A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine

A method was developed for the separation and quantification of the anti-nerve agent pyridostgmine bromide (PB;3-dimethylaminocarbonytoxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid. and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1 H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C-18 Sep-Pak(R) cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C-18 column, and UV detection at 280 nm. The compounds were separated using gradient of 1-85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min, The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9 +/- 9.5, 73.7 +/- 9.8, 88.6 +/- 9.3, 83.9 +/- 7.8, and from urine 69.1 +/- 8.5, 74.5 +/- 8.7, 85.9 +/- 9.8, 83.2 +/- 9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats. (C) 2001 Elsevier Science B.V, All rights reserved.