Validation and application of a liquid chromatography-tandem mass spectrometric method for quantification of the drug transport probe fexofenadine in human plasma using 96-well filter plates

Abstract

A rapid method to determine fexofenadine concentrations in human plasma using protein precipitation in 96-well plates and liquid chromatography-tandem mass spectrometry was validated. Plasma proteins were precipitated with acetonitrile containing the internal standard fexofenadine-d6, mixed briefly, and then filtered into a collection plate. The resulting filtrate was diluted and injected onto a Phenomenex Gemini C18 (50 mm x 2.0 mm, 5 microm) analytical column. The mobile phase consisted of 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (35:65, v/v). The flow rate was 0.2 ml/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization and high resolution multiple reaction monitoring mode (H-SRM). The linear standard curve ranged from 1 to 500 ng/ml and the precision and accuracy (intra- and inter-run) were within 4.3% and 8.0%, respectively. The method has been applied successfully to determine fexofenadine concentrations in human plasma samples obtained from subjects administered a single oral dose of fexofenadine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving fexofenadine.

Fig. 1

Chemical structures of (A) fexofenadine and (B) fexofenadine-d6 (internal standard).

Fig. 2

Representative extracted ion chromatograms of: (A) blank plasma (B) fexofenadine lower limit of quantitation (LLOQ; 1.00 ng/ml); (C) plasma sample from a subject obtained 48 hours after oral administration of fexofenadine 60 mg (concentration = 1.62 ng/ml); and (D) fexofenadine-d6 (ISTD).

Fig. 3

Concentration-time profile for a study subject administered single oral dose of fexofenadine (60 mg).