HPLC method validation and pharmacokinetic study of alendronate sodium in human urine with fluorescence detection

The purpose of this study was to validate a reliable analytical method for the pharmacokinetic study of alendronate sodium in human urine by a high performance liquid chromatography (HPLC) system with fluorescence detection. Alendronate sodium was extracted by using diethylamine (DEA) solid phase extraction (SPE) and derivatized with 9-fluorenylmethyl chloroformate (FMOC). Pamidronate was used as the internal standard. The sample was precipitated with sodium hydroxide and derivatized with FMOC in sodium carbonate buffer at pH 11.9. Separation was performed on a Capcell Pak UG C₁₈ column (4.6mm x 150mm, 5 μm particles), using a gradient method starting with mobile phase acetonitrile/methanol-citrate/pyrophosphate buffer (28:72, v/v). The fluorometric detector was operated at 260 nm (excitation) and 310 nm (emission). The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The limit of quantification was 25 ng/mL of alendronate sodium using 5 mL of urine. The calibration curve was linear in the concentration range of 25-5000 ng/mL (r² = 0.9999). Following the oral administration of 70 mg alendronate to volunteers, the cumulative amount of alendronate excreted (A_et) and peak excretion rate (U_max) were 198.39 ± 81.16 μg and 65.67 ± 20.83 μg/mL, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies of alendronate sodium in seven volunteers after oral administration (70 mg as alendronate).