High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs.
A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-microm Capcell Pak C18 column (150 x 4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05-100 mg/l for rat blood (r2>0.9995, n=6) and dog blood (r2>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-microl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1-104.1% and 1.5-6.8% for rat blood and 95.4-105.7% and 1.4-5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8-105.3% and 3.7-12.6% for rat blood and 87.5-107.1% and 2.9-15.3% for dog blood, respectively. The absolute recoveries were 82.4-93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.
Lee
S H
SH
LG Chem Biotech Research Institute, Yu Sung, Taejon, South Korea.
Choi
Y J
YJ
Jeong
Y N
YN
Kim
H S
HS
Lee
S H
SH
Kim
I C
IC
Oh
Y S
YS
Lee
Y H
YH
D000284:Administration, Oral; D000577:Amides / Q000097:blood* / Q000493:pharmacokinetics; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D008297:Male; D009281:Naphthalenes / Q000097:blood* / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013917:Thrombin / Q000037:antagonists & inhibitors*
D000577:Amides; C116020:N-cyclopentyl-N-methyl-3-(4-amidrazonophenyl)-2-(2-naphthylsulfonylamino)-propionamide; D009281:Naphthalenes; D013917:Thrombin