DEVELOPMENT OF A SENSITIVE RADIOIMMUNOASSAY FOR THE POTENTIAL ANTIMIGRAINE DRUG GR151004 - INCLUDING CROSS-VALIDATION TO LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY MASS-SPECTROMETRY AND APPLICATION TO PHARMACOKINETIC STUDIES

The development of a sensitive radioimmunoassay (RIA) for the determin ation of the potential anti-migraine drug GR151004 in human plasma is described. The validated method was used for the determination of the drug in plasma samples collected during a Phase I safety tolerability study in healthy volunteers, Synthetic routes for the hapten and radio ligand are presented. Immunogen synthesis was performed using the 2-ch loro-1-methylpyridinium iodide coupling agent (Mukaiyama reaction); th is method gave the highest hapten incorporation ratios during a compar ison of possible conjugation reactions Antisera were generated in Soay sheep using two different immunisation regimens. One of these regimen s was a 'fast-track' method that was also used to compare antisera qua lity using immunogens prepared with three different carrier proteins. The antiserum used for the assay was produced with a GR151004 hapten - bovine thyroglobulin immunogen during a 19 week immunisation schedule . The potential specificity of the RIA was assessed by performing HPLC -RIA analysis on urine samples collected during toxicokinetic studies, and subsequently cross-reactivity measurements with a major GR151004 metabolite in man, Final confirmation of method specificity was achiev ed by cross-validating the RIA to an established method employing HPLC with tandem mass spectrometry (LC-MSMS). This latter study establishe d that the two techniques afforded equivalent results. The reliable qu antification range of the RIA is from 0.1 to 25.6 ng GR151004 ml(-1) i n human plasma, using a 0.1 mi undiluted sample. Over this concentrati on range the intra- and inter-assay bias is < +/-9% and imprecision is <12%. The assay drift, measured as the difference in concentration va lues for quality control samples assayed immediately before and after the sequence of test plasma samples, is <+/-3% (batch size 30-40 sampl es).