Development of a liquid chromatography/negative-ion electrospray tandem mass spectrometry assay for the determination of cilnidipine in human plasma and its application to a bioequivalence study.

A simple method using a one-step liquid-liquid extraction (LLE) with methyl-t-butyl ether (MTBE) followed by high-performance liquid chromatography (HPLC) with negative-ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of cilnidipine in human plasma using benidipine as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 491.1>121.8 for cilnidipine and m/z 504.2>122.1 for IS, respectively. Analytes were chromatographed on a CN column by isocratic elution using 10mM ammonium acetate buffer-methanol (30:70, v/v; adjusted with acetic acid to pH 5.0). Results were linear (r2=0.99998) over the studied range (0.1-20ng/ml) with a total LC-MS/MS analysis time per run of 3min. The developed method was validated and successfully applied to a cilnidipine bioequivalence study in 24 healthy male volunteers.