A Method for Diagnosing Organophosphate Pesticide Exposure in Humans using Liquid Chromatography Coupled Tandem Mass...

Organophosphate (OP) pesticides are commonly utilized worldwide for agricultural purposes and pose a health threat through air, ground, and water contamination. Here, we present a convenient method for diagnosing exposure to OP pesticides in humans. This immunoprecipitation method relies on extraction of butyrylcholinesterase (BChE), a biomarker of OP poisoning that adducts OP compounds, from human serum using agarose beads conjugated to anti-BChE antibodies. Extracted BChE was then digested with pepsin and analyzed for unadducted and OP-adducted peptides by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). To characterize and validate this method, pooled human plasma was exposed to parathion and dichlorvos to form diethoxyphospho, aged ethoxyphospho and dimethoxyphospho adducts with BChE. Untreated plasma was also analyzed for unadducted peptides. Additionally, samples were analyzed using Ellman's assay to measure BChE functional activity. The percent inhibition of BChE was 53.5±5.76 and 95.2±0.37%, respectively, for plasma treated with parathion for 1 hour and 24 hours. The percent inhibition was 97.2±0.98 for plasma treated with dichlorvos for 1 hour. The percent inhibition was 97.9±0.41% when the plasma treated with parathion for 1 hour, parathion for 24 hour and dichlorvos for 1 hour were mixed. Individual adducts were quantified in a single chromatographic run. Untreated plasma contained 26.4±1.87 ng/mL of unadducted BChE and no adducted peptides. In contrast, the plasma sample treated with both pesticides contained no unadducted BChE, but did contain 9.46±1.10, 10.9±0.98 and 14.1±1.10 ng/mL of diethoxyphospho, aged-ethoxy, and dimethoxyphospho peptides, respectively. The ability to identify and measure BChE and BChE adducts to parathion and dichlorvos is expected to be useful for diagnosing human exposure to multiple OP pesticides.