Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with liquid chromatography-tandem mass spectrometry

Fingolimod (Gilenyae (R): FTY720), has been recently approved for the treatment of multiple sclerosis in Europe and in the USA. In the present study, we have developed and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify FTY720 and FTY720-P in human blood. The sample preparation involves the sample dilution with a solution made of dimethylhexylamine (DMHA), ortho-phosphoric acid and methanol prior to the on-line solid phase extraction (SPE) on a C(18) cartridge. The samples were then eluted on a C(18) column with a gradient elution of DMHA solution and acetonitrile and analyzed by LC-MS/MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.5 min. Standard curves were linear over the ranges of 0.0800 ng/mL (LLOQ) to 16.0 ng/mL for FTY720 and 0.100 ng/mL (LLOQ) to 20.0 ng/mL for FTY720-P with correlation coefficient (r(2)) greater than 0.997. The method selectivities for FTY720 and FTY720-P were demonstrated in six different batches of human blood. Intra-run and inter-run precision and accuracy within +/- 20% (at the LLOQ) and +/- 15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). In addition, stability data obtained during freeze-thaw (3 cycles), at room temperature (24 h), and in an auto-sampler were determined and reported. The method robustness was demonstrated by the consistent data obtained by reanalyzing human blood samples for several clinical studies. In addition comparative data for FTY720 and FTY720-P were obtained between our current method and those of two available separate LC-MS/MS assays. The results of the present work demonstrated that our bioanalytical LC-MS/MS method is rapid, sensitive, specific and reliable for the simultaneous quantitative analysis of FTY720 and FTY720-P in human blood. (C) 2011 Elsevier B.V. All rights reserved.