Determination of imatinib (Gleevec ®) in human plasma by solid-phase extraction–liquid chromatography–ultraviolet absorbance detection

Abstract

A sensitive HPLC method has been developed for the assay of imatinib in human plasma, by off-line solid-phase extraction followed by HPLC coupled with UV-Diode Array Detection. Plasma (750 μl), with clozapine added as internal standard, is diluted 3+1 with water and subjected to a solid-phase extraction on a C18 cartridge. After matrix components elimination with 2000 μl of water (in two aliquots of 1000 μl), imatinib is eluted with 3×500 μl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 180 μl 50% methanol. A 50 μl volume is injected onto a Nucleosil 100–5 μm C18 AB column. Imatinib is analyzed using a gradient elution program with solvent mixture constituted of methanol and water containing both 0.05% ammonium acetate. Imatinib is detected by UV at 261 nm. The calibration curves are linear between 0.1 and 10 μg/ml. The limit of quantification and detection are 0.05 and 0.01 μg/ml, respectively. The mean absolute recovery of imatinib is 96%. The method is precise with mean inter-day CVs within 1.1–2.4%, and accurate (range of inter-day deviations −0.6 to +0.7%). The method has been validated and is currently being applied in a clinical study assessing the imatinib plasma concentration variability in a population of chronic myeloid leukemia- and gastro-intestinal stromal tumor-patients.