Abstract
l-3-Hydroxybutyrate ( l-3HB), the enantiomer of d-3-hydroxybutyrate ( d-3HB), has traditionally been regarded the “unnatural” ketone body in mammals, although there is suspicion that it is a more-favorable energy fuel for mammalian tissues than d-3HB. In this study, we demonstrated that l-3HB is an original substance in rat serum by applying fluorescence derivatization and a column-switching high-performance liquid chromatography system as the analysis technique. Racemic 3HB in rat serum derivatized using 4-nitro-7-piperazino-2,1,3-benzoxadiazole was first separated by an ODS column and was further confirmed by verifying the disappearance of the racemic 3HB peak after pretreating rat serum with d-3-hydroxybutyryl dehydrogenase. A switching valve was used to simultaneously introduce isolated racemic 3HB to the enantiomeric separation by two CHIRALCEL OD-RH columns connected in tandem. An l isomer was found to accompany the d isomer, which were quantified to be 3.98 μM (3.61%) and 106.20 μM (96.39%), respectively. Using the present analytical method, the dubious interpretation of the existence of l-3HB was clarified.
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