Chiral bioanalysis by normal phase high-performance liquid chromatography-atmospheric pressure ionization tandem mass spectrometry

A highly sensitive and specific chiral high-performance liquid chromatographic tandem mass spectrometric (HPLC-MS-MS) technique has been developed for the determination of several chiral drugs and their metabolites. Examples include enantiomers of verapamil and its most abundant active metabolite, norverapamil, enantiomers of oxybutynin and its metabolite, N-desethyloxybutynin, enantiomers of doxazosin and those of sotalol. These drugs are marketed as racemates although their optical isomers possess different pharmacological activities. Drug enantiomers and their respective chiral metabolites were separated by isocratic normal-phase chiral chromatography on a small-bore (100X2.1 mm I.D.) Chiralpak AD column. Post-column reagent addition of an aqueous mobile phase was introduced to enable direct coupling of hexane-based mobile phases chiral chromatography with the heated nebulizer interface of the SCIEX API 300 tandem mass spectrometer. Quantification of individual drug and metabolite enantiomers was obtained by multiple reaction monitoring. This novel approach was developed and used in bioanalytical studies. It successfully combines the convenience of normal-phase chiral separations on small-bore HPLC columns with high specificity and low limits of quantification achieved by atmospheric pressure ionization MS-MS detection.