In vitro plasma protein binding determination of flunarizine using equilibrium dialysis and liquid chromatography–tandem mass spectrometry

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A highly sensitive method was developed and validated for determining the free fraction of flunarizine in human plasma. Equilibrium dialysis was used for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry (LC–MS/MS) was used for quantitation. Post-dialysis plasma or buffer samples of 0.2 mL were extracted using a liquid–liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Supelco Supelcosil ABZ + Plus column, ionized using a positive ion atmospheric pressure electrospray ionization source, and analyzed using multiple reaction monitoring. The ion transitions monitored were m/ z 405 → 203 for flunarizine and m/ z 409 → 207 for flunarizine-d4 (internal standard, IS). The chromatographic run time was 3.5 min per injection, with retention times of 2.1 min for both flunarizine and IS. The calibration curve for flunarizine was linear over the concentration range of 0.25–2000 ng/mL ( r 2 > 0.9989) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v) with the lower limit of quantitation of 0.25 ng/mL. The inter-assay coefficient of variability (CV) for the quality control samples was less than 13.5%, and the inter-assay percent nominal was greater than 98.2%. In vitro protein binding of flunarizine was determined at concentrations of 5, 10 and 100 μg/mL using the validated method. Flunarizine was extensively bound to plasma protein with a 0.083 ± 0.005% overall percent free drug in plasma and a CV value less than 7.8%. This validated method will be used for the ex vivo assessment of flunarizine protein binding in human plasma from a drug–drug interaction clinical study.