Quantitative determination of thalidomide in human serum with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and ultraviolet detection.

A validated and precise reversed-phase high-performance liquid chromatographic method for the determination of thalidomide in serum, with phenacetin as an internal standard, is described. Protein precipitation, using trichloroacetic acid, was used for clean-up. The aliquot was chromatographed on a octadecyl column, using an eluent composed of 250 ml 0.01 M potassium dihydrogenphosphate, adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 750 ml methanol. Ultraviolet detection was used at an operation wavelength of 220 nm. Hydrolytic degradation was prevented during analysis by acidification of samples with the precipitation reagent. Thalidomide and phenacetin were found to have retention times of 7.9 and 15.0 min, respectively. Recoveries ranging from 79 to 84% were found for both components, with reproducibility relative standard deviations of 0.8-3% and repeatability coefficients of 1.2-3%. A mean correlation coefficient of 0.9995 was found for the linear calibration curve (n=2) of thalidomide with limits of quantitation of 0.222-21 mg/l. The method appeared to be feasible for pharmacokinetic studies with thalidomide.