Chiral capillary electrophoresis–mass spectrometry of 3,4-dihydroxyphenylalanine: Evidence for its enantioselective metabolism in PC-12 nerve cells
2011
Yuan, Baiqing | Wu, Hao | Sanders, Talia | McCullum, Cassandra | Zheng, Yi | Tchounwou, Paul B. | Liu, Yi-Ming
A fully automated chiral capillary electrophoresis–tandem mass spectrometry (CE–MS/MS) method was developed for enantiomeric quantification of 3,4-dihydroxyphenylalanine (DOPA) and its precursors, phenylalanine (Phe) and tyrosine (Tyr). To avoid MS source contamination, a negatively charged chiral selector, sulfated β-cyclodextrin (sulfated β-CD), that migrated away from the detector was used in combination with the partial filling technique. The six stereoisomers were simultaneously quantified in less than 12min. Detection limits were 0.48 and 0.51μM for l- and d-DOPA enantiomers, respectively. Assay reproducibility values (relative standard deviations [RSDs], n=6) were 4.43, 3.15, 4.91, 5.16, 3.96, and 3.25% for l- and d-DOPA, l- and d-Tyr, and l- and d-Phe at 10μM, respectively. Thanks to the high enantioseparation efficiency, detection of trace d-DOPA in l-/d-DOPA mixtures could be achieved. The assay was employed to study the metabolism of DOPA, a well-known therapeutic drug for treating Parkinson’s disease. It was found that l-DOPA was metabolized effectively in PC-12 cells. Approximately 88% of l-DOPA disappeared after incubation at a cell density of 2×10⁶cells/ml for 3h. However, d-DOPA coexisting with l-DOPA in the incubation solution remained intact. The enantiospecific metabolism of DOPA in this neuronal model was demonstrated.
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