Development of HPLC-MS/MS method for the simultaneous determination of metabolites of organophosphate pesticides, synthetic pyrethroids, herbicides and DEET in human urine

ABSTRACT We developed an isotopic dilution high-performance liquid chromatography (HPLC)/tandem mass spectrometer (MS/MS) method to rapidly and accurately quantify nine metabolites of several classes of pesticide in 1 mL human urine specimens. The analytes covered in the method are two organophosphate (OP) pesticide metabolites: 3,5,6-trichloro-2-pyridinol (TCPy), 2-isopropyl-6-methyl-4-pyrimidinol (IMPY); three synthetic pyrethroid metabolites: 3-phenoxy benzoic acid (3-PBA), 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA) and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-1(1-cyclopropane) carboxylic acid (t-DCCA); three herbicide metabolites: 2,4-dichlorophenoxyacetic acid (DCPAA), 2,4,5-trichlorophenoxyacetic acid (TCPAA) and atrazine mercapturate; and one insect repellent: N,N-diethyl-meta-toluamide (DEET). The analytes are first deconjugated by incubating with acetate/β-glucuronidase buffer at 37°C for 17 h. The deconjugated analytes are extracted and concentrated from the urine matrix using solid-phase extraction cartridges, separated through C18 reversed phase HPLC, and analysed on MS/MS. The MS/MS was operated in positive and negative electrospray ionisation switch mode. Two ions from each analyte and one from each labelled internal standard are monitored for quantification and confirmation. The limit of detections (LODs) for all the analytes are in the low parts-per-trillion (0.05 ng/mL) except TCPy where it was 0.5 ng/mL) with a wide linear range (0.05 up to 40 ng/mL) and provides high accuracy (recoveries: 90–118%) and high precision (coefficient of variation <15%). The method accuracy was also verified by the analysis of proficiency testing urine samples. We analysed 101 urine samples for a recent California study cohort, and detection frequencies ranged from ~100% to 0%: 3-PBA (98%), IMPY (91%), TCPy, (89%), DCPAA (66%), 4-F-3-PBA (11%), TCPAA (0%).