Evaluation of immunoassays for semiquantitative detection of cocaine and metabolites or heroin and metabolites in extracts of sweat patches.

Abstract

Two types of immunoassays, radioimmunoassay (RIA) and microplate enzyme immunoassay (EIA), were compared for their ability to detect and quantitate cocaine and metabolites or heroin and metabolites in extracts of sweat patches. Experiments used sweat patches that had been fortified with cocaine, benzoylecgonine (BE), and ecgonine methyl ester (EME) or 6-acetylmorphine (6-AM), heroin, and morphine. Assays were first evaluated for sensitivity in detection of the analyte(s) known to be excreted in sweat (cocaine >> BE and EME; 6-AM > heroin > morphine). The cocaine metabolite RIA had cross-reactivity for cocaine > BE > EME, and the cocaine metabolite EIA had cross-reactivity for BE > cocaine >> EME. The RIA, having greater sensitivity for COC, was studied further. Optimal linearity was 4 to 200 ng/patch, and quantitation within these limits at 4, 75, and 150 ng/patch had intrarun %CVs within 7.8% and percent targets within 15% and inter-run %CVs within 13.5% and % targets within 13%. The opiate RIA had cross-reactivities for morphine >> 6-AM and heroin. The opiate EIA had cross-reactivities for 6-AM and heroin of 42 and 28% relative to morphine, respectively. The EIA, having greater sensitivity for 6-AM and heroin, was studied further. The limits of detection ranged from 1.7 to 24.7 ng/patch, and the lower limits of quantitation ranged from 7.3 ng/patch to beyond the linear range. The assay, however, had consistently good precision at 4 and 5 ng/patch, and optimal linearity was established from 4 to 100 ng/patch. With controls at 5, 25, and 90 ng/patch, both intrarun and inter-run precision were acceptable. Quantitation was accurate at 5 and 25 ng/patch, but the 90 ng/patch controls were consistently < 70% of target. Because our studies focused on the assays that had greater sensitivity for the analytes excreted in sweat, we did not fully evaluate the cocaine metabolite EIA or the RIA opiate screen and therefore cannot make any comment on the usefulness of these assays for detecting analytes in extracts of sweat patches beyond predicting that they will have less sensitivity. Both the cocaine metabolite RIA and opiate EIA had the ability to detect analytes known to be extracted from sweat patches.