A reversed-phase high performance liquid chromatography method using solid phase extraction to quantitate thalidomide in human serum

Thalidomide, a drug developed in the mid 1950s for sedation and used in the treatment of leprosy for several years, has recently been shown to have anti-angiogenic activity in the rabbit cornea micropocket model. Phase II clinical trials have been initiated in several tumor types, including prostate, brain, breast, and Kaposiʹs sarcoma. Thus, we developed a high performance liquid chromatography (HPLC) assay to monitor thalidomide serum concentrations. A Hewlett-Packard 1090 Series II Liquid Chromatograph equipped with a photodiode-array detector was used for the Chromatographic analysis. A Waters Nova-Pak C-18 (3.9 × 300 mm) column was used. Thalidomide and phenacetin (internal standard) were detected at UV wavelengths of 220 and 248 nm, respectively, with a run time of 16 min. A gradient mobile phase of water, acetonitrile, and a 0.5 M NaH2PO4 buffer (pH 3.0) was run at a flow rate of 1 ml min−1. Thalidomide was isolated from serum by solid phase extraction. 10% H2SO4 (7.5 μl) was added to the serum to halt degradation. Standard curves were prepared between 50 and 10000 ng ml−1 and linearity was demonstrated in this range of concentrations (r2 = 0.9996 ± 0.0011) (n = 6). Intra-assay and inter-assay imprecision was <4.0% with an error of accuracy of <7.0%. In conclusion, the assay was shown to be reproducible and acceptable for clinical monitoring of thalidomide concentrations in human serum.