A high throughput flow gradient LC-MS/MS method for simultaneous determination of fingolimod, fampridine and prednisone in rat plasma, application to in vivo perfusion study.

In this study a selective and high throughput liquid chromatography-mass spectrometry method was developed and validated for the simultaneous quantification of fingolimod (FLD), fampridine (FMP) and prednisone (PDN) in rat plasma using imipramine (IMP) as internal standard (ISTD). In this LC-MS method, following protein precipitation extraction (PPE), the analytes and ISTD were run on XBridge C18 column (150×4.6mm, 5μm) using gradient mobile phase consisting of 5mM ammonium formate in water (pH 9.0) and acetonitrile in a flow gradience program. The drug precursor and product ions were monitored on a triple quadrupole instrument that was operated in positive ionization mode. The method was validated over a concentration range of 0.1-100ng/mL for all the three analytes with relative recoveries ranging from 69 to 82%. The intra and inter batch precision (% CV) across four validation runs were less than 13.4%. The accuracy determined at four QC levels (LLOQ, LQC, MQC and HQC) were within ±6.5% of CV values. The method proved to be highly reproducible and sensitive that was successfully applied in a pharmacokinetic study after single dose oral administration to the rats and also in perfusion study sample analysis.