Two fast, simple, selective and economical sample preparation methods for the determination of entecavir in biological materials available at low amounts are reported. The choice of optimal extraction techniques was performed with regard to analyte hydrophilicity, sample volumes, selectivity, method recovery and rapidity. The compatibility of the eluate with the hydrophilic interaction chromatography (HILIC) mobile phase was crucial to allow the elimination of the evaporation and reconstitution steps and to obtain acceptable peak shapes. Different types of sorbents were employed for the extraction of two biological materials (plasma and plasma ultrafiltrate). The mixed-mode polymeric sorbent MCX was chosen as a suitable one for the solid phase extraction (SPE) of plasma samples. The analytes were eluted with 1ml of the mixture of 5 % ammonium hydroxide in ACN:water (95:5). Protein precipitation (PP) with 1ml of ACN was used to remove proteins from 500μl of plasma sample prior to SPE extraction. The microextraction by packed sorbent (MEPS) was employed for the cleaning up of plasma ultrafiltrate samples due to very small volumes available for the analysis. MEPS implemented a novel sorbent based on porous graphitic carbon, semi-automatic analytical syringe and a small volume of sample (50μl). The elution step was performed using 100μl of the mixture of 5mM ammonium acetate pH 4.0:ACN (25:75). The MEPS eluate was fully compatible with HILIC mobile phase subsequently used for the analysis of entecavir, unlike SPE eluate, which had to be evaporated and reconstituted in mobile phase. Both analytical methods were validated and demonstrated good linearity in a range 1-100ng/ml (r(2)>0.9992) for plasma samples and in a range 0.5-100ng/ml (0.9991) for the plasma ultrafiltrate samples. Intra-day accuracy expressed as recovery was within the range from 80-98% for the plasma samples and 97-106% for the plasma ultrafiltrate samples. Inter-day accuracy ranged within 81-106% for the plasma and 95-101% for the plasma ultrafiltrate samples. The intra-day precision expressed as the % of RSD was lower than 4% for both matrices and inter-day precision was lower than 7% for plasma and lower than 17% for plasma ultrafiltrate. Method sensitivity reached LLOQ of 1ng/ml in plasma and 0.5ng/ml in plasma ultrafiltrate samples. The method was applied for the determination of concentration-time profiles of entecavir in plasma of the perfusate for rat kidney perfusion and for the measurement of concentration of entecavir in plasma ultrafiltrate samples. The results should be helpful in the evaluation of excretion mechanism of entecavir.

Department of Analytical Chemistry Faculty of Pharmacy Charles University Prague Heyrovského 1203 500 05 Hradec Králové Czech Republic

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