P17: Analytical evaluation of newly developed enzymelinked immunosorbent assay kit for the detection of tramadol and the metabolite O-desmethyl tramadol in urine and blood

Abstract

Introduction Tramadol, a synthetic analogue of codeine, is used clinically as an analgesic for the treatment of moderate to moderately- severe pain. Tramadol expresses dual mechanism of action that includes weak agonistic effects at the μ-opioid receptors as well as inhibition of serotonin and norepinephrine uptake. Tramadol has two primary metabolites, N-desmethyl and O-desmethyl tramadol, the latter presenting greater affinity for μ-opioid receptors and twice the analgesic potency of the parent compound. Impairing side effects from the usage of tramadol include dizziness, confusion, light-headedness or fainting spells, drowsiness, seizures and respiratory depression. Much of the toxicity in tramadol overdose appears to be attributable to the monoamine uptake inhibition. This study reports the analytical evaluation of a newly developed enzyme-linked immunosorbent assay (ELISA) for the sensitive detection of tramadol and the active metabolite O-desmethyltramadol in urine and blood. This represents a useful analytical tool for applications in toxicology. Methods A colorimetric competitive immunoassay was employed. The capture antibody was immobilised and stabilised on the 96-well microtitre plate surface. The analyte, if present in the sample, competes with the horseradish peroxidase labelled conjugate for antibody binding sites. Absorbances were read at 450nm. The signal is inversely proportional to the concentration of the analyte in the sample. Results The assay was standardised to tramadol and the metabolite O-desmethyl tramadol was also detected with a % cross-reactivity of 57 %. The limit of detection values were 1.40 ng/ml (urine) and 0.86 ng/ml (blood) for an assay range 0–100 ng/ml. The intraassay precision (n=12), expressed as % CV, was Conclusion The results indicate that the newly developed ELISA kit is applicable to the detection of tramadol and the metabolite Odesmethyl tramadol in urine and blood. The reagents are liquid and ready to use. This represents a useful analytical tool for applications in toxicology.