Validation of an ELISA method for screening methadone in postmortem blood

In this study, we evaluate Venture Labs' enzyme-linked immunosorbent assay (ELISA) for the detection of methadone in postmortem specimens. Sixty-one postmortem specimens that previously screened positive for methadone along with 59 specimens which screened negative for methadone were included. All specimens were screened using the Venture Labs methadone assay in conjunction with a liquid-liquid basic extraction and gas chromatographic-mass spectrometric (GC-MS) analysis. All cases screening positive by either method were confirmed for methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3- diphenylpyrrolidine by a solid-phase extraction utilizing deuterated internal standards and GC-MS-SIM. Twenty-four postmortem samples that screened negative by both methods were also extracted and analyzed using the confirmation method to demonstrate the validity of both screening methods. The intra- and interassay precision for the ELISA method was evaluated at the cut-off concentration used for the analysis (50 ng/mL). True positives, true negatives, false positives, and false negatives were calculated for the ELISA results as compared to the GC-MS screening data. The Venture Labs methadone assay demonstrated a sensitivity of 96.7% ± 2.3% and a specificity of 98.3% ± 1.7% relative to the GC-MS method.