A D-carnitine dehydrogenase electrode for the assessment of enantiomeric purity of L-carnitine preparations

The enantiomeric purity of pharmaceutical L-carnitine preparations can be assessed within 60 seconds using a highly selective bienzyme electrode. D-carnitine dehydrogenase from Agrobaterium is highly specific for the nonphysiological D-enantiomer and was therefore used as the recognition element. NADH produced in the primary reaction was oxidized by salicylate hydroxylase (EC 1.14. 13.1) in an oxygen and salicylate dependent reaction. The consumption of oxygen was monitored with a miniature Clark-electrode. A linear calibration graph from 0.01 mM through 0.6 mM D-carnitine was obtained in phosphate buffer pH 8 comprising 0.5 mM concentrations of the cosubstrates NAD and salicylate. The sensitivity for DL-carnitine was exactly 50% of the respective value for pure D-carnitine, while L-carnitine and ascorbic acid, a common interferent, gave no response at all. Mixtures of both enantiomers containing 1% and 3% D-carnitine, respectively, could be distinguished from each other and from pure (i.e. >98 %) L-carnitine preparations with the new sensor. The biosensor method is faster and less laborious than established HPLC and H-1-NMR methods since it requires no chemical derivatization. The lower detection limit was 10-fold reduced as compared with a recently published enzymatic assay.