High-throughput chiral analysis of albuterol enantiomers in dog plasma using on-line sample extraction/polar organic mode chiral liquid chromatography with tandem mass spectrometric detection.

Abstract

An automated chiral chromatography/tandem mass spectrometry bioanalytical method for the determination of albuterol in dog plasma was developed. The method employed on-line sample extraction using turbulent flow chromatography coupled to a Chirobiotic T column for chiral separation using a polar organic mobile phase consisting of methanol, 0.02% formic acid, and 0.1% ammonium formate. The analytes were detected by a tandem mass spectrometer operated in positive ion mode. The (S)- and (R)-isomers were resolved chromatographically with retention times of 5.1 and 5.6 min, respectively. The analytical run time was 8 min. The enantiomers did not interconvert either in mobile phase or in dog plasma at room temperature over the course of at least 2 h. The assay has a linear dynamic range from 2.5-2500 nM for both enantiomers. The lower limit of quantitation (LLOQ) was 2.5 nM for both enantiomers using 50 microL of plasma. The accuracy and precision of intraday validation were determined at five concentration levels of six replicates. The accuracy of the method for the (R)-isomer ranged from 94-103% of nominal concentrations, and the precision (%CV) ranged from 3.6-12%. The accuracy of the method for the (S)-isomer ranged from 94.5-108% of nominal concentrations, and the precision ranged from 3.2-9.3%. Interday accuracy and precision were evaluated for three days at five concentrations for one replicate. The accuracy of the method for the (R)-isomer ranged from 98-110% of nominal concentrations, and the precision ranged from 1.5-10.6%. The accuracy of the method for the (S)-isomer ranged from 96-104% of nominal concentrations, and the precision ranged from 1.5-8.7%. The combination of turbulent flow on-line sample extraction with polar organic mode chiral chromatography provided a specific, rugged, and high-throughput method for the chiral analysis of albuterol in biological fluids.