High performance liquid chromatographic determination of thalidomide in patients affected by hepatocellular carcinoma

The present study developed a validate and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of thalidomide (T) in plasma, to quantify T in patients affected by hepatocellular carcinoma. Twelve male subjects aging from 62 to 82 years and weighting 66-88 kg, were orally administered with single dose of T (200 mg/BW). Two ml of stabilizer-solution (CH3OH/CH3CN, 1/1 (v/v) + CH3COOH 2%) were added to 1 ml of human plasma and stoked to −80 °C until analyses. This moisture (1.38 μl) was added with 20 μl of CF3COOH and 100 μl of phthalimide (IS) 1.75 μg/ml, vortexed and centrifuged. Surnatant (800 μl) was dried under vacuum at room temperature, added with 50 μl of appropriate solution and injected onto HPLC. T and IS were detected at UV wavelength of 220 nm with a run time of 10 min. Mobile phase was 10 mM pH 5.5 NH4+CH3COO−/CH3CN, 75/25 (v/v) buffer at flow rate of 1.5 ml/min. Inter-day and intra-day variation coefficient was <10% with an error of accuracy <10%. The present detection method was able to quantify T to every withdrawal time period (LOD 0.05 μg/ml). The IS used in the present study had the same wavelength maximum absorption of T, differently from early UV detection methods reported in literature where phenacetin was used. Pharmacokinetic parameters belonging from the present study are not significantly different from those calculated in previously studies performed in human health subjects and patients affected by other pathology.