Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma

Chlorpyrifos oxon (CPO) is the active metabolite of the pesticide chlorpyrifos that inhibits cholinesterases at high reaction rates. Chlorpyrifos is of major concern because it causes some ten thousand fatalities each year, mostly due to suicidal attempts. Notwithstanding, toxicokinetic studies on chlorpyrifos in humans are scarce and CPO has not been detected hitherto in human blood. Knowledge of the concentration and the time course of CPO in poisonings would be helpful to better design antidotal strategies, particularly with oximes. Owing to the exceptionally fast covalent binding to butyrylcholinesterase we searched for an enzyme-based assay for CPO determination. We succeeded in a simple procedure where CPO is titrated with purified equine butyrylcholinesterase. The assay requires less than 0.2 mL EDTA plasma and allows the quantification of CPO down to 0.5 nM. CPO is first extracted from plasma with n-pentane, thereby largely excluding the majority of the more hydrophilic pesticide oxons from possible cross-reactions. When chlorpyrifos incorporation is ascertained the assay may be considered largely specific. The new procedure enabled the assessment of the extent of reversible binding of CPO to human albumin, amounting to 85% under physiological conditions. The assay allowed the quantification of CPO in the plasma of a poisoned patient, where the active metabolite was about two orders of magnitude lower than chlorpyrifos. Similar to the parent compound its oxon showed the same tendency to persist for longer periods, thus calling for a change of the usual oxime dosage regimen.