Development, validation and application of assays to quantify metrifonate and 2,2-dichlorovinyl dimethylphosphate in human body fluids

Gas chromatographic procedures [GC with electron-capture detection (ECD) an d GC-MS] for the quantitative analysis of metrifonate and its active metabo lite 2,2-dichlorovinyl dimethylphosphate (DDVP) in human blood and urine we re developed, validated, and applied to the analysis of clinical study samp les. Analysis of metrifonate involved extraction of acidified blood with et hyl acetate followed by solid-phase clean-up of the organic extract. Acidif ied urine was extracted with dichloromethane and the residue of evaporated organic phase was reconstituted in toluene, ECD and diethyl analogue of met rifonate internal standard (I.S.) were used for quantitation of metrifonate . The metrifonate lower limit of quantitation (LOQ) was 10.0 mu g/l. The DD VP metabolite was chromatographed separately after cyclohexane extraction o f acidified blood and urine using d(6)-DDVP I.S. and MS detection. The LOQ of DDVP was 1 mu g/l. Stability studies have confirmed that the matrix shou ld be acidified prior to storage at -20 degrees C or -80 degrees C to inhib it chemical and enzymatic degradation of the analytes and to avoid overesti mation of DDVP concentrations. Metrifonate was found to be stable in acidif ied human blood after 20 months of storage at -20 degrees C and after 23 mo nths of storage at -80 degrees C, Under these conditions DDVP was found to be stable after 12 months of storage. Both assay procedures were cross-vali dated by different world-wide laboratories and found to be accurate and rob ust during analyses of clinical study samples. (C) 2000 Elsevier Science B. V. All rights reserved.