Determination of Racemic Thalidomide in Human Plasma by use of an Avidin Column and Solid Phase Extraction

Abstract

A HPLC method was developed to determine racemic thalidomide in plasma on a avidin protein column using solid phase extraction. The enantiomers were separated isocratically using a mobile phase of 2:98 v/v 2-propanol: phosphate buffer (0.1M, pH 4) at a flow rate of 0.6 mL/min at ambient temperature with detection at 300 nm. The chromatographic run time was less than 13 min. Calibration curves were prepared for each enantiomer in the range 100–5000ng/mL. The correlation coefficients for each enantiomer were greater than 0.999. The method is sensitive and can be used to measure plasma blood levels (100–1000ng/mL) of each enantiomer. The % RSD for quality control samples for both enantiomers were less than 10%. A C-18 SPE cartridge was used to extract the drug from plasma and recoveries of the enantiomers were greater than 95%. The limits of quantitation and detection were 100ng/mL and 50 ng/mL (s/n <3), respectively, for each enantiomer. Two of the commercially available metabolites of thalidomide were found not to interfere with the assay procedure.