyl-O-D¬glucoside has also been shown to have potential for the production of a medium specifically for the isolation of Serratia sp. This dual substrate system also has applications for the production of media for the specific isolation and detection of numerous clinically important pathogens e.g Enterococcus sp, C.perfringens and Yersinia enterocolitica.

The destruction of hazardous chemicals, and in particular halogenated compounds, has become of significant interest in the last twenty years, as public awareness of Green issues has become more prominant. This work investigates the use of the DuPont process, for the non-oxidative destruction of organohalogens, as an alternative method of disposal for these environmentally hazardous compounds. A small-scale bath was constructed for the generation of a 2% sodium hydride solution in molten sodium hydroxide, by the reaction of hydrogen and metallic sodium. A variety of different halogenated compounds, both aliphatic and aromatic, were immersed in the molten salt of interest, along with respective non-halogenated analogues. The reaction products were analysed using a variety of different analytical techniques including FTIR, IC, GC and GCMS. By identifying these reaction products, possible reaction mechanisms have been postulated. For PVC, it is shown that the immersed polymer undergoes some form of base-accelerated thermal degradation, whilst the immersion of 4-chlorostyrene and poly-4-chlorostyrene apparently leads to an elimination-addition reaction. This results in the formation of the dechlorinated monomer, styrene, which then undergoes further reduction, giving ethyl benzene. The immersion of volatile halogenated compounds, such as chlorobenzene, leads to nucleophilic substitution. However, the presence of the hydride ion is shown to be essential, as the substitution reaction is in competition with volatilisation: no substitution is observed in molten sodium hydroxide alone. The rate of nucleophilic substitution in the presence of hydride is comparable to the rate of volatilisation, resulting in only 50% dechlorination. With non-volatile compounds, 100% dechlorination is observed in both the presence and absence of the hydride ion. However, the rate of dechlorination is significantly faster when the hydride ion is present. The ability of the 2% sodium hydride bath to dechlorinate poly-halogenated compounds is of particular interest, as the DuPont process could be used for the destruction of the more hazardous halogenated compounds such as PCBs and dioxins. Furthermore, due to the non-oxidative properties of the molten salt, no chlorinated gaseous products are observed, including HCl. This would be of major benefit to a method of disposal for halogenated hydrocarbons.