Simultaneous analysis of sarin, pyridostigmine bromide and their metabolites in rat plasma and urine using HPLC

Summary A method was developed for the separation and quantification of the warfare nerve agent sarin (O-isopropylmethylphosphonoflouridate), its metabolite methylphosphonic acid, the anti nerve agent drug pyridostigmine bromide (PB;3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metaboliteN-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The method involved using solid phase extraction and high performance liquid chromatography (HPLC) with reversed phase C₁₈ column, and UV detection at 280 nm. The compounds were separated using gradient of 1% to 55% acetonitrile in 0.1% triflouroacetic acid water solution (pH 3.20) at flow rate of 0.9 ml/min in a period of 15 min. The retention times ranged from 4.4–12.1 min. The limits of detection were 50 ng mL⁻¹ for PB andN-methyl-3-hydroxypyridinium bromide, and 10 μg mL⁻¹ for sarin and methylphosphonic acid, while limits of quantitation were between 100 ng mL⁻¹–12 μg mL⁻¹. Average percentage recovery of five spiked samples from plasma were 84.6±8.4, 86.5±9.0, 76.4±8.5, 81.3±8.2, and from urine 78.5±7.9, 76.4±7.8, 74.4±8.4, 80.6±6.8 for sarin, methylphosphonic acid, pyridostigmine bromide andN-methyl-3-hydroxypyridinium bromide, respectively. This method was applied to analyze the above chemicals and metabolites following combined administration in rats.