Determination of Thalidomide in Transport Buffer for Caco-2 Cell Monolayers by High-Performance Liquid Chromatography with Ultraviolet Detectionr Caco-2 Cell Monolayers by High-Performance Liquid Chromatography with Ultraviolet Detection

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Caco-2 cell monolayers

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We report simple validated HPLC methods for the determination of thalidomide in the transport buffer for the human colonic cell line (Caco-2) cell monolayers. An aliquot of 50 μl of the mixture was injected onto a Spherex C18 column (150×4.6 mm; 5 μm) at a flow-rate of 0.5 ml/min of mobile phase consisting of acetonitrile–10 mM ammonium acetate buffer (24:76, v/v, pH 5.5), and thalidomide was detected by ultraviolet detector at a wavelength of 220 nm. Calibration curves for thalidomide were constructed at the concentration range of 0.025–1.0 and 1.0–50 μM in transport buffer. The validated methods were used to determine the transport of thalidomide by Caco-2 monolayers. The transport across the monolayers from the apical (A) to basolateral (B) side was similar to that from B to A side. The apparent permeability coefficient (Papp) values of thalidomide at 10–300 μM from the A to B and from B to A side was 2–6×10−5 cm/s, with a marked decrease in Papp values from A to B side at increased thalidomide concentration. The A to B transport appears to be dependent on temperature and sodium ion. Sodium azide, 2,4-dinitrophenol (both ATP inhibitors), 5-fluorouracil, cytidine and glutamic acid significantly inhibited the transport of thalidomide. These results indicate that the transport of thalidomide by Caco-2 monolayers was rapid, which might involve an energy-dependent mechanism.

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Journal of Chromatography B, v. 785, issue 1, p. 165-173