Quantification of piperazine phosphate in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry employing precolumn derivatization with dansyl chloride

This paper describes a novel method that combines dansyl chloride (DNS-CL) derivatization with high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS/MS) for the sensitive and selective determination of piperazine phosphate in human plasma. After addition of ondansetron hydrochloride as internal standard (IS), piperazine phosphate was derivatized and then extracted with ethyl acetate. After being evaporated and reconstituted, the sample was analyzed using LC–ESI/MS/MS. Separation was achieved using an Agilent ZORBAX SB-C18 (150 mm × 2.1 mm I.D., 3.5 μm) column and isocratic elution with 10 mM ammonium acetate solution (pH 3.0)–methanol (50: 50, v/v). Detection was performed on a triple-quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 320 → 171 for DNS-CL–piperazine phosphate and m/z 294 → 170 for the IS. The method was fully validated for its selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability. The coefficient (r) of piperazine phosphate with a linear range of 0.1–15 μg mL−1 was 0.9974–0.9995. The limit of detection and lower limit of quantification in human plasma were 0.01 and 0.1 μg mL−1, respectively. The validated LC–ESI/MS/MS method has been successfully applied to a bioequivalence study of piperazine phosphate trochiscus in Chinese healthy male volunteers.