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

Emotte, Corinne and Deglave, Fanny and Heudi, Olivier and Picard, Franck and Kretz, Olivier (2011) Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with tandem liquid chromatography mass spectrometry. Journal of Pharmaceutical and biomedical analysis, 58. pp. 102-112. ISSN 0731-7085

Abstract

Fingolimod (Gilenya®; FTY720), has been recently approved for the treatment of multiple sclerosis in Europe and in the USA. In vivo, FTY720 is phosphorylated by sphingosine kinase 2 to FTY720-phosphate (FTY720-P) which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. The quantitative analysis of FTY720 and that of FTY720-P in blood represents a great challenge. Two separate methods are commonly performed because of the different chemical properties of these two compounds and due the fact that low concentrations have to be measured. 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 C18 cartridge. The samples were then eluted on a C18 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 minutes. 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. The method was validated for selectivity, dilution and the accuracy and precision data were in accordance with those specified by the FDA guidance. 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.

Item Type: Article
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Keywords: FTY720, FTY720-P, LC-MS/MS, on-line SPE and human blood
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Date Deposited: 13 Oct 2015 13:15
Last Modified: 13 Oct 2015 13:15
URI: https://oak.novartis.com/id/eprint/5157

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