A liquid chromatographic–mass spectrometric (LC–MS) method for the analysis of the bis-pyridinium oxime ICD-585 in plasma: Application in a guinea pig model

In recent animal studies, several novel oxime compounds that are better than 2-PAM as antidotes against selected organophosphate (OP) nerve agents have been identified. The purpose of this study was to develop and validate a liquid chromatographic–mass spectrometric (LC–MS) method for analysis of the bis-pyridinium oxime ICD-585 (1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-propane) in plasma and to establish the utility of the method in a guinea pig model. Calibration curves were prepared using ICD-585-spiked plasma at concentrations from 0.156 to 10μg/ml. Curves were run over a 1-month time frame and a total of 13 (n=13) were generated. The lower limit of quantification (LLOQ) was determined to be 0.216μg/ml. Intra- and inter-day variability was assessed by studying precision and accuracy. For intra-day studies, data from the precision determinations indicated that the % CV's ranged from 4.28 to 14.98%. The % error in the accuracy assessments ranged from −8.73 to 4.61%. For inter-day studies, precision data ranged from 3.53 to 13.20%. The % error in the accuracy assessments ranged from 0.39 to 13.77%. Room temperature, freeze–thaw and autosampler stability was also examined. For all 3 stability studies, the compound remained within ±15% of the initial analysis. Application of the method was demonstrated by analyzing samples from guinea pigs challenged with sarin (GB) or cyclosarin (GF) (1× LD50) followed with intramuscular ICD-585 (58 μM/kg, 21.8mg/kg). At 55min after oxime administration, mean (±SD) plasma concentrations were 15.98 (±4.88)μg/ml and 14.57 (±3.70)μg/ml in GB- and GF-exposed animals, respectively. In summary, studies have been carried out to verify the sensitivity, precision and accuracy of the assay as well as the stability of the analyte under various conditions. The method has been demonstrated to be applicable to the analysis of plasma from nerve agent-exposed guinea pigs.