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

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 metabolite N-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 C-18 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 and N-methyl-3-hydroxypyridinivm bromide, and 10 mug ml- I for sarin and methylphosphonic acid, while limits of quantitation were between 100 ng mL(-1) - 12 mug 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 +/- 78, 74.4 +/- 8.4, 80.6 +/- 6.8 for sarin, methylphosphonic acid, pyridostigmine bromide and N-methyl-3-hydroxypyridinium bromide, respectively. This method was applied to analyze the above chemicals and metabolites following combined administration in rats.