A validated enantioselective assay for the simultaneous quantitation of ( R)-, ( S)-fluoxetine and ( R)-, ( S)-norfluoxetine in ovine plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS)

Abstract

A liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantitation of ( R)-, ( S)-fluoxetine, and ( R)-, ( S)-norfluoxetine in ovine plasma. The analytes were extracted from ovine plasma at a basic pH using a single-step liquid–liquid extraction with methyl-tert-butyl ether. Chromatographic separation of all enantiomers was achieved using an AGP-chiral column with a run time of 10 min. ( R)-, ( S)-fluoxetine, and ( R)-, ( S)-norfluoxetine were quantitated at the total ion current (TIC) of multiple reaction monitoring (MRM) transitions of m/ z 310.2 → 44.1, m/ z 310.2 → 147.7 for ( R)-, ( S)-fluoxetine, and m/ z 296.2 → 30.3, m/ z 296.2 → 133.9 for ( R)-, ( S)-norfluoxetine. This method was validated for accuracy, precision, linearity, range, limit of quantitation (LOQ), selectivity, recovery, dilution integrity, matrix effect, and evaluation of carry-over. Observed accuracy ranges were as follows: ( R)-fluoxetine −8.82 to 3.75%; ( S)-fluoxetine −10.8 to 1.46%; ( R)-norfluoxetine −7.50 to 0.37% and ( S)-norfluoxetine −8.77% to −1.33%. Observed precision ranges were as follows: ( R)-fluoxetine 5.29–11.5%; ( S)-fluoxetine 3.91–11.1%; ( R)-norfluoxetine 4.32–7.67% and ( S)-norfluoxetine −8.77% to −1.33%. The calibration curves were weighted (1/ X 2, n = 4) and observed to be linear for all analytes with the following r 2 values: ( R)-fluoxetine ≥ 0.997; ( S)-fluoxetine ≥ 0.996; ( R)-norfluoxetine ≥ 0.989 and ( S)-norfluoxetine ≥ 0.994. The analytical range of the method was 1–500 ng/ml with an LOQ of 1 ng/ml for all analytes, using a sample volume of 300 μL.