Chromatographic method for the determination of diazepam, pyridostigmine bromide, and their metabolites in rat plasma and urine.
This study describes a chromatographic method for the determination of diazepam, an anxiolytic drug that is also used as an antidote against nerve agent seizures, its metabolites N-desmethyldiazepam, and temazepam, 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 compounds were extracted using C18 Sep-Pak Vac 3cc (500 mg) cartridges and separated using isocratic mobile phase of methanol, acetonitrile and water (pH 3.2) (10:40:50) at a flow-rate of 0.5 ml/min in a period of 12 min, and UV detection ranging between 240 and 280 nm. The limits of detection for all analytes ranged between 20 and 50 ng/ml, while limits of quantitation were 100 ng/ml. Average percentage extraction recoveries of five spiked plasma samples were 79.1+/-7.7, 83.5+/-6.4, 83.9+/-5.9, 71.3+/-6.0 and 77.7+/-5.6, and from urine 79.4+/-7.9, 83.1+/-6.9, 73.6+/-7.7, 74.3+/-7.1 and 77.6+/-5.9 for diazepam, N-desmethyldiazepam, temazepam, pyridostigmine bromide, and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 1000 ng/ml. This method was applied to determine the above analytes following a single oral administration in rats as a tool to study the pharmacokinetic profile of each compound, alone and in combination.
Abu-Qare
A W
AW
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
Abou-Donia
M B
MB
D000818:Animals; D014151:Anti-Anxiety Agents / Q000032:analysis / Q000097:blood / Q000378:metabolism / Q000652:urine; D002138:Calibration; D002800:Cholinesterase Inhibitors / Q000032:analysis / Q000097:blood / Q000378:metabolism / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003975:Diazepam / Q000032:analysis* / Q000097:blood / Q000378:metabolism / Q000652:urine; D011729:Pyridostigmine Bromide / Q000032:analysis* / Q000097:blood / Q000378:metabolism / Q000652:urine; D011786:Quality Control; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards
D014151:Anti-Anxiety Agents; D002800:Cholinesterase Inhibitors; D011729:Pyridostigmine Bromide; D003975:Diazepam