Method validation for simultaneous determination of atropine, pralidoxime and 12 organophosphorus compounds in blood samples by means of high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

Organophosphorus (OP) compounds stand out as one of the most extensively used pesticides' class worldwide, being associated with a high prevalence of voluntary and accidental poisoning in humans. These compounds promote inhibition of the acetylcholinesterase (AChE) leading to the accumulation of acetylcholine in the central and peripheral cholinergic synapses. Standard treatment after acute OP poisoning employ the antidotes, atropine (ATR), to prevent cholinergic effects and oximes, such as the pralidoxime (2-PAM), to reactivate the inhibited AChE. In cases of OP poisoning, identifying the toxic agent, as well as monitoring antidotes and toxic agent's concentration along time may be of great importance for therapeutic success and survival, particularly in view of the marked hemodynamic changes present in the poisoning state, which could interfere with OPs and antidotes levels achieved. Despite the profusion of methods described for analysis of either OP compounds or antidotes, to our knowledge, there is no method available that allows simultaneous identification and monitoring of these compounds. This study aimed to develop a fully validated LC-MS/MS method for combined analysis of multiple OP compounds and antidotes in blood samples. Method validation was achieved using blank blood samples from rats, followed by deproteinization with cold acetonitrile. Linearity results as well as limits of detection and quantification were within the desired range for all analytes tested. Intra-assay precision ranged from 1.2% (atropine) to 14.6% (chlorpyrifos), while inter-assay precision ranged from 4.4% (pyrazophos) to 17.2% (chlorpyrifos). The intra-assay accuracy results were within 82.8% (pirimiphos-methyl) to 111.8% (malathion) and inter-assay accuracy ranged from 84.9% (pirimiphos-methyl) to 111.6% (malathion). The validated method was applied to blood samples from rats acutely intoxicated with an OP, chlorpyrifos (CPF) and treated 1 h after poisoning with the antidotes, ATR and 2-PAM, separately or in combination. Samples were collected in different periods after the antidote treatment (15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 24 h). The method successfully detected and measured the CPF as well as the antidotes concentrations along the different times tested. The method was also tested in human blood samples from forensic cases of pesticides poisoning, being also effective in detecting and quantifying OP compounds. The present method proved to be reliable and suitable in identifying and quantifying OP pesticides together with antidotes in blood samples. The promising application of the method in the forensic and clinical field in cases of poisoning is also rationalized.