Simultaneous determination of cloricromene and its active metabolite in rabbit aqueous humor by high-performance liquid chromatography.
A rapid and simple method was developed for the simultaneous separation and quantification of cloricromene, a coumarine derivative, and its active metabolite, cloricromene acid, in rabbit aqueous humor. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Hypersil ODS) with UV detection at 318 nm. The mobile phase consisted of acetonitrile-water containing 1% triethylamine pH 3.5, adjusted with orthophosphoric acid. An acetonitrile gradient was necessary to achieve good separation within 13 min. Timolol was found to be a suitable internal standard. The retention times ranged from 5.72 to 11.25 min. A simple pre-treatment with acetonitrile containing 0.6% HCIO4 was used to deproteinize aqueous humor samples. The limit of quantitation ranged between 10 and 20 ng/ml. The recovery was >90%. The relationship between peak areas and concentration was linear over the range between 0.01 and 3.8 microg/ml, with r2 > 0.99. The assay provided good reproducibility and accuracy for both analytes and proved to be suitable for pharmacokinetic studies of cloricromene.
Maltese
Adriana
A
Fidia Oftal Research Laboratories, Catania, Italy.
Bucolo
Claudio
C
D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002866:Chromonar / Q000031:analogs & derivatives* / Q000378:metabolism*; D008297:Male; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
C025945:cloricromen; D002866:Chromonar