Determination of thalidomide in transport buffer for Caco-2 cell monolayers by high-performance liquid chromatography with ultraviolet detection.
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 microl of the mixture was injected onto a Spherex C(18) column (150 x 4.6 mm; 5 microm) 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 microM 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 (P(app)) values of thalidomide at 10-300 microM from the A to B and from B to A side was 2-6 x 10(-5) cm/s, with a marked decrease in P(app) 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.
Zhou
Shufeng
S
Division of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, the University of Auckland, 85 Park Road, Grafton Private Bag 92019, New Zealand. phazsf@nus.edu.sg
Li
Yan
Y
Kestell
Phillip
P
Paxton
James W
JW
D002021:Buffers; D018938:Caco-2 Cells; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013792:Thalidomide / Q000032:analysis*
D002021:Buffers; D013792:Thalidomide