Ultrasound-assisted chiral derivatization of etodolac with (1R)-(−)-menthyl chloroformate for the determination of etodolac enantiomers

Abstract

This study presents the first report of an ultrasound-assisted derivatization reaction between a carboxylic acid of etodolac (ETO) and a chiral derivatization reagent, (1R)-(−)-menthyl chloroformate (R-MCF). FiftyμL of deproteinated mouse serum containing ETO enantiomers was derivatized with 125μL of 200mM R-MCF and 17μL of pyridine (a catalyst), with the reaction facilitated by ultrasonic radiation for 13min, which were the optimal conditions as determined by response surface methodology. After quenching the reaction by adding an aqueous L-proline solution, the mixture was subjected to salting-out assisted liquid–liquid extraction (SA-LLE), which provided phase separation for sample concentration as well as cleanup. The ETO diastereomers were separated on a Phenomenex Gemini C18 column (150×4.6mm, 5μm) under a simple gradient elution of a mobile phase containing a mixture of methanol: acetonitrile (10:1, V/V) and 10mM acetic acid at a flow rate of 1.0mLmin−1, followed by fluorescence detection with excitation and fluorescence emission wavelengths of 235nm and 345nm, respectively. The developed method was validated for specificity, sensitivity, linearity, accuracy, precision, stability, and matrix effect. A good linearity in the range of 0.5–50.0μgmL−1 for each ETO enantiomer with r2>0.998 and acceptable values for the intra-day and inter-day accuracy and precision as well as negligible matrix effects supported the suitability and reliability of the method. Finally, this method was used to analyze real samples taken from mice treated with (±)-ETO.