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

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.

Conclusions

Normal phase HPLC was successfully interfaced into an API 3000 LC/MS/MS System using an APCI io
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