Determination of alendronate in human urine as 9-fluorenylmethyl derivative by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of alendronate as the 9-fluorenylmethyl derivative (FMOC) in human urine is presented. The sample preparation involved coprecipitation with calcium phosphate, separation on diethylamine (DEA) solid-phase extraction (SPE) cartridge and derivatization with 9-fluorenylmethyl chloroformate in citrate buffer pH 11.9. Liquid chromatography was performed on an octadecylsilica column (150×4.6 mm, 3 μm particles); a gradient method with starting mobile phase acetonitrile–methanol–citrate/pyrophosphate buffer (20:15:65 v/v) was employed. The total run time was 21 min. The fluorimetric detector was operated at the following wavelengths: 260 nm (excitation) and 310 nm (emission). Pamidronate was used as the internal standard. The limit of quantitation was 3.5 ng/ml using 5 ml of urine. Within-day and between-day precision expressed by relative standard deviation was less than 8% and inaccuracy did not exceed 9%. The assay was applied to the analysis of samples from a pharmacokinetic study.