A LC-MS/MS method validation for the simultaneous determination of thalidomide and its two metabolites in rabbit plasma, semen and uterine contents

<p>A determination method for thalidomide, 5-hydroxythalidomide and 5'-hydroxythalidomide in rabbit plasma, semen and uterine contents (embryo, yolk sac and placenta) was developed and validated using pomalidomide as an internal standard (IS). The uterine contents were homogenized using Shake Master NEO. All matrices were stabilized with Sorensen’s citrate buffer (pH 1.5). The analytes were extracted from biological samples by solid-phase extraction with Oasis HLB. The mobile phase consisted of acetonitrile-water (50:50) containing 0.1% acetic acid. The detection was performed using a triple quadrupole mass spectrometer TQ5500 in electrospray ionization mode. The precursor-to-product ion transitions m/z 258.9>186.0 for thalidomide and m/z 273.9>201.0 for IS at positive mode, m/z 272.9>160.9 for 5-hydroxythalidomide, m/z 272.9>146.0 for 5'-hydroxythalidomide and m/z 271.9>161.0 for IS at negative mode were used. The calibration curves were obtained in the concentrations of 0.400-400 ng/mL for thalidomide, and 0.0400-40.0 ng/mL for two metabolites in plasma. The concentration range of calibration curves was 4.00-4000 ng/g for thalidomide, and 0.400-400 ng/g for two metabolites in semen, and 0.800-800 ng/g for thalidomide, and 0.0800-80.0 ng/g for two metabolites in uterine contents. The method was validated with respect to linear, within-batch precision and accuracy. It was then applied to measure the concentrations of thalidomide and two metabolites in biological samples collected from rabbits after administration of thalidomide.</p>