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Title: Applications of liquid chromatography-tandem mass spectrometry to cytochrome P450 inhibiton screening and the measurement of plasma free metanephrines
Author: Graham, K. S.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 2005
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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 free 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available