An improved high performance liquid chromatography-fluorescence detection method for the analysis of Pimozide in human plasma samples
Alessandra Barassi1,*, Francesca Ghilardi1, Alessandra Flaminio2, Roberta Marotta1, GianVico Melzi d'Eril1
1Laboratorio di Analisi, Ospedale San Paolo, Dipartimento di Scienze della Salute, Università degli Studi di Milano, 2Laboratorio di Analisi, Ospedale San Paolo, Milan, Italy
Introduction: Tourette Syndrome (TS) is a chronic neurodevelopmental disorder characterised by combined motor and vocal tics. Neuroleptic drugs are considered the first choice for the treatment of TS. Pimozide represents one of the alternative therapies and it is included in the atypical neuroleptic drugs. Since Pimozide has cardiotoxic and neurologic adverse effects, the therapeutic drug monitoring is essential.
Objectives: The aim of this study was to validate a sensitive, specific and reproducible method using HPLC with fluorescence detection and both small volumes of plasma sample and reduced quantities of reagents.
Methods: The chromatographic separation of Pimozide and internal standard (IS), i.e. dextromethorphan hydrobromide monohydrate, was performed under isocratic conditions on a ZORBAX Eclipse XDB-C18 column (4,5x150 mm, 5 μm particle size, Agilent Tecnologies, USA) using acetonitrilethe-20 mM monobasic sodium phosphate mixture (65:35, v/v) as a mobile phase with a flow rate of 1 mL/min. The fluorescence detection was performed at excitation wavelength of 285 nm and emission wavelength of 320 nm. Working solutions of Pimozide and IS, were obtained from Sigma (Sigma-Aldrich, Germany). The method was validated according to the European Medicines Agency Guidelines (EMA, 2012).
Results: IS and Pimozide retention times were at 2.66 and 11.56 min, respectively. Linearity was confirmed into the range 0-100 ng/mL. The precision evaluation, based on low, medium, high as well as the lower and upper limit of quantification, was satisfactory in the range tested, with relative standard deviation of 2.5-15.5% for intra-assay and 0.3-7.6% for inter-assay. The within-assay accuracy was found between -6.16 and +2.00 and the between-assay accuracy was between -5.14 and +5.90.
Conclusion: The procedures described here provide a specific, reproducible, sensitive and linear method also useful for the limited volume of plasma and reagents employed. Then it is recommended for therapeutic drug monitoring in TS patients.