Measurement of Sediment (MESED) and LC-MS-MS for Cellular Kinetics Studies of STI-571(Imatinib, Gleevec® , Glivec®) in Cells with Different Pgp- 170 Expression

Abstract

During long-term treatment with STI-571 (Gleevec®, Glivec®, Imatinib), it is possible that tumor cells change their expression of proteins in order i.a. to reduce their exposure to STI-571. This would eventually result in reduced activity. In order to evaluate this possibility, we have developed an analytical technique to determine intrinsic cellular concentrations of STI-571 in tumor cells which have been manipulated to generate lines with differential expression of the multi drug resistance protein Pgp-170. A high-speed isocratic high-performance liquid chromatographic method coupled to tandem mass spectrometry for the quantification of the new anticancer agent STI-571 in cultured tumor cells is described. The method involves Measurement of Sediment (MESED) technology used for the first time in the quantitative isolation of cells from a cultured cell suspension. The sample mixture was centrifuged (10 min x 3600g), and the supernatant filtered through as HPLC filter (0,20 μm). The compounds of interest, STI-571 and the internal standard STI-571-d8 were eluted on a Waters Symmetry C18 column (50 x 2.1 mm ID, 3.5 μm particle size) using a 85/15 methanol/0.05% NH4-acetate (v/v) mixture. STI-571 and STI-571-d8 were detected by electrospray tandem mass spectrometry in the positive mode and monitored in the MRM transitions 494 > 394 and 502 > 394 respectively. The limit of quantitation of STI-571 was 5,0 ng/100 μL isolated cells containing 95 % tumor cells and 5 % medium. The recovery from the cultured tumor cells is comparable with that from RBC, i.e. ≥ 82%. The method proved to be rugged, allowing simultaneous quantification of STI-571 in cultured tumor cells and medium with sufficient precision, accuracy and sensitivity. The assay is of interest in cellular kinetic studies such as with STI-571 in CC-531 cells with different Pgp-170 expression.