HPLC ASSAY FOR INTRACELLULAR ACCUMULATION OF VERAPAMIL IN VER-RESISTANT HUMAN LEUKEMIC CELL SUSLINES AND THEIR PARENTAL CELL LINES
Abstract
Objective: To establish a HPLC method using fluorometric detection for quantitatively determinating intracellular accumulation of verapamil (VER).
Methods: Chromatography column was packed with spherisorb ODS (250 x4.6 mm, 10 μm). The mobile phase consisted of the mixture of methanol:NaAC (0.01 mol/L): diethylamine (65:35:0.25). The detect wavelength was 280/310 nm (Ex/Em).
Results: The standard curve showed a good correlation between concentration and peak area within the range of 5-50 ng/ml. RSD was 0.86%, and recovery radio of loading sample, 100%. The detection limit for cell sample was 0.2-148 ng/ml. Intracellalar accumulation of VEIl was observed to decrease from a 13-fold to 5-fold in K562/ADM cells, and from a 3.5-fold to 4.3-fold in K562/VER cells and from a 2.1-fold to 6.5-fold in K562/ADM/VER cells, compared with the relevant control cells.
Conclusion: HPLC method was proved to be sensitive and specific for using to quantitatively determine the intracellular accumulation of VER.
Methods: Chromatography column was packed with spherisorb ODS (250 x4.6 mm, 10 μm). The mobile phase consisted of the mixture of methanol:NaAC (0.01 mol/L): diethylamine (65:35:0.25). The detect wavelength was 280/310 nm (Ex/Em).
Results: The standard curve showed a good correlation between concentration and peak area within the range of 5-50 ng/ml. RSD was 0.86%, and recovery radio of loading sample, 100%. The detection limit for cell sample was 0.2-148 ng/ml. Intracellalar accumulation of VEIl was observed to decrease from a 13-fold to 5-fold in K562/ADM cells, and from a 3.5-fold to 4.3-fold in K562/VER cells and from a 2.1-fold to 6.5-fold in K562/ADM/VER cells, compared with the relevant control cells.
Conclusion: HPLC method was proved to be sensitive and specific for using to quantitatively determine the intracellular accumulation of VER.