Determination of chlorpyrifos metabolites in human urine by reversed-phase/weak anion exchange liquid chromatography–electrospray ionisation–tandem mass spectrometry

A liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) method for the quantification of major chlorpyrifos (CP) metabolites, i.e. diethyl thiophosphate (DETP), diethyl phosphate (DEP), and 3,5,6-trichloro-2-pyridinol (TCP), in human urine was developed. Simultaneous separation of the parent compound and its primary biotransformation products was achieved within 20 min in gradient elution mode employing a mixed-mode reversed-phase/weak anion exchange (RP/WAX) separation principle. The analytical method was developed for a toxicokinetic study of an acute poisoning incidence with a CP containing pesticide formulation. An initial mass spectrometric screening performed with unprocessed urine samples revealed that CP is not excreted unchanged by the kidney. Hence, the quantitative assay was validated for DETP (quantifier transition: m/z 169 → 95, qualifier transition: m/z 169 → 141), DEP (m/z 153 → 79, 153 → 125), and TCP (m/z 196 → 35, 198 → 35) taking dibutyl phosphate (DBP) (m/z 209 → 79, 209 → 153) as internal standard. Clean-up of urine samples prior to LC–ESI–MS/MS analysis was carried out by a liquid–liquid extraction step with a mixture of ethylacetate and acetonitrile (70:30; v/v). Linearity was observed between 0.25 and 75 mg L−1, and the signal-to-noise ratio at 0.25 mg L−1 was better than six for the individual analytes. Recoveries, precision, and accuracies were all adequate across the validated range of 1–75 mg L−1 for the present toxicological case study.