Abstract:

A new HPLC method has been developed for determining donepezil in human plasma. To find the optimum conditions, a derivatization reaction was performed in different media, and the reaction product was identified by NMR and GC-MS after a semi-preparative HPLC separation. Under optimized conditions, donepezil was derivatized by 9-fluorenylmethyl chloroformate in chloroform and carbonate buffer at pH 9.5 in the presence of NaI after solid-phase extraction from a plasma sample. The reaction product was quantified on a reversed-phase TRACER EXCEL ODS-A, 5 µm column using a mixture of acetonitrile–10 mM acetate buffer (pH 6.0)–THF (60:35:5, v/v/v) as the mobile phase with fluorescence detection at 264 nm (ex) and 313 nm (em). Fluoxetine was used as the internal standard. The total run-time of the analysis was about 10 min, and a clean chromatogram was obtained. The developed method was linear over the range of 1–100 ng/mL in 500 µL of plasma samples (r²>0.998). The intra-day and inter-day precision values were in the range of 2.6%–11.6%. The limit of quantification was 1 ng/mL.

Key words: 9-Fluorenylmethyl chloroformate, Derivatization, HPLC