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Studies on the impacts of prenatal exposure of Alderley Park strain mice to benzodiazepine receptor-influencing drugs on behavioural, biophysical and biochemical changes were examined in early neonatal life, with a view to throwing light on the molecular mechanisms underpinning the 'floppy infant syndrome'. Three benzodiazepine agonists (chlordiazepoxide, diazepam and bromazepam), the antagonist flumazenil (Ro 15-1788) or combinations of agonist (chlordiazepoxide or diazepam) with flumazenil or ethanol, were administered to mice in their third week of pregnancy. Pups exposed to 9-10 days of drugs gestationally were selected and cross-fostered to non-treated lactating dams. Pups exposed in these ways had slowed righting reflexes (hypotonia) and hypothermia. Both effects are classic symptoms of the 'floppy infant syndrome'. Both the agonists chlordiazepoxide and diazepam and the antagonist, flumazenil increased membrane fluidity in neural tissue. Flumazenil was, however, more effective in this respect than either of the agonists. When the agonists were administered in combination with either flumazenil or ethanol, their ability to fluidise the membrane was increased. Such changes in fluidity may result from biochemical changes in neural tissue. The most profound changes in neural cholesterol and phospholipid resulted after prenatal exposure to flumazenil, an action evident even when combinations of antagonist and agonist were administered. The biochemical and biophysical changes in neural tissue may account for the flacidity common in infants suffering from the 'floppy infant syndrome'. The model and the approach provide a fruitful way of examining the lasting behavioural impact of prenatal exposure to psychoactive drugs.

A more detailed knowledge of the inhibition profiles for new chemical entities with respect to the major cytochrome P450 enzymes is prerequisite for their registration. The use of recombinant P450 enzymes is intended to limit interactions with other minor P450 enzymes and their reductases that are present in human liver microsomes. Using combined substrates (phenacetin, diclofenac, S-mephenytoin, bufuralol and midazolam) with combined recombinant P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4), a high-throughput LC/MS/MS method for the determination of IC₅₀ values using six concentrations for up to thirteen compounds in a single assay has been developed. Kinetic analysis of each enzyme-substrate pair under single and combined conditions highlighted the poor selectivity of phenacetin. Subsequent manipulation of relative P450 enzyme concentrations reduced the cross-reactivity of this substrate. IC₅₀ determinations with α-naphthoflavone (0.04µM), sulphapenazole (0.26µM), tranylcypromine (9µM), quinidine (0.02µM) were also similar under single and combined conditions. Further evaluation of the assay was carried out using 11 known inhibitors of cytochromes P450 and 52 new drug candidates. Using the high sensitivity and selectivity offered by tandem mass spectrometry in conjunction with hydrophilic interaction liquid chromatography (HILIC), a method capable of measuring nanomolar levels of unconjugated or <i>free </i>metanephrines in plasma has been developed. Elevated levels of metanephrine, normetanephrine and 3-methoxytyramine can be indicative of a rare tumour of the chromaffin cells termed phenochromocytoma. Limits of quantitation for metanephrine (0.02nmol/L), normetanephrine (0.21nmol/L) and 3-methoxytryaramine (0.14nmol/L) are within or below published reference ranges where documented. Linearity of response has been shown to at least 10.0 nmol/L for all analytes with intra- and inter-assay variability shown to be <15%CV across the range of expected physiological concentrations.

Six drugs currently used in the treatment of schizophrenia were examined for their effects upon membranes and second messenger related enzymes. The drugs investigated in this thesis were selected on the basis of their pharmacological effects and the extent of their side effects. The effects of these drugs on the membranes and enzyme activity of Ca-ATPase, adenylyl cyclase and phosphodiesterase were studied, using different techniques, and an attempt was made to establish whether the effects of drugs exerted on the enzyme activities are direct or indirect i.e. as a result of alterations in the membrane fluidity, hence correlating the two phenomena. Chlorpromazine and trifluoperazine had ordering effects on artificial membranes at clinically therapeutic concentrations. Phosphorescence polarisation measurements indicated restrictions in protein mobility and aggregation as concentrations of these two drugs increased, hence supporting the previous observations. Meanwhile, sulpiride, remoxipride and procyclidine did not cause any apparent changes on the membrane fluidity, yet remoxipride still demonstrated a degree of restriction in the protein mobility, indicating a direct interaction of drug with the protein. While investigation of the effects of clozapine on membrane were not successful, the octanol/buffer partition coefficient obtained, suggests that clozapine partitions into the membrane. Similar observations, as remoxipride, were obtained for the phosphorescence polarisation measurements, again suggesting a direct interaction with the protein. Chlorpromazine and trifluoperazine were found to be potent inhibitors of enzyme activity. Sulpiride, remoxipride and procyclidine also inhibited the enzyme but to a much lesser extent than chlorpromazine and trifluoperazine. Clozapine demonstrated a general inhibition of the enzyme activities, although an initial stimulation was observed for Ca-ATPase and adenylyl cyclase of rat brain and liver at low concentrations. These data are discussed in the content of recent research into the action of these drugs.

The objectives of these studies were to develop multidimensional chromatographic techniques and combine them, in some cases with mass spectrometry, in order to provide improved methods for the determination of an anabolic steroid in plasma extracts and for the identification of by-products in drug process waste streams and bulk products. Combination of high pressure liquid chromatography (HPLC) with high resolution capillary gas chromatography (HRGG) was studied using an autosampler as an interface device. Detailed investigations were carried out using liquid chromatography solute preconcentration and optimal conditions for injections of large volumes (1 ml) of polar solutions of solutes in both normal and reverse phase HPLC were established. This resulted in enhanced detection limits whilst maintaining chromatographic resolution with reduction in interferences from endogenous materials. The system was extended by direct interfacing of the HPLC-HRGC to a mass selective mass spectrometer to provide HPLC-HRGC-MS. The normal and reverse phase HPLC preconcentration methods were also inferfaced to a supercritical fluid chromatograph and the HPLC-SFC system was used with ultraviolet and mass spectrometric detection. In addition, a simple headspace focussing procedure was developed and combined with HRGC-MS. The systems developed were used to investigate the analysis of stanozolol in plasma extracts. HPLC-HRGC gave useful results, but problems were encountered due to adsorptive properties of the drug. Studies with HPLC-SFC-MS gave good data which indicated that this form of analysis is worthy of further assessment. Use of HPLC-HRGC-MS, SFC-MS and HPLC-SFC-MS extended the positive identification of by-products in the paracetamol manufacturing process. In particular only these combined methods have enabled benzenediazonium innersalts to be identified. These positive identifications have considerably enhanced understanding of the mass balance study of the chemical process.