Quantification of pralidoxime (2-PAM) in urine by ion pair chromatography-diode array detection: application to in vivo samples from minipig Harald John, a * Michael Eddleston, b R. Eddie Clutton, c Franz Worek a and Horst Thiermann a 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, ap- propriate 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 ml, 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 (1ml/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 mg/ml, a limit of quantification at 9.8 mg/ml, and a broad calibration range from 4.9–2500 mg/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. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: chromatography; nerve agents; oxime; pesticides; pharmacokinetics Introduction Pralidoxime (2-PAM, N-methyl-2-aldoximinopyridinium, Figure 1A) is a monopyridinium oxime administered for the causal therapy of poisoning with organophosphorus (OP) compounds; for example, pesticides and nerve agents. The abbreviation 2-PAM is used to address the pure oxime structure, not specifying any of the counterions typically present in pharmaceutical formulations; for example, chloride, iodide, methanesulfonate. OP poisons react (phosphylation) with the serine residue of the active site of acetylcholinesterase (AChE) thus inhibiting the enzyme, sometimes irreversibly, and preventing regulatory ester-cleavage of the neurotransmitter acetylcholine (ACh). Consequently, ACh accumulates in the synaptic cleft and over- stimulates effector cells, leading initially to miosis, fasciculation, and increased secretion of body fluids (saliva, tears, sweat) and ultimately to death by respiratory paralysis. [1] To reactivate the enzyme and thus restore its physiological function, 2-PAM is administered intravenously (i.v.) or intramus- cularly (i.m.) typically as solutions of its completely dissociated chloride, iodide or methanesulfonate salts. [2] In vivo 2-PAM is partly deprotonated and forms the active zwitterionic oximate that is capable of cleaving the OP-moiety from the enzyme by nucleophilic substitution. [3] Free active AChE and the unstable intermediate phosphylated oxime (POX) are produced. [4–6] Even though 2-PAM was introduced as the first oxime antidote against OP poisoning in 1955, [3] there remains ongoing debate over its optimum use and poison-specific efficacy. [2,7–13] Although human in vivo data have been obtained from OP pesticide poisoned patients [8–10,12,13] controlled animal studies are required to elaborate indications and limitations of therapy. Present in vitro approaches are helpful and promising but cannot replace in vivo studies entirely. [14,15] Therefore, studies were performed using the Göttingen minipig, which is a good model for biomedical research having many physiological, anatomical, nutritional, and metabolic similarities to humans. [16–18] Therapy optimization necessitates analytical approaches for quantification of 2-PAM in body fluids and distinct compartments. * Correspondence to: Harald John, Bundeswehr Institute of Pharmacology and Toxicology; Neuherbergstr. 11, 80937 Munich, Germany. E-mail: HaraldJohn@bundeswehr.org a Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany b Clinical Pharmacology Unit, Centre for Cardiovascular Science, University of Edinburgh, and National Poisons Information Service - Edinburgh, Royal Infirmary of Edinburgh, UK c Royal (Dick) School of Veterinary Sciences, University of Edinburgh, UK Drug Test. Analysis 2012, 4, 169–178 Copyright © 2011 John Wiley & Sons, Ltd. Research article Drug Testing and Analysis Received: 31 May 2011 Revised: 1 July 2011 Accepted: 3 July 2011 Published online in Wiley Online Library: 18 November 2011 (wileyonlinelibrary.com) DOI 10.1002/dta.340 169