synchronized determination of fingolimod and its primary metabolite fingolimod phosphate in whole blood: bio-equivalence approach

A novel tandem mass spectrometry (lc-ms/ms) bio-analytical method was developed and validated for the synchronized quantitation of fingolimod (fin) and its active metabolite, fingolimod-phosphate (fph), in human whole blood using a unified sample preparation protocol. this method was successfully applied in a bio-equivalence study comparing a 0.5 mg test capsule to the reference product, gilenya™, in twenty-eight healthy male subjects under fasting conditions. the study employed a randomized, single-dose, two-period, two-treatment, two-sequence crossover design with a seven-week washout period. fin and its internal standard (is) were extracted using liquid-liquid extraction, while fph was isolated from the residual blood via protein precipitation. quantitative analysis of both analytes was performed using positive electrospray ionization in multiple reaction monitoring mode (+esi-mrm), with ion transitions of m/z 388.2 → 255.3 (is: 392.12 → 259.2) for fin, and m/z 308.4 → 255.3 (is: 312.4 → 259.3) for fph. the method was validated over dynamic calibration ranges of 10-800 pg/ml for fin and 50-5000 pg/ml for fph, meeting all acceptance criteria of emea bioanalytical method validation guidelines. pharmacokinetic parameters including cmax and auc0t were determined for both fin and fph and compared between the test and reference formulations. the results confirmed that both products are bio-equivalent.