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Separation and Quantitation of R and S Enantiomers using Normal Phase Chromatography on an API 3000 LC/MS/MS System


Normal phase chromatography can be used for the analysis of chiral compounds using the API 3000 LC/MS/MS System.


In recent years, pharmaceutical companies have placed greater importance on examining the stereoisomeric composition of drugs having a chiral center. Isomerspecific pharmaceuticals often exhibit increased potency, higher bioavailability and reduced side effects when compared to racemic pharmaceutical compounds. Previously, chiral compounds could not be separated by conventional HPLC techniques without derivatizing enantiomers into diastereomers. Todays improved chiral stationary phases allow for separation and quantitation of these enantiomers. These chiral HPLC columns are amenable to normal phase chromatography. This technique was not often utilized in LC/MS in the past as there is a serious non-polar mobile phase incompatibility with atmospheric pressure ionization mass spectrometry. Mobile phases primarily consisting of hexane present an explosion hazard when paired with the extreme temperatures of atmospheric pressure chemical ionization (APCI).

The experiment outlined here demonstrates that it is possible to safely and effectively perform normal phase chromatography by APCI LC/MS/MS using an API 3000 triple quadrupole mass spectrometer.

Key Features

Excellent APCI performance across a wide range of flow rates and mobile phase compositions

Simplified MS/MS tuning

Analyst quantitation software for fast and easy data acquisition and reduction


The goal was to separate and quantitate the precise amount of R and S enantiomers of three commonly used drugs found in biological fluids of animals dosed with racemic mixtures. These drugs include the an tidepressant fluoxetine (Prozac), methylphenidate (Ritalin)used to treat attention deficit disorder, and the coronary vasodilator, verapamil.

These compounds were administered to lab rats at a dose of 1 mg/kg body weight for verapamil and 10 mg/kg for fluoxetine and methylphenidate. Blood samples were drawn at specific time intervals and centrifuged. Samples containing fluoxetine and verapamil were prepared using a simple protein precipitation whereas methylphenidate required a liquidliquid extraction.

Results and Discussion

Analyst software was used for data acquisition and instrument control. Calibration curves were prepared for the R and S entantiomers of each drug. Verapamil and methylphenidate showed excellent response and linearity over the concentration range of 01,000 ng/mL. Fluoxetine demonstrated linearity over a larger range, 010,000 ng/mL. Verapamil and methylphenidate both exhibited good response and separation of the R and S enantiomers with relatively short chromatographic runs. Both enantiomers of verapamil were separated in under 8 minutes. Methylphenidate enantiomers were separated in just under 9 minutes. Fluoxetine required a significantly longer retention time for enantiomer separation, approximately 65 minutes. The extensive retention time and higher Diethylamine (DEA) concentration adversely affected assay sensitivity for fluoxetine. To overcome this mobile phase incompatibility, a switching valve was incorporated to replace DEA containing mobile phase with mobile phase void of DEA over the analyte elution period, resulting in a 20-fold sensitivity increase. Improving HPLC column selectivity and stability could potentially address the need for a long retention time and further improve sensitivity.


Normal phase HPLC was successfully interfaced into an API 3000 LC/MS/MS System using an APCI io n source without any hardware modifications or post-flow column addition. To eliminate potential explosion hazards associated with normal phase APCI LC/MS/MS, nitrogen gas was utilized for the auxiliary and nebulizer gas. This experiment resulted in a sensitive chiral analysis of verapamil and methylphenidate with an assay sensitivity for each enantiomer of 0.05 ng/mL. Fluoxetine did not exhibit similar sensitivity due to poor column performance requiring long retention times and high DEA levels to achieve a reproducible enantiomer separation.



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