Development and validation of a UPLC-MS/MS analytical method for dofetilide in mouse plasma and urine, and its application to pharmacokinetic study.

• We first developed a LC/MS-MS analytical method for the quantification of dofetilide in plasma and urine. • This analytical method is more accurate and precise which requires very small sample volume (10 μL for plasma, and 2 μL for urine). • We used isotope labeled dofetilide-D4 as an internal standard drug which increases method's stability and accuracy. • We first reported the pharmacokinetic profile of dofetilide in FVB strain mice administered by oral and intravenous route. • The effective extraction of dofetilide is 93.7% and 97.4% in plasma and urine samples. A novel method using UPLC with tandem mass-spectrometric detection (UPLC-MS/MS) with positive electrospray ionization was developed for the detection of the antiarrhythmic drug, dofetilide, in mouse plasma and urine. Protein precipitation was performed on 10 μL of plasma and 2 μL of urine samples using dofetilide-D4 as an internal standard, and separation of the analyte was accomplished on a C18 analytical column with the flow of 0.40 mL/min. Subsequently, the method was successfully applied to determine the pharmacokinetic parameters of dofetilide following oral and intravenous administration. The calibration curve was linear over the selected concentration range (R2 ≥ 0.99), with a lower limit of quantitation of 5 ng/mL. The intra-day and inter-day precisions, and accuracies obtained from a 5-day validation ranged from 3.00 to 7.10%, 3.80–7.20%, and 93.0–106% for plasma, and 3.50–9.00%, 3.70–10.0%, 87.0–106% for urine, while the recovery of dofetilide was 93.7% and 97.4% in plasma and urine, respectively. The observed pharmacokinetic profiles revealed that absorption is the rate-limiting step in dofetilide distribution and elimination. Pharmacokinetic studies illustrate that the absolute bioavailability of dofetilide in the FVB strain mice is 34.5%. The current developed method allows for accurate and precise quantification of dofetilide in micro-volumes of plasma and urine, and was found to be suitable for supporting in vivo pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

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