Introduction. Vardenafil belongs to inhibitors of the 5th type phosphodiesterase and used in therapy of erectile dysfunction.
Objective. Study of the influence of the solvent nature on intensity of vardenafil fluorescence in solutions. Development of optimal conditions for the quantitative determination of preparation by fluorescence spectroscopy method in plasma and blood.
Materials and Methods. Fluorescence characteristics of vardenafil in water, ethanol, and acetonitrile solutions with a concentration of 10 mg/ml was studied.
The calibration curves were built for vardenafil concentrations in range 5–100 ng/ml and 0.1–5.0 mg/ml. The drug solutions were prepared in acetonitrile.
The optimal conditions of the vardenafil definition in plasma and blood by the fluorescence method were developed based on model mixtures, which made different amounts of the preparation (from 0.05 to 10 mcg per 1 ml sample).
Purification of biological probes were carried out on the Oasis HLB cartridges (30 mg, Waters, USA). For deproteinization of blood, it was used the 10% solution of ZnSO4.
Results. It was found that lex of vardenafil in aqueous and ethanolic solutions is 270 nm, and lem is 460–470 nm. The lex of vardenafil in acetonitrile solution is 285 nm and the fluorescence maximum is at 448–452 nm. Acetonitrile is the best solvent for fluorometric determination of vardenafil.
The detection limit of vardenafil by fluorescence spectroscopy in acenitrile solution is 4 ng/ml, and the limit of quantification is 5 ng/ml. The limit of vardenafil detection in a blood by fluorescence spectroscopy is 9 ng/ml and in plasma — 7 ng/ml. The limit of quantification in given biological fluids is 10 ng/ml. In plasma it can be determined up to 79% of the preparation by above mentioned method, and in blood (after protein precipitation with 10% solution of ZnSO4 and purification on Oasis HLB cartridges) — up to 55.0%. The relative error of vardenafil quantification in plasma is 1.72%, and in the blood — 1.68%.
Conclusions:
1. Dependence of vardenafil fluorescence intensity on the solvent nature was studied.
2. Conditions of vardenafil quantification in blood and plasma by fluorescence spectroscopy were developed.