Validation of the Cozart® microplate ELISA for detection of opiates in hair

The purpose of this study was to determine the performance characteristics of the Cozart Opiates Microplate ELISA assay for the detection of opiates in hair specimens. One hundred and six hair specimens were collected from volunteers and from drug-related deaths. The hair samples were extracted by sonication followed by overnight extraction in methanol at 60°C. The methanol extract was dried, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture and 0.1M HCl were added to 20 mg of specimen or spiked blank hair and sonicated for 1 h. The opiates were extracted by solid-phase and derivatized with BSTFA + 1% TMS for GC-MS analysis. Fifty-one hair specimens were confirmed positive by GC-MS. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results as the reference method. The optimum cutoff for the Cozart Opiate Microplate ELISA was determined to be between 200 and 300 pg morphine equivalents/mg hair using a 20-mg hair sample. The Cozart Opiates Microplate EIA for opiates in hair using a cutoff of 200 pg/mg hair with a 20-mg hair sample had a sensitivity of 98% ± 2% and a specificity of 92.7% ± 3.5% versus GC-MS.