Quantitative determination of a potent lipopolysaccharide antagonist, E5564, in rat and dog plasma by high-performance liquid chromatography with fluorescence detection.
The assay method was established for the quantification of a potent lipopolysaccharide (LPS) antagonist, E5564, in rat and dog plasma using HPLC. E5564 and the I.S. (an analogue of E5564) were extracted and derivatized with 9-Anthryldiazomethane (ADAM reagent) to be given fluorescence. LC-MS analysis indicated that single molecule of E5564 was coupled with two molecules of ADAM reagent at one on each of the phosphorus groups. After solid-phase extraction, ADAM derivatives of E5564 and the I.S. were separated on an ODS column using methanol/ethanol containing sodium acetate as a mobile phase at 1.2 ml/min (gradient elution), and detected by a fluorescence detector (excitation: 254 nm, emission: 415 nm). The intra-day and inter-day precision were less than 14.4%, and accuracy were within +/-13.0% in the concentration range of 30 to 20,000 ng/ml plasma in both species. E5564 was stable for at least 13 days in rat and dog plasma at -20 degrees C, and the processed sample was stable for up to 14 days at 4 degrees C. This validated method was successfully applied to the evaluation of the pharmacokinetics of E5564 in rats and dogs after single bolus intravenous doses.
Kaneko
K
K
Tsukuba Research Laboratories, Eisai Co. Ltd., Ibaraki, Japan.
Ueda
R
R
Kikuchi
K
K
Sano
Y
Y
Yoshimura
T
T
D000818:Animals; D015925:Cryopreservation; D004285:Dogs; D004355:Drug Stability; D008050:Lipid A / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000493:pharmacokinetics; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
C404742:E5564; D008050:Lipid A; D008070:Lipopolysaccharides