Immunological analysis of drugs of abuse with reference to anhydroecgonine methyl ester.

The field of forensic drug testing is continually changing with analytical methodology being developed for an increasing number of drugs in a variety of biological matrices. The aim of this thesis was to develop a novel enzyme immunoassay (EIA) for screening oral fluid specimens for the presence of anhydroecgonine methyl ester (AEME), a pyrolysis product of cocaine. A confirmatory method was also to be developed to accurately quantify the levels of cocaine, its metabolites and pyrolysis products in oral fluid samples. The immunoassay development was started by synthesising an immunogen using anhydroecgonine (AE) and thyroglobulin. Following immunisation the antisera were screened by enzyme linked immunosorbent assay (ELISA) to enable selection of the antibody with the highest specificity and sensitivity. An enzyme labelled drug was synthesised and the titres of antibody and enzyme were optimised. A series of validation experiments were carried out which concluded that the EIA was sensitive, highly specific, and precise.Gas chromatography-mass spectrometry (GC-MS) was investigated for the quantitation of cocaine and its metabolites. A temperature program was selected which allowed for the simultaneous analysis of all the analytes. A solid phase extraction (SPE) method was developed to extract cocaine and its metabolites from oral fluid. The SPE method provided high recovery for all analytes apart from the highly polar AE. Degradation of the GC column had a detrimental effect on the analysis of AEME, and so the confirmation method was switched to liquid chromatography-tandem mass spectrometry (LC-MS/MS). A series of LC columns and mobile phases were tested for optimum separation and ionisation. The instrument parameters such as capillary voltage, drying gas temperature, shield voltage, and needle voltage, were optimised. Following a number of validation experiments the method was found to be highly sensitive, precise, accurate and robust.Both the EIA and LC-MS/MS methods were applied to the analysis of clinical samples from self declared crack cocaine users. The EIA showed good correlation to LC-MS/MS. It was evident that the presence of AEME can positively identify smoking as the route of cocaine administration however its absence does not necessarily mean the individual has not smoked cocaine.