Indirect enantioseparation of fluoxetine in mouse serum by derivatization with 1 R ‐(–)‐menthyl chloroformate followed by ultra high performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry

Here we describe a simple and sensitive analytical method for the enantioselective quantification of fluoxetine in mouse serum using ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry. The sample preparation method included a simple deproteinization with acetonitrile in 50 μL of serum, followed by derivatization of the extracts in 50 μL of 2 mM 1 R ‐(–)‐menthyl chloroformate at 45ºC for 55 min. These conditions were statistically optimized through response surface methodology using a central composite design. Under the optimized conditions, neither racemization nor kinetic resolution occurred. The derivatized diastereomers were readily resolved on a conventional sub‐2 μm C 18 column under a simple gradient elution of aqueous methanol containing 0.1% formic acid. The established method was validated and found to be linear, precise, and accurate over the concentration range of 5.0–1000.0 ng/mL for both R and S enantiomers ( r 2 > 0.993). Stability tests of the prepared samples at three different concentration levels showed that the R ‐ and S ‐fluoxetine derivatives were relatively stable for 48 h. No significant matrix effects were observed. Last, the developed method was successfully used for enantiomeric analysis of real serum samples collected at a number of time points from mice administered with racemic fluoxetine.