title: Quantification of pralidoxime (2-PAM) in urine by ion pair chromatography-diode array detection: application to in vivo samples from minipig.
abstract: Pralidoxime (2-PAM) is a monopyridinium oxime used as an antidote for the treatment of poisoning with organophosphorus (OP) compounds, for example, pesticides and nerve agents, reactivating OP-inhibited acetylcholinesterase. However, appropriate dosing and efficacy remains a matter of discussion requiring experimental data. Therefore, we developed and validated an ion pair chromatography-diode array detection (IPC-DAD) method suitable for quantitative analysis of 2-PAM in human and porcine urine. Before injection of 20 µl, urine was acidified with trichloroacetic acid, mixed with internal standard (pyridine-4-aldoxime, 4-PAO), and diluted with IPC solvent yielding a total dilution of 1:49.5 and a 100% recovery. Isocratic separation was carried out at 25 °C on a LiChrospher 60 RP-select B column (125 x 4.0 mm I.D.) using phosphate buffer (7.5 mM Na(2) HPO(4) , 7.5 mM KH(2) PO(4) , pH 2.6) mixed with octanesulfonate (2.5 mM) as ion pair reagent and acetonitrile (6% v/v) as organic modifier (1 ml/min). 2-PAM was detected at 293 nm and 4-PAO at 275 nm. The method is rugged, selective, and characterized by good intra-day and inter-day precision (RSD, 1.3-6.0%) and accuracy (88-100%) with a limit of detection at 4.9 µg/ml, a limit of quantification at 9.8 µg/ml, and a broad calibration range from 4.9-2500 µg/ml. The procedure was applied to urine samples obtained from dimethoate poisoned minipigs receiving 2-PAM therapy (intravenous bolus injection and infusion). Results indicate that 60-80% of infused 2-PAM is rapidly (within 1-2 h) excreted in the urine.
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, Munich, Germany. HaraldJohn@bundeswehr.org
authors.forename: Harald
authors.initials: H
authors.lastname: John
authors.forename: Michael
authors.initials: M
authors.lastname: Eddleston
authors.forename: R
authors.initials: R
authors.lastname: Eddie Clutton
authors.forename: Franz
authors.initials: F
authors.lastname: Worek
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
mesh_terms: D000818:Animals; D000931:Antidotes / Q000032:analysis; D002138:Calibration; D002800:Cholinesterase Inhibitors / Q000506:poisoning; D002801:Cholinesterase Reactivators / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004117:Dimethoate / Q000506:poisoning; D006801:Humans; D057230:Limit of Detection; D011220:Pralidoxime Compounds / Q000652:urine*; D013552:Swine
chemical_list: D000931:Antidotes; D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D011220:Pralidoxime Compounds; C028797:pralidoxime; D004117:Dimethoate