Review of UV spectroscopic, chromatographic, and electrophoretic methods for the cholinesterase reactivating antidote pralidoxime (2‐PAM)

Abstract

Pralidoxime (2-PAM) belongs to the class of monopyridinium oximes with reactivating potency on cholinesterases inhibited by phosphylating organophosphorus compounds (OPC), for example, pesticides and nerve agents. 2-PAM represents an established antidote for the therapy of anticholinesterase poisoning since the late 1950s. Quite high therapeutic concentrations in human plasma (about 13 µg/ml) lead to concentrations in urine being about 100 times higher allowing the use of less sensitive analytical techniques that were used especially in the early years after 2-PAM was introduced. In this time (mid-1950s until the end of the 1970s) 2-PAM was most often analyzed by either paper chromatography or simple UV spectroscopic techniques omitting any sample separation step. These methods were displaced completely after the establishment of column liquid chromatography in the early 1980s. Since then, diverse techniques including cation exchange, size-exclusion, reversed-phase, and ligand-exchange chromatography have been introduced. Today, the most popular method for 2-PAM quantification is ion pair chromatography often combined with UV detection representing more than 50% of all column chromatographic procedures published. Furthermore, electrophoretic approaches by paper and capillary zone electrophoresis have been successfully used but are seldom applied. This review provides a commentary and exhaustive summary of analytical techniques applied to detect 2-PAM in pharmaceutical formulations and biological samples to characterize stability and pharmacokinetics as well as decomposition and biotransformation products. Separation techniques as well as diverse detectors are discussed in appropriate detail allowing comparison of individual preferences and limitations. In addition, novel data on mass spectrometric fragmentation of 2-PAM are provided.

Repetitive obidoxime treatment induced increase of red blood cell acetylcholinesterase activity even in a late phase of a severe methamidophos poisoning: A case report

“…Enzyme activity assays (a-c) were based on modifications of the colorimetric Ellman assay following established protocols (Thiermann et al, 2007Worek et al, 2004. Obidoxime was analyzed by liquid chromatography online coupled to UV-detection and atropine by online coupling to tandem mass spectrometry as described earlier (John et al, 2010;John and Blum, 2012). Treatment with obidoxime was continued until day 3.…”

Quantification of pralidoxime (2‐PAM) in urine by ion pair chromatography‐diode array detection: application to in vivo samples from minipig

“…HPLC analysis was performed at different column temperatures controlled by the column oven (10,15,20,25,30,35, and 40 C). HPLC analysis was performed at different column temperatures controlled by the column oven (10,15,20,25,30,35, and 40 C).…”

“…[23,24] But most frequently ion pair chromatography (IPC) coupled to electrochemical detection (ECD), [25,26] radio detection (RD), [27] and UV detection [14,28,29] has been applied. [30] Although more modern RPC-tandem mass spectrometric (RPC-MS/MS) methods provide better sensitivity and selectivity, [23,24] UV-detection appears to be sufficient for the quantification of high micromolar 2-PAM concentrations present in plasma and urine samples. [30] Although more modern RPC-tandem mass spectrometric (RPC-MS/MS) methods provide better sensitivity and selectivity, [23,24] UV-detection appears to be sufficient for the quantification of high micromolar 2-PAM concentrations present in plasma and urine samples.…”

In vitro and in vivo efficacy of PEGylated diisopropyl fluorophosphatase (DFPase)

“…Due to the phosphoroamidate structure in tabun, AChE inhibited by this agent is hard to reactivate by currently fielded oximes like obidoxime or 2-PAM. [7] This aging reaction is especially rapid in case of soman with in vivo reaction half-lives of only a few minutes. Aging denotes the loss of an O-alkyl sidechain from the phosphylated moiety resulting in a negative charge at this group hindering the nucleophilic attack of a reactive oximate anion.…”