METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTITATIVE DETERMINATION OF TAFAMIDIS IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY

Srinivasa Reddy, Nirmala Nayak, Licto Thomas, Imran Ahmed M, Arindam Mukhopadhyay*, Saral Thangam

Tafamidis is a selective transthyretin (TTR) stabilizer indicated for the treatment of transthyretin amyloidosis in adult patients presenting with early-stage symptomatic polyneuropathy. In the present study, a rapid, sensitive, and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of tafamidis in human plasma. Plasma samples were processed using a liquid–liquid extraction procedure employing tert-butyl methyl ether. Chromatographic separation was achieved on a reversed-phase C18 column using a mobile phase comprising 5mM Ammonium acetate in 0.025% Formic acid and organic mixture (Acetonitrile: Methanol:: 75:25 v/v) at a flow rate of 1.2 mL/min. Detection was performed using multiple reaction monitoring (MRM) in negative electrospray ionization mode, monitoring the transitions m/z 305.4 → 261.4 for tafamidis and m/z 312.0 → 267.7 for the 13C6 containing internal standard. The method demonstrated a lower limit of quantification (LLOQ) of 10 ng/mL and exhibited linearity over the concentration range of 10–10,000 ng/mL with a correlation coefficient (R²) greater than 0.99. Validation parameters, including selectivity, precision, accuracy, matrix effect, extraction recovery, and stability, complied with established regulatory acceptance criteria. The validated method was successfully applied to the pharmacokinetic evaluation of tafamidis in human subjects.