How to address the sample preparation of hydrophilic compounds: Determination of entecavir in plasma and plasma ultrafiltrate with novel extraction sorbents
Abstract
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 1 ml of the mixture of 5% ammonium hydroxide in ACN:water (95:5).
Protein precipitation (PP) with 1 ml of ACN was used to remove proteins from 500 RI 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 (50 mu l). The elution step was performed using 100 mu l of the mixture of 5 mM 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.