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A rapid and sensitive High-Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC/MS/MS) method for determining apremilast in beagle dog plasma and urine samples was developed and validated using clopidogrel as the internal standard (IS). Apremilast was extracted from the plasma and urine samples by liquid–liquid extraction using methyl tert-butyl ether. Chromatographic separation was performed using a C 8  column with gradient elution and a mobile phase containing methanol and 0.1% formic acid. Quantification was achieved in multiple reaction monitoring (MRM) mode with a transition of m/z 461.3→178.2 for apremilast and m/z 322.2→184.1 for clopidogrel (IS). This method was validated regarding its specificity, linearity, precision, accuracy, and stability. The lower limit of quantification (LLOQ) for this method was 5 ng/mL, and the calibration curve was linear over 5–1,000 ng/mL. The intra- and inter-run coefficients of variance (CV) of aprelimast in plasma samples were less than 12.92% and 10.64%, respectively, while in urine samples, the CV were less than 11.84% and 10.20%, respectively. The samples were stable under the tested conditions. This method was successfully applied to a pharmacokinetic study in beagle dogs following oral administration of 10 mg of apremilast.

acta chromatographica

A rapid and sensitive High-Performance Liquid Chromatography-tandem Mass Spectrometry method for determining apremilast in beagle dog plasma and urine: Application in a pharmacokinetic study