title: Development, validation and application of a sensitive LC-MS/MS method for the quantification of thalidomide in human serum, cells and cell culture medium.
abstract: A simple, robust, sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of thalidomide was developed and validated. The method was applied to thalidomide quantification in three different types of biological samples. Thalidomide was extracted from human serum (100 μL), cells (2.5 × 10(5)), or cell culture media (100 μL) by LLE and separated on a Prodigy C18 (150 mm × 4.0 mm, 5 μm i.d.) column with isocratic elution using water/acetonitrile (70/30, v/v) 0.1% formic acid, at a flow rate of 0.5 mL/min, with umbelliferone (600 ng/mL) as an internal standard. Thalidomide was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring mode using positive electrospray ionisation. The method was validated in two separate thalidomide concentration ranges; human serum (0.05-20 μg/mL) and in vitro cells (0.78-50 ng) with an inter-day precision of 1.8% and 1.9% and average accuracy of 100% and 101% in serum and cells respectively. Despite the use of small sample volume, the limit of quantification for thalidomide in serum was determined to be 3 ng/mL. The method was successfully employed to measure levels of thalidomide in cancer patient serum and cell culture model systems. Although cellular levels were quantifiable, thalidomide was shown to be unstable under in vitro conditions with a half life of approximately 2 h. In patient samples, circulating serum levels showed a broad correlation with dose and uncovered some patient compliance issues.
authors: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. sandra.roche26@mail.dcu.ie
authors: Sandra
authors: S
authors: Roche
authors: Louise
authors: L
authors: Sewell
authors: Justine
authors: J
authors: Meiller
authors: Kasper
authors: K
authors: Pedersen
authors: Rajesh
authors: R
authors: Rajpal
authors: Peter
authors: P
authors: O'Gorman
authors: Martin
authors: M
authors: Clynes
authors: Robert
authors: R
authors: O'Connor
mesh_terms: D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D004355:Drug Stability; D006801:Humans; D059625:Liquid-Liquid Extraction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013792:Thalidomide / Q000032:analysis* / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D002457:Cell Extracts; D003470:Culture Media; D013792:Thalidomide