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Fast gas chromatography/mass spectrometric assay for the validated quantitative determination of methadone and the primary metabolite EDDP in whole blood



A toxicological analysis was developed and validated for simultaneous screening and quantification of methadone (METH) and its primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The method employs microscale liquid-liquid extraction (µLLE) and direct injection of a separated aliquot of the organic layer into a gas chromatography/mass spectrometric (GC/MS) system without any other pre-treatment stages. A fast GC/MS runtime (total 5.8 min; METH, Rt = 3.55 min; EDDP, Rt = 3.40 min) combined with rapid sample preparation allowed cost-efficient and routinely applicable performance with a low amount of manual work. The validated parameters included: linearity (25–1000 ng mL−1 both; Rmath image = 0.998 and Rmath image = 0.997), accuracy (BiasMETH: from −0.05 to 11.3%, BiasEDDP: from 1.11 to 4.37%); intra and inter-assay precision (RSDMETH: from 2.4 to 3.9%, from 4.89 to 10.3%; RSDEDDP: from 4.50 to 6.20%, from 4.57 to 15.2%), extraction efficiency (METH = 95.5%; EDDP = 90.6%), LOQMeth,EDDP = 25 ng mL−1. Samples were stable for at least 25 h and no selectivity problems or baseline interference were observed. The method should be applicable for identifying and quantitative confirmation of possible misuse and/or illegal use of METH in toxicological cases. Copyright © 2006 John Wiley & Sons, Ltd.